The Small Gtpase Rap1 in Platelets Is Critical for Arterial but Not Venous Thrombosis in Mice

Background: Platelets and their main adhesion receptors, integrins, are critical in hemostasis and arterial thrombosis, i.e., in situations involving severe insult to the vasculature and elevated shear stress, respectively. We recently demonstrated that integrin activation under both of these conditions depends on the small GTPase Rap1 directly activating the integrin adapter protein, Talin1. Our studies further suggested that the Rap1-talin1 axis is less important for platelet function at sites of inflammation, i.e., in situations of mild endothelial insult and low shear stress.

Aim: To investigate the contribution of the platelet Rap1-Talin1-integrin signaling axis in the pathogenesis of venous thrombosis (VT).

Methods: The following mice with documented deficiencies in platelets were subjected to the inferior vena cava (IVC) stenosis VT model: Rap1amKO (Rap1a ^fl/fl pf4-Cre+), Rap1bmKO (Rap1b ^fl/fl pf4-Cre+), or both isoforms (Rap1a ^fl/flRap1b ^fl/fl pf4-Cre+ [Rap1dKO]), and Talin1mKO (Tln1 ^fl/flpf4-Cre+). Thrombus weight was determined 48 hours after flow restriction. Clot contraction and tissue plasminogen activator (tPA)-mediated lysis of whole blood clots were studied ex vivo to characterize effects on clot consolidation and stability .

Results: Compared to control mice, thrombus weight was markedly reduced in Talin1mKO mice (13.1±2 and 2.2±1.2 mg, p<0.05), but not Rap1dKO or single knockout mice. Ex vivo clot contraction was significantly impaired in whole blood from Talin1mKO (clot weight: control 13.35±3.4 mg vs. 34.63±4.2 mg), Rap1amKO (control 18.0±3.9 mg vs. 25.6±6.0 mg), Rap1bmKO (control 18.0±3.9 vs 23.0±6.6 mg) and in Rap1dKO (control 18.03±3.9 mg vs. 30.02±4.6 mg). Clot weights were not significantly different between Rap1dKO and Talin1mKO mice. However, ex vivo clot lysis assays revealed that compared to controls, clots formed in whole blood from Talin1mKO mice lysed faster, whereas clots from Rap1dKO did not.

Conclusions: Platelet Talin1 is critical for VT in mice, and this role is partially independent of its best-known upstream regulator, Rap1. Future studies will identify the alternative mechanism allowing for Rap1-independent Talin1 signaling and platelet function in VT and how these signaling affect various phases of VT.