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1
Beaulieu, Lea, Kahraman Tanriverdi, Jane Freedman, and Lauren Clancy. "The role of RNA uptake in platelet heterogeneity." Thrombosis and Haemostasis 117, no. 05 (2017): 948–61. http://dx.doi.org/10.1160/th16-11-0873.
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SummaryThe role of platelets in regulating vascular homeostasis has expanded beyond mediation of haemostasis and thrombosis. The discovery of platelet RNA and the presence of subpopulations of platelets containing varying amounts of RNA suggest a role for platelet transcripts in vascular function. As the RNA in anucleated platelets is biologically functional and may transfer to other vascular cells, we hypothesised that platelet RNA diminishes over the lifespan of the platelet with diminishing platelet size due to horizontal cellular transfer. The purpose of this study is to determine if platelet RNA variance is the result of horizontal cellular transfer between platelets and other vascular cells. Utilising platelet sorting and RNA sequencing, we found that smaller platelets contained a more diverse set of transcripts than larger platelets. Further investigation using fluorescence imaging, gene expression analyses and in vitro and in vivo modelling revealed that platelets take up RNA from other vascular cells in a complex manner, revealing a dynamic role for platelets in modulating vascular homeostasis through bidirectional RNA transfer. The resultant RNA profile heterogeneity suggests unique functional roles for platelets dependent on size and complexity. This study expands our basic understanding of platelet function and heterogeneity and is the first to evaluate endogenous vascular RNA uptake and its relation to platelet processes. Our findings describe a novel endogenous phenomenon that can help elucidate the platelet’s role in these non-thrombotic and haemostatic fields, as well as present potential for diagnostic and therapeutic development.Supplementary Material to this article is available online at www.thrombosis-online.com.
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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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Krisch, Linda, Gabriele Brachtl, Sarah Hochmann, André Cronemberger Andrade, Michaela Oeller, Patricia Ebner-Peking, Katharina Schallmoser, and Dirk Strunk. "Improving Human Induced Pluripotent Stem Cell-Derived Megakaryocyte Differentiation and Platelet Production." International Journal of Molecular Sciences 22, no. 15 (July 30, 2021): 8224. http://dx.doi.org/10.3390/ijms22158224.
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Several protocols exist for generating megakaryocytes (MKs) and platelets from human induced pluripotent stem cells (hiPSCs) with limited efficiency. We observed previously that mesoderm induction improved endothelial and stromal differentiation. We, therefore, hypothesized that a protocol modification prior to hemogenic endothelial cell (HEC) differentiation will improve MK progenitor (MKP) production and increase platelet output. We further asked if basic media composition affects MK maturation. In an iterative process, we first compared two HEC induction protocols. We found significantly more HECs using the modified protocol including activin A and CHIR99021, resulting in significantly increased MKs. MKs released comparable platelet amounts irrespective of media conditions. In a final validation phase, we obtained five-fold more platelets per hiPSC with the modified protocol (235 ± 84) compared to standard conditions (51 ± 15; p < 0.0001). The regenerative potency of hiPSC-derived platelets was compared to adult donor-derived platelets by profiling angiogenesis-related protein expression. Nineteen of 24 angiogenesis-related proteins were expressed equally, lower or higher in hiPSC-derived compared to adult platelets. The hiPSC-platelet’s coagulation hyporeactivity compared to adult platelets was confirmed by thromboelastometry. Further stepwise improvement of hiPSC-platelet production will, thus, permit better identification of platelet-mediated regenerative mechanisms and facilitate manufacture of sufficient amounts of functional platelets for clinical application.
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Campbell, Robert A., Thomas H. Fischer, and Alisa S. Wolberg. "Rehydrated, Lyophilized Platelets Generate Thrombin in the Presence of Recombinant Factor VIIa." Blood 106, no. 11 (November 16, 2005): 4057. http://dx.doi.org/10.1182/blood.v106.11.4057.4057.
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Abstract The anti-bleeding therapy, recombinant factor VIIa (rFVIIa), is thought to bind to the platelet’s surface and increase thrombin generation in hemophilia. However, high plasma levels of rFVIIa are required, in part, due to the weak binding of rFVIIa to platelets. We hypothesized that the efficacy of the therapy could be improved by administering rFVIIa already bound to platelets. A recently described protocol involving pretreatment of platelets with paraformaldehyde permits platelets to be lyophilized while preserving many platelet functions. Such platelets could be used for binding rFVIIa ex vivo and then administered to hemophilic patients during a bleeding event. In this study, we have characterized the ability of reconstituted, lyophilized (RL) platelets to support thrombin generation under normal and hemophilic conditions and in the presence of rFVIIa. First, freshly-isolated (control) or RL platelets were incubated with factors IXa, VIII(a), X, V and II in the presence of 3 mM CaCl2 and assayed for thrombin generation. In these assays, both freshly-isolated and RL platelets supported thrombin generation (1.15x10−4 +/− 5.37x10−5 mOD/min2/platelet and 8.46x10−3 +/− 4.78x10−3 mOD/min2/platelet, respectively). In the absence of factor IX (hemophilia B), thrombin generation was significantly reduced on both freshly-isolated and RL platelets (4.19x10−6 +/− 4.50x10−6 mOD/min2/platelet and 8.25x10−4 +/− 1.13x10−6 mOD/min2/platelet, respectively). Interestingly, RL platelets supported 10 – 100-fold higher thrombin generation rates than fresh thrombin-activated platelets. Second, we examined the activity of rFVIIa on RL platelets in the absence of factors IX and VIII. RFVIIa increased thrombin generation on RL platelets in a rFVIIa-concentration dependent manner (between 1nM and 150nM), similar to that seen when using fresh platelets. An inhibitory anti-tissue factor (TF) antibody did not affect rFVIIa-mediated thrombin generation on RL platelets, indicating that the activity of rFVIIa on RL platelets is independent of TF. Finally, we examined the effect of different platelet agonists (thrombin, convulxin, and A23187) on fresh and RL platelets. When fresh platelets are stimulated with A23187 or co-stimulated with thrombin and convulxin, they become more procoagulant than platelets activated with thrombin alone. However, stimulation of RL platelets with A23187 or co-stimulation with thrombin and convulxin did not increase thrombin generation versus thrombin alone. RL platelets stimulated with thrombin, alone, had 3.1-fold higher activity than thrombin- and convulxin-costimulated fresh platelets, but 1.4-fold lower activity than A23187-stimulated fresh platelets. These data suggest that RL platelets are in a maximally active state prior to the addition of platelet agonists. We conclude that RL platelets are procoagulant and can support rFVIIa-mediated thrombin generation in the absence of factor IX. We hypothesize that co-administration of RL platelets with rFVIIa may increase the efficacy of rFVIIa treatment.
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Yang, Linlin, Roger Ottenheijm, Paul Worley, Marc Freichel, and Juan E. Camacho Londoño. "Reduction in SOCE and Associated Aggregation in Platelets from Mice with Platelet-Specific Deletion of Orai1." Cells 11, no. 20 (October 14, 2022): 3225. http://dx.doi.org/10.3390/cells11203225.
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Calcium signalling in platelets through store operated Ca2+ entry (SOCE) or receptor-operated Ca2+ entry (ROCE) mechanisms is crucial for platelet activation and function. Orai1 proteins have been implicated in platelet’s SOCE. In this study we evaluated the contribution of Orai1 proteins to these processes using washed platelets from adult mice from both genders with platelet-specific deletion of the Orai1 gene (Orai1flox/flox; Pf4-Cre termed as Orai1Plt-KO) since mice with ubiquitous Orai1 deficiency show early lethality. Platelet aggregation as well as Ca2+ entry and release were measured in vitro following stimulation with collagen, collagen related peptide (CRP), thromboxane A2 analogue U46619, thrombin, ADP and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin, respectively. SOCE and aggregation induced by Thapsigargin up to a concentration of 0.3 µM was abrogated in Orai1-deficient platelets. Receptor-operated Ca2+-entry and/or platelet aggregation induced by CRP, U46619 or thrombin were partially affected by Orai1 deletion depending on the gender. In contrast, ADP-, collagen- and CRP-induced aggregation was comparable in Orai1Plt-KO platelets and control cells over the entire concentration range. Our results reinforce the indispensability of Orai1 proteins for SOCE in murine platelets, contribute to understand its role in agonist-dependent signalling and emphasize the importance to analyse platelets from both genders.
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Crawford, N., A. Chajara, G. Pfliegler, B. EI Gamal, L. Brewer, and L. Capron. "Targeting Platelets Containing Electro-encapsulated lloprost to Balloon Injured Aorta in Rats." Thrombosis and Haemostasis 73, no. 03 (1995): 535–42. http://dx.doi.org/10.1055/s-0038-1653809.
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SummaryDrugs can be electro-encapsulated within platelets and targeted to damaged blood vessels by exploiting the platelet’s natural haemostatic properties to adhere to collagen and other vessel wall constituents revealed by injury. A rat aorta balloon angioplasty model has been used to study the effect on platelet deposition of giving iloprost loaded platelets i.v. during the balloon injury. After labelling the circulating platelets with 111-Indium before balloon injury, time course studies showed maximum platelet deposition on the injured aorta occurred at about 1 h post-injury and the deposition remained stable over the next 2-3 h. When iloprost-loaded platelets were given i.v. during injury and the circulating platelet pool labelled with 111-Indium 30 min later, platelet deposition, measured at 2 h postinjury, was substantially and significantly reduced compared with control platelet treatment. Some antiproliferative effects of iloprost-loaded platelets given i.v. during injury have also been observed. Whereas the incorporation of [3H]-thymidine into aorta intima-media DNA at 3 days post injury was 62-fold higher in balloon injured rats than in control sham operated rats, thymidine incorporation into intima/media of rats which had received iloprost loaded platelets during injury was reduced as compared with rats subjected only to the injury procedure. The reduction was only of near significance, however, but at 14 days after injury the total DNA content of the aorta intima/media of rats given iloprost loaded platelets during injury was significantly reduced. Although iloprost loaded platelets can clearly inhibit excessive platelet deposition, other encapsulated agents may have greater anti-proliferative effects. These studies have shown that drug loaded platelets can be targeted to injured arteries, where they may be retained as depots for local release. We believe this novel drug delivery protocol may have therapeutic potential in reducing the incidence of occlusion and restenosis after angioplasty and thrombolysis treatment. Electro-encapsulation of drugs into platelets is a simple procedure and, using autologous and fully biocompatible and biodegradable platelets as delivery vehicles, might overcome some of the immunological and toxicological problems which have been encountered with other delivery vectors such as liposomes, microbeads, synthetic microcapsules and antibodies.
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Battinelli, Elisabeth M. "Platelet and Megakaryocytic Regulation of Tumor Progression." Blood 130, Suppl_1 (December 7, 2017): SCI—26—SCI—26. http://dx.doi.org/10.1182/blood.v130.suppl_1.sci-26.sci-26.
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Traditionally viewed as the bandaids of the blood, the contribution of platelets to the progression of malignancy is emerging as a compelling focus for therapeutic intervention. Complex interactions between tumor cells, and circulating platelets play an important role in tumor growth and dissemination, and a growing body of data supports a role for platelet activation and release of chemokines in metastases and neovascularization. Supporting this concept is the evidence that elevated platelet counts (thrombocytosis) at time of diagnosis with malignancy is a harbinger of an aggressive cancer with a poor prognosis. One very interesting and provocative connection between cancer and platelets is the increasing evidence that tumor cells hijack platelets to promote a more pro-malignant phenotype to drive disease progression. Our laboratories have been instrumental in establishing the pro-malignant role of platelets in metastasis and neovascularization. We have demonstrated that tumor cells can instruct platelets to release key cytokines that promote angiogenesis and metastasis of tumor cells. Perhaps the most compelling clinical evidence of the link between platelets and malignancy is the finding that anti-platelet agents can have a profound impact on malignancy. We have demonstrated previously, anti-platelet agents such as aspirin and anticoagulants suppress release of key neovascularization factors from platelets and suppress the neovascularization potential. Aspirin also suppresses the invasive properties of platelets in mouse metastasis models as well as in vitro metastasis assays. Similarly, we have also demonstrated that tamoxifen, a selective estrogen receptor modulator often used to treat breast cancer, can also diminish the ability of platelets to support malignancy by diminishing the platelet's role in promoting neovascularization as well as metastasis. Although much is understood regarding how tumors communicate with platelets less is understood about how platelets manipulate tumor cells. Our laboratory has elucidated the role of key chemokines released from platelets in response to tumor cells and how these factors promote tumor growth and metastasis. We have recently discovered that tumor cells can instruct platelets to release CCL5, a known driver of tumor cell invasion and metastasis, and have expanded the role of CCL5 not only as a regulator of metastasis but also as a central controller of platelet production. Despite this progress many questions still remain regarding the interaction between tumor cells and platelets. We are particularly interested in how tumor cells instruct megakaryocytes to increase platelet production. In addition malignancy may also reprogram megakaryocytes thereby manipulating the platelet phenotype to support tumor growth and metastasis. Because most cancer therapies focus on the tumor itself, the idea of targeting platelets in the tumor microenvironment to arrest tumor growth and metastatic spread represents a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.
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Puricelli, Chiara, Elena Boggio, Casimiro Luca Gigliotti, Ian Stoppa, Salvatore Sutti, Mara Giordano, Umberto Dianzani, and Roberta Rolla. "Platelets, Protean Cells with All-Around Functions and Multifaceted Pharmacological Applications." International Journal of Molecular Sciences 24, no. 5 (February 26, 2023): 4565. http://dx.doi.org/10.3390/ijms24054565.
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Platelets, traditionally known for their roles in hemostasis and coagulation, are the most prevalent blood component after erythrocytes (150,000–400,000 platelets/μL in healthy humans). However, only 10,000 platelets/μL are needed for vessel wall repair and wound healing. Increased knowledge of the platelet’s role in hemostasis has led to many advances in understanding that they are crucial mediators in many other physiological processes, such as innate and adaptive immunity. Due to their multiple functions, platelet dysfunction is involved not only in thrombosis, mediating myocardial infarction, stroke, and venous thromboembolism, but also in several other disorders, such as tumors, autoimmune diseases, and neurodegenerative diseases. On the other hand, thanks to their multiple functions, nowadays platelets are therapeutic targets in different pathologies, in addition to atherothrombotic diseases; they can be used as an innovative drug delivery system, and their derivatives, such as platelet lysates and platelet extracellular vesicles (pEVs), can be useful in regenerative medicine and many other fields. The protean role of platelets, from the name of Proteus, a Greek mythological divinity who could take on different shapes or aspects, is precisely the focus of this review.
9
SANTORO, S. A. "Platelets: Platelet Immunobiology." Science 245, no. 4915 (July 21, 1989): 314–15. http://dx.doi.org/10.1126/science.245.4915.314.
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Cheepala, Satish B., Kazumasa Takenaka, Tamara I. Pestina, Carl W. Jackson, and John D. Schuetz. "The Role of ABC Transporter Abcc4 in Platelets Physiologic Function and Its Impact On Collagen Meditated Platelet Aggregation." Blood 120, no. 21 (November 16, 2012): 1063. http://dx.doi.org/10.1182/blood.v120.21.1063.1063.
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Abstract Abstract 1063 Platelet activation is a highly regulated process, and cyclic nucleotide mediated signaling pathways are crucial to effective platelet activation. Vascular injury produces, exposed collagen which binds circulating platelets through the platelet's “collagen” receptor, GPVI, resulting in the activation of guanyly/adenlyl cyclases. These interactions result in the rapid alterations in the cyclic nucleotide concentration inside the platelets leading to activation of protein kinase A and G signaling pathways to modulate platelet function. While, ABCC4 functions as a plasma membrane transporter for cyclic nucleotides its contribution to platelet activation has been obscured because it was reportedly as primarily intracellular in the platelets dense granules. This original report (Jedlitschky, Tirschmann et al. 2004) evaluated ABCC4 localization by immune-fluorescence of platelets attached to collagen coated coverslips. However, attachment via collagen produces platelet activation leading to mobilization and fusion of alpha and dense granules to the plasma membrane, thus under these conditions distinguishing between plasma membrane and dense granules is not possible. We resolved this problem by labeling quiescent platelets with a cell impermeable biotinylating agent (EZ-Link Sulfo-NHS-LC-LC Biotin). Isolation of membrane and internal fraction demonstrated that of over ninety percent of Abcc4 localizes to the plasma membrane. Furthermore, confocal microscopy of platelets stained with specific antibodies against Abcc4 confirmed Abcc4 localization to the plasma membrane. We extended these studies to the Abcc4- knockout (KO) mouse model. The Abcc4- KO mouse does not have any change in the number of platelet or dense granules compared to the wild type mouse. Platelet activation in vivo can be initiated by interaction with collagen through the GPVI receptor that is expressed at the plasma membrane of the platelets. At the molecular level, the initiation of platelet activation by collagen results in an increase in the cyclic nucleotide concentration leading to activation of signaling cascade through protein kinase A or G. Expose of Abcc4-KO platelets to collagen and revealed impaired activation in response to collagen. However, Abcc4-KO platelets activated by either thrombin or ADP (which activate either G-coupled PAR receptors or P2Y12 receptor respectively) shows an aggregation profile almost identical to wildtype platelets, thus indicating the defect in Abcc4 -KO platelet aggregation is specific to the collagen pathway. To understand the basis for the impaired collagen aggregation of Abcc4-KO platelets, we investigated the collagen receptor (GPVI) signaling pathway in Abcc4-KO platelets. Interestingly, in the Abcc4-KO platelets after the platelet activation with collagen, cyclic nucleotide dependent phosphorylation of VASP through protein kinase A or G at Ser-157 or Ser-239 respectively is reduced compared to the wildtype. Notably, Abcc4-KO platelets had reduced GPVI surface expression that correlated with the reduced phosphorylation of VASP after collagen stimulation. The similar, protein levels of Syk and Plcg2, (downstream signaling molecules of GPVI signaling pathway), in the Abcc4 wildtype and KO platelets implies that GPVI expression is the primary defect in Abcc4 deficiency. These results suggest that Abcc4 plays a crucial role in regulating cyclic nucleotides in response to GPVI activation by collagen. These findings suggest ABCC4/Mrp4 loss of function or inhibition (by drugs) may disrupt platelet aggregation under conditions of vascular injury. As, many antiplatelet drugs are potent inhibitors of Abcc4 (e.g., Dipyridamole and Sildenafil) these conclusions have strong implications for not just the development of antiplatelet drugs, but also for further exploring the role of Abcc4 in regulating intracellular nucleotide levels and platelet biology. Disclosures: No relevant conflicts of interest to declare.
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Ismail, M. Taufik. "Distinct Mechanism between Arterial and Venous Thrombosis: Impact for Clinical Manifestations." ACI (Acta Cardiologia Indonesiana) 5, no. 1 (P) (July 18, 2019): 47. http://dx.doi.org/10.22146/aci.47683.
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Hemostasis is a complex physiological process aiming to keep the integrity of a closed circulatory system after an occurrence of vessel wall injury. Hemostasis involving the role of circulating platelets and coagulation cascade.1 There are two major pathways that act independently to activate the platelet. The first pathway is mediated by collagen and the other by tissue factor. After intimal layer injury, platelets are recruited through the interaction between platelet’s surface glycoprotein (GPVI and GPIb/V/IX) with collagen and von Willebrand factor. This process results in adhesion of platelets in the site of injury. Further recruitment of platelets is achieved by secretion of aggregatory mediators such as thromboxane A2 and adenosine diphosphate.
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Battinelli, Elisabeth M., Rajesh Kulenthirarajan, Joseph E. Italiano, and Kelly Johnson. "Tamoxifen Directly Disrupts Platelet Angiogenic Potential and Inhibits Platelet-Mediated Metastasis." Blood 124, no. 21 (December 6, 2014): 4169. http://dx.doi.org/10.1182/blood.v124.21.4169.4169.
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Abstract Platelets, which are mainly known for their role in hemostasis, are now known to play a crucial role in metastasis. Metastatic disease is the cause of roughly 90% of all cancer-related deaths and understanding the mechanisms leading to dissemination of tumor cells to distant sites remains one of the main challenges of cancer research. Tamoxifen is a selective estrogen receptor modulator that is widely used for the treatment and prevention of breast cancer. Interestingly, tamoxifen has demonstrated anti-cancer efficacy in estrogen negative breast cancers suggesting that this drug has additional mechanisms of action. Previously tamoxifen and its metabolites have been shown directly impact platelet function; however, the reported effects have been varied (Vitseva et al,. 2005, Nayak et al., 2011). Because platelets are critical for metastatic spread, we posited that tamoxifen may exert anti-tumor effects by directly altering platelet function. To explore this, we first examined the effect of tamoxifen on platelet activation. P-selectin expression was determined by flow cytometry for platelets pretreated with or without tamoxifen and then activated with ADP or MCF-7 cells. Both ADP and exposure to MCF-7 cells resulted in platelet activation and treatment with tamoxifen lead to a partial inhibition of activation. Platelets are a reservoir for angiogenic proteins that are secreted in a differentially regulated process. We have previously shown that we can manipulate the angiogenic potential of the platelet releasate through physiological (platelet agonists) and pathological activation (MCF-7 tumor cells) (Battinelli et al., 2011). We hypothesized that tamoxifen may impact malignancy by altering the release of angiogenesis regulatory proteins from platelets. To explore the impact of tamoxifen on the angiogenic potential of platelets, we analyzed the releasates from platelets exposed to tamoxifen alone or activated with ADP or MCF-7 cells in conjunction with tamoxifen. Our data reveals that platelets exposed to tamoxifen release significantly decreased amounts of VEGF in response to activation by either the platelet agonist ADP or interaction with tumor cells (MCF-7 cells). Next, in vitro angiogenesis assays were performed to further examine the effect of tamoxifen on the angiogenic potential of platelets. We observed dramatically diminished capillary tube branch point formation and decreased migration in endothelial cell cultures exposed to releasates generated from tamoxifen treated platelets compared to control releasates, demonstrating that tamoxifen inhibits the ability of activated platelets to promote angiogenesis. Platelets play a critical role in aiding the intravasation and extravasation of tumor cells in the circulation and therefore we postulated that tamoxifen could alter the ability of platelets to aid tumor cells in crossing the vascular endothelium. We performed transendothelial migration assays in which platelets were pretreated with tamoxifen or vehicle control, washed and mixed with MCF-7 tumor cells in endothelialized transwells. Platelets significantly increased the number of tumor cells that crossed the endothelial barrier; however this increase was lost when platelets were pretreated with tamoxifen. Overall our results demonstrate that tamoxifen directly alters platelet function, leading to decreased angiogenic and metastatic potential. These studies highlight a previously unknown mechanism of action for tamoxifen and may shed light on the efficacy of tamoxifen in estrogen-receptor negative cancers. Furthermore our work stresses the importance of crosstalk between platelets and cells within the tumor microenvironment for successful angiogenesis and metastatic spread and, ultimately, support the idea of utilizing targeted platelet therapies to inhibit the platelet’s role in malignancy. Disclosures No relevant conflicts of interest to declare.
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He, Ao-Di, Ming-Lu Liang, Gang Liu, Xing-Wen Da, Guang-Qiang Yao, Wen Xie, Ji-Zhou Xiang, Cunji Gao, and Zhang-Yin Ming. "The Role of FcγRIIa and TGF-β1/KLF6 Pathway in Platelet's Promoting Hepatocellular Carcinoma Cells Growth." Blood 124, no. 21 (December 6, 2014): 1429. http://dx.doi.org/10.1182/blood.v124.21.1429.1429.
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Abstract Background: Platelet in the primary tumor microenvironment plays a crucial role in tumor cells angiogenesis, growth, and metastasis. Clinical and experimental evidences support that platelets and their extracts influence hepatocellular carcinoma (HCC) growth and biology. But the mechanism is still not fully clarified. The aim of present study was to elucidate an unperceived mechanism of the proliferative effect of platelet on HCC cells. Methods: Human blood was collected from health volunteers, washed platelets were prepared and resuspended by fresh medium. The ability of HepG2 cells to induce platelet aggregation was analyzed using a Chrono-Log Lumi-aggregometer. HepG2 cells were incubated with platelets activated by thrombin (0.08 U/ml) and collagen-related peptide (CRP, 0.8μg/ml), or releasates isolated from CRP-stimulated platelets. The effect of platelet releasate on HepG2 cell proliferation was determined with the colorimetric 3-(4, 5-dimethylthiazol)-2, 5-diphenyltetrazolium bromide (MTT) assay. Western blot was used to measure expression of Krüppel-like factor 6 (KLF6) in HepG2 cells. Anti-FcγRIIa monoclonal antibody IV.3 (10μg/ml) and transforming growth factor beta 1 (TGF-β1) receptor inhibitor SB431542 (10μM) were used. Furthermore, KLF6 gene silence was also conducted in HepG2 cells by transfected with KLF6 siRNA. Results: Our data showed HepG2 cells (1.0×105/ml) could induce human washed platelet (3.0×108/ml) aggregation in vitro, indicating that HepG2 cells could activate platelets. We further verified that releasate from CRP-activated platelets could promote the proliferation of HepG2 cells. Importantly, this effect exhibits on the down expression of KLF6 in HepG2 cells. In presence and absence of platelet stimulator thrombin (0.08 U/ml) or collagen-related peptide (CRP, 0.8μg/ml), washed platelets could reduce KLF6 expression in HepG2 cells after incubated for 12 and 24 hours. Meanwhile, supernatant from CRP-activated platelets exhibited the same effect. On the other hand, the resuspended CRP-activated platelet pellet showed no significant influence on KLF6 expression. And platelets incubated with anti- FcγRIIa monoclonal antibody IV.3 (10μg/ml) and transforming growth factor beta 1 (TGF-β1) receptor inhibitor SB431542 (10μM) abolished the effects. Furthermore, the platelet’s promoting proliferation effect was attenuated in HepG2 cells with silencing KLF6 expression. Conclusion: Tumor cells could activate platelet, and activated platelet could regulate cancer cell progression in turn. We further verified that platelet, a main source of bioavailable TGF-β1, has a promoting effect on the proliferation of HepG2 cells. Importantly, this effect exhibits on the down expression of KLF6 in HepG2 cells, in which FcγRIIa and TGF-β1 involved. These results extend our understanding of mechanisms by which platelets contribute to tumor progression, which may provide a new therapeutic target for the prevention and treatment of HCC. Disclosures No relevant conflicts of interest to declare.
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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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Sakurai, Yumiko, Jennifer L. Fitch-Tewfik, Yongzhi Qiu, David R. Myers, Byungwook Ahn, Robert Flaumenhaft, and Wilbur A. Lam. "“Self-Deposition” of Matrix Proteins from Platelet α-Granules Enable Extended Adhesion and Spreading on Micron/Submicron-Scale Fibrinogen and Collagen Substrates." Blood 124, no. 21 (December 6, 2014): 2764. http://dx.doi.org/10.1182/blood.v124.21.2764.2764.
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Abstract Introduction: While the platelet’s role in achieving hemostasis in the context of bleeding is well-characterized, how platelets support and maintain vascular integrity during homeostatic, non-thrombotic conditions remains unclear. Previous studies have shown that single platelets fill micron/submicron-sized “gaps” of small discontinuities in the endothelium. In this context, however, as the overall force of adhesion correlates with the area of exposed subendothelial matrix, how platelets establish sufficient adhesion to resist the dynamic shear forces of the circulation are unknown. In this study, we investigate, using protein microcontact printing, how platelets sense the geometry of the matrix micro/nano-environment and transduce those cues to affect platelet function. Our results reveal that platelets, upon sensing spatially-limited (<25 µm2 area) micro/nanopatterns of fibrinogen or collagen, spatially regulate actin-rich filopodia extension, via Rac1, beyond the geometric constraints of the matrix pattern, redistribute α-granules to enable “self-deposition” of fibrinogen/fibronectin matrix on those regions, and spread onto those newly deposited matrix via integrin αIIbβ3-mediated interactions. Furthermore, this phenomenon is markedly attenuated in Gray Platelet Syndrome platelets, which lack α-granules, and Wiskott-Aldrich Syndrome platelets, which have impaired integrin αIIbβ3-mediated activation, decreased α-granules, and cytoskeletal defects. Materials and Methods: Human blood was collected in ACD and platelets were isolated via two-step centrifugation or gel filtration. Platelets were suspended in Tyrode’s buffer containing 2 mM MgCl2 and exposed to various pharmacologic agents such as eptifibatide, Y27632, NSC23766. Fibrinogen and collagen micro/nanostamps were prepared on glass coverslips after which platelets were incubated onto. A plasma membrane dye, phalloidin, anti-P-selectin, anti-fibronectin and PAC-1, were used to visualize platelet morphology, f-actin, α-granule secretion, matrix depositions, and activated integrin αIIbβ3, respectively. Results and Discussion: Upon contact with the protein microstamps, platelets formed filopodia and lamellipodia and spread to conform to the micropattern boundaries with high fidelity. However, on micropatterned circles (microdots) of less than 25 µm2 area, platelets spread beyond the geometric constraints (Fig.1A). Anti P-selectin staining revealed α-granule secretion at the extended lamellipodia (Fig.1B left), and interestingly, this phenomenon was not detected with GPS platelets. Also we observed direct deposition of fibronectin from the platelets granules via anti-fibronectin staining (Fig.1B center). In addition, immunostaining with PAC-1 revealed a predominance of activated αIIbβ3 on regions of platelets at and beyond the borders of the microdots (Fig.1B right). When exposed to eptifibatide, an αIIbβ3 antagonist, platelet spreading beyond the microdot boundaries was completely eliminated. Various cytoskeleton inhibition experiments provided insight into how actin rearrangement is involved in this processes of platelet extension beyond matrix boundaries. Interestingly, disruption of cytoplasmic stress fibers by inhibiting Rho kinase with Y25632 facilitated platelet extension beyond the microdot boundaries and strong actin polymerization at the margins. On the other hand, platelet extension beyond the microdot boundaries was completely inhibited with NSC23766, a Rac-1 inhibitor (Fig.1C). We also confirmed that WAS platelets had significantly less f-actin, α-granule secretion, and overall extension beyond microdot boundaries. Overall, our results indicate that the matrix geometry, in and of itself, dictates platelet α-granule secretion, αIIbβ3 distribution and activation, and Rac1 activation, all of which work in concert to extend adhesion and spreading on matrix substrates of <25 µm2. In addition, we show that during this process, directionally directed α-granule secretion enables a platelet to “self-deposit” fibrinogen/fibronectin matrix to accommodate increased spreading. This phenomenon likely endows platelets a certain amount of “spatial flexibility” to establish firm adhesion upon contact with small sites of exposed subendothelium and may provide insight into how platelets help support vascular integrity. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Parker, RI, and HR Gralnick. "Effect of aspirin on platelet-von Willebrand factor surface expression on thrombin and ADP-stimulated platelets." Blood 74, no. 6 (November 1, 1989): 2016–21. http://dx.doi.org/10.1182/blood.v74.6.2016.2016.
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Abstract Platelets contain a pool of endogenous platelet-von Willebrand factor (vWF) that becomes expressed on the platelet surface when platelets are stimulated by a variety of agonists. Maximal platelet-vWF expression occurs in concert with platelet alpha-granule secretion. Aspirin (ASA) is known to impair platelet activation and alpha-granule secretion by irreversible inhibition of platelet cyclo-oxygenase. We studied native and ASA-treated platelets for their ability to mobilize and to express platelet-vWF in response to adenosine diphosphate (ADP) or thrombin. We found that each agonist was effective in promoting increased platelet- vWF surface expression on native and ASA-treated platelets. ASA-treated platelets responded identically to native platelets to low (0.01 U/mL) and high (1.0 U/mL) concentrations of thrombin, while the ADP-induced increase in ASA-treated platelets was only 50% to 60% of that for control platelets. Measurement of secreted platelet-vWF and beta- thromboglobulin indicated that the increase seen with ADP was largely independent of alpha-granule secretion. Using monoclonal antibodies (MoAbs) against the platelet glycoproteins (GP) IIb/IIIa and Ib (MoAbs 10E5 and 6D1, respectively), we demonstrated that the ADP-induced increase in platelet-vWF expression on control platelets primarily involved the binding of secreted platelet-vWF to the platelet GPIIb/IIIa. In contrast, the increase in platelet-vWF that occurred following ADP stimulation of ASA-treated platelets was largely insensitive to GPIIb/IIIa blockade. No effect of GPIb blockade in platelet-vWf expression was noted for either control or ASA-treated platelets. When platelet shape change was prevented by the addition of cytochalasin D, ADP-induced platelet-vWf surface expression on ASA- treated platelets was reduced by more than 80%. Our data indicate that platelets in which the cyclooxygenase pathway is blocked by the action of aspirin can increase surface expression of platelet-vWf as a consequence of platelet shape change. We speculate that this process exposes platelet-vWf bound to GPIIb/IIIa, or possibly GPIb, within the surface connected canalicular system.
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Parker, RI, and HR Gralnick. "Effect of aspirin on platelet-von Willebrand factor surface expression on thrombin and ADP-stimulated platelets." Blood 74, no. 6 (November 1, 1989): 2016–21. http://dx.doi.org/10.1182/blood.v74.6.2016.bloodjournal7462016.
Full textAbstract:
Platelets contain a pool of endogenous platelet-von Willebrand factor (vWF) that becomes expressed on the platelet surface when platelets are stimulated by a variety of agonists. Maximal platelet-vWF expression occurs in concert with platelet alpha-granule secretion. Aspirin (ASA) is known to impair platelet activation and alpha-granule secretion by irreversible inhibition of platelet cyclo-oxygenase. We studied native and ASA-treated platelets for their ability to mobilize and to express platelet-vWF in response to adenosine diphosphate (ADP) or thrombin. We found that each agonist was effective in promoting increased platelet- vWF surface expression on native and ASA-treated platelets. ASA-treated platelets responded identically to native platelets to low (0.01 U/mL) and high (1.0 U/mL) concentrations of thrombin, while the ADP-induced increase in ASA-treated platelets was only 50% to 60% of that for control platelets. Measurement of secreted platelet-vWF and beta- thromboglobulin indicated that the increase seen with ADP was largely independent of alpha-granule secretion. Using monoclonal antibodies (MoAbs) against the platelet glycoproteins (GP) IIb/IIIa and Ib (MoAbs 10E5 and 6D1, respectively), we demonstrated that the ADP-induced increase in platelet-vWF expression on control platelets primarily involved the binding of secreted platelet-vWF to the platelet GPIIb/IIIa. In contrast, the increase in platelet-vWF that occurred following ADP stimulation of ASA-treated platelets was largely insensitive to GPIIb/IIIa blockade. No effect of GPIb blockade in platelet-vWf expression was noted for either control or ASA-treated platelets. When platelet shape change was prevented by the addition of cytochalasin D, ADP-induced platelet-vWf surface expression on ASA- treated platelets was reduced by more than 80%. Our data indicate that platelets in which the cyclooxygenase pathway is blocked by the action of aspirin can increase surface expression of platelet-vWf as a consequence of platelet shape change. We speculate that this process exposes platelet-vWf bound to GPIIb/IIIa, or possibly GPIb, within the surface connected canalicular system.
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Gordy, Dominique, and Elizabeth Stone. "Mouse Model for Platelet Aggregation using Flow Cytometry." American Journal of Clinical Pathology 158, Supplement_1 (November 1, 2022): S8—S9. http://dx.doi.org/10.1093/ajcp/aqac126.014.
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Abstract Platelets play crucial roles in hemostasis, bleeding, and thrombosis. Within the United States, an estimated 7,000 platelet units are transfused daily. The gold standard to measure platelet survival in humans is to determine post-transfusion recovery after an autologous transfusion of radiolabeled platelet units, however, the ability to detect platelets circulating after transfusion does not provide information on how well these platelets function in hemostasis. Clinically, platelet unit function is routinely measured using aggregometry, which requires large volumes of platelet concentrates and normal platelet counts. While the clinical demand for platelets continues to increase with advances in medical care, there remains a lack of in vitro measures to properly assess platelet function in animal models. Here we outline an in vitro method for characterizing platelet function using flow cytometry to assess platelet activation and aggregation simultaneously in a mouse model. Using two commercially available transgenic mouse lines, one with platelets expressing red fluorescent protein (RFP) and the other with platelets expressing green fluorescent protein (GFP), whole blood is collected from each transgenic mouse line by aseptic cardiac puncture, and leukocyte-reduced platelet rich plasma (LRPRP) is isolated after two consecutive centrifuge spins and leukocyte reduction. RFP- and GFP-labeled platelets can be visually separated by flow cytometry. Platelets are activated after incubation with 4 mM GPRP peptide followed by exposure to 0.5U/mL high activity bovine thrombin for 2 minutes. Platelets are then stained with anti-CD41a, labelling all platelets, and anti-CD62P, labelling activated platelets; these platelet populations can be analyzed by flow cytometry. When RFP- and GFP-LRPRP are combined prior to activation with thrombin, RFP- and GFP-labeled platelet aggregation can be measured as the number of cells doubly expressing RFP and GFP by flow cytometry. Thrombin stimulation activated 93.31% of the total number of RFP- and GFP-platelets. Of the activated, CD62P-positive platelets, 11.03% expressed both RFP and GFP, indicating that these platelets were aggregated. Of the inactivated, CD62P-negative platelets, 0.81% expressed both RFP and GFP. These results suggest that thrombin exposure activates platelets and causes platelet aggregation, while <1% of inactivated platelets aggregate. Additionally, platelet aggregation is easily measured by flow without differentially labeling the two separate platelet populations. These data indicate that our model is a viable measure of platelet function in vitro. Our model of platelet activation and aggregation provides a baseline measure of in vitro platelet function in RFP and GFP mice. This in vitro method of assessing platelet function can be applied to various experimental populations and used to quantify the effects of experimental conditions on platelet function in mice. Using this model of platelet function, factors influencing platelet storage can be characterized and ultimately used to improve platelet transfusion therapies.
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Stolla, Moritz, Renata Grozovsky, Melissa M. Lee-Sundlov, Herve Falet, and Karin M. Hoffmeister. "Effects of Platelet Circulatory Age on Platelet Function." Blood 128, no. 22 (December 2, 2016): 413. http://dx.doi.org/10.1182/blood.v128.22.413.413.
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Abstract The human body produces and removes 1011 platelets daily to maintain a normal steady-state platelet count. However, the regulatory mechanisms remain elusive. We have shown that platelets lacking sialic acid (desialylated platelets) are removed by the hepatic Ashwell-Morell receptor (AMR or asialoglycoprotein receptor type 2), thereby regulating platelet survival and hepatic TPO levels. Platelet counts and lifetime were increased in Asgr2-/- mice (AMR-null mice), compared to wild type (WT) mice. Platelet volume and immature platelet fraction (IPF) are decreased in AMR-null mice, consistent with the notion that platelets in AMR-null mice (AMR-null platelets) circulate longer and are older. By contrast, deficiency of the sialyltransferase St3gal4 gene induces a marked thrombocytopenia (St3gal4-null platelets), due to rapid platelet clearance by the hepatic AMR. Consistent with the rapid platelet clearance, platelet volume and IPF were increased in St3gal4-/- mice, reflecting high platelet turnover and younger platelets. While both AMR-null and St3gal4-null platelets are desialylated, they differ substantially in their time, i.e. age, in circulation. Here we investigated the effects of in vivo and in vitro aging on platelet function. Freshly isolated St3gal4-null platelets showed significantly increased integrin activation when stimulated with convulxin and thrombin, while AMR-null platelets showed a significantly lower response compared to WT platelets. Secretion of α-granule was significantly increased in St3gal4-null platelets. By contrast no significant difference was measured between WT and AMR-null platelets. Despite increased platelet counts, the tail-bleeding time was significantly prolonged in AMR-null mice, compared to WT mice, suggesting that increased circulatory time (age) negatively affects platelet function in vivo. We next performed in vitro storage for up to 72 hours at room temperature to stress platelet aging. Stored St3gal4-deficient platelets had increased integrin activation, α-granule secretion in response to convulxin and thrombin and showed increased phosphatidyl serine exposure as detected by Annexin V binding in response to calcium ionophore, compared to stored control platelets. By contrast, stored AMR-null platelets had significantly impaired integrin activation, α-granule secretion and Annexin V binding compared to controls. To further evaluate the propensity to undergo apoptosis, we tested caspase-3 activation and mitochondrial membrane potential. Surprisingly, we found that both St3gal4-null (young) and AMR-null (old) platelets showed a significantly lower caspase-3 activation in response to calcium ionophore and ABT-737 compared to WT platelets. Furthermore, the mitochondrial membrane potential was lower in both St3gal4-deficient and AMR-null platelets compared to WT platelets, indicating functionally impaired mitochondria. Taken together, our data indicate that younger St3gal4-null platelets have an increased baseline function while by contrast older AMR-null platelets have decreased function in vitro and in vivo. Interestingly, younger and older (longer circulating platelets) had reduced propensity to undergo apoptosis and impaired mitochondrial function. Overall, younger platelets represent a highly favorable profile during storage. Identification of donors with a larger fraction of younger platelets could result in safer and more efficacious platelet transfusions. Disclosures No relevant conflicts of interest to declare.
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Fager, Ammon M., and Paula B. Tracy. "“Young” Platelets Regulate Thrombus Formation by Expression of Increased Adhesive Properties and Procoagulant Activity." Blood 108, no. 11 (November 16, 2006): 1527. http://dx.doi.org/10.1182/blood.v108.11.1527.1527.
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Abstract Previous studies from our lab and others have demonstrated that thrombin mediated activation of platelets results in the formation of a distinct procoagulant subpopulation capable of assembling the components of both the prothrombinase complex and intrinsic tenase complex at its surface. A similar subpopulation has been identified following activation of platelets with the combination of thrombin and convulxin, a glycoprotein VI agonist, and has been found to be enriched in young platelets. In addition, young zebrafish thrombocytes were shown to have a higher density of adhesive receptors and greater functional activity than their more mature counterparts. Since the membrane properties that distinguish the platelet’s ability to assemble these procoagulant complexes are unknown, the purpose of this study was to elucidate these important differences. Following their isolation from whole, anticoagulated blood, human platelets were activated with 50 nM thrombin (2 min, ambient temperature) followed by the addition of hirudin (75 nM) to quench the reaction. Equimolar concentrations of factors Va and Xa were added (5 nM, 10 min) to saturate the functional Prothrombinase binding sites determined previously using kinetics of prothrombin activation, prior to platelet fixation with 2% v/v paraformaldehyde. Those platelets capable of binding factors Va and Xa were identified by flow cytometry using specific, non-inhibitory fluorophore-conjugated MoAbs directed against the required cofactor and serine protease, respectively. Analyses of the membrane density of well-known and characterized platelet receptors including glycoprotein IIb/IIIa and glycoprotein Ib-IX-V, were also assessed using specific MoAbs conjugated with the appropriate fluorophore to determine whether expression of any or all of these adhesive receptors defined the platelet subpopulation capable of assembling a competent Prothrombinase. Thiazole orange (TO), a dye frequently used to stain nucleic acid, and previously shown to identify the youngest platelets in circulation, was used to evaluate the effect of platelet age on these various parameters. Using this approach, we demonstrated a 7–8-fold increase in both CD62P (P-selectin) and CD61 (glycoprotein β3) expression in TO-positive platelets, while expression of the platelet marker CD42b (glycoprotein 1bα) increased as much as 14-fold. The subpopulation capable of assembling Prothrombinase likewise demonstrated a 3–4-fold increase in CD62P, CD61, and CD41 (glycoprotein IIb) expression, and a ~7-fold increase in CD42b expression. Dual-labeling studies demonstrated that ~60% of TO-positive platelets are able to bind factor Xa while ~85% of platelets able to bind Xa are TO-positive. Similar results were seen when platelets were evaluated for their ability to bind high levels of factor Va. These data suggest that it is predominantly the younger platelets with increased density of adhesive receptors that are capable of assembling Prothrombinase on their activated surface. Since platelet adhesion is the initiating step in both normal hemostasis and pathological thrombus formation, the observed increase in adhesive receptor density places this platelet subpopulation in a unique position of significance in the regulation of the hemostatic response, and indicates a potential role for these platelets, in the initiation of thrombus formation.
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Hoffmann, Johannes J. M. L., Nicole M. A. van den Broek, and Joyce Curvers. "Reference Intervals of Reticulated Platelets and Other Platelet Parameters and Their Associations." Archives of Pathology & Laboratory Medicine 137, no. 11 (November 1, 2013): 1635–40. http://dx.doi.org/10.5858/arpa.2012-0624-oa.
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Context.—Reticulated platelets are platelets recently released from the bone marrow, and they can serve as a noninvasive indicator of recent megakaryopoietic activity. Widespread clinical use has been hampered by laborious methods and lack of standardization. Recently, a fully automated method was released on the Abbott CELL-DYN Sapphire hematology analyzer. Objective.—To establish reference ranges for reticulated platelets. Secondary aims were to investigate associations between reticulated platelets and other platelet parameters like mean platelet volume, plateletcrit, and platelet distribution width. Design.—Reticulated platelets and other platelet parameters were measured in an unselected cohort of 8089 subjects visiting a primary health care laboratory. The reticulated platelet data were analyzed using the Bhattacharya technique. In addition, a nonparametric method was used in selected subjects with normal platelet counts for providing reference ranges. Results.—Reticulated platelets ranged from 0.4% to 6.0% or from 1 to 18 × 103/μL. Reticulated platelets increased significantly with the subjects' age. Statistically, males had slightly higher values than females, but the differences were negligible. Reticulated platelets were positively correlated with platelet count and negatively with mean platelet volume. Conclusions.—Reference ranges have been established for reticulated platelets as measured on the CELL-DYN Sapphire hematology analyzer. There were no relevant differences between the sexes, but there was a clear effect of age. An individual's reticulated platelets are associated with the platelet count as well as mean platelet volume.
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Patel, Dipti, Heikki Väänänen, Markéta Jiroušková, Thomas Hoffmann, Carol Bodian, and Barry S. Coller. "Dynamics of GPIIb/IIIa-mediated platelet-platelet interactions in platelet adhesion/thrombus formation on collagen in vitro as revealed by videomicroscopy." Blood 101, no. 3 (February 1, 2003): 929–36. http://dx.doi.org/10.1182/blood.v101.3.929.
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Abstract The conventional description of platelet interactions with collagen-coated surfaces in vitro, based on serial static measurements, is that platelets first adhere and spread to form a monolayer and then recruit additional layers of platelets. To obtain dynamic information, we studied gravity-driven platelet deposition in vitro on purified type 1 collagen by video phase-contrast microscopy at 22°C. With untreated human and wild-type mouse platelets, soon after the initial adhesion of a small number of “vanguard” platelets, “follower” platelets attached to the spread-out vanguard platelets. Follower platelets then adhered to and spread onto nearby collagen or over the vanguard platelets. Thus, thrombi formed as a concerted process rather than as sequential processes. Treatment of human platelets with monoclonal antibody (mAb) 7E3 (anti–GPIIb/IIIa (αIIbβ3) + αVβ3) or tirofiban (anti–GPIIb/IIIa) did not prevent platelet adhesion but nearly eliminated the deposition of follower platelets onto vanguard platelets and platelet thrombi. Similar results were obtained with Glanzmann thrombasthenia platelets. Wild-type mouse platelets in the presence of mAb 1B5 (anti–GPIIb/IIIa) and platelets from β3-null mice behaved like human platelets in the presence of 7E3 or tirofiban. Deposition patterns of untreated human and wild-type mouse platelets were consistent with random distributions under a Poisson model, but those obtained with 7E3- and tirofiban-treated human platelets, 1B5-treated mouse platelets, or β3-null platelets demonstrated a more uniform deposition than predicted. Thus, in this model system, absence or blockade of GPIIb/IIIa receptors interferes with thrombus formation and alters the pattern of platelet deposition.
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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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Liu, Fang, Graciela Gamez, David R. Myers, Wilbur Lam, and Shawn M. Jobe. "Mitochondrially-Mediated Platelet Integrin AlphaIIbbeta3 Inactivation Limits Platelet Recruitment and Thrombus Growth,." Blood 118, no. 21 (November 18, 2011): 3251. http://dx.doi.org/10.1182/blood.v118.21.3251.3251.
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Abstract Abstract 3251 Platelet stimulation with strong agonist(s) results in the formation of a phenotypically unique platelet subpopulation known as procoagulant platelets. All platelets stimulated in strongly-activating conditions undergo granule release, activation of integrin αIIbβ3, and spreading. However, minutes later a subpopulation of these activated platelets undergo additional phenotypic changes including phosphatidylserine (PS) exposure, adoption of a vesiculated and balloon-like morphology, and an altered integrin αIIbβ3 conformation that has a decreased binding affinity for activation-dependent antibodies such as PAC-1 or JON/A. Deletion of the mitochondrial protein cyclophilin D (CypD), a critical regulator of mitochondrial permeability transition pore (mPTP) formation, results in marked abrogation of procoagulant platelet formation. We have previously demonstrated remarkable impairment of dual-agonist-initiated PS exposure and procoagulant activity in CypD−/− platelets (Jobe et al, Blood 2008). In this study the functional importance of the morphological and adhesive changes that occur in procoagulant platelets were examined using CypD−/− platelets and mice with platelet-specific deficiency of CypD. (CypDplt−/−). The morphology and phenotype of fibrinogen-adherent CypD+/+ (WT) and CypD−/− platelets were examined following dual stimulation with thrombin and the GPVI agonist convulxin. In dual-stimulated WT platelets, platelets initially spread; then after three minutes, nearly half of the platelets demonstrated lamellopodial and filipodial retraction with platelet rounding and vesiculation, and these changes were associated with increased PS exposure and decreased binding of JON/A. These changes were delayed in CypD−/− platelets, but eventually started to be seen 30 minutes subsequent to stimulation. Adhesion and platelet recruitment to strongly-stimulated platelets were studied in flow conditions. Increased platelet accumulation was noted when unstimulated platelets were flowed over strongly-stimulated adherent CypD−/− platelets relative to WT platelets. Similarly, increased platelet accumulation of CypD−/− platelets was noted on a collagen surface compared to WT platelets. In platelet aggregation assays, dual stimulated WT platelets demonstrated a chaotic pattern of aggregation two to three minutes after activation consistent with aggregate disruption, a pattern that was not observed in CypD−/− platelets. Together these studies indicate that procoagulant platelet formation results in integrin inactivation. Since this process is impaired in the absence of CypD, CypD−/− platelets demonstrate increased platelet accumulation in flow assays and stable aggregate formation. Adhesive and cytoskeletal proteins were investigated in strongly-stimulated platelets. Western blot analysis demonstrated significant proteolytic cleavage of both talin and the cytoplasmic domain of integrin β3 in WT platelets stimulated with thrombin plus convulxin, and these events were only minimally observed in single-agonist stimulated platelets. Proteolytic cleavage of both talin and integrin β3 were markedly decreased in CypD−/− platelets and in WT platelets treated with calpain inhibitors. CypD is a ubiquitously expressed protein; therefore, in vivo thrombosis and hemostasis were tested in CypDplt−/− mice. Following photochemically mediated mesenteric endothelial injury, time to occlusion was shortened in CypDplt−/− mice (723 ± 111 sec in CypDplt+/− vs. 371 ± 106 sec in CypDplt−/− mice), and complete arterial occlusion was increased in CypDplt−/− mice (55% in CypDplt+/− vs. 86% in CypDplt−/−) (p=0.02). Our in vitro and in in vivo results are consistent with the hypothesis that in strongly-stimulated platelets mitochondrially-mediated events initiate proteolytic cleavage of talin and integrin β3 accompanied by platelet integrin αIIbβ3 inactivation, and that this process limits platelet recruitment and thrombus growth. Since these events are limited to platelets stimulated by strong agonist(s), these results suggest a novel negative feedback mechanism initiated by accumulation of multiple or strong agonist(s) that limits thrombotic occlusion in vivo. Disclosures: No relevant conflicts of interest to declare.
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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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Lominadze, David G., Jack T. Saari, Frederick N. Miller, James L. Catalfamo, David E. Justus, and Dale A. Schuschke. "Platelet Aggregation and Adhesion during Dietary Copper Deficiency in Rats." Thrombosis and Haemostasis 75, no. 04 (1996): 630–34. http://dx.doi.org/10.1055/s-0038-1650334.
Full textAbstract:
SummaryThe role of dietary copper deficiency in platelet-to-endothelial cell adhesion and in platelet-to-platelet aggregation was studied in vitro. Platelets were obtained from male, weanling Sprague-Dawley rats fed purified diets which were either copper-adequate (CuA, 6.3 μg copper/g of diet) or copper-deficient (CuD, 0.3 μg copper/g of diet) for 4 weeks. The platelet adhesion study was performed by adding CuA or CuD platelets either suspended in homologous plasma or in Tyrode buffer salt solution (TBSS) to cultured rat endothelial cells. After a one hour incubation at 37° C non-adhered platelets were removed and counted in a microcytometer. Platelet aggregation in platelet rich plasma (PRP) samples was induced by adding ADP (2 × 10−4 M) and measured in a turbidometric platelet aggregometer. The content of von Willebrand factor (vWF) in platelets and in plasma and the content of fibrinogen in platelets was determined. Platelet adhesion to rat endothelial cells was significantly lower for platelets from CuD rats than for platelets from CuA rats. ADP induced platelet aggregation from CuD rats was significantly higher than platelet aggregation from CuA rats. The content of vWF in platelets and in plasma from CuD rats was significantly lower than in platelets and plasma from CuA rats. However, the amount of fibrinogen in platelets from CuD rats was about 4-fold higher than that in platelets from CuA rats while the plasma fibrinogen was lower in CuD rats than in CuA rats. These studies illustrate that copper deficiency diminishes platelet adhesion to endothelial cells but increases platelet aggregability. The results suggest that these physiological alterations may be the result of decreased platelet vWF and increased platelet fibrinogen during dietary copper deficiency.
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Savage, B., CS Hunter, LA Harker, VL Jr Woods, and SR Hanson. "Thrombin-induced increase in surface expression of epitopes on platelet membrane glycoprotein IIb/IIIa complex and GMP-140 is a function of platelet age." Blood 74, no. 3 (August 15, 1989): 1007–14. http://dx.doi.org/10.1182/blood.v74.3.1007.1007.
Full textAbstract:
Abstract Platelets are heterogeneous in the content of membrane glycoprotein (GP)IIb/IIIa complex. To determine whether this heterogeneity is related to changes associated with platelet aging in the circulation, newly released platelets, obtained during recovery from nonimmune- mediated acute experimental thrombocytopenia in baboons, were studied. Monoclonal antibody (MoAb) binding to epitopes expressed on GPIIb/IIIa complex (LJ-CP8), GMP-140 (S12), and GPIa/IIa (12F1) was measured on control platelets (comprising platelets with a normal age distribution; mean age 60 to 72 hours) and newly formed platelets (mean age 12 hours), both in the resting state and after thrombin stimulation. Whereas LJ-CP8 binding to resting control platelets increased by 34% upon stimulation by gamma-thrombin from 30,885 +/- 1,171 to 41,458 +/- 1,311 molecules/platelet at saturating concentrations of antibody, LJ- CP8 binding to resting young platelets did not increase significantly upon thrombin stimulation (31,878 +/- 3,330 and 33,791 +/- 3,486 molecules/platelet, respectively). Similarly, binding of antibody S12 in response to maximal thrombin stimulation was reduced by 42% from 10,246 +/- 834 molecules/platelet at saturating concentrations of S12 for control platelets to 5,971 +/- 665 molecules/platelet for young platelets (P = .001). S12 binding to unstimulated platelets was less than 10% of the binding observed after thrombin stimulation at all concentrations of S12 for both control and young platelets. However, maximal binding of antibody 12F1 to resting control platelets did not differ significantly from that observed with resting young platelets (2,926 +/- 167 and 2,857 +/- 208 molecules/platelet, respectively), and 12F1 binding was unchanged after thrombin stimulation for both control and young platelets. We conclude that the thrombin-induced increase in the expression of epitopes on platelet membrane GPIIb/IIIa complex and GMP-140 is a function of platelet age.
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Savage, B., CS Hunter, LA Harker, VL Jr Woods, and SR Hanson. "Thrombin-induced increase in surface expression of epitopes on platelet membrane glycoprotein IIb/IIIa complex and GMP-140 is a function of platelet age." Blood 74, no. 3 (August 15, 1989): 1007–14. http://dx.doi.org/10.1182/blood.v74.3.1007.bloodjournal7431007.
Full textAbstract:
Platelets are heterogeneous in the content of membrane glycoprotein (GP)IIb/IIIa complex. To determine whether this heterogeneity is related to changes associated with platelet aging in the circulation, newly released platelets, obtained during recovery from nonimmune- mediated acute experimental thrombocytopenia in baboons, were studied. Monoclonal antibody (MoAb) binding to epitopes expressed on GPIIb/IIIa complex (LJ-CP8), GMP-140 (S12), and GPIa/IIa (12F1) was measured on control platelets (comprising platelets with a normal age distribution; mean age 60 to 72 hours) and newly formed platelets (mean age 12 hours), both in the resting state and after thrombin stimulation. Whereas LJ-CP8 binding to resting control platelets increased by 34% upon stimulation by gamma-thrombin from 30,885 +/- 1,171 to 41,458 +/- 1,311 molecules/platelet at saturating concentrations of antibody, LJ- CP8 binding to resting young platelets did not increase significantly upon thrombin stimulation (31,878 +/- 3,330 and 33,791 +/- 3,486 molecules/platelet, respectively). Similarly, binding of antibody S12 in response to maximal thrombin stimulation was reduced by 42% from 10,246 +/- 834 molecules/platelet at saturating concentrations of S12 for control platelets to 5,971 +/- 665 molecules/platelet for young platelets (P = .001). S12 binding to unstimulated platelets was less than 10% of the binding observed after thrombin stimulation at all concentrations of S12 for both control and young platelets. However, maximal binding of antibody 12F1 to resting control platelets did not differ significantly from that observed with resting young platelets (2,926 +/- 167 and 2,857 +/- 208 molecules/platelet, respectively), and 12F1 binding was unchanged after thrombin stimulation for both control and young platelets. We conclude that the thrombin-induced increase in the expression of epitopes on platelet membrane GPIIb/IIIa complex and GMP-140 is a function of platelet age.
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Battinelli, Elisabeth M., Kelly Elizabeth Johnson, Jodi A. Forward, Mason Tippy, Rajesh Kulenthirarajan, Erica L. Mayer, and Joseph E. Italiano. "Tamoxifen Directly Inhibits Platelet Activation, Angiogenic Potential and Platelet-Mediated Metastasis." Blood 128, no. 22 (December 2, 2016): 3723. http://dx.doi.org/10.1182/blood.v128.22.3723.3723.
Full textAbstract:
Abstract Objective - Platelets, which are mainly known for their role in hemostasis, are now known to play a crucial role in tumor metastasis and neovascularization. Metastatic disease is the cause of roughly 90% of all cancer-related deaths and understanding the mechanisms leading to dissemination of tumor cells to distant sites remains one of the main challenges of cancer research. Platelets carry a plethora of potent angiogenic and metastatic mediators within their alpha-granules and exposure to breast tumor cells induces platelet activation, leading to release of these mediators. Tamoxifen is a selective estrogen receptor modulator that is widely used for the treatment and prevention of breast cancer; its use is associated with a 50% reduction in the risk of invasive and noninvasive breast cancer in women who utilized the drug for at least 5 years. Interestingly, tamoxifen has demonstrated anti-cancer efficacy in estrogen negative breast cancers suggesting that this drug has additional mechanisms of action. Previously tamoxifen and its metabolites have been shown to directly impact platelet function. Because platelets are critical for metastatic spread, we posited that tamoxifen or its metabolites may exert anti-tumor effects through direct platelet inhibition. To test this hypothesis, we examined the impact of tamoxifen on platelet activation, angiogenic potential and metastasis both ex vivoand in breast cancer patients utilizing tamoxifen therapy. Approach and Results - We found that ex vivo pretreatment with tamoxifen or its metabolite 4-hydroxytamoxifen (4-OH) lead to a significant inhibition of platelet activation in response to TRAP, ADP or the breast tumor cell lines MDA-MB-231 and MCF-7 in platelets from healthy human donors. Platelets, known to promote tumor angiogenesis, are a reservoir for angiogenic proteins that are secreted in a differentially regulated process. Activated platelets exposed to tamoxifen or 4-OH released significantly lower amounts of the pro-angiogenic regulator VEGF in while anti-angiogenic endostatin release is unaffected, thus shifting the balance of angiogenic regulators that are released. To examine the impact of this alteration in angiogenic protein release, we performed functional angiogenesis assays using releasates generated from tamoxifen or 4-OH treated platelets. These in vitroangiogenesis assays confirmed that tamoxifen pretreatment led to dramatically diminished capillary tube formation and decreased endothelial migration. Tamoxifen also significantly inhibited the ability of platelets to promote metastasis in vitro, causing a dramatic decrease in breast tumor cell invasion and transendothelial migration. Next, we utilized membrane-based cytokine arrays to further interrogate the effect of tamoxifen on the release of stored platelet factors. Using this method, we identified several key proteins known to be associated with metastasis that were lower in releasate from tamoxifen treated platelets including TGF-b, IL-6 and IGF-1 while anti-angiogenic angiopoietin-1 was elevated. Platelets isolated from patients on tamoxifen maintenance therapy were also found to have decreased activation responses, diminished VEGF release and lower angiogenic potential. Conclusions - Overall we demonstrate that tamoxifen directly influences the release of specific stored platelet factors leading to decreased tumor cell support. The mechanism is directly linked to tamoxifen's inhibitory role in platelet activation, causing altered of release of key platelet-derived angiogenic and metastatic factors including VEGF, angiopoietin-1, TGF-b, IL-6 and IGF-1 during tumor cell and platelet crosstalk. Furthermore, translational studies confirmed that platelet activation and angiogenic potential are significantly suppressed in breast cancer patients utilizing tamoxifen therapy. Our work stresses the importance of platelets for successful angiogenesis and metastatic spread and, ultimately, supports the idea of utilizing targeted platelet therapies to inhibit the platelet's role in malignancy. Disclosures No relevant conflicts of interest to declare.
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Campbell, Robert A., Thomas H. Fischer, and Alisa S. Wolberg. "A Novel Use of Rehydrated, Lyophilized Platelets Increases Recombinant FVIIa Procoagulant Activity in a Model of Hemophilia." Blood 108, no. 11 (November 16, 2006): 1754. http://dx.doi.org/10.1182/blood.v108.11.1754.1754.
Full textAbstract:
Abstract The anti-bleeding therapy recombinant factor VIIa (rFVIIa) is used to abrogate bleeding in hemophiliacs with inhibitors, bypassing the need for replacement factors. RFVIIa is hypothesized to work by increasing Xa generation on the platelet’s surface. However, high plasma levels of rFVIIa are required, in part due to the weak binding of rFVIIa to platelets. We hypothesized that the efficacy of the therapy could be improved by administering rFVIIa already bound to platelets. One platelet preparative that may be used in this application is rehydrated, lyophilized (RL) platelets. RL platelets are fixed with paraformaldehyde, which allows them to be frozen and lyophilized while retaining their hemostatic capabilities. Previously, we have shown RL platelets are capable of supporting rFVIIa-mediated thrombin generation and that thrombin generation is increased in a rFVIIa dose-dependent manner (Blood, 106:4057, 2005). In this current study, we have characterized the ability of RL platelets to modulate rFVIIa-mediated thrombin generation and fibrin clot formation in a cell-based complete model of hemophilia. The addition of RL platelets with 50 nM rFVIIa increased the thrombin generation rate in hemophilia 2.8-fold more than 50 nM rFVIIa, alone. Further, the addition of RL platelets with 50 nM rFVIIa normalized clot formation and stability in a fibrinolytic environment, which did not occur in the presence of rFVIIa, alone. In contrast, the addition of RL platelets, alone, to hemophilic conditions had minimal to no effect on thrombin generation rate or the onset of clot formation, suggesting that the effects were due to a specific interaction between rFVIIa and RL platelets. When rFVIIa plus RL platelets were added to platelet-rich plasma from patients with hemophilia A in the presence of tissue-type plasminogen activator, clot formation and stability were improved more than the addition of either agent alone. To examine the mechanism of RL platelets’ augmentation of rFVIIa activity, we titrated the phosphatidylserine (PS) binding protein, annexin V, into reactions with RL platelets in the presence of factors Xa, Va, and prothrombin and measured thrombin generation. The addition of annexin V reduced thrombin generation equally in reactions that contained RL platelets stimulated with or without SFLLRN. Further, thrombin generation was similar on RL platelets simulated with or without SFLLRN in the absence of annexin V. These data suggest that RL platelets already have PS exposed on their surface. We conclude that RL platelets can support rFVIIa-mediated thrombin generation in the absence of factor IX and may enhance rFVIIa activity in part due to PS exposure on the RL platelet surface. We hypothesize that co-administration of RL platelets with rFVIIa may increase the efficacy of rFVIIa, at lower doses of rFVIIa than are currently required to achieve hemostasis.
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Savage, B., PR McFadden, SR Hanson, and LA Harker. "The relation of platelet density to platelet age: survival of low- and high-density 111indium-labeled platelets in baboons." Blood 68, no. 2 (August 1, 1986): 386–93. http://dx.doi.org/10.1182/blood.v68.2.386.386.
Full textAbstract:
Abstract The relationship between platelet density and platelet age has been studied using continuous linear Percoll density gradients and 111In- labeling of autologous platelets in baboons. To investigate changes in platelet density during senescence in the circulation, baboons were infused with 111In-labeled autologous platelets, and blood was collected at one hour postinfusion and twice daily thereafter for six days. Platelets were isolated from these samples in high yield (greater than 95%) and separated in continuous linear Percoll density gradients following density equilibrium centrifugation. Although at one hour postinfusion the density distribution of radiolabeled platelets coincided closely with the distribution of the total platelet population, a detectable symmetrical shift toward higher densities was observed after five days. The relative specific radioactivity (RSR) of high-density platelets (1.064 to 1.067 g/mL) decreased at a slower rate than that of the total platelet population (platelets of all densities), whereas the RSR of low-density platelets (1.053 to 1.056 g/mL) showed a more immediate and rapid decrease. These results give rise to one of two interpretations: (1) low-density platelets have a shorter survival time than more dense platelets and are therefore cleared from the circulation at a faster rate, or (2) platelets of all densities increase in density upon aging in the circulation. To determine the explanation for changing RSR of different density fractions we studied the in vivo disappearance characteristics of low- and high-density 111In-labeled platelets. There were no significant differences between the mean survival times of low-density platelets (5.0 +/- 0.49 days, +/- 1 SD, n = 6), high-density platelets (4.9 +/- 0.56 days, n = 6), or control platelets representing platelets of all densities (4.9 +/- 0.38 days, n = 6). Although a slight increase in the density of all platelets during platelet senescence is indicated by these studies, we conclude that platelet density heterogeneity is not primarily a consequence of age-related changes in platelet density.
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Savage, B., PR McFadden, SR Hanson, and LA Harker. "The relation of platelet density to platelet age: survival of low- and high-density 111indium-labeled platelets in baboons." Blood 68, no. 2 (August 1, 1986): 386–93. http://dx.doi.org/10.1182/blood.v68.2.386.bloodjournal682386.
Full textAbstract:
The relationship between platelet density and platelet age has been studied using continuous linear Percoll density gradients and 111In- labeling of autologous platelets in baboons. To investigate changes in platelet density during senescence in the circulation, baboons were infused with 111In-labeled autologous platelets, and blood was collected at one hour postinfusion and twice daily thereafter for six days. Platelets were isolated from these samples in high yield (greater than 95%) and separated in continuous linear Percoll density gradients following density equilibrium centrifugation. Although at one hour postinfusion the density distribution of radiolabeled platelets coincided closely with the distribution of the total platelet population, a detectable symmetrical shift toward higher densities was observed after five days. The relative specific radioactivity (RSR) of high-density platelets (1.064 to 1.067 g/mL) decreased at a slower rate than that of the total platelet population (platelets of all densities), whereas the RSR of low-density platelets (1.053 to 1.056 g/mL) showed a more immediate and rapid decrease. These results give rise to one of two interpretations: (1) low-density platelets have a shorter survival time than more dense platelets and are therefore cleared from the circulation at a faster rate, or (2) platelets of all densities increase in density upon aging in the circulation. To determine the explanation for changing RSR of different density fractions we studied the in vivo disappearance characteristics of low- and high-density 111In-labeled platelets. There were no significant differences between the mean survival times of low-density platelets (5.0 +/- 0.49 days, +/- 1 SD, n = 6), high-density platelets (4.9 +/- 0.56 days, n = 6), or control platelets representing platelets of all densities (4.9 +/- 0.38 days, n = 6). Although a slight increase in the density of all platelets during platelet senescence is indicated by these studies, we conclude that platelet density heterogeneity is not primarily a consequence of age-related changes in platelet density.
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Zhao, Xi, Yangchao Zhao, Yanzhong Ding, Yongjuan Ruan, Xiaowei Li, Qi Zhou, Yangfan Zhou, et al. "Autophagy Ameliorates Reactive Oxygen Species-Induced Platelet Storage Lesions." Oxidative Medicine and Cellular Longevity 2022 (April 5, 2022): 1–11. http://dx.doi.org/10.1155/2022/1898844.
Full textAbstract:
Platelet transfusion is a life-saving therapy to prevent bleeding; however, the availability of platelets for transfusion is limited by the markedly short shelf life owing to the development of platelet storage lesions (PSLs). The mechanism of PSLs remains obscure. Dissection of the intracellular biological changes in stored platelets may help to reduce PSLs and improve platelet transfusion efficiency. In the present study, we explore the changes of stored platelets at room temperature under constant agitation. We found that platelets during storage showed an increased reactive oxygen species (ROS) generation accompanied with receptor shedding, apoptosis, and diminished platelet aggregation. ROS scavenger reduced platelet shedding but also impaired platelet aggregation. Autophagy is a conserved catabolic process that sequesters protein aggregates and damaged organelles into lysosomes for degradation and platelets’ own intact autophagic system. We revealed that there exist a stable autophagic flux in platelets at the early stage of storage, and the autophagic flux in platelets perished after long-term storage. Treatment stored platelets with rapamycin, which stimulates autophagy in eukaryotic cells, markedly ameliorated PSLs, and improved platelet aggregation in response to extracellular stimuli.
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Pedersen, Oliver Buchhave, Leonardo Pasalic, Erik Lerkevang Grove, Steen Dalby Kristensen, Anne-Mette Hvas, and Peter H. Nissen. "Advanced Flow Cytometry Using the SYTO-13 Dye for the Assessment of Platelet Reactivity and Maturity in Whole Blood." Methods and Protocols 6, no. 1 (January 13, 2023): 8. http://dx.doi.org/10.3390/mps6010008.
Full textAbstract:
Newly produced immature platelets are larger, contain higher amounts of residual RNA, and are more reactive than mature platelets. Flow cytometry using the SYTO-13 dye is a method for the subdivision of immature platelets from mature platelets based on the labelling of intracellular platelet RNA, enabling the simultaneous investigation of the reactivity of each platelet population. This method provides detailed information on several aspects of platelet physiology using a combination of platelet surface markers and agonists. Currently, no standardized protocol exists across laboratories. Here, we describe a flow cytometry protocol in detail to investigate platelet reactivity and its relation to platelet maturity. We analyzed 20 healthy individuals with the protocol and compared the platelet subpopulation with the highest SYTO-13 labelling (in the first quintile, “SYTO-high”) corresponding to the most immature platelets (highest RNA content) with the platelet subpopulation with the lowest SYTO-13 labelling (in the fifth quintile, “SYTO-low”) corresponding to the mature platelets with the lowest RNA content. SYTO-high platelets had overall significantly increased platelet reactivity compared with that of SYTO-low platelets. The presented method may be a valuable research tool for the analysis of platelet reactivity and its relation to platelet maturity.
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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.
Full textAbstract:
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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Marjanovic, Jasna A., Aleksandra Stojanovic, Viktor Brovkovych, Randal A. Skidgel, and Xiaoping Du. "Inducible Nitric Oxide Synthase Plays a Stimulatory Role in Platelet Activation." Blood 106, no. 11 (November 16, 2005): 1650. http://dx.doi.org/10.1182/blood.v106.11.1650.1650.
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Abstract Platelets generate nitric oxide (NO) in response to agonist stimulation. Previous reports have shown that the endothelial nitric oxide synthase (eNOS) plays a role in agonist-stimulated platelet NO production and in platelet activation. Here we show that platelets from eNOS knockout mice (eNOS−/−) showed only partial reduction in thrombin-induced NO production compared to wild type platelets (50% reduction), indicating the presence of another NOS isoform in platelets. More importantly, we show that resting platelets express functional inducible nitric oxide synthase (iNOS), which participates in platelet activation. Compared to wild type platelets, thrombin-induced NO production was reduced by 54% in platelets isolated from iNOS knockout mice (iNOS−/−), indicating an iNOS-dependent NO production in platelets induced by thrombin. Since thrombin-induced NO production occurs during the first 3 min of thrombin stimulation, our findings provide the first evidence for a short-term regulation of iNOS activity independent of transcription regulation. In contrast, previous description of iNOS activation was primarily at the transcriptional level and required much longer time of induction. To determine the role of iNOS in platelet activation, platelets from wild type and iNOS−/− mice were stimulated with low concentrations of agonists. iNOS−/− platelets exhibited lower aggregation and secretion response compared to wild type control, indicating that iNOS plays a stimulatory role in platelet activation. We also examined the effect of iNOS inhibitors on platelet activation. Human and mouse platelets preincubated with iNOS specific inhibitors, 1400W and aminoguanidine, exhibited a dose-dependent inhibition of platelet secretion and aggregation induced by either low-dose thrombin or collagen. Furthermore, the inhibitory effect of iNOS-specific inhibitors was only shown in wild type mouse platelets, but was lacking in iNOS−/− platelets. Thus, activation of both iNOS and eNOS is important in agonist-induced NO production which stimulates platelet secretion and aggregation.
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Yuhki, Koh-ichi, Fumiaki Kojima, Takehiro Yamada, Takayuki Fujino, Akiyoshi Hara, Koji Takayama, Takayuki Maruyama, et al. "Selective activation of the prostaglandin E2 receptor subtype EP2 or EP4 leads to inhibition of platelet aggregation." Thrombosis and Haemostasis 104, no. 10 (2010): 796–803. http://dx.doi.org/10.1160/th10-01-0043.
Full textAbstract:
SummaryThe effect of selective activation of platelet prostaglandin (PG) E2 receptor subtype EP2 or EP4 on platelet aggregation remains to be determined. In platelets prepared from wild-type mice (WT platelets), high concentrations of PGE2 inhibited platelet aggregation induced by U-46619, a thromboxane receptor agonist. However, there was no significant change in the inhibitory effect of PGE2 on platelets lacking EP2 (EP2 –/– platelets) and EP4 (EP4 –/– platelets) compared with the inhibitory effect on WT platelets. On the other hand, AE1–259 and AE1–329, agonists for EP2 and EP4, respectively, potently inhibited U-46619 -induced aggregation with respective IC50 values of 590 ± 14 and 100 ± 4.9 nM in WT platelets, while the inhibition was significantly blunted in EP2 –/– and EP4 –/– platelets. In human platelets, AE1–259 and AE1–329 inhibited U-46619-induced aggregation with respective IC50 values of 640 ± 16 and 2.3 ± 0.3 nM. Notably, the inhibitory potency of AE1–329 in human platelets was much higher than that in murine platelets, while such a difference was not observed in the inhibitory potency of AE1–259. AE1–329 also inhibited adenosine diphosphate-induced platelet aggregation, and the inhibition was almost completely blocked by AE3–208, an EP4 antagonist. In addition, AE1–329 increased intracellular cAMP concentrations in a concentration- and EP4-dependent manner in human platelets. These results indicate that selective activation of EP2 or EP4 can inhibit platelet aggregation and that EP4 agonists are particularly promising as novel anti-platelet agents.
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Goodnough, Lawrence T., David J. Kuter, Jeffrey McCullough, Sherrill J. Slichter, John DiPersio, John Romo, Randolph Peterson, et al. "Prophylactic platelet transfusions from healthy apheresis platelet donors undergoing treatment with thrombopoietin." Blood 98, no. 5 (September 1, 2001): 1346–51. http://dx.doi.org/10.1182/blood.v98.5.1346.
Full textAbstract:
Many patients receiving dose-intensive chemotherapy acquire thrombocytopenia and need platelet transfusions. A study was conducted to determine whether platelets harvested from healthy donors treated with thrombopoietin could provide larger increases in platelet counts and thereby delay time to next platelet transfusion compared to routinely available platelets given to thrombocytopenic patients. Community platelet donors received either 1 or 3 μg/kg pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) or placebo and then donated platelets 10 to 15 days later. One hundred sixty-six of these platelet concentrates were then transfused to 120 patients with platelets counts 25 × 109/L or lower. Pretransfusion platelet counts (11 × 109/L) were similar for recipients of placebo-derived and PEG-rHuMGDF–derived platelets. Early after transfusion, the median platelet count increment was higher in patients receiving PEG-rHuMGDF–derived platelets: 19 (range, −12-66) × 109/L, 41 (range, 5-133) × 109/L, and 82 (range, −4-188) × 109/L for placebo-, 1-μg/kg–, and 3-μ/kg–derived platelets, respectively. This difference was maintained 18 to 24 hours after transfusion. Transfusion-free intervals were 1.72, 2.64, and 3.80 days for the recipients of the placebo-, 1-μg/kg–, and 3-μ/kg–derived platelets, respectively. The rate of transfusion-related adverse events was not different in recipients of placebo-derived and PEG-rHuMGDF–derived platelets. Therefore, when transfused into patients with thrombocytopenia, platelets collected from healthy donors undergoing thrombopoietin therapy were safe and resulted in significantly greater platelet count increments and longer transfusion-free intervals than platelets obtained from donors treated with placebo.
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Schmaier, AH, HN Bradford, D. Lundberg, A. Farber, and RW Colman. "Membrane expression of platelet calpain." Blood 75, no. 6 (March 15, 1990): 1273–81. http://dx.doi.org/10.1182/blood.v75.6.1273.1273.
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Abstract Platelet calpain has many platelet substrates, including external membrane proteins. We thus investigated whether platelet calpain II was associated with platelet membranes in unstimulated and thrombin- activated platelets. A monospecific, goat polyclonal antibody was reared to purified platelet calpain II. Sixteen whole platelet lysates were found to contain 4.5 +/- 0.7 micrograms calpain antigen II per 10(8) platelets (mean +/- SEM) as determined by a competitive enzyme- linked immunosorbent assay. Using the dipeptide fluorogenic substrate, Suc-Leu-Tyr-MCA, 17 human platelet lysates contained 3.6 +/- 0.4 micrograms calpain activity per 10(8) platelets. Platelet calpain II was associated with the Triton X-100 insoluble platelet cytoskeletons from both unstimulated and thrombin-activated platelets. When compared with the total cell content of platelet calpain II, calpain antigen (10% to 13%) and calpain activity (24% to 28%) was associated with platelet cytoskeletons in unstimulated and thrombin-activated platelets, respectively. On immunoblot, the heavy chain (80 Kd) of calpain II was detected in platelet cytoskeletons. Subcellular fractionation studies on both unstimulated and thrombin-activated platelets, revealed that half of the total platelet calpain II antigen was associated with cytosol, and the other half was associated with the membrane fraction. Platelet calpain II was not seen on the surface of unstimulated, paraformaldehyde fixed platelets by immunofluorescence. However, on thrombin-activated platelets, rim immunofluorescence was seen, indicating that activated platelets externalize their calpain. This observation was confirmed by the finding that about 2,000 molecules per platelet of an 125I-anti-calpain II Fab' specifically bound to thrombin-activated but not unstimulated platelets. Both dibucaine (1 mmol/L) and platelet activating factor (1.86 mumol/L) in the absence of external Ca++, but not collagen (5 micrograms/mL) or ionophore A23187 (2.5 mumol/L) in the absence of external Ca++, were also able to externalize platelet calpain II antigen, as indicated by a similar level of specific 125I-anti-calpain II Fab'-platelet binding. These combined studies indicate that platelet calpain II is a major protein, comprising 2% of total platelet protein, a substantial portion of which is membrane-associated. When platelets are activated by thrombin and platelet activating factor, calpain II antigen also becomes present on the external platelet surface.
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Schmaier, AH, HN Bradford, D. Lundberg, A. Farber, and RW Colman. "Membrane expression of platelet calpain." Blood 75, no. 6 (March 15, 1990): 1273–81. http://dx.doi.org/10.1182/blood.v75.6.1273.bloodjournal7561273.
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Platelet calpain has many platelet substrates, including external membrane proteins. We thus investigated whether platelet calpain II was associated with platelet membranes in unstimulated and thrombin- activated platelets. A monospecific, goat polyclonal antibody was reared to purified platelet calpain II. Sixteen whole platelet lysates were found to contain 4.5 +/- 0.7 micrograms calpain antigen II per 10(8) platelets (mean +/- SEM) as determined by a competitive enzyme- linked immunosorbent assay. Using the dipeptide fluorogenic substrate, Suc-Leu-Tyr-MCA, 17 human platelet lysates contained 3.6 +/- 0.4 micrograms calpain activity per 10(8) platelets. Platelet calpain II was associated with the Triton X-100 insoluble platelet cytoskeletons from both unstimulated and thrombin-activated platelets. When compared with the total cell content of platelet calpain II, calpain antigen (10% to 13%) and calpain activity (24% to 28%) was associated with platelet cytoskeletons in unstimulated and thrombin-activated platelets, respectively. On immunoblot, the heavy chain (80 Kd) of calpain II was detected in platelet cytoskeletons. Subcellular fractionation studies on both unstimulated and thrombin-activated platelets, revealed that half of the total platelet calpain II antigen was associated with cytosol, and the other half was associated with the membrane fraction. Platelet calpain II was not seen on the surface of unstimulated, paraformaldehyde fixed platelets by immunofluorescence. However, on thrombin-activated platelets, rim immunofluorescence was seen, indicating that activated platelets externalize their calpain. This observation was confirmed by the finding that about 2,000 molecules per platelet of an 125I-anti-calpain II Fab' specifically bound to thrombin-activated but not unstimulated platelets. Both dibucaine (1 mmol/L) and platelet activating factor (1.86 mumol/L) in the absence of external Ca++, but not collagen (5 micrograms/mL) or ionophore A23187 (2.5 mumol/L) in the absence of external Ca++, were also able to externalize platelet calpain II antigen, as indicated by a similar level of specific 125I-anti-calpain II Fab'-platelet binding. These combined studies indicate that platelet calpain II is a major protein, comprising 2% of total platelet protein, a substantial portion of which is membrane-associated. When platelets are activated by thrombin and platelet activating factor, calpain II antigen also becomes present on the external platelet surface.
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Charania, Roseleen, James W. Smith, Sara K. Vesely, George L. Dale, and Jennifer L. Holter. "Collection and Storage of Apheresis Platelets Results in a Significant Decline in the Percentage of Coated-Platelets." Blood 114, no. 22 (November 20, 2009): 23. http://dx.doi.org/10.1182/blood.v114.22.23.23.
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Abstract Abstract 23 Introduction: Platelets initiate hemostasis by adhering to the site of vascular injury and providing a negatively charged surface for thrombin generation. A subset of platelets, referred to as coated-platelets, has an enhanced ability to generate thrombin. Coated-platelets, observed in vitro upon dual agonist stimulation with collagen and thrombin, retain several serotonin-derivatized proteins on their surface and express phosphotidylserine, creating a highly active prothrombinase complex. Experimental studies have concluded that dogs deficient in coated-platelets manifest with excessive nasal, intraocular and post-operative bleeding, suggesting a key role for coated-platelets in hemostasis. The fraction of coated-platelets made by normal individuals is approximately 30% with a range of 15% – 55%. Platelet concentrates are stored at room temperature to preserve their viability and must be transfused within five days of collection secondary to a high risk of contamination. We conducted a pilot study in healthy platelet donors to evaluate how the process of apheresis and in vitro storage affect the ability to generate coated-platelets. Secondarily, we aimed to evaluate the coated-platelet potential of apheresis platelet units transfused in clinical practice. Methods: Healthy voluntary platelet donors presenting at the Oklahoma Blood Institute were enrolled and demographic data such as age, sex, smoking status, donation history, past medical history and medications were noted. A Pre-donation blood sample was obtained. Thereafter, donors underwent apheresis using the Caridian BCT Trima system via continuous centrifugation in a closed system per guidelines outlined by the American Association of Blood Banks (AABB). A Post-donation sample was drawn from the platelet concentrate bag 30 minutes after apheresis. Units were packaged, processed and stored per routine procedures. A third platelet sample was obtained when the unit was released for transfusion. Platelet rich plasma was prepared and coated-platelets were assayed as previously described (J. Thromb. Haemostasis 6:609, 2008). Statistical analysis was performed using paired t-test to compare the change in percentage of coated-platelets between the three samples. Results: Nine donors were enrolled. The mean percentage of coated-platelets in the Pre-donation samples was 40.4% (20.7%–59.7%). Thirty minutes after the apheresis collection procedure, the mean percentage of coated-platelets in the concentrate was 34.3% (17.3%–49.0%) suggesting a 15% decline in percentage of coated-platelets with the process of apheresis (p<0.01). Eight units were transfused within one to four days of collection. The mean percentage of coated-platelets in the transfused product was 17.1 % (10.1%–28.4%); a 52% decline in the percentage of coated-platelets during the process of storage was noted (p<0.001). [Figure A and B]. Conclusion: Coated-platelets are a recently recognized component of hemostasis, and the ability of stored platelets to retain coated-platelet potential is unknown. Our preliminary results indicate that the process of platelet apheresis and storage significantly decreases the ability to generate coated-platelets. Current quality control guidelines for platelet storage measure platelet viability with factors such as pH, pCO2, pO2, lactate and platelet morphology; however, there is no evaluation of the hemostatic function of stored platelets relative to coated-platelets. Our results reveal that during storage following current transfusion guidelines, the percentage of coated-platelets decreases. Part of this decline appears to be associated with the apheresis process itself and further progresses with storage. Our study provides the first evidence that retention of coated platelet levels during preparation and storage is problematic and may be an important factor in evaluating the hemostatic potential of infused platelets. 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.
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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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Liu, Xiao, Li Liu, Ana-Maria Zaske, Zhou Zhou, Yuanyuan Fu, Xi Yang, Jodie Conyers, et al. "Contact- and agonist-regulated microvesiculation of human platelets." Thrombosis and Haemostasis 110, no. 08 (2013): 331–99. http://dx.doi.org/10.1160/th12-11-0853.
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SummaryAfter exposure to an agonist, platelets are activated and become aggregated. They also shed membrane microparticles that participate in the pathogenesis of thrombosis, hyper-coagulation and inflammation. However, microvesiculation can potentially disrupt the integrity of platelet aggregation by shedding the membrane receptors and phosphatidylserine critical for forming and stabilising a platelet clot. We tested the hypothesis that adhesion and microvesiculation are functions of different subsets of platelets at the time of haemostasis by real-time monitoring of agonist-induced morphological changes and microvesiculation of human platelets. We identified two types of platelets that are adherent to fibrinogen: a high density bubble shape (HDBS) and low-density spread shape (LDSS). Adenosine diphosphate (ADP) predominantly induced HDBS platelets to vesiculate, whereas LDSS platelets were highly resistant to such vesiculation. Thrombin-receptor activating peptide (TRAP) stabilised platelets against microvesiculation by promoting a rapid HDBS-to-LDSS morphological transition. These activities of ADP and TRAP were reversed for platelets in suspension, independent of an engagement integrin αIIbβ 3. As the result of membrane contact, LDSS platelets inhibited the microvesiculation of HDBS platelets in response to ADP. Aspirin and clopidogrel inhibited ADP-induced microvesiculation through different mechanisms. These results suggest that platelet aggregation and microvesiculation occur in different subsets of platelets and are differently regulated by agonists, platelet-platelets and platelet-fibrinogen interactions.
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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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Dubois, Christophe, Laurence Panicot-Dubois, Barbara C. Furie, and Bruce Furie. "Dynamics of Calcium Mobilization in Platelets during Thrombus Formation in a Living Mouse." Blood 106, no. 11 (November 16, 2005): 649. http://dx.doi.org/10.1182/blood.v106.11.649.649.
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Abstract Platelet accumulation at sites of vascular injury arrests bleeding but also plays a critical role in the pathogenesis of thrombosis, leading to ischemia in myocardial infarction or stroke. Intracellular calcium mobilization in platelets is a critical step in the activation of platelets and formation of the platelet thrombus. Here we show the relationship of the dynamics of intracellular calcium mobilization with platelet accumulation into the developing thrombus in a living mouse. Following injection of 100 x 106 fura-2 loaded platelets into a living mouse we used high speed intravital multi-channel digital fluorescence microscopy to monitor calcium status in circulating and thrombus-bound platelets during thrombus development. One population of platelets binds transiently to the developing thrombus but does not mobilize calcium. The mean duration of platelet-thrombus interaction for these platelets is 11 sec. Another population of platelets undergoes calcium mobilization after binding to the developing thrombus. The time interval from attachment to calcium mobilization for individual platelets varied from 1.0 to 12 sec, with a median of 3.5 sec. More than 90% of platelets that undergo calcium mobilization do so with in 5 sec of attachment. The calcium mobilization in the thrombus bound platelets is reversible. About two thirds of the platelets return rapidly to the basal Ca2+ state while the remaining thrombus bound platelets maintain an elevated Ca2+ level for an extended period. The mean duration of platelet-thrombus interaction is 35 sec with a range of 1.5 sec to 284 sec (median duration 39.5 sec) as calculated from multiple independent observations of single platelets. In each platelet studied, only one calcium peak is detected per platelet. There is a close correlation between the duration of calcium mobilization in an individual platelet and the time that the platelet remains attached to the developing thrombus, suggesting a relationship of calcium-dependent events and platelet-thrombus affinity. A population of platelets binds to the thrombus, mobilizes calcium and remains associated with the thrombus. Using widefield deconvolution techniques to obtain planar images and increased numbers of dye-loaded platelets, individual platelets could be observed undergoing sustained calcium elevation within the thrombus. As the platelet thrombus reaches maximal size at about 120 sec, calcium mobilization continues in the stable core of the thrombus for several minutes, then decreases. These studies describe thrombus formation in a living animal under conditions in which the endothelium and vessel wall, blood cells and plasma components, and flowing blood are preserved in the absence of anticoagulants. Our results indicate that stable platelet thrombus formation is dependent upon durable calcium mobilization, and that intracellular calcium regulates thrombus development and maturation in vivo.
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Tanoue, K., Y. Mano, K. Kuroiwa, H. Suzuki, M. Shibayama, and H. Yamazaki. "Consumption of platelets in decompression sickness of rabbits." Journal of Applied Physiology 62, no. 5 (May 1, 1987): 1772–79. http://dx.doi.org/10.1152/jappl.1987.62.5.1772.
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Platelet behavior was studied in rabbit decompression sickness which was brought about by the exposure to 6 ATA for 40 min (bottom time) followed by rapid decompression. Platelet counts significantly decreased after the decompression. Kinetic studies with 111In-oxine-labeled platelets revealed shortened survivals of circulating platelets, and audioradiograms indicated the accumulation of radioactivity in the lungs after the decompression. Although there was no change in the mode volume of platelets after the decompression, the transient appearance of circulating smaller or fragmented platelets suggested a random overdestruction of platelets. Whole and releasable adenine nucleotide contents of platelets were decreased significantly after the decompression. There were no significant changes in cytoplasmic adenine nucleotide contents. Therefore, in decompression sickness, the circulating platelets behaved similarly to those in acquired storage pool disease. Platelet thrombi were found in the pulmonary arteries, compatible with the accumulation of 111In-oxine-labeled platelets. These findings suggest that circulating air bubbles interact with platelets, causing the platelet release reaction, and these activated platelets participate in the formation of thrombi in experimental decompression sickness.
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Schmaier, AH, S. Amenta, T. Xiong, GD Heda, and AM Gewirtz. "Expression of platelet C1 inhibitor." Blood 82, no. 2 (July 15, 1993): 465–74. http://dx.doi.org/10.1182/blood.v82.2.465.465.
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Abstract Human platelets contain a pool of C1 inhibitor (C1 INH) distinct from that in plasma. Twelve normal platelet samples washed by centrifugation had a mean platelet C1 INH antigen level of 19.3 +/- 2.8 ng (mean +/- SEM) per 10(8) platelets. These values contrast with the mean +/- SEM platelet C1 INH antigen level of 6.1 +/- 0.9 per 10(8) platelets from 12 C1 INH-deficient patients. The level of platelet C1 INH correlated (r = .7) with the level of plasma C1 INH in normal individuals and patients with classic hereditary angioedema. Platelet C1 INH, like plasma C1 INH, was a 105-Kd protein on immunoblots of solubilized platelets and in thrombin- or collagen-induced platelet releasates. On indirect immunofluorescence, morphologically and immunochemically identifiable elutriated human megakaryocytes had C1 INH antigen. Using nested primer polymerase chain reaction, C1 INH mRNA was detected in megakaryocytes. When activated, human platelets expressed a portion of their total pool of C1 INH antigen on their membrane. Using a competitive enzyme-linked immunosorbent assay for C1 INH as a quantitative, indirect antibody consumption assay, the surface of unstimulated platelets had 0.55 +/- 0.4 ng C1 INH/10(8) platelets (mean +/- SEM). When activated with thrombin, platelets secreted 7.37 +/- 2.2 ng C1 INH/10(8) platelets into the suspension buffer and simultaneously expressed 4.4 +/- 1.2 ng C1 INH/10(8) platelets on their external membrane. These studies showed that activated platelets secreted 38% of their C1 INH and externalized another 23% of the total platelet C1 INH on their membrane. Furthermore, in 125I-anti-C1 INH Fab' binding experiments to platelets, about 8 ng of the antibody fragment specifically bound to 10(8) activated platelets. These data suggest that level of platelet C1 INH packaged into platelet alpha-granules is modulated by the amount of protein produced in megakaryocytes. Platelet alpha-granule C1 INH can both be secreted from platelets and expressed on their activated membranes. The cell membrane expression of C1 INH may be important to modulate the activity of the proteases of the complement and contact systems of plasma proteolysis in the microenvironment of the inflammatory response.
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Schmaier, AH, S. Amenta, T. Xiong, GD Heda, and AM Gewirtz. "Expression of platelet C1 inhibitor." Blood 82, no. 2 (July 15, 1993): 465–74. http://dx.doi.org/10.1182/blood.v82.2.465.bloodjournal822465.
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Human platelets contain a pool of C1 inhibitor (C1 INH) distinct from that in plasma. Twelve normal platelet samples washed by centrifugation had a mean platelet C1 INH antigen level of 19.3 +/- 2.8 ng (mean +/- SEM) per 10(8) platelets. These values contrast with the mean +/- SEM platelet C1 INH antigen level of 6.1 +/- 0.9 per 10(8) platelets from 12 C1 INH-deficient patients. The level of platelet C1 INH correlated (r = .7) with the level of plasma C1 INH in normal individuals and patients with classic hereditary angioedema. Platelet C1 INH, like plasma C1 INH, was a 105-Kd protein on immunoblots of solubilized platelets and in thrombin- or collagen-induced platelet releasates. On indirect immunofluorescence, morphologically and immunochemically identifiable elutriated human megakaryocytes had C1 INH antigen. Using nested primer polymerase chain reaction, C1 INH mRNA was detected in megakaryocytes. When activated, human platelets expressed a portion of their total pool of C1 INH antigen on their membrane. Using a competitive enzyme-linked immunosorbent assay for C1 INH as a quantitative, indirect antibody consumption assay, the surface of unstimulated platelets had 0.55 +/- 0.4 ng C1 INH/10(8) platelets (mean +/- SEM). When activated with thrombin, platelets secreted 7.37 +/- 2.2 ng C1 INH/10(8) platelets into the suspension buffer and simultaneously expressed 4.4 +/- 1.2 ng C1 INH/10(8) platelets on their external membrane. These studies showed that activated platelets secreted 38% of their C1 INH and externalized another 23% of the total platelet C1 INH on their membrane. Furthermore, in 125I-anti-C1 INH Fab' binding experiments to platelets, about 8 ng of the antibody fragment specifically bound to 10(8) activated platelets. These data suggest that level of platelet C1 INH packaged into platelet alpha-granules is modulated by the amount of protein produced in megakaryocytes. Platelet alpha-granule C1 INH can both be secreted from platelets and expressed on their activated membranes. The cell membrane expression of C1 INH may be important to modulate the activity of the proteases of the complement and contact systems of plasma proteolysis in the microenvironment of the inflammatory response.
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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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Packham, MA, MA Guccione, and KM O'Brien. "Duration of the effect of aspirin on the synthesis of thromboxane by density subpopulations of rabbit platelets stimulated with thrombin." Blood 66, no. 2 (August 1, 1985): 287–90. http://dx.doi.org/10.1182/blood.v66.2.287.287.
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Abstract The controversy concerning the relationship between platelet buoyant density and platelet age is unresolved. Our earlier results with rabbit platelets indicate that the most-dense subpopulations are enriched in young platelets and that some platelets become less dense as they age. Other investigators have concluded that platelets either do not change in density upon aging or become more dense. In the present experiments, rabbit platelets were separated on discontinuous gradients of Stractan. Most-dense platelets synthesized significantly more thromboxane B2 (TXB2) (1.27 ng per 10(6) platelets) in response to thrombin (0.75 U/mL) than did least-dense platelets (0.70 ng per 10(6) platelets), indicating that the arachidonate pathway in most-dense platelets is more active than in least-dense platelets. After aspirin administration to rabbits, most-dense platelets recovered their ability to synthesize thromboxane B2 significantly more quickly than did least-dense platelets. Because the platelet cyclooxygenase that is responsible for TXB2 formation is permanently inhibited by aspirin, it is only the new platelets entering the circulation that will be able to form TXB2. These results indicate that, at least in rabbits, the most-dense platelets are enriched in young platelets, and that platelets decrease in density as they age in the circulation.