Identification of the epidermal growth factor-like domains of thrombomodulin essential for the acceleration of thrombin-mediated inactivation of single-chain urokinase-type plasminogen activator.
Single-chain urokinase-type plasminogen activator (scu-PA) can be cleaved by thrombin into a virtually inactive form called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T), a process accelerated by thrombomodulin, which contains six epidermal growth factor (EGF)-like domains. In this study, we identified the EGF-like domains of thrombomodulin required for the acceleration of the inactivation of scu-PA by thrombin using various forms of thrombomodulin (TM). scu-PA was treated with thrombin in the absence and presence of full-length rabbit TM (containing EGF1-6), recombinant TM comprising all of the extracellular domains including EGF1-6 (TMLEO) and recombinant TM comprising EGF4-6 plus the interconnecting region between EGF3 and EGF4 (TMEi4-6), and the tcu-PA/T generated was quantitated in each case. Rabbit TM accelerated the inactivation of scu-PA approximately 35-fold, while both recombinant forms accelerated it only threefold due to the absence of a critical chondroitin sulfate moiety. Subsequently, TME5-6 was prepared by cyanogen bromide digestion of TMEi4-6. TME5-6 bound to thrombin but did not accelerate the activation of protein C. In contrast, the inactivation of scu-PA by thrombin was accelerated to the same extent as that induced by TMLEO and TMEi4-6. This study demonstrates that, in addition to the chondroitin sulfate moiety, only EGF-like domains 5 and 6 are essential for the acceleration of the inactivation of scu-PA by thrombin. This differs from the domains that are critical for activation of protein C (EGF-like domains i4-6) and thrombin activatable fibrinolysis inhibitor (EGF-like domains 3-6).
Schenk-Braat
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EA
TNO Prevention and Health, Gaubius Laboratory, Leiden, the Netherlands.
Morser
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Rijken
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D000911:Antibodies, Monoclonal / Q000494:pharmacology; D025901:Carboxypeptidase B2 / Q000378:metabolism; D003488:Cyanogen Bromide / Q000737:chemistry; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D011486:Protein C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*; D018180:Thrombomodulin / Q000737:chemistry / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D011486:Protein C; D018180:Thrombomodulin; D004815:Epidermal Growth Factor; D025901:Carboxypeptidase B2; D013917:Thrombin; D014568:Urokinase-Type Plasminogen Activator; D003488:Cyanogen Bromide