Endothelial topography, phosphatidylserine, and procoagulant activity

凝血酶只有在含有磷脂酰丝氨酸的膜上才能有效发挥作用。 然而，磷脂酰丝氨酸通常不是可用的，因为血细胞将其隔离在 细胞膜。血小板对损伤组织中的胶原蛋白产生反应，暴露出大量的 磷脂酰丝氨酸在膜和这些血小板的泡状突起上被称为 “促凝血剂血小板”，被认为是防止出血所必需的。然而，最近 来自血小板磷脂酰丝氨酸暴露缺陷患者的临床信息表明，这些 患者仅有轻至中度的粘膜出血。这就提出了一个问题，即 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确定了几个可能有助于解释 明显的矛盾。首先，凝血复合体识别凸膜曲率。 此外还有磷脂酰丝氨酸的含量。有突起和内陷的膜可能有 凝血复合体高度定位于凸起。第二，血小板和内皮细胞 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 对于大多数磷脂酰丝氨酸分析来说，暴露在检测阈值以下。因此，低水平 存在磷脂酰丝氨酸暴露，可支持凝血复合体，但未被检测到。 第三，富含磷脂酰丝氨酸的膜还能在一定程度上支持抗凝蛋白 抑制或消除促凝血潜能。净抗凝作用，就像促凝剂支架， 取决于磷脂酰丝氨酸含量和膜曲率。这些见解，以及 获得它们的方法，给了我们独特的机会来研究血小板和 内皮细胞磷脂酰丝氨酸暴露使促凝血酶活性局部化。 我们假设，血液抗凝剂通常会抑制血管紧张素转换酶促凝血的潜能。 用富含磷脂酰丝氨酸的气泡刺激血小板。血小板在体内获得真正的促凝活性 抗凝血剂被微环境中的蛋白质衰减或绕过的环境。 这项提案将侧重于深入了解通常抑制血液的抗凝蛋白。 促凝血剂复合体上的凝血反应，特别是与磷脂酰丝氨酸暴露有关的反应 和膜曲率。接下来我们将探讨抗凝剂作用减弱的程度。 或者被粘膜中的纤溶酶作用所绕过。此外，我们还将研究 条件性内皮细胞支持局部高度凝血反应的方式 复杂的膜突起。我们还将评估内皮促凝剂在多大程度上 黏附的血小板和内皮细胞产生的Xa因子是否绕过 血小板滤过泡的抗凝血活性。 血小板研究将提供与治疗外科和创伤性出血相关的见解， 与血友病患者的止血有关。血管内皮细胞研究与凝血障碍有关 称为弥散性血管内凝血，与血管损伤和炎症有关 新冠肺炎。

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.