Nobuo Sakuragawa

Clinical evaluation of low-molecular-weight heparin (FR-860) on disseminated intravascular coagulation (DIC) - a multicenter co-operative double-blind trial in comparison with heparin -

This study was evaluated the effectiveness, safety and utility of FR-860 and to compare those with heparin in patients with Disseminated intravascular coagulation (DIC). A diagnosis of DIC was made based on the criteria proposed by the Research Committee on DIC in the Ministry of Health and Welfare of Japan. FR-860 (FR group, 75 anti-factor Xa international units/kg/day) and Heparin (HP group, 240 units/kg/day) were administered for 5 days by continuous intravenous infusion. The total number of enrolled patients was 126 cases, and after excluding 1 case a total of 125 cases. Moderate or higher improvement of bleeding symptoms was 33.3% in the FR group and 18.5% in the HP group. On the organic symptoms, FR group showed a significantly higher improvement rate than the HP group, 20.5% and 8.2% respectively. On the overall efficacy of cases with pretreatment plasma AT III levels of less than 21 mg/dl or less than 70%. FR group showed a significantly higher improvement rate than the HP group. The safety rate of FR-860 (93.4%) was a significantly higher than that of the HP group (79.7%). Our report demonstrates that FR-860, as a therapeutic agent for the treatment of patients with DIC, is significantly higher safety and clinical utility as compared with heparin.

Hypoproduction of erythropoietin contributes to anemia in chronic cadmium intoxication: clinical study on Itai-itai disease in Japan

Itai-itai disease is a condition caused by longterm exposure of the inhabitants of Toyama prefecture, Japan, to cadmium intoxication. The characteristic clinical features of this disease include renal tubular dysfunction, osteomalacia, and anemia. In order to clarify the pathogenesis of the anemia, the red blood cell count, hemoglobin concentration, hematocrit, serum iron level, total ironbinding capacity, serum ferritin level, serum erythropoietin level, creatinine clearance, fractional excretion of β2-microglobulin, and bone marrow morphology were determined in ten patients with Itai-itai disease. Low serum iron or ferritin levels were not observed, and bone marrow aspiration did not reveal any specific hematological disorders. A close relationship was observed between the decrease in the hemoglobin level and the progression of renal dysfunction. Low serum erythropoietin levels were detected despite the presence of severe anemia. These results suggest an important role of renal damage in the anemia which develops in Itai-itai disease.

Inhibition of thrombin by sulfated polysaccharides isolated from green algae.

Eight different sulfated polysaccharides were isolated from Chlorophyta. All exhibited thrombin inhibition through a heparin cofactor II (HCII)-dependent pathway, and their effects on the inhibition of thrombin were more potent than those of heparin or dermatan sulfate. In particular, remarkably potent thrombin inhibition was found for the sulfated polysaccharides isolated from the Codiales. In the presence of these sulfated polysaccharides, both the recombinant HCII (rHCII) variants Lys(173)-->Leu and Arg(189)-->His, which are defective in interactions with heparin and dermatan sulfate, respectively, inhibited thrombin in a manner similar to native rHCII. This result indicates that the binding site of HCII for each of these eight sulfated polysaccharides is different from the heparin- or dermatan sulfate-binding site. All the sulfated polysaccharides but RS-2 significantly stimulated the inhibition of thrombin by an N-terminal deletion mutant of HCII (rHCII-Delta74). Furthermore, hirudin(54-65) decreased only 2-5-fold the rate of thrombin inhibition by HCII stimulated by the sulfated polysaccharides, while HD22, a single-stranded DNA aptamer that binds exosite II of thrombin, produced an approximately 10-fold reduction in this rate. These results suggest that, unlike heparin and dermatan sulfate, the sulfated polysaccharides isolated from Chlorophyta activate HCII primarily by an allosteric mechanism different from displacement and template mechanisms.

Antithrombin III toyama: A hereditary abnormal antithrombin III of a patient with recurrent thrombophlebitis

A familial abnormal antithrombin III (AT-III) is reported. The characteristic of the abnormality in this family is low heparin cofactor activity with normal progressive antithrombin activity and normal or rather increased level of AT-III antigen. The patient is a 23-year-old female who had suffered from recurrent thrombophlebitis involving her lower extremities. Her plasma AT-III antigen concentration was 54 mg/dl and progressive antithrombin and factor Xa inhibitory activities were of normal level. However, the heparin cofactor activity of her plasma was as low as 26% of normal control. On crossed immunoelectrophoresis (CIE) containing heparin in the first dimension agarose, patients AT-III showed no increase in electrophoretic mobility compared to that in the absence of heparin, suggesting that the patients AT-III has no affinity for heparin. From CIE pattern in the presence of heparin, the patient was found to be a homozygote, and parents and one of her younger sisters were heterozygotes. Thus, the mode of inheritance is proposed to be autosomal dominant.

Purification and biological property of heparin cofactor II: Activation of heparin cofactor II and antithrombin III by dextran sulfate and various glycosaminoglycans

Heparin cofactor II (HC II) has been purified from human plasma by a modification of the method described by Tollefsen et al. (J. Biol. Chem., 257, 2162, 1982) and abilities of dextran sulfate and various glycosaminoglycans to activate the antithrombin activities of HC II and antithrombin III (AT III) were studied. By the purification method described here, highly purified HC II with the same specific activity as reported by Tollefsen et al. was obtained with a higher yield and in a shorter purification time. Heparin, dextran sulfate and chondroitin polysulfates 1 and 5 activated both HC II and AT III, while dermatan sulfate activated only HC II. Dextran sulfate was almost as active as heparin in the activation of HC II and AT III, indicating that in the interactions of heparin with HC II and AT III, sulfate groups of heparin are more important than carboxyl groups. When mixed with thrombin in the presence of dermatan sulfate, normal human plasma showed antithrombin activity which was not due to AT III but to HC II only. HC II did not inhibit factor Xa or plasmin in the presence of any glycosaminoglycans or dextran sulfate, suggesting that HC II would be a specific inhibitor of thrombin.

Hemostatic Abnormalities and Liver Diseases

Professor Eberhard F. Mammen greatly contributed to the understanding of the relationship between hemostatic abnormalities and liver diseases. The physiology of the hemostatic system is closely linked to liver function because the liver parenchymal cells produce most of the factors of the clotting and fibrinolytic systems. Acute or chronic hepatocellular diseases and hepatic failure including liver cirrhosis, vitamin K deficiency, liver surgery including liver transplantation, and sclerotherapy of bleeding esophageal varices, which were classified by Prof. Mammen, show various hemostatic abnormalities in the coagulation system, fibrinolytic system, platelets, and the reticuloendothelial system. Hemostatic abnormalities in patients with hepatic failure or in those that have undergone liver surgery are similar to those in disseminated intravascular coagulation. Prof. Mammen also contributed to the study of vitamin K-dependent clotting factors, antithrombin, and hemostatic molecular markers. Partly based on this work, the prothrombin time-international normalized ratio, several hemostatic molecular markers, and antithrombin therapy have been recently developed for the diagnosis and treatment of thrombosis.

Hemostatic Molecular Markers Before the Onset of Disseminated Intravascular Coagulation

We retrospectively measured various hemostatic markers in 240 patients with disseminated intravascular coagulation (DIC) before the onset of DIC and in 110 non‐DIC patients, and examined their usefulness for the diagnosis of pre‐DIC. Changes in prothrombin time ratio and fibrinogen levels were not significant before the onset of DIC. The plasma levels of fibrinogen and fibrin degradation products before the onset of DIC were increased and the platelet count was gradually reduced in nonleukemic patients; these changes were already significant in the non‐DIC state. The plasma levels of thrombin–antithrombin complex (TAT), plasmin–plasmin inhibitor complex (PPIC), D‐dimer, and soluble fibrin monomer (sFM) were increased before the onset of DIC. In leukemic patients, the plasma levels of sFM on day 5, those of TAT on day 3, and D‐dimer on day 1, were significantly increased before the onset of DIC. The levels of most hemostatic markers 7 days before the onset of DIC were not different from those observed in the non‐DIC state. In nonleukemic patients, only D‐dimer, sFM, and TAT levels were significantly increased 7 days before the onset of DIC compared with values in the non‐DIC state. The positive rate of hemostatic markers for the diagnosis of DIC, TAT, and PPIC were high during the pre‐DIC and non‐DIC groups. The plasma levels of sFM and D‐dimer were low in non‐DIC and increased gradually during the pre‐DIC state. These findings suggest that hemostatic molecular markers such as sFM, D‐dimer, and TAT are useful for the diagnosis of pre‐DIC, although their cutoff values were different among various diseases. Am. J. Hematol. 60:273–278, 1999.

Pathologic cells as procoagulant substance of disseminated intravascular coagulation syndrome in acute promyelocytic leukemia

Abstract Pathologic cells of acute promyelocytic leukemia were studied as a potential trigger substance for disseminated intravascular coagulation syndrome (DIC). The pathologic cells had both procoagulant and fibrinolytic activity. When this substance was separated by Sephadex G-200 column chromatography, procoagulant activity and fibrinolytic activity were observed in the same peak. Procoagulant activity was proven by prothrombin conversion activity, and fibrinolytic activity was proven by plasminogen free and plasminogen rich fibrin plate method. This procoagulant activity was inhibited by Trasylol and heparin, and fibrinolytic activity was inhibited by Trasylol and soybean trypsin inhibitor (STI). From the point of the effect of these inhibitors on the proteolytic action of the pathologic cells, Trasylol administration should be the best treatment for DIC of acute promyelocytic leukemia because Trasylol inhibited both hypercoagulation and hyperfibrinolysis. When the lysate of the pathologic cells of acute promyelocytic leukemia was infused into a rabbit, typical DIC was observed in laboratory studies, and many fibrin thrombi were observed in kidneys and lungs.

The extract from the tissue of gastric cancer as procoagulant in disseminated intravascular coagulation syndrome.

Abstract Tissue extracts from two histopathologic types of surgically removed gastric cancer were investigated for procoagulant and fibrinolytic activities. Procoagulant activity was found to be due to a thromboplastin-like substance; 1 mg of the extract protein was equivalent to 0.014 mg of Lyoplastin (thromboplastin). It is inhibited by heparin, Trasylol and is partially inactivated by heating at 100°C for 10 min, but not at 56°C and 37°C. The extracts had both plasminogen activator and plasmin activities. The fibrinolytic activity was inhibited by soybean trypsin inhibitor, Trasylol, and t-AMCHA. In 12 cases studied, nine were of adenocarcinoma and three were carcinoma simplex. Both procoagulant and fibrinolytic activities appeared to be greatest in the extracts from adenocarcinoma, as compared to those from carcinoma simplex or the normal tissue surrounding the lesions.

Heparin cofactor II-dependent antithrombin activity of calcium spirulan

Calcium spirulan (Ca-SP), a novel sulfated polysaccharide isolated from the blue-green alga Spirulina platensis, enhanced the antithrombin activity of heparin cofactor II (HC II) more than 10000-fold. The apparent second-order rate constant of thrombin inhibition by HC II was calculated to be 4.2 x 10(4) M-1 min-1 in the absence of Ca-SP, and it increased in the presence of 50 micrograms/ml Ca-SP to 4.5 x 10(8) M-1 min-1. Ca-SP effectively induced the formation of a thrombin-HC II complex in plasma. In the presence of Ca-SP, both the recombinant HC II variants Lys173-->Leu and Arg 189-->His, which are defective in interactions with heparin and dermatan sulfate, respectively, inhibited thrombin in a manner similar to native rHC II. This result indicates that the binding site of HC II for Ca-SP is different from the heparin- or dermatan sulfate-binding site. When we removed the calcium from the Ca-SP, the compound did not exert any antithrombin activity. Furthermore, Na-SP, which was prepared by replacement of the calcium in Ca-SP with sodium, accelerated the antithrombin activity of HC II as Ca-SP did. We therefore suggest that the molecular conformation maintained by Ca or Na is indispensable to the antithrombin activity of Ca-SP. The HC II-dependent antithrombin activity of Ca-SP was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, but not by treatment with chondroitinase ABC and chondroitinase AC II, suggesting that a heparin- or dermatan sulfate-like structure is not responsible for the activation of HC II by Ca-SP. Ca-SP is therefore thought to be a unique sulfated polysaccharide which shows a strong antithrombin effect in an exclusively HC II-dependent manner.