EFFECT OF THE BUFFER COMPOSITION AND THE PRESENCE OF ORG 10172 AND SOME GLYCOSAMINOGLYCANS ON THE INHIBITION OF ALPHA-THROMBIN BY HEPARIN COFACTOR II

Heparin cofactor II (HCII) inhibits alpha-thrombin (Ila) with formation of a 1:1 complex.The inhibition is accelerated by heparin and dermatan sulfate (DS).The present study describes the pH and ionic strength dependence of the second order inactivation constant k_2nd for the simple IIa-HCII interaction and for the interaction catalysed by heparin, dermatan sulfate and the antithrombotic Org 10172 containing low molecular DS.In all cases we found a bell-shaped pH profile for k_2nd with characteristic maxima and pK values. Org 10172 accelerates the HCII mediated inhibition of Ila though not to the same extent as heparin and DS. Substantial narrowing of the pH profile occurs under influence of glycosaminoglycans , the alkaline limb of the profiles being displaced towards a more acidic pH. The ionic strength is critical for the determination of k_2nd. A nonlinear decrease is observed with increasing ionic strength, due to the screening effect of the ions in solution. Ca⁺⁺ ions do not accelerate the inactivation process in absence of GAG, in contrast to the findings for the Ila-ATIII interaction. The pseudo-first order model for Ila inactivation is only valid for : a) the uncatalysed reaction irrespective of ionic strength, b) the inactivation in buffers of intermediate ionic strength and in the presence of low GAG concentration and c) the inactivation in media of high ionic strength irrespective of GAG concentration. In all other reaction conditions the inactivation rate substantially deviates from a pseudo-first order plot.Suicide substrates are known to exhibit a comparable behavior. However this model appeared inadequate for describing our experiments. An appropriate model for these aberrant inactivation profiles is not evident.Deviation from pseudo-first order kinetics occurs more readily with heparin than with dermatan sulfate and is probably related with their difference in charge density.