The effect of extracellular Ca⁺⁺ and Na⁺ on the intracellular pH ([pH]_i) of human umbilical vein endothelial cells (HUVEC) was investigated using a fluorescent pH indicator, 2′, 7′-bis (carboxyethyl) carboxyfluorescein (BCECF) with reference to C-kinase and PGI₂ generation. Thrombin increased PGI₂ generation or intracellular Ca⁺⁺ concentration ([Ca⁺⁺]_i). Pretreatment with EGTA or H-7 decreased thrombin-induced PGI₂ generation or [Ca⁺⁺]_i, but did not affect on the baseline PGI₂ generation and [Ca⁺⁺]_i. Na⁺-free buffer did not affect on the enhancement effect. Addition of Ca⁺⁺ or thrombin alkalified [pH]_i, and pretreatment with EGTA decreased basal or thrombin-induced alkalinization. Pretreatment with H-7 did not affect on the baseline [pH]_i, but decreased thrombininduced alkalinization. Na⁺/H⁺ antiporter, amiloride remarkably decreased basal or thrombin-induced alkalinization. Nat-free buffer decreased both alkalinization. These results suggested that thrombin activated the Na⁺/H⁺ exchange system, and that this activation was mediated by an increase in [Ca⁺⁺]_i or by C-kinase.

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Five Ca²⁺-dependent monoclonal antibodies against human protein C: HPC 1 to 5, have been produced. Calcium binding to the light chain (HPC 1, 2) or to the heavy chain (HPC 3, 4) elicited the epitopes for these antibodies. HPC 5 lost its binding activity upon disulfide bridge reduction. HPC 1 and 5 did not react with γ-carboxyglutamic acid (Gla)-domainless protein C nor with isolated Gla-domain while others retained the reactivity even after the Gla-domain had been removed. Half maximal binding of these antibodies were observed 0.35-1.5mM Ca²⁺. Other divalent cations including Mg²⁺ and Mn²⁺ could substitute for Ca²⁺. Thus the low affinity or secondary metal binding sites rather than the high affinity Ca²⁺-binding site may have participated in the epitope induction. Furthermore, HPC 1, 4 and 5 reacted with prothrombin and factor X. The cross-reactivities to prothrombin were lost in accordance with the extent of modification of the Gla residues, indicating that certain Gla-residues had been involved in the expression of conserved epitopes. It is notable that the conformational change in the Gla-domain also influenced the induction of a Ca²⁺-dependent epitope in the serine protease portion of the molecule (HPC 4). The antibodies did not affect the reactions in the fluid phase, but they, except HPC 2, interfered with the cell surface-mediated activation of protein C by the thrombin-thrombomodulin complex and inactivation of factor Va by activated protein C (APC). We therefore conclude that certain concerted conformational changes induced by Ca²⁺ in both the light and heavy chain of protein C are important for the functions of protein C on the surface of endothelial cells.

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Plasma levels of thrombomodulin antigen in various diseases were measured by ETA. Patients with renal insufficiency were not included in this study. In normal subjects the thrombomodulin levels were 16.3±4.1ng/ml (mean±SD). Thrombomodulin levels were elevated in patients with carcinoma, thrombotic disease, aneurysm and collagen disease. In patients with hematopoietic malignancy and carcinoma, there was no relationship between thrombomodulin levels and the presence or absence of DIC. In one patient with protein C deficiency complicated by DIC and thrombophlebitis, plasma thrombomodulin levels were in the normal range. In another patient with carcinoma of the cervix and without vascular complication, plasma thrombomodulin levels were very high. Our data suggest that DIC dose not always cause elevated plasma levels of thrombomodulin antigen, and that high level of plasma thrombomodulin dose not always indicate the existence of vascular damage.

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Factor VII has been purified from bovine plasma with an over yield of 60%. The isolation procedure involves barium citrate adsorption and elution, ammonium sulfate fractionation (40-70%), DEAE-Sepharose Fast Flow column chromatography, and affinity column chromatography using anti-factor VII monoclonal antibody. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Using this rapid purification method, 24.6mg and 20mg of factor VII was obtained from 70 liters and 75.3 liters of bovine plasma, respectively.

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We have investigated the use of synthetic thrombin inhibitors in assays of thrombin-antithrombin III complexes (TAT). In a purified system, when antithrombin III (AT-III) was incubated with thrombin, a complex of 90KD was formed gradually during the first 60 minutes and this complex was converted to lower molecular weight species (78-86×10³) within 48 hours. Additionally, the interaction of thrombin and AT-III was studied in the presence of heparin. A 93KD complex was formed immediately and then lower molecular weight complexes (78-86×10³) were formed during 60 minutes of incubation. In these circumstances, therefore, heparin seemed to accelerate the breakdown of TAT complexes. Plasma values of TAT were measured by ELISA in the presence of heparin and were reduced by approximately 55per cent during 48 hours of incubation. These findings suggested that the biochemical nature of TAT was modified during incubation, and that the assays for TAT in plasma might not detect lower molecular weight forms of the complex. However, a synthetic thrombin inhibitor, argatroban (0.1mM/l) prevented these changes. The levels of TAT in plasma were increased by adding tissue factor or contact factor activator to the assay mixture. Argatroban (0.01mM/l) or another synthetic protease inhibitor, FOY (4.3mM/l), inhibited this generation of TAT. These observations suggested that argatroban minimized the changes of TAT molecular weight and prevented generation of TAT in vitro. The data indicated that the addition of argatroban (final concentration 0.1mM/l) to plasma would permit more accurate measurements of circulating levels of TAT.

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Successful resection of a cerebellar tumor found in a patient with severe hemophilia A using RCG-11 (monoclonal purified F VIII concentrates) is reported. Pt (I. S.) is a 43-yr.-severe hemophilia A with numerous bleeding episodes but no inhibitor to F VIII. Since Jan 1991, he has been suffering from headache, vertigo, nausea and a diagnosis of cerebellar tumor was made by Computer Tomography sand Magnetic Resonance Imaging. Complete resection of cerebellar tumor was performed after infusion of RCG-11 in dose of 60U/kg and total blood loss was 210gram. Neither HIV antibody nor HBs antigen in his serum turned positive 3 months later.
Pharmacokinetic analysis of RCG-11 administered in dose of 50U/kg showed that t_1/2 of RCG-11 was 12.8 hrs and in vivo recovery rate was 80%, which were similar to those of commercially available monoclonal purified F VIII concentrates. From these results, RCG-11 is safe and offers effective control of major operations in patients with severe type of hemophilia A.

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