Blocking αIIbβ3-fibrinogen interactions prevents spreading of human platelets and thrombus formation over immobilized IgG.

<p><b>(A)</b> Washed human platelets spread on BSA- or IgG-coated coverslips for 30 minutes in the presence or absence of the integrin αIIbβ3 antagonist abciximab (6.7 μg/ml) or Fab fragments of the FcγRIIa blocking antibody mAb IV.3 (10 μg/ml). Spread platelets were fixed, permeabilized and stained with rhodamine-phalloidin. Scale bar, 5μm. Images are representative of three independent experiments. <b>(B)</b> Platelet spreading was quantified using Metamorph software and shown as the mean μm² ± SEM of at least 200 platelets/group from one of 3 representative experiments. (*<i>P</i><0.05). Statistically significant differences were identified by performing a one-way ANOVA followed by a two-tailed Student’s t test. Note that abciximab or IV.3 Fab significantly inhibited platelet spreading on immobilized IgG. <b>(C)</b> mAb IV.3 immunoprecipitates of lysed spread platelets were analyzed by Western blot with the indicated antibodies. The blots for P-FAK and FAK were performed using whole cell lysates. Note that platelet binding to immobilized IgG elicits strong activation of FcγRIIa and Fak, as well as enhanced recruitment of Syk, and that both abciximab and mAb IV.3 inhibit platelet spreading-induced phosphorylation. <b>(D)</b> Mepacrine-labeled whole blood was perfused at a shear rate 5 dynes/cm² over 100 μg/ml IgG-coated microchannels in the presence or absence of 10 μg/ml mAb IV.3 Fabs, 6.7 μg/ml abciximab, or an isotype-matched control Fab. Epifluorescence microscopic images of platelet adhesion and thrombus formation shown are representative of three separate experiments. Note that abciximab inhibits thrombus formation over immobilized IgG under conditions of flow.