Platelet spreading and clot retraction are regulated by 2 distinct α_IIbβ₃ outside-in signaling pathways

Bidirectional signaling through platelet integrin α_IIbβ₃ is essential in hemostasis and thrombosis. In quiescent platelets, α_IIbβ₃ is in a low-affinity ligand binding state. However, on platelet activation by agonists through inside-out signaling, a rapid switch in the conformation of the integrin results in a high affinity ligand binding state capable of binding soluble fibrinogen. Ligand binding to the α_IIbβ₃ induces a signaling termed outside-in signaling that regulate platelet spreading and clot retraction. These events are often interchangeably used to represent outside-in signaling pathway. Using pharmacological inhibitors of known signaling molecules that have been implicated to regulate outside-in signaling, we assessed human platelet spreading and clot retraction. We found that inhibition of phosphoinositide-3-kinase, phospholipase C, protein kinase C, and focal adhesion kinase strongly attenuated both platelet spreading and clot retraction suggesting that they are essential for both clot retraction and platelet spreading, whereas inhibition of Rac1, rho-associated, coiled-coil containing protein kinase, p38, and MEK did not affect platelet spreading but significantly delayed clot retraction suggesting that these signaling molecules do not participate in platelet spreading. Interestingly, Src family kinases are required for platelet spreading and FAK activation but suppress clot retraction because their inhibition causes faster clot retraction. Thus, it becomes evident that platelet spreading, and clot retraction are differently regulated through α_IIbβ₃ outside-in signaling and should not be used interchangeably as readout for α_IIbβ₃ outside-in signaling assessment. SIGNIFICANCE STATEMENT: Current antiplatelet drugs have increased risk of bleeding and low efficacy. There is an increased effort to identify novel antiplatelet agents that have improved efficacy with reduced risk of bleeding. It is increasingly felt that inhibition of α_IIbβ_3-induced outside-in signaling may inhibit thrombosis without compromising hemostasis. However, the signaling entities regulating outside-in signaling are poorly understood. The work included in this article delineates the distinct signaling pathways involved in outside-in signaling and identify potential novel targets for intervention of thrombosis.