Figure 4.

Platelet tyrosine phosphorylation, fibrin clot retraction, and binding properties of β3 cytoplasmic domain mutants. (A) Platelets were incubated in suspension in the presence of 250 μg/mL fibrinogen plus or minus 0.5 mM MnCl₂ for 20 minutes at room temperature and then lysed. Protein tyrosine phosphorylation was assessed by immunoblotting with antiphosphotyrosine antibodies (pTyr). Blots were stripped and reprobed with an antibody to c-Src. (B) Phosphorylation of β3 Tyr⁷⁴⁷ was detected with a phospho-specific antibody. Blots were reprobed with an antibody to total β3 and phosphorylation data were normalized for β3 expression. The bar graph depicts the fold-increase in β3 Tyr⁷⁴⁷ phosphorylation induced by MnCl₂ + fibrinogen. Data are means (± range) for 2 independent experiments. (C) Pull-down of c-Src and talin from mouse platelet lysate (PltLys) by recombinant wild-type and mutant β3 cytoplasmic domain model proteins. An αIIb cytoplasmic domain protein was used as a negative control. Bound c-Src and talin were detected by immunoblotting. Loading of pull-down beads with recombinant cytoplasmic domains was monitored by staining with Coomassie brilliant blue. (D) Fibrin clot retraction using platelet-rich plasma from wild-type or mutant mice. Clotting was initiated with 9.4 U/mL thrombin and 1.9 mM CaCl₂. Data represent mean (± SEM) for least 3 separate experiments performed in duplicate on at least 4 animals with each genotype. Experiments with wild-type platelets in the presence of 10 μM cytochalasin D (cyto D) or vehicle (dimethyl sulfoxide [DMSO]) were performed in duplicate on platelets from 4 animals to confirm that clot retraction was dependent on actin polymerization in this assay.