Heparin-activated antithrombin interacts with the autolysis loop of target coagulation proteases.
A unique pentasaccharide fragment of heparin can enhance the reactivity of antithrombin with coagulation proteases factors IXa and Xa by 300- to 600-fold through a conformational activation of the serpin, without having a significant effect on the reactivity of antithrombin with thrombin. In this study, it was hypothesized that differences in the structure of the autolysis loop of coagulation proteases (residues 143-154 in chymotrypsin numbering) may be responsible for their differential reactivity with the native and heparin-activated antithrombin. To test this hypothesis, the autolysis loops of both thrombin and the anticoagulant serine protease-activated protein C were replaced with the corresponding loop of factor Xa. Inhibition studies revealed that in contrast to the approximately 1.5-fold difference in the reactivity of thrombin with antithrombin in the absence and presence of pentasaccharide, the difference in reactivity was increased to approximately 37-fold for the mutant thrombin. In the case of the activated protein C mutant, similar to factor Xa, pentasaccharide accelerated the reaction 375-fold. These results suggest that structural differences in the autolysis loop of coagulation proteases play a key role in their differential reactivity with the native and heparin-activated conformations of antithrombin.
Yang
Likui
L
Edward A. Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, 1402 S Grand Blvd, St Louis, MO 63104, USA.
Manithody
Chandrashekhara
C
Rezaie
Alireza R
AR
D000818:Animals; D000991:Antithrombins / Q000819:agonists* / Q000378:metabolism*; D001777:Blood Coagulation; D002460:Cell Line; D006224:Cricetinae; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005170:Factor X / Q000235:genetics / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009154:Mutation / Q000235:genetics; D010974:Platelet Aggregation; D011485:Protein Binding; D011486:Protein C / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011516:Prothrombin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology
D000991:Antithrombins; D004792:Enzyme Precursors; D046911:Macromolecular Substances; D011486:Protein C; D011516:Prothrombin; D005170:Factor X; D006493:Heparin; C012007:prethrombins; D010450:Endopeptidases; D013917:Thrombin