Both G protein–coupled and immunoreceptor tyrosine-based activation motif receptors mediate venous thrombosis in mice

Platelets are critical in hemostasis and a major contributor to arterial thrombosis (AT). (Pre)clinical studies suggest platelets also contribute to venous thrombosis (VT), but the mechanisms are largely unknown. We hypothesized that in VT, platelets utilize a signaling machinery distinct from AT. Here we aimed to characterize the contributions of platelet G protein-coupled (GPCR) and immunoreceptor tyrosine-based activation motif (ITAM) receptor signaling to VT. Wild type (WT) and transgenic mice were treated with inhibitors to selectively inhibit platelet signaling pathways: ITAM - CLEC2 (Clec2mKO), GPVI (JAQ1 antibody), and Bruton's tyrosine kinase (ibrutinib); GPCR - cyclooxygenase 1 (aspirin) and P2Y12 (clopidogrel). VT was induced by inferior vena cava stenosis. Thrombin generation in platelet-rich plasma and whole blood clot formation were studied ex vivo. Intravital microscopy was used to study platelet-leukocyte interactions after flow restriction. Thrombus weights were reduced in WT mice treated with high dose aspirin+clopidogrel (DAPT), but not in mice treated with either inhibitor alone or low dose DAPT. Similarly, thrombus weights were reduced in mice with impaired ITAM signaling (Clec2mKO+JAQ1; WT+ibrutinib), but not in Clec2mKO or WT+JAQ1 mice. Both aspirin and clopidogrel, but not ibrutinib, protected mice from FeCl3-induced AT. Thrombin generation and clot formation were normal in blood from high dose DAPT- or ibrutinib-treated mice; however, platelet adhesion and platelet-neutrophil aggregate formation at the vein wall were reduced in high dose DAPT- or ibrutinib-treated mice. In summary, VT initiation requires platelet activation via GPCRs and ITAM receptors. Strong inhibition of either signaling pathway reduces VT in mice.