We developed a new thrombosis model using a microvasculature of hamster cheek pouch for the kinetic evaluation of thrombotic process in relation to endothelial damage. The thrombus was produced in the venule with excellent reproducibility by the irradiation of the filtered light with the intravascular administration of fluorescein sodium. The thrombotic process, assessed by two parameters such as Ti (initiation time of thrombus formation) and Ts (stopping time of blood flow by thrombus), was dependent on the light intensity and the dye concentration.
In the initial stage of thrombus formation, cytoplasmic organella swelling, vacuole formation, luminal membrane rupture and swelling of the nuclear envelope were observed in endothelium. With progress of thrombus formation, these changes became more obvious and focal endothelial denudation was observed at the advanced stage. The thrombus was developed only on the irradiated vascular endothelium without exposure of basement membrane, which suggested that primary endotherial damage could lead to the thrombus formation.
Superoxide dismutase (SOD), scavenger of oxygen radicals, was intravenously applied to evaluate the cytotoxic effects of oxygen radicals on endothelium. Continuous infusion of SOD of 5, 000 and 10, 000U/kg/min dose dependently prolonged Ti from 66.8±9.6 to 121.0±46.1 and 186.2±40.0sec, and Ts from 405.9±32.5 to 792.0±190.5 and 921.8±197.2sec (mean±SD, n=10), respectively. Furthermore, morphological changes of endothelium and platelet were markedly reduced by administration of SOD. From these data, oxygen radicals were suggested to cause the injury of endothelium and lead to thrombus formation.

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We investigated the relationship between IL-6 and hemostatic markers regarding surgery in two groups of abdominal surgery: Group I, hepatectomy (20 cases); Group II, gastrectomy (10 cases) and colectomy (10 cases). IL-6 increased during operation and early postoperative period. There is no significant difference between two groups during these periods. But IL-6 increased late postoperatively in 5 patients with complications in Group I. Thrombin-antithrombin III complex (TAT) in both groups increased similarly during operation and continued until early postoperative period. In the fibrinolytic system, tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) increased, but not plasmin α 2 plasmin inhibitor cimplex (PIC) in both groups during operation. Significant correlation was noted between the levels of IL-6 and TAT (r=0.482), PAI-1 (r=0.405) in all patients during operation. Following the peak levels of IL-6, Fibrinogen, α 1 antitrypsin and α 2 plasmin inhibitor increased significantly on the 3rd or 5th postoperative day. These increases were prominent and tended to correlate with IL-6 levels in Group II, but not in Group I. In a hepatectomy patient with severe septic complication and disseminated intravascular coagulation, marked increase of IL-6 was seen and suggested a poor prognosis.

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The purpose of this study is to evaluate the platelet functions in 11 male (66±4 years old) with arteriosclerosis obliterans (ASO) and the effects of Ticlopidine and Cilostazol on the clinical and laboratory features of the patients using crossover test. Clinical effects were evaluated based on subjective symptoms and pressure index (PI), the ratio of systolic blood pressure between the affected limb and the unaffected upper extremity. Platelet aggregability was expressed by maximum aggregation rate induced with both 1 (MA1) and 2μM (MA2) of ADP (%), and by collagen aggregability (CA) (μg/ml) which was expressed by the lowest collagen concentration among 7 different ones which could induce more than 50% in the maximal amplitude of aggregation. Plasma β thromboglobulin (βTG) and platelet factor 4 (ng/ml) were also measured. Platelet aggregability was also examined in 8 male without ASO whose clinical profiles were similar to those in 8 of 11 in this study. (1) Platelet aggregability was significantly higher in patients with ASO (MA1: 34±28, MA2: 59±33, CA: 0.8±0.4) than those without ASO (MA1: 18±8, MA2: 50±19, CA: 1.4±0.7). (2) More patients were subjectively improved with Cilostazol (7 of 11) compared with Ticlopidine (2 of 11) (p<0.05). PI was also improved more common with Cilostazol (7 of 11 cases) than with Ticlopidine (3 of 11 cases) (p<0.05). (3) Platelet aggregability was significantly lower with Ticlopidine (MA1: 14±10, MA2: 30±13, CA: 2.0±1.6) than with Cilostazol (MA1: 29±16, MA2: 50±23, CA: 1.4±0.9), although both of agents significantly suppressed platelet aggregability compared with the control value (MA1: 39±31, MA2: 68±31, CA: 0.8±0.4). βTG was also significantly lower with Ticlopidine (36±15) than with Cilostazol (55±10). Thus there was a considerable discrepancy between clinical and laboratory effects of these agents. This might be accounted for by vasodilating action and microcirculatory effect of Cilostazol.
In conclusion, enhanced platelet aggregability was found in patients with ASO, and both Ticlopidine and Cilostazol seemed to be useful strategy for them, but further study is necessary for clarifying the relationship between the clinical improvements and the degree of suppressive effects on platelet functions with these agents.

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Megakaryocytopoiesis was immunohistochemically investigated by staining paraffin-embedded sections of BrdU in vitro treated bone marrow cells (CML 8 cases, MDS 13 cases, ITP 16 cases and Control 6 cases) with antibodies to Bromodeoxyuridine (BrdU) and von Willebrand factor (vWf) or Platelet glycoprotein IIIa (GPIIIa). The percent of BrdU-labeled cells (BrdU⁺) and that of cells recongnized with vWf (vWf⁺), or GPIIIa (GPIIIa⁺) were microscopically calculated. No significant difference in the percent of BrdU⁺ cells and that of megakaryocytes among these diseases was found. In cases with ITP, the number of immature megakaryocytes and BrdU⁺ megakaryocytes did not increase compared with that of control. In cases with CML and MDS, however, increases of immature megakaryocytes and BrdU⁺ megakaryocytes were observed. In MDS, cases transformed to acute leukemia were found to show increased number of immature megakaryocytes and BrdU⁺ megakaryocytes. It was concluded that the increases of immature megakaryocytes or BrdU⁺ megakaryocytes might reflect the clinical progress of MDS.

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An inflammation mediator interleukin-6 (IL-6) acts on various types of cells including vascular endothelial cells. Since inflammation is associated with locally enhanced coagulation, and the endothelium is actively involved in coagulation, it is of interest to investigate whether IL-6 excerts effects on procoagulant and fibrinolytic activities of endothelial cells. This paper describes a study in this matter using cultured bovine endothelial cells (CBEC).
IL-6 did not affect the CBEC proliferation, as shown by ineffectiveness in incorporation of ³H-thymidine into the cells. IL-6 suppressed the secretion of plasminogen activator (PA) of CBEC: both PA activity and tissue-type PA antigen in the culture medium were reduced in the presence of IL-6. In contrast, IL-6 increased the secretion of plasminogen activator inhibitor 1 (PAI-1). Moreover, the cytokine caused CBEC to produce tissue factor (TF) temporarily, which did not occur in the control culture. When IL-1 coexisted, these effects of IL-6 were enhanced.
These findings indicate that IL-6 implicates in generation of hypercoagulability in the inflammatory lesions by modulating the coagulation and fibrinolytic activities of endothelial cells.

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Effects of green tea catechins on thrombin activity were investigated. The major part of catechins in green tea are epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECg) and epigallocatechin gallate (EGCg). ECg and EGCg except others were found to be more potent inhibitors for thrombin by mode of noncompetitive inhibition. These 50% inhibition of thrombin amidolysis by ECg and EGCg were 1.2×10^-6M and 1.1×10^-6M, and fibrin formation were 2.5×10^-5M and 8.8×10^-6M.
These facts suggested that thrombosis may be prevented by ECg and EGCg in green tea.

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We investigated the effect of histidine-rich glycoprotein (HRG) and platelet factor 4 (PF4) on the antithrombin III (ATIII) cofactor action of parnaparin sodium (LHG), a type of low molecular weight heparin (LMWH), and unfractionated heparin. The anti-Xa actvity of both LHG and heparin was inhibited by these proteins, but the degree of inhibition was significantly less in case of LHG. In contrast, the anti-thrombin actvity of both LHG and heparin was inhibited to almost the same degree by these heparin-neutralizing proteins. Moreover, studies with crossed immunoelectrophoresis of ATIII revealed that both HRG and PF 4 more strongly inhibited the complex formation between heparin and ATIII compared to that of LHG and ATIII. These findings show that HRG and PF 4 inhibit the ATIII cofactor activity of larger size heparin molecules. In addition, the inhibitory action of HRG and PF 4 on the anti-Xa activity of the fraction of LHG with high affinity for ATIII (HA-LHG) was reduced by the low-affinity fraction (LA-LHG). Therefore, this shows that while the LA-LHG itself is “inactive”, it plays an important role in protecting the “active” molecules from heparin neutralizing proteins. LHG has a longer half-life compared to heparin and has been demonstrated to possess sustained anticoagulant activity. Since LHG is less affected by proteins like HRG and PF 4 compared to heparin, the anticoagulant activity can probably be thought of as one of its results.

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In order to evaluate the safety and efficacy of recombinant factor VIII (BL-160) in treating bleeding episodes, a multicenter clinical study was conducted in twenty-six hemophilia A patients. This product is produced by recombinant technology using the human factor VIII hosted in Chinese hamster ovary cells followed by anti-factor VIII immunoaffinity and ionexchange chromatographic purification steps. The twenty-six patients have been treated with BL-160 for at least six months after the initial infusion. The mean t 1/2 value and the mean recovery rate at entry were 13.6±6.6 hrs (n=8) and 87.7±16.7% (n=24) respectively. The t 1/2 and recovery observed with BL-160 werecomparable to those with plasma-derived factor VIII. Five hundred and ninety-nine bleeding episodes were assessed during the study period, hemostatic effects were excellent in 419, good in 155 and fair in 25 bleeding episodes, the efficacy rate was 95.8%. No patient has developed evidence of an inhibitor to factor VIII. Also, there is no evidence of neoantigenicity resulting from potential contaminating proteins used in production steps. No adverse reactions were reported. These results indicate that recombinant factor VIII (BL-160) is safe and efficacious for the treatment of bleeding episodes in hemophilia A.

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