Allosteric modification of factor XIa functional activity upon binding to polyanions.

The effects of several polyanions on the hydrolysis of the chromogenic substrate L-pyroglutamyl-L-prolyl-L-arginyl-p-nitroaniline (S-2366) and on the activation of factor IX by factor XIa have been investigated. Two forms of dextran sulfate (M(r) approximately 500000 and M(r) approximately 10000, DX10) and two forms of heparin (64 disaccharide units, M(r) approximately 14000, and hypersulfated heparin, S-Hep, M(r) approximately 12000) inhibited both factor XIa amidolytic activity and factor IX activation in a concentration-dependent manner. The inhibitory effect was not due to binding of either substrate by the polyanions since only a decrease in V(max) without any effect on K(m) was observed in kinetic assays. Steric inhibition is unlikely since the concentrations of polyanions required for inhibition of small peptide hydrolysis were lower than those required for macromolecular substrate cleavage. In contrast, an allosteric inhibitory mechanism was supported by an enhancement of the dansyl fluorescence of 5-(dimethylamino)-1-(naphthalenesulfonyl)glutamylglycylarginyl- (DEGR-) factor XIa observed when the fluorophore was in complex with either DX10 or S-Hep. Moreover, in the presence of a polyanion the fluorophore was far more resistant to quenching by acrylamide. These results provide compelling evidence that factor XIa binding to the polyanions, dextran sulfate and heparin, results in inhibition of the enzyme by an allosteric mechanism.