Linear diffusion of thrombin and factor Xa along the heparin molecule explains the effects of extended heparin chain lengths

Question: How does the size of the heparin moiety in the anti-thrombin (AT)-heparin complex influence its anticoagulant properties? Approach: Of 52 heparin fractions of precise Mr between 2800 and 37,000 we determined the dissociation constant (K-d) of the binding of the enzyme to the AT-heparin complex and the decay constant (k(dec)) of thrombin and factor Xa at 1 mu M of that complex. Results: The K-d of thrombin or factor Xa is constant when expressed in terms of the concentration of sugar units, i.e. the enzymes bind the better the longer the heparin. Thrombin (K-d = 1.86 +/- 0.13 mu M) binds 11 times tighter than factor Xa (K-d = 20.2 +/- 1.5 mu M). Factor Xa inactivation velocity is proportional to the concentration of pentasaccharide-bound AT if Mr<10,000 but decreases at higher Mr. Thrombin inactivation is constant per pentasaccharide with twelve adjacent monosaccharides (C-domain). Conclusion: The data fit a model in which thrombin and factor Xa bind at a random site on the heparin chain and, via one-dimensional diffusion, reach the AT that is bound to its specific binding site on the heparin. Factor Xa, but not thrombin, can dissociate from heparin before reaching bound AT. (C) 2007 Elsevier Ltd. All rights reserved.