Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada.
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada.
Biochem J. 2024 Oct 17;481(20):1395-1410. doi: 10.1042/BCJ20240114.
Platelets are critical mediators of hemostasis and thrombosis. Platelets circulate as discs in their resting form but change shape rapidly upon activation by vascular damage and/or soluble agonists such as thrombin. Platelet shape change is driven by a dynamic remodeling of the actin cytoskeleton. Actin filaments interact with the protein myosin, which is phosphorylated on the myosin light chain (MLC) upon platelet activation. Actin-myosin interactions trigger contraction of the actin cytoskeleton, which drives platelet spreading and contractile force generation. Filamin A (FLNA) is an actin cross-linking protein that stabilizes the attachment between subcortical actin filaments and the cell membrane. In addition, FLNA binds multiple proteins and serves as a critical intracellular signaling scaffold. Here, we used platelets from mice with a megakaryocyte/platelet-specific deletion of FLNA to investigate the role of FLNA in regulating platelet shape change. Relative to controls, FLNA-null platelets exhibited defects in stress fiber formation, contractile force generation, and MLC phosphorylation in response to thrombin stimulation. Blockade of Rho kinase (ROCK) and protein kinase C (PKC) with the inhibitors Y27632 and bisindolylmaleimide (BIM), respectively, also attenuated MLC phosphorylation; our data further indicate that ROCK and PKC promote MLC phosphorylation through independent pathways. Notably, the activity of both ROCK and PKC was diminished in the FLNA-deficient platelets. We conclude that FLNA regulates thrombin-induced MLC phosphorylation and platelet contraction, in a ROCK- and PKC-dependent manner.
血小板是止血和血栓形成的关键介质。血小板在其静止状态下以圆盘形式循环，但在血管损伤和/或可溶性激动剂（如凝血酶）的激活下会迅速改变形状。血小板形状的变化是由肌动蛋白细胞骨架的动态重塑驱动的。肌动蛋白丝与肌球蛋白相互作用，肌球蛋白在血小板激活时在肌球蛋白轻链（MLC）上磷酸化。肌动蛋白-肌球蛋白相互作用触发肌动蛋白细胞骨架的收缩，从而驱动血小板扩展和收缩力的产生。细丝蛋白 A（FLNA）是一种肌动蛋白交联蛋白，可稳定皮质下肌动蛋白丝与细胞膜之间的附着。此外，FLNA 结合多种蛋白质，并作为关键的细胞内信号支架。在这里，我们使用巨核细胞/血小板特异性缺失 FLNA 的小鼠血小板来研究 FLNA 在调节血小板形状变化中的作用。与对照相比，FLNA 缺失的血小板在凝血酶刺激下表现出应力纤维形成、收缩力产生和 MLC 磷酸化缺陷。用抑制剂 Y27632 和双吲哚马来酰亚胺（BIM）分别阻断 Rho 激酶（ROCK）和蛋白激酶 C（PKC）也减弱了 MLC 磷酸化；我们的数据进一步表明，ROCK 和 PKC 通过独立途径促进 MLC 磷酸化。值得注意的是，FLNA 缺陷血小板中的 ROCK 和 PKC 活性均降低。我们得出结论，FLNA 通过 ROCK 和 PKC 依赖性方式调节凝血酶诱导的 MLC 磷酸化和血小板收缩。
