PB0470 - Platelet spreading/clot contraction phenotype correlates with venous thrombosis response in integrin signaling mutant mice

Background: Venous thrombosis (VT) affects over a million individuals annually. Platelets modulate VT pathogenesis through the recruitment and activation of inflammatory cells during VT initiation and facilitating clot contraction/resistance to fibrinolysis. Integrin receptors, especially αIIbβ3, regulate adhesion of platelets to the endothelium, other blood cells, and other platelets. αIIbβ3 inside-out activation, mediated by small GTPase Rap1 and cytoskeletal adapter protein Talin1, is critical for integrin-mediated adhesion, and mice lacking either of these proteins are protected from arterial thrombosis (AT). In addition, integrins induce cytoskeletal changes (outside-in signaling) to regulate platelet spreading and clot contraction.

Aims: To evaluate the role of platelet Rap1, Talin1, and αIIbβ3 in VT pathogenesis.

Methods: Talin1fl/flPF4-Cre+(Talin1mKO), Talin1fl/R35,118EPF4-Cre+(Talin1R35,118E), Rap1a/bfl/flPF4-Cre+(Rap1mKO) and wild-type (WT) mice treated with anti-αIIbβ3 antibody were used. Whole blood (WB) was collected via inferior vena cava (IVC). Platelet aggregation and spreading on fibrinogen were studied with platelets activated with physiological agonists. Clot contraction and tissue plasminogen activator (tPA)-mediated clot lysis were studied by thromboelastography and tPA-induced lysis assay. VT was studied using IVC stenosis model. Data were analyzed using paired t-test or one-way ANOVA; P < 0.05 was considered significant.

Results: Integrin inside-out activation and platelet aggregation were dramatically impaired in Rap1mKO, Talin1mKO, Talin1R35,118E (disabled Rap1-talin interaction), and WT/anti-αIIbβ3 mice. Platelet spreading and clot contraction were abolished in Talin1 and WT/anti-αIIbβ3 mice, but only partially impaired in Rap1mKO mice. Consistent with clot contraction phenotype, tPA-induced lysis was observed in Talin1mKO but not Rap1mKO clots. Talin1mKO and WT/anti-αIIbβ3 mice, but not Rap1mKO or Talin1R35,118E mice were protected from IVC stenosis-induced VT.

Conclusion(s): We here demonstrate that platelet integrin signaling machinery required in VT is different from that in AT. Furthermore, our studies suggest that in vitro assays monitoring outside-in signaling (spreading, clot contraction) may be useful to identify proteins regulating talin1-αIIbβ3 function in the context of VT.