Analysis of inhibition rate enhancement by covalent linkage of antithrombin to heparin as a potential predictor of reaction mechanism.
Antithrombin (AT) inhibition of coagulation enzymes is catalyzed by unfractionated heparin (UFH) and other heparinoids. Reaction proceeds either via conformational activation of the inhibitor or template-mediated binding of both inhibitor and protease. We investigated if the relative inhibition rates of AT + UFH and covalent AT-heparin conjugate (ATH) with coagulation factors might be indicative of the mechanism involved. Rates were determined by discontinuous assay and mechanisms were probed by a variety of binding studies with UFH or ATH heparin chains. Rates were increased more than 2-fold with ATH over AT + UFH in reactions with thrombin, factor (F) VIIa + tissue factor + Ca2+ + lipid, FIXa and FXIa, but not with FXa or FXIIa. In comparison, UFH or ATH heparin binding (evidence of a template mechanism) was only observed with thrombin, tissue factor, FIXa and FXIa. Thus, inhibition rate enhancement by conjugation of AT with heparin were predictive of inhibitor.enzyme template bridging by heparin. Rationales behind this novel concept are discussed.
Patel
Sanjay
S
Henderson Research Centre, 711 Concession Street, Hamilton, Canada L8V 1C3.
Berry
Leslie R
LR
Chan
Anthony K C
AK
D000991:Antithrombins / Q000737:chemistry* / Q000378:metabolism*; D001779:Blood Coagulation Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015949:Factor IXa / Q000037:antagonists & inhibitors / Q000378:metabolism; D015942:Factor VIIa / Q000037:antagonists & inhibitors / Q000378:metabolism; D015945:Factor XIa / Q000037:antagonists & inhibitors / Q000378:metabolism; D006493:Heparin / Q000737:chemistry* / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism; D013925:Thromboplastin / Q000037:antagonists & inhibitors / Q000378:metabolism
D000991:Antithrombins; D001779:Blood Coagulation Factors; D006493:Heparin; D013925:Thromboplastin; D015942:Factor VIIa; D015949:Factor IXa; D015945:Factor XIa; D013917:Thrombin