Subcellular Mechanisms of Platelet Function

血小板活化通常始于内皮下胶原基质的暴露，并随着更多的血小板被可溶性试剂招募到不断增长的血小板中而继续。这些事件背后的信号转导途径一直是人们密切关注的主题，尤其是导致整合素激活的途径。在上一个资金周期观察的基础上，拟议的研究将检验这样一个假设，即即使在发生血小板聚集之后，细胞内信号仍在继续，有助于保持血小板堵塞的生长和稳定性。我们的主要关注点是Eph激酶，这是一个受体酪氨酸激酶家族，它的突起，也就是肾上腺素，也是细胞表面的。 分子。一个细胞上的Eph激酶之间的相互作用！而在另一种情况下，麻翅可以在这两种情况下产生信号。尽管Eph蛋白和Ephin最为人所知的是它们在发育过程中的作用，但我们已经发现，人类血小板至少表达两种Eph蛋白(EpEA4和EphB1)和一种作为两者的配体的Eph蛋白(EPhin BI)。我们还表明，通过Eph激酶和Ephin发出的信号促进了血小板的黏附，而阻断Eph/Ephin的相互作用将导致血小板解聚。我们的机制研究表明，EphA4在血小板聚集过程中形成信号复合体，包括激酶Fyn和Lyn以及细胞黏附分子L1。与他们所提议的作用一致的是，在血小板聚集开始之前，预计肾上腺素与Eph激酶的结合不会发生。基于这些观察，我们现在提出以下四个具体目标。目的#1将重点介绍在血小板聚集过程中，由于Eph/Ephin相互作用而形成的信号复合体的组成，并研究Eph和Ephn磷酸化在这一过程中的作用。目的#2将从我们已经确定的两个信号通路开始，重点放在血小板中由表皮化蛋白相互作用激活的信号通路：Ras家族成员Rap 1B的激活和AlphalIb/beta3β链细胞质结构域的磷酸化。目的#3将使用小鼠模型在体内测试Eph激酶和ephins的拟议作用。目的#4验证EphA4和L1之间的相互作用通过与相邻血小板表面的L1和β3整合素的反式相互作用来影响L1支持血小板聚集的能力的假设。综上所述，拟议的研究应该会为血小板激活的晚期事件提供新的见解，这是一个相对未被探索的研究领域。

Platelet activation typically begins with the exposure of the subendothelial collagen matrix and continues as additional platelets are recruited into the growing platelet mass by soluble agordsts. The signal transduction pathways that underlie these events have been the subject of intense scrutiny, with particular emphasis on pathways leading to integrin activation. Building upon observations made during the last funding cycle, the proposed studies will test the hypothesis that intracellular signaling continues even after platelet aggregation has occurred, helping to perpetuate the growth and stability of the platelet plug. Our primary focus will be upon Eph kinases, a family of receptor tyrosine kinases whose tigands, the ephrins, are also cell surface molecules. Interactions between Eph kinases on one cel! and ephfins on another can produce signaling in both. Although Eph kinases and ephrins are best known for their role during development, we have found that human platelets express at least two Eph kinases (EpEA4 and EphB1) and an ephrin (ephrin BI) that is a ligand for both. We have also shown that signaling through Eph kinases and ephrins promotes platelet adhesion, and that blockade of Eph/ephrin interactions will cause platelets to disaggregate. Our investigations of mechanism show that EphA4 forms signaling complexes during platelet aggregation that include the kinases, Fyn and Lyn, and the cell adhesion molecule, L1. Consistent with their proposed role, the binding of ephrins to Eph kinases would not be expected to occur until after platelet aggregation has begun. Drawing on these observations, we now propose the following four specific aims. Aim #1 will focus on the composition of the signaling complexes that form as a consequence of Eph/ephrin interactions during platelet aggregation, and examine the role of Eph and epbdn phosphorylation in this process. Aim #2 will focus on the signaling pathways activated by Eptdephrin interactions in platelets, starting with two that we have already identified: activation of the Ras family member, Rap 1B, and phosphorylation of the cytoplasmic domain of the beta chain of alphalIb/beta3. Aim #3 will test the proposed role of Eph kinases and ephrins in vivo using mouse models. Aim #4 will examine the hypothesis that interactions between EphA4 and L1 affect the ability of L1 to support platelet aggregation by interacting in trans with L1 and beta3 integrins on the surface of adjacent platelets. Taken together, the proposed studies should provide new insights into the late events of platelet activation, an area of investigation that is relatively unexplored.