Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
Thromb Res. 2022 Oct;218:112-129. doi: 10.1016/j.thromres.2022.08.018. Epub 2022 Aug 20.
The collagen receptor glycoprotein VI (GPVI) drives strong platelet activation, however its role at later stages of clotting remains less clear. Controlled timing of addition of anti-human GPVI Fab (clone E12) with microfluidic venous whole blood flow over collagen (± lipidated tissue factor, TF) produced distinct effects on platelets, fibrin, P-selectin exposure, and phosphatidylserine (PS) exposure. On collagen alone, Fab present initially potently reduced platelet deposition on collagen, while Fab added 90 s after initial platelet deposition, stopped subsequent platelet accumulation (despite the absence of fibrin). With thrombin generation via TF, Fab added at either t = 0 or 90 s had no effect on platelet deposition. However, Fab added initially, but not at 90-s, blocked fibrin formation. Gly-Pro-Arg-Pro ablated fibrin formation without effect on platelet accumulation (regardless of Fab added at t = 0 or 90 s), indicating thrombin signaling can suffice over GPVI signaling. Still, Fab moderately reduced P-selectin exposure with thrombin present and fibrin absent. On collagen/TF, Fab present initially ablated PS exposure, but had no effect when added 30 to 90-s later. The thrombin generated via PS exposure had an important role in driving platelet deposition in the presence of Fab, since inhibition of PS via annexin V binding in the presence of Fab significantly inhibited platelet deposition. We conclude GPVI signaling in the first platelet layer on collagen dictates thrombin and fibrin production, but the role of GPVI at subsequent times after formation of the first monolayer is obscured by thrombin-induced signaling.
胶原受体糖蛋白 VI（GPVI）可驱动血小板强烈激活，但在血栓形成的后期阶段，其作用尚不清楚。在微流控静脉全血流过胶原（±脂质化组织因子 TF）的过程中，控制添加抗人 GPVI Fab（克隆 E12）的时间，对血小板、纤维蛋白、P-选择素暴露和磷脂酰丝氨酸（PS）暴露有不同的影响。在单独的胶原上，Fab 最初大量减少血小板在胶原上的沉积，而 Fab 在初始血小板沉积 90 秒后添加，则阻止了随后的血小板聚集（尽管没有纤维蛋白）。通过 TF 生成凝血酶时，在 t = 0 或 90 秒时添加 Fab 对血小板沉积没有影响。然而，最初添加的 Fab，但不是在 90 秒时添加的 Fab，阻止了纤维蛋白的形成。甘氨酰-脯氨酰-精氨酰-脯氨酸（Gly-Pro-Arg-Pro）阻断纤维蛋白形成，而不影响血小板聚集（无论 Fab 是在 t = 0 还是 90 秒添加），表明凝血酶信号足以替代 GPVI 信号。尽管如此，在存在凝血酶和不存在纤维蛋白的情况下，Fab 适度减少了 P-选择素的暴露。在胶原/TF 上，最初存在的 Fab 消除了 PS 的暴露，但在 30 至 90 秒后添加时则没有效果。通过 PS 暴露生成的凝血酶在有 Fab 存在的情况下对驱动血小板沉积有重要作用，因为在有 Fab 存在的情况下通过与 annexin V 结合抑制 PS 显著抑制了血小板沉积。我们得出结论，在胶原上的第一层血小板中 GPVI 信号决定了凝血酶和纤维蛋白的产生，但在形成第一层单层后的后续时间内，GPVI 的作用被凝血酶诱导的信号所掩盖。
