Platelet contraction cytometry as a novel assay of platelet function

项目总结/摘要 尽管在许多疾病状态（包括原发性和非原发性）中， 血液系统疾病、癌症4和脓毒症5，6，我们对出血和血栓性并发症的了解 由于缺乏工具，这一工作还远远没有完成。在血凝块形成过程中，血小板经历肌肉样收缩， 通过新生纤维蛋白聚合物的收缩来动态收缩和稳定凝块以阻止出血。 然而，血小板是如何通过微环境信号来介导收缩和改变凝块力学的， 由于过度软化和硬化的血栓与血栓相关，因此在很大程度上仍然未知，但具有临床相关性 出血7和血栓性疾病8。批量测定不足以用于这些研究（作为个体） 血小板表现出依赖于局部凝血生化浓度的高度可变的行为， 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 不同微环境中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，我们从患者中获得的初步数据 表明血小板收缩细胞计数器可能代表一种全新的诊断类别， 确定了异常血小板收缩力和出血之间的联系。30-60%的患者 出血仍然未被诊断，这可能是一个重要的和有影响力的途径，以更好地了解和 识别异常止血。我们发现，在健康人中存在高度收缩的血小板亚群， 在小样本的未确诊出血性疾病患者中，对照明显缺失。同样地， 我们发现，在免疫缺陷患者的背景下，活动性瘀伤和血小板收缩力之间存在相关性。 血小板减少症（ITP）。此外，血小板收缩细胞计数似乎与已知的 血小板活化标志物（磷脂酰丝氨酸、P-选择素和PAC-1）。 这个NIBIB开拓者奖的新的和早期阶段的研究人员（R21）的研究目标是 研究一个新的假设：血小板的收缩力独立预测出血。我们将 首先定义健康血小板对胶原蛋白和纤维蛋白（原）的收缩反应， 凝块中存在的生理硬度。我们假设这些不同的配体会引发不同的力 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血小板表型，我们以前发现， 诊断潜力此外，我们将分析血小板收缩力，来自：健康志愿者， 未确诊的有出血症状的患者，以及免疫性血小板减少性紫癜的患者进行检测 血小板收缩力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.