■血小板的主动和被动生物力学特性基本上有助于血栓形成。肌动球蛋白收缩性驱动稳定止血栓所需的凝块收缩。收缩力受损导致出血,但使用血小板功能试验难以检测。
■为了确定肌球蛋白活性降低如何影响血小板功能,包括和超过凝块收缩。
■使用肌球蛋白IIA特异性药物抑制剂blebbistatin,我们调节了来自健康供体的血小板中肌球蛋白的活性,并通过使用一系列互补检测方法询问血栓形成的每个阶段不同的血小板功能,对不同抑制水平的血小板反应进行了系统表征.
■部分肌球蛋白IIA抑制既不影响动脉剪切下的血小板血管性血友病因子相互作用,也不影响血小板扩散和纤维蛋白原的细胞骨架重排。然而,它影响了应力纤维的形成和细胞-基质粘连的纳米结构,急剧减少和限制牵引力。较高的blebbistatin浓度会损害流动下的血小板粘附,改变了层片边缘的机械传感,消除牵引力而不影响血小板扩散,α-颗粒分泌,或促凝血小板形成。出乎意料的是,肌球蛋白IIA抑制减少钙流入,致密的颗粒分泌物,和糖蛋白(GP)VI下游的血小板聚集,限制了GPVI在细胞膜上的重新分布,而二磷酸腺苷或花生四烯酸诱导的聚集不受影响。
■我们的发现强调了肌球蛋白IIA在血小板细胞骨架中的主动收缩和被动交联作用的重要性。他们支持止血需要高度收缩的血小板的假说,并进一步表明肌球蛋白IIA在GPVI信号传导中的支持作用。
UNASSIGNED: Active and passive biomechanical properties of platelets contribute substantially to thrombus formation. Actomyosin contractility drives clot contraction required for stabilizing the hemostatic plug. Impaired contractility results in bleeding but is difficult to detect using platelet function tests.
UNASSIGNED: To determine how diminished myosin activity affects platelet functions, including and beyond clot contraction.
UNASSIGNED: Using the myosin IIA-specific pharmacologic inhibitor
blebbistatin, we modulated myosin activity in platelets from healthy donors and systematically characterized platelet responses at various levels of inhibition by interrogating distinct platelet functions at each stage of thrombus formation using a range of complementary assays.
UNASSIGNED: Partial myosin IIA inhibition neither affected platelet von Willebrand factor interactions under arterial shear nor platelet spreading and cytoskeletal rearrangements on fibrinogen. However, it impacted stress fiber formation and the nanoarchitecture of cell-matrix adhesions, drastically reducing and limiting traction forces. Higher
blebbistatin concentrations impaired platelet adhesion under flow, altered mechanosensing at lamellipodia edges, and eliminated traction forces without affecting platelet spreading, α-granule secretion, or procoagulant platelet formation. Unexpectedly, myosin IIA inhibition reduced calcium influx, dense granule secretion, and platelet aggregation downstream of glycoprotein (GP)VI and limited the redistribution of GPVI on the cell membrane, whereas aggregation induced by adenosine diphosphate or arachidonic acid was unaffected.
UNASSIGNED: Our findings highlight the importance of both active contractile and passive crosslinking roles of myosin IIA in the platelet cytoskeleton. They support the hypothesis that highly contractile platelets are needed for hemostasis and further suggest a supportive role for myosin IIA in GPVI signaling.