Pro32Pro33 Mutations in the Integrin b3 PSI Domain Result in aIIbb3 Priming and Enhanced Adhesion: Reversal of the Hypercoagulability Phenotype by the Src Inhibitor SKI-606 s

The plasma-membrane integrin aIIbb3 (CD41/CD61, GPIIbIIIa) is a major functional receptor in platelets during clotting. A common isoform of integrin b3, Leu33Pro is associated with enhanced platelet function and increased risk for coronary thrombosis and stroke, although these findings remain controversial. To better understand the molecular mechanisms by which this sequence variation modifies platelet function, we produced transgenic knockin mice expressing a Pro32Pro33 integrin b3. Consistent with reports utilizing human platelets, we found significantly reduced bleeding and clotting times, as well as increased in vivo thrombosis, in Pro32Pro33 homozygous mice. These alterations paralleled increases in platelet attachment and spreading onto fibrinogen resulting from enhanced integrin aIIbb3 function. Activation with protease-activated receptor 4– activating peptide, the main thrombin signaling receptor in mice, showed no significant difference in activation of Pro32Pro33 mice as compared with controls, suggesting that inside-out signaling remains intact. However, under unstimulated conditions, the Pro32Pro33 mutation led to elevated Src phosphorylation, facilitated by increased talin interactions with the b3 cytoplasmic domain, indicating that the aIIbb3 intracellular domains are primed for activationwhile the ligand-binding domain remains unchanged. Acute dosing of animals with a Src inhibitor was sufficient to rescue the clotting phenotype in knockin mice to wild-type levels. Together, our data establish that the Pro32Pro33 structural alteration modifies the function of integrin aIIbb3, priming the integrin for outside-in signaling, ultimately leading to hypercoagulability. Furthermore, our data may support a novel approach to antiplatelet therapy by Src inhibition where hemostasis is maintained while reducing risk for cardiovascular disease.