TxA2 internalization by human platelets is inhibited by picotamide

Abstract Picotamide has previously been shown to inhibit the platelet binding of thromboxane A2 analogues. Picotamide has been also reported to inhibit thromboxane A2 synthase so that an intracellular target for its action has been postulated. A carrier-mediated active transport for thromboxane A2 (TxA2) was previously reported in platelets. In the present paper the effect of picotamide on the internalization process was assessed. The kinetics of the TxA2 binding to and internalization by human platelets from 6 male healthy volunteers were investigated by a radiolabeled structural TxA2 analogue,125I-PTA-OH, in the absence and in the presence of increasing concentrations of picotamide. The kinetic constants (kobs, k1, and k−1) were calculated by time course experiments according to Weiland and Molinoff. The affinity constant (Km) and maximal uptake velocity (Vmax) values for each subject were determined by the double reciprocal plot of Lineweaver-Burk. The kinetically determined dissociation constant Kd was 10 nmol/L. In the uptake experiments the association rate of the nondisplaceable binding was found to be saturable at increased ligand concentration with a Michaelis-Menten type of kinetics. Picotamide was able to inhibit the internalization process with a Ki of 7100µmol/L