The Heparin Binding Properties of Heparin Cofactor II Suggest an Antithrombin -Like Activation Mechanism.

Heparin cofactor II(HCII) is a serpin which specifically inhibits thrombin. Like antithrombin(AT) it requires glycosaminoglycan(GAG) binding before full activation. However unlike antithrombin no specific activators have been identified. What currently prevents selective therapeutic activation of HCII is the lack of knowledge of the determinants of GAG binding specificity. We have investigated the heparin binding properties of HCII using size fractionated heparins. The affinity of heparins for HCII, ranging in size from 8 to 26 saccharide units, were determined by following change in fluorescence of the extrinsic probe TNS. The effect of heparin concentration(from 1 to 100nM) on the rate of thrombin inhibition was determined for the same heparins under psuedo first-order conditions. Based on these studies we can conclude that binding is non specific with a minimal heparin length of 13 monosaccaride units required. The observed dependence of catalytic activity on heparin length is caused entirely by the size dependence of heparin affinity for HCII. By studying affinity at different ionic strengths it is clear that binding is critically dependent on ionic strength with a very weak non-ionic contribution to binding. Rapid binding experiments were conducted under psuedo first-order conditions in heparin using a stopped-flow fluorometer. These studies of heparin binding indicate an induced- fit mechanism which involves a conformational change in HCII. The low affinity of HCII can now be understood as a failure of interactions to reduce the rate of the reverse conformational, in a manner analogous to the interaction of AT with low affinity heparin. HCII is activated by a fully allosteric mechanism which produces a 2000 fold increase in activation on heparin binding. We conclude that the heparin binding mechanism of HCII is closely analogous to that of AT, and that the design or discovery of high affinity, HCII-specific GAGs is thus feasible.