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1
Filipovic, N., M. Kojic, and A. Tsuda. "Modelling thrombosis using dissipative particle dynamics method." Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 366, no. 1879 (July 2008): 3265–79. http://dx.doi.org/10.1098/rsta.2008.0097.
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Aim . Arterial occlusion is a leading cause of cardiovascular disease. The main mechanism causing vessel occlusion is thrombus formation, which may be initiated by the activation of platelets. The focus of this study is on the mechanical aspects of platelet-mediated thrombosis which includes the motion, collision, adhesion and aggregation of activated platelets in the blood. A review of the existing continuum-based models is given. A mechanical model of platelet accumulation onto the vessel wall is developed using the dissipative particle dynamics (DPD) method in which the blood (i.e. colloidal-composed medium) is treated as a group of mesoscale particles interacting through conservative, dissipative, attractive and random forces. Methods . Colloidal fluid components (plasma and platelets) are discretized by mesoscopic (micrometre-size) particles that move according to Newton's law. The size of each mesoscopic particle is small enough to allow tracking of each constituent of the colloidal fluid, but significantly larger than the size of atoms such that, in contrast to the molecular dynamics approach, detailed atomic level analysis is not required. Results . To test this model, we simulated the deposition of platelets onto the wall of an expanded tube and compared our computed results with the experimental data of Karino et al . ( Miscrovasc. Res. 17 , 238–269, 1977). By matching our simulations to the experimental results, the platelet aggregation/adhesion binding force (characterized by an effective spring constant) was determined and found to be within a physiologically reasonable range. Conclusion . Our results suggest that the DPD method offers a promising new approach to the modelling of platelet-mediated thrombosis. The DPD model includes interaction forces between platelets both when they are in the resting state (non-activated) and when they are activated, and therefore it can be extended to the analysis of kinetics of binding and other phenomena relevant to thrombosis.
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Kim, Dongjune A., Katrina J. Ashworth, Jorge Di Paola, and David N. Ku. "Platelet α-granules are required for occlusive high-shear-rate thrombosis." Blood Advances 4, no. 14 (July 22, 2020): 3258–67. http://dx.doi.org/10.1182/bloodadvances.2020002117.
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Abstract von Willebrand factor (VWF) is essential for the induction of arterial thrombosis. In this study, we investigated the critical role of platelet VWF in occlusive thrombosis formation at high shear in mice that do not express platelet VWF (Nbeal2−/−). Using in silico modeling, in vitro high-shear microfluidics, and an in vivo Folts model of arterial thrombosis we reproduced the platelet dynamics that occur under pathological flow in a stenosed vessel. Computational fluid dynamics (CFDs) simulated local hemodynamics in a stenosis based on arterial geometries. The model predicted shear rates, time course of platelet adhesion, and time to occlusion. These predictions were validated in vitro and in vivo. Occlusive thrombosis developed in wild-type control mice that had normal levels of plasma VWF and platelet VWF in vitro and in vivo. Occlusive thrombosis did not form in the Nbeal2−/− mice that had normal plasma VWF and an absence of platelet VWF. Occlusive thrombosis was corrected in Nbeal2−/− microfluidic assays by the addition of exogenous normal platelets with VWF. Combining model and experimental data, we demonstrated the necessary requirement of platelet VWF in α-granules in forming an occlusive thrombus under high shear. These results could inspire new pharmacological targets specific to pathological conditions and prevent arterial thrombosis.
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Stritt, Simon, Inga Birkholz, Sarah Beck, Simona Sorrentino, K. Tanuj Sapra, Julien Viaud, Johannes Heck, et al. "Profilin 1–mediated cytoskeletal rearrangements regulate integrin function in mouse platelets." Blood Advances 2, no. 9 (May 8, 2018): 1040–45. http://dx.doi.org/10.1182/bloodadvances.2017014001.
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Key Points Profilin 1–mediated cytoskeletal dynamics regulate platelet β1- and β3-integrin function and turnover. Profilin 1 deficiency in platelets impairs hemostasis and results in a marked protection from arterial thrombosis.
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Yangfan Zhou, Mengjiao Hu, Xiaoyan Chen, Shuai Wang, Jingke Li, Lina Sa, Li Li, Jiaqi Huang, Hongqiang Cheng, and Hu Hu. "Migfilin supports hemostasis and thrombosis through regulating platelet αIIbβ3 outside-in signaling." Haematologica 105, no. 11 (December 26, 2019): 2608–18. http://dx.doi.org/10.3324/haematol.2019.232488.
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Elucidating the regulation mechanism of integrin αIIbβ3 is key to understand platelet biology and thrombotic diseases. Previous in vitro studies have implicated a role of migfilin in the support of platelet αIIbβ3 activation, however, contribution of migfilin to thrombosis and hemostasis in vivo and a detailed mechanism of migfilin in platelets are not known. In this study, with migfilin deletion (migfilin-/-) mice, we report that migfilin is a pivotal positive regulator of hemostasis and thrombosis. Migfilin-/- mice showed a nearly doubled tail-bleeding time and a prolonged occlusion time in Fecl3-induced mesenteric arteriolar thrombosis. Migfilin deficiency impedes platelet thrombi formation on collagen surface and impairs platelet aggregation and dense-granule secretion. Supported by characteristic functional readings and phosphorylation status of distinctive signaling molecules in the bidirectional signaling processes of αIIbβ3, the functional defects of migfilin-/- platelets appear to be mechanistically associated with a compromised outside-in signaling, rather than inside-out signaling. A synthesized cell-permeable migfilin peptide harboring filamin A binding sequence rescued the defective function and phosphorylation of signaling molecules of migfilin-/- platelets. Finally, migfilin does not influence the binding of filamin A and β3 subunit of αIIbβ3 in resting platelets, but hampers the re-association of filamin A and β3 during the conduct of outside-in signaling, suggesting that migfilin functions through regulating the interaction dynamics of αIIbβ3 and filamin A in platelets. Our study enhances the current understanding of platelet integrin αIIbβ3-mediated outside-in signaling and proves that migfilin is an important regulator for platelet activation, hemostasis and thrombosis.
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Belyaev, Aleksey V. "Computer modelling of initial platelet adhesion during microvascular thrombosis." Russian Journal of Numerical Analysis and Mathematical Modelling 34, no. 5 (October 25, 2019): 241–51. http://dx.doi.org/10.1515/rnam-2019-0020.
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Abstract Hemostasis is one of the most important protective mechanisms that functions to maintain vascular integrity and prevent bleeding. In arterial and microvascular circulation, where the near-wall shear stress is relatively high, the hemostatic response begins with aggregation of platelets on the injured endothelium or collagen. Regulation of hemostasis and thrombosis is immensely complex, as it depends on the blood cell adhesion and fluid dynamics. A possible regulatory mechanism relies on the coil-stretch transitions in a plasma protein — von Willebrand factor — that serves as a ligand to platelet adhesive membrane receptors. In this work, the initial stages of thrombus growth are studied using a 3D computer model that explicitly accounts for the shear-dependent vWf conformation.
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Grande Gutiérrez, Noelia, Mark Alber, Andrew M. Kahn, Jane C. Burns, Mathew Mathew, Brian W. McCrindle, and Alison L. Marsden. "Computational modeling of blood component transport related to coronary artery thrombosis in Kawasaki disease." PLOS Computational Biology 17, no. 9 (September 7, 2021): e1009331. http://dx.doi.org/10.1371/journal.pcbi.1009331.
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Coronary artery thrombosis is the major risk associated with Kawasaki disease (KD). Long-term management of KD patients with persistent aneurysms requires a thrombotic risk assessment and clinical decisions regarding the administration of anticoagulation therapy. Computational fluid dynamics has demonstrated that abnormal KD coronary artery hemodynamics can be associated with thrombosis. However, the underlying mechanisms of clot formation are not yet fully understood. Here we present a new model incorporating data from patient-specific simulated velocity fields to track platelet activation and accumulation. We use a system of Reaction-Advection-Diffusion equations solved with a stabilized finite element method to describe the evolution of non-activated platelets and activated platelet concentrations [AP], local concentrations of adenosine diphosphate (ADP) and poly-phosphate (PolyP). The activation of platelets is modeled as a function of shear-rate exposure and local concentration of agonists. We compared the distribution of activated platelets in a healthy coronary case and six cases with coronary artery aneurysms caused by KD, including three with confirmed thrombosis. Results show spatial correlation between regions of higher concentration of activated platelets and the reported location of the clot, suggesting predictive capabilities of this model towards identifying regions at high risk for thrombosis. Also, the concentration levels of ADP and PolyP in cases with confirmed thrombosis are higher than the reported critical values associated with platelet aggregation (ADP) and activation of the intrinsic coagulation pathway (PolyP). These findings suggest the potential initiation of a coagulation pathway even in the absence of an extrinsic factor. Finally, computational simulations show that in regions of flow stagnation, biochemical activation, as a result of local agonist concentration, is dominant. Identifying the leading factors to a pro-coagulant environment in each case—mechanical or biochemical—could help define improved strategies for thrombosis prevention tailored for each patient.
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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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de Vrij, Edwin L., Hjalmar R. Bouma, Maaike Goris, Ulrike Weerman, Anne P. de Groot, Jeroen Kuipers, Ben N. G. Giepmans, and Robert H. Henning. "Reversible thrombocytopenia during hibernation originates from storage and release of platelets in liver sinusoids." Journal of Comparative Physiology B 191, no. 3 (March 4, 2021): 603–15. http://dx.doi.org/10.1007/s00360-021-01351-3.
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AbstractImmobility is a risk factor for thrombosis due to low blood flow, which may result in activation of the coagulation system, recruitment of platelets and clot formation. Nevertheless, hibernating animals—who endure lengthy periods of immobility—do not show signs of thrombosis throughout or after hibernation. One of the adaptations of hemostasis in hibernators consists of a rapidly reversible reduction of the number of circulating platelets during torpor, i.e., the hibernation phase with reduction of metabolic rate, low blood flow and immobility. It is unknown whether these platelet dynamics in hibernating hamsters originate from storage and release, as suggested for ground squirrel, or from breakdown and de novo synthesis. A reduction in detaching forces due to low blood flow can induce reversible adhesion of platelets to the vessel wall, which is called margination. Here, we hypothesized that storage-and-release by margination to the vessel wall induces reversible thrombocytopenia in torpor. Therefore, we transfused labeled platelets in hibernating Syrian hamster (Mesocricetus auratus) and platelets were analyzed using flow cytometry and electron microscopy. The half-life of labeled platelets was extended from 20 to 30 h in hibernating animals compared to non-hibernating control hamsters. More than 90% of labeled platelets were cleared from the circulation during torpor, followed by emergence during arousal which supports storage-and-release to govern thrombocytopenia in torpor. Furthermore, the low number of immature platelets, plasma level of interleukin-1α and normal numbers of megakaryocytes in bone marrow make platelet synthesis or megakaryocyte rupture via interleukin-1α unlikely to account for the recovery of platelet counts upon arousal. Finally, using large-scale electron microscopy we revealed platelets to accumulate in liver sinusoids, but not in spleen or lung, during torpor. These results thus demonstrate that platelet dynamics in hibernation are caused by storage and release of platelets, most likely by margination to the vessel wall in liver sinusoids. Translating the molecular mechanisms that govern platelet retention in the liver, may be of major relevance for hemostatic management in (accidental) hypothermia and for the development of novel anti-thrombotic strategies.
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Haley, Kristina M., Joseph E. Aslan, Garth W. Tormoen, Sandra M. Baker, Cassandra P. Loren, Jonathan Chernoff, and Owen J. T. McCarty. "The PAK Signaling System Links Rho Gtpase Activation to Platelet Lamellopodia Formation, Aggregation and Aggregate Stability Under Shear." Blood 120, no. 21 (November 16, 2012): 1060. http://dx.doi.org/10.1182/blood.v120.21.1060.1060.
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Abstract Abstract 1060 Platelets serve as the primary mediators of hemostasis and thrombosis, circulating as surveyors for gaps in vascular integrity. As platelets encounter exposed extracellular matrix proteins, receptors on the platelet surface trigger intracellular signaling events that result in rapid platelet activation and a complex rearrangement of platelet morphology to form filopodia and lamellipodia. Rac1, a member of the Rho GTPase family, has emerged as a key regulator in platelet actin dynamics. However, the specific downstream events following Rac1 activation that mediate platelet actin cytoskeleton reorganization remain ill-defined. The Rho GTPase, Rac, supports the autocatalytic activation of the p21 activated kinases, or PAKs, to mediate actin reorganization processes in focal adhesion formation and cell migration. Upon activation by GTP-bound Rac, the PAKs phosphorylate a number of substrates to coordinate actin dynamics. Platelets express a number of PAK isoforms, and like Rac, PAK has been shown to be activated as platelets spread on collagen in a Src and PI3K dependent manner. Furthermore, the adaptor protein SLP-76 has been proposed to potentiate PAK activity downstream of Rac activation to mediate platelet lamellipodia formation. However, the specific roles of PAK in platelet function have yet to be characterized. Thus we set out to elucidate the role of PAK in platelet function and to define the connection between Rac activation, PAK, and platelet cytoskeletal reorganization. Our initial experiments with mass spectrometry revealed that following platelet activation, Rac1 associates with a set of PAK effectors, GIT1, GEFH1, LIMK1, and Merlin. We next demonstrated a co-localization of Rac1 and PAK with actin at the leading edge of spread platelets on fibrinogen. In addition, inhibition of PAK signaling by two different pharmacologic inhibitors blocked platelet focal adhesion and lamellopodia formation on both fibrinogen and collagen. Inhibition of PAK signaling abrogated intracellular calcium mobilization in platelets, prevented platelet aggregation to the GPVI-agonist, CRP, and destabilized platelet lamellipodia, resulting in the retraction of lamellipodia in spread platelets. Finally, inhibition of PAK resulted in the disaggregation of platelet aggregates formed under shear flow conditions. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation to PAK effector function and platelet lamellopodia formation, thus filling an important gap in the understanding of platelet actin cytoskeletal organization. In addition, these data characterize the integral role of PAK in platelet spreading, aggregation, and aggregate stability. Elucidating the mechanisms that mediate platelet spreading and aggregate formation may highlight important steps in the platelet activation cascade at which to pharmacologically intervene in order to inhibit or treat pathologic thrombi formation. Disclosures: No relevant conflicts of interest to declare.
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Stalker, Timothy J., Jie Wu, and Lawrence F. Brass. "Platelet Expression of a Genetically Encoded Calcium Indicator Reveals Platelet Activation Gradients in Real Time during Thrombus Formation in Vivo." Blood 124, no. 21 (December 6, 2014): 96. http://dx.doi.org/10.1182/blood.v124.21.96.96.
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Abstract Introduction: Recent studies have demonstrated that platelet activation during hemostatic thrombus formation in vivo is heterogeneous in time and space. We have previously shown that in response to a penetrating injury in the microcirculation platelet accumulation and activation are driven by gradients of soluble agonists emanating from the site of injury. These gradients result in a characteristic thrombus architecture in which a core of fully activated platelets that have released their alpha granules is overlaid by a shell of less activated platelets that frequently embolize. While dependent on ADP/P2Y12 signaling, the extent and dynamics of platelet activation in the shell region remain poorly understood. Here, we used intravital imaging of platelet cytosolic calcium concentration to examine platelet activation in real time following vascular injury in vivo. Methods: We generated transgenic mice expressing a genetically encoded calcium indicator (GCaMP3) specifically in megakaryocytes and platelets by crossing PF4-Cre mice with Ai38 mice carrying the GCaMP3 transgene. GCaMP3 fluorescence was visualized in platelets following laser-induced injury in mouse cremaster arterioles using spinning disk confocal intravital microscopy. Results: Rapid and dynamic changes in GCaMP3 fluorescence were observed during platelet accumulation following vascular injury. Platelets immediately adjacent to the site of injury exhibited a rapid and sustained increase in cytosolic calcium. Peak cytosolic calcium levels in the core region occurred within the first minute post-injury, prior to peak P-selectin expression. Platelets in the shell region were characterized by dynamic changes in cytosolic calcium at the level of individual platelets, with the appearance of transient “waves” of calcium signaling propagating among groups of platelets. At the population level, cytosolic calcium concentration was substantially lower in the shell region as compared to the core. Conclusions: Expression of a genetically encoded calcium indicator in platelets allows for the visualization of platelet activation events in real time in vivo. Platelets within the core region of a thrombus exhibit a rapid and sustained increase in cytosolic calcium concentration, indicating robust activation. In contrast, platelets in the shell region exhibit transient increases in cytosolic calcium indicating weak activation, consistent with their increased likelihood of embolization. The examination of platelet calcium signaling in vivo provides a sensitive readout of platelet activation that will shed new light on mechanisms regulating hemostasis and thrombosis, and may be useful in assessing the impact of anti-thrombotic agents on platelet function. Research support was provided by the American Heart Association and National Heart, Lung and Blood Institute. Disclosures Stalker: Medicines Company: Research Funding. Brass:Medicines Company: Research Funding.
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Pula, Giordano, Kai Schuh, Ulrich Walter, Keiichi I. Nakayama, Keiko Nakayama, and Alastair W. Poole. "PKCδ Regulates Platelet Activity through the Inhibition of VASP-Mediated Filopodia Formation." Blood 108, no. 11 (November 16, 2006): 1513. http://dx.doi.org/10.1182/blood.v108.11.1513.1513.
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Abstract Filopodia are finger-like protrusions that play a crucial role in the guidance of cells through their extracellular and cellular environment. Blood platelets provide an essential early response system in haemostasis and arterial thrombosis and display a rapid and dramatic generation of filopodia upon activation, which play an essential role regulating their adhesive events leading to platelet aggregation. Here we report a novel and unexpected control mechanism for this explosive cellular rearrangement. Using pharmacological and genetic approaches we show that the novel isoform of protein kinase C, PKCδ, is a critical negative regulator of actin polymerization, filopodia formation and platelet aggregation. We show that PKCδ interacts with vasodilator-stimulated phosphoprotein (VASP), a major regulator of actin cytoskeleton dynamics, and regulates VASP phosphorylation. VASP is shown to be necessary for the PKCδ-dependent regulation of actin polymerization, filopodia formation and platelet aggregation. We therefore propose a causative link between the functional interaction of PKCδ with VASP and the role of the kinase as modulator of platelet activity.
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Flaumenhaft, Robert C., Secil Koseoglu, James R. Dilks, Christian G. Peters, Nathalie A. Fadel, Reema Jasuja, and Christy L. Haynes. "Dynamin-Related Protein-1 Controls Fusion Pore Dynamics During Platelet Granule Secretion and Thrombus Formation In Vivo." Blood 118, no. 21 (November 18, 2011): 361. http://dx.doi.org/10.1182/blood.v118.21.361.361.
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Abstract Abstract 361 Platelet granule secretion serves a central role in hemostasis and thrombosis. During platelet secretion, fusion of granule membranes with those of the plasma membrane results in the release of granule contents. Recently electrochemical techniques using single-cell amperometry have shown that platelet membrane fusion results in the formation of a fusion pore. The fusion pore subsequently expands to enable the complete extrusion of granule contents. However, the molecular mechanisms that control platelet fusion pore formation and expansion are not known. To discover novel components of the platelet secretory machinery, we tested >300,000 compounds in a forward chemical genetic screen designed to identify inhibitors of dense granule secretion. A compound, ML160, was found that blocked dense granule release with an IC50 of approximately 0.5 μM. ML160 was also identified in an unrelated high throughput screen designed to detect inhibitors of dynamin-related protein-1 (Drp-1). Although best known as mediators of membrane fission, dynamins also contribute to granule exocytosis by controlling fusion pore expansion. Immunoblot analysis of platelet pellets and supernatants confirmed the presence of Drp-1 in platelets and demonstrated nearly equal distribution between platelet membranes and cytosol. mDivi-1, a well-characterized small molecule inhibitor of Drp-1 that acts outside of the GTP binding site, blocked PAR1-mediated platelet dense granule and α-granule release with an IC50 of approximately 20 μM. mDivi-1 also inhibited granule release induced by the thromboxane receptor agonist U46619, PMA, or Ca2+ ionophore, indicating that Drp-1 acts distally in the secretory pathway. To assess whether Drp-1 functions in platelet fusion pore dynamics, we tested the effect of mDivi-1 on the release of dense granules from rabbit platelets using single-cell amperometry. This technique monitors the release of serotonin from single granules in real-time with sub-millisecond temporal resolution. mDivi-1 exposure (10 μM) retarded each release event, resulting in a prolonged spike width of 23.00 ± 1.702 msec compared to the control value of 14.71 ± 1.194 msec. Although this concentration of mDivi-1 did not change the overall percentage of the fusion pore events or the amount of serotonin released through the fusion pore, it showed a distinct effect on the transition from stable fusion pore to maximal fusion pore dilation (% foot= 17.46 ± 1.809%, 9.464 ± 2.014% for control and mDivi-1 conditions, respectively). Evaluation of fluorescein-dextran incorporation into activated platelets by fluorescence microscopy enabled visualization of fusion pore dynamics and confirmed the effect of mDivi-1 on fusion pore expansion. To assess whether Drp-1 participates in platelet function in vivo, we determined the effect of mDivi-1 on thrombus formation following laser-induced injury of mouse cremaster arterioles. mDivi-1 inhibited platelet accumulation at the site of vascular injury by 74%. In contrast, mDivi-1 had no significant effect on fibrin formation under the same conditions. These results identify Drp-1 in platelets, demonstrate a role for Drp-1 in fusion pore dynamics, and indicate that pharmacological regulation of platelet fusion pore expansion can be used to control thrombus formation in vivo. Disclosures: No relevant conflicts of interest to declare.
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Li, Jian, Lin Zhang, and Yan Sun. "Molecular basis of the initial platelet adhesion in arterial thrombosis: Molecular dynamics simulations." Journal of Molecular Graphics and Modelling 37 (July 2012): 49–58. http://dx.doi.org/10.1016/j.jmgm.2012.04.002.
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Stiehm, Michael, Carolin Wüstenhagen, Stefan Siewert, Hüseyin Ince, Niels Grabow, and Klaus-Peter Schmitz. "Impact of strut dimensions and vessel caliber on thrombosis risk of bioresorbable scaffolds using hemodynamic metrics." Biomedical Engineering / Biomedizinische Technik 64, no. 3 (May 27, 2019): 251–62. http://dx.doi.org/10.1515/bmt-2017-0101.
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AbstractBioresorbable scaffolds (BRS) promise to be the treatment of choice for stenosed coronary vessels. But higher thrombosis risk found in current clinical studies limits the expectations. Three hemodynamic metrics are introduced to evaluate the thrombosis risk of coronary stents/scaffolds using transient computational fluid dynamics (CFD). The principal phenomena are platelet activation and effective diffusion (platelet shear number, PSN), convective platelet transport (platelet convection number, PCN) and platelet aggregation (platelet aggregation number, PAN) were taken into consideration. In the present study, two different stent designs (thick-strut vs. thin-strut design) positioned in small- and medium-sized vessels (reference vessel diameter, RVD=2.25 mm vs. 2.70 mm) were analyzed. In both vessel models, the thick-strut design induced higher PSN, PCN and PAN values than the thin-strut design (thick-strut vs. thin-strut: PSN=2.92/2.19 and 0.54/0.30; PCN=3.14/1.15 and 2.08/0.43; PAN: 14.76/8.19 and 20.03/10.18 for RVD=2.25 mm and 2.70 mm). PSN and PCN are increased by the reduction of the vessel size (PSN: RVD=2.25 mm vs. 2.70 mm=5.41 and 7.30; PCN: RVD=2.25 mm vs. 2.70 mm=1.51 and 2.67 for thick-strut and thin-strut designs). The results suggest that bulky stents implanted in small caliber vessels may substantially increase the thrombosis risk. Moreover, sensitivity analyses imply that PSN is mostly influenced by vessel size (lesion-related factor), whereas PCN and PAN sensitively respond to strut-thickness (device-related factor).
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Tokuue, Junya, Junichi Hayashi, Yoshiya Hata, Kazuhiko Nakahara, and Yasuo Ikeda. "Enhanced Platelet Aggregability under High Shear Stress after Treadmill Exercise in Patients with Effort Angina." Thrombosis and Haemostasis 75, no. 05 (1996): 833–37. http://dx.doi.org/10.1055/s-0038-1650375.
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SummaryPlatelet activation induced by shear forces occurring in a stenosed coronary artery is one of the mechanisms of coronary thrombosis. We evaluated the shear-induced platelet aggregation (SIPA) dynamics in patients with effort angina during treadmill exercise. SIPA was measured by a rotational cone-plate aggregometer. SIPA was markedly increased by exercise from 71.2 ± 8.9% to 81.9 ± 7.6% (p <0.01) in the patient group. Although epinephrine concentrations were elevated, its rate of increase was not correlated with that of SIPA. Yohimbine partially inhibited the exercise-induced increase in SIPA. In contrast, a significant correlation between the changing rate of plasma von Willebrand factor (vWF) larger multimers and that of SIPA (r = 0.74, p <0.05) was observed. Exercise-augmented SIPA is probably dependent on an increase in vWF larger multimers rather than platelet alpha2-receptor activation. Prevention of the interaction between vWF and its platelet receptors may play some role in decreasing the risk of coronary thrombosis during exercise.
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Becker, Richard, and Paul Gurbel. "Platelet P2Y12 receptor antagonist pharmacokinetics and pharmaco -dynamics: A foundation for distinguishing mechanisms of bleeding and anticipated risk for platelet-directed therapies." Thrombosis and Haemostasis 103, no. 03 (2010): 535–44. http://dx.doi.org/10.1160/th09-07-0491.
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SummaryThe platelet P2Y12 receptor is involved in all aspects of arterial thrombosis, including adhesion, activation, aggregation, secretion and development of a stable aggregate on which coagulation proteins can assemble and fibrin strands can mesh. Inhibition of the P2Y12 receptor has been shown convincingly to reduce cardiovascular events among patients with acute coronary syndromes (ACS) and in patients undergoing percutaneous intervention (PCI). Current studies are exploring whether there is a threshold of platelet aggregation below which only more bleeding occurs, without a concomitant reduction in clinical events. The following review considers the potential relevance of reversible and irreversible mechanisms of P2Y12 inhibition to bleeding risk, posing the question, “Is it not only how much but how a platelet P2Y12 receptor is inhibited that determines the attributable safety profile?”
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Urban, Alexander E., Erin O. Quick, Kaylie P. Miller, Jennifer Krcmery, and Hans-Georg Simon. "Pdlim7 Regulates Arf6-Dependent Actin Dynamics and Is Required for Platelet-Mediated Thrombosis in Mice." PLOS ONE 11, no. 10 (October 28, 2016): e0164042. http://dx.doi.org/10.1371/journal.pone.0164042.
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Nishimura, Satoshi, Koji Eto, and Ryozo Nagai. "Thrombus Development Processes Are Dependent On Endothelial Injuries: Examined By In Vivo Molecular Imaging." Blood 122, no. 21 (November 15, 2013): 1070. http://dx.doi.org/10.1182/blood.v122.21.1070.1070.
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Abstract The thrombotic cellular mechanisms associated with cardiovascular events remains unclear, largely because of an inability to visualize thrombus formation. In addition, the contribution of endothelial cell (EC) injuries to thrombus formation processes are unclear, and we developed in vivo imaging technique based on single- and multi-photon microscopy to revealed the multicellular processes during thrombus development (Figure a,b). We visualized the cell dynamics including single platelet behavior, and assessed dynamic cellular interplay in two thrombosis models. First, we visualized that rapidly developing thrombi composed of discoid platelets without EC disruption was triggered by ROS photochemically induced by moderate power laser irradiation (Figure c). In this model, thrombus consisted by discoid platelet aggregations without leukocyte recruitment. The second model is, thrombus with EC disruption. High power laser induced EC erosion and extravasations of circulating leukocytes with thrombus development. Inflammatory cytokine, adhesion molecules dynamically control these two processes. (Figure d)Figure.Figure. Using this technique, we elucidated that Lnk (adapter protein) regulates integrin signaling leading to stabilization of developing thrombus without EC disruption. Specifically, adhesion molecules dynamically control these processes. Thrombus formation was initiated by the binding of platelet GPIb-alpha to endothelial von Willebrand Factor in this model, and actin linker talin-dependent activation of alphaIIb-beta3 integrin in platelets was required for late phase thrombus stability. As for the thrombus formation with EC disruption, chemokine expressions in endothelium and leukocyte (especially neutrophils) recruitment played a significant role in these processes. TLR4 signaling also contributed to these steps. In sum, using our imaging system can be a powerful tool to analyze thrombus formation and evaluate the therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.
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Diamond, Scott L. "Systems Biology to Predict Platelet Function." Blood 116, no. 21 (November 19, 2010): SCI—38—SCI—38. http://dx.doi.org/10.1182/blood.v116.21.sci-38.sci-38.
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Abstract Abstract SCI-38 Systems Biology seeks to provide patient-specific prediction of dynamic cellular response to multiple stimuli, critical information toward predicting risk, disease progression, or response to therapy. We deployed two distinct approaches, bottom-up and top-down analyses, to gain insight into platelet signaling. The bottom-up approach required a definition of reaction network and kinetic equations (topology), kinetic parameters, and initial concentrations in order to simulate platelet signaling. We developed a computational platelet model – assembled from 24 peer-reviewed platelet studies to yield 132 measured kinetic rate constants – that accurately predicts resting levels of cytosolic calcium, IP3, diacylglycerol, phosphatidic acid, phosphoinositol, PIP, and PIP2. The model accurately predicts the full transient calcium dynamics in response to increasing levels of ADP. In the first full stochastic simulation of single platelet response to ADP, the model provides an extremely accurate prediction of the statistics of the asynchronous [Ca]i spikes observed in single platelets. Specifically, this is the first work to provide a quantitative molecular explanation of the asynchronous calcium spiking observed in ADP-activated human platelets. We show the asynchronous spiking is a result of the fundamentally stochastic nature of signal transduction in cells as small as human platelets. Specific testable predictions have emerged about the requirement of high SERCA/IP3R ratios in functional platelets, limits on the concentration of calcium in the DTS, and relative potencies of PAR peptides and ADP. For functional phenotyping platelets, a top-down approach linking multiple inputs to functional outputs was used to understand how human platelets integrate diverse signals encountered during thrombosis. We developed a high-throughput platform that measures the human platelet calcium mobilization in response to all pairwise combinations of six major agonists. Agonists tested in this study were: convulxin (CVX; GPVI activator), ADP, the thromboxane analog U46619, PAR1 agonist peptide (SFLLRN), PAR4 agonist peptide (AYPGKF), and PGE2 (activator of IP and EP receptor). The calcium responses to single agonists at 0.1, 1, 10′ EC50 and 135 pairwise combinations trained a neural network (NN) model to predict the entire 6-dimensional platelet response space. The NN model successfully predicted responses to sequential additions and 27 ternary combinations of [ADP], [convulxin], and [SFLLRN] (R=0.881). With 4077 NN simulations spanning the 6-dimensional agonist space, 45 combinations of 4–6 agonists (ranging from synergism to antagonism) were selected and confirmed experimentally (R=0.883), revealing a highly synergistic condition of high U46619/PGE2 ratio, consistent with the risk of COX-2 therapy. Furthermore, pairwise agonist scanning (PAS) provided a direct measurement of 135 synergy values, thus allowing a unique phenotypic scoring of 10 human donors. Patient-specific training of NNs represent a compact and robust approach for prediction of cellular integration of multiple signals in a complex disease milieu. Either bottom-up models or top-down NN models are ideal for incorporation into systems biology simulations of thrombotic pathways under flow conditions. Disclosures: No relevant conflicts of interest to declare.
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Markou, Christos, Nicolas Chronos, and Stephen Hanson. "Antithrombotic Effects of Ionic and Non-ionic Contrast Media in Nonhuman Primates." Thrombosis and Haemostasis 85, no. 03 (2001): 488–93. http://dx.doi.org/10.1055/s-0037-1615610.
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SummaryThromboembolic complications have been attributed to the use of radiographic contrast media (CM) during interventional procedures for arterial revascularization. However, due to the low frequency of adverse events, comparisons between different CM have been difficult to perform, although it has been suggested that ionic (vs. non-ionic) CM may be associated with fewer thrombotic events. The present study was undertaken using well-characterized baboon thrombosis models in order to compare different CM under physiologically relevant and controlled conditions of blood flow, exposure time, and CM concentration. Three CM were studied: ioxaglate, iohexol, and iodixanol. CM were locally infused into the proximal segment of femoral arteriovenous shunts. Palmaz-Schatz stents (4 mm i.d.) and expanded tubular segments (9 mm i.d.), which exhibited venous-type flow recirculation and stasis, were deployed into the shunts distally. Saline was infused in identical control studies. Blood flow was maintained at 100 ml/min. Thrombosis was measured over a blood exposure period of 2 hours by gamma camera imaging of 111In-platelets and by gamma counting of deposited 125I-fibrin. CM concentrations within the flowfield were predicted using computational fluid dynamics. At infusion rates of 0.1 and 0.3 ml/min, the low-osmolar ionic CM ioxaglate reduced both platelet and fibrin deposition on the stents by 75-80% (p <0.005), while both iohexol and iodixanol reduced platelet deposition by 30-50% (p <0.05). In the regions of low shear flow, ioxaglate (0.3 ml/min) also reduced platelet deposition significantly (by 52% vs. control results; p <0.05). Thus the three agents evaluated – ioxaglate, iohexol, and iodixanol – all produced anticoagulant and antiplatelet effects and were inherently antithrombotic in vivo. The most striking effects were seen with the low osmolarity, ionic contrast agent ioxaglate.
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Orfeo, T., S. Butenas, A. Undas, K. Brummel-Ziedins, and K. G. Mann. "Blood coagulation dynamics in haemostasis." Hämostaseologie 29, no. 01 (2009): 7–16. http://dx.doi.org/10.1055/s-0037-1616932.
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SummaryOur studies involve computational simulations, a reconstructed plasma/platelet proteome, whole blood in vitro and blood exuding from microvascular wounds. All studies indicate that in normal haemostasis, the binding of tissue factor (TF) with plasma factor (F) VIIa (extrinsic FXase complex) results in the initiation phase of the procoagulant response. This phase is negatively regulated by tissue factor pathway inhibitor (TFPI) in combination with antithrombin (AT) and the protein C (PC) pathway. The synergy between these inhibitors provides a threshold-limited reaction in which a stimulus of sufficient magnitude must be provided for continuation of the reaction. With sufficient stimulus, the FXa produced activates some prothrombin. This initial thrombin activates the procofactors and platelets required for presentation of the intrinsic FXase (FVIIIa- FIXa) and prothrombinase (FVa-FXa) complexes which drive the subsequent propagation phase; continuous downregulation of which is provided by AT and the thrombinthrombomodulin- PC complex. FXa generation during the propagation phase is largely (>90%) provided by the intrinsic FXase complex. TF is required for the initiation phase of the reaction but becomes non-essential once the propagation phase has been achieved. The propagation phase catalysts (FVIIIa-FIXa and FVa-FXa) continue to drive the reaction as blood is resupplied to the wound site by flow. Ultimately, the control of the reaction is governed by the pro- and anticoagulant dynamics and the supply of blood reactants to the site of a perforating injury. Our systems have been utilized to examine the qualities of hypothetical and novel antihaemorrhagic and anticoagulation agents and in epidemiologic studies of venous and arterial thrombosis and haemorrhagic pathology.
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Faes, Camille, Anton Ilich, Erica M. Sparkenbaugh, Alisa S. Wolberg, Brian C. Cooley, Kenneth I. Ataga, Nigel S. Key, and Rafal Pawlinski. "Alteration of the Structure and Dynamics of Venous Clot Formation in Human and Murine Sickle Cell Disease." Blood 128, no. 22 (December 2, 2016): 2478. http://dx.doi.org/10.1182/blood.v128.22.2478.2478.
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Abstract Sickle cell disease (SCD) is associated with chronic activation of coagulation and an increased risk of venous thromboembolism. Traditionally, it is believed that during venous thrombosis, red blood cells (RBC) are simply trapped within fibrin-rich thrombi and do not actively affect thrombosis. However, a study from our group showed that factor XIII (FXIII) activity is critical for the retention of RBC within clots and directly affects thrombus size. Others reported that during clot contraction polyhedral shaped RBC formed a densely packed core and that SCD alters the formation of polyhedrocytes which may affect clot stability (Strauss et al, 2015, ASH abstract). We further investigated if SCD affects the structure and the dynamics of clot formation. Ex vivo clot retraction was performed using blood from sickle cell patients and Townes mice (a model of SCD). Citrated blood was added to siliconized wells of 96-well plates containing CaCl2 (10mM) and tissue factor (1pM) and incubated for 2 hours at 37°C. The number of RBC extruded from the clot was counted in serum by Hemavet™ and expressed as a percentage of initial RBC number in the anticoagulated blood. Morphology of the clots was evaluated using scanning and transmission electron microscopy (EM). Thrombosis in Townes SS (sickle) and AA (control) mice was studied using femoral vein thrombosis induced by electrolytic injury and inferior vena cava stenosis models. The number of mouse SS RBC in the serum extruded during clot contraction was dramatically reduced compared to the number of AA RBC (0.8±0.8% vs. 19.4±0.8%, n=3, p<0.0001, Fig. A). A similar result was observed for human RBC. Since SS mice and sickle patients have a lower hematocrit (HCT), we investigated if the number of RBC affects the extrusion of these cells during clot retraction. Indeed, lowering HCT in AA mouse blood reduced RBC extrusion from the clots. However, increasing HCT in SS mouse blood to that of AA blood did not increase the number of SS RBC extruded from the clot. Furthermore, inhibition of FXIIIa activity with T101 (10µM) increased the release of AA (by 64%, n=6, p<0.05) but not SS RBC from mouse clots. These data indicate that the entrapment of SS RBCs within the clot is not simply caused by lower RBC number, and is FXIII-independent. Mixing the platelet poor plasma (PPP) and cellular fraction of AA and SS mouse blood revealed that the entrapment of SS RBC is not mediated by PPP (AA blood recombined = 6.9±3.6%; SS blood recombined = 0.4±0.4%; AA cells/SS PPP = 6.7±6.4%; SS cells/AA PPP = 0.1±0.2%, n=3 per group). Clots formed ex vivo from AA blood had a gel-like, soft structure, whereas SS clots were more firm and stiff. EM demonstrated that RBC within AA clots had polyhedral shapes and were tightly packed in the central part of the clot. In contrast, most of the mouse SS RBC did not have polyhedral shapes, underwent sickling and were not compacted within the clot. They also formed long "spicule-like" processes that intertwined with fibrin fibers (Fig. C). Similar results were observed in blood of sickle patients; however, the sickled RBC phenotype was less prominent. Importantly, sickling of RBC was observed in clots formed in the inferior vena cava of SS mice, 2 hours after vessel stenosis (Fig. D). We also performed a tPA challenge assay on clots formed ex vivo from human blood and showed that SS clots challenged with low tPA concentration (0.6nM) were more resistant to fibrinolysis compared to AA clots (clot lysis time, 714±6 vs. 388.3±120.7 minutes, n=6, p=0.08). The electrolytic injury model of venous thrombosis was used to investigate the dynamics of clot formation in SS mice in vivo. Mice were infused with fluorescently labeled antibodies for fibrin (green) and platelets (red). Electrolytic injury was applied to the femoral vein; a relative intensity of fibrin and platelet accumulation was assessed by fluorescence microscopy for one hour at 10-minute intervals. SS mice have increased platelet and fibrin accumulation compared to AA mice (~ 2 fold, n=5-7, p<0.05 for 40, 50 and 60 minute time points). Interestingly, in the AA clots, platelets were mostly localized on the surface, in contrast to their widespread distribution throughout the clot in SS mice (Fig. E, yellow color). Our data demonstrated that SCD alters the structure and dynamics of venous clot formation. Experiments investigating the consequence of these observations in mouse models of stroke and pulmonary embolism are currently ongoing. Figure Figure. Disclosures No relevant conflicts of interest to declare.
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Ting, Lucas, Shirin Feghhi, Ari Karchin, Wes Tooley, Nathan J. White, and Nathan Sniadecki. "Clot-On-A-Chip: A Microfluidic Device To Study Platelet Aggregation and Contractility Under Shear." Blood 122, no. 21 (November 15, 2013): 2363. http://dx.doi.org/10.1182/blood.v122.21.2363.2363.
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Abstract Introduction In primary hemostasis, platelets adhere, activate, and aggregate at the wall of an injured vessel to form a hemostatic plug for the cessation of bleeding. After activation, platelets generate myosin-driven contractile forces to compact the size of the plug in order to reduce the space between platelets and prevent their disaggregation. Hemodynamic shear can be a major effector of platelet function in hemostasis, but its effect on the ability of platelets to produce contractile forces is an open question. Studying the dynamics of platelet aggregation and platelet force generation under hemodynamic shear can provide important insights into hemostasis and thrombosis. Method We have developed a microfluidic device that uses microscale blocks to induce platelet aggregation and microscale posts to measure platelet forces in a hemostatic plug. Whole human blood in heparin or citrate is pumped through a microfabricated chip containing microchannels with arrays of blocks and posts arranged along the bottom of a microchannel (Fig. 1). The surface of the blocks and posts are pre-coated with von Willebrand factor and type I collagen to allow for platelet adhesion. As blood is passes over a block, its rectangular shape induces a high shear rate that causes platelets to aggregate on its surface. A flexible micropost is situated behind each block. As platelets aggregate between the block and post, their contractile forces causes the post to bend toward the block. The deflection of the post is recorded under fluorescence microscopy and analyzed using quantitative image analysis of the videos. Since a microscale post bends like a cantilever beam, its deflection can be used to quantify the forces of platelets. Results Blebbistatin, a myosin inhibitor, was used to confirm that deflection of the posts by the platelets in heparinized blood was due to myosin activity. When blood was incubated with 2-MeSAMP, a P2Y12 antagonist, platelets were able to aggregate, but their ability to generate contractile forces was substantially reduced. This finding indicates that ADP activation is needed for platelet contractility under shear. The rate of hemodynamic shear was found to influence platelet function, for the rate of platelet aggregation and force generation were found to increase for blood sheared from 2000 to 12,000 s-1. Moreover, platelet aggregation and contractile forces were reduced when glycoprotein Ib-V-IX complex and integrin αIIbβ3 were inhibited with antibody AK2 and antibody fragment c7E3 Fab, respectively. When citrated blood was incubated with tissue plasminogen activator, platelets aggregate and produced contractile forces that increased steadily within the first ten minutes, but then the forces began to subside. Conclusions Our device can be used to study the role of hemodynamic shear in platelet function and gives insights into the role of platelet forces during hemostasis. Its microscale dimensions also allow us the study the biomechanics involved in the formation of a hemostatic plug during its early stages of growth and stability. Disclosures: White: Vidacare Corp: Honoraria; Stasys Medical Corp: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; NIH: Research Funding; Coulter Foundation: Research Funding; Washington State Life Sciences Discovery Fund: Research Funding. Sniadecki:Stasys Medical Corporation: Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees.
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Sikora, Joanna, Aleksandra Karczmarska-Wódzka, Joanna Bugieda, and Przemysław Sobczak. "The Use of Total Thrombus Formation Analysis System as a Tool to Assess Platelet Function in Bleeding and Thrombosis Risk—A Systematic Review." International Journal of Molecular Sciences 22, no. 16 (August 10, 2021): 8605. http://dx.doi.org/10.3390/ijms22168605.
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Background. Today there are many devices that can be used to study blood clotting disorders by identifying abnormalities in blood platelets. The Total Thrombus Formation Analysis System is an automated microchip flow chamber system that is used for the quantitative analysis of clot formation under blood flow conditions. For several years, researchers have been using a tool to analyse various clinical situations of patients to identify the properties and biochemical processes occurring within platelets and their microenvironment. Methods. An investigation of recent published literature was conducted based on PRISMA. This review includes 52 science papers directly related to the use of the Total Clot Formation Analysis System in relation to bleeding, surgery, platelet function assessment, anticoagulation monitoring, von Willebrand factor and others. Conclusion. Most available studies indicate that The Total Thrombus Formation Analysis System may be useful in diagnostic issues, with devices used to monitor therapy or as a significant tool for predicting bleeding events. However, T-TAS not that has the potential for diagnostic indications, but allows the direct observation of the flow and the interactions between blood cells, including the intensity and dynamics of clot formation. The device is expected to be of significant value for basic research to observe the interactions and changes within platelets and their microenvironment.
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Souvenir, Rhonda, Melissa J. Jensen, Trevor P. L. Fidler, Joshua A. Volker, Sanjana Dayal, Andrew S. Weyrich, and E. Dale Abel. "The Effects of Optic Atrophy Protein (OPA)-1 Deletion on Platelet Function Is Regulated By the Hormonal Milieu." Blood 128, no. 22 (December 2, 2016): 410. http://dx.doi.org/10.1182/blood.v128.22.410.410.
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Abstract Cardiovascular diseases (CVD) are the leading cause of death in the United States. Thrombosis represents one of the most common underlying pathologies in CVD and disproportionately plagues females. Analyses of transcripts from the Framingham offspring cohort, revealed enrichment in transcripts encoding for the mitochondrial dynamics protein: optic atrophy-1 in females that positively correlated with coronary heart disease and diabetes. Optic atrophy (OPA)-1 regulates mitochondrial fusion, electron transport chain (ETC), complex assembly and apoptosis. The correlation suggests that altered mitochondrial dynamics may contribute to platelet dysfunction. Thus, to determine whether the enrichment in OPA1 transcripts in females that correlates with increased CVD risk represents a physiological adaptation or a pathophysiological response, mice with platelet-specific deletion of OPA1 (pOPA-1 KO) were generated. Male pOPA-1 KO mice exhibited compromised cristae morphology and a 50% reduction in mitochondrial DNA. Additionally, mitochondrial function assessed by an extracellular flux analyzer (XF24 Seahorse biosciences) was reduced by 50%. Mitochondrial dysfunction in male pOPA-1 KO mice was associated with heightened agonist-mediated platelet activation (a hyperactive phenotype). Male pOPA-1 KO mice had a shortened time to stable occlusion of the carotid artery as assessed in vivo by (rose Bengal) photochemical injury (~25 min knockout vs ~ 35 min control), and were more prone to develop a thrombus (14/15 knockouts vs. 4/8 controls) following permanent ligation of the inferior vena cava. In contrast, cristae morphology was normal and mitochondrial DNA unchanged in females. Females revealed no evidence of heightened agonist response or increased incidence of thrombus formation. In contrast, female pOPA-1 KO mice had increased time to stable occlusion of the carotid artery as assessed by photochemical injury (~75 min knockout vs ~35 min control). Additionally, when platelets from pOPA-1 KO or control males were transferred into female mice harboring the human interleukin 4 receptor (HIL4R) transgene that were depleted of their native platelets by HIL4R antibodies, the reconstituted male platelets acquired the phenotype of female pOPA-1 KO mice. Thus, the time to stable occlusion of the carotid artery following photochemical injury was increased. In parallel, gonadectomized pOPA-1 KO male mice were protected from the prothrombotic phenotype. Additionally, gonadectomized female pOPA-1 KO no longer exhibited increased time to stable carotid artery occlusion. These findings suggest a direct interaction between platelet genotype and the systemic milieu, which determines platelet function in pOPA-1 KO mice and the modulation by gonadectomy suggests a role for sex steroids. Thus, whereas preserving OPA-1 expression might be a protective adaptation in males, OPA-1 induction in platelets of females that correlates with CVD in humans, might promote platelet hyper-activation. Disclosures No relevant conflicts of interest to declare.
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Bur, Andres M., Karen P. Fong, Keith B. Neeves, Sean F. Maloney, Scott L. Diamond, Mark L. Kahn, and Lawrence F. Brass. "Microfluidic Flow Studies of Murine Thrombus Formation and Stability on Collagen." Blood 112, no. 11 (November 16, 2008): 1835. http://dx.doi.org/10.1182/blood.v112.11.1835.1835.
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Abstract Glycoprotein (GP) VI and integrin α2β1 are platelet receptors for collagen which, along with GPIb and von Willebrand factor, mediate platelet adhesion to sites of vascular injury. In pathologic states, these receptors may cause inappropriate platelet activation resulting in thrombosis. The primary objective of this study was to examine in real time, the relative contribution of α2β1 and GPVI, in platelet aggregation and thrombus stability on collagen under flow. A novel microfluidic flow chamber device was used which allowed the effects of a single agonist to be studied under varying shear rates using just 50μL of whole blood. In addition, real-time imaging of the flow experiment allowed the dynamics of thrombus formation to be observed in response to a platelet agonist, thus simulating vascular injury. In the microfluidic flow experiments, we used whole blood anti-coagulated with the thrombin inhibitor PPACK to study the accumulation of mouse platelets in response to fibrillar and acid-soluble collagen patterned in a 100μm square on a glass slide. By tagging platelets with fluorescent anti-CD41 antibodies, it was possible to quantify the degree to which platelets adhere and aggregate by measuring the fluorescence intensity, corresponding to thrombus size, with time. After allowing a thrombus to form for 300 seconds at a shear of 400s−1, the shear in the chamber was increased to 8000s−1 to assess the stability of the platelet aggregate under high shear. This served as a method for evaluating the strength of platelet-platelet and platelet-collagen interactions. The three types of knockout mice used in the evaluation of the roles of these receptors in platelet-collagen interaction were α2−/− GPVI−/− and α2−/−/FcRγ−/−. The α2 knockout eliminates the α2β1 receptor while the FcRγ knockout deletes FcRγ and prevents its partner GPVI from being expressed on the cell surface. The data from these knockout mice were compared to measurements from wild type mice and major differences in thrombus formation and stability were identified. Platelets from α2−/−/FcRγ−/− double knockout mice showed a markedly diminished ability to adhere to both acid-soluble and fibrillar types of collagen as compared to wild type mice. The small aggregates that formed washed away rapidly in response to an increase in shear. Platelets from α2−/− mice exhibited significantly less thrombus formation than wild type, but adhered and aggregated to a greater extent than platelets from α2−/−/FcRγ−/− double knockout mice. There was no significant difference in aggregation of α2−/− platelets on both types of collagen. Additionally, α2−/− thrombi were slightly more stable than α2−/−/FcRγ−/− thrombi, as evidenced by the small aggregate that remained following an increase in shear. GPVI−/− platelets also produced smaller aggregates than wild type, but they were larger than those produced by either α2−/−/FcRγ−/− or α2−/− platelets. Although GPVI−/− thrombi were less stable than wild type, significant thrombus remained following an increase in shear suggesting that α2β1, not GPVI, may be the most important collagen receptor for maintaining thrombus strength. While these findings differ from those commonly supported by the literature, the small diameter of the microfluidic flow chamber may better approximate the flow conditions in arterioles than traditional flow chambers. In conclusion, these data show a significant decrease in thrombus formation and stability for α2−/− and α2−/−/FcRγ−/− platelets and a moderate decrease in aggregation of GPVI−/− platelets with minimal decrease in thrombus stability as compared to wild type. These results suggest that α2β1 plays a critical role in adhesion and aggregation of platelets to Type I collagen under flow.
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Zhu, Jieqing, Jianghai Zhu, Ana Negri, Davide Provasi, Marta Filizola, Barry S. Coller, and Timothy A. Springer. "Closed headpiece of integrin αIIbβ3 and its complex with an αIIbβ3-specific antagonist that does not induce opening." Blood 116, no. 23 (December 2, 2010): 5050–59. http://dx.doi.org/10.1182/blood-2010-04-281154.
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Abstract The platelet integrin αIIbβ3 is essential for hemostasis and thrombosis through its binding of adhesive plasma proteins. We have determined crystal structures of the αIIbβ3 headpiece in the absence of ligand and after soaking in RUC-1, a novel small molecule antagonist. In the absence of ligand, the αIIbβ3 headpiece is in a closed conformation, distinct from the open conformation visualized in presence of Arg-Gly-Asp (RGD) antagonists. In contrast to RGD antagonists, RUC-1 binds only to the αIIb subunit. Molecular dynamics revealed nearly identical binding. Two species-specific residues, αIIb Y190 and αIIb D232, in the RUC-1 binding site were confirmed as important by mutagenesis. In sharp contrast to RGD-based antagonists, RUC-1 did not induce αIIbβ3 to adopt an open conformation, as determined by gel filtration and dynamic light scattering. These studies provide insights into the factors that regulate integrin headpiece opening, and demonstrate the molecular basis for a novel mechanism of integrin antagonism.
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Nandurkar, Harshal, Warwick Nesbitt, Rose Brazilek, Francisco Tovar-Lopez, Angus Wong, Huyen Tran, Amanda Davis, and Arnan Mitchell. "A Shear Micro-Gradient Microfluidic to Monitor Platelet Aggregation Dynamics in the Context of Von Willebrand Disease." Blood 128, no. 22 (December 2, 2016): 3753. http://dx.doi.org/10.1182/blood.v128.22.3753.3753.
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Abstract Background: Virchow's triad identifies the three principle parameters driving haemostasis and thrombosis as: i. Changes in vessel wall properties and exposure of subendothelial matrix proteins; ii. Presentation of blood borne chemical activators (hypercoaguability); and iii.Blood-flow dependent mechanical factors (haemodynamics). Studies identifying a key role for micro-scale shear gradients in driving the earliest stages of platelet thrombus formation have informed the development of a novel set of microfluidic devices that have potential utility as rapid and efficient screening tools of shear dependent platelet function. Aim: The aim of this project was to undertake a small scale clinical and laboratory based characterisation study of a microfluidic platform designed by us and to assess its ability to identify differences in platelet aggregation dynamics in citrated whole blood taken from control subjects and subjects with clinically diagnosed or undiagnosed von Willebrand disease (Types 1, 2 and 3). Method: Patients with VWD were recruited from the haemophilia outpatient clinic, Alfred hospital. Whole blood samples (250mL) or samples treated ex vivo to block the canonical platelet amplification loop pathways were perfused at a defined flow rate (45ml/mL) through a set of well defined micro-shear gradient geometries pre-coated with purified VWF to initiate platelet capture. Microfluidic geometries were characterised by an entry shear rate of 1,800.s-1, that was accelerated to a peak shear rate of either 45,000.s-1 or 150,000.s-1, returning to an exit shear rate of 1,800.s-1. The rate of initial shear acceleration was varied using a series of geometries with contraction entry angles varying from 15 - 85o. Results: The microfluidic platform was able to identify patients with Types 1 (VWF antigen < 30%), 2A and 3 VWD. ROC analysis of control versus VWD samples determined that the device sensitivity approached 94.4%, with a specificity of 100% for VWD. A statistically significant difference (p< 0.05) was observed when comparing control blood samples to type 1VWD (p< 0.001) and type 2A VWD samples (p=0.004), with both subtypes showing minimal to no platelet aggregation in the device. Patients presenting with bleeding symptoms but found not to have VWD (normal VWF:Ag levels) showed no significant difference (p=0.907) to controls. Furthermore, exogenous titration of Type 3 (n=2) VWD blood samples with purified VWF (10 - 100mg/mL) recapitulated platelet aggregation in a concentration dependent manner. Head to head comparison with standard laboratory based tests including, VWF:Ag, VWF:CB and VWF:RCo demonstrated a strong linear correlation with device output. In addition, time-course (0, 2 and 4 Hrs) trials demonstrate that the device is a sensitive measure of DDAVP treatment of VWD. Conclusion: The Studies presented demonstrate that haemodynamically sensitive platelet aggregation within our prototype platform is critically dependent on blood VWF antigen levels and demonstrates good proof-of-concept that the microfludic can selectively identify VWD dependent defects in whole blood platelet aggregation. Taken together these data suggest that a microfluidic platform with discrete haemodynamic control can operate as a sensitive screen for VWD. Future studies will focus on defining the particle and key haemodynamic parameters that shift both device selectivity and sensitivity. Disclosures No relevant conflicts of interest to declare.
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Chen, Xinsheng, Yanfeng Wang, Edward K. Williamson, Timothy J. Stalker, Lawrence F. Brass, Morris J. Birnbaum, John H. Harwig, and Charles S. Abrams. "Loss of PIP5KIβ Causes a Defect in Lamellipodia Formation and Shear Resistant Adhesion." Blood 108, no. 11 (November 16, 2006): 141. http://dx.doi.org/10.1182/blood.v108.11.141.141.
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Abstract Phosphatidylinositol 4,5-bisphosphate (PIP2) is widely known for the production of lipid second messengers after its hydrolysis by phospholipase C or phosphorylation by phosphatidylinositol 3-kinase. PIP2 also regulates cytoskeletal dynamics by directly interacting with actin-binding proteins. Three isoforms of PIP5KI (α, β, and γ) are all capable of phosphorylating PI4P to synthesize PIP2. However, these isoforms have different primary structures, expression levels in various tissues, and intracellular localization. Our previous studies have demonstrated that PIP5KIβ and PIP5KIγ are the dominant isoforms present in platelets. We generated and bred mice heterozygous for a null mutation into the murine PIP5KIβ gene, and crossed these mice to determine the phenotype of mice lacking this protein. PIP5KIβ-null mice were born, appeared developmentally normal, had normal platelet counts, and exhibited no spontaneous hemorrhage. Compared to platelets derived from wild type littermates, platelets lacking PIP5KIβ had PIP2 concentrations that were 61% of normal under basal conditions (p<0.01), and 51% of normal 45 seconds following thrombin stimulation (p<0.01). Similarly, maximum IP3 levels were only 65% of normal in the knockout platelets (p<0.01). Consistent with this second messenger defect, PIP5KIβ −/− platelets had impaired aggregation in response to submaximal doses of thrombin, ADP, collagen, and a thromboxane analogue (U46619). PIP5KIβ-null platelets exhibited disaggregation suggesting that sustained second messenger formation is critical for a sustained aggregation response. Since PIP2 can directly associate with, and thereby regulate actin-binding proteins, we analyzed platelet spreading upon fibrinogen. PIP5KIβ knockout platelets start to spread, but ultimately spread less well than platelets derived from wild type littermates. Imaging this process with real time differential interference contrast microscopy, we found that PIP5KIβ-null platelets extend filopodia as efficiently as wild type platelets, but have difficulty anchoring down these extended membranes. When a filopod on a PIP5KIβ −/− platelet does ultimately adhere to the matrix, a normal lamellipod is rapidly formed. The cytoskeletal organization of PIP5KIβ knockout platelets spread upon fibrinogen was further studied in the electron microscope. This higher resolution analysis verified the profound defect in lamellipodia formation. We speculated that this process of lamellipodia formation is critical for adhesion under the shear conditions found within the arterial system. To test this hypothesis, we analyzed the ability of PIP5KIβ knockout platelets to adhere to collagen in a flow chamber. At all shear conditions between 200 and 1100/s, platelets lacking PIP5KIβ consistently adhered less than wild type platelets. To further analyze the necessity of PIP5KIβ in adhesion of platelets under conditions of arterial shear, we compared PIP5KIβ −/− and PIP5KIβ +/+ mice in a ferric chloride carotid injury model. Under conditions that induced thrombosis in 75% of wild type mice (n=4), we only detected thrombi in 20% of PIP5KIβ-null mice (n=5). Together, these data demonstrate that PIP5KIβ is required for sustained PIP2 and second messenger synthesis, the formation of actin-rich lamellipodia, and stable ex vivo and in vivo platelet adhesion under shear.
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Gorar, Suheyla, Bulent Alioglu, Esranur Ademoglu, Seyit Uyar, Handan Bekdemir, Zehra Candan, Beylan Saglam, Gonul Koc, Cavit Culha, and Yalcin Aral. "Is There a Tendency for Thrombosis in Gestational Diabetes Mellitus?" Journal of Laboratory Physicians 8, no. 02 (July 2016): 101–5. http://dx.doi.org/10.4103/0974-2727.180790.
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ABSTRACT Context: Impact of gestational diabetes mellitus (GDM) on the coagulation system, dynamics involved at a pathophysiological level and the exact mechanism remain unclear. Aims: To evaluate the association between diabetes-related parameters and hemostatic factors to search for a tendency of thrombosis in GDM. Settings and Design: Nineteen pregnant women who had GDM, 16 healthy pregnant and 13 healthy nonpregnant controls admitted to the Endocrinology outpatient clinics were enrolled in the study. Subjects and Methods: Fasting and postprandial glucose, hemoglobin A1c and insulin levels, and insulin resistance; fructosamine, thrombin activatable fibrinolysis inhibitor (TAFI), tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor Type-1 (PAI-1), tissue-type plasminogen activator (t-PA), fibrinogen, plasminogen and hemoglobin levels, platelet counts, prothrombin time (PT), and activated partial thromboplastin time (aPTT) were studied. Statistical Analysis Used: One-way analysis of variance, Kruskal–Wallis, and post hoc Tukey honestly significant difference or Conover’s nonparametric multiple comparison tests for comparison of the study groups. Results: PT and aPTT were significantly lower in GDM patients compared to controls (P < 0.05), whereas fibrinogen and plasminogen levels were significantly higher in this group compared to both nonpregnant and healthy pregnant controls (P < 0.05 for each). TAFI, TFPI, PAI-1, and tissue t-PA levels were not significantly different among groups. Conclusions: Our findings indicate tendency to develop thrombosis in GDM similar to diabetes mellitus; but more comprehensive studies with larger sample size are needed to determine the relationship between GDM and hemostasis.
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Mousa, Shaker A., Ahmad Aljada, Shyam Patil, and Shymaa S. Mousa. "Inhibition of Cancer and Drug-Associated Thrombosis Using Novel Inhibitors of NF-κB and Oxidative Stress Pathways." Blood 110, no. 11 (November 16, 2007): 3937. http://dx.doi.org/10.1182/blood.v110.11.3937.3937.
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Abstract Background: Venous thromboemblism is associated with a systemic hypercoagulable state that is secondary to tumor or proinflammatory activation of procoagulant mechanisms, as well as down-regulation of anticoagulant mechanisms. In the present investigation, the potential antithrombotic efficacy and mechanism for OT-304 analogs, novel small molecules targeting NFkB and oxidative stress pathways, was examined in various cellular systems. Methods: Human monocytic cells were incubated with OT-304 analogs for 3 hours and then stimulated with LPS (25 ng/ml) for another 3 hours. Real-time PCR for NF-κB (P50/P65 complex) nuclear translocation dynamics, TNF-alpha, EGR1, and tissue factor (TF) was then performed. Additionally, the effect of OT-304 analogs on oxidative stress, inflammation, and procoagulant pathways in human monocytes and endothelial cells were performed using Affymatrix Microarray. Furthermore, the anticoagulant efficacy of OT-304 analogs as compared to classical anticoagulants such as Low Molecular weight Heparins (LMWH) was determined in proinflammatory-mediated hypercoagulable state, cancer, and/or chemotherapy-associated platelet/fibrin clot formation assays using thrombelastography (TEG). TEG was performed using blood from healthy volunteers. Clot dynamics were initiated using different activators of coagulation that included different cancer types, different chemotherapeutic agents, and standard anti-angiogenesis agents such as bevacizumab, ranibizumab and others in human blood. Additionally, effects of OT-304 analogs as compared to unfractionated heparin and the LMWH enoxaparin on activated partial thromboplastin time (aPTT) in human plasma was carried out using a fibrometric method. Results: OT-304 analogs effectively inhibited NF-κB and oxidative stress pathways leading to down-regulation of cytokines and chemokines, including TNF-alpha. OT-304 analogs effectively inhibited EGR1, which was associated with down-regulation of the key procoagulant mediator tissue factor (TF). In human blood, OT-304 effectively inhibited hypercoagulation and prothrombotic states mediated by proinflammatory stimuli including LPS, NF-κB activators such as ceramide, different cancer types such as pancreatic, glioma and lung cancer, different chemotherapeutic agents such as doxorubicin and etopocide, and angiogenesis inhibitors such as bevacizumab (Avastin) or ranibizumab (Lucentis) as evident from the inhibition of platelet-fibrin clot dynamics. The IC50 required for blocking cancer or drug-associated thrombosis ranged from 1–3 uM. OT-304 analogs did not have any effect on aPTT up to concentrations ranging from 1– 100 uM in human plasma. In contrast, heparin or LMWH resulted in a dose-dependent (0.001 – 1 ug/ml) increase in aPTT. These data suggest OT-304 effectively inhibits prothrombotic events, regardless of the stimulus, with potentially no effect on hemostasis. Conclusions: OT304 analogs, by virtue of their ability to target and modulate more than one pathway, may represent a promising therapeutic strategy for inhibiting thrombosis associated with inflammation, cancer, and chemotherapy plus other adjunct therapies without compromising hemostasis.
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Lu, Ye, and Shu Sheng Zhang. "Analysis on Fluid Characteristics in Y-Shape Bifurcate Coronary Artery." Materials Science Forum 857 (May 2016): 452–58. http://dx.doi.org/10.4028/www.scientific.net/msf.857.452.
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This article analyzes the bifucate Y-type coronary artery based on hemodynamics and computational fluid dynamics to study the optimal location for thrombosis. Mathematical derivation is used to analyze the fluid machanics. The issue of fully developed flow is derivated. The optimal location for thrombus is identified. According to the mathematical derivation and numerical modeling results, the optimal location for thrombus is at the bifurcation of coronary artery. Vortex and turbulent fluctuation will take place. Three bifurcate angle (60, 90 and 120) is chosen to analyze by numarical stimulation. The result shows that vortex at bifurcation area grows when the bifrucate angle increases. It may cause the sediment of blood platelet, fibrous protein and lipochondrion [1]. It is also concluded that the wall shear stress is so high that it may injure the vascular wall around bifurcation.
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Havlovska, Ya Yu. "HEMOSTASIOLOGICAL PARAMETERS AND SEVERITY OF STROKE AMONG PATIENTS WITH ATHEROTROMBOTIC AND CARDIEMOBOLIC SUBTYPES." Актуальні проблеми сучасної медицини: Вісник Української медичної стоматологічної академії 21, no. 2 (June 17, 2021): 34–38. http://dx.doi.org/10.31718/2077-1096.21.2.34.
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The aim of this study is to investigate the differences in hemostasiological parameters among patients with atherotrombotic and cardiemobolic subtypes of ischemic stroke and the relationship between the parameters and the severity of the disease in the first day. The study included 68 patients who were examined on the first day of the disease with a diagnosis of acute cerebrovascular disorders on ischemic type, among them 47 (69%) men and 21 (31%) women aged from 42 to 75 years (the average age was 61,85 ± 2,33 years old). We quantified the stroke severity by using the National Institutes of Health Stroke Scale, findings of magnetic resonance tomography and / or computer tomography of the brain; ultrasound scan of intra- and extracranial vessels of the brain was performed to verify the diagnosis. Patients were divided into 2 groups: Group 1 included atherotrombotic subtype of ischemic stroke (n = 51 individuals), group 2 included cardiembolic subtype of ischemic stroke (n = 17 individuals. The state of the hemostasis system was studied by the analysis of complete coagulograms. The patients with ischemic stroke were found to have a thrombin time reduction compared to the control group. The dynamics of this indicator in the coagulogram points out an increased risk of thrombosis in the patients of both groups with a significant predominance among the patients with an atherotrombotic stroke. In both groups of the patients with ischemic stroke, there was a decrease in intensity, time and rate of aggregation in 30 seconds compared to the control group, indicating the imbalance of platelet response to adenosine diphosphate-induced aggregation. When the rate and intensity of aggregation (the lowering of platelet aggregation function) for 30 seconds decreased, the aggregation time (the activation of platelet function) also reduced. The analysis of coagulogram indicators points out the possibility of developing the syndrome of disseminated intravascular coagulation among patients with ischemic stroke. In this case, the decrease in the platelet aggregation properties indicates the development of thrombocytopathy under a preserved platelet number among the patients with ischemic stroke. The degree of the severity of atherotrombotic ischemic stroke is associated with indicators of coagulation hemostasis and platelet aggregation characteristics. The severity of cardioembolic ischemic stroke is associated with processes of platelet aggregation processes.
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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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Skewis, Lynell R., Tatiana Lebedeva, Vyacheslav Papkov, Edward C. Thayer, Walter Massefski, Adam Cuker, Chandrasekaran Nagaswami, et al. "T2 Magnetic Resonance: A Diagnostic Platform for Studying Integrated Hemostasis in Whole Blood—Proof of Concept." Clinical Chemistry 60, no. 9 (September 1, 2014): 1174–82. http://dx.doi.org/10.1373/clinchem.2014.223735.
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Abstract BACKGROUND Existing approaches for measuring hemostasis parameters require multiple platforms, can take hours to provide results, and generally require 1–25 mL of sample. We developed a diagnostic platform that allows comprehensive assessment of hemostatic parameters on a single instrument and provides results within 15 min using 0.04 mL of blood with minimal sample handling. METHODS T2 magnetic resonance (T2MR) was used to directly measure integrated reactions in whole blood samples by resolving multiple water relaxation times from distinct sample microenvironments. Clotting, clot contraction, and fibrinolysis stimulated by thrombin or tissue plasminogen activator, respectively, were measured. T2MR signals of clotting samples were compared with images produced by scanning electron microscopy and with standard reference methods for the following parameters: hematocrit, prothrombin time, clot strength, and platelet activity. RESULTS Application of T2MR methodology revealed conditions under which a unique T2MR signature appeared that corresponded with the formation of polyhedral erythrocytes, the dynamics and morphology of which are dependent on thrombin, fibrinogen, hematocrit, and platelet levels. We also showed that the T2MR platform can be used for precise and accurate measurements of hematocrit (%CV, 4.8%, R2 = 0.95), clotting time (%CV, 3.5%, R2 = 0.94), clot strength (R2 = 0.95), and platelet function (93% agreement with light transmission aggregometry). CONCLUSIONS This proof-of-concept study demonstrates that T2MR has the potential to provide rapid and sensitive identification of patients at risk for thrombosis or bleeding and to identify new biomarkers and therapeutic targets with a single, simple-to-employ analytic approach that may be suitable for routine use in both research and diverse clinical settings.
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Schmidtke, David W., and Scott L. Diamond. "Direct Observation of Membrane Tethers Formed during Neutrophil Attachment to Platelets or P-Selectin under Physiological Flow." Journal of Cell Biology 149, no. 3 (May 1, 2000): 719–30. http://dx.doi.org/10.1083/jcb.149.3.719.
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Adhesion and subsequent aggregation between neutrophils and platelets is dependent upon the initial binding of P-selectin on activated platelets to P-selectin glycoprotein ligand 1 (PSGL-1) on the microvilli of neutrophils. High speed, high resolution videomicroscopy of flowing neutrophils interacting with spread platelets demonstrated that thin membrane tethers were pulled from neutrophils in 32 ± 4% of the interactions. After capture by spread platelets, neutrophil membrane tethers (length of 5.9 ± 4.1 μm, n = 63) were pulled at an average rate of 6–40 μm/s as the wall shear rate was increased from 100–250 s−1. The average tether lifetime decreased significantly (P < 0.001) from 630 to 133 ms as the shear rate was increased from 100 s−1 (Fbond = 86 pN) to 250 s−1 (Fbond = 172 pN), which is consistent with P-selectin/PSGL-1 bond dynamics under stress. Tether formation was blocked by antibodies against P-selectin or PSGL-1, but not by anti-CD18 antibodies. During neutrophil rolling on P-selectin at 150 s−1, thin membrane tethers were also pulled from the neutrophils. The characteristic jerking motion of the neutrophil coexisted with tether growth (8.9 ± 8.8 μm long), whereas tether breakage (average lifetime of 3.79 ± 3.32 s) caused an acute jump in the rolling velocity, proving multiple bonding in the cell surface and the tether surface contact area. Extremely long membrane tethers (>40 μm) were sometimes pulled, which detached in a flow-dependent mechanism of microparticle formation. Membrane tethers were also formed when neutrophils were perfused over platelet monolayers. These results are the first visualization of the often hypothesized tethers that shield the P-selectin/PSGL-1 bond from force loading to regulate neutrophil rolling during inflammation and thrombosis.
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Stevens, Hannah, and James D. McFadyen. "Platelets as Central Actors in Thrombosis—Reprising an Old Role and Defining a New Character." Seminars in Thrombosis and Hemostasis 45, no. 08 (October 17, 2019): 802–9. http://dx.doi.org/10.1055/s-0039-1698829.
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AbstractPlatelets have long been considered simple anucleate cells that rapidly adhere and aggregate at sites of vascular injury. However, recent in vivo experimental data have shed new light on the platelet response to vascular injury. These data have unexpectedly revealed that platelet thrombus formation is a highly dynamic process and yields a platelet thrombus with a distinct hierarchical structure composed of a “core” of highly activated platelets and a “shell” of platelets in a low activation state. This has given rise to the concept that therapeutic targeting of the propagating thrombus shell may hold promise as a means to target thrombosis while sparing hemostasis. While platelets have been historically considered central to arterial thrombosis, they have been traditionally viewed as minor contributors to the formation of venous thrombosis. However, this concept has recently been challenged with the emergence of a large body of evidence highlighting the important proinflammatory function of platelets. The proinflammatory function of platelets is afforded by their ability to induce neutrophil extracellular trap formation, enhance leucocyte recruitment, and secrete granular contents such as high mobility group protein B1 and polyphosphate. These proinflammatory processes trigger coagulation, via the intrinsic pathway, and are central to the formation of venous thrombosis, a condition now appreciated to be a form of sterile inflammation. These data now place platelets at the center stage in orchestrating the thromboinflammatory response underpinning venous thrombosis and have provided new hope that novel platelet-targeted therapeutics may represent a safe and effective approach to prevent venous thrombosis.
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Silverstein, Roy L. "Activation of Vascular Cells by Microparticles and Other Danger Signals Via the CD36 Scavenger Receptor." Blood 118, no. 21 (November 18, 2011): SCI—12—SCI—12. http://dx.doi.org/10.1182/blood.v118.21.sci-12.sci-12.
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Abstract SCI-12 Clinically significant thrombotic events are common complications of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes mellitus, cancer, and chronic autoimmune rheumatologic disorders. Many of these potentially life-threatening thrombi occur in the arterial circulation and likely relate to inappropriate platelet activation and/or vessel wall injury. CD36 is a type B scavenger receptor expressed on platelets, monocytes, microvascular endothelial cells (MVEC), and vascular smooth muscle cells (VSMC) that recognizes and responds to endogenous and exogenous “danger signals” including advanced glycated proteins (AGE), microbial cell wall components, and oxidized phospholipids that are found on the surface of oxidized LDL (oxLDL) and cell-derived microparticles (MP). Mouse FeCl3 thrombosis model systems revealed that genetic deletion of cd36 rescued the prothrombotic phenotypes associated with hyperlipidemia, oxidant stress, or diabetes/hyperglycemia. Interestingly, compared to wild type mice, fewer MP were incorporated into thrombi formed in cd36 null mice after FeCl3-induced vascular injury. Cd36 deficiency also provides partial protection from atherosclerosis in experimental mouse models. On a cellular level, the prothrombotic, pro-atherogenic, and anti-angiogenic activities of CD36 relate to ligand-dependent alterations of platelet, macrophage, and vessel wall cellular functions. For example, CD36 binding to MP, AGE, or oxLDL “sensitizes” platelets to activation by low doses of agonists, such as ADP. In macrophages, CD36 interactions with oxLDL leads to lipid internalization, foam cell formation, and inhibition of migration. In the vessel wall CD36 promotes VSMC oxidant stress and MVEC apoptosis. Mechanistically, CD36 transmits intracellular signals in response to ligand engagement that involve recruitment of specific Src family kinases to its short cytoplasmic tail, with subsequent activation of specific MAP kinase pathways and Vav family guanine nucleotide exchange factors. These signaling pathways remain incompletely understood, but impact many important cellular processes, including cytoskeletal dynamics, cell polarity, transcription of antioxidant genes controlled by the transcription factor Nrf2, and activation of NADPH oxidases. Specific components of CD36 signaling pathways depend on the cellular context and in some cases involve engagement of co-receptors, including integrins, TLRs, and tetraspanins. Recent targeted genome-wide association studies suggest that cd36 polymorphisms associate with CD36 expression levels on platelets and monocytes as well as with risk of thrombotic, metabolic, and atherosclerotic disorders. Targeting CD36 may therefore provide novel approaches to many important human vascular diseases. Disclosures: No relevant conflicts of interest to declare.
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Zhu, Jieqing, Won-Seok Choi, Joshua G. McCoy, Ana Negri, Jianghai Zhu, Sarasija Naini, Jihong Li, et al. "Structure-Guided Design of A Novel High Affinity Integrin αIIbβ3 Receptor Antagonist (RUC-2) That Displaces Mg2+ From the β3 MIDAS,." Blood 118, no. 21 (November 18, 2011): 3255. http://dx.doi.org/10.1182/blood.v118.21.3255.3255.
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Abstract Abstract 3255 The platelet αIIbβ3 integrin receptor plays a central role in hemostasis and thrombosis. Small molecule inhibitors of αIIbβ3 based on the RGD cell recognition sequence block ligand binding by interacting with αIIb D224 via their positively-charged (R-like) group and coordinating the Mg2+ ion in the metal ion adhesion site (MIDAS) via their carboxyl (D-like) group. We recently reported a novel inhibitor of αIIbβ3 (RUC-1) that binds exclusively to αIIb and we now report the structure-based design and synthesis of RUC-2 [2-amino-N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)acetamide; MW 385], a RUC-1 derivative with ∼100-fold higher affinity and an IC50= ∼90 nM for ADP-induced platelet aggregation. RUC-2, like RUC-1 shows specificity for αIIbβ3 compared to αVβ3 and produces much less exposure of the β3 LIBS1 epitope than does eptifibatide (eptifibatide=100%, untreated platelets=22±3 %; RUC-2=21±3%). RUC-2 also produces less of a global conformational change in αIIbβ3 compared to eptifibatide as measured by dynamic light scattering, gel permeation chromatography, and electron microscopic imaging of αIIbβ3 in nanodiscs. X-ray crystallography of RUC-2 soaked into the αIIbβ3 headpiece in 1 mM Ca2+ and 5 mM Mg2+ at 2.6 Å revealed that RUC-2 binds to αIIb much the way RUC-1 does, but in addition it binds to one of the sidechain carboxyl oxygens of the β3 MIDAS residue Glu-220, thus displacing Mg2+ from the MIDAS. When RUC-2 was soaked into the crystal in the presence of 20 mM Mg2+, however, the Mg2+ was identified in the MIDAS and RUC-2 was absent from the pocket. Molecular dynamics simulations were in accord with the X-ray crystallographic data. Support for competition between RUC-2 and Mg2+ for binding to the MIDAS came from studies showing that increasing the Mg2+ concentration significantly decreased RUC-2's ability to inhibit PAC-1 binding to CHO cells expressing αIIbβ3, platelet adhesion to fibrinogen, and thrombin receptor activating peptide-induced platelet aggregation. We conclude that RUC-2 inhibits ligand binding with high affinity and specificity by a novel mechanism in which it competes with Mg2+ for Glu-220, and as such may offer advantages as a therapeutic agent. The binding pocket of RUC-2 in the closed αIIbβ3 headpiece crystal structure. αIIb and β3 are shown as solvent accessible surfaces. Ca2+ ions of SyMBS or ADMIDAS (yellow) are shown as spheres. RUC-2 and selected αIIbβ3 sidechain and backbone atoms are shown as sticks with green (RUC-2), light blue (αIIb), or wheat carbons (β3), red oxygens, blue nitrogens, and yellow sulphurs. Water molecules are small red spheres. Hydrogen and metal coordination bonds are shown as dashed blue lines. Disclosures: Coller: Centocor/Accumetrics/Rockefeller University: Royalty interests in abciximab/VerifyNow assays/RUC-1 and RUC-2.
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Cooley, Brian C. "In Vivo Imaging of Large-Vessel Thrombosis." Blood 112, no. 11 (November 16, 2008): 690. http://dx.doi.org/10.1182/blood.v112.11.690.690.
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Abstract In vivo thrombosis imaging systems have been developed that show localized smallscale and short-acting thrombotic events in arterioles and venules; however, these microvascular models may not accurately simulate thrombogenesis in large vessels. A new quantitative imaging system was developed for evaluation of electrolytic-injury models of thrombus induction in large vessels of mice (carotid arteries and femoral veins). Laser-induced fluorophore emissions of membrane-labeled platelets and labeled fibrin-specific monoclonal antibodies were captured with time-lapse video recordings. Platelets were seen to accrue slowly after thrombus induction in both arteries and veins, peaking at approximately 15–45 minutes. In arteries, platelets accumulated more rapidly and to a greater extent than fibrin. Surprisingly, platelet accumulation in veins was substantial, occurring diffusely throughout the thrombus. Arterial thrombus dissolution occurred most often via large embolic events, sometimes occurring cyclically every 5–20 minutes by regrowth and re-embolization. In contrast, the rate of thrombus dissolution in veins was more gradual and appeared as micro-embolic events. Standard heparin anticoagulation inhibited venous thrombus growth by reducing fibrin development by 75% without affecting platelet accumulation. In contrast, blocking platelet aggregation at the αIIb/β3 receptor (with RGD-based mimetics) resulted in a 43% reduction in platelets and a surprising 5-fold increase in fibrin accumulation, suggesting the possibility of temporal competition between platelets and fibrin under venous conditions of thrombus formation. In established thrombi, newly activated platelets, identified by exposure of the active site of the αIIb/β3 receptor (using JON/A, a fluorophore-labeled antibody specific to activated αIIb/β3), were seen to roll in small aggregates across the outer margin of the thrombus mass in both arteries and veins for over 30 minutes, indicating continual dynamic platelet aggregation in both vessel types. Fibrin accrual was most intense at the margins of acute thrombotic stimuli and co-localized with fluorophore-labeled anti-Factor Xa and with Factor Va/VIIIa (identified using labeled activated protein C), which suggests that fibrin polymerization predominates adjacent to sites of these assembled coagulation complexes. In summary, the time-lapse nature of this video imaging system provides a unique and quantifiable method for determining how coagulation and platelet aggregation develop and are modulated in vivo within the same thrombus. Its use for the study of large vessel thrombosis often corroborated long-held tenets of platelet activity and coagulation, while revealing several novel findings: platelets are major contributors to venous thrombosis; arteries and veins have profound differences in thrombus dissolution/embolization patterns; inhibiting platelet aggregation may augment fibrin clot formation; platelet aggregation is a dynamic process in both arteries and veins, continually in flux on the thrombus surface for at least 30 minutes; and fibrin forms local to sites of coagulation complex assembly. Figure Figure Graph illustrating single traces for representative electrolytically-induced, free-radical-mediated thrombi in mouse carotid arteries (EAT) and femoral veins (EVT) over 60 minutes, for anti-fibrin (FBN) and platelet (Plt) labels. The y-axis displays arbitrary units for the product of total thrombus area and average fluorophore intensity within the thrombus.
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Leiderman, Karin, Suzanne S. Sindi, Dougald M. Monroe, Aaron L. Fogelson, and Keith B. Neeves. "The Art and Science of Building a Computational Model to Understand Hemostasis." Seminars in Thrombosis and Hemostasis 47, no. 02 (February 26, 2021): 129–38. http://dx.doi.org/10.1055/s-0041-1722861.
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AbstractComputational models of various facets of hemostasis and thrombosis have increased substantially in the last decade. These models have the potential to make predictions that can uncover new mechanisms within the complex dynamics of thrombus formation. However, these predictions are only as good as the data and assumptions they are built upon, and therefore model building requires intimate coupling with experiments. The objective of this article is to guide the reader through how a computational model is built and how it can inform and be refined by experiments. This is accomplished by answering six questions facing the model builder: (1) Why make a model? (2) What kind of model should be built? (3) How is the model built? (4) Is the model a “good” model? (5) Do we believe the model? (6) Is the model useful? These questions are answered in the context of a model of thrombus formation that has been successfully applied to understanding the interplay between blood flow, platelet deposition, and coagulation and in identifying potential modifiers of thrombin generation in hemophilia A.
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Li, Da, Xiaosong Zhang, Honggang Zhang, and Xiaoqiang Li. "Synergic effect of GPIBA and von Willebrand factor in pathogenesis of deep vein thrombosis." Vascular 28, no. 3 (January 6, 2020): 309–13. http://dx.doi.org/10.1177/1708538119896446.
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Objectives In cardiovascular disease, deep vein thrombosis is one of the vital symptoms causing pulmonary thromboembolism. However, the pathogenesis of deep vein thrombosis is still not clear. One of the critical factors leading to deep vein thrombosis is the platelet aggregation that is mediated by a set of key genes including platelet membrane protein coded by platelet glycoprotein Ib alpha chain (GPIBA). Methods Deep vein thrombosis model was established according to the previous protocol, and venous blood and thrombi were collected for further analysis. Results The dynamic changes of GPIBA and coagulation factor, von Willebrand factor, were observed in deep vein thrombosis models. Meanwhile, critical proteins participating in adhesion and binding of platelets such as epithelial membrane protein 2 (EMP2), vascular cell adhesion protein 1 (VCAM1), immunoreceptor tyrosine-based activation motif 1 (ITAM1), integrin subunit alpha M (ITGAM), or fibronectin were also differentially expressed in deep vein thrombosis models. Conclusions Application of heparin could reverse these dynamic changes in deep vein thrombosis models. Thus, we explained the potential synergic role of GPIBA and von Willebrand factor in regulating the occurrence of deep vein thrombosis and provide therapeutic target against cardiovascular disease.
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Nicoleau, Salina, and Beata Wojciak-Stothard. "Beyond Thrombosis: the Role of Platelets in Pulmonary Hypertension." SciMedicine Journal 2, no. 4 (December 1, 2020): 243–71. http://dx.doi.org/10.28991/scimedj-2020-0204-7.
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Pulmonary Hypertension (PH) is a multifactorial and lethal disease, characterised by elevated pulmonary arterial pressure and progressive right heart failure. PH pathobiology rests on four pillars: vascular remodelling, vasoconstriction, inflammation and thrombosis. While vascular and inflammatory cells have been the focus of PH research over the past decades, platelets have received relatively less attention, despite their associations with key pathophysiological processes of the disease. Platelets contain a wide range of vasoactive, inflammatory and pro-thrombotic mediators, likely to promote PH development and progression. There is currently no cure for PH, and platelet-associated pathways may help identify new therapeutic strategies. This review summarises available evidence on the role of platelets in different forms of PH, and comments on the current state of platelet-targeting therapies. It also describes the latest advances in the in vitro technologies that enable exploration of platelet function under dynamic and physiologically relevant conditions. Doi: 10.28991/SciMedJ-2020-0204-7 Full Text: PDF
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de Oliveira, Diana C., David G. Owen, Shuang Qian, Naomi C. Green, Daniel M. Espino, and Duncan E. T. Shepherd. "Computational fluid dynamics of the right atrium: Assessment of modelling criteria for the evaluation of dialysis catheters." PLOS ONE 16, no. 2 (February 25, 2021): e0247438. http://dx.doi.org/10.1371/journal.pone.0247438.
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Central venous catheters are widely used in haemodialysis therapy, having to respect design requirements for appropriate performance. These are placed within the right atrium (RA); however, there is no prior computational study assessing different catheter designs while mimicking their native environment. Here, a computational fluid dynamics model of the RA, based on realistic geometry and transient physiological boundary conditions, was developed and validated. Symmetric, split and step catheter designs were virtually placed in the RA and their performance was evaluated by: assessing their interaction with the RA haemodynamic environment through prediction of flow vorticity and wall shear stress (WSS) magnitudes (1); and quantifying recirculation and tip shear stress (2). Haemodynamic predictions from our RA model showed good agreement with the literature. Catheter placement in the RA increased average vorticity, which could indicate alterations of normal blood flow, and altered WSS magnitudes and distribution, which could indicate changes in tissue mechanical properties. All designs had recirculation and elevated shear stress values, which can induce platelet activation and subsequently thrombosis. The symmetric design, however, had the lowest associated values (best performance), while step design catheters working in reverse mode were associated with worsened performance. Different tip placements also impacted on catheter performance. Our findings suggest that using a realistically anatomical RA model to study catheter performance and interaction with the haemodynamic environment is crucial, and that care needs to be given to correct tip placement within the RA for improved recirculation percentages and diminished shear stress values.
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Ross, JM, and LV McIntire. "Molecular Mechanisms of Mural Thrombosis Under Dynamic Flow Conditions." Physiology 10, no. 3 (June 1, 1995): 117–22. http://dx.doi.org/10.1152/physiologyonline.1995.10.3.117.
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Mural adhesion and aggregation of blood platelets are crucial for hemostasis but in pathological situations can lead to myocardial infarction and stroke. The specific platelet surface receptors employed are determined by the local fluid dynamic conditions and extracellular matrix components exposed at the site of a vascular wound.
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Justo Sanz, Raul, Elena Monzón Manzano, Ihosvany Fernandez-Bello, Teresa Álvarez-Roman, Mónica Martín, María Isabel Rivas Pollmar, Miguel A. Canales, Victor Jimenez-Yuste, and Nora Butta. "Platelet Apoptosis and PAI-1 Content Are Involved in the Procoagulant Profile of Immune Thrombocytopenia Patients Responders to Agonists of Thrombopoietin Receptor." Blood 132, Supplement 1 (November 29, 2018): 3738. http://dx.doi.org/10.1182/blood-2018-99-115898.
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Abstract Background: The treatment goal for patients with immune thrombocytopenia (ITP) is to raise platelet counts to levels that minimize or stop bleeding. Thrombopoietin receptor agonists (TPO-RAs) have been successfully and extensively employed as second-line therapy for ITP. TPO-RAs, however, have a small but significant increase in the risk of thrombosis. Aim: The aim of this study was to elucidate the mechanisms involved in the procoagulant effect of TPO-RAs. Methods: This is a prospective, observational and transversal study. Eighty-two patients with chronic primary ITP, 40 without treatment for at least six months (UT-ITP) and 42 responders to TPO-RA therapy (64.3% with eltrombopag and 35.7 % with romiplostim) were recruited. One hundred and twelve healthy participants were also included. ROTEM® (naTEM test: only recalcification) was performed on platelet rich plasma adjusted to a platelet count of 25 x 109/L. Clotting time (CT, time from start of measurement until 2 mm of amplitude [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2 mm amplitude [in degrees]), maximum clot firmness, which reflects the maximum tensile strength of the thrombus (MCF, [in mm]) and LI60, which describes the percentage of maximum clot strength present at 60 min (in %), were recorded. Surface exposure of phosphatidylserine (PS), active caspase-3, -8 or -9 and prothrombinase complex binding to platelets were assessed by flow cytometry. Plasma and platelet levels of PAI-1 were determined by ELISA (eBioscience Ltd., Hatfield, United Kingdom). The effect of TPO and romiplostim on PAI-1 content of MEG-01 cells was evaluated by Western blot. Three MEG-01 cell cultures were initiated simultaneously: control without drugs and treated with either TPO (100 ng/mL) or romiplostim (53 μg/mL). Samples were collected at the start and after 24, 48 and 72 hours to determine the PAI-1 content. The statistical analysis was performed using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA). Results: The ROTEM® studies showed significant differences in the dynamics of clot formation when comparing the control with ITP samples. There was a delay in clot formation in the UT-ITP group, as observed by a prolonged CT [expressed as median (p25-p75): control: 516 (490- 633) s; UT-ITP: 938 (914-1348) s, p<0.001], and a diminished alpha angle (mean±SD; control: 61.7±5.6 degrees; UT-ITP: 49.2±7.3 degrees, p<0.05). Nevertheless, samples from patients with UT-ITP reached the same MCF as those from healthy controls (control: 45.3±2.4 mm; UT-ITP: 46.9±3.7 mm). On the other hand, patients with ITP undergoing TPO-RA therapy presented an initial clot formation similar to that of the control group [expressed as median (p25-p75): CT, 672 (598-928) s; alpha angle, 55.8±5.8 degrees] but achieved a higher MCF (53.1±4.5 mm, p<0.05) and a reduced clot lysis after 60 min (control: 91.8±4.0%; UT-ITP: 93.7±4.0%, TPO-RA ITP: 97.6±1.7, p<0.05). Higher values of MCF observed with platelets from ITP patients treated with TPO-RAs might be a consequence of their augmented apoptosis signs: platelets from this group exposed more PS than controls and this situation was accompanied by an increased activity of caspases-3,7, -8 and -9 (Figure 1 A and B). Moreover, platelets from ITP patients on treatment with TPO-RAs bound more prothrombinase complex than platelets from UT-ITP patients and healthy controls (Figure 1 C). Reduced clot lysis observed in ITP patients treated with TPO-RA was due, at least in part, to increased plasma and platelet levels of PAI-1 (Table 1). Increase in platelet content of PAI-1 might be the result of the effect of TPO-RAs during megakaryopoiesis since treatments of MEG-01 cells with TPO or romiplostim induced a 3-fold increase in their endogenous PAI-1 content after an incubation period of 48 hs. Conclusion: The patients with ITP undergoing TPO-RAs therapy presented a procoagulant profile due to the formation of a more fibrinolysis-resistant clot because of increased platelet and plasma PAI-1 levels. Moreover, platelets from this group of patients showed more signs of apoptosis that causes a higher exposure of PS and, consequently, a larger surface for the binding of the prothrombinase complex. Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). Disclosures Álvarez-Roman: SOBI: Consultancy; NovoNordisk: Consultancy; Shire: Consultancy. Jimenez-Yuste:Grifols: Consultancy, Research Funding; Octapharma: Consultancy, Research Funding; CSL Behring: Consultancy; Bayer: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Shire: Consultancy, Research Funding; Sobi: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; NovoNordisk: Consultancy, Research Funding. Butta:FIS-Fondos FEDER: Research Funding.
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Jain, Nishank, Adam L. Corken, Amudha Kumar, Clayton L. Davis, Jerry Ware, and John M. Arthur. "Role of Platelets in Chronic Kidney Disease." Journal of the American Society of Nephrology 32, no. 7 (June 17, 2021): 1551–58. http://dx.doi.org/10.1681/asn.2020121806.
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Platelet-dependent mechanisms for excessive clotting and bleeding in CKD remain undefined. Moreover, platelets’ contribution to inflammation, and specifically to CKD, are equally elusive. To date, descriptions of changes in the functional properties of circulating platelets during CKD have provided confusing interpretations. Experimental approaches that can advance our understanding of platelet dysfunction in CKD are needed, and studies that provide mechanistic insights into the dynamic relationships between thrombosis, bleeding, and inflammation associated with CKD will be essential to improve clinical management and outcomes for this vulnerable population. This article summarizes existing literature characterizing platelets in CKD and identifies areas that need further investigation.
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Suárez-Rivero, Juan M., Carmen J. Pastor-Maldonado, Suleva Povea-Cabello, Mónica Álvarez-Córdoba, Irene Villalón-García, Marta Talaverón-Rey, Alejandra Suárez-Carrillo, Manuel Munuera-Cabeza, and José A. Sánchez-Alcázar. "From Mitochondria to Atherosclerosis: The Inflammation Path." Biomedicines 9, no. 3 (March 5, 2021): 258. http://dx.doi.org/10.3390/biomedicines9030258.
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Inflammation is a key process in metazoan organisms due to its relevance for innate defense against infections and tissue damage. However, inflammation is also implicated in pathological processes such as atherosclerosis. Atherosclerosis is a chronic inflammatory disease of the arterial wall where unstable atherosclerotic plaque rupture causing platelet aggregation and thrombosis may compromise the arterial lumen, leading to acute or chronic ischemic syndromes. In this review, we will focus on the role of mitochondria in atherosclerosis while keeping inflammation as a link. Mitochondria are the main source of cellular energy. Under stress, mitochondria are also capable of controlling inflammation through the production of reactive oxygen species (ROS) and the release of mitochondrial components, such as mitochondrial DNA (mtDNA), into the cytoplasm or into the extracellular matrix, where they act as danger signals when recognized by innate immune receptors. Primary or secondary mitochondrial dysfunctions are associated with the initiation and progression of atherosclerosis by elevating the production of ROS, altering mitochondrial dynamics and energy supply, as well as promoting inflammation. Knowing and understanding the pathways behind mitochondrial-based inflammation in atheroma progression is essential to discovering alternative or complementary treatments.
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Tsai, Rachel, Yiming Li, Olga Yakovenko, An-Yue Tu, Nichole S. Tyler, and Gianluca Interlandi. "Molecular Basis for the Pro-Thrombotic Activity of Von Willebrand Factor Under Inflammatory Conditions." Blood 128, no. 22 (December 2, 2016): 554. http://dx.doi.org/10.1182/blood.v128.22.554.554.
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Abstract Inflammation is a ubiquitous phenomenon, resulting from such common disease states as obesity, injury, and sepsis, and it is associated with an elevated risk of thrombosis. During inflammation, neutrophils are activated and produce oxidants, such as hydrogen superoxide, which is then converted to hypochlorous acid (HOCl) through the action of the enzyme myeloperoxidase. This leads to modifications in the structure of coagulation factors, which is mainly due to the conversion of methionine residues to methionine sulfoxide. Recent experimental evidence indicated that oxidative conditions increase the ability of von Willebrand Factor (VWF) to tether platelets. This was linked to the oxidation of methionine residues in the A1, A2 and A2 domains of VWF (Fu et al. Blood 2011). These domains are key in regulating the platelet-tethering function of VWF. The A3 domain attaches to the exposed collagen at the site of injury, while the A1 domain binds to the platelet surface receptor GpIbα. On the other hand, the A2 domain, sandwiched between the two, contains a proteolytic target site for the metalloprotease ADAMTS13 (Figure 1), which cleaves it once the A2 domain is unfolded under tensile force. The molecular mechanism behind the oxidation-induced activation of VWF is currently not understood. In particular, it is not clear whether oxidation activates the A1 domain itself or whether oxidation removes the inhibitory function of neighboring domains. In this work, we used an assay based on a parallel plate flow chamber to explore which inhibitory mechanisms of A1 are removed by oxidation. Then, we used molecular dynamics simulations and free energy perturbation methods to propose a model how oxidation affects the structure of the A2 domain favoring activation of VWF. Experimental evidence points out that neighboring domains of A1, in particular the A2 and A3 domains, inhibit its platelet-binding function (Auton et al. J. Biol. Chem. 2010). For this reason, constructs of VWF containing the A1 domain with or without the neighboring A2 or A3 domains were expressed in Chinese hamster ovary cells and anchored on a surface. Oxidation was induced by incubating the surface with HOCl. Beads coated with GpIbα were then washed over the surface at different shear rates while their trajectories were monitored. The results indicated that oxidation has little effect on the rolling behavior of beads when washed over the isolated A1 domain. However, oxidation caused beads to roll slower and accumulate more on surfaces coated with constructs where the A1 domain was flanked by the A2 and A3 domains. The molecular mechanism how oxidation removes the inhibitory function of the A2 domain was explored through molecular dynamics simulations. The A2 domain presents a globular fold consisting of a central β sheet surrounded by α helices (Figure 1). Oxidation of methionine residues causes the C-terminal helix to separate from the rest of the protein at a lower tensile force. Furthermore, free energy perturbation calculations indicated that the A2 domain is more likely to be found in the denatured state when its methionine residues are oxidized. Although under normal conditions the A2 domain is cleaved by ADAMTS13 when unfolded, oxidizing agents increase its resistance towards proteolysis due to the oxidation of a methionine residue in the cleavage site (Chen et al. Blood 2010). Taken together, oxidation destabilizes the A2 domain, which causes its separation from the A1 domain, thus removing its inhibitory function. Understanding the molecular basis of how oxidation increases the pro-thrombotic activity of VWF could provide a target for future therapeutics aimed specifically at mitigating the thrombotic complications seen in many inflammatory conditions. Figure 1 VWF A2 domain Figure 1. VWF A2 domain Disclosures No relevant conflicts of interest to declare.
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Gianazza, Erica, Maura Brioschi, Roberta Baetta, Alice Mallia, Cristina Banfi, and Elena Tremoli. "Platelets in Healthy and Disease States: From Biomarkers Discovery to Drug Targets Identification by Proteomics." International Journal of Molecular Sciences 21, no. 12 (June 25, 2020): 4541. http://dx.doi.org/10.3390/ijms21124541.
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Platelets are a heterogeneous small anucleate blood cell population with a central role both in physiological haemostasis and in pathological states, spanning from thrombosis to inflammation, and cancer. Recent advances in proteomic studies provided additional important information concerning the platelet biology and the response of platelets to several pathophysiological pathways. Platelets circulate systemically and can be easily isolated from human samples, making proteomic application very interesting for characterizing the complexity of platelet functions in health and disease as well as for identifying and quantifying potential platelet proteins as biomarkers and novel antiplatelet therapeutic targets. To date, the highly dynamic protein content of platelets has been studied in resting and activated platelets, and several subproteomes have been characterized including platelet-derived microparticles, platelet granules, platelet releasates, platelet membrane proteins, and specific platelet post-translational modifications. In this review, a critical overview is provided on principal platelet proteomic studies focused on platelet biology from signaling to granules content, platelet proteome changes in several diseases, and the impact of drugs on platelet functions. Moreover, recent advances in quantitative platelet proteomics are discussed, emphasizing the importance of targeted quantification methods for more precise, robust and accurate quantification of selected proteins, which might be used as biomarkers for disease diagnosis, prognosis and therapy, and their strong clinical impact in the near future.