Background: The small GTPase Rap1 is a central regulator of platelet function and hemostatic plug formation. Rap1 is best known for its crucial role in integrin inside-out activation and cellular adhesion. Previous studies also suggest a role for Rap1 signaling in phosphatidylserine (PS) exposure and platelet procoagulant response.

Aims: To determine if and how Rap1 contributes to platelet procoagulant response in vitro and in vivo.

Methods: PS exposure response in vitro was determined by flow cytometry (annexin V). In vivo, PS exposure, platelet adhesion and fibrin formation were monitored in hemostatic plugs by spinning disk confocal microscopy. Three-dimensional stacks of hemostatic plugs were obtained every 10 seconds and analyzed with ImageTank software.

Results: In vitro, deficiency in both Rap1 isoforms in the megakaryocyte lineage (Rap1^mKO) resulted in markedly decreased platelet PS exposure following cellular stimulation. The defect in PS exposure was partially compensated by concomitant inhibition of RhoA/ROCK signaling. In vivo, the importance of procoagulant platelets for fibrin formation was demonstrated in mice lacking cyclophilinD (CypD) in platelets only, generated by adoptive transfer of CypD^-/- platelets into thrombocytopenic mice. To determine the procoagulant function of Rap1^mKO platelets in vivo, a mixture of WT/CypD^-/- or Rap1^mKO/CypD^-/- platelets was transfused into thrombocytopenic mice. Co-transfusion with CypD^-/-platelets was required to prevent prolonged bleeding and to create a situation where only WT or Rap1^mKO platelets could provide a procoagulant platelet surface. Compared to WT/CypD^-/- mice, fibrin formation was  reduced in hemostatic plugs of Rap1^mKO/CypD^-/- mice.

Conclusions: In summary, we provide the first evidence for a Rap1-RhoA-PS connection in platelets, and proof that platelet Rap1 signaling affects hemostatic plug formation independent of its key role in integrin-mediated adhesion processes.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/the-role-of-rap1-signaling-in-platelet-coagulation-interplay/