The coagulation and fibrinolytic systems are central to the hemostatic mechanism, which works promptly on vascular injury and tissue damage. The rapid response is generated by specific molecular interactions between components in these systems. Thus, the regulation mechanism of the systems is programmed in each component, as exemplified by the elegant processes in zymogen activation. This review describes recently identified small molecules that modulate the activation of zymogens in the fibrinolytic and coagulation systems.

Using a ¹H NMR metabolomics approach, the effects of dietary exposure of bunker A heavy oil (0.01, 0.1, 1, and 5% in diet) on freshwater carp, Cyprinus carpio, were examined. Statistical analysis by PCA score plots showed that the amount of metabolites in exposure groups 0.1, 1, and 5% differed from those in the control group. Although no discernible effects on metabolites were noted in the 0.1% exposure group as well as in the lowest concentration (0.01%) group, several metabolites such as amino acid (e.g., leucine, isoleucine, valine, glutamine, histidine, proline, and methionine), 3-D-hydroxybutyrate, and glycerol were elevated, while another metabolite such as formate was reduced in 1 and 5% groups. These changes in the metabolites associated with the tri-carboxylic-acid (TCA) cycle suggest that oil exposure resulted in the disturbance of the TCA cycle in the liver of the carp. Isobutyrate, a marker of anoxia, was also increased in 1 and 5% exposures groups and was directly related to low hemoglobin concentrations leading to reduced oxygen transport by blood. In addition, significant elevation of creatinine in the plasma of carps exposed to 5% heavy oil suggests disturbance in kidney function. Thus, metabolomics approach can detect toxic effects of hazardous pollutants on fish.

A novel peptaibol, designated clonostachin, was isolated from cultures of Clonostachys sp. F5898 by HP-20 and silica gel column chromatographies and reverse-phase HPLC.The structure ofclonostachin was determined by Edmanand chemical degradations, positive ion FAB-MS,EI-MS, and NMR analyses. Clonostachin was a linear tetradecapeptide with an TV-terminal acetyl group and a C-terminal sugar alcohol. Clonostachin inhibited ADP-induced aggregation of human platelets by 80% at 150/jm. 'During the course of screening for blood coagulation inhibitors of microbial origin, we have found a novel peptaibol, clonostachin, as an active compound. In this paper, we describe the isolation, structure elucidation and biological activity of clonostachin. Materials and Methods MaterialsHumanvenous blood was drawn from healthy volunteers in 13mM sodium citrate. L-Isovaline (Iva) was purchased from Acros, a-amino isobutyric acid (Aib) and ADPwere obtained from Sigma. Malt extract agar, oatmeal agar and corn meal agar were purchased from Difco, U.S.A. Czapek-Dox agar was obtained from Eiken chemical, Japan. YpSs agar was prepared as follows: soluble starch (15.0 g) and agar (20.0g) was dissolved in 400ml of distilled water by heating in a boiling bath, followed by adding 600 ml of distilled water containing malt extract (4.0g), K2HPO4 (1.0g) andMgSO4-7H2O (0.5g). MicroorganismThe producing strain Clonostachys sp. F5898 was isolated from a soil sample collected in Koganei-shi, Tokyo and subcultured on potato glucose agar slants at 25°C.Amino Acid Analysis Clonostachin (0.5mg) was hydrolyzed in 6n HC1 at After incubation at 50°C for 10minutes under nitrogen gas, the mixture was evaporated to dryness at 50°C under high vacuum. The residue was dissolved in 30fi\ of methanol-HC1 (1 : 1, by volume) and incubated at 50°C for 5minutes under nitrogen gas. The mixture was evaporated to dryness and the resulting PTH-amino acids were fractionated by HPLC on an Inertsil PREP-ODS column (6 x 250mm, GL Sciences, Japan) using CH3CN-10mM aqueous sodium acetate, pH 4.5 (39.5 : 60.5). The fraction containing PTH-Iva was extracted with ethyl acetate and the organic extract was applied to HPLCon a CHIRAL-CEL OJ-R column (4.6x 150mm, Daicel Chemical, Japan). The column was developed with CH3CN-10 mM aqueous sodium acetate, pH 4.5 (39.5 : 60.5) at 40°C at a rate of0.5 ml/minute. The identification of the absolute configuration of Iva was carried out by the co-chromatography with the PTH-derivatives of authentic land D,L-Iva.Platelet-rich Plasma and Platelet-poor Plasma Venous blood drawn from healthy volunteers who had not taken any drugs for 2 weeks prior to the study was mixed with 1/9 vol of 0.llm trisodium citrate.

We recently discovered several nonlysine-analog conformational modulators for plasminogen. These include SMTP-6, thioplabin B and complestatin that are low molecular mass compounds of microbial origin. Unlike lysine-analog modulators, which increase plasminogen activation but inhibit its binding to fibrin, the nonlysine-analog modulators enhance both activation and fibrin binding of plasminogen. Here we show that some nonlysine-analog modulators promote autoproteolytic generation of plasmin(ogen) derivatives with its catalytic domain undergoing extensive fragmentation (PMDs), which have angiostatin-like anti-endothelial activity. The enhancement of urokinase-catalyzed plasminogen activation by SMTP-6 was followed by rapid inactivation of plasmin due to its degradation mainly in the catalytic domain, yielding PMD with a molecular mass ranging from 68 to 77 kDa. PMD generation was observed when plasmin alone was treated with SMTP-6 and was inhibited by the plasmin inhibitor aprotinin, indicating an autoproteolytic mechanism in PMD generation. Thioplabin B and complestatin, two other nonlysine-analog modulators, were also active in producing similar PMDs, whereas the lysine analog 6-aminohexanoic acid was inactive while it enhanced plasminogen activation. Peptide sequencing and mass spectrometric analyses suggested that plasmin fragmentation was due to cleavage at Lys615-Val616, Lys651-Leu652, Lys661-Val662, Lys698-Glu699, Lys708-Val709 and several other sites mostly in the catalytic domain. PMD was inhibitory to proliferation, migration and tube formation of endothelial cells at concentrations of 0.3-10 lgAEmL )1 . These results suggest a possible application of nonlysine-analog modulators in the treatment of cancer through the enhancement of endogenous plasmin(ogen) fragment formation.

Plactin, a family of cyclopentapeptides, enhances fibrinolytic activity by elevating the activity of cellular urokinase‐type plasminogen activator (u‐PA), a protease involved in a variety of extracellular proteolytic events. Factor(s) in the blood plasma is an absolute requirement for this plactin activity. In this study, we found that plactin promoted plasma cofactor‐dependent conversion of inactive single‐chain u‐PA to active two‐chain u‐PA on U937 cells. Using plactin‐affinity chromatography, we identified prothrombin as one of the plasma cofactors. In incubations of U937 cells with prothrombin and Xa, plactin increased the formation of thrombin, which cleaved single‐chain u‐PA to afford the inactive two‐chain form. Thrombin‐cleaved two‐chain u‐PA was alternatively activated by cellular cystatin‐sensitive peptidase activity, yielding fully active two‐chain u‐PA. In a purified system, plactin bound to prothrombin, altered its conformation and dually modulated factor Xa‐mediated proteolytic activation of prothrombin to α‐thrombin. Plactin inhibited the activation catalyzed by Xa in complex with Va, Ca2+ and phospholipids (prothrombinase), whereas the activations catalyzed by nonmembrane‐associated Xa were enhanced markedly by plactin. Plactin inhibited in vitro plasma coagulation, which involved prothrombinase formation. Plactin did not cause prothrombin activation or thrombosis in normal mice at doses that produced a protective effect in a thrombin‐induced pulmonary embolism mouse model. Therefore, the dual modulation of prothrombin activation by plactin may be interpreted as leading to anticoagulation under physiological coagulating conditions.