Src family kinases (SFKs) play a central role in mediating the rapid response of platelets to vascular injury. They transmit activation signals from a diverse repertoire of platelet surface receptors, including the integrin αIIbβ3, the immunoreceptor tyrosine–based activation motif–containing collagen receptor complex GPVI-FcR γ-chain, and the von Willebrand factor receptor complex GPIb-IX-V, which are essential for thrombus growth and stability. Ligand-mediated clustering of these receptors triggers an increase in SFK activity and downstream tyrosine phosphorylation of enzymes, adaptors, and cytoskeletal proteins that collectively propagate the signal and coordinate platelet activation. A growing body of evidence has established that SFKs also contribute to Gq- and Gi-coupled receptor signaling that synergizes with primary activation signals to maximally activate platelets and render them prothrombotic. Interestingly, SFKs concomitantly activate inhibitory pathways that limit platelet activation and thrombus size. In this review, we discuss past discoveries that laid the foundation for this fundamental area of platelet signal transduction, recent progress in our understanding of the distinct and overlapping functions of SFKs in platelets, and new avenues of research into mechanisms of SFK regulation. We also highlight the thrombotic and hemostatic consequences of targeting platelet SFKs.

Platelets are highly reactive cell fragments that adhere to exposed extracellular matrix (ECM) and prevent excessive blood loss by forming clots. Paradoxically, megakaryocytes, which produce platelets in the bone marrow, remain relatively refractory to the ECM-rich environment of the bone marrow despite having the same repertoire of receptors as platelets. These include the ITAM (immunoreceptor tyrosine-based activation motif)-containing collagen receptor complex, which consists of glycoprotein VI (GPVI) and the Fc receptor γ-chain, and the ITIM (immunoreceptor tyrosine-based inhibition motif)-containing receptor G6b-B. We showed that mice lacking G6b-B exhibited macrothrombocytopenia (reduced platelet numbers and the presence of enlarged platelets) and a susceptibility to bleeding as a result of aberrant platelet production and function. Platelet numbers were markedly reduced in G6b-B-deficient mice compared to those in wild-type mice because of increased platelet turnover. Furthermore, megakaryocytes in G6b-B-deficient mice showed enhanced metalloproteinase production, which led to increased shedding of cell-surface receptors, including GPVI and GPIbα. In addition, G6b-B-deficient megakaryocytes exhibited reduced integrin-mediated functions and defective formation of proplatelets, the long filamentous projections from which platelets bud off. Together, these findings establish G6b-B as a major inhibitory receptor regulating megakaryocyte activation, function, and platelet production.

Platelets play a fundamental role in hemostasis and thrombosis. They are also involved in pathologic conditions resulting from blocked blood vessels, including myocardial infarction and ischemic stroke. Platelet adhesion, activation, and aggregation at sites of vascular injury are regulated by a diverse repertoire of tyrosine kinase–linked and G protein–coupled receptors. Src family kinases (SFKs) play a central role in initiating and propagating signaling from several platelet surface receptors; however, the underlying mechanism of how SFK activity is regulated in platelets remains unclear. CD148 is the only receptor-like protein tyrosine phosphatase identified in platelets to date. In the present study, we show that mutant mice lacking CD148 exhibited a bleeding tendency and defective arterial thrombosis. Basal SFK activity was found to be markedly reduced in CD148-deficient platelets, resulting in a global hyporesponsiveness to agonists that signal through SFKs, including collagen and fibrinogen. G protein–coupled receptor responses to thrombin and other agonists were also marginally reduced. These results highlight CD148 as a global regulator of platelet activation and a novel antithrombotic drug target.

Key Points• The protein-tyrosine phosphatases Shp1 and Shp2 are critical regulators of megakaryocyte development, platelet production, and function.• Shp1 and Shp2 perform mainly distinct functions in megakaryocytes and platelets, with little functional overlap.The SH2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2 have been implicated in regulating signaling from a variety of platelet and megakaryocyte receptors.In this study, we investigate the functions of Shp1 and Shp2 in megakaryocytes and platelets. Megakaryocyte/platelet (MP)-specific deletion of Shp1 in mice resulted in platelets being less responsive to collagen-related peptide due to reduced GPVI expression and signaling via the Src family kinase (SFK)-Syk-PLCg2 pathway, and fibrinogen due to reduced SFK activity. By contrast, deletion of Shp2 in the MP lineage resulted in macrothrombocytopenia and platelets being hyper-responsive to anti-CLEC-2 antibody and fibrinogen. Shp1-and Shp2-deficient megakaryocytes had partial blocks at 2N/4N ploidy; however, only the latter exhibited reduced proplatelet formation, thrombopoietin, and integrin signaling. Mice deficient in both Shp1 and Shp2 were severely macrothrombocytopenic and had reduced platelet surface glycoprotein expression, including GPVI, aIIbb3, and GPIba. Megakaryocytes from these mice were blocked at 2N/4N ploidy and did not survive ex vivo. Deletion of the immunoreceptor tyrosine-based inhibition motif-containing receptor G6b-B in the MP lineage phenocopied multiple features of Shp1/2-deficient mice, suggesting G6b-B is a critical regulator of Shp1 and Shp2. This study establishes Shp1 and Shp2 as major regulators of megakaryocyte development, platelet production, and function. (Blood. 2013;121(20):4205-4220)