Outside-In Signaling by Integrin αIIbβ3 Is Dramatically Affected by the Density of Adsorbed Fibrinogen and Results in Differences in Calcium Mobilization, Integrin Engagement, and Platelet Morphology.

Abstract

αIIbβ3-mediated outside-in signaling following platelet adhesion to fibrinogen affects platelet adhesion, degranulation, and spreading, but the precise mechanisms involved remain incompletely understood. In this study, we demonstrate that the character of αIIbβ3 signaling is dependent on the surface density of the adsorbed ligand. Time-lapse total internal reflection fluorescence microscopy (TIR-FM) allowed us to visualize the interactions between fibrinogen and the αIIbβ3 integrins on the basal membrane of adhering platelets using a fluorescently-labeled antibody specific for β3. We observed significant morphological and biochemical differences in the initial interaction between αIIbβ3 and fibrinogen depending on the fibrinogen surface density. These included differences in the kinetics of filopodia and lamellipodia formation. Although filopodia started to form at the same time after initial platelet contact with both low-density (adsorbed from 3 μg/ml) fibrinogen (~ 40–50 s), new filopodia formed for a significantly shorter period of time on low- than on high-density fibrinogen [120 s (n = 79) vs. 235 s (n = 62); medians; p< 0.001]. Lamellipodia started to form in platelets adhering to low-density fibrinogen significantly later than in platelets on high-density fibrinogen (140 s vs. 110 s; p = 0.04). Moreover, once lamellipodia started to appear, formation of new filopodia ceased in platelets adhering to low-density fibrinogen. In contrast, in platelets on high-density fibrinogen new filopodia continued to form even in the presence of lamellipodia for another 155 s. We also observed that a higher percentage of platelets adherent to high-density fibrinogen (52 ± 6 %) failed to develop a cytoplasmic Ca2+ signal compared to platelets adherent to low-density fibrinogen (13 ± 11 %; n = 3; p<0.001). In addition, only 8 ± 6 % of platelets on high-density fibrinogen developed sustained Ca2+ oscillations compared to 39 ± 14 % of platelets on low-density fibrinogen (n = 3; p = 0.01). TIR-FM analysis of platelets stained with a monoclonal antibody specific for a ligand-induced binding site in the PSI domain of β3 (AP5), showed that on low-density fibrinogen only receptors in a very thin and stationary ring at the edge of fully spread platelets were stained, whereas on high-density fibrinogen AP5 stained a thicker, more diffuse, and more mobile ring. These observations suggested that αIIbβ3 interactions with high-density fibrinogen are more transient than those with low-density fibrinogen. To test this directly, platelets adherent to fibrinogen for 1 h were treated with αIIbβ3 antagonists or EDTA and the loss of adhesion was then measured. Treatement with c7E3, tirofiban, or EDTA resulted in loss of 66 ± 13%, 57 ± 21%, and 63 ± 25%, respectively, of platelets on high density fibrinogen. In contrast, the same treatments resulted in loss of only 25 ± 3%, 34 ± 14%, and 31 ± 21% of platelets on low-density fibrinogen (n = 3; p ≤ 0.01 for each treatment). Thus, we provide evidence that the density of a single immobilized ligand can profoundly affect the morphological and biochemical nature of events leading to platelet adhesion, spreading, and outside-in signaling. These observations have potential significance for hemostasis, thrombosis, and the development of biocompatible materials.