Figure 3. Functional responses of γ−/− platelets.

Release of ATP (A) and aggregation [measured using absorbance (‘optical density’)] (B) in WT and γ−/− platelets. Platelets were stimulated with collagen (50 μg/ml), CRP (5 μg/ml), U46619 (1 μM), thrombin (1 units/ml) or a combination of collagen and U46619. The effects of the αIIbβ3 antagonist lotrafiban (10 μM) and the anti-α2 antibody Ha1/29 (20 μg/ml) are shown. Negative values for aggregation indicate shape change. The apparent inability of lotrafiban to inhibit thrombin-induced aggregation is due to thrombin-induced release of fibrinogen from alpha granules and subsequent generation of fibrin, which causes platelet trapping. This can be blocked by the fibrin polymerization inhibitor Gly-Pro-Arg-Pro (results not shown). The apparent residual CRP-induced aggregation in the presence of lotrafiban is caused by the release of platelet granules, which reduces the absorbance of suspensions of platelets [43]. The unexpectedly weak aggregation induced by the combination of U46619, collagen and Ha1/29 in γ−/− platelets was caused by a non-specific inhibitory effect of Ha1/29 on aggregation induced by U46619 alone. A similar effect was observed with the antibodies HMα2 and Ha31/8 (results not shown). Data are means±S.E.M. for responses measured 6 min after addition of the agonist (n=3–6).