Platelet aggregation assays

Five healthy subjects and five patients diagnosed with primary ITP were recruited for the study. Citrated whole blood was drawn from donors and centrifuged to isolate platelet-rich plasma (PRP). PRP platelet counts were adjusted to 200,000–300,000 in healthy subjects and >125,000 in ITP patient samples. PRP was pretreated with 0.3 or 1 μM of rilzabrutinib, or controls ibrutinib, 1% DMSO, and 10 μM roxifiban for 15 min. Samples were then transferred to platelet aggregation test wells and a panel of platelet aggregation agonists were added: 10 μM ADP, 5 μM TRAP, 5 μM U46619, saline, 1.5 mg/ml ristocetin, 5 μg/ml collagen, and 2.5 μg collagen. Platelet aggregation was measured in a lumiaggregometer for 6 min for all agonists except saline, where aggregation was measured for 15 min. Agonist-induced platelet aggregation was indicated by maximum aggregation caused by agonist. The effect of rilzabrutinib was analyzed by conducting a two-sample (equal variance), two-tailed t test between the DMSO samples and each inhibitor-treated group.