Flow cytometry has recently been used to determine the characterization of platelet surface membrane. However, the utilization of this technique to detect the platelet binding IgG (PBIgG) has been limited, because a high frequency of non-specific reaction has been observed on the platelet membranes when reacted with sera from healthy individuals. One reason for this may be related to the surface bound HLA-A, B, C. To overcome this problem we have used the method of acid treatment of platelets. The platelet pellets prepared by albumin density gradient separation were first treated with 0.123M citric acid-Na₂HPO₄, pH 3.0, for 10min on ice, followed by formalin fixation. In the present study we have performed the flow cytometry analysis and have used the parameter, delta mean fluorescence intensity channel number (Δ MFI) which corresponds to the relative amounts of antigens on the platelet membrane. By acid treatment of platelets, Δ MFI for HLA-A, B, C markedly reduced. ΔMFI for platelet surface IgG also reduced. Then, the levels of PBIgG in healthy adults and patients with chronic idiopathic thrombocytopenic purpura (ITP) were determined. For healthy adults, the Δ MFI reduced significantly by acid treatment. We then determined the levels of PBIgG (Δ MFI) in 20 patients with chronic ITP using acid-treated platelets. The levels of PBIgG in chronic ITP was significantly higher than healthy controls. In the ITP patients, a clearly inverted correlation was observed between the platelet count and the levels of PBIgG (r=-0.7242, P<0.01). The captured enzyme linked immunosorbent assay (captured-ELISA) was also performed in the same 20 ITP patients. Monoclonal antibodies agaist GPIb or IIb/IIIa complex were used. Anti GPIb antibody activity was detected in seven of 20 patients. Anti GPIIb/IIIa complex antibody activity was detected in two patients. These results clearly indicate that acid treatment of platelets increases the availability and specificity for the detection of anti-platelet antibodies.

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Stable prostacyclin (PGI₂) analogue, beraprost sodium (sodium (±)-(1R*, 2R*, 3aS*, 8bS*)-2, 3, 3a, 8b-tetrahydro-2-hydroxy-1-[(E)-(3S*)-3-hydroxy-4-methyl-1-octen-6-ynyl]-1H-cyclopenta [b] benzofuran-5-butyrate, TRK-100) had been reported to inhibit aggregation of platelets and dilate blood vessels. In this report, the effect of beraprost sodium (beraprost) on haemorheological parameters such as erythrocyte deformability and blood viscosity, and activity of blood coagulation system such as activated partial thromboplastin time and prothrombin time were measured in normal rats. Effect of orally administered beraprost on experimentally induced disorders of peripheral circulation such as femoral artery occlusion and venous thrombosis model in rats were assessed.
Erythrocyte deformability was increased by beraprost from the concentration of 23.8 up to 2380nM in vitro (Fig. 1), and from 176 up to 560μg/kg/day after 7 days consecutive administration to normal rats ex vivo (Fig. 2).
Occlusion of rat femoral artery by insertion of polyethylene tube induces reproducible lesions in the toe (Fig. 7) and elevation of blood and plasma viscosity (Tab. 1). Blood viscosity of normal rats was not affected by beraprost up to 10μM in vitro (Fig. 3) or up to 100μg/kg ex vivo (Fig. 4). However, at the dose of 30μg/kg, beraprost normalized blood viscosity of femoral artery occluded rats (Fig. 8).
In the model of stasis induced venous thrombosis, beraprost decreased the weight of thrombus in the inferior vena cava of rats. The effect was dose-dependent at range from 30 to 100μg/kg (Fig. 9). It, however, showed no effects on blood coagulation system of rats ex vivo at the dose which decreased thrombus weight (Fig. 6), and at the concentration range of 23.8nM to 238μM in human plasma in vitro (Fig. 5).
Above results suggest that beraprost may contribute to the improvement of haemorheological impairment and prevention of thrombus formation in the disorder of peripheral circulation.

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Activation of fibrinolysis in patients with diabetic nephropathy was determined by the plasma levels of α₂ plasmin inhibitor-plasmin complexes (α₂ PIPC). The levels of α₂ PIPC in plasma were measured by the one-step sandwich enzyme immunoassay (EIA). This quantitative assay of α₂ PIPC could be valuable for the assessment of fibrinolysis. Plasma levels of α₂ PIPC in diabetic patients with overt proteinuria were significantly higher than those in diabetic patients without proteinuria or in chronic glomerulonephritis patients with proteinuria (P<0.01, respectively). Plasma levels of α₂ PIPC in diabetic patients with intermittent proteinuria were also significantly higher than those in diabetic patients without proteinuria or in chronic glomerulonephritis patients with proteinuria (P<0.05, respectively). These findings indicated that the activation of fibrinolysis as well as the coagulation system might play a role in the development and/or progression of diabetic nephropathy.

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We studied the effects of noneyzymatic glycation on human antithrombin III (AT-Ill). In vitro, purified human AT-III (15U/ml) was incubated with glucose (0, 100, 400, 1, 000mg/dl) at 37°C for 2 weeks. AT-III was significantly glycated and then progressive activity and heparin cofactor activity of glycated AT-III decreased, while the levels of AT-III antigen had not changed. When glycated AT-III (1U/ml) was incubated with thrombin (250NIHU/ml) for various times, the amount of complex formed was less than control (control: 4.3-10.6mg/ml, glycated AT-III: 0.4-6.2mg/ml, p<0.01). In the presence of heparin, glycated AT-III (treated with 1g/dl glucose) was only formed a complex of high molecular weight (M.W.) from SDS-PAGE. The amount of complex formed was the same degree both control and glycated AT-III (treated with 0-400mg/dl glucose). But in the glycated AT-III (treated with 1g/dl glucose), the pattern of complex formation was different from control because of measurable complexes of M.W from 78, 000 to 81, 500 were detected in this method.
On crossed immunoelectrophoresis containing heparin in the first dimension agarose, control AT-III demonstrated AT-III as 2 peaks including the fast moving main peak. On the contrary, glycated AT-III showed 2 main peaks including the slow moving peak, suggesting that the glycated AT-III has little affinity for heparin. We attached a guanidine group to the lysyl residues of AT-III by treating it with O-methylisourea. The modified AT-III was not glycated and then progressive activity had not changed and heparin cofactor activity decreased.
From these observations, it is suggested that lysyl residues of AT-III are essential in nonenzymatic glycation and nonenzymatic glycation and nonenzymatic glycation causes a conformational change which results in a poor formation of complex, decreasing AT-III activity.

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In order to evaluate precisely the fibrinolytic state in clinical disorders, plasma levels of fibrinogenolysis products (FgDP), fibrinolysis products (FbDP) and fibrinogenolysis plus fibrinolysis products (TDP) were measured by newly developed sandwich ELISA in patients with hematological malignancies, diabetes mellitus, thrombotic disease, liver disease and disseminated intravascular coagulation (DIC). The mean plasma levels of FgDP, FbDP and TDP in 27 healthy subjects were 389±SD 127, 246±98 and 685±205ng/ml, respectively. Not only FbDP but also FgDP were elevated in a variety of diseases except for liver disease and diabetes. Marked elevations in both FbDP and FgDP were found in DIC followed by hematological malignancies and thrombotic disease. Among several disease categories underlying DIC, patients with acute promyelocytic leukemia and vascular disease had a high FgDP/TDP ratio and patients with sepsis had the lowest. On the whole, FgDP and FbDP values were positively correlated with plasmin-α₂-plasmin inhibitor complex and thrombin-antithrombin III complex, and negatively correlated with α₂-plasmin inhibitor. These findings indicate that primary fibrinogenolysis is rare in liver disease, but fibrinolysis is usually accompanied by fibrinogenolysis in DIC, thrombotic disease and hematological malignancies. Thus measurements of FgDP, FbDP and TDP would be valuable for the precise assessment of fibrinolysis in selected disease states.

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Activities of clotting and fibrinolytic factors in golden hamsters under acute alcohol consumption were measured with fluorogenic peptide substrate. The obtained data were as follows;
1) Concentration of blood alcohol reached to the maximum values on 1hr after administration of alcohol.
2) Prothrombin and factor X activities began to decrease on 1hr after administration. But their activated form, thrombin like and factor Xa like activities significantly increased on 1hr, then returned to initial value on 4hrs after administration.
3) Although plasminogen activity began to decrease on 1hr, activated form of it, plasmin like, and t-PA like activities increased on 1hr and returned to the initial values or decreased on 4hrs. Both prekallikrein and kallikrein like activities significantly increased from 1hr.
Under acute alcohol consumption, activities of clotting and fibrinolytic factors (prothrombin, factor X, plasminogen) in golden hamster were changed into their activated form (thrombin, factor Xa, plasmin) at the corresponding time that concentration of blood alcohol reached to the maximum values.

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A 15-year-old Japanese boy with severe haemophilia A developed ulcerative colitis. In spite of the absence of factor VIII inhibitor, factor VIII replacement and/or sulfasalazine resulted in no improvement of his bloody stools. The symptoms were successfully managed with oral prednisolone.
This is an extremely rare case with haemophilia A developing ulcerative colitis. The findings indicate that when it is difficult to control gastrointestinal haemorrhage in a haemophiliac using factor VIII concentrate, factor VIII inhibitor or other organic lesions including ulcerative colitis must be taken into consideration.

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In this paper, we described a case of thrombosed Hall-Kaster valve who suffered from recurrent coronary thromboemboli.
The patient was a 39 year-old man who received an operation of aortic valve replacement (Hall-Kaster valve) and mitral valve replacement (St. Jude Medical valve) for his rheumatic heart disease 10 years ago (Fig. 1). Since then he had been chronically anticoagulated using warfarin and remained symptom free. He suffered from acute myocardial infarction on January, 1989 and unstable angina on April, 1989. Coronary angiogram at the time of those two episodes revealed thrombi in left coronary artery system (middle portion of left anterior descending artery, left main trunk) (Fig. 4, 5, respectively). Although we suspected of thrombosed prosthetic valve, the diagnosis could not be made by auscultation, echocardiogram including Doppler technique, cardiac catheterization and angiogram before the operation. Patient's blood sample revealed high level of D-dimer at the time when the coronary artery thrombi disappeared on angiogram. It was speculated that thrombus derived from elsewhere (Table). On November 21, 1989 he suffered from acute left-sided heart failure due to thrombosed valve with chest pain. Although emergency re-operation of valve replacement was carried out, he died during the operation. The inspection at the time of the operation revealed thrombosed Hall-Kaster prosthetic valve (Fig. 6).

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