Fig. 1.

The Src kinase inhibitors and α_IIbβ₃ blockade by GRGDS peptide reduced time course of clot retraction and inhibited protein tyrosine phosphorylation during clot retraction.

(A) Human washed platelets (5 × 10⁸/ml) were preincubated with DMSO, 50 μM PP2, 40 μM PD173952. Clot retraction assays were started by adding 250 μl of 2 U/ml thrombin to 250 μl of platelets in the presence of 2 mg/ml fibrinogen and 2 mM CaCl₂ (final concentrations: 2.5 × 10⁸/ml of platelets, 1 U/ml of thrombin, 1 mg/ml of fibrinogen, 1 mM CaCl₂). The volume of remaining fluid was measured to assay the degree of clot retraction. The volume was expressed mean±SE (n=5-18 from 2-5 experiments). Results were analyzed using unpaired Student's t-test. One asterisk denotes p<0.05 and two denote p<0.005 between control and PP2/PD173952. (B) Human washed platelets in the presence or absence of 1 mM GRGDS peptide were stimulated by 250 μl of 2 U/ml thrombin as described. Reactions were terminated by addition of 2× lysis buffer. Samples were sonicated 3 periods of 15 sec and insoluble debris removed by centrifugation at 15,000 g for 10 min. The supernatant was solubilized by addition of 4× SDS sample buffer. The platelet proteins were separated by SDS-PAGE and blotted with anti-phosphotyrosine antibody (4G10). (C) Human washed platelets (5×10⁸/ml) were preincubated with 50 μM PP3 or PP2 before starting assays of clot retraction. Protein tyrosine phosphorylation was analysed as described above.