Platelet contraction cytometry as a novel assay of platelet function

项目摘要/摘要 尽管在包括初级疾病在内的许多疾病状态1-3中存在显著的死亡率和发病率 血液系统疾病，癌症4，败血症5，6，我们对出血和血栓并发症的了解 由于缺乏工具，仍远未完成。在血液凝块形成过程中，血小板会经历肌肉样 使用新生纤维蛋白聚合物进行收缩，以动态收缩和稳定血栓，以阻止出血。 然而，血小板如何传递微环境信号来调节收缩和改变凝血机制 大部分尚不清楚，但临床上相关，因为过度软化和僵硬的血栓与 出血性疾病7和血栓性疾病8。对于这些研究来说，批量分析是不够的。 血小板表现出高度可变的行为，这取决于局部凝血生化浓度和 机械刚度。在这里，一种新开发的高通量血小板收缩细胞仪现在能够 不同微环境下单个血小板收缩力的平行测量。 除了能够对个体血小板行为进行详细研究外，我们还从患者那里获得了初步数据 提示血小板收缩细胞仪可能代表了一种全新的诊断类别 确定了异常的血小板收缩力和出血之间的联系。30%-60%的患者患有 出血仍未确诊，这可能是一种重要而有效的途径，可以更好地了解和 识别异常止血。我们发现健康人群中存在高度收缩的血小板亚群 在一小部分未确诊的出血性疾病患者中，明显缺乏对照。同样， 我们发现，在免疫性疾病患者中，主动瘀伤和血小板收缩力量之间存在相关性。 血小板减少症(ITP)。此外，血小板收缩细胞术似乎不依赖于已知的 血小板活化的标志物(磷脂酰丝氨酸、P-选择素和PAC-1)。 NIBIB新近和早期研究人员开拓者奖(R21)的研究目标是 研究一个新的假设：血小板的收缩力独立地预测出血。我们会 首先定义健康的血小板对胶原和纤维蛋白(原)的收缩反应 凝块中存在的生理僵硬。我们假设这些不同的配体会产生不同的力。 血小板的反应，我们将分析这些反应并寻找微环境条件 协同作用产生高度收缩的血小板表型，我们之前发现 潜在的诊断能力。此外，我们还将分析来自健康志愿者的血小板收缩力量， 对未确诊的症状性出血患者和免疫性血小板减少性紫癜患者进行检测 认为血小板收缩功能受损与出血症状相关的假设。

PROJECT SUMMARY/ABSTRACT Despite accounting for significant mortality and morbidity in many disease states1–3, including primary hematologic disorders, cancer4, and sepsis5,6, our knowledge of bleeding and thrombotic complications remains far from complete due to a lack of tools. During blood clot formation, platelets undergo muscle-like contraction with nascent fibrin polymers to dynamically contract and stabilize the clot to stem hemorrhage. However, how platelets transduce microenvironmental cues to mediate contraction and alter clot mechanics remains largely unknown yet clinically relevant, as overly softened and stiffened clots are associated with bleeding7 and thrombotic disorders8, respectively. Bulk assays are insufficient for these studies as individual platelets exhibit highly variable behavior that depends on the local clotting biochemical concentration and mechanical stiffness. Here, a newly developed high-throughput platelet contraction cytometer now enables parallel measurements of single platelet contraction forces in varied microenvironments. In addition to enabling detailed studies of individual platelet behavior, our preliminary data from patients suggests that the platelet contraction cytometer may represent an entirely new category of diagnostic that identifies a link between aberrant platelet contractile force and bleeding. As between 30-60% of patients with bleeding remain undiagnosed, this may represent a significant and impactful avenue to better understand and identify aberrant hemostasis. We found that highly contractile platelet subpopulations present in healthy controls are conspicuously absent in a small sample patients with undiagnosed bleeding disorders. Similarly, we found correlations with active bruising and platelet contractile forces in the context of patients with immune thrombocytopenia (ITP). Moreover, platelet contraction cytometery appears to be independent of the known markers of platelet activation (phosphatidylserine, P-selectin, and PAC-1). The research objective of this NIBIB Trailblazer Award for New and Early Stage Investigators (R21) is to investigate a novel hypothesis: that the contractile force of platelets independently predicts bleeding. We will first define the healthy platelet contractile response to both collagen and fibrin(ogen) over the range of physiological stiffnesses present in a clot. We hypothesize that these various ligands will elicit different force responses from platelets, and we will analyze these responses and look for microenvironmental conditions that synergize to produce a highly contractile platelet phenotype, which we have previously found to have diagnostic potential. In addition, we will analyze the platelet contractile forces from: healthy volunteers, undiagnosed patients with symptomatic bleeding, and patients with immune thrombocytopenic purpura to test the hypothesis that impaired platelet contractile force will correlate with bleeding symptoms.