Abstract
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.
Highlights
PROCEDURESMaterials-Restriction endonucleases, T4 polynucleotide kinase, Escherichia coli DNA polymerase I (Klenow fragment), T4 DNA ligase, deoxynucleotide triphosphates, M13mp, and the 7-deaza sequencing kit were obtained from Takara Shuzo (Kyoto, Japan). [y-
We found that the fourth and fifth epidermal growth factor (EGF) domains are essential for expressing protein C-activating cofactor activity
We reported [11] that recombinant EGF456-N24 without the 24 NH2-terminal amino acids of EGF456 completely lacks cofactor activity. These findings suggest that the complete fourth EGF domain (Val-348 to Met-388) is required for interaction with protein C through Ca2+ions
Summary
Materials-Restriction endonucleases, T4 polynucleotide kinase, Escherichia coli DNA polymerase I (Klenow fragment), T4 DNA ligase, deoxynucleotide triphosphates, M13mp, and the 7-deaza sequencing kit were obtained from Takara Shuzo (Kyoto, Japan). [y-. Diisopropyl fluorophosphate-thrombin-coupled Sepharose 4B was prepared as described previously [16]. (lo), carrying the coding sequence for the first to sixth EGF domains (EGFl-6) of human thrombomodulin was subcloned into M13mp. (lo), carrying the coding sequence for the first to sixth EGF domains (EGFl-6) of human thrombomodulin was subcloned into M13mp19 Assay of Cofactor Actiuity for Protein C Actiuation-Enhancement by thrombomodulin of thrombin-catalyzed protein C activation was determined using Boc-Leu-Ser-Thr-Arg-MCA [10]. After washing the well with the same buffer, 100 ~1 of 200 )IM H-o-Phe-Pip-Arg-pNA in 0.05 M Tris-HCl, pH 8.0, 0.1 M NaCl was added and incubated at 37 “C. To determine the effects of the recombinant proteins (EGF45 or EGF56) or the synthetic peptide on thrombin binding to attached D123, thrombin (0.1 pg/ml) was incubated with various concentrations of EGF45 or EGF56 or with the synthetic peptide at 37 “C for 30 min
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