Abstract
125I-labeled heparin cofactor II (HCII) was mixed with plasma and coagulation was initiated by addition of CaCl2, phospholipids, and kaolin or tissue factor. In the presence of 67 micrograms/ml of dermatan sulfate, radioactivity was detected in a band which corresponded to the thrombin-HCII complex (Mr = 96,000) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No other complexes were observed. The thrombin-HCII complex was undetectable when 5 units/ml of heparin was present or when prothrombin-deficient plasma was used. In experiments with purified proteases, HCII did not significantly inhibit coagulation factors VIIa, IXa, Xa, XIa, XIIa, kallikrein, activated protein C, plasmin, urokinase, tissue plasminogen activator, leukocyte elastase, the gamma-subunit of nerve growth factor, and the epidermal growth factor-binding protein. HCII inhibited leukocyte cathepsin G slowly, with a rate constant of 8 X 10(4) M-1 min-1 in the presence of dermatan sulfate. These results indicate that the protease specificity of HCII is more restricted than that of other plasma protease inhibitors and suggest that the anticoagulant effect of dermatan sulfate is due solely to inhibition of thrombin by HCII.
Highlights
From the Division of Hematology-Oncology, Departments of internal Medicine and Biological Chemistry, Washington University School of Medicine, St
Tivity was detected in a band which corresponded to the thrombin-HCII comple(xM.=96,000)upon sodium inhibits a broad range of proteolytic enzymes, including the coagulation factors thrombin, Xa, IXa, XIa, XIIa, and kallidodecyl sulfate-polyacrylamidgeel electrophoresis.No krein, and the fibrinolytic enzyme plasmin [9, 11].We have othercomplexes were observed.Thethrombin-HCII complex was undetectable when5 units/ml of heparin was present or whenprothrombin-deficientplasma wasused
We reported that ' " ~ - t ~ r o ~ baidnded to plasma containing dermatan sulfate becomes bound to HCII [5].We have shown that thromkocyte elastase, the r-subunit of nerve growth factor, bin generated in plasma during coagulation is inhibited by and the epidermgarl owthfactor-binding proteinH
Summary
From the Division of Hematology-Oncology, Departments of internal Medicine and Biological Chemistry, Washington University School of Medicine, St. The enzyme,HCII, heparin, and dermatan sulfate were mixed at thefinal concentrations specified in Table I in buffer containing0.15 M NaCl, 0.02 M Tris-HC1, pH 7.4, and 1mg/ml bovine serum albumin. Assay for factor VIIa-Factor VIIa was incubated with HCII and heparin or dermatan sulfate in the presence of 5 mM CaClz and 1mg/ ml human brain thromboplastin in 0.15 M NaC1,0.02 M Tris-HC1, pH 7.4, for 15 min at 37 "C. Assay for Factor IXa-Factor IXa, HCII, and heparinor dermatan sulfate were incubated for min at 37 "C in 55pl of buffer containing 0.15 M NaCI, 0.02 M Tris-HC1, pH 7.4, and 1 mg/ml bovine serum albumin.
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