Immunolocalization of β1 integrins in platelets and platelet-derived microvesicles

Abstract

Human platelets contain several adhesion receptors belonging to the integrin super-family. At least three β1 integrins are present on platelets and have been shown to mediate platelet adhesion to collagen, fibronectin, and laminin. To study the cellular localization of the β1 integrins in platelets, we produced a polyclonal antibody by immunization of goat 172 with purified β1 subunit from HPB-ALL cells. Antibody 172 (Ab172) specifically immunoblotted a 135-Kd protein in a lysate of whole platelets. The reactivity of Ab172 with platelet membrane proteins was further determined by immunoprecipitation of lysates of surface-radioiodinated platelets. Ab172 immunoprecipitates, resolved by nonreducing/reducing two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis consisted of three labeled proteins with migrational properties of platelet glycoprotein (GP)Ia, GPIc and GPIIa. Neither GPIIb/IIIa nor the vitronectin receptor were immunoprecipitated by Ab172, confirming a lack of cross-reactivity with the β3 integrins in platelets, lmmunofluorescence studies using Ab172 were performed to investigate the cellular distribution of β1 integrins in platelets. Fluorescent labeling of intact cells demonstrated the presence of β1 antigen on the surface of resting cells. Permeabilization of platelets with Triton X-100 showed the presence of an intracellular pool of β1 antigen. Double-label experiments using Ab172 and AP-2 (anti-GPIIb/IIIa) showed identical labeling patterns, suggesting a similar subcellular distribution for these integrins. Following thrombin stimulation, permeabilized cells showed a centralized clearing of both β1 antigen and GPIIb/IIIa as well as an intensification of surface labeling for β1 antigen. These findings suggest the translocation of intracellular β1 antigen to the platelet surface as a result of thrombin stimulation. Because platelet-derived microvesicles have been reported to contain GPIIb/ IIIa, we investigated the possible distribution of β1 integrins in these structures. Microvesicles, produced as a result of platelet activation, were labeled with Ab172, suggesting the distribution of β1 integrins in these structures as well as in intact cells. © 1993 by The American Society of Hematology.