Stimulated Platelets but Not Endothelium Generate Thrombin Via a Factor XIIa-Dependent Mechanism Requiring Phosphatidylserine Exposure

Abstract

To inhibit pathological thrombus formation without impairing hemostasis is the holy grail of anticoagulant therapy. Recent data from animal models have indicated that factor XII (FXII) may be a promising new antithrombotic target that is particularly intriguing due to the longstanding clinical observation that severe congenital FXII deficiency is not associated with a bleeding diathesis in humans. FXII is thought to participate in thrombus formation after being activated in high shear arterial environments. FXIIa then initiates downstream activation of the contact pathway, culminating in thrombin generation. However, the relevant cell surface for FXII activation remains unclear. Here we compare the role of platelets versus endothelial cells in FXII activation and study the function of surface phospholipids in this process.To explore the effect of FXII inhibition on thrombus formation in vivo, we used antibody X210-C01, a novel human IgG1 developed using phage-display technology that blocks both mouse and human FXIIa. Using a mouse laser injury model of arterial thrombosis, we showed that X210-C01 inhibited both fibrin formation and platelet accumulation at sites of vascular injury. Plasma removed from animals after completion of these experiments was used to quantify the concentrations of X210-C01 achieved in vivo at a given dose. FXII inhibition was somewhat more potent in preventing platelet accumulation (IC50 dose = 27 mg/kg, R2=0.93) than fibrin formation (IC50 dose = 43 mg/kg, R2=0.95). Importantly, treatment with X210-C01 at 100 mg/kg did not prolong bleeding times or increase total blood loss in a tail bleeding assay.To evaluate the mechanism underlying our in vivo observations, we studied the differential role of FXII in thrombin generation by stimulated platelets and endothelium. X210-C01 did not globally inhibit SFLLRN-induced platelet aggregation or granule release. We next performed a fluorogenic thrombin generation assay (TGA) using human platelets treated with the peptide agonist SFLLRN. X210-C01 inhibited platelet-based thrombin generation in a dose-dependent fashion, whereas anti-tissue factor (TF) and anti-factor VIIa (FVIIa) antibodies did not. By contrast, in a similar TGA using SFLLRN-stimulated endothelial cells, X210-C01 had no effect, while anti-TF antibodies abrogated thrombin generation. These results indicate that stimulated endothelium generates thrombin by a mechanism distinct from that of platelets.FXII is known to be activated in vitro by anionic surfaces. Because phosphatidylserine (PS) is a negatively-charged phospholipid expressed on the surface of stimulated platelets, we reasoned that PS may serve as the platelet-based activator of FXII. To test this hypothesis, we used lactadherin, a potent and specific inhibitor of PS, in the platelet-based TGA and showed that PS blockade inhibited platelet-based thrombin generation at concentrations as low as 10 nM. We then used a chromogenic FXIIa activity assay to test the ability of PS-containing liposomes to activate FXII. Liposomes containing 80% phosphatidylcholine (PC) and 20% PS (PC-PS 80/20) failed to activate FXII at concentrations as high as 100 µM. In this assay, SFLLRN-stimulated platelets led to significantly greater FXII activation than either resting platelets or the PC-PS 80/20 liposomes tested.In summary, we have made the unexpected finding that thrombin generation on the surface of stimulated platelets proceeds by a FXIIa-dependent pathway and does not require FVIIa or TF. By contrast, thrombin generation on endothelium requires TF but not FXIIa. Additionally, surface PS is necessary but not sufficient for platelet-based FXII activation and thrombin generation, pointing to the involvement of a second platelet component. Further studies will be directed towards investigating the in vivo role of platelet-based FXII activation in arterial thrombus formation. DisclosuresMason:Shire Pharmaceuticals: Employment. Kenniston:Shire Pharmaceuticals: Employment.