Platelet factor 4-containing immune complexes induce platelet activation followed by calpain-dependent platelet death

Platelets were incubated for 15？min (light bars) and 60？min (dark bars) under the following conditions: untreated platelets (negative control), platelets treated with the KKO/PF4 and RTO/PF4 complexes, KKO, RTO, and PF4 alone as well as Ca2+-ionophore A23187 (positive control). Final concentrations: 10？μg/ml PF4, 50？μg/ml KKO, and RTO, 10？μM Ca2+-ionophore. a Annexin V-positive platelets as a fraction of all gated platelets taken as 100%: untreated platelets (n？=？17/13 for 15？min/60？min, respectively), KKO/PF4 (n？=？16/13), RTO/PF4 (n？=？3/3), KKO (n？=？16/13), RTO (n？=？3/3), PF4 (n？=？16/13), and A23187 (n？=？14/12). b CD62P-positive platelets as a fraction of all gated platelets taken as 100%: untreated platelets (n？=？8/7), KKO/PF4 (n？=？8/7), RTO/PF4 (n？=？3/3), KKO (n？=？8/7), RTO (n？=？3/3), PF4 (n？=？8/7), and A23187 (n？=？8/7). c Number of CD41- and Annexin V-double-positive microvesicles normalized by the total number of gated platelets in the corresponding sample: untreated platelets (n？=？8/9), KKO/PF4 (n？=？8/9), RTO/PF4 (n？=？3/4), KKO (n？=？8/9), RTO (n？=？3/4), PF4 (n？=？8/9), and A23187 (n？=？8/9). “n” is the number of experiments with platelets from independent donors. *P？<？0.05 compared to a corresponding negative control. The differences between 15 and 60？min are insignifican