Implication of fibrin and myosin translocation to DRM raft fraction in clot retraction. (A) Impairment of clot retraction in type I Glanzmann’s thrombasthenia. Time-dependent clot retraction of Glanzmann’s thrombasthenia PRP (circles) and normal PRP (triangles). PRP was incubated with 1 U/mL thrombin and 2 mM CaCl₂. The extent of clot retraction was assessed at the indicated times by measuring clot area. Photo image of clot retraction assay after 120 minutes: N, normal PRP; G, Glanzmann’s thrombasthenia PRP (inset). (B) Inhibition of clot retraction by fibrinogen γ-chain 400-411 dodecapeptide. PRP was incubated with 1 U/mL thrombin, 2 mM CaCl₂ with no peptide (circles), and 1 mM (squares), 5 mM (triangles), and 15 mM (diamonds) fibrinogen γ-chain 400-411 dodecapeptide. Data are presented as means plus or minus SD of quadruplicates. *Statistically significant difference (P < .01). **Statistically significant difference (P < .05). (C) Inhibition of clot retraction by cystamine or blebbistatin. PRP was incubated with 1 U/mL thrombin, 2 mM CaCl₂ with buffer (circles), 10 mM cystamine (squares), or 100 μM blebbistatin (triangles). Data are presented as means plus or minus SD of triplicates. *Statistically significant difference (P < .001). (D) Inhibition of clot retraction by raft disruption by MBCD. Mixture of washed platelets and purified fibrinogen was incubated with 1U/ml thrombin in the presence (circle) and absence (triangle) of 2% MBCD. The photograph was taken after 30 and 60 minutes. (E) Raft disruption by MBCD inhibits transient increase in degree of tyrosine phosphorylation (a) and phosphorylation of myosin light chain at serine residue 19 (b). A mixture of washed platelets and purified fibrinogen was incubated with 1 U/mL thrombin for 0 minutes (lanes 1 and 4), 5 minutes (lanes 2 and 5), and 60 minutes (lanes 3 and 6) in the presence (lanes 4-6) and absence (lanes 1-3) of 2% MBCD. Arrows indicate the tyrosine phosphorylation of 125-kDa and 100-kDa proteins.