Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To elucidate the binding sites for thrombin and protein C in the six epidermal growth factor (EGF) domains of human thrombomodulin, recombinant mutant proteins were expressed in COS-1 cells. Mutant protein EGF456, which contains the fourth, fifth, and sixth EGF domains from the NH2 terminus of thrombomodulin, showed complete cofactor activity in thrombin-catalyzed protein C activation, as did intact thrombomodulin or elastase-digested thrombomodulin. EGF56, containing the fifth and sixth EGF domains, did not have cofactor activity; but EGF45, containing the fourth and fifth EGF domains, had about one-tenth of the cofactor activity of EGF456. Thrombin binding to attached recombinant thrombomodulin (D123) was inhibited by EGF45 as well as by EGF56. A synthetic peptide (ECPEGYILDDGFICTDIDE), corresponding to Glu-408 to Glu-426 in the fifth EGF domain, inhibited thrombin binding to attached thrombomodulin (D123) with an apparent Ki of 95 microM. At Ca2+ concentrations of 0.25-0.3 mM, intact protein C was maximally activated by thrombin in the presence of EGF45, EGF456, or EGF1-6, which contains the first to sixth EGF domains; but such maximum cofactor activity was not observed when gamma-carboxyglutamic acid-domainless protein C was used. These findings suggest that: 1) thrombin binds to the latter half of the fifth EGF domain; and 2) protein C binds to the fourth EGF domain of thrombomodulin through Ca2+ ions.
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PMID:Further localization of binding sites for thrombin and protein C in human thrombomodulin. 217 98

We have identified a minimum functional domain of human thrombomodulin for anticoagulant activity using deletion analysis. Four mutants were constructed by site-directed deletion mutagenesis to delete one or more epidermal growth factor (EGF)-like structures from the domain of human thrombomodulin containing six repeated EGF-like structures. These deletion mutants were expressed transiently in COS-1 cells, and their protein C-activating cofactor activities in the culture medium were examined. One mutant protein, E456, which contains the fourth, fifth, and sixth EGF-like structures expresses apparent cofactor activity. However, neither E456-N24 (24 NH2-terminal-residue deletion) nor E456-C16 (16 COOH-terminal-residue deletion) have cofactor activity. E456 was partially purified and its anticoagulant effects on plasma clotting time and platelet aggregation examined. E456 expressed almost the same anticoagulant activities as D123 which contains six consecutive EGF-like structures of thrombomodulin. It was concluded that E456 is the minimum functional domain for both protein C-activating cofactor activity and anticoagulant activity.
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PMID:The last three consecutive epidermal growth factor-like structures of human thrombomodulin comprise the minimum functional domain for protein C-activating cofactor activity and anticoagulant activity. 254 66

Thrombomodulin (TM) is composed of five domains. We investigated the roles of the sixth epidermal growth factor (EGF)-like structure (E6) in the second domain (D2) and of an O-glycosylation site rich domain (D3) in the function of TM in more detail using deletion analysis. Two soluble mutants of TM, TMD123 and TMD12, and three deletion mutants lacking respectively 6, 16, and 38 C-terminal residues of the E6 portion, TMD12 delta 6, TMD12 delta 16, and TMD12 delta 38, were expressed in COS cells and purified. The results of protein C-activating cofactor assay showed that TMD12 delta 6, TMD12 delta 16, and TMD12 delta 38, which lack the C-terminal region, had remarkably weak cofactor activities in comparison with TMD123 (9.1, 1.4, and 1.1% of TMD123 activity, respectively). Similar findings were obtained for anticoagulant activity. These findings indicate that the last loop structure in E6 is required for full activity of recombinant human TM. We also determined in vivo stabilities of TMD12, TMD123, and TMD12 delta 6 in a pharmacokinetic study in rats. TMD12 and TMD12 delta 6, which lack the D3 domain, exhibited increased clearance (about twice that of D123). This finding suggested that the D3 domain of TM plays an important role in stabilizing TM in vivo.
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PMID:The roles played by the D2 and D3 domains of recombinant human thrombomodulin in its function. 874 23