Gpibα Engagement Induces Activation of Human Platelet TF and Association with Constitutively Platelet Surface-Bound FVIIa

The membranes of activated platelets assemble clotting protein complexes and enzymatic reactions needed for hemostasis and causing pathological thrombosis. Several reports have shown that human platelets contain TF, but controversies remain on the functional contributions of platelet TF to coagulation initiation. Here, we delineate the mechanism that converts platelet-associated TF to a procoagulant molecule. By combining specific inhibitors with human platelets from normal probands or patients with Glanzmann thrombasthenia or homozygous GPVI deficiency, we analyzed the procoagulant activation of platelet TF by biochemical methods, confocal microscopy, platelet adhesion under flow on VWF and fibrinogen surfaces, and functional coagulation readouts. Platelet stimulation with VWF-ristocetin (VWF-R) did not induce δ- and α-granule release, but translocated phosphatidylserine to the outer membrane. Engagement of GPIbα was sufficient to trigger TF-FVIIa mediated activation of FX with a peak 5 minutes after stimulation. TF activation was not seen upon platelet activation with ADP and strong agonists, such as collagen or TRAP. GPIbα stimulation rapidly increased the co-immunoprecipitation of TF with GPIbα as well as of GPIbα-associated FVIIa with TF. FVIIa was detected on the cell surface of washed platelets by confocal microscopy and GPIbα-induced FXa generation did not require exogenous addition of FVIIa, confirming that FVIIa was constitutively bound to the platelet surface. GPIbα engagement also induced TF cytoplasmic domain phosphorylation dependent on PI3 kinase, Src kinases, and Akt and inhibition of these signaling pathways attenuated GPIbα-dependent induction of TF-FVIIa activity. In contrast, preincubation with PDI inhibitors rutin, RL90, PACMA and 16F16 did not prevent the GPIbα-dependent activation of FXa generation, indicating that thiol-disulfide exchange-dependent activation of TF typical for myelomonocytic cells was not required. We further analyzed a series of individual donors and found that antibody blockade of GPIbα, TF and FVIIa suppressed induction of FXa generation on platelets. In addition, TFPI and its cofactor Protein S suppressed TF-FVIIa-dependent FXa generation, whereas pre-incubation of platelets with α-TFPI and α-Protein S increased activity. Stimulation of platelets from patients with Glanzmann thrombasthenia or homozygous GPVI deficiency as well as of normal platelets treated with tirofiban responded normally to VWF-R with increased FXa generation. Moreover, clotting times of platelet-rich plasma after activation with ristocetin alone were significantly shorter than the control stirred in the aggregometer with added saline. Also, thrombin generation of platelet-rich plasma with no added TF was significantly higher after stimulation with ristocetin than with TRAP, confirming the relevance of the identified TF activation mechanism in a plasma milieu. Taken together, these data link engagement of GPIbα specifically to signaling-dependent activation of platelet-associated TF and FVIIa.