Effects of anti-β2GPI–β2GPI–PF4 complex. (A) Effects of anti-β2GPI–β2GPI–PF4 complex on platelet TXB2 production in the presence or absence of thrombin priming. Washed platelets were either stimulated or not with 0.005 U/mL thrombin and were then exposed to immobilized (1) anti-β2GPI + BSA + PF4, (2) isotype control + β2GPI + PF4, (3) anti-β2GPI + β2GPI + PF4, (4) β2GPI + BSA, and (5) β2GPI + PF4. TXB2 was measured in the supernatants as described in “Measurement of platelet thromboxane B2 metabolite production.” A negative control (no. 6) comprising platelets treated with 0.005 U/mL thrombin alone, as well as a positive control (no. 7) comprising platelets treated with 1 U/mL thrombin alone, were also included. Values are the mean ± SD; n = 7. (B) Effects of anti-β2GPI–β2GPI–PF4 complex on phosphorylation of p38 MAPK. Washed platelets were treated with anti-β2GPI–β2GPI–PF4 or control combinations (see “Methods” for details) and stimulated with 0.005 U/mL thrombin. A positive control comprising platelets treated with 1 U/mL thrombin alone was also included in the experiments. Lysates of the platelets were immunoblotted by using specific antibodies for the unphosphorylated form of p38 MAPK (i) and the phosphorylated form of p38 MAPK (ii). The effect of anti-FcγRII blocking mAb IV.3 is presented (iii).