GPVI-specific platelet aggregation and tyrosine phosphorylation is reduced in S14 platelets. (A) Washed platelets were prepared from a control donor (Cont or C) and patient S14 (S14 or S) and aggregated with continuous stirring at 1200 rpm, with collagen (1 μg/mL; left) or TRAP (3 μM; right). Platelet aggregation was demonstrated by a change in light transmission. Representative aggregation tracings are shown. (B) Washed platelets were treated with lotrafiban (10 μM) and allowed to rest (Basal) or were stimulated with CRP (5 μg/mL for 90 seconds, CRP) or TRAP (30 μM for 90 seconds, TRAP) prior to lysis with 2 × Lysis buffer. Proteins were separated on SDS-PAGE, using 4% to 12% precast gradient gels, transferred onto PVDF membranes, and blotted with anti-phosphotyrosine antibody (Ab), 4G10. (C) Proteins PLCγ2 (Ci), Syk (Ciii), and LAT (Civ) were immunoprecipitated from whole platelet lysates samples and analyzed by immunoblotting with a monoclonal anti-phosphotyrosine antibody (4G10). Immunoblots were then stripped and reprobed with an antibody to PLCγ2, Syk, or LAT (lower). (Cii) Lysates were immunoblotted with 4G10 and then stripped and reprobed with an antibody to FcRγ chain. Numbers on left side of blot are molecular weight markers.