TRANSMISSION OF CONFORMATIONAL CHANGE FROM THE HEPARIN-BINDING SITE TO THE REACTIVE CENTER OF ANTITHROMBIN

Heparin greatly increases the rates at which antithrombin inhibits tar get proteinases. An important part of this rate acceleration is a hepa rin-induced conformational change in antithrombin. To answer the quest ion of whether or not this change is transmitted to the reactive cente r, we have prepared a recombinant P1 mutant of antithrombin, R393C, la beled the cysteine with nitrobenzofuran (NBD) fluorophore, and examine d the perturbation of NBD fluorescence intensity as a function of boun d sulfated oligosaccharide. Two high-affinity heparins, low-affinity h eparin, and dextran sulfate were used. We found (i) that binding to an tithrombin of all these oligosaccharides resulted in transmission of c onformational change to P1 in the reactive center, (ii) that these oli gosaccharides all gave enhancements of the rate of inhibition of facto r Xa beyond any contribution from surface approximation, and (iii) tha t the degree of perturbation of P1 correlated with the enhancement of the rate of factor Xa inhibition that was not due to surface approxima tion.