A sensitive platelet activation-based functional assay for the antileukemic agent bryostatin 1

Bryostatin 1, a macrocyclic lactone activator of protein kinase C (PKC) currently in phase I evaluation, is a biologic response modifier which exhibits significant antitumor activity in several experimental systems. Clinical trials have been hampered by the absence of a sensitive assay for bryostatin 1 blood levels. The purpose of these studies was to exploit the exquisite sensitity of human platelets to bryostatin 1-induced aggregation in order to develop an assay capable of detecting plasma bryostatin 1 levels in the nanomolar range. Addition of bryostatin 1 (5-100 nM) to platelet-rich plasma resulted in complete platelet aggregation. A highly linear relationship was observed between low bryostatin 1 concentrations (i.e. 2-25 nM) and (i) reduction in the lag phase prior to aggregation and (ii) maximal rate of aggregation (R = 0.976). At higher bryostatin 1 concentrations (i.e. 10-100 nM), platelet aggregation was accompanied by detectable ATP release; both the extent and maximal rate of ATP secretion were highly linear functions of bryostatin 1 levels (R = 0.992). Bryostatin 1 concentrations in anticoagulated human blood samples could also be determined by mixing platelet poor plasma obtained from such samples with normal platelet-rich plasma. Notably, measurement of the delay in the aggregation lag phase permitted quantitation of bryostatin 1 concentrations of 5 nM or below. The capacity to detect bryostatin 1 plasma levels of 10 nM or lower should facilitate the conduct of pharmacokinetic and pharmacodynamic studies in conjunction with ongoing phase 1 trials.