Abstract 42: A Talin Mutant that Disrupts Talin-integrin Binding in Platelets Decelerates aIIbß3 Activation Without Pathological Bleeding

Abstract

Tight regulation of integrin affinity is a critical component of hemostasis. The final step of integrin inside-out activation is talin binding to two distinct sites within the integrin β cytoplasmic tail, an NPLY sequence and a membrane-proximal region (MPR). Selective deletion of talin1 from platelets (Tln1fl/flPf4-Cre+) completely prevents integrin activation and thrombus formation, and results in profound defects in hemostasis. Interestingly, platelet-specific expression of a talin1 mutant (L325R) that lacks the ability to bind the MPR, but can still bind the NPLY sequence, phenocopies talin1-knockout platelets. In this study we sought to analyze the effects on thrombosis and hemostasis of a platelet-specific talin1 mutation (W359A) that dramatically impairs binding to the NPLY sequence and does not rescue the talin1 deficiency in transfected cells. Homozygous knock-in of talin(W359A) is embryonic lethal in mice. Thus, Tln1W359A/wt mice were crossed with Tln1fl/flPf4-Cre+ mice to generate Tln1W359A/flPf4-Cre+ (TlnWA) and Tln1wt/flPf4-Cre+ (Tlnwt) control mice. Expression of talin(W359A) in platelets partially rescued talin1 deficiency. Compared to Tlnwt controls, agonist-induced αIIbβ3 activation was reduced by ~50% and spreading on fibrinogen was only moderately impaired in TlnWA platelets. However, kinetic studies demonstrated decelerated αIIbβ3 activation in TlnWA platelets, which resulted in delayed aggregation under static conditions and reduced thrombus size at low shear rates. Interestingly, adhesion of TlnWA platelets to collagen at high shear rates was not significantly better than that of talin-deficient cells and TlnWA mice were completely protected from FeCl3-induced carotid artery occlusion. Lastly, in contrast to platelet-specific talin-knockout or TlnLR mice, TlnWA mice showed no detectable gastrointestinal bleeding and only modestly increased tail-bleeding times compared to littermate controls. In conclusion, we demonstrate that the talin(W359A) mutation decelerates, but does not abolish, talin-dependent integrin activation in platelets. Our studies further suggest that this delay in integrin activation is protective from pathological vessel occlusion while it does not affect hemostasis in mice.