Psi Domain Of Beta 3 Integrin Has Endogenous Thiol Isomerase Function And Is A Novel Antithrombotic Therapeutic Target

Integrin αIIbβ3 plays a critical role in platelet aggregation and adhesion, key events in hemostasis and thrombosis. Integrin activation involves complex signalling events that lead to conformational changes exposing ligand-binding sites; however, mechanisms underlying integrin activation remain poorly understood. The β subunit contains a PSI domain that is highly conserved across integrins and species, though its function is unknown. Integrin β subunits are cysteine-rich and endogenous thiol isomerase activity in integrin β3 has been reported. The PSI domain contains two CXXC sequences, the active site motif of protein disulfide isomerase (PDI). Based on this observation and the location of this domain at the knee region of the integrin, we hypothesized that integrin PSI domain has endogenous thiol isomerase function, which plays a key regulatory role in integrin conformation and function. Targeting the PSI domain may have therapeutic potential. Using reduced, denatured RNase, a recombinant murine integrin β3 PSI domain demonstrated endogenous PDI-like activity. This PDI-like activity was dose-dependently inhibited by the PDI inhibitor, bacitracin. Mutation of either CXXC motif within the integrin β3 PSI domain reduced PDI-like activity, while removal of both CXXC motifs completely abolished this activity. We developed unique mouse anti-mouse/anti-human β3 PSI domain monoclonal antibodies (anti-PSI mAbs) that inhibited the PDI-like activity of both the murine recombinant integrin β3 PSI domain and purified human platelet β3 integrin, in a dose-dependent manner. Interestingly, the anti-PSI mAbs blocked fibrinogen to human platelet β3 integrin in a cell free system. Furthermore, anti-PSI mAbs inhibited murine and human platelet aggregation in vitro and ex vivo and inhibited murine thrombus formation in vivo without significantly changing bleeding time or platelet count. In conclusion, we identified that the PSI domain has PDI function, is a fundamental regulator of platelet β3 integrin activation, and is a potential novel target for anti-thrombotic therapies. Since PSI domain is conserved in all integrin β subunits, our discovery may have broad implications for the role of integrins in cell biology of many human diseases.