We tested a new system TS-4000 which simulated primary hemostasis in vitro in diabetic patients, in an attempt to examine the relationship between primary hemostasis in vitro and diabetic retinopathy and anti-platelet medication effect.
1) Initial flow rate of the blood (INF) tended to be higher in diabetics compared with normal controls, but the time until blood flow stopped (BT) was significantly shorter and blood volume flowing out (BV) was significantly smaller in diabetics than in normal controls. 2) INF was higher, BT was almost equivalent, and BV tended to be larger in diabetics who were given anti-platelet drugs compared with those who were not given anti-platelet drugs. 3) Although it was statistically insignificant, the effect of anti-platelet drugs was different between patients without retinpathy and with simple retinopathy, and patients with proliferative retinopathy. For patients without retinopathy and with simple retinopathy, INF was higher, BT was somewhat longer and BV was larger in diabetics who were given anti-platelet drugs compared with those who were not given anti-platelet drugs. For patients with proliferative retinopathy, INF was higher but BT was shorter in diabetics who were given anti-platelet drugs compared with those who were not given anti-platelet drugs, so BV was not different between treated or untreated patients.

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In order to investigate a possibility that polymerization of platelet cytoskeletal proteins may contribute to an association of cytosolic phospholipase A₂ (PLA₂) with the plasma membranes, the present study was undertaken to examine whether or not cytosolic PLA₂ is increased in Triton-insoluble fractions containing cytoskeletal components upon stimulation of rabbit platelets. Apparent PLA₂ activity could be detected in the Triton-insoluble residue obtained from thrombin or collagen-stimulated platelets, and the enzyme activity increased in a dose-dependent manner of the agonists. The specific activity of the enzyme in the residue from the stimulated cells was higher than that from unstimulated cells. The enzyme in the former residue was significantly activated with 0.5-10μM Ca²⁺ and had a substrate preference for hydrolyzing phospholipids having an arachidonoyl residue more effectively than the ones with a linoleoyl residue. Furthermore, 70% of the enzyme activity was immunoprecipitated with an antibody against cytosolic PLA₂ of rabbit platelets, while only 20% of that was neutralized by an antibody against group II PLA₂. In addition, the specific activity of PLA₂ in Triton-insoluble residue obtained from membrane fractions of platelets also increased upon stimulation with thrombin, while such an increase was not observed when cytochalasin E-treated platelets were stimulated with thrombin. These results suggest that the interaction of cytosolic PLA₂ with cytoskeletal proteins increases upon stimulation of rabbit platelets, and that the cytoskeletal reassembly might be involved in an association of the enzyme with plasma membranes.

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Two monoclonal antibodies AI18 (IgM) and AI51 (IgG₁) were generated against the Factor X activating coagulant serine protease which extracted from LK52 human squamous cell carcinoma cell line. Both antibodies inhibit the activity of LK52 coagulant serine protease, but not of Russel's viper venom. AI18 and AI51 react with human lung and colon cancer cell lines, but not react with mouse cancer cell lines, rabbit cancer cell line or human fibroblast cell lines from lung and colon. From these results, LK52 coagulant serine protease was named as coagulant cancer antigen 1 (CCA-1). This is a first report of a novel CCA-1 expression on various human cancer cell lines. CCA-1 may play a important role in unbalanced haemostasis of cancer patients.

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Antiphospholipid syndrome patients often suffer from thrombotic episodes, but the mechanism of thrombosis remains unclear. β2-Glycoprotein I (β2-GPI) is known to play an important role in the reaction between antiphospholipid antibodies (APA) and cardiolipin, but the true antigen for APA is not known. Although there are many laboratory tests to detect APA, none of them shows good correlation with the clinical severity of thrombosis. Moreover, cases of antiphospholipid syndrome vary in their APA types. To elucidate the interaction among APA, β2-GPI and phospholipids, we investigated the effects of the IgG fraction of APA on the activation of factor X by the using the chromogenic substrate S-2222.
The IgG fraction from 2 of 4 patients dose-dependently inhibited the activation of factor X. β2-GPI also dose-dependently inhibited phospholipids and decreased the generation of Xa. To investigate the interaction between β2-GPI and IgG fractions, they were incubated together before adding phospholipids. The inhibitory effects in Xa generation were increased, and the effects did not seem to neutralize each other.

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Recently, polycations have been reported to activate the erythrocyte multicatalytic proteinase and release lactate dehydrogenase from endothelial cells. Furthermore, polycations seem to affect vascular permeability and prostaglandin synthesis in cultured glomerular mesangial cells. We previously reported the application of poly-L-lysine for the purification of leukocyte cathepsin G (CLP) by affinity chromatogaphy and the effect of polycations on the amidolytic activities of leukocyte elastase (ELP) and CLP.
In this study, we also found that at a low concentration, poly-L-lysine stimulated the release of ELP and CLP from leukocytes.
1) At a concentration of 1.5μM, poly-L-lysine (mw. 290, 000) stimulated the release of ELP and CLP from separated leukocytes in the same extent as 2M NaCl.
2) Poly-L-lysine (mw. 290, 000) stimulated the release of CLP and ELP from leukocytes in diluted blood at a concentration of 5 and 10nM, respectively. On the other hand, a several times higher concentration of poly-L-lysine was required for release of both enzymes from leukocytes in non-diluted blood.
3) In proportion to molecular weight, poly-L-lysine enhanced amidolytic activities of ELP and CLP and the fibrinolytic activity of ELP. Thus, poly-L-lysine enhanced the activity of ELP at a lower concentration than activity of CLP.

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The effects of argatroban (a selective thrombin inhibitor) and unfractionated heparin (UFH) on t-PA induced thrombolysis were compared in an experimental thrombosis using the microvasculature of hamster cheek pouch. The thrombus was produced by irradiation of ultraviolet light (intensity; 20mW/mm², wave length; 400-500nm) in combination with the intravascular administration of fluorescein sodium (50mg/kg), until the development of thrombus stenosed 99% of the luminal area of the venule (50-70μm). Thrombolytic process mediated by t-PA was observed on a color television through a high gain video camera and recorded on videotape. The effects of these anticoagulants administered with t-PA were evaluated by the percent stenosis of lumen and the percent area of thrombus, which were calculated by the computed video analyzer. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were measured on plasma samples obtained one hour after the injection of anticoagulants. Bleeding time was measured on the shaved left medial hind leg with blade before sampling the blood. Argatroban (0.1-0.3mg/kg·hr) significantly enhanced thrombolysis by t-PA (72×10⁴ IU/kg·hr) in one and two hours after infusion of t-PA. Argatroban (0.1-0.3mg/kg·hr) prolonged PT, aPTT and bleeding time dose dependently and significantly at the dose of 0.3mg/kg/·hr. Argatroban (0.3mg/kg·hr) enhanced thrombolysis by t-PA (72×10⁴ IU/kg·hr), but UFH (12.5 anti-X U/kg·hr) delayed it. Argatroban (0.3mg/kg·hr) did not significantly prolonged PT and aPTT as compared with UFH (12.5 anti-X U/kg·hr). From these results, argatroban was suggested to be a useful additional anticoagulant in thrombolytic therapy using t-PA.

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Plasma levels of plasmin-α₂-plasmin inhibitor complex (PPI) were measured by a rapid semi-quantitative method based on the latex agglutination immunoassay in patients with a variety of diseases such as hematological malignancies, infection, thrombotic disease and disseminated intravascular coagulation (DIC). As compared with healthy subjects, plasma levels of PPI were markedly elevated in the patients with DIC. In addition, some patients with thrombotic disease and hematological malignancies had high PPI levels. Serial determinations of PPI demonstrated a dynamic fluctuation of PPI during the course of DIC and during fibrinolytic therapy. On the whole, PPI values obtained by this assay were correlated well with those measured by an enzyme-linked immunosorbent assay. In addition, the plasma PPI level was correlated positively with concurrently assayed FDP and D-dimer levels, and negatively with plasma levels of fibrinogen, α₂-plasmin inhibitor and plasminogen. No correlation was found between the PPI level and antithrombin III, protein C or thrombin-antithrombin III complex values. These findings indicate that a rapid measurement of the plasma PPI level with this method would be useful for the assessment of activation of fibrinolysis in these disease states.

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