Figure 2. Inhibition and potentiation of collagen-induced whole-cell tyrosine phosphorylation in γ−/− platelets.

Washed platelets were stimulated with collagen (50 μg/ml) plus U46619 (1 μM) in the presence or absence of the αIIbβ3 antagonist lotrafiban (10 μM), the anti-α2 antibodies Ha1/29 (20 μg/ml) or HMα2 (20 μg/ml), or the anti-α1 antibody Ha31/8 (20 μg/ml). Tyrosine phosphorylation was measured in whole-cell lysates using the anti-phosphotyrosine antibody 4G10. (A) Collagen-induced tyrosine phosphorylation was unaffected by lotrafiban, whereas it was substantially inhibited in the presence of Ha1/29 or HMα2. The isotype control Ha31/8 (Armenian hamster IgG2, λ) had no effect on the response. (B) In the presence of lotrafiban, the thromboxane A₂ analogue U46619 induced a weak response when added alone. However, when added in combination with collagen, the response was greatly potentiated, in particular for a band at 100 kDa. Both Ha1/29 and HMα2 inhibited the response, although the former antibody was slightly more effective. Ha31/8 had no effect. In order to highlight the increase in response in the presence of U46619 in particular, the films shown in this Figure were exposed to a much lesser extent than those shown in Figure 1. This accounts for the apparently lower levels of phosphorylation in basal and collagen-stimulated lanes, and the lighter background in this Figure compared with Figure 1. MM, molecular mass (kDa).