Endothelial topography, phosphatidylserine, and procoagulant activity

血液凝固酶仅在含有磷脂酰丝氨酸的膜上有效地起作用。 然而，磷脂酰丝氨酸通常是不可用的，因为血细胞将其隔离在血管的内部。 细胞膜血小板对受伤组织中的胶原蛋白有反应， 磷脂酰丝氨酸在膜的水泡样突起上，这些血小板被称为 血小板是“促凝血血小板”，并且被认为是预防出血所必需的。但最近的 来自血小板磷脂酰丝氨酸暴露缺陷患者的临床信息表明， 患者仅有轻度-中度粘膜出血。这就提出了一个问题， 血小板磷脂酰丝氨酸暴露是否确实是血液凝固的关键成分。 我们实验室的初步研究已经确定了几个可能有助于解释 明显的矛盾。首先，血液凝固复合物识别出血管膜的凸曲率， 磷脂酰丝氨酸含量。具有突起和内陷的膜可能具有 凝固复合物高度定位于凸形突起。第二，血小板和内皮细胞 具有有限的膜磷脂酰丝氨酸暴露模式。在这些模式中，磷脂酰丝氨酸 暴露低于大多数磷脂酰丝氨酸测定的检测阈值。因此，低水平 存在磷脂酰丝氨酸暴露，并且可以支持凝血复合物，但未被检测到。 第三，富含磷脂酰丝氨酸的膜也支持抗凝蛋白， 抑制或消除促凝血潜能。净抗凝作用，如促凝血支持， 依赖于磷脂酰丝氨酸含量和膜曲率。这些见解，以及 用于获得它们的方法，为我们提供了独特的机会来研究血小板和 内皮细胞磷脂酰丝氨酸暴露定位促凝血酶活性。 我们假设，血液抗凝剂通常抑制促凝血剂的潜力， 富含磷脂酰丝氨酸的气泡刺激血小板。血小板获得真正的促凝血活性， 抗凝剂被微环境中的蛋白质减弱或绕过的环境。 这项建议将集中在深入了解抗凝蛋白，通常抑制血液 促凝血复合物的凝血反应，特别是磷脂酰丝氨酸暴露 和膜曲率。接下来我们将探讨抗凝剂作用减弱的程度 或者在粘膜环境中被纤溶酶的作用绕过。此外，我们会研究 条件内皮支持限制局灶性、高度 复杂的膜突起。我们还将评估内皮促凝剂在多大程度上 粘附的血小板和内皮生成因子Xa是否绕过 对血小板泡的抗凝活性。 血小板研究将提供与治疗手术和创伤性出血相关的见解， 与血友病患者的止血相关。内皮检查与凝血功能障碍有关 称为弥散性血管内凝血，与血管损伤和炎症有关， 2019冠状病毒病。

Blood coagulation enzymes function efficiently only on membranes containing phosphatidylserine. However, phosphatidylserine is not ordinarily available because blood cells sequester it on the interior of cell membranes. Blood platelets respond to collagen in injured tissue, exposing abundant phosphatidylserine on bleb-like protrusions of the membrane and these platelets have been called “procoagulant platelets” and are thought to be essential for prevention of bleeding. However, recent clinical information from patients with platelet phosphatidylserine-exposing defects indicate that these patients have only mild-moderate bleeding from mucous membranes. This raises the question as to whether platelet phosphatidylserine exposure is, indeed, a critical component of blood coagulation. Preliminary studies from our laboratory have identified several factors that may help to explain the apparent contradiction. First, blood coagulation complexes recognize convex membrane curvature in addition to phosphatidylserine content. A membrane with protrusions and invaginations may have coagulation complexes highly localized to the convex protrusions. Second, platelets and endothelial cells have modes of limited membrane phosphatidylserine exposure. In these modes, phosphatidylserine exposure is below the threshold of detection for most phosphatidylserine assays. Thus, low level phosphatidylserine exposure is present and can support coagulation complexes, yet goes undetected. Third, phosphatidylserine-rich membranes also support anticoagulant proteins to a degree that can suppress or eliminate the procoagulant potential. The net anticoagulant effect, like procoagulant support, is dependent on both phosphatidylserine content and on membrane curvature. These insights, and the methods used to gain them, give us the unique opportunity to study the manner in which platelet and endothelial cell phosphatidylserine exposure localizes procoagulant enzyme activity. We have hypothesized that blood anticoagulants ordinarily suppress the procoagulant potential of stimulated platelets with phosphatidylserine-rich blebs. The platelets gain true procoagulant activity in environments where anticoagulants are attenuated or bypassed by proteins in the micro-environment. This proposal will focus on gaining insight into the anticoagulant proteins that ordinarily suppress blood coagulation reactions on procoagulant complexes, particularly in regard to phosphatidylserine exposure and membrane curvature. We will next probe the extent to which the anticoagulants effect is attenuated or bypassed by the effects of plasmin in the context of mucous membranes. In addition, we will study the manner in which conditioned endothelial support limited blood coagulation reactions on focal, highly complex membrane projections. We will also evaluate the extent to which the endothelial procoagulant activity may be amplified by adherent platelets and whether the endothelial generated factor Xa bypasses anticoagulant activity on platelet blebs. The platelet studies will provide insights relevant to treating surgical and traumatic bleeding, relevant to hemostasis for hemophilia patients. The endothelial studies are relevant to the coagulopathy known as disseminated intravascular coagulation and relevant to the vascular injury and inflammation of COVID-19.