Journal articles on the topic 'Blood platelets'
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1
Franco, Aime T., Adam Corken, and Jerry Ware. "Platelets at the interface of thrombosis, inflammation, and cancer." Blood 126, no. 5 (July 30, 2015): 582–88. http://dx.doi.org/10.1182/blood-2014-08-531582.
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Abstract Although once primarily recognized for its roles in hemostasis and thrombosis, the platelet has been increasingly recognized as a multipurpose cell. Indeed, circulating platelets have the ability to influence a wide range of seemingly unrelated pathophysiologic events. Here, we highlight some of the notable observations that link platelets to inflammation, reinforcing the platelet’s origin from a lower vertebrate cell type with both hemostatic and immunologic roles. In addition, we consider the relevance of platelets in cancer biology by focusing on the hallmarks of cancer and the ways platelets can influence multistep development of tumors. Beyond its traditional role in hemostasis and thrombosis, the platelet’s involvement in the interplay between hemostasis, thrombosis, inflammation, and cancer is likely complex, yet extremely important in each disease process. The existence of animal models of platelet dysfunction and currently used antiplatelet therapies provide a framework for understanding mechanistic insights into a wide range of pathophysiologic events. Thus, the basic scientist studying platelet function can think beyond the traditional hemostasis and thrombosis paradigms, while the practicing hematologist must appreciate platelet relevance in a wide range of disease processes.
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Heazlewood, Shen Y., Tanveer Ahmad, Monika Mohenska, Belinda B. Guo, Pradnya Gangatirkar, Emma C. Josefsson, Sarah L. Ellis, et al. "The RNA-binding protein SRSF3 has an essential role in megakaryocyte maturation and platelet production." Blood 139, no. 9 (March 3, 2022): 1359–73. http://dx.doi.org/10.1182/blood.2021013826.
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Abstract RNA processing is increasingly recognized as a critical control point in the regulation of different hematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that serine-arginine–rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterized by megakaryocyte maturation arrest, dramatically reduced platelet counts, and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs, demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.
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D’Ambrosi, Silvia, R. Jonas Nilsson, and Thomas Wurdinger. "Platelets and tumor-associated RNA transfer." Blood 137, no. 23 (May 3, 2021): 3181–91. http://dx.doi.org/10.1182/blood.2019003978.
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Abstract Until recently, the nucleic acid content of platelets was considered to be fully determined by their progenitor megakaryocyte. However, it is now well understood that additional mediators (eg, cancer cells) can intervene, thereby influencing the RNA repertoire of platelets. Platelets are highly dynamic cells that are able to communicate and influence their environment. For instance, platelets have been involved in various steps of cancer development and progression by supporting tumor growth, survival, and dissemination. Cancer cells can directly and/or indirectly influence platelet RNA content, resulting in tumor-mediated “education” of platelets. Alterations in the tumor-educated platelet RNA profile have been described as a novel source of potential biomarkers. Individual platelet RNA biomarkers as well as complex RNA signatures may be used for early detection of cancer and treatment monitoring. Here, we review the RNA transfer occurring between cancer cells and platelets. We explore the potential use of platelet RNA biomarkers as a liquid biopsy biosource and discuss methods to evaluate the transcriptomic content of platelets.
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Delgado Lagos, Fredy, Amro Elgheznawy, Anastasia Kyselova, Dagmar Meyer zu Heringdorf, Corina Ratiu, Voahanginirina Randriamboavonjy, Alexander W. Mann, Beate Fisslthaler, Mauro Siragusa, and Ingrid Fleming. "Secreted modular calcium-binding protein 1 binds and activates thrombin to account for platelet hyperreactivity in diabetes." Blood 137, no. 12 (March 25, 2021): 1641–51. http://dx.doi.org/10.1182/blood.2020009405.
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Abstract Secreted modular calcium-binding protein 1 (SMOC1) is an osteonectin/SPARC-related matricellular protein, whose expression is regulated by microRNA-223 (miR-223). Given that platelets are rich in miR-223, this study investigated the expression of SMOC1 and its contribution to platelet function. Human and murine platelets expressed SMOC1, whereas platelets from SMOC1+/− mice did not present detectable mature SMOC1 protein. Platelets from SMOC1+/− mice demonstrated attenuated responsiveness to thrombin (platelet neutrophil aggregate formation, aggregation, clot formation, Ca2+ increase, and β3 integrin phosphorylation), whereas responses to other platelet agonists were unaffected. SMOC1 has been implicated in transforming growth factor-β signaling, but no link to this pathway was detected in platelets. Rather, the SMOC1 Kazal domain directly bound thrombin to potentiate its activity in vitro, as well as its actions on isolated platelets. The latter effects were prevented by monoclonal antibodies against SMOC1. Platelets from miR-223–deficient mice expressed high levels of SMOC1 and exhibited hyperreactivity to thrombin that was also reversed by preincubation with monoclonal antibodies against SMOC1. Similarly, SMOC1 levels were markedly upregulated in platelets from individuals with type 2 diabetes, and the SMOC1 antibody abrogated platelet hyperresponsiveness to thrombin. Taken together, we have identified SMOC1 as a novel thrombin-activating protein that makes a significant contribution to the pathophysiological changes in platelet function associated with type 2 diabetes. Thus, strategies that target SMOC1 or its interaction with thrombin may be attractive therapeutic approaches to normalize platelet function in diabetes.
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Sen Gupta, Anirban. "Synthetic Platelets for Treatment of Traumatic Hemorrhage and Thrombocytopenia." Blood 134, Supplement_1 (November 13, 2019): SCI—37—SCI—37. http://dx.doi.org/10.1182/blood-2019-121079.
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Platelets are primarily responsible for staunching bleeding by forming a 'platelet plug' and further amplifying thrombin generation on its surface to facilitate fibrin formation, leading to hemostatic clot formation at the site of vascular breach. Therefore, platelet transfusions are clinically used to mitigate bleeding risks in thrombocytopenia (prophylactic transfusion) and to mitigate hemorrhage in traumatic injuries (emergency transfusion). Currently these transfusions utilize donor-derived platelets, stored at 20-24oC with gentle agitation. In this condition, platelets have high risk of bacterial contamination and very short shelf-life (~ 5 days), which severely limit their logistical availability and use. Several parallel strategies are currently undergoing research to address these issues, including platelet storage at reduced temperatures (chilled or freeze-dried), pathogen reduction technologies and bioreactor-based in vitro platelet production from precursor cells. An alternative (and complimentary) approach that is the focus of our research is the engineering of I.V.-administrable synthetic hemostat nanoparticles that functionally mimic platelet's clotting mechanisms. These 'synthetic platelet' nanoparticle systems can be manufactured at large scale, sterilized without compromising functions and stored for long periods of time (6-9 months), thereby allowing significant logistical advantages in transfusion applications. Here we present in vitro and in vivo evaluation of such technology. For these studies, the 'synthetic platelet' nanoparticles were manufactured by decorating liposomes with a combination of VWF-binding, collagen-binding and fibrinogen-mimetic peptides, for integrative mimicry of platelet's hemostasis-relevant adhesive and aggregatory mechanisms. The nanoparticles were stored at room temperature in aqueous suspension as well as lyophilized powder, and particle stability was assessed over 6-9 months by dynamic light scattering (DLS). The nanoparticles were also exposed to E-beam sterilization, and particle stability as well platelet-mimetic bioactivity was assessed by DLS, aggregometry, microfluidics and rotational thromboelastometry (ROTEM). The systemic safety and targeted hemostatic efficacy of I.V.-administered nanoparticles were evaluated in mouse model of thrombocytopenia, and in mouse, rat and pig models of traumatic hemorrhage. DLS and electron microscopy confirmed that the synthetic platelet nanoparticles have a size of 150-200 nm diameter, and they remain stable over 6-9 months in storage. Microfluidic studies showed that these nanoparticles could rapidly adhere to 'vWF + collagen'-coated surfaces and enhance the recruitment and aggregation of active platelets on these surfaces. Aggregometry studies showed that the nanoparticles did not affect resting platelets but enhanced aggregation of ADP- or collagen-activated platelets (i.e. no thrombotic risk towards resting platelets). Flow cytometry studies confirmed this specificity of nanoparticle binding to active platelets. ROTEM studies showed that the 'synthetic platelet' nanoparticles significantly improved clot kinetics and firmness. In vivo, in all animal models, the nanoparticles showed no systemic pro-thrombotic effects, as assessed by hemodynamics as well as organ histology. In thrombocytopenic mice, prophylactically administered 'synthetic platelet' nanoparticles dose-dependently reduced tail bleeding time. In mouse, rat and pig trauma models, post-injury administration of 'synthetic platelet' nanoparticles reduced blood loss, stabilized blood pressure, delayed hypotension and thereby significantly improved survival. The nanoparticles could be further utilized as a platform for targeted presentation of phosphatidylserine (PS) to augment thrombin generation, or targeted delivery of tranexamic acid (TXA) for anti-fibrinolytic effect or delivery of inorganic polyphosphate (PolyP) to augment clot stability. These studies not only establish the potential of these nanoparticles as a platelet surrogate for transfusion applications, but also demonstrate their utilization as a platform for modular augmentation of various hemostatic outputs in prophylactic and emergency applications. Figure Disclosures Sen Gupta: Haima Therapeutics LLC: Equity Ownership.
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Maurya, Preeti, Sara Ture, Kathleen E. McGrath, James Palis, and Craig N. Morrell. "Adult, but Not Neonatal, Platelet Transfusions Drive a Monocyte Trafficking Phenotype in Vitro and In Vivo." Blood 138, Supplement 1 (November 5, 2021): 2144. http://dx.doi.org/10.1182/blood-2021-152913.
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Abstract Although, thrombocytopenia can affect all age groups, neonates, especially pre-term, have an increased incidence of thrombocytopenia. Platelet transfusions may reduce the bleeding risk in neonates, but are also associated with adverse short and long-term immune and inflammatory outcomes. A randomized trial of platelet transfusions in neonates found that transfusion was associated with an increased risk of necrotizing enterocolitis, unilateral/bilateral retinopathy, and bronchopulmonary dysplasia. Past work from our research team found that neonatal platelets expressed lower levels of mRNA for many immune related molecules compared to adult platelets. We therefore sought to determine whether the transfusion of adult platelets to neonates resulted in developmental immune dysregulation, with a focus on platelet and monocyte interactions. To explore the interactions between monocytes and platelets, we isolated monocytes from adult mouse bone marrow and co-incubated monocytes with adult (>8 weeks old) or neonatal mouse platelets (7 days old mice) and determined inflammatory and trafficking monocyte phenotypes by flow cytometry and qRT-PCR. Monocytes treated with adult platelets had an increased inflammatory (Ly6C hi) and trafficking phenotype (CCR2 hi), while monocytes treated with neonatal platelets adopted an inflammatory, but not trafficking phenotype. As expected, adult platelets increased the expression of monocyte inflammatory (Nos2, Cxcl1, Ccl2) and trafficking (Ccr2) mRNA, while neonatal platelets also increased inflammatory mRNA expression, but did not increase Ccr2 expression. Adult platelets express more Selp (P-selectin) than neonatal platelets and P-selectin is a major mediator of platelet and monocyte interactions. We confirmed that adult platelets expressed more P-selectin protein compared to neonatal platelets, and found that blocking P-selectin decreased adult platelet induced CCR2 expression to levels similar to monocytes treated with neonatal platelets. Using a transwell chamber we assessed adult and neonatal platelet effects on monocyte migration towards the CCR2 ligand CCL2. Monocytes were treated with adult platelets had significantly greater monocyte migration compared to monocytes co-incubated with neonatal platelets. To model platelet transfusions in the setting of thrombocytopenia, we used 14d old thrombopoietin receptor knockout mice (TPOR -/-) that have low platelet counts, and infused adult or neonatal platelets. We observed a significant increase in inflammatory and trafficking monocytes in mice transfused with adult platelets compared to those transfused with neonatal platelets. Using an in vivo model of monocyte chemotaxis, mice were treated with CCL2 intraperitoneal after platelet transfusion. Adult platelet transfusions, but not neonatal, increased monocyte peritoneal trafficking to CCL2. These data provide comparative insights as to how adult and neonatal platelet transfusions regulate monocyte functions. Adult platelet transfusions to neonates are associated with an inflammatory and trafficking monocyte phenotype that is platelet P-selectin dependent and may have a major impact on neonatal platelet transfusion complications. Disclosures Palis: Rubius Therapeutics: Consultancy.
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Denorme, Frederik, Bhanu Kanth Manne, Irina Portier, Alicia S. Eustes, Yasuhiro Kosaka, Benjamin T. Kile, Matthew T. Rondina, and Robert A. Campbell. "Platelet necrosis mediates ischemic stroke outcome in mice." Blood 135, no. 6 (February 6, 2020): 429–40. http://dx.doi.org/10.1182/blood.2019002124.
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Abstract Dysregulated platelet functions contribute to the development and progression of ischemic stroke. Utilizing mice with a platelet-specific deletion of cyclophilin D (CypD), a mediator of necrosis, we found that platelet necrosis regulates tissue damage and outcomes during ischemic stroke in vivo. Mice with loss of CypD in platelets (CypDplt−/−mice) exhibited significantly enhanced cerebral blood flow, improved neurological and motor functions, and reduced ischemic stroke infarct volume after cerebral ischemia-reperfusion injury. These effects were attributable, at least in part, to platelet-neutrophil interactions. Twenty-four hours after stroke, significantly more circulating platelet-neutrophil aggregates (PNAs) were found in CypDplt+/+ mice. Underscoring the role of platelet necrosis in PNA formation, we observed a significant number of phosphatidylserine (PS)+ platelets in PNAs in CypDplt+/+ mice. In contrast, significantly fewer platelets in PNAs were PS+ in CypDplt−/− counterparts. Accordingly, mice with CypD-deficient platelets had fewer neutrophils and PNAs recruited to their brain following stroke relative to wild-type counterparts. Neutrophil depletion in wild-type mice conferred protection from ischemic stroke to a similar degree as observed in mice with CypD-deficient platelets. Neutrophil depletion in CypDplt−/− mice did not further reduce infarct size. Transmission electron microscopy of ex vivo–formed PNAs revealed a propensity of necrotic platelets to interact with neutrophils. These results suggest that necrotic platelets interact with neutrophils to exacerbate brain injury during ischemic stroke. Because inhibiting platelet necrosis does not compromise hemostasis, targeting platelet CypD may be a potential therapeutic strategy to limit brain damage following ischemic stroke.
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Jacob, Shancy, Marina Tistao, Abigail Ajanel Gomez, Frederik Denorme, Yasuhiro Kosaka, Bhanu Kanth Manne, Li Guo, Robert A. Campbell, Matthew T. Rondina, and Jesse W. Rowley. "Mitochondrial Fission Protein Drp1 Regulates Platelet Function." Blood 142, Supplement 1 (November 28, 2023): 1201. http://dx.doi.org/10.1182/blood-2023-187556.
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Background: Mitochondria in platelets contribute to platelet function and are altered by diseases that involve dysregulated hemostasis or thrombosis. Mitochondrial integrity, governed by its size, number and distribution is modulated by fusion and fission proteins. Pharmacological inhibition of the mitochondrial fission protein Dynamin-related protein 1 (Drp1) has been shown to inhibit granule release in human platelets and impair thrombus formation in a murine model of thrombosis. However, the mitochondrial roles for Drp1 in platelet function are still largely unknown. Objective: To determine the mitochondrial roles of Drp1 in regulating platelet function. Methods: PF4-Cre mediated MK/platelet specific Drp1 null mice (Drp1 -/-) were used. Platelet morphology in Drp1 -/- and wild type (WT) platelets was analyzed by confocal microscopy. Procoagulant platelet formation (PS +ve platelets) after thrombin and the GPVI agonist convulxin (THR+CVX) stimulation was measured using Annexin V staining. Mitochondrial potential (ΔΨm) and mass at baseline and after stimulation was measured using TMRM and mitotracker green. Washed platelet activation in WT and Drp1 -/- platelets in response to convulxin was measured by flow cytometry. Platelet cytoplasmic calcium transients were measured using FURA-2AM after stimulation with THR+CVX. Phosphorylation of Drp1 (pDrp1 S616) in washed WT platelets in response to dual agonist THR+CVX was measured by western blotting. Results: Drp1 -/- platelets were bigger in size than WT platelets as measured by microscopy and had increased mitochondrial mass as determined by flow cytometry. Absence of Drp1 reduced activation of the αIIb/β3 fibrinogen binding receptor (P=0.035; n=5-6 per group) and reduced alpha granule release (P=0.025; n=5-6 per group) in response to CVX.Dual agonist stimulation resulted in reduced PS +ve platelets in Drp1 -/- compared to WT platelets (P=0.0035; n=7 per group). Since depolarization precedes PS exposure, we measured ΔΨm using TMRM in resting and dual agonist stimulated WT and Drp1 -/- platelets. No difference in ΔΨm was observed in resting Drp1 -/- vs WT platelets whereas, stimulated Drp1 -/- platelets exhibited significantly higher ΔΨm (P=0.038; n=4 per group) than stimulated WT platelets. Interestingly, in response to dual agonists, Drp1 -/- platelets also showed a significant increase (P<0.001) in cytoplasmic calcium transients than WT platelets. Western blot analyses of washed WT platelets stimulated with dual agonist showed phosphorylation of Drp1 at S616 (S616), indicating Drp1 activation. Conclusion: These findings highlight a role for Drp1 in murine platelet mitochondrial function, GPVI activation, and procoagulant platelet formation.
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Rinder, HM, JL Bonan, CS Rinder, KA Ault, and BR Smith. "Dynamics of leukocyte-platelet adhesion in whole blood." Blood 78, no. 7 (October 1, 1991): 1730–37. http://dx.doi.org/10.1182/blood.v78.7.1730.1730.
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Abstract The dynamics of leukocyte-platelet adhesion and platelet-platelet interaction in whole blood are not well understood. Using different platelet agonists, we have studied the whole blood kinetics of these heterotypic and homotypic interactions, the relative abilities of different leukocyte subsets to participate in platelet adhesion, and the ligands responsible for adhesion. When platelet aggregation was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide, thrombin stimulation of whole blood resulted in platelet expression of granule membrane protein 140 (GMP-140) and, simultaneously, a marked increase in the percentage of monocytes and neutrophils (PMN) binding platelets, as well as an increase in the number of platelets bound per monocyte and PMN. Lymphocytes were unaffected. Monocytes bound more platelets and at an initially faster rate than PMN. This increase in monocyte and PMN adhesion to platelets was completely inhibited by the blocking monoclonal antibody (MoAb), G1, to GMP-140. When the combination of epinephrine and adenosine diphosphate (epi/ADP) was used as a less potent agonist in the presence of RGDS, GMP-140 expression per platelet was less, and while monocyte-platelet conjugates formed, PMN-platelet conjugates did not. With epi/ADP in the absence of RGDS, there was an immediate, marked decrease in the percentage of all leukocytes with bound platelets, simultaneous with an increase in the percentage of unbound platelet aggregates. As these platelet aggregates dissociated, the percentage of monocytes and PMN with adherent platelets increased, with monocytes again binding at a faster initial rate than PMN. This recovery of monocyte and PMN adhesion to platelets was also inhibited by the G1 MoAb. We conclude that: (1) monocytes and PMN bind activated platelets in whole blood through GMP-140; (2) monocytes have a competitive advantage over PMN in binding activated platelets, particularly when less potent platelet agonists are used; and (3) platelet aggregate formation initially competes unactivated platelets off leukocytes; subsequent aggregate dissociation allows the now activated platelets to readhere to monocytes and PMN through GMP-140. These studies further elucidate the dynamic interaction of blood cells and possible links between coagulative and inflammatory processes.
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Rinder, HM, JL Bonan, CS Rinder, KA Ault, and BR Smith. "Dynamics of leukocyte-platelet adhesion in whole blood." Blood 78, no. 7 (October 1, 1991): 1730–37. http://dx.doi.org/10.1182/blood.v78.7.1730.bloodjournal7871730.
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The dynamics of leukocyte-platelet adhesion and platelet-platelet interaction in whole blood are not well understood. Using different platelet agonists, we have studied the whole blood kinetics of these heterotypic and homotypic interactions, the relative abilities of different leukocyte subsets to participate in platelet adhesion, and the ligands responsible for adhesion. When platelet aggregation was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide, thrombin stimulation of whole blood resulted in platelet expression of granule membrane protein 140 (GMP-140) and, simultaneously, a marked increase in the percentage of monocytes and neutrophils (PMN) binding platelets, as well as an increase in the number of platelets bound per monocyte and PMN. Lymphocytes were unaffected. Monocytes bound more platelets and at an initially faster rate than PMN. This increase in monocyte and PMN adhesion to platelets was completely inhibited by the blocking monoclonal antibody (MoAb), G1, to GMP-140. When the combination of epinephrine and adenosine diphosphate (epi/ADP) was used as a less potent agonist in the presence of RGDS, GMP-140 expression per platelet was less, and while monocyte-platelet conjugates formed, PMN-platelet conjugates did not. With epi/ADP in the absence of RGDS, there was an immediate, marked decrease in the percentage of all leukocytes with bound platelets, simultaneous with an increase in the percentage of unbound platelet aggregates. As these platelet aggregates dissociated, the percentage of monocytes and PMN with adherent platelets increased, with monocytes again binding at a faster initial rate than PMN. This recovery of monocyte and PMN adhesion to platelets was also inhibited by the G1 MoAb. We conclude that: (1) monocytes and PMN bind activated platelets in whole blood through GMP-140; (2) monocytes have a competitive advantage over PMN in binding activated platelets, particularly when less potent platelet agonists are used; and (3) platelet aggregate formation initially competes unactivated platelets off leukocytes; subsequent aggregate dissociation allows the now activated platelets to readhere to monocytes and PMN through GMP-140. These studies further elucidate the dynamic interaction of blood cells and possible links between coagulative and inflammatory processes.
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Lievens, Dirk, Alma Zernecke, Tom Seijkens, Oliver Soehnlein, Linda Beckers, Imke C. A. Munnix, Erwin Wijnands, et al. "Platelet CD40L mediates thrombotic and inflammatory processes in atherosclerosis." Blood 116, no. 20 (November 18, 2010): 4317–27. http://dx.doi.org/10.1182/blood-2010-01-261206.
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Abstract CD40 ligand (CD40L), identified as a costimulatory molecule expressed on T cells, is also expressed and functional on platelets. We investigated the thrombotic and inflammatory contributions of platelet CD40L in atherosclerosis. Although CD40L-deficient (Cd40l−/−) platelets exhibited impaired platelet aggregation and thrombus stability, the effects of platelet CD40L on inflammatory processes in atherosclerosis were more remarkable. Repeated injections of activated Cd40l−/− platelets into Apoe−/− mice strongly decreased both platelet and leukocyte adhesion to the endothelium and decreased plasma CCL2 levels compared with wild-type platelets. Moreover, Cd40l−/− platelets failed to form proinflammatory platelet-leukocyte aggregates. Expression of CD40L on platelets was required for platelet-induced atherosclerosis as injection of Cd40l−/− platelets in contrast to Cd40l+/+ platelets did not promote lesion formation. Remarkably, injection of Cd40l+/+, but not Cd40l−/−, platelets transiently decreased the amount of regulatory T cells (Tregs) in blood and spleen. Depletion of Tregs in mice injected with activated Cd40l−/− platelets abrogated the athero-protective effect, indicating that CD40L on platelets mediates the reduction of Tregs leading to accelerated atherosclerosis. We conclude that platelet CD40L plays a pivotal role in atherosclerosis, not only by affecting platelet-platelet interactions but especially by activating leukocytes, thereby increasing platelet-leukocyte and leukocyte-endothelium interactions.
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Murphy, Lauren, Jeanne Inchauspe, Nikolaos Sousos, Natalie Jooss, Hayley L. Belnoue-Davis, Pamela Holland, Rong Li, et al. "Platelets Sequester Extracellular DNA, Capturing Tumour-Derived and Free Fetal DNA." Blood 142, Supplement 1 (November 28, 2023): 277. http://dx.doi.org/10.1182/blood-2023-182697.
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Introduction & Hypothesis Platelets are small, multi-functional cells that lack a nucleus but contain RNA and translational machinery for protein synthesis. Platelet RNA mostly derives from parent megakaryocytes, but they also sense and sequester endogenous and pathogen-derived nucleic acids during circulation (D'Ambrosi, 2021; Koupenova, 2019). Nucleated cells release DNA after cell death or aberrant mitosis, resulting in ‘cell-free’ DNA in plasma (cfDNA). Excess cfDNA is deleterious. cfDNA isolated from platelet-poor plasma is emerging as a major liquid biopsy tool in cancer and antenatal screening, but a major limitation is its low abundance, especially in early-stage disease. Given their role in sensing pathogen-derived nucleic acids, we hypothesized that platelets may clear cfDNA from plasma, and that clinically-relevant insights may be derived from the analysis of DNA fragments contained in platelets. Methods Live/fixed imaging using specific DNA probes and fluorescence in-situ hybridization (FISH), droplet digital PCR (ddPCR), NGS and flow cytometry were applied to platelets isolated from Streck, EDTA tubes or apheresis bags from healthy donors, pregnant women, patients with cancer and murine platelets from cancer models. DNA was extracted from platelet pellets or platelet-depleted plasma (cfDNA) using the Qiagen QIAamp Circulating Nucleic Acid Kit. Results Using live cell imaging and flow cytometry, significant DNA content (Draq5+) was detected in ~8% of platelets from healthy donors, including in platelets with low RNA content (SYTO-13-negative). Overall Draq5+ platelets were larger and primed for agonist activation. FISH and ddPCR of platelets from pregnant women carrying male fetuses detected Y-chromosome fragments (n=10), confirming that platelet DNA is not solely derived from parent megakaryocytes but also sequestered during circulation (Fig. 1a). In contrast, Y-chromosome genes were not detected in DNA isolated from mononuclear cells or red blood cells pellets. Acute platelet depletion in an ITP mouse model (n=20) led to a >2-fold increase in cfDNA extracted from platelet-poor plasma, suggesting a role for platelets in plasma DNA clearance. Platelet uptake in vitro was rapid, visible using live cell microscopy within 2 minutes of co-culture with cancer cells labelled with a probe that irreversibly intercalates to nuclear DNA. ddPCR of platelet DNA following co-culture of healthy donor platelets with cancer cell lines detected a range of canonical cancer driver mutations, including in PI3K, BRAF and JAK2. DNase treatment of co-cultured platelets did not reduce mutant allele content, indicating that platelets encapsulate DNA and protect it from degradation. We explored the mechanism of platelet DNA uptake. Platelets internalized DNA contained in extracellular vesicles (EVs). Platelet uptake of “free” DNA was also confirmed, with uptake of synthetic DNA fragments of varying lengths (120 bp to 650 bp). Importantly, platelet DNA detection was enhanced following exocytosis inhibition, confirming that platelet DNA uptake and release is an active process. Whole genome sequencing revealed that platelets contain a repertoire of DNA fragments that map across the nuclear genome, similar to cfDNA, and that the majority of platelet DNA is of nuclear not mitochondrial origin. We also found tumor-derived DNA showing multiple copy number aberrations in a patient with pancreatic carcinoma. In a transgenic mouse model of colorectal cancer, mutant KRASG12D alleles were readily detectable in platelet DNA and, notably, in higher abundance in platelets than platelet-poor plasma (cfDNA) in 11/16 mice (Fig. 1b). Similarly, KRASG12D was detected in platelets of mice with orthotopic pancreatic adenocarcinoma (n=4). Finally, to explore utility in cancer screening, we analysed patients with high-risk, pre-malignant colonic lesions (serrated polyps). Remarkably, the driver mutation BRAFV600E was detected in platelets in 16% patients despite the small-size lesions (5/30, Fig.1b). Conclusions This study establishes a role for platelets in sequestration of cfDNA, an aspect of platelet biology that has not previously been highlighted and is of substantial clinical relevance. Their abundance, ease of isolation, and continuous tissue perfusion make platelets ideal ‘sentinels’ for genetic perturbations including in early stage/pre-malignant disease.
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Butta, Nora V., María Teresa Alvarez Román, Elena Monzón Manzano, Paula Acuña, Mónica Martín, María Isabel Rivas Pollmar, Raul Justo Sanz, et al. "Glycoside Residues on Platelet's Surface Regulate Platelet Function, Apoptosis and Binding of Coagulation Complexes in Patients with Immune Thrombocytopaenia." Blood 136, Supplement 1 (November 5, 2020): 10–11. http://dx.doi.org/10.1182/blood-2020-141299.
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Introduction: Platelet surface glycoproteins (GPs) are highly glycosylated and are key elements for platelet function since most of them constitute receptors for adhesion ligands. However, exact role of their glycan composition is not clear. Under normal conditions, platelets contain sialic acid in the carbohydrate side chains of their GPs, and it has been described that alterations in the degree of their sialinization can affect the clearance of platelets. This mechanism has been proposed as involved in etiopathogenesis of immune thrombocytopaenia (ITP), mainly in those patients who do not respond to treatments. Thus, after the loss of sialic acid, there would be a greater exposure of galactose and of N-acetyl-glucosamine residues on the surface of circulating platelets to hepatic Ashwell-Morell receptors, which could induce their phagocytosis and platelet clearance. On the other hand, procoagulant platelets, defined as the platelet subpopulation that binds functional prothrombinase, exposed on their surface increased levels of P-selectin and GPIb, two glycan rich GPs. So, it is tempting to speculate that changes in glycan residues on platelet surface may induce changes in their function. Aim: We aimed to assess in ITP patients whether changes in platelet glycosylation, mainly the loss of sialic acid, may condition platelet function, apoptosis and binding of prothrombinase complex. Methods: This is an observational, prospective and transversal study approved by Ethics Committee from La Paz University Hospital. One hundred and eight patients with chronic primary ITP (68 with a platelet count ≥30x103 platelets/µL and 40 with a platelet count <30x103 platelets/µL) and 132 healthy controls were included after signing the informed consent. Platelet activation markers were determined in platelet rich plasma; whereas platelet glycosylation, binding of prothrombinase, annexin V and caspase's activities were assayed in washed platelets. Samples were analyzed by flow cytometry. Table 1 shows lectins tested and their sugar-binding specificity. Data were analyzed with GraphPad Prism 6.0 software. Results: Platelets from ITP patients with a platelet count <30x103/µL exposed less sialic acid in correspondence to an enhanced binding of lectins to non-sialylated residues. Moreover, levels of α1,6-Fucose, a glycan residue which could directly regulate antibody-dependent cellular cytotoxicity, and of α-Mannose, which could be recognized by the mannose binding lectin and activate complement pathway, were increased in platelets from these ITP patients. In accordance, sialic acid loss and consequent platelet surface exposure of other glycoside residues were inversely related to platelet count and ability to be activated (Table 1). These differences in glycosylation observed in ITP patients with a platelet count <30x103/µL were accompanied by a less ability of platelets to be activated (Figure 1), an increased exposure of phosphatidylserine and higher caspase activites (Figure 2). Moreover, increased exposure of phosphatidylserine and of N-acetyl-glucosamine residues (measured through the binding of WGA) enhanced binding of prothrombinase complex (Figure 3). Conclusion: Changes in glycoside composition of GPs on platelet's surface impaired their functional capacity, increases their apoptosis and modifies conditions for the binding of coagulation proteins. These modifications in platelet's glycoside residues seem to be related to severity of ITP. This work was supported by grants from FIS-FONDOS FEDER (PI19/00772) and and Platelet Disorder Support Association. EMM holds a predoctoral fellowship from Fundación Española de Trombosis y Hemostasia (FETH-SETH). Disclosures Butta: Grifols: Research Funding; Novartis: Speakers Bureau; ROCHE: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau; SOBI: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk: Speakers Bureau. Alvarez Román:Grifols: Research Funding; Bayer: Consultancy; Novartis: Speakers Bureau; Roche: Speakers Bureau; Pfizer,: Research Funding, Speakers Bureau; SOBI,: Consultancy, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk,: Research Funding, Speakers Bureau. Martín:SOBI: Research Funding; Pfizer: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Novartis: Speakers Bureau; NovoNordisk: Speakers Bureau. Rivas Pollmar:Novartis: Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau. Justo Sanz:Takeda: Current Employment. García Barcenilla:NovoNordisk: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Pfizer,: Speakers Bureau; Roche: Speakers Bureau; Bayer: Speakers Bureau; Novartis: Speakers Bureau. Canales:Celgene: Honoraria; Janssen: Speakers Bureau; Novartis: Honoraria; Roche: Honoraria; Gilead: Honoraria; Sandoz: Honoraria; iQone: Honoraria; Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Roche: Speakers Bureau; Janssen: Speakers Bureau; Sandoz: Honoraria; Roche: Honoraria; Takeda: Speakers Bureau; Novartis: Honoraria; Sandoz: Speakers Bureau; Karyopharm: Honoraria; Roche: Speakers Bureau; Janssen: Honoraria; Karyopharm: Honoraria; Janssen: Honoraria. Jimenez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer: Honoraria; F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer: Consultancy; Grifols, Novo Nordisk, Takeda, Sobi, Pfizer: Research Funding.
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Roweth, Harvey G., and Elisabeth M. Battinelli. "Lessons to learn from tumor-educated platelets." Blood 137, no. 23 (May 3, 2021): 3174–80. http://dx.doi.org/10.1182/blood.2019003976.
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Abstract Platelets have long been known to play important roles beyond hemostasis and thrombosis. Now recognized as a bona fide mediator of malignant disease, platelets influence various aspects of cancer progression, most notably tumor cell metastasis. Interestingly, platelets isolated from cancer patients often display distinct RNA and protein profiles, with no clear alterations in hemostatic activity. This phenotypically distinct population, termed tumor-educated platelets, now receive significant attention for their potential use as a readily available liquid biopsy for early cancer detection. Although the mechanisms underpinning platelet education are still being defined, direct uptake and storage of tumor-derived factors, signal-dependent changes in platelet RNA processing, and differential platelet production by tumor-educated megakaryocytes are the most prominent scenarios. This article aims to cover the various modalities of platelet education by tumors, in addition to assessing their diagnostic potential.
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Jacob, Shancy P., Yasuhiro Kosaka, Seema Bhatlekar, Alexandra Moody, Victoria Moody, Emilia A. Tugolukova, Grayson A. Hull, et al. "Mitofusin2 (MFN2) Preserves Mitochondrial Integrity and Function in Megakaryocytes and Platelets." Blood 138, Supplement 1 (November 5, 2021): 3137. http://dx.doi.org/10.1182/blood-2021-154329.
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Abstract Genome wide association studies (GWAS) have associated mitochondria related loci with platelet numbers, function, and CVD. However, causality has not been established for many of these variants, and their mechanism and functional consequences are unknown. One such variant, MFN2 eQTL rs1474868 (T/T), has been associated with reduced platelet counts and reduced expression (5 fold) of MFN2 RNA in platelets. We show here that the MFN2 T/T variant corresponds with significantly reduced MFN2 protein in platelets. This difference contributes to a significant correlation between MFN2 RNA levels and mitochondrial load and potential in platelets. MFN2 RNA is also reduced by T/T in human cord blood derived megakaryocytes resulting in unfused mitochondria and impaired megakaryopoiesis. Using platelet/megakaryocyte specific Mfn2-/- (Mfn2 KO) mice, we show that Mfn2 impacts platelet numbers, activation and function by regulating mitochondrial energetics. Platelets without Mfn2 had reduced mitochondrial membrane potential and significantly reduced platelet lifespan (P<0.01) that was attributed to an increased rate of phosphatidylserine (PS) flipping (P=0.01). Increased RNA expression with conversely reduced protein expression of Ndufb8 (P<0.01), an index nuclear encoded complex I subunit that is stable only in a fully assembled complex I, suggested a defect in complex I assembly in Mfn2 KO platelets. Furthermore, complex I activity was reduced in Mfn2 KO platelets compared to WT platelets (P<0.01). Both basal and thrombin triggered mitochondrial oxygen consumption rate as assessed by Seahorse analyzer was significantly reduced in Mfn2 KO platelets (1.28 pmol/min/µg protein) compared to WT control platelets (3.06 pmol/min/µg protein). Platelet activation was subtly, yet significantly, decreased in Mfn2 KO platelets compared to WT platelets as assessed by surface expression of activated integrin alpha2b/beta3 and P-selectin. In addition, Mfn2 KO platelets had impaired Ca 2+ signaling, ROS generation, and procoagulant platelet formation (PS +ve platelets), and formed fewer platelet-neutrophil aggregates (PNAs) compared to WT platelets (P=0.01). Consistent with this, we observed significantly prolonged bleeding times in Mfn2 KO mice compared to their WT control littermates (P=0.001). Finally, mice with loss of platelet Mfn2 exhibited a modest reduction in ischemic stroke infarct size after cerebral ischemia-reperfusion that was statistically significant (P<0.01). Taken together these results suggest that MFN2 preserves mitochondrial functions necessary for platelet survival and activity, and that loss of MFN2 leads to accelerated platelet death, dysfunction, and altered hemostasis and thrombosis. Disclosures Rondina: Novartis: Research Funding; Platelet Biogenesis: Membership on an entity's Board of Directors or advisory committees; Acticor Biotech: Membership on an entity's Board of Directors or advisory committees; Platelet Transcriptomics: Patents & Royalties.
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DeHelian, Daniel, Shuchi Gupta, Jie Wu, Chelsea Thorsheim, Brian Estevez, Matthew Cooper, Kelly Litts, et al. "RGS10 and RGS18 differentially limit platelet activation, promote platelet production, and prolong platelet survival." Blood 136, no. 15 (October 8, 2020): 1773–82. http://dx.doi.org/10.1182/blood.2019003251.
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Abstract G protein–coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor–activating peptide, an increased maximum response to adenosine 5′-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10−/− platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18−/− mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18−/− and RGS10−/−18−/− mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.
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Strebel, Anja, Fabrizio Pennacchio, Sebastian Lickert, Kateryna Selcuk, Konstantin Wolf, and Viola Vogel. "Impact of Recombinant Factor VIII and Platelet Interaction on Platelet Functionality and Hemophilia a Treatment." Blood 142, Supplement 1 (November 28, 2023): 1244. http://dx.doi.org/10.1182/blood-2023-180651.
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Background: Platelets play an important role throughout the various stages of hemostasis. After vascular injury, platelets become activated in a pro-aggregatory state, and subsequently a subpopulation undergoes a phenotype shift to a pro-coagulant state. Pro-coagulant platelets bind to factor VIII (FVIII) and ensure efficient localization of FVIII at the site of injury. It is unclear whether modifications in recombinant FVIII (rFVIII) products impact FVIII-platelet binding and subsequent platelet signaling. Aims: To examine binding of different rFVIII products to platelets in vitro, and the impact of rFVIII on platelet phenotype and intracellular signaling. Methods: Platelet activation: Platelets isolated from healthy donors were activated with thrombin and cross-linked collagen-related peptide (CRP-XL). Platelet-FVIII binding: Activated platelets were incubated with simoctocog alfa, efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol. Binding was quantified by flow cytometry either by immunostaining with an anti-FVIII antibody conjugated to Alexa fluor (AF)647 or direct measurement of rFVIII concentrates labelled with AF647. Phenotype shift: During the shift to a pro-coagulant state, phosphatidylserine (PS) becomes exposed on the platelet membranes, with the extent of PS exposure correlating with the level of FVIII binding. To assess PS exposure, activated platelets were incubated with Annexin V-BV421, a molecular probe with high affinity for PS. Inhibition of integrin αIIbβ3 signaling: Activated platelets were incubated with the integrin αIIbβ3 inhibitor antibody 10E5. Results: Binding to pro-coagulant platelets was significantly higher with simoctocog alfa than with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol irrespective of immunostaining method (p<0.05; Figure 1 shows detection with anti-FVIII antibody conjugated with AF647).To validate the variability in rFVIII-platelet interactions observed, we performed a dynamic platelet-binding assay with the two FVIII products exhibiting the strongest binding (simoctocog alfa and efmoroctocog alfa). The results of the dynamic assay confirmed the results of the static experiments (Figure 1). Exposure of activated platelets to simoctocog alfa and efmoroctocog alfa resulted in increased PS exposure, with a greater effect observed with simoctocog alfa, consistent with the results of the binding experiments. Immunostaining experiments revealed co-localization of simoctocog alfa and clusters of integrin αIIbβ3 in pro-aggregatory platelets. Inhibition of integrin αIIbβ3 by 10E5 decreased binding of simoctocog alfa to platelets and subsequent PS exposure in a dose-dependent manner (Figure 2). Analysis of platelet binding and phenotype shift in patients with hemophilia A are ongoing. Conclusion: Simoctocog alfa demonstrated higher binding to activated platelets in vitro compared with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol, resulting in an increased phenotype shift of platelets from the pro-aggregatory to the pro-coagulant state. The increased binding of simoctocog alfa was associated with a phenotypic shift in platelets as evidenced by increased exposure of PS on the platelet membranes. The binding of simoctocog alfa to platelets was disrupted when integrin αIIbβ3 activation was inhibited, suggesting a role of integrin αIIbβ3 signaling following binding of FVIII to platelets. Variations in platelet binding and signaling between different rFVIIIs might impact their efficacy for the prevention of bleeds. Further experiments are ongoing to assess the impact of FVIII on the release of von Willebrand factor and thrombin generation by activated platelets, and to repeat the FVIII-binding experiments using platelets from healthy donors vs hemophilia A patients.
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Zhou, Junsong, Yi Wu, Lubica Rauova, Gavin Koma, Lu Wang, Mortimer Poncz, Hong Li, et al. "A novel role for endoplasmic reticulum protein 46 (ERp46) in platelet function and arterial thrombosis in mice." Blood 139, no. 13 (March 31, 2022): 2050–65. http://dx.doi.org/10.1182/blood.2021012055.
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Abstract Although several members of protein disulfide isomerase (PDI) family support thrombosis, other PDI family members with the CXYC motif remain uninvestigated. ERp46 has 3 CGHC redox-active sites and a radically different molecular architecture than other PDIs. Expression of ERp46 on the platelet surface increased with thrombin stimulation. An anti-ERp46 antibody inhibited platelet aggregation, adenosine triphosphate (ATP) release, and αIIbβ3 activation. ERp46 protein potentiated αIIbβ3 activation, platelet aggregation, and ATP release, whereas inactive ERp46 inhibited these processes. ERp46 knockout mice had prolonged tail-bleeding times and decreased platelet accumulation in thrombosis models that was rescued by infusion of ERp46. ERp46-deficient platelets had decreased αIIbβ3 activation, platelet aggregation, ATP release, and P-selectin expression. The defects were reversed by wild-type ERp46 and partially reversed by ERp46 containing any of the 3 active sites. Platelet aggregation stimulated by an αIIbβ3-activating peptide was inhibited by the anti-ERp46 antibody and was decreased in ERp46-deficient platelets. ERp46 bound tightly to αIIbβ3 by surface plasmon resonance but poorly to platelets lacking αIIbβ3 and physically associated with αIIbβ3 upon platelet activation. ERp46 mediated clot retraction and platelet spreading. ERp46 more strongly reduced disulfide bonds in the β3 subunit than other PDIs and in contrast to PDI, generated thiols in β3 independently of fibrinogen. ERp46 cleaved the Cys473-Cys503 disulfide bond in β3, implicating a target for ERp46. Finally, ERp46-deficient platelets have decreased thiols in β3, implying that ERp46 cleaves disulfide bonds in platelets. In conclusion, ERp46 is critical for platelet function and thrombosis and facilitates αIIbβ3 activation by targeting disulfide bonds.
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Gehrie, Eric A., and Aaron A. R. Tobian. "PATCHing platelet data to improve transfusion." Blood 135, no. 16 (April 16, 2020): 1309–10. http://dx.doi.org/10.1182/blood.2020005384.
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The Platelet Transfusion in Cerebral Hemorrhage (PATCH) trial, a trial of platelet transfusion in patients on antiplatelet agents with cerebral hemorrhage, suggested that platelets worsened outcomes. Reanalysis of the data by the investigators revealed that despite randomization, some, if not all, of the adverse effects of platelets reflected worse baseline hemorrhage in patients in the platelet arm.
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Storrie, Brian, Sung W. Rhee, Irina D. Pokrovskaya, Kenny Ling, Yajnesh Vedanaparti, Maria Aronova, Timothy J. Stalker, Lawrence F. Brass, and Richard Leapman. "Platelet Activation State Intermixing in a Venous Puncture Model Indicates Novel Patterns of Thrombus Formation." Blood 134, Supplement_1 (November 13, 2019): 9. http://dx.doi.org/10.1182/blood-2019-130805.
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Introduction: Platelet recruitment to generate a thrombus is key to bleeding cessation. That recruitment is dependent on a series of platelet activation processes that include adhesion to the exposed vessel matrix, platelet-platelet adhesion and platelet granule release. How platelet activation is patterned to generate a thrombus has previously been studied by intravital light microscopy, two-photon microscopy and scanning electron microscopy at resolutions insufficient to infer platelet activation at the level of the individual platelet. Here, we present a collaborative effort to stratify spatially the extent of platelet activation at the cellular level in a mouse jugular vein puncture model. We used wide-area transmission electron microscopy (WA-TEM) and serial block face scanning electron microscopy (SBF-SEM) at a resolution of 3 to 100 nm across whole thrombi to determine activation state of individual platelets. Our results, indicate a pattern of platelet stratification within the puncture wound that varies in a time-dependent manner with distinct structural stages in the formation of the thrombus. Methods: Jugular vein thrombi from C57BL/6 mice were collected 1, 5, or 20 min after a 300 µm needle puncture and prepared for EM imaging. For WA-TEM, hundreds of overlapping 3 nm resolution images were acquired using a gondimeter stage. The images sets were aligned using NIH Fiji software to create a single high-res, thrombus-wide image. Individual platelets were stratified into morphologically defined activation states (1: no activation, 2: decreased granule number, 3: no visible granules left, 4: hollow inside). The spatial distribution of platelet stratification was analyzed using iVision software. For SBF-SEM, 100-nm XY-resolution SEM images were collected every 200 nm and 20 nm XY-resolution images every 20 µm. Semi-automated stratification of platelet activation state in individual slices of the thrombus were combined into a 3-D representation using Amira software. Volumetric distributions of platelets with respect to the puncture hole and the vascular wall were quantified. Results: Thrombus Formation Stage 1 (anchor and extend) -- One min post puncture, platelets were anchored in clumps along the exposed vessel wall. Near the damaged vessel wall was a peripheral layer of activated or degranulated platelets (states 3 and 4) covered by additional layers of less-activated platelets (state 1 and 2). Short cylindrical ingrowths extended into the 300 µm hole. Unexpectedly, large aggregates of platelets with a mixture of activation states (states 1 - 4) were found extending from these anchor points into the hole and vertically into the intravascular space. Aggregate surface layers were composed mostly of degranulated platelets (states 3 and 4). Less than 40% of neighboring platelets were of the same activation state as their neighbor. Surprisingly, <2% of platelet-occupied volume within the puncture hole contained largely degranulated platelets (aggregates of only states 3 and 4). Stage 2 (cap and erect) -- At 5 min after injury, the puncture hole was capped. ~70% of platelets neighboring degranulated platelets (3 and 4) formed visible aggregates within the thrombus. These aggregates were found along the exposed vessel wall and encasing vertical platelet aggregate towers containing a mixture of platelets in different states (1 - 4). Towers were typically separated by large cavities. SBF-SEM images, a machine-based, unbiased sampling of the underlying platelet distribution, revealed that ~10% of the platelet volume in the puncture hole of the thrombus and the intravascular towers contained largely degranulated platelets, similar to the data from WA-TEM. Stage 3 (infill and remodeling) - At 20 min post-puncture, the thrombus was filled with a mixture of platelets of varying activation states, which surrounded central, vertical aggregates (towers) of degranulated platelets seen at 5 min. Only minor cavity space was apparent. The intravascular surface of the thrombus was covered with an ~10 platelet-thick layer of loosely packed, variably activated platelets (states 1 - 4). Conclusions: Our results demonstrate dynamic spatial patterns of platelet activation within a forming puncture-wound thrombus. Such patterns yield insights into thrombus formation and suggest the need to reference platelets defects and anti-thrombotics drugs against new models. Figure Disclosures No relevant conflicts of interest to declare.
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Faruque, Md Omar, Irina D. Pokrovskaya, Michael W. Webb, and Brian Storrie. "Screening for Key Structural Differences in Thrombosis Versus Hemostasis through Single Platelet Analysis." Blood 142, Supplement 1 (November 28, 2023): 3950. http://dx.doi.org/10.1182/blood-2023-191005.
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INTRODUCTION: Taking a single platelet analysis (SPA) approach to score platelet activation states distribution within a thrombus provides a unique opportunity to examine cellular difference in the process of thrombotic versus hemostatic clot formation. Potentially, it may reveal platelet activation states are dispensable for hemostasis but critical for thrombosis, e.g., platelet infill within the interior cavities of thrombus. Hence, our goal of determining the distribution of platelet activation states and of a-granule content release in an occlusive clot compared to a hemostatic thrombus. METHODS: We used a femoral artery model in mice in which occlusive clots were induced by ferric chloride and hemostatic processes occurred within a puncture wound. Images were produced by wide area transmission electron microscopy of full thrombi cross sections, 3.185 nm XY pixel size, at various depths. We mapped and analyzed 2D images of ferric chloride induced clotting using automated SPA with locally run Zeiss Arivis Pro software. Through automated SPA, we segmented and mapped the distribution of tens of thousands of platelets and trapped red blood cells versus the vessel wall and associated vascular damage. The incidence of condensed and decondensed a-granules, an indicator of platelet secretion activity, was scored through Cloud implemented APEER models. Occlusive clot cross sections were analyzed at multiple scales to reveal the distribution of platelet activation states based on platelet shape or to reveal a-granule release state (condensed versus decondensed granules). For the simpler problem of SPA of puncture wound hemostasis, we manually scored platelet activation states across tens of thousands of platelets. RESULTS AND CONCLUSIONS: With respect to occlusive clotting, several significant results and conclusions emerged from the platelet shape analysis. In the occlusive clot, we observed that activated platelets adhering directly to the damaged area of the vessel wall, exposed intima, lost their shape, becoming oval with extended pseudopods to more compressed, compacted tightly adherent shape compared to circulating, discoid platelets. Tightly adherent platelets formed column-like projections that structured the interior of the clot. Alpha-granule secretion was apparent. Most interestingly, the interior space between columns was filled with loosely adherent elongated, discoid-shaped platelets that frequently contained decondensed a-granules indicative of granule release (Figure 1). To the best of our knowledge, these data are the first indications that discoid platelets can secrete at least partially, their a-granule contents. In addition to platelets in various activation states, ~7% of the clot cross-sectional area was occupied by shape distorted red blood cells trapped in clusters. Any morphological effect of the red blood cells on platelet activation was restricted to a short distance of ~5 microns. Our SPA analysis of the femoral artery, puncture wound thrombi, support at least two conclusions: 1) Decondensed a-granules were abundant in discoid platelets coating the intravascular “crown” of the 20 min post puncture thrombus, i.e., a-granule secretion by discoid platelet is found in both hemostasis and thrombosis. 2) Circular folding of the hemostatic thrombus to give complete infill of the artery would place discoid platelets within central interior portions of an occlusive clot. We suggest that there may well be shared principles of assembly between the two situations. Importantly, the intravascular accumulation of discoid platelets in hemostasis occurs post-bleeding cessation. Therefore, drugs selectively affecting this state would target preferentially thrombotic clots in which clot infill results in the accumulation of discoid platelets. Figure 1: Distribution of platelets within a cross section through the middle of a ferric chloride induced, occlusive femoral clot. A)Platelet rich clot showing extensive platelet adhesion to the intima, white line encircling the platelet rich clot, inset blowup of the centrally located discoid platelets B) Distribution of red blood cells (red) and elongated, discoid shaped platelets (yellow). C) Cloud based SPA of red blood cells, platelets (yellow), condensed a-granules (light blue) and decondensed a-granules (magenta). D) Global distributions of granules (light blue and magenta) relative to platelets.
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Annarapu, Gowtham K., Deirdre Nolfi-Donegan, Anuradha Bharara Singh, Michael Reynolds, and Sruti Shiva. "Heme Induced Platelet Mitochondrial Oxidant Production Regulates Thrombospondin-1 Release from Platelets." Blood 136, Supplement 1 (November 5, 2020): 30. http://dx.doi.org/10.1182/blood-2020-142640.
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Background: Sickle Cell Disease (SCD) patients develop chronic vasculopathy leading to conditions such as pulmonary hypertension (PH), a major cause of morbidity and mortality. Vasculopathy is tightly associated with hemolysis in SCD. We and others have shown that in SCD, hemolysis activates platelets, and activated platelets are implicated in vasculopathy through their release of vasoactive molecules, such as thrombosponding-1 (TSP1), from storage granules. In SCD, free heme is accumulated in the circulation as a consequence of oxidative degradation of hemoglobin secondary to intravascular hemolysis. Under homeostatic conditions, albumin, haptoglobin, hemopexin, and high- and low-density lipoproteins clear plasma heme from the circulation. However excessive hemolysis can overwhelm these clearance mechanisms, resulting in elevated levels of free heme, which can lead to platelet activation, aggregation and thrombus formation. The role of activated platelets in these thrombotic events is well studied, but the mechanisms by which heme induces platelet activation and platelet-dependent signaling to the vessel wall remain unclear. We previously showed that hemolysis induces platelet mitochondrial reactive oxygen species (mtROS) production, but the mechanism by which heme induces mtROS production and downstream consequences are less clear. We hypothesize that heme induces platelet mtROS through Toll-like Receptor 4 (TLR4) signaling, which regulates platelet activation and granule secretion. Methods: Washed platelets were prepared from whole blood collected from healthy human volunteers in 0.32% sodium citrate anticoagulant (n=6). Platelets were treated with heme (2.5µM) in presence or absence of TLR4 neutralizing antibody, IRAK1/4 inhibitor, TBK1/IKKε inhibitor, MitoTEMPO or DS16570511 (Mitochondrial calcium uniporter inhibitor). Platelet activation was measured using flow cytometry by staining for CD62P (P-selectin) on the platelet surface and the active confirmation of GPIIb/IIIa. Platelet mtROS was measured by fluorescence spectroscopy using MitoSOX Red. The granule secretion function of platelets was measured by assessing TSP-1 and Platelet Factor 4 (PF4) levels in platelet releasates by ELISA. Results: We found that heme activates platelets and induces mtROS production, which can be mitigated by TLR4 neutralizing antibody, a molecule that makes platelet surface TLR4 unavailable to heme. In heme-treated platelets, pharmacological inhibition of the interleukin-1 receptor kinase-4 (IRAK4), a key kinase recruited by the TLR4 adaptor protein MyD88, had no significant effect on platelet activation. In contrast, inhibition of tank binding kinase-1 (TBK1), a TLR4 effector molecule independent of MyD88 signaling, significantly attenuated heme-mediated platelet activation and mtROS production. Scavenging mtROS with MitoTEMPO did not alter platelet activation, instead it decreased the TSP1 release from platelets, indicating heme induced platelet mtROS tightly regulates the granule secretion function of platelets. In addition, blocking mitochondrial calcium uptake by Mitochondrial calcium uniporter (MCU) inhibitor significantly decreased mtROS production and TSP-1 release from heme treated platelets. Conclusion: Our data demonstrate that heme activates human platelets and induces mtROS production via a TLR4 -MyD88-independent signaling pathway. In addition, heme also induces mitochondrial calcium uptake that enhances mtROS production in platelets. Heme induced platelet mtROS tightly regulates platelet granule secretion function. Ongoing studies are further elucidating this pathway and determining the role of mtROS in heme-dependent platelet function. These studies will provide a mechanistic link between hemolysis and thrombosis, as well as platelet-vascular wall signaling. These studies suggest that platelet mtROS may potentially be a novel therapeutic target to prevent thrombotic events and vascular dysfunction in hemolytic disorders including SCD. Disclosures No relevant conflicts of interest to declare.
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Thom, Christopher S., Patricia Ellen Davenport, Hossein Fazelinia, Zhi-Jian Liu, Haorui Zhang, Hua Ding, Jennifer Roof, Lynn Spruce, Harry Ischiropoulos, and Martha Sola-Visner. "Quantitative Label-Free Mass Spectrometry Identifies Molecular Content and Signaling Differences between Neonatal and Adult Platelets." Blood 142, Supplement 1 (November 28, 2023): 1196. http://dx.doi.org/10.1182/blood-2023-179711.
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Observational and randomized studies have associated platelet transfusions with increased morbidity and mortality in preterm neonates. Clinical practice changes have been hindered by a limited understanding of why adult donor-derived platelets might be inappropriate or harmful to infants. Neonatal and adult platelets differ in reactivity and function, but we lack an understanding of underlying molecular differences. As proteins and kinase-driven phosphorylation signaling dictate platelet biology, we aimed to characterize altered protein and phosphoprotein content between neonatal and adult platelets to help explain differences in platelet reactivity and function. We isolated resting platelets from full term cord blood (n=9) and adult peripheral blood (n=7) and used mass spectrometry-based platforms to ascertain protein and phosphoprotein content. We identified 4745 high-confidence proteins in adult and neonatal platelets, including 331 differentially abundant proteins (≥1.5-fold change, p<0.05). Neonatal platelets had increased metabolic and ribosomal proteins, consistent with transcriptional data. Adult platelets were enriched for inflammatory proteins, including complement components, consistent with pro-inflammatory function. We also identified 2115 phospho-proteins (17852 unique phosphopeptides) across all samples. These impacted actin cytoskeletal biology, cell adhesion, and GTPase signaling that regulate platelet functions. Kinase enrichment analysis identified FYN, SRC, ABL1, and AKT1 as kinases most responsible for platelet phosphorylation activities. Granule trafficking and degranulation proteins were among 1183 differentially abundant phosphoproteins in neonatal vs adult samples (p<0.05). For example, 31 different Reticulon 1 (RTN1) phosphopeptides were enriched in adult platelets (p<0.05). RTN1 regulates membrane trafficking and participates in SNARE-mediated exocytosis. Increased RTN1-directed membrane trafficking may promote increased activation and degranulation in adult vs neonatal platelets. Indeed, SNARE complex protein deficiency was previously suggested to mediate hyporeactive degranulation in neonatal platelets. We detected 2 phosphopeptides in adult samples that were absent from any neonatal sample. We reasoned that these could represent developmental stage-specific differences that exist only in adult platelets. One of the adult-specific phosphopeptides was in Rap1GAP2 (phospho-S588). Rap1GAP2 is a highly phosphorylated GTPase-activating protein that inhibits Rap1, a small guanine-nucleotide-binding protein that facilitates integrin activation in platelets. Rap1GAP2 binds synaptotagmin-like protein 1 (Slp1) to regulate dense granule secretion. These findings may link altered developmentally regulated Rap1GAP2 activities with platelet degranulation. A Platelet Cell Adhesion Molecule (PECAM) phosphopeptide (phospho-S726) was also found exclusively in adult platelets. PECAM facilitates inflammation and leukocyte transendothelial migration. PECAM phosphorylation in adult platelets may reflect an enhanced ability to participate in these processes. These findings reveal key differences in protein content and signaling that underlie differential neonatal vs adult platelet reactivity and function, including pro-inflammatory activities and enhanced activation and/or degranulation propensity in adult platelets. RTN1-mediated SNARE complex function and Rap1 signaling activities represent novel and potentially modifiable targets to manipulate platelet activation and/or degranulation. Our results also help provide a biological rationale for the higher morbidity and mortality observed in preterm infants transfused with adult platelets, including the inadvertent introduction of serum complement and other pro-inflammatory molecules within transfusions of donated adult platelets.
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Bhatlekar, Seema, Shancy Jacob, Marina Tistao, Molly Y. Mollica, Nicole Rhoads, Lili Chen, Bhanu Kanth Manne, et al. "The Actin Bundling Protein L-Plastin Mediates Platelet Force Generation and Thrombosis." Blood 142, Supplement 1 (November 28, 2023): 2560. http://dx.doi.org/10.1182/blood-2023-178052.
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Within minutes of activation, platelets dramatically change their shape, forming filopodia and lamellipodia, aggregate with other platelets or different cells, and can increase their surface area up to 4 times. This relies on a dynamic actin cytoskeleton, regulated by many actin binding proteins in a time- and spatial-specific manner. Deficiency or inhibition of several actin binding proteins in platelets have been shown with impaired platelet spreading and reduced thrombosis. Recently, we found that an actin bundling protein, L-plastin, is expressed in platelets. Its function in platelets has not been studied. In megakaryocytes, we showed that L-plastin is responsible for maintaining cytoskeletal stiffness; in this study we sought to determine L-plastin's role in platelet functions, including thrombosis. We examined the role of L-plastin in platelets in vivo and ex vivo using L-plastin deficient ( Lcp1 -/-) male and female mice. First, we used the collagen/epinephrine induced pulmonary embolism (PE) model and found that survival times were significantly shorter in Lcp1 -/- mice than in their littermate wildtype (WT) controls (Mean±SEM WT 202±12 sec, Lcp1 -/- 139.5±14 sec, P=0.004), suggesting accelerated thrombosis upon L-plastin deficiency in vivo. Second, we examined L-plastin's role in arterial thrombosis in vivo using the laser-induced cremaster arteriole thrombosis mode. Platelets were labeled with anti-GPIb IgG derivative X488 and the thrombosis formation after laser injury was recorded. Preliminary data indicate that thrombi in Lcp1 -/- mice are larger than those in WT mice. Third, we examined L-plastin's role in platelet adhesion using a microfluidics assay with DiOC6-labelled whole blood. At a shear rate of 200/s, the Lcp1 -/- mice had significantly increased platelet adhesion to both collagen and fibrinogen matrices. Fourth, to evaluate the effect of L-plastin on platelet aggregation in vitro, we stimulated washed platelets with 3 different concentrations of thrombin and measured the platelet aggregation (i.e., 0.1, 0.5 and 1 U/mL). When platelets are stimulated with 0.5U/mL or 1U/mL thrombin, platelets from WT and Lcp1 -/- mice showed comparable aggregation kinetics. At 0.1U/mL thrombin, however, preliminary data showed increased aggregation in the Lcp1 -/- group. The difference between the low and high concentration of thrombin indicates potential regulation of the GPIba mediated actin cytoskeleton rearrangement during platelet aggregation. We next examined under low thrombin concentration, the platelet actin cytoskeleton reorganization and platelet spreading. We seeded washed platelets on a fibrinogen matrix with the presence of thrombin (0.1U/mL). Platelets were then fixed and stained for F-actin and imaged using immunofluorescent confocal microscopy. The spreading area per platelet was calculated using FIJI software. Platelets from Lcp1 -/- mice showed significantly increased average membrane area at 30 min (Median WT 689 pixels, Lcp1 -/- 866 pixels, P<0.0001). This suggests that the actin rearrangement in platelets upon fibrinogen activation and thrombin stimulation is inhibited by L-plastin. Lastly, we examined whether L-plastin is involved in platelet force generation during the actin reorganization. We used a recently developed “black dot” assay. Briefly, a fluorescent pattern (i.e., black dots) was microcontact printed onto a flexible polydimethylsiloxane substrate and coated with fibrinogen. Platelets were seeded onto the matrix and fixed after 30 min. The contractile forces of individual platelets were measured based on the deformation of the fluorescent micropattern as illustrated in Fig 1A. The contractile force in platelets from Lcp1 -/- mice is decreased with altered actin distribution pattern compared to WT mice (Fig 1. Contractile force per platelet, Mean±SEM WT 14.78±0.86nN, Lcp1 -/- 12.03±0.67nN, P=0.01). In summary, we found accelerated thrombosis in Lcp1 -/- mice, associated with accelerated platelet adhesion and spreading ex vivo. Mechanistically, our preliminary data suggest this is due to impaired actin bundle formation and decreased platelet contractile force. L-plastin may be unique among actin binding proteins.
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Novakovic, Valerie Anne, Madhumouli Chatterjee, and Gary E. Gilbert. "Cryopreserved Platelets Retain Agonist Responsiveness and Support for Factor VIII Function." Blood 136, Supplement 1 (November 5, 2020): 3–4. http://dx.doi.org/10.1182/blood-2020-143366.
Full textAbstract:
Platelet activation supports procoagulant activity through phosphatidylserine exposure, secretion of procoagulant factors, and receptor conformational change. For example, thrombin-stimulated platelets bind factor VIII (fVIII) via a macromolecular complex including oligomeric fibrin and the active αIIbβ3 receptor (Phillips et al, JTH 2004; Gilbert et al, Blood 2015). Thus, coagulation assays in which phospholipid vesicles are substituted for platelets do not fully emulate modulators of fVIII activity. Indeed, inhibition of platelet-supported fVIII activity by a panel of mAbs against the C2 domain was not correlated to inhibition of vesicle-dependent activity (Chatterjee et al, JTH 2020). An obstacle to adoption of a platelet-based assay for fVIII is the need for fresh platelets. Therefore we asked whether cryopreserved platelets might support fVIII activity similarly to fresh platelets. Apheresis platelets were mixed with cryopreservatives with or without calcium chelators, in various aliquot sizes, and frozen on various cooling media. Cryopreserved platelets were compared to non-preserved apheresis platelets with regard to agonist response and support of procoagulant activity. Cryopreservation resulted in an increase in subcellular debris and an unresponsive fraction of platelets with decreased forward scatter judged by flow cytometry. Optimized results were obtained when platelet rich plasma with 5% DMSO, in 1 mL aliquots was frozen on powdered dry ice, and stored at -150C. Purification of thawed platelets using a density gradient removed debris and decreased unresponsive platelets resulting in a forward and side scatter profile comparable to fresh platelets. We refer to these as cryopreserved platelets (CryoPlts). CryoPlts were compared to control and outdated apheresis platelets. As with fresh platelets, procoagulant activity of CryoPlts increased with thrombin receptor agonist peptides (TRAP) 1 & 4 and supported a log-linear relationship between time to initial fibrin strand formation and fVIII activity over a range of 0.0001 - 1 u/mL (Fig 1). Further, the degree of inhibition of fVIII activity by mAbs ESH4 and G99 against the fVIII C2 domain, was the same on control and CryoPlts, but markedly different from inhibition in an aPTT-based inhibitor assay. In contrast, outdated apheresis platelets had increased procoagulant activity, minimal agonist response and a shallow curve with varying fVIII concentration. Flow cytometry studies with lactadherin-FITC indicated that 33 ± 14% of CryoPlts had high PS exposure, and the size of this population was minimally affected by TRAP 1+4. In contrast, the main platelet population had a small, uniform, increment in PS exposure comparable to control platelets. Surprisingly, the PS-rich platelets did not significantly affect the time to fibrin formation, confirming that the viable platelets, with limited PS exposure, provide much of the support for fVIII-related procoagulant activity. Flow cytometry indicated αIIbβ3 activation (PAC1-FITC) and α-granule release (anti-P-selectin-PE) were qualitatively intact on CryoPlts, although staining was decreased 70% for PAC1 and 57% for Psel. We also tested whether CryoPlts may be utilized for evaluating response to anti-PF4-heparin antibodies, relevant to heparin-induced thrombocytopenia (HIT). We evaluated platelet response to platelet factor 4 (PF4) and a platelet-activating anti-PF4 antibody (KKO), a combination that induces activation similar to authentic autoimmune antibodies for HIT. The non-activating PF4 antibody RTO served as a negative control. Geometric mean response, corrected for background, was normalized to response to thrombin activation. Both fresh and CryoPlts responded with increases in PAC1 (Fig 2A) and anti-Psel (Fig 2B) binding in response to KKO/PF4 compared to RTO/PF4 . This data demonstrates that the qualitative αIIbβ3 and P-selectin response to HIT-like antibodies is intact. Our results demonstrate a refined cryopreservation protocol of apheresis platelets. These platelets maintain qualitative agonist responsiveness with near-normal support for factor VIII activity, suggesting that they could be used for other platelet-based laboratory or diagnostic assays. Further, our results suggest that major procoagulant activity is provided by platelets with very limited PS exposure, an area for further investigation. Disclosures No relevant conflicts of interest to declare.
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Nasimuzzaman, Md. "Purification Platelets from Mouse Blood with Sickle Cell Disease Using Iohexol Gradient Medium." Blood 134, Supplement_1 (November 13, 2019): 4888. http://dx.doi.org/10.1182/blood-2019-128459.
Full textAbstract:
Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. Since RBC and WBC contain significantly more RNA and proteins than platelets, the presence of even a small number of these cells can interfere with transcriptomic and proteomic analyses of RNA and proteins derived from platelets. Several protocols have described the isolation of platelets from human, dog, rat, and non-human primate by various methods. Some of the methods require multiple steps such as collection of platelet-rich plasma by centrifugation, filtration by separation column, negative selection of platelets with RBC- and WBC-specific antibody conjugated to magnetic beads, and so on, which are time-consuming and may degrade platelets and their contents. Moreover, mouse blood with sickle cell disease contains a significant level of fragments of RBC which should be removed from the platelet preparation. However, the mouse yields a relatively smaller volume of blood, which makes it difficult to purify platelets. If the same small volume of gradient medium and blood samples are used, the platelet layer cannot be clearly separated from RBC-WBC layer after centrifugation. We describe a simple method for purification of platelets from mouse blood with sickle cell disease using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 °C. The platelet layer is collected and centrifuged again at 200 x g for 20 min at 20 °C to remove the residual fragments of RBC. The recovery/yield of the purified platelets were 10-17%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets were activated with thrombin indicating their viability. We confirmed that the purified platelets were sufficiently pure using flow cytometric and microscopic evaluation. Although flow cytometric analysis of purified platelet from sickle cell disease mice showed a few RBC events after staining with anti-TER119 antibody, the microscopic study did not show any intact RBC or larger fragments indicating that these are smaller fragments of RBC which do not interfere with the biochemical and functional studies. This method can be used for purification of platelets from the blood of other species and disease models as well. Disclosures No relevant conflicts of interest to declare.
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Zhao, Xuefei, Matthew Cooper, Yanki Yanman, Aiden Baltz, James Michael, Steven Edward McKenzie, Timothy J. Stalker, Li Zhu, and Peisong Ma. "GRK2 Regulates ADP Signaling in Platelets Via P2Y 1 and P2Y 12." Blood 138, Supplement 1 (November 5, 2021): 578. http://dx.doi.org/10.1182/blood-2021-148615.
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Abstract Abstract Venous thromboembolism (VTE), heart attack, and stroke are all diseases in which platelets play a role, through inappropriate platelet activation and subsequent thrombus formation. Most platelet agonists activate platelets via G protein-coupled receptors (GPCRs), which are targeted by many antiplatelet drugs. Along with thrombin and TxA 2, ADP has long been recognized for its important role in hemostasis and thrombosis. It activates platelets via GPCRs, P2Y 1 and P2Y 12. However, little is known about the negative feedback mechanisms governing P2Y receptor-mediated platelet activation and thrombus formation. Here, we provide the first evidence that GPCR kinase 2 (GRK2) serves this regulatory role during platelet activation and thrombus formation by using a platelet-specific GRK2 deletion mouse model and a GRK2-specific inhibitor in human platelets. Deletion of GRK2 in mouse platelets causes increased platelet accumulation following laser-induced injury in cremaster muscle arterioles, particularly in the shell region of thrombi. In addition, this deletion increases ADP-induced pulmonary thromboembolism. GRK2 -/- platelets also have increased platelet aggregation in response to ADP, but not to PAR4 receptor agonist, TxA 2, or convulxin. Underlying these changes in GRK2 -/- platelets is an increase in Ca 2+ mobilization, Akt phosphorylation, and Rap1 activation in response to ADP, and an attenuated rise of cAMP levels in response to ADP in the presence of prostaglandin I 2. Furthermore, platelet aggregation can be restored in GRK2 -/- platelets in response to ADP re-stimulation, indicating that GRK2 contributes to ADP receptor desensitization. To further assess the role of GRK2 in the P2Y 12 signaling pathway in vivo, we examine laser-induced thrombus formation in WT and GRK2 -/- mice treated with the P2Y 12 antagonist, cangrelor. Cangrelor treatment eliminates the phenotypic difference in platelet accumulation between WT and GRK2 -/- mice in response to injury. Using a specific GRK2 inhibitor, pharmacologic inhibition of GRK2 activity in human platelets results in an increase in platelet activation in response to ADP. Finally, our biochemical studies show that GRK2 binds to endogenous Gβγ subunits during platelet activation. Taken together, we have demonstrated for the first time that 1) GRK2 plays a negative regulatory role in platelet activation by attenuating ADP-dependent signaling, 2) it does this by limiting P2Y 1 and P2Y 12-mediated signaling, 3) GRK2 interacts with Gβγ and functions as a signaling hub in platelets for fine-tuning GPCR signaling, and 4) although the potential inhibition of GRK2 can be beneficial for treatment of heart diseases, maintaining GRK2 activity in platelets could be beneficial for prevention of thrombotic diseases. Disclosures No relevant conflicts of interest to declare.
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Jobe, Shawn M., Lorie Leo, Joshua S. Eastvold, Gerhard Dickneite, Timothy L. Ratliff, Steven R. Lentz, and Jorge Di Paola. "Role of FcRγ and factor XIIIA in coated platelet formation." Blood 106, no. 13 (December 15, 2005): 4146–51. http://dx.doi.org/10.1182/blood-2005-03-1223.
Full textAbstract:
Platelet activation in response to dual stimulation with collagen and thrombin results in the formation of a subpopulation of activated platelets known as coated platelets. Coated platelets are characterized by high surface levels of α-granule proteins and phosphatidylserine, which support the assembly of procoagulant protein complexes. Using murine models, we tested the hypothesis that the collagen receptor-associated molecule FcRγ and the transglutaminase factor XIIIA are required for the formation of coated platelets. Following dual stimulation with the collagen receptor agonist convulxin and thrombin, 68% of platelets from C57BL/6 mice acquired the coated platelet phenotype, defined by high surface levels of fibrinogen and von Willebrand factor and decreased binding of the αIIbβ3 activation-dependent antibody PE-JON/A. In FcRγ-/- mice, only 10% of platelets became “coated” after dual stimulation with convulxin plus thrombin (P < .05 vs C57BL/6 platelets). Decreased coated platelet formation in FcRγ-/- platelets was accompanied by decreased annexin V binding (P < .01) and decreased platelet procoagulant activity (P < .05). Platelets from FXIIIA-/- mice did not differ from control platelets in coated platelet formation or annexin V binding. We conclude that FcRγ, but not factor XIIIA, is essential for formation of highly procoagulant coated platelets following dual stimulation with collagen and thrombin.
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Guo, Li, Sikui Shen, Jesse W. Rowley, Neal D. Tolley, Wenwen Jia, Bhanu Kanth Manne, Kyra N. McComas, et al. "Platelet MHC class I mediates CD8+ T-cell suppression during sepsis." Blood 138, no. 5 (April 25, 2021): 401–16. http://dx.doi.org/10.1182/blood.2020008958.
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Abstract Circulating platelets interact with leukocytes to modulate host immune and thrombotic responses. In sepsis, platelet-leukocyte interactions are increased and have been associated with adverse clinical events, including increased platelet–T-cell interactions. Sepsis is associated with reduced CD8+ T-cell numbers and functional responses, but whether platelets regulate CD8+ T-cell responses during sepsis remains unknown. In our current study, we systemically evaluated platelet antigen internalization and presentation through major histocompatibility complex class I (MHC-I) and their effects on antigen-specific CD8+ T cells in sepsis in vivo and ex vivo. We discovered that both human and murine platelets internalize and proteolyze exogenous antigens, generating peptides that are loaded onto MHC-I. The expression of platelet MHC-I, but not platelet MHC-II, is significantly increased in human and murine platelets during sepsis and in human megakaryocytes stimulated with agonists generated systemically during sepsis (eg, interferon-γ and lipopolysaccharide). Upregulation of platelet MHC-I during sepsis increases antigen cross-presentation and interactions with CD8+ T cells in an antigen-specific manner. Using a platelet lineage–specific MHC-I–deficient mouse strain (B2Mf/f-Pf4Cre), we demonstrate that platelet MHC-I regulates antigen-specific CD8+ T-cell proliferation in vitro, as well as the number and functional responses of CD8+ T cells in vivo, during sepsis. Loss of platelet MHC-I reduces sepsis-associated mortality in mice in an antigen-specific setting. These data identify a new mechanism by which platelets, through MHC-I, process and cross-present antigens, engage antigen-specific CD8+ T cells, and regulate CD8+ T-cell numbers, functional responses, and outcomes during sepsis.
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Tao, Derrick L., Samuel Tassi Yunga, Craig D. Williams, and Owen J. T. McCarty. "Aspirin and antiplatelet treatments in cancer." Blood 137, no. 23 (May 3, 2021): 3201–11. http://dx.doi.org/10.1182/blood.2019003977.
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Abstract Platelets have been hypothesized to promote certain neoplastic malignancies; however, antiplatelet drugs are still not part of routine pharmacological cancer prevention and treatment protocols. Paracrine interactions between platelets and cancer cells have been implicated in potentiating the dissemination, survival within the circulation, and extravasation of cancer cells at distant sites of metastasis. Signals from platelets have also been suggested to confer epigenetic alterations, including upregulating oncoproteins in circulating tumor cells, and secretion of potent growth factors may play roles in promoting mitogenesis, angiogenesis, and metastatic outgrowth. Thrombocytosis remains a marker of poor prognosis in patients with solid tumors. Experimental data suggest that lowering of platelet count may reduce tumor growth and metastasis. On the basis of the mechanisms by which platelets could contribute to cancer growth and metastasis, it is conceivable that drugs reducing platelet count or platelet activation might attenuate cancer progression and improve outcomes. We will review select pharmacological approaches that inhibit platelets and may affect cancer development and propagation. We begin by presenting an overview of clinical cancer prevention and outcome studies with low-dose aspirin. We then review current nonclinical development of drugs targeted to platelet binding, activation, and count as potential mitigating agents in cancer.
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Amer, Johnny, and Eitan Fibach. "Oxidative Status of Platelets in Normal and Thalassemic Blood." Blood 104, no. 11 (November 16, 2004): 3765. http://dx.doi.org/10.1182/blood.v104.11.3765.3765.
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Abstract Thromboembolic complications, possibly involving chronic platelet activation, are an important cause of morbidity and mortality in beta-thalassemia. Oxidative stress, with the generation of reactive oxygen species (ROS), has been suspected to play a role in the pathophysiology of thalassemia and cardiovascular disorders. Previous investigations demonstrated that ROS profoundly affect platelet function and promote platelet activation. Other studies have shown that platelets themselves produce ROS upon activation. In the present study, we adapted flow cytometric techniques to measure oxidative-state markers, ROS generation and reduced glutathione (GSH), using 2′-7′-dichlorofluorescin diacetate and mercury orange, respectively, in platelets. GSH is the major intracellular antioxidant - an important scavenger of ROS. To exclude non-platelets from analysis, a two-parameter (side light scatter and forward light scatter) gate was set. The identity of the gated cells was verified by immunofluorescence staining for CD41 - a platelet-specific antigen. Using these techniques, the average Mean Fluorescence Channel (MFC) values of platelets from 46 normal donors and 22 beta-thalassemic donors were 176 ± 99 vs. 314 ± 81, respectively, for ROS and 319 ± 87 vs. 113 ± 47, respectively, for GSH. These results show that thalassemic platelets contain higher ROS and lower GSH levels than do normal platelets, indicating a state of oxidative stress. The relationship between platelet activation and oxidative status was determined by treating platelets with thrombin (0.1 U/ml), calcium ionophore (5 μM) or phorbol myristate acetate (400 ng/ml). All these treatments caused platelet activation as well as ROS generation; thalassemic platelets were more responsive than platelets from normal controls. In the absence of any known inherent abnormality in thalassemic platelets, the increased oxidative status was attributable to continuous exposure to oxidative insults from extra-platelet sources. Indeed, further investigation indicated that the oxidative status of normal platelets was increased by thalassemic plasma and was inhibited by the iron-chelator Desferoxamin. Iron and hemin, whose levels are increased in thalassemic plasma, stimulated the platelets’ oxidative stress. This was also affected by RBC: it was higher in normal platelets incubated with thalassemic RBC than when incubated with normal RBC. Normal RBC stimulated with hydrogen peroxide, a treatment which results in an elevated oxidative status, increased platelet ROS to a greater extent (3.3-fold) than did unstimulated RBC. These results suggest that thalassemic RBC, having higher than normal ROS, mediate oxidative stress in platelets directly, probably by contact or close proximity. Platelet oxidative stress was ameliorated by antioxidants such as N-acetyl-L-cysteine and vitamin C. Treatment with these agents of oxidant-stimulated platelets reduced ROS and enhanced the GSH level. The present results indicate that in thalassemia, platelets are in a state of oxidative stress, causing their chronic activation and possibly thromboembolic consequences. This situation may also prevail in other RBC anomalies, such as sickle cell anemia, Polycythemia Vera and Paroxysmal Nocturnal Hemoglobinuria, which are also associated with thromboembolic phenomena. Our findings raise the possibility of using antioxidants in addition to antithrombotic drugs as prophylactic treatment in these diseases.
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Stratiievska, Anastasiia, Olga Filippova, Hasan Tahsin Ozpolat, Daire Byrne, S. Lawrence Bailey, Molly Mollica, Jeffrey Harris, et al. "TRPM8 Contributes to Early Temperature-Induced Platelet Activation." Blood 142, Supplement 1 (November 28, 2023): 5566. http://dx.doi.org/10.1182/blood-2023-174122.
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Background: Like sensory neurons, platelets are activated by a decrease in temperature. However, the molecular mechanism of this temperature-sensing ability is unknown. Platelet activation by temperature could contribute to numerous clinical observations, most importantly to the well-known reduced lifespan of ex vivo-stored platelets for transfusion. In this study, we sought to dissect the molecular basis of the cold-induced platelet activation response and specifically, evaluate the role of the thermosensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in platelets and their precursor cells. Methods: We tested for the presence of TRPM8 on the megakaryocytic cell line MEG-01 and human platelets by immunoblotting, confocal microscopy, and imaging flow cytometry. The effect of TRPM8 on platelets was evaluated using the commercially available TRPM8 inhibitor PF-05105679. In addition, we used TRPM8 agonists such as menthol, WS-12, and icilin. Platelet shape change and αIIbβ3 integrin activation were assessed by imaging flow cytometry and conventional flow cytometry. We tested for platelet aggregation using light transmission aggregometry and for intracellular calcium by reading fluorescence on a plate reader after loading platelets with Fura-2-AM. Calcium release in response to temperature decrease was assessed after loading platelets or TRPM8-transfected HEK293T/17 and reading fluorescence in a Real-Time PCR Detection System. Results: We detected TRPM8 mRNA and protein in MEG-01 cells and platelets but there was a remarkable variability among donors. Further evidence for TRPM8's presence in platelets came from the fact that inhibitors of TRPM8 prevented early temperature-induced platelet activation events, such as αIIbβ3 integrin activation and shape change. Unlike temperature changes, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. Platelets exposed to below-normal body temperature displayed a cytosolic calcium increase, but this was independent of TRPM8 and entirely dependent on the calcium release from the endoplasmic reticulum. Conclusion: We found evidence for TRPM8 expression in platelets but the expression varies greatly between individuals and is of unclear physiologic significance. Our study suggests that the cold response of platelets is complex, and TRPM8 appears to play a calcium-independent role in the early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies.
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Kim, Oleg V., Tatiana A. Nevzorova, Elmira R. Mordakhanova, Izabella A. Andrianova, Giang Le Minh, Mark S. Alber, Olga Vagin, Rustem I. Litvinov, and John W. Weisel. "Fatal Dysfunction and Fragmentation of Thrombin-Stimulated Platelets." Blood 132, Supplement 1 (November 29, 2018): 521. http://dx.doi.org/10.1182/blood-2018-99-112703.
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Abstract Introduction: Platelets play a key role in formation of protective hemostatic blood clots and pathological obstructive thrombi. Under (patho)physiological conditions, chemically activated platelets change their morphology, express adhesive molecules, undergo aggregation, secrete procoagulant substances, and induce mechanical contraction (retraction) of the blood clots. Despite the vital importance of these platelet functions, the subsequent fate of activated platelets is largely unknown. We hypothesize that activated platelets undergo late alterations that determine their fate and may have a pathogenic importance in thrombotic and hemostatic disorders. Methods: We used a combination of confocal microscopy, immunofluorescence, scanning and transmission electron microscopy, flow cytometry, biochemical and biomechanical measurements to study deferred structural, metabolic, and functional consequences of thrombin-induced activation of viable human platelets, either suspended in platelet-rich plasma or isolated by gel-filtration. Results: Visualized by confocal microscopy, fluorescently labeled platelets in thrombin-induced plasma clots initially underwent shape changes characteristic of platelet activation, but in about 30 min many platelets and platelet aggregates broke up into organelle-containing vesicular fragments. There were two types of platelet-derived vesicles differing in their size and cellular origin: one smaller type was shedding from the tips of filopodia, while the other type resulted from fragmentation of platelet bodies. Concurrently with the fragmentation, thrombin-activated platelets displayed dramatically altered intracellular distribution of F-actin and septins detected as intense fluorescent clusters with a ~2-fold increase in the intensity of septins 2 and 9 and a ~300-fold increase in the F-actin staining. Synchronously with the structural alterations, thrombin induced a time-dependent reduction of the mitochondrial membrane potential (Δψm) in platelets. The overall fluorescence intensity of the Δψm-sensitive MitoTracker dye in freshly formed thrombin-initiated plasma clots dropped 2- and 4-fold after 60 min and 90 min, respectively. A drop of Δψm inversely correlated with an increase of the fraction of disintegrated platelets (r=-0.93, p<0.01). Flow cytometry showed enhanced phosphatidylserine exposure in thrombin-activated platelets, either with or without mitochondrial depolarization. Thrombin caused a significant 59% decrease of the average ATP content in activated platelets relative to untreated platelets after 60 min of incubation. Remarkably, the initial drop of Δψm and ATP content was almost concurred with the termination of contraction of the platelet-rich plasma clot measured as a 90%-decrease of platelet-generated contractile stress. Unexpectedly, no activation of caspase 3/7 was detected in platelets after 90 min of treatment with thrombin. Meanwhile, calpain activity detected in platelets 90 min after thrombin treatment was 6.5-fold higher compared to untreated platelets. Moreover, calpain inhibition caused a ~30-min delay in the commencement of thrombin-induced platelet fragmentation. Conclusions: Our findings indicate that following thrombin-induced platelet activation, a substantial fraction of platelets undergo time-dependent dysfunction and structural disintegration into subcellular particles. The fragmentation of platelets is accompanied by dramatic rearrangements of platelet cytoskeletal components, including polymerization, clustering, and redistribution of actin and septins. Thrombin-induced platelet fragmentation is concurrent with severe impairment of platelet functionality, including mitochondrial depolarization, ATP depletion, and loss of platelet contractility. The lack of caspase activity and increased calpain activity in energetically exhausted thrombin-treated platelets undergoing fragmentation suggests a calpain-dependent platelet death pathway. These studies indicate that such a form of platelet death may be an underappreciated mechanism for enhanced elimination of platelets from the circulation in (pro)thrombotic conditions or under other conditions once they have performed their functions. Work supported by the Program for Competitive Growth at KFU and AHA grant 17SDG33680177. Disclosures No relevant conflicts of interest to declare.
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Bat, Taha, Eunice S. Wang, Joanne Becker, Orla Maguire, Hans Minderman, and Jordan Cinquino. "Feasibility of Flow Cytometric Identification and Isolation of Pre-Platelets to Understand Its Function and Role in Cancer Metastasis." Blood 134, Supplement_1 (November 13, 2019): 3688. http://dx.doi.org/10.1182/blood-2019-132241.
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Background:It is known that platelets interact with tumors to support malignant cell growth and stimulate epithelial to mesenchymal transition (Li, et al. "The Role of Platelets in Tumor Growth, Metastasis, and Immune Evasion", Platelets, pp. 547-561, Academic Press, 2019). P-selectin which is secreted from alpha granules has been implicated as the major perpetrator of this tumor cell and platelet communication. (Kim et al, PNAS 95(16): 9325-9330, 1998). Higher numbers of alpha granules are found in newly released young platelets from megakaryocytes, also known as pre-platelets, than mature platelets. Given their potential role in cancer metastasis, the ability to identify and isolate pre-platelets from mature plateletsforassessment of overall functionality and biology is important. Of note, identification and measurement of pre-platelets based on higher RNA content has been in clinical use for various clinical conditions for several years. Here we evaluated the feasibility of identifying and sorting pre-platelets by flow cytometric analyses in multiple platelet apheresis samples from heathy donors. Study Design and Methods Apheresis samples from adult healthy donors at Roswell Park Comprehensive Cancer Center were obtained and analyzed in accordance with an IRB approved protocol. Platelets sampled from apheresis products were collected with the use of an apheresis device known to yield a leuko-reduced platelet concentrate. All components were irradiated with 2500 cGy immediately after the collection and stored on platelet agitator at 22°C. A 4 mL aliquot was withdrawn from each bag for sorting essays.Pre-platelets were distinguished from mature platelets based on the presence of higher RNA levels. The cell permeant RNA dye TO (BD Biosciences, San Jose, CA) was used to identify pre-plapletes by flow cytometry according to published protocol (Ando et al Blood97 (4): p915-921, 2001).The optimal concentration of TO dye was determined by titration at decreasing dilutions and acquisition on a LSR-II flow cytometer (BD Biosciences, San Jose, CA). Confirmation of flow cytometry-based assay was determined by processing 5 healthy donor whole blood samples by both flow cytometry and using an XE-5000 automated hematology analyzer (Sysmex America, Mundelein, IL). The percentage of pre-platelets was measured as a percent of total platelets by both techniques. Platelet sorting was performed on 12 leuko-reduced apheresis platelet products on a FACSAria II (BD Biosciences, San Jose, CA). Platelets were identified by co-staining with CD41a. Platelet activation was assessed before and after sorting based on P-selectin (CD62P) surface expression. Flow cytometry analysis was performed using FCSExpress v6. Results: We determined that flow cytometric sorting of pre-platelets optimally required TO (0.5 µg/mL) for optimal staining for gating of pre-platelets. A two way, paired simple t test resulted in a p value of 0.09 when compared to standard gating strategy per XE-5000 automated hematology analyzer. Among all 14 samples evaluated, the percentage of preplatelet to apheresis sample pre-platelet ratio was estimated between 2.8% to 8.2%. Interestingly P-selectin ratio of pre-platelets was significantly higher than mature platelets (median: 67% vs 52%). ConclusionThis study is the first to demonstrate the feasibility of identifying and sorting pre-platelets using a novel flow cytometric method with TO staining. Isolation of these rare platelet precursor cells (pre-platelets) with known higher P-selectin expression are an essential first step to understanding the potential pre-eminent role of these cells in cancer evasion and metastasis. Disclosures Wang: Abbvie: Other: Advisory role; Kite: Other: Advisory role; Jazz: Other: Advisory role; Astellas: Other: Advisory role, Speakers Bureau; celyad: Other: Advisory role; Pfizer: Other: Advisory role, Speakers Bureau; Stemline: Other: Advisory role, Speakers Bureau; Daiichi: Other: Advisory role; Amgen: Other: Advisory role; Agios: Other: Advisory role.
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Prasetya, Hieronymus Rayi, Maria Irena Dentri, and Sistiyono Sistiyono. "Perbedaan Hitung Jumlah Trombosit Menggunakan Darah Vena dan Darah Kapiler." Journal of Health 3, no. 2 (July 31, 2016): 81. http://dx.doi.org/10.30590/vol3-no2-p81-84.
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Background: Platelets play a role in hemostasis which is the body's mechanisms to prevent and stop the bleeding. Platelets participate in the effort to close the wound, so that the body does not experience a loss of blood and protected from foreign cells. Examination of the platelet count is very important in the diagnosis of diseases, one of which is the diagnosis of dengue hemorrhagic fever (DHF). Examination of blood counts, especially platelets in clinical laboratories causes blood samples in use are not always the venous blood but could use capillary blood. Capillary blood samples are used primarily in pediatric patients, because the venous blood sampling is difficult, patient loads, and also shorten the time when taking blood. The purpose of this study was to determine whether there is a difference in counting the number of platelets using samples of blood veins and capillaries.
Methods: Quantitative research with observational approach using a cross sectional study design in the 30 samples of student D3 Health Analyst STIKes To Nation Yogyakarta. Statistical methods in use are independent T test.
Results: The research subjects were 30 samples of student D3 Health Analyst STIKes To Nation Yogyakarta. The results of the examination of venous blood platelet count and blood capillaries have different average values are 247 530 cells / ml of blood, for blood platelets veins and 184 270 cells / ml of blood for capillary blood platelets. Spearman correlation analysis Obtained results of the examination of venous blood platelet count and blood capillaries normal distribution (p> 0.05). 0.129 venous blood platelet counts, while the number of blood platelets kapilernya 0.089.
Conclusion: There is a significant difference from the results of counting the number of blood platelets using veins and capillaries, where the use of capillary blood samples showed that lower platelet counts.
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Guo, Qiuchen, Michael W. Malloy, Harvey G. Roweth, Joseph E. Italiano, and Elisabeth Battinelli. "Platelets up-Regulate Tumor Cell Programmed Death-Ligand 1 through Epidermal Growth Factor Receptor Signal Transduction." Blood 138, Supplement 1 (November 5, 2021): 1006. http://dx.doi.org/10.1182/blood-2021-151339.
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Abstract Introduction: Programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) are important immune checkpoint proteins in cancer immunotherapy and targeted therapies against PD-L1 have significantly prolonged many patients' lives. Recently, high baseline platelet to lymphocyte ratio was reported to be associated with decreased patient response rate to immune checkpoint inhibition (ICI) therapies, including anti-PD-L1 therapy, suggesting the potential role of platelets in tumor immunity. Platelets express PD-L1 on their surface, and platelets binding to PD-L1 negative tumor cells can "decorate" tumor cells with PD-L1 and protect against T cell-mediated cytotoxicity. However, whether platelet can affect PD-L1 expression on tumor cells is still unknown. Methods: In this study, we designed platelet-tumor cell co-culture systems to investigate whether direct or indirect exposure to platelets affects tumor cell PD-L1 surface expression. Considering platelets can be artificially activated by commonly used cell culture medium, the co-culture was performed in platelet resuspension buffer (HEPES, NaCl, KCl, MgCl2, NaHCO3, Glucose, pH7.4) supplied with fetal bovine serum and L-glutamine. After 24 hours of co-culture, platelets were washed out and fresh culturing medium was added to tumor cells and cultured for another 24 hours. At the end of the experiments, tumor cells were harvested and the PD-L1 expression analyzed by flow cytometry and RT-qPCR. Results and discussion: Here we report that direct co-culture of platelets with either breast cancer cell line MDA-MB-468 or lung cancer cell line A549 increased tumor cell PD-L1 surface expression by up-regulating PD-L1 transcription. This platelet-induced tumor cell PD-L1 up-regulation can be partly reduced by pre-treating platelets with antiplatelet agents such as aspirin and ticagrelor, suggesting platelet activation contributes to platelet induced tumor cell PD-L1 up-regulation. The up-regulation of tumor cell PD-L1 by platelets was not due to abundant platelet cytokines such as C-C Motif Chemokine Ligand 5 (CCL5) and C-X-C motif chemokine 5 (CXCL5). However, both an epidermal growth factor (EGF) neutralizing antibody and cetuximab (EGFR neutralizing monoclonal antibody) decreased the platelet-induced increase in tumor cell PD-L1, suggesting that platelets initiate tumor cell PD-L1 transcription through the EGF signaling pathway. Our data indicate a novel function of platelets in tumor immunity and warrant further investigation to determine if targeting platelets offers a novel adjuvant approach to improve ICI therapy. Disclosures Italiano: Sierra Oncology: Consultancy; PlateletBio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Carrick Therapeutics: Consultancy.
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Nolfi-Donegan, Deirdre, Sruti Shiva, and Cheryl A. Hillery. "HMGB1 As a Novel Platelet Agonist That Acts Synergistically with ADP to Activate Platelets in Sickle Cell Disease." Blood 132, Supplement 1 (November 29, 2018): 1073. http://dx.doi.org/10.1182/blood-2018-99-110269.
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Abstract Background: Sickle cell disease (SCD) is a proinflammatory and prothrombotic disorder that exhibits increased platelet activation. High mobility group box 1 (HMGB1) is a nuclear protein that can mediate inflammation when released from inflammatory or ischemic cells. HMGB1 is increased in many inflammatory disease states including SCD. Recent data suggests HMGB1 activates platelets and may work synergistically with potent platelet agonists such as collagen and thrombin, but little is known regarding HMGB1-platelet interactions in combination with weaker agonists like ADP, or in isolated platelets. Moreover, the effect of HMGB1 on platelet activation has not been evaluated in SCD. We hypothesized that the in vitro addition of low-dose recombinant HMGBI (rHMBG1) to isolated platelets will lower the threshold dose of physiologic agonists required to achieve platelet activation, and that this effect is exaggerated in SCD. Methods: Platelets were isolated from healthy controls (n=4) and patients with hemoglobin SS disease (SCD; n=5). The level of platelet activation was assessed after treatment with ADP at concentrations of 0 μM, 0.5 μM, 2 μM, and 5 μM with the addition of either low-dose rHMGB1 (10 μg/mL) or the same volume of vehicle. Percent platelet activation was measured via flow cytometry using PE antibody to GPIIb (CD41) to select for platelets, and PAC1 to detect the activation-dependent conformational change in integrin αIIbβ3 (GP IIb-IIIa). Platelet activation was interpreted as percent of platelets that bound PAC1. Data was analyzed using FlowJo software and nonparametric statistical tests. Results: Mean baseline platelet activation was 1.5% (range 0.4-3.3%) for control platelets and 7.3% (1.4-17.7%) for SCD platelets (p=0.19). In the SCD group, the addition of low-dose rHMGB1 (10 μg/mL) increased the mean percent of activated platelets from 7.3% to 26.5% (10.9-43%) (p=0.01). In comparison, mean activation of control platelets increased from only 1.5% to 19.5% (8.3-42.7%) after addition of rHMGB1 (p=0.12). Having illustrated that rHMGB1 can activate washed SCD platelets, we then compared the synergistic effect of rHMGB1 with ADP. There was increased platelet activation observed when ADP was added to rHMGB1 in SCD platelets: ADP 0.5 μM increased mean platelet activation from 13.8% (range 0.3-25.3%) to 54.4% (6.7-84.9%) with the addition of rHMGB1 (p=0.02); ADP 2 μM increased platelet activation from 14.1% (2.8-23.8%) to 56.2% (22.2-88.6%) with rHMGB1 (p=0.006); and ADP 5 μM increased platelet activation from 21.4% (2.5-30.1%) to 65.3% (31.7-85.9%) after adding rHMGB1 (p=0.004) (Fig 1; * and **, diamonds vs stars). We did not find a similar statistically significant synergistic effect in the control samples treated with ADP compared to combined HMGB1 + ADP, except at ADP dose 2 μM where platelet activation increased from 14.94% (4.6-28.6%) to 39.1% (19.5-56.0%) after the addition of rHMGB1 (p=0.04; Fig 1; #, square vs circle). Activation of platelets with just ADP was not different comparing control with SCD platelets (Fig 1; circles vs stars). Similarly, activation of platelets with both ADP and rHMGB1 was not significantly different comparing control with SCD platelets except for a trend at 0.5 μM ADP + rHMGB1 10 μg/mL with 19.29% (6.6-38.7) in controls vs 54.44% (6.7-84.9) in the SCD group (p=0.07) (Fig 1; diamonds vs squares). Summary: We found that rHMGB1 acts both independently and synergistically with ADP to increase platelet activation in SCD platelets. In our small cohort, SCD platelets had increased responsiveness to low dose-rHMGB1 compared to control platelets. Moreover, combining rHMGB1 with ADP greatly enhanced platelet activation in SCD but not control platelets. Our data suggest that SCD platelets are sensitized to HMGB1 in the presence of weaker agonists such as ADP. This heightened responsiveness of SCD platelets to HMGB1 may explain the enhanced platelet activation and inflammation associated with SCD in vivo. With further study, HMGB1 could be a target of clinical drug-directed therapy in SCD patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Rajasekhar, Damodara, Anita S. Kestin, Francis J. Bednarek, Patricia A. Ellis, Marc R. Barnard, and Alan D. Michelson. "Neonatal Platelets Are Less Reactive than Adult Platelets to Physiological Agonists in Whole Blood." Thrombosis and Haemostasis 72, no. 06 (1994): 957–63. http://dx.doi.org/10.1055/s-0038-1648990.
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SummaryPrevious studies have reported that the platelets of healthy term neonates have either diminished or normal reactivity compared to the platelets of adults. To circumvent the methodologic problems of previous studies, we used a whole blood flow cytometric method to study neonatal platelet reactivity to thrombin, a combination of ADP and epinephrine, and U46619 (a stable thromboxane A2 analogue). Inclusion in the assay of the peptide GPRP (an inhibitor of fibrin polymerization) enabled us to study platelet reactivity to human α-thrombin in whole blood. Umbilical cord blood and day 1 peripheral blood were collected from 30 healthy term neonates and compared to peripheral blood from 20 normal adults. In whole blood samples without added agonist, there were no significant differences between neonates and adults in the platelet binding of monoclonal antibodies 6D1 (GPIb-specific) or 7E3 (GPIIb-IIIa complex-specific). As determined by S12 (a P-selectin-specific monoclonal antibody), neither neonates nor adults had circulating degranulated platelets. However, in both cord and peripheral whole blood samples, neonatal platelets were significantly less reactive than adult platelets to thrombin, ADP/epinephrine, and U46619, as determined by the extent of increase in the platelet surface expression of P-selectin and the GPIIb-IIIa complex, and the extent of decrease in the platelet surface expression of the GPIb-IX complex. For example, as compared to maximal platelet surface P-selectin in adults (with thrombin 10 U/ml), thrombin 1 U/ml resulted in platelet surface P-selectin of 95 ± 2% (mean ± S.E.M.) in adult peripheral blood, but only 70 ± 4% in cord blood and 70 ± 3% in neonatal peripheral blood (p <0.0001). Thrombin 0.1 U/ml resulted in platelet surface P-selectin of 49 ± 4% in adult peripheral blood, but only 10 ± 2% in cord blood and 17 ± 2% in neonatal peripheral blood (p α0.0001). Similar results were obtained in a washed platelet system. In summary: 1) Compared to adult controls, neonatal platelets are hyporeactive to thrombin, a combination of ADP and epinephrine, and a thromboxane A2 analogue in the physiologic milieu of whole blood. 2) The hyporeactivity of neonatal platelets compared to adult platelets is the result of a defect intrinsic to neonatal platelets. 3) Whole blood flow cytometry is particularly advantageous for neonatal studies because only 5 μl of blood per assay is required.
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Miles, Jeffrey, Shawn Lawrence Bailey, Lydia Fang, Barbara Osborne, Jill Corson, Johnathan P. Mack, and Moritz Stolla. "Evaluation of Efficacy and Safety of Cold-Stored Platelets in Healthy Human Subjects Treated with Dual Antiplatelet Therapy." Blood 134, Supplement_1 (November 13, 2019): 718. http://dx.doi.org/10.1182/blood-2019-124122.
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Background: Platelets are currently stored at 22°C (room temperature-stored) for clinical purposes. This approach has numerous downsides including limited storage time due to risk of bacterial growth, and increased costs caused by bacterial testing or pathogen reduction. Platelets stored at 4ºC (cold-stored) were the standard of care in the 1960s and 70s, and while they generally perform better than room temperature-stored platelets in in vitro assays, functional studies in vivo have yielded contradictory results. Methods: Eight healthy human participants were included in the analysis of this randomized, cross-over study. A double unit of apheresis platelets was collected from each subject for autologous transfusion. Platelets were stored for 5 days at either 4ºC, or 22ºC, based on randomization. On day 5, platelets were transfused 12-24 hours after participants received a loading dose of acetylsalicylic acid and clopidogrel. An array of platelet function testing was done at baseline, after loading dose, and after transfusion (1h, 4h, 24h). The first round was followed by a wash-out period of at least 10 days, and a second sequence of collection, antiplatelet loading dose, and transfusion with platelets stored under alternate conditions from the first sequence. The primary endpoint was defined as post-transfusion platelet αIIbβ3 integrin activation in response to agonists for pathways inhibited by dual antiplatelet therapy, measured by the VerifyNOW assay. Secondary endpoints included platelet function testing by bleeding time, VASP-phosphorylation, light transmission aggregometry, and flow cytometric analysis of platelet αIIbβ3 integrin activation and α-granule secretion. Results: All cold-stored platelet units passed quality control analyses, and no platelet aggregates were observed at the end of the storage period. One room temperature-stored unit had to be discarded due to quality control failure. Transfusion of a double unit with 5 day cold-stored platelets was tolerated well by all recipients. The absolute number of transfused platelets did not differ significantly between the cold-storage arm and the room temperature storage arm. The corrected count increment did not differ significantly at 1h after transfusion, but was significantly lower in the cold-stored group at the 4h, and 24h time points. The primary endpoint, platelet function testing by VerifyNOW, showed reversal of the effect of acetylsalicylic acid with both products at 1h and 4h post transfusion, but platelet inhibition re-appeared after 24h post transfusion in the cold-storage arm, presumably due to accelerated clearance of cold-stored platelets. No significant differences were observed between the two products in the VerifyNOW assay for clopidogrel. The bleeding time (a secondary endpoint) did not differ significantly at any time point between the cold-stored and room temperature-stored transfusion group. However, the bleeding time improved or remained unchanged in all recipients transfused with room temperature-stored platelets, whereas, transfusion of cold-stored platelets caused prolongation of the bleeding time in 50% of recipients. Surprisingly, platelets isolated from recipients of room temperature-stored platelets aggregated significantly better in response to collagen compared to platelets isolated from recipients of cold-stored platelets at 1h and 4h post transfusion. Other secondary endpoints, including VASP-phosphorylation, αIIbβ3 integrin activation and α-granule secretion by flow cytometry, and platelet aggregation in response to arachidonic acid and ADP did not differ significantly between the two treatment groups. Conclusion: We report the first safety and efficacy data of 5 day cold stored platelets in plasma. Unexpectedly, some endpoints demonstrate agonist- and assay-dependent inferiority of cold-stored platelets at early and late time points. Further studies are needed to determine the maximum storage time, and the efficacy of cold-stored platelets in actively bleeding patients and patients with platelet dysfunction. Disclosures No relevant conflicts of interest to declare.
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Davizon-Castillo, Pavel, Brandon McMahon, Sonia Aguila, David Bark, Katrina Ashworth, Ayed Allawzi, Robert A. Campbell, et al. "TNF-α–driven inflammation and mitochondrial dysfunction define the platelet hyperreactivity of aging." Blood 134, no. 9 (August 29, 2019): 727–40. http://dx.doi.org/10.1182/blood.2019000200.
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Abstract Aging and chronic inflammation are independent risk factors for the development of atherothrombosis and cardiovascular disease. We hypothesized that aging-associated inflammation promotes the development of platelet hyperreactivity and increases thrombotic risk during aging. Functional platelet studies in aged-frail adults and old mice demonstrated that their platelets are hyperreactive and form larger thrombi. We identified tumor necrosis factor α (TNF-α) as the key aging-associated proinflammatory cytokine responsible for platelet hyperreactivity. We further showed that platelet hyperreactivity is neutralized by abrogating signaling through TNF-α receptors in vivo in a mouse model of aging. Analysis of the bone marrow compartments showed significant platelet-biased hematopoiesis in old mice reflected by increased megakaryocyte-committed progenitor cells, megakaryocyte ploidy status, and thrombocytosis. Single-cell RNA-sequencing analysis of native mouse megakaryocytes showed significant reprogramming of inflammatory, metabolic, and mitochondrial gene pathways in old mice that appeared to play a significant role in determining platelet hyperreactivity. Platelets from old mice (where TNF-α was endogenously increased) and from young mice exposed to exogenous TNF-α exhibited significant mitochondrial changes characterized by elevated mitochondrial mass and increased oxygen consumption during activation. These mitochondrial changes were mitigated upon TNF-α blockade. Similar increases in platelet mitochondrial mass were seen in platelets from patients with myeloproliferative neoplasms, where TNF-α levels are also increased. Furthermore, metabolomics studies of platelets from young and old mice demonstrated age-dependent metabolic profiles that may differentially poise platelets for activation. Altogether, we present previously unrecognized evidence that TNF-α critically regulates megakaryocytes resident in the bone marrow niche and aging-associated platelet hyperreactivity and thrombosis.
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O'Sullivan, Brian P., Matthew D. Linden, Andrew L. Frelinger, Marc R. Barnard, Michele Spencer-Manzon, James E. Morris, Raneem O. Salem, Michael Laposata, and Alan D. Michelson. "Platelet activation in cystic fibrosis." Blood 105, no. 12 (June 15, 2005): 4635–41. http://dx.doi.org/10.1182/blood-2004-06-2098.
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Abstract Cystic fibrosis (CF) is caused by a mutation of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). We examined platelet function in CF patients because lung inflammation is part of this disease and platelets contribute to inflammation. CF patients had increased circulating leukocyte-platelet aggregates and increased platelet responsiveness to agonists compared with healthy controls. CF plasma caused activation of normal and CF platelets; however, activation was greater in CF platelets. Furthermore, washed CF platelets also showed increased reactivity to agonists. CF platelet hyperreactivity was incompletely inhibited by prostaglandin E1 (PGE1). As demonstrated by Western blotting and reverse-transcriptase-polymerase chain reaction (RT-PCR), there was neither CFTR nor CFTR-specific mRNA in normal platelets. There were abnormalities in the fatty acid composition of membrane fractions of CF platelets. In summary, CF patients have an increase in circulating activated platelets and platelet reactivity, as determined by monocyte-platelet aggregation, neutrophil-platelet aggregation, and platelet surface P-selectin. This increased platelet activation in CF is the result of both a plasma factor(s) and an intrinsic platelet mechanism via cyclic adenosine monophosphate (cAMP)/adenylate cyclase, but not via platelet CFTR. Our findings may account, at least in part, for the beneficial effects of ibuprofen in CF. (Blood. 2005;105:4635-4641)
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Baugh, Robert F. "Platelets and whole blood coagulation." Perfusion 15, no. 1 (January 2000): 41–50. http://dx.doi.org/10.1177/026765910001500107.
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In our early work in developing activated clotting time (ACT) assays, it became apparent that changes occurred in coagulation times as a whole blood sample aged (0-6 h). Subsequent studies showed that the coagulation parameters of plasma obtained from the samples remained stable during this time frame. These changes in whole blood clotting times during storage were eventually traced to the platelets. Several years of work demonstrated that this change was due to the removal of the blood from the vascular lining. This recalled a mechanism that was originally put forth in the 1970s with the discovery of prostacyclin. In this postulated mechanism, platelets are ‘time-bombs’. They are kept under control by prostacyclin (PGI2) secreted by the vascular lining. Without this prostacyclin, platelets ‘preactivate’. Since that time, additional substances secreted by the vascular endothelium have been identified, such as nitric oxide, that also influence platelet activity. The ‘preactivation’ of platelets in a blood sample can be followed using an ACT. In the same donor, the preactivation is uniform and reproducible over an extended period (months). There is, however, considerable variability between donors. Some donors’ platelets preactivate dramatically, while other donors show hardly any change. Prostacyclin, added to the blood sample when it is collected, prevents this preactivation. The clinical significance of these observations has yet to be clearly established, but these observations raise a number of questions with respect to methods for improving platelet function during bypass and in evaluating the risk of platelet-mediated cardiovascular disease.
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Lambert, Michele P., Spencer K. Sullivan, Rudy Fuentes, Deborah L. French, and Mortimer Poncz. "Challenges and promises for the development of donor-independent platelet transfusions." Blood 121, no. 17 (April 25, 2013): 3319–24. http://dx.doi.org/10.1182/blood-2012-09-455428.
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Abstract Platelet transfusions are often a life-saving intervention, and the use of platelet transfusions has been increasing. Donor-derived platelet availability can be challenging. Compounding this concern are additional limitations of donor-derived platelets, including variability in product unit quality and quantity, limited shelf life and the risks of product bacterial contamination, other transfusion-transmitted infections, and immunologic reactions. Because of these issues, there has been an effort to develop strategies to generate platelets from exogenously generated precursor cells. If successful, such platelets have the potential to be a safer, more consistent platelet product, while reducing the necessity for human donations. Moreover, ex vivo–generated autologous platelets or precursors may be beneficial for patients who are refractory to allogeneic platelets. For patients with inherited platelet disorders, ex vivo–generated platelets offer the promise of a treatment via the generation of autologous gene-corrected platelets. Theoretically, ex vivo–generated platelets also offer targeted delivery of ectopic proteins to sites of vascular injury. This review summarizes the current, state-of-the-art methodologies in delivering a clinically relevant ex vivo–derived platelet product, and it discusses significant challenges that must be overcome for this approach to become a clinical reality.
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Lagarrigue, Frederic, David S. Paul, Alexandre R. Gingras, Andrew J. Valadez, Hao Sun, Jenny Lin, Monica N. Cuevas, et al. "Talin-1 is the principal platelet Rap1 effector of integrin activation." Blood 136, no. 10 (September 3, 2020): 1180–90. http://dx.doi.org/10.1182/blood.2020005348.
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Abstract Ras-related protein 1 (Rap1) is a major convergence point of the platelet-signaling pathways that result in talin-1 binding to the integrin β cytoplasmic domain and consequent integrin activation, platelet aggregation, and effective hemostasis. The nature of the connection between Rap1 and talin-1 in integrin activation is an important remaining gap in our understanding of this process. Previous work identified a low-affinity Rap1-binding site in the talin-1 F0 domain that makes a small contribution to integrin activation in platelets. We recently identified an additional Rap1-binding site in the talin-1 F1 domain that makes a greater contribution than F0 in model systems. Here we generated mice bearing point mutations, which block Rap1 binding without affecting talin-1 expression, in either the talin-1 F1 domain (R118E) alone, which were viable, or in both the F0 and F1 domains (R35E,R118E), which were embryonic lethal. Loss of the Rap1–talin-1 F1 interaction in platelets markedly decreases talin-1–mediated activation of platelet β1- and β3-integrins. Integrin activation and platelet aggregation in mice whose platelets express only talin-1(R35E, R118E) are even more impaired, resembling the defect seen in platelets lacking both Rap1a and Rap1b. Although Rap1 is important in thrombopoiesis, platelet secretion, and surface exposure of phosphatidylserine, loss of the Rap1–talin-1 interaction in talin-1(R35E, R118E) platelets had little effect on these processes. These findings show that talin-1 is the principal direct effector of Rap1 GTPases that regulates platelet integrin activation in hemostasis.
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Scharf, Rüdiger E. "In Vitro Thromboxane Synthesis of Depleted Blood Platelets Following Renal Transplantation." Thrombosis and Haemostasis 64, no. 01 (1990): 161–64. http://dx.doi.org/10.1055/s-0038-1647274.
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SummaryRenal transplant rejection is associated with platelet activation in vivo which may lead to partially α- and γ-granule-depleted platelets that continue to circulate. These “exhausted” platelets are hemostatically defective. Tb quantitate the extent of platelet granule depletion following kidney transplantation, we determined intraplatelet levels of β-thromboglobulin (βTG), platelet factor 4 (PF4), and serotonin (5-hydroxytryptamine, 5-HT) ex vivo in Tiiton X-1O0-treated platelet lysates. To explore biochemical alterations of partially depleted platelets, we studied platelet thromboxane A2 (TXA2) synthesis in citrated plateletrich plasma (PRP) upon stimulation with thrombin or collagen in 45 recipients of renal allografts and 10 healthy volunteers. The patients were divided into subjects with acute and chronic allograft rejection (N = 15), those with compensated renal failure after kidney transplantation but without evidence of allograft rejection (N = 15), and those with functioning renal transplant (N = 15). The mean intraplatelet content of βTG (38.6 ± 4.2 μE/109 platelets), PF4 (11.8 ± 1.8 μg/109 platelets), and 5-HT (274 ± 31 μg/109 platelets) in patients with acute or chronic renal allograft rejection was significantly lower than in other recipients off < idney transplants or healthy volunteers (βTG: 59.9±4.7 μgl 109 platelets; PF4: 20.4±2.3 ¼g/n platelets; s-rrr: 46lraB ngl 10e platelets; p < 0.ffi5 in all casls). Platelet TxB2 formation upon stimulation with thrombin (10 U/ml) or collagen (6.25 ¼g/ml) for 5 min was significantly reduced in patients with acute or chronic renal allograft rejection (2.25±0.29 and 0.641 0.08 nmoUl0e platelets for thrombin- and collagen-stimulated platelets, respectively) compared to that of healthy volunteers (4.72± 0.60 and 1.35 ± 0.12 nmol/109 platelets, respectively; p <0.05 in all cases). In contrast, platelet TXB2 formation of patients with functioning kidney transplant or those with compensated renal failure but without evidence of transplant rejection did not differ significantly from that of normals. These results confirm that platelets with reduced levels of α- and β-granular constituents are detectable in the circulation following kidney transplantation when acute or chronic renal allograft rejections occur. These platelets are incapable of forming normal amounts of thromboxane upon stimulation with thrombin and collagen in vitro. This dysfunction of thromboxane synthesis, due to alterations in the platelet arachidonate pathway, may reflect the previous activation of platelets in vivo associated with acute or chronic renal allograft rejection.
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Olsson, Mattias, Pierre Bruhns, William A. Frazier, Jeffrey V. Ravetch, and Per-Arne Oldenborg. "Platelet homeostasis is regulated by platelet expression of CD47 under normal conditions and in passive immune thrombocytopenia." Blood 105, no. 9 (May 1, 2005): 3577–82. http://dx.doi.org/10.1182/blood-2004-08-2980.
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Abstract Interaction between target cell CD47 and the inhibitory macrophage receptor signal regulatory protein α (SIRPα) counteracts macrophage phagocytosis of CD47-expressing host cells. As platelets also express CD47, we asked whether inhibitory CD47/SIRPα signaling regulates normal platelet turnover and clearance of platelets in immune thrombocytopenic purpura (ITP). CD47-/- mice had a mild spontaneous thrombocytopenia, which was not due to a decreased platelet half-life as a result of increased expression of P-selectin, CD61, or phosphatidylserine. In contrast, CD47-/- platelets were rapidly cleared when transfused into CD47+/+ recipients, whereas CD47+/- platelets had a nearly normal half-life in CD47+/+ mice under nonautoimmune conditions. CD47-/- mice were more sensitive to ITP, as compared with CD47+/+ mice. In vitro, macrophage phagocytosis of immunoglobulin G (IgG)–opsonized CD47-/- platelets was significantly higher than that for equally opsonized CD47+/+ platelets. However, when SIRPα was blocked, phagocytosis of CD47+/+ platelets increased to the level of CD47-/- platelets. Phagocytosis of opsonized CD47+/- platelets was higher than that for CD47+/+ platelets, but lower than that for CD47-/- platelets, suggesting a gene-dose effect of CD47 in this system. In conclusion, we suggest that inhibitory CD47/SIRPα signaling is involved in regulating platelet phagocytosis in ITP, and that targeting SIRPα may be a new means of reducing platelet clearance in ITP.
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Davenport, Patricia Ellen, Christopher S. Thom, Erin L. Soule-Albridge, Emily Nolton, Zhi-Jian Liu, Henry A. Feldman, and Martha Sola-Visner. "Developmental Differences between Neonatal and Adult Platelets in Regard to Immune Protein Content and Regulation of Monocyte Inflammatory Responses." Blood 142, Supplement 1 (November 28, 2023): 699. http://dx.doi.org/10.1182/blood-2023-177993.
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Preterm neonates frequently receive platelet transfusions (PTx), owing to their high incidence of thrombocytopenia and bleeding. However, a recent large randomized trial found a significantly higher incidence of death or major bleeding among more liberally transfused preterm neonates. The mechanisms underlying these deleterious effects are unknown, but it has been suggested that they are related to the developmental differences between adult and neonatal platelets, and to a potential “developmental mismatch” occurring when adult platelets (comparatively hyperreactive) are transfused into neonates. While most studies so far have focused on the hemostatic effects of PTx, it is now clear that platelets are key modulators of immune and inflammatory responses. We hypothesized that adult platelets would be more pro-inflammatory than neonatal platelets. To test this hypothesis, we first compared the proteome of resting cord blood (CB, n=9) and adult (n=7) platelets. Using data-independent acquisition mass spectrometry, we identified 4173 high-confidence proteins present in both adult and neonatal platelets. In a principal component analysis, neonatal and adult platelets clustered separately, and 359 proteins were present at significantly different relative abundances between the two groups (≥1.5-fold change, p<0.05). Pathway analysis of the 169 proteins significantly more abundant in the adult platelet proteome revealed enrichment for immune and inflammatory pathways, while neonatal platelets were enriched for pathways involved in metabolic processes. Next, we compared the effects of neonatal and adult platelets on the inflammatory response of human neonatal monocytes. Platelets and monocytes were isolated from healthy adult blood or full-term CB (n=6/group). Platelet P-selectin surface expression was determined by flow cytometry, and key platelet-derived immune regulators (RANTES, β-2 Microglobulin [β2M], and TGF-β) were quantified in the supernatant of resting and activated platelets by ELISA. After washing, neonatal and adult platelets had similarly low P-selectin surface expression levels. Following thrombin activation, P-selectin surface expression was significantly higher in adult than in neonatal platelets (MFI 4010±1645 vs. 1987±816, p=0.04). Thrombin activation increased RANTES and TGF-β release from adult and neonatal platelets to similar levels. In contrast, β2M levels were significantly higher in the supernatant from activated adult compared to neonatal platelets (37.9±10.2 vs 17.9±5.0 ng/mL, p=0.0004). Next, we co-cultured activated adult or neonatal platelets with neonatal monocytes and quantified the pro-inflammatory cytokines IL-8 and MCP-1 (CCL2) in the supernatants after 18 hours. The presence of activated platelets significantly increased IL-8 production from neonatal monocytes compared to monocytes alone, 2.9±1.2-fold when neonatal platelets were added and 4.2±1.8-fold with addition of adult platelets (p=0.01 and p=0.001, respectively). MCP-1 increased to similar levels with the addition of either neonatal or adult platelets. In a regression model, both P-selectin expression and platelet-released β2M levels were significantly associated with monocyte-produced IL-8 levels (p=0.001 and p=0.04, respectively). In summary, we found that proteins related to inflammatory and immune responses are more abundant in adult compared to neonatal platelets, and that activated adult platelets express more P-selectin and release more β2M than neonatal platelets, resulting in higher IL8 production by neonatal monocytes. These findings support the hypothesis that the pro-inflammatory effects of adult platelets might contribute to the increased morbidity and mortality associated with neonatal PTx.
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Li, Chen, Preeti Maurya, Benjamin Nieves-Lopez, Sara Ture, and Craig N. Morrell. "Platelet Mediated Monocyte/Macrophage Immune Training." Blood 138, Supplement 1 (November 5, 2021): 3127. http://dx.doi.org/10.1182/blood-2021-151243.
Full textAbstract:
Abstract Platelets are essential mediators of vascular and immune homeostasis as well as mediators of thrombosis. Platelet functions in hemostasis and thrombosis have received great attention in both basic and clinical research, however, emerging research indicates platelets are also critical components of the immune system. Platelets have important roles in many inflammation-associated diseases including sepsis, atherosclerosis, and peripheral artery disease. In recent years, much interest has focused on the acute outcomes of interactions between platelets and monocytes/macrophages in immune responses to bacterial infections, as several studies have demonstrated that platelets contribute to bacteria clearance and inflammation resolution by regulating monocyte/macrophage responses and their immune differentiation. However, macrophages are long-lived cells and past stimulation and immune interactions can change their responses to future stimuli - a concept termed 'innate immune memory'. Whether platelet interactions with monocytes/macrophages alters their inflammatory responses to secondary stimuli in a long-lasting manner remains unclear. We have now found that platelet interactions with monocytes and macrophages as a first stimulus, changed their responses to secondary stimuli, such as lipopolysaccharide (LPS) and unmethylated cytosine-phosphate-guanine (CpG). We incubated monocytes or macrophages with control buffer or platelets overnight and then removed the platelets prior to the addition of LPS or CpG as secondary stimulation. LPS and CpG induced IL-6 and TNFα were reduced by platelet pre-incubation compared to platelet naïve macrophages. Despite reduced cytokine secretion, platelet pre-incubation increased monocyte and macrophage proliferation in response to secondary stimuli. Furthermore, the platelet mediated macrophage secondary stimuli phenotype was preserved for several days after platelet exposure indicating a genetic reprogramming mediated mechanism. Circulating monocytes from platelet deficient TPOR -/- mice and adult mice made platelet deficient also had higher Il6 expression and secrete more IL-6 in response to stimulation compared to monocytes from WT mice indicating in vivo platelet mediated monocyte immune-training. Furthermore, the genetic reprogramming may be dependent on a HIF1α dependent process, as platelet pre-incubation increased macrophage Hif1α expression, a known mediator of trained immunity. Monocytes and macrophages, are 'immune plastic' and adapt to their environment in a functional manner. These novel data indicate that platelets 'reprogram' monocytes/macrophages and shape their responses to future stimulation. Disclosures No relevant conflicts of interest to declare.
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Manne, Bhanu Kanth, Frederik Denorme, Elizabeth A. Middleton, Irina Portier, Jesse W. Rowley, Chris Stubben, Aaron C. Petrey, et al. "Platelet gene expression and function in patients with COVID-19." Blood 136, no. 11 (September 10, 2020): 1317–29. http://dx.doi.org/10.1182/blood.2020007214.
Full textAbstract:
Abstract There is an urgent need to understand the pathogenesis of coronavirus disease 2019 (COVID-19). In particular, thrombotic complications in patients with COVID-19 are common and contribute to organ failure and mortality. Patients with severe COVID-19 present with hemostatic abnormalities that mimic disseminated intravascular coagulopathy associated with sepsis, with the major difference being increased risk of thrombosis rather than bleeding. However, whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection alters platelet function to contribute to the pathophysiology of COVID-19 remains unknown. In this study, we report altered platelet gene expression and functional responses in patients infected with SARS-CoV-2. RNA sequencing demonstrated distinct changes in the gene-expression profile of circulating platelets of COVID-19 patients. Pathway analysis revealed differential gene-expression changes in pathways associated with protein ubiquitination, antigen presentation, and mitochondrial dysfunction. The receptor for SARS-CoV-2 binding, angiotensin-converting enzyme 2 (ACE2), was not detected by messenger RNA (mRNA) or protein in platelets. Surprisingly, mRNA from the SARS-CoV-2 N1 gene was detected in platelets from 2 of 25 COVID-19 patients, suggesting that platelets may take-up SARS-COV-2 mRNA independent of ACE2. Resting platelets from COVID-19 patients had increased P-selectin expression basally and upon activation. Circulating platelet-neutrophil, -monocyte, and -T-cell aggregates were all significantly elevated in COVID-19 patients compared with healthy donors. Furthermore, platelets from COVID-19 patients aggregated faster and showed increased spreading on both fibrinogen and collagen. The increase in platelet activation and aggregation could partially be attributed to increased MAPK pathway activation and thromboxane generation. These findings demonstrate that SARS-CoV-2 infection is associated with platelet hyperreactivity, which may contribute to COVID-19 pathophysiology.
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Wandall, Hans H., Karin M. Hoffmeister, Anne Louise Sørensen, Viktoria Rumjantseva, Henrik Clausen, John H. Hartwig, and Sherrill J. Slichter. "Galactosylation does not prevent the rapid clearance of long-term, 4°C-stored platelets." Blood 111, no. 6 (March 15, 2008): 3249–56. http://dx.doi.org/10.1182/blood-2007-06-097295.
Full textAbstract:
AbstractCold storage of platelets for transfusion is desirable to extend platelet storage times and to prevent bacterial growth. However, the rapid clearance of cold-stored platelets prevents their use. A novel method for preventing the rapid clearance of cold-stored platelets has previously been developed in a murine model. Cold storage induces the clustering and recognition of exposed β-N-acetylglucosamine (βGlcNAc) on platelet surfaces. Glycosylation of βGlcNAc residues with uridine 5′-diphosphogalactose (UDP-galactose) results in the normal survival of short-term (2 h) 0°C-stored murine platelets. Based on this finding, we developed a similar glycosylation process by adding UDP-galactose to human apheresis platelets. A phase 1 clinical trial was conducted transfusing radiolabeled autologous apheresis platelets stored for 48 hours at 4°C with or without pretreatment with UDP-galactose. In contrast to the murine study, galactosylation of human platelets did not prevent the accelerated platelet clearance routinely observed after 4°C storage. We next developed a murine model of platelet storage for 48 hours at 4°C and showed that UDP-galactose treatment of murine platelets also did not prevent their rapid clearance, in agreement with the human platelet study. We conclude that different mechanisms of clearance may exist for short- and long-term cold-stored platelets.