TxA2internalization by human platelets is inhibited by picotamide

Picotamide has previously been shown to inhibit the platelet binding of thromboxane A 2 analogues. Picotamide has been also reported to inhibit thromboxane A 2 synthase so that an intracellular target for its action has been postulated. A carrier-mediated active transport for thromboxane A 2 (TxA 2 ) was previously reported in platelets. In the present paper the effect of picotamide on the internalization process was assessed. The kinetics of the TxA 2 binding to and internalization by human platelets from 6 male healthy volunteers were investigated by a radiolabeled structural TxA 2 analogue, 125 I-PTA-OH, in the absence and in the presence of increasing concentrations of picotamide. The kinetic constants (k obs , k 1 , and k −1 ) were calculated by time course experiments according to Weiland and Molinoff. The affinity constant (Km) and maximal uptake velocity (V max ) 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 7100 μ mol/L