Yacine Boulaftali

Platelets play a critical role in maintaining vascular integrity during inflammation, but little is known about the underlying molecular mechanisms. Here we report that platelet immunoreceptor tyrosine activation motif (ITAM) signaling, but not GPCR signaling, is critical for the prevention of inflammation-induced hemorrhage. To generate mice with partial or complete defects in these signaling pathways, we developed a protocol for adoptive transfer of genetically and/or chemically inhibited platelets into thrombocytopenic (TP) mice. Unexpectedly, platelets with impaired GPCR signaling, a crucial component of platelet plug formation and hemostasis, were indistinguishable from WT platelets in their ability to prevent hemorrhage at sites of inflammation. In contrast, inhibition of GPVI or genetic deletion of Clec2, the only ITAM receptors expressed on mouse platelets, significantly reduced the ability of platelets to prevent inflammation-induced hemorrhage. Moreover, transfusion of platelets without ITAM receptor function or platelets lacking the adapter protein SLP-76 into TP mice had no significant effect on vascular integrity during inflammation. These results indicate that the control of vascular integrity is a major function of immune-type receptors in platelets, highlighting a potential clinical complication of novel antithrombotic agents directed toward the ITAM signaling pathway.

Platelets play a central role in primary hemostasis by forming aggregates that plug holes in injured vessels. Half a century ago, detailed studies of the microvasculature by electron microscopy revealed that under inflammatory conditions that do not induce major disruption to vascular structure, individual platelets are mobilized to the vessel wall, where they interact with leukocytes and appear to seal gaps that arise between endothelial cells. Recent developments in genetic engineering and intravital microscopy have allowed further molecular and temporal characterization of these events. Surprisingly, it turns out that platelets support the recruitment of leukocytes to sites of inflammation. In parallel, however, they exercise their hemostatic function by securing the integrity of inflamed blood vessels to prevent bleeding from sites of leukocyte infiltration. It thus appears that platelets not only serve in concert as building blocks of the hemostatic plug but also act individually as gatekeepers of the vascular wall to help preserve vascular integrity while coordinating host defense. Variants of this recently appreciated hemostatic function of platelets that we refer to as "inflammation-associated hemostasis" are engaged in different contexts in which the endothelium is challenged or dysfunctional. Although the distinguishing characteristics of these variants and the underlying mechanisms of inflammation-associated hemostasis remain to be fully elucidated, they can differ notably from those supporting thrombosis, thus presenting therapeutic opportunities.

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.