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 (k_obs, k₁, 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 μmol/L