Abstract：
Bi-directional signaling through platelet integrin αIIbβ3 is essential in hemostasis and thrombosis. In quiescent platelets αIIbβ3 is in a low-affinity ligand binding state. However, upon platelet activation by agonists through inside-out signaling, a rapid switch in the conformation of the integrin results in a high affinity ligand binding state capable of binding soluble fibrinogen. Ligand binding to the αIIbβ3 induces a signaling termed outside-in signaling that regulate platelet spreading and clot retraction. These events are often interchangeably used to represent outside-in signaling pathway. Using pharmacological inhibitors of known signaling molecules that have been implicated to regulate outside-in signaling, we assessed human platelet spreading and clot retraction. We found that inhibition of PI3K, PLC, PKC, and FAK strongly attenuated both platelet spreading and clot retraction suggesting that they are essential for both clot retraction and platelet spreading. Whereas inhibition of Rac1, ROCK, p38, and MEK did not affect platelet spreading but significantly delayed clot retraction suggesting that these signaling molecules do not participate in platelet spreading. Interestingly, Src family kinases (SFKs) are required for platelet spreading and FAK activation but suppresses clot retraction since their inhibition causes faster clot retraction. Thus, it becomes evident that platelet spreading, and clot retraction are differently regulated through αIIbβ3 outside-in signaling and should not be used interchangeably as readout for αIIbβ3 outside-in signaling assessment. Significance Statement Current anti-platelet drugs have increased risk of bleeding and low efficacy. There is an increased effort to identify novel anti-platelet agents that have improved efficacy with reduced risk of bleeding. It is increasingly felt that inhibition of αIIbβ3-induced outside-in signaling may inhibit thrombosis without compromising hemostasis. However, the signaling entities regulating outside-in signaling is poorly understood. Our work included in this manuscript delineates the distinct signaling pathways involved in outside-in signaling and identify potential novel targets for intervention of thrombosis.
摘要：
通过血小板整合素αIIbβ3的双向信号传导在止血和血栓形成中至关重要。在静止的血小板中,αIIbβ3处于低亲和力配体结合状态。然而,在激动剂通过由内而外的信号激活血小板后，整联蛋白构象的快速转换导致能够结合可溶性纤维蛋白原的高亲和力配体结合状态。与αIIbβ3结合的配体诱导称为外-内信号传导的信号传导,其调节血小板扩散和凝块收缩。这些事件通常可互换地用于表示外-内信号传导途径。使用已知信号分子的药理学抑制剂，这些信号分子涉及调节外向内信号，我们评估了人血小板扩散和凝块回缩.我们发现抑制PI3K，PLC,PKC,和FAK强烈减弱血小板扩散和凝块回缩,表明它们对于凝块回缩和血小板扩散都是必需的。而抑制Rac1，ROCK，p38和MEK不影响血小板扩散，但显著延迟凝块收缩，表明这些信号分子不参与血小板扩散。有趣的是,Src家族激酶(SFK)是血小板扩散和FAK活化所必需的,但由于它们的抑制导致更快的凝块回缩,因此抑制凝块回缩。因此，很明显，血小板扩散，和凝块回缩通过αIIbβ3外-内信号传导受到不同的调节，不应互换用作αIIbβ3外-内信号传导评估的读数。重要声明当前的抗血小板药物具有增加的出血风险和低疗效。越来越努力鉴定具有改善的功效和降低的出血风险的新型抗血小板剂。越来越感到抑制αIIbβ3诱导的内外信号传导可以抑制血栓形成而不损害止血。然而,对调节内外信令的信令实体知之甚少。本手稿中我们的工作描述了参与内外信号传导的不同信号通路，并确定了干预血栓形成的潜在新目标。
