Functional redundancy between RAP1 isoforms in murine platelet production and function.

RAP GTPases, important regulators of cellular adhesion, are abundant signaling molecules in the platelet/megakaryocytic lineage. However, mice lacking the predominant isoform, RAP1B, display a partial platelet integrin activation defect and have a normal platelet count, suggesting the existence of a RAP1-independent pathway to integrin activation in platelets and a negligible role for RAP GTPases in megakaryocyte biology. To determine the importance of individual RAP isoforms on platelet production and on platelet activation at sites of mechanical injury or vascular leakage, we generated mice with megakaryocyte-specific deletion (mKO) of Rap1a and/or Rap1b Interestingly, Rap1a/b-mKO mice displayed a marked macrothrombocytopenia due to impaired proplatelet formation by megakaryocytes. In platelets, RAP isoforms had redundant and isoform-specific functions. Deletion of RAP1B, but not RAP1A, significantly reduced α-granule secretion and activation of the cytoskeleton regulator RAC1. Both isoforms significantly contributed to thromboxane A2 generation and the inside-out activation of platelet integrins. Combined deficiency of RAP1A and RAP1B markedly impaired platelet aggregation, spreading, and clot retraction. Consistently, thrombus formation in physiological flow conditions was abolished in Rap1a/b-mKO, but not Rap1a-mKO or Rap1b-mKO, platelets. Rap1a/b-mKO mice were strongly protected from experimental thrombosis and exhibited a severe defect in hemostasis after mechanical injury. Surprisingly, Rap1a/b-mKO platelets were indistinguishable from controls in their ability to prevent blood-lymphatic mixing during development and hemorrhage at sites of inflammation. In summary, our studies demonstrate an essential role for RAP1 signaling in platelet integrin activation and a critical role in platelet production. Although important for hemostatic/thrombotic plug formation, platelet RAP1 signaling is dispensable for vascular integrity during development and inflammation.

Loss of P2Y1 receptor desensitization does not impact hemostasis or thrombosis despite increased platelet reactivity in vitro.

BACKGROUND: The hemostatic plug formation at sites of vascular injury is strongly dependent on rapid platelet activation and integrin-mediated adhesion and aggregation. However, to prevent thrombotic complications, platelet aggregate formation must be a self-limiting process. The second-wave mediator adenosine diphosphate (ADP) activates platelets via Gq-coupled P2Y1 and Gi-coupled P2Y12 receptors. After ADP exposure, the P2Y1 receptor undergoes rapid phosphorylation-induced desensitization, a negative feedback mechanism believed to be critical for limiting thrombus growth. OBJECTIVE: The objective of this study was to examine the role of rapid P2Y1 receptor desensitization on platelet function and thrombus formation in vivo. METHODS: We analyzed a novel knock-in mouse strain expressing a P2Y1 receptor variant that cannot be phosphorylated beyond residue 340 (P2Y1340-0P), thereby preventing the desensitization of the receptor. RESULTS: P2Y1340-0P mice followed a Mendelian inheritance pattern, and peripheral platelet counts were comparable between P2Y1340-0P/340-0P and control mice. In vitro, P2Y1340-0P/340-0P platelets were hyperreactive to ADP, showed a robust activation response to the P2Y1 receptor-selective agonist, MRS2365, and did not desensitize in response to repeated ADP challenge. We observed increased calcium mobilization, protein kinase C substrate phosphorylation, alpha granule release, activation of the small GTPase Rap1, and integrin inside-out activation/aggregation. This hyperreactivity, however, did not lead to increased platelet adhesion or excessive plug formation under physiological shear conditions. CONCLUSION: Our studies demonstrate that receptor phosphorylation at the C-terminus is critical for P2Y1 receptor desensitization in platelets and that impaired desensitization leads to increased P2Y1 receptor signaling in vitro. Surprisingly, desensitization of the P2Y1 receptor is not required for limiting platelet adhesion/aggregation at sites of vascular injury, likely because ADP is degraded quickly or washed away in the bloodstream.

Protein kinase C signaling dysfunction in von Willebrand disease (p.V1316M) type 2B platelets.

von Willebrand disease (VWD) type 2B is characterized by gain-of-function mutations in von Willebrand factor (VWF), enhancing its binding affinity for the platelet receptor glycoprotein (GP)Ibα. VWD type 2B patients display a bleeding tendency associated with loss of high-molecular-weight VWF multimers and variable thrombocytopenia. We recently demonstrated that a marked defect in agonist-induced activation of the small GTPase, Rap1, and integrin αIIbß3 in VWD (p.V1316M) type 2B platelets also contributes to the bleeding tendency. Here, we investigated the molecular mechanisms underlying impaired platelet Rap1 signaling in this disease. Two distinct pathways contribute to Rap1 activation in platelets: rapid activation mediated by the calcium-sensing guanine nucleotide exchange factor CalDAG-GEF-I (CDGI) and sustained activation that is dependent on signaling by protein kinase C (PKC) and the adenosine 5'-diphosphate receptor P2Y12. To investigate which Rap1 signaling pathway is affected, we expressed VWF/p.V1316M by hydrodynamic gene transfer in wild-type and Caldaggef1-/- mice. Using αIIbß3 integrin activation as a read-out, we demonstrate that platelet dysfunction in VWD (p.V1316M) type 2B affects PKC-mediated, but not CDGI-mediated, activation of Rap1. Consistently, we observed decreased PKC substrate phosphorylation and impaired granule release in stimulated VWD type 2B platelets. Interestingly, the defect in PKC signaling was caused by a significant increase in baseline PKC substrate phosphorylation in circulating VWD (p.V1316M) type 2B platelets, suggesting that the VWF-GPIbα interaction leads to preactivation and exhaustion of the PKC pathway. Consistent with PKC preactivation, VWD (p.V1316M) type 2B mice also exhibited marked shedding of platelet GPIbα. In summary, our studies identify altered PKC signaling as the underlying cause of platelet hypofunction in p.V1316M-associated VWD type 2B.

Deletion of the Arp2/3 complex in megakaryocytes leads to microthrombocytopenia in mice.

Actin reorganization regulates key processes in platelet activation. Here we examined the role of the Arp2/3 complex, an essential component in actin filament branching, in platelet function. The Arpc2 gene, encoding the p34 subunit of the Arp2/3 complex, was deleted in the megakaryocyte lineage (Arpc2fl/flPF4-Cre). Deletion of the Arp2/3 complex resulted in marked microthrombocytopenia in mice, caused by premature platelet release into the bone marrow compartment and impaired platelet survival in circulation. Arpc2fl/flPF4-Cre platelets exhibited alterations in their actin cytoskeleton and their peripheral microtubule coil. Thrombocytopenia was alleviated following clodronate liposome-induced macrophage depletion in Arpc2fl/flPF4-Cre mice. Arpc2fl/flPF4-Cre platelets failed to spread and showed a mild defect in integrin activation and aggregation. However, no significant differences in hemostasis or thrombosis were observed between Arpc2fl/flPF4-Cre and control mice. Thus, Arp2/3 is critical for platelet homeostasis but plays only a minor role for vascular hemostasis.