Two distinct roles of integrin ??llb????? ???outside-in??? signaling: platelet spreading and clot retraction

Naik, Ulhas P.Platelet activation is central, in the process of physiological hemostasis and pathological thrombosis. In quiescent platelets the megakaryocyte/platelet lineage specific integrin ??IIb??3 is found in a low-affinity ligand binding state. However, upon platelet activation there is a rapid switch in the conformation of the integrin to a high affinity ligand binding state (a.k.a. ???inside-out??? signaling) ultimately facilitating platelet aggregate formation. Ligand occupied integrin then sends another signal into the platelet (a.k.a. ???outside-in??? signaling) regulating processes of platelet spreading and clot retraction. Previously, we had identified Calcium and integrin binding protein ??? 1 (CIB1) as a binding partner of the cytoplasmic tail of integrin subunit ??IIb. Former work on CIB1 done at other laboratories had implicated CIB1 as an activator as well as an inhibitor of integrin ??IIb??3 activation. Furthermore, experimental evidences from our lab indicated CIB1 as an effector molecule regulating integrin signaling. Thus, amidst contradictory literature, I sought to investigate the actual role of CIB1 in integrin ???inside-out??? signaling. Using Cib1 knock-out murine platlelets, my results show that Cib1 is not required for agonist induced integrin activation as well as dense granular ATP release. Later in my study I went ahead to characterize the integrin ???outside-in??? signaling pathway using pharmacological inhibitors and human platelet spreading and clot retraction as my read-outs. The results obtained show that the enzymes PI3K, PLC, PKC and FAK are all important regulators of integrin ???outside-in??? signaling and control both platelet spreading as well as clot retraction. However, p38 MAPK and ROCK are only required for clot retraction and are dispensable for platelet spreading. Furthermore, Src tyrosine kinase on one hand is required for platelet spreading and FAK activation, whereas, its inhibition causes faster clot retraction. Thus, it becomes evident that integrin mediated outside-in signaling regulates two distinct processes viz., clot retraction and platelet spreading, and either of them cannot be used interchangeably while studying integrin ???outside-in??? signaling.University of Delaware, Department of Biological SciencesM.S