Anja Maag1,2, Priyanka Sharma1, Tim J Schuijt3, Wil F Kopatz4, Daniëlle Kruijswijk1, J Arnoud Marquart5, Tom van der Poll1, Tilman M Hackeng6, Gerry A F Nicolaes6, Joost C M Meijers4,5, Mettine H A Bos2, Cornelis van 't Veer1. 1. Amsterdam UMC, University of Amsterdam, Center for Experimental and Molecular Medicine, Amsterdam Infection and Immunity Institute, Amsterdam, The Netherlands. 2. Division of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, The Netherlands. 3. Hospital Gelderse Vallei Ede, Clinical Chemistry and Hematology Laboratory, Ede, The Netherlands. 4. Department of Experimental Vascular Medicine, Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands. 5. Department of Molecular and Cellular Hemostasis, Sanquin Research, Amsterdam, The Netherlands. 6. Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM) Maastricht University, Maastricht, The Netherlands.
Abstract
BACKGROUND: The prothrombinase complex consists of factors Xa (FXa) and Va (FVa) on an anionic phospholipid surface and converts prothrombin into thrombin. Both coagulation factors require activation before complex assembly. We recently identified TIX-5, a unique anticoagulant tick protein that specifically inhibits FXa-mediated activation of FV. Because TIX-5 inhibited thrombin generation in blood plasma, it was concluded that FV activation by FXa contributes importantly to coagulation. OBJECTIVE: We aimed to unravel the structure-function relationships of TIX-5. METHOD: We used a structure model generated based on homology with the allergen Der F7. RESULTS: Tick inhibitor of factor Xa toward FV was predicted to consist of a single rod formed by several beta sheets wrapped around a central C-terminal alpha helix. By mutagenesis we could show that two hydrophobic loops at one end of the rod mediate the phospholipid binding of TIX-5. On the other end of the rod an FV interaction region was identified on one side, whereas on the other side an EGK sequence was identified that could potentially form a pseudosubstrate of FXa. All three interaction sites were important for the anticoagulant properties of TIX-5 in a tissue factor-initiated thrombin generation assay as well as in the inhibition of FV activation by FXa in a purified system. CONCLUSION: The structure-function properties of TIX-5 are in perfect agreement with a protein that inhibits the FXa-mediated activation on a phospholipid surface. The present elucidation of the mechanism of action of TIX-5 will aid in deciphering the processes involved in the initiation phase of blood coagulation.
BACKGROUND: The prothrombinase complex consists of factors Xa (FXa) and Va (FVa) on an anionic phospholipid surface and converts prothrombin into thrombin. Both coagulation factors require activation before complex assembly. We recently identified TIX-5, a unique anticoagulant tick protein that specifically inhibits FXa-mediated activation of FV. Because TIX-5 inhibited thrombin generation in blood plasma, it was concluded that FV activation by FXa contributes importantly to coagulation. OBJECTIVE: We aimed to unravel the structure-function relationships of TIX-5. METHOD: We used a structure model generated based on homology with the allergen Der F7. RESULTS: Tick inhibitor of factor Xa toward FV was predicted to consist of a single rod formed by several beta sheets wrapped around a central C-terminal alpha helix. By mutagenesis we could show that two hydrophobic loops at one end of the rod mediate the phospholipid binding of TIX-5. On the other end of the rod an FV interaction region was identified on one side, whereas on the other side an EGK sequence was identified that could potentially form a pseudosubstrate of FXa. All three interaction sites were important for the anticoagulant properties of TIX-5 in a tissue factor-initiated thrombin generation assay as well as in the inhibition of FV activation by FXa in a purified system. CONCLUSION: The structure-function properties of TIX-5 are in perfect agreement with a protein that inhibits the FXa-mediated activation on a phospholipid surface. The present elucidation of the mechanism of action of TIX-5 will aid in deciphering the processes involved in the initiation phase of blood coagulation.
Authors: Anja Maag; Nienke van Rein; Tim J Schuijt; Wil F Kopatz; Danielle Kruijswijk; Stella Thomassen; Tilman M Hackeng; Rodney M Camire; Tom van der Poll; Joost C M Meijers; Mettine H A Bos; Cornelis van 't Veer Journal: J Thromb Haemost Date: 2021-11-23 Impact factor: 16.036