Fibrinolytic and antithrombotic protease from Spirodela polyrhiza

A fibrinolytic protease was purified from a Chinese herb (Spirodela polyrhiza). The protease has a molecular mass of 145 kDa and 70 kDa in gel filtration and SDS-polyacrlamide gel electrophoresis (PAGE), respectively, implying it is a dimer. Its optimum pH was 4.5-5.0. The enzyme was stable below 42 degrees C and after lyophilization. The enzyme activity was inhibited significantly by leupeptin and aprotinin. The protease hydrolyzed not only fibrin but also fibrinogen, cleaving Aalpha and Bbeta without affecting the gamma chain of fibrinogen. It preferentially cleaved the peptide bond of Arg or Lys of synthetic substrates (P1 position). The enzyme had an anticoagulating activity measured with activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT) tests. It delayed APTT, TT, and PT two times at the concentration of 36, 39, and 128 nM, respectively and this was drastically reduced after heat treatment.     

Prothrombin activation by immobilized thrombin]

The ability of thrombin, immobilized on BrCN-activated Sepharose 4B, to split prothrombin, was studied. Immobilized thrombin retained up to 70% of its esterase activity and about 5% of its coagulating activity; it was also found to induce partial proteolysis of prothrombin. Two products of prothrombin degradation isolated, i.e. P1 (m. w. 50.000-52.000) and P2 (m. w. 22.000-24.000), did not show either the thrombin or the prothrombin activities. P1 was converted into thrombin under the action of tripsin or Factor Xa. The rate of conversion was considerably increased after addition of Factor V, thromboplastin and Ca2+ ions. Intravenous administration of P1 to rats resulted in changes in the coagulating system of blood, which may be probably indicative of the stimulation of the anticoagulating system. P2 possessed no thrombogenic activity.     

Fibrinolytic and hypoglycemic effects of the Pro-Gly-Pro-Leu peptide during development of insulin-dependent diabetes in rats

Repeated (over 7 days) intranasal introduction of the Pro-Gly-Pro-Leu peptide into animals at a dose of 1 mg/kg before injection of the diabetogenic metabolite alloxan provided effective protection of an organism against development of insulin-dependent diabetes mellitus and prevented development of hyper-coagulating alterations in the system of hemostasis. An increasing in the anticoagulating and fibrinolytic activities in rat blood plasma was detected. The peptide under study also showed antidiabetogenic action: repeated intranasal introduction of the Pro-Gly-Pro-Leu peptide into animals for 7 days inhibited development of diabetes symptoms in rats pretreated with alloxan.     

Fibrin monomer complexes (SFMC) after substitution of antithrombin III (AT III) in consumption coagulopathy

The report refers to substitution therapy of 33 patients who suffered consumption coagulopathy. Various blood coagulation and fibrinolytic variables were measured. After successful AT III donation to patients suffering DIC, soluble fibrin monomer complexes (SFMC) disappeared within 0.5-12 hours. If AT III decreases SFMC proves positive again. In addition to analysis of AT III we recommend to analyse SFMC to detect thrombin induced consumption reaction (DIC). Furthermore we found the fibrin split product D-dimer was a particularly sensitive indicator of DIC in case of hyperfibrinolysis (D-dimer was analysed in six patients). The reactions of fibronectin and SFMC proved inversely proportional.     

Effects of carboxymethyl chitosan on the blood system of rats

Carboxymethyl chitosan (CM-chitosan), a derivative of chitosan, was extensively studied in the biomedical materials field for its beneficial biological properties of hemostasis and stimulation of healing. However, studies examining the safety of CM-chitosan in the blood system are lacking. In this study CM-chitosan was implanted into the abdominal cavity of rats to determine blood indexes at different times and to evaluate the effects of CM-chitosan on the blood system of rats. Coagulation function was reflected by thrombin time (TT), prothrombin time (PT), activated partial thromboplatin time (APTT), fibrinogen (FIB) and platelet factor 4 (PF4) indexes; anti-coagulation performance was assessed by the index of antithrombinIII (ATIII); fibrinolytic function was reflected by plasminogen (PLG) and fibrin degradation product (FDP) indexes; and blood viscosity (BV) and plasma viscosity (PV) indexes reflected hemorheology. Results showed that CM-chitosan has no significant effects on the blood system of rats, and provides experimental basis for CM-chitosan to be applied in the field of biomedical materials.     

Antithrombin III inhibition of the reaction to thrombin excitation of the anticoagulation system

It has been established that intravenous administration of alpha-thrombin-antithrombin III preparations (1 mkM) has practically no effect on anticoagulation parameters (thrombin time, additive fibrinolytic activity, nonenzymatic fibrinolysis and nonenzymatic fibrinolytic activity). Administration of 1 mkM of alpha-thrombin caused a statistically significant increase of all the parameters. The experiments on perfusion of the humorally isolated sinocarotid area of the rabbit with alpha-thrombin-antithrombin III preparations (1.25 mkM) showed no changes peculiar to the induction of anticoagulation response with thrombin. It is concluded that antithrombin III blocks the ability of thrombin to activate anticoagulation system function.     

Fibrinolytic complexes of low-molecular heparin and acetylsalicylic acid]

Complexes of low-molecular heparin with acetylsalicilic acid was formed in vitro when the weight ratio of components was 1:1, 1:5 and 5:1, respectively. All the complexes possessed fibrinolytic and anticoagulating activities. The complex possessed the highest activity when the ratio of heparin to acetylsalicilic acid was 5:1. This complex at a dose 1 mg/200 g of rat weight had the largest thrombolytic effect on the experimental fresh thrombus.     

Thrombin increases proliferation and decreases fibrinolytic activity of kidney glomerular epithelial cells.

Human glomerular epithelial cells (GECs) in culture synthesize single-chain, urokinase-type plasminogen activator (SC-uPA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor 1 (PAI-1) and possess specific membrane-binding sites for u-PA. Using purified 125I-alpha thrombin, we demonstrate here the presence of two populations of specific binding sites for thrombin on GECs (1.Kd = 4.3 +/- 1.0 x 10(-10) M, 5.4 +/- 1.4 x 10(4) M sites per cell, 2. Kd = 1.6 +/- 0.5 x 10(-8) M, 7.9 +/- 1.8 x 10(5) sites per cell). Purified human alpha thrombin promoted the proliferation of GECs and induced a time- and dose-dependent increase of SC-uPA, t-PA, and PAI-1 antigens released by GECs. Thrombin-mediated increase in antigen was paralleled by an increase in the levels of corresponding u-PA and PAI-1 messenger RNA. In contrast, thrombin decreased u-PA activity in conditioned medium. This discrepancy between u-PA antigen and u-PA activity was explained by a limited proteolysis of SC-uPA by thrombin, leading to a two-chain form detected by immunoblotting and that could not be activated by plasmin. Thrombin also decreased the number of u-PA binding sites on GECs (p less than 0.05) without changing receptor affinity. Hirudin inhibited the binding and the cellular effects of thrombin, whereas thrombin inactivated by diisopropylfluorophosphate had no effect, indicating that both membrane binding and catalytic activity of thrombin were required. We conclude that thrombin, through specific membrane receptors, stimulates proliferation of GECs and decreases the fibrinolytic activity of GECs both at the cell surface and in the conditioned medium. These results suggest that thrombin could be involved in the pathogenesis of extracapillary proliferation and persistency of fibrin deposits in crescentic glomerulonephritis.     

Anticoagulant and nonenzymatic fibrinolytic activity of plasma in animals after intravenous injection of a heparin-antithrombin III complex

The heparin-antithrombin III complex with a weight ratio 1:3 had a powerful anticoagulant and non-enzymatic fibrinolytic activity in vitro. Seven and 30 min after intravenous injection of the complex the animal blood plasma showed a considerable prolongation of the thrombin time and an increase in non-enzymatic fibrinolytic activity. The effect of heparin injected in a dose equivalent to its content in the complex on the parameters under study was appreciably less and disappeared by the 30th minute of the observation period.     

内生多粘类芽胞杆菌纤溶酶基因PPFE-I在大肠杆菌中融合表达及活性分析

以内生多粘类芽胞杆菌EJS-3基因组DNA为模板,PCR扩增PPFE-I基因,并克隆到pMD19-T载体上,构建克隆载体pMD-PPFE-I,经测序正确后,将PPFE-I基因克隆至表达载体pET-DsbA上构建重组表达质粒pET-DsbA/PPFE-I,将其转化至E. coli BL21(DE3),在IPTG诱导下实现了融合蛋白DsbA-PPFE-I的表达,表达产物酶活性达228 IU/mL。表达产物用SDS-PAGE和Western blotting进行鉴定。SDS-PAGE电泳检测表明融合蛋白主要以可溶形式表达,占菌体总蛋白的18.4％。Western blotting结果表明在相应分子量处有一条特异性条带,证实该蛋白为DsbA-PPFE-I融合蛋白。表达产物通过Ni亲和柱、凝血酶酶切及Sephadex G-100等步骤进行分离纯化,并用 MALDI-TOF 质谱对重组酶进行了鉴定。纯化后的表达产物在纤维蛋白平板上表现出明显的纤溶活性。     

Changes in the hemostatic function of muscle tissue in phylogenesis

Studies have been made on coagulating and fibrinolytic activity of muscle tissue from various animals (earthworm, clam, car, frog, pigeon, rat). It was shown that extracts from muscles of these animals contain activators and inhibitors of hemocoagulation and fibrinolysis, exhibiting also antiheparin activity. It is concluded that progressive development of hemostatic function of muscle tissue involves the decrease in anticoagulating activity and the increase of thromboplastic activity.     

The role of a contaminant in thrombin in the human plasmin assay system

Many of the anomalous results obtained in the fibrinolytic assay of human plasmin systems were shown to be simply explained if bovine plasminogen had been introduced into the assay system on the addition of thrombin. Experimental investigation of the proteolytic and fibrinolytic activity of systems containing plasmin and thrombin showed that enzyme activity was influenced by the presence and quantity of thrombin. The quantity of bovine plasminogen present as a contaminant in bovine fibrinogen was shown to be responsible for only 1/25th of the observed enhanced activity. Thrombin in the amounts commonly used for clotting contained sufficient proenzyme to account for all this activity. A highly purified thrombin preparation obtained from another laboratory, and thrombin purified in this laboratory by starch electrophoresis brought about no enhancement of activity. The material separated from thrombin by starch electrophoresis was shown to be enzymatically identical with bovine plasminogen and, on labelling with radioactive iodine, was shown to behave physically like bovine plasminogen. Several experiments reported in the literature were reinterpreted in the light of this observation.     

A novel anticoagulant protein from Scapharca broughtonii

An anticoagulant protein was purified from the edible portion of a blood ark shell, Scapharca broughtonii, by ammonium sulfate precipitation and column chromatography on DEAE-Sephadex A-50, Sephadex G- 75, DEAE-Sephacel, and Biogel P-100. In vitro assays with human plasma, the anticoagulant from S. broughtonii, prolonged the activated partial thromboplastin time (APTT) and inhibited the factor IX in the intrinsic pathway of the blood coagulation cascade. But, the fibrin plate assay did not show that the anticoagulant is a fibrinolytic protease. The molecular mass of the purified S. broughtonii anticoagulant was measured to be about 26.0 kDa by gel filtration on a Sephadex G-75 column and SDSPAGE under denaturing conditions. The optimum activity in the APTT assay was exhibited at pH 7.0-7.5 and 40-45 degrees C in the presence of Ca(2+).     

Studies on the different modes of action of the anticoagulant protease inhibitors DX-9065a and Argatroban. II. Effects on fibrinolysis

The accompanying paper (Nagashima, H. (2002) J. Biol. Chem. 277, 50439-50444) has demonstrated that argatroban can yield a stronger inhibitory effect on thrombin generation than DX-9065a during extrinsic pathway-stimulated human plasma coagulation, while these anticoagulant compounds have comparable abilities to prolong clot time. Since thrombin generation is known to be an important determinant for fibrinolytic resistance of clots formed during coagulation, the two compounds are compared by tissue plasminogen activator-induced clot lysis assays. The results demonstrated that, in the presence of thrombomodulin, argatroban dose dependently accelerated fibrinolysis of the clots, whereas DX-9065a did not. The activation of thrombin activatable fibrinolysis inhibitor (TAFI) determined in separate assays reflected the differential influence on thrombin generation by these compounds. Moreover, TAFI activation correlated closely with the fibrinolytic resistance observed during tissue plasminogen activator-induced clot lysis. This study demonstrates the differential effects of DX-9065a and argatroban on thrombin generation, which in turn results in a differential acceleration of fibrinolysis as well as TAFI activation in the clots formed under the influence of these compounds. The data implicate a possible difference in the antifibrinolytic properties of clots formed during treatment with these compounds.     

Aldosterone synthase cytochrome P-450 expressed in the adrenals of patients with primary aldosteronism

A human cytochrome P-450 with aldosterone synthase activity was purified from the mitochondria of an aldosterone-producing adenoma. It was recognized by an anti-bovine cytochrome P-450(11 beta) IgG and by a specific antibody raised against a portion of the CYP11B2 gene product, one of the two putative proteins encoded by human cytochrome P-450(11 beta)-related genes (Mornet, E., Dupont, J., Vitek, A., and White, P. C. (1989) J. Biol. Chem. 264, 20961-20967). A similar and probably the same aldosterone synthase cytochrome P-450 was detected in the adrenal of a patient with idiopathic hyperaldosteronism. These aldosterone synthases were distinguishable from cytochrome P-450(11 beta), the product of another cytochrome P-450(11 beta)-related gene, i.e. CYP11B1, by their catalytic, molecular, and immunological properties and also by their localization. The latter enzyme was unable to produce aldosterone and did not react with the specific antibody against the CYP11B2 gene product. It was present both in tumor and non-tumor portions of the adrenals carrying the adenoma and in normal adrenal cortex. On the other hand, aldosterone synthase cytochrome P-450 localized in the tumor portions of the adrenals or in the adrenal of a patient with idiopathic hyperaldosteronism. Thus aldosterone synthase cytochrome P-450, a distinct species from cytochrome P-450(11 beta), is responsible for the biosynthesis of aldosterone in the human, at least in patients suffering from primary aldosteronism.     

Effects of exercise and conditioning on clotting and fibrinolytic activity in men

Sixty healthy men in three physical fitness categories (sedentary, on no organized fitness program; joggers, running 5-15 miles/wk; and marathoners, running greater than 50 miles/wk) were evaluated for changes in blood clotting and fibrinolytic activity before and after maximum exercise on a treadmill according to the Bruce protocol. The rate of blood clotting, as measured by prothrombin times, partial thromboplastin times and thrombin times, was accelerated by exercise (all P less than 0.005). The ability of euglobulin clots and plasma clots to lyse incorporated 125I-fibrin, termed 125I-euglobulin clot lysis (IEL) and 125I-plasma clot lysis (IPCL), were used as indexes of fibrinolytic activity. Marathoners had greater increases in fibrinolytic activity with exercise (76% compared with 63% for joggers and 55% for sedentary subjects by IEL; 427% compared with 418% for joggers and 309% for sedentary subjects by IPCL; all P less than 0.05). Fibrin degradation products increased with exercise (P less than 0.005 for the total group of 60 subjects). The absolute concentrations of alpha 2-plasmin inhibitor, alpha 2-macroglobulin, and antithrombin III increased with exercise (all P less than 0.005), but when concentrations were corrected for acute shifts of plasma water during exercise, the quantity of these inhibitors actually decreased (all P less than 0.005). The changes in clotting assays with exercise were not significantly correlated with changes in whole blood lactate, blood pyruvate, or rectal temperatures. Fibrinolytic assays before and after exercise correlated poorly to moderately with blood lactates (IEL: r = 0.441 and r = 0.425, respectively; IPCL: r = 0.294 and r = 0.544, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

High density fermentation and activity of a recombinant lumbrokinase (PI239) from Pichia pastoris

A system for the expression of recombinant lumbrokinase (rPI239) was developed in the yeast Pichia pastoris. A total supernatant protein content of 0.174 g/L of high density fermentation broth was obtained. The rPI239 exhibited in vitro fibrinolytic activity. The in vivo activity of rPI239 was measured by prothrombin time, kaolin part thrombin time, thrombin time, and fibrinolytic activity. This work presents the high-density fermentation of rPI239 from P. pastoris and shows that the recombinant protein has similar fibrinolytic activity both in vivo and in vitro.     

Hemostasis and fibrinolysis in patients with intermittent claudication: effects of prostaglandin E1

There is evidence that the coagulation system is activated in patients with peripheral arterial occlusive disease (PAOD). The beneficial effects of the vasoactive drug prostaglandin E1 (PGE1) may rely in part on the modulation of the coagulation system. The study was designed to evaluate the effects of PGE1 on hemostatic and fibrinolytic variables in patients with intermittent claudication. Therefore molecular markers of thrombin (prothrombin fragment 1+2, PTF 1+2; thrombin-antithrombin III complexes, TAT) and fibrin formation (fibrinopeptide A, FPA) and markers of the fibrinolytic activity (fibrin degradation products, D-dimers) were determined before and immediately after the first PGE1 dose (60 microg in 100 ml NaCl over 2 h i.v.) as well as after 4 weeks of daily infusion therapy in 12 PAOD patients and in eight control patients before and after a single placebo infusion. Plasma levels of PTF1+2, TAT, FPA and D-dimers tended to decrease after the initial dose of PGE1. Infusion therapy with PGE1 for 4 weeks led to a decrease of all hemostatic and fibrinolytic parameters with most pronounced changes for PFT1+2, D-dimers and plasminogen activator inhibitor-1 decreasing by 11% (P<0.05), 20% (P<0.05), and 7% (P<0.05), respectively. These variables remained unchanged in controls with placebo infusion.In summary, infusion therapy with PGE1 in patients with PAOD reduces thrombin formation and results in a decrease of fibrin degradation. PGE1 may thus reduce fibrin deposition involved in the pathogenesis of atherosclerosis.     

Mapping the interaction of bradykinin 1-5 with the exodomain of human protease activated receptor 4

The angiotensin converting enzyme breakdown product of bradykinin, bradykinin 1-5 (RPPGF), inhibits thrombin-induced human or mouse platelet aggregation. RPPGF binds to the exodomain of human protease-activated receptor 1 (PAR1). Studies determined if RPPGF also binds to the exodomain of human PAR4. RPPGF binds to a peptide of the thrombin cleavage site on PAR4. Recombinant wild-type and mutated exodomain of human PAR4 was prepared. The N-terminal arginine on RPPGF binds to the P2 position or proline46 on PAR4 to block thrombin cleavage. These data indicate that RPPGF influences thrombin activity by binding to the thrombin cleavage site on both PAR4 and PAR1.     

Involvement of SNARE proteins in thrombin-induced platelet aggregation: evidence for the relevance of Ca2+ entry

Thrombin induces platelet activation through a variety of intracellular mechanisms, including Ca(2+) mobilization. The protein of the exocytotic machinery SNAP-25, but not VAMPs, is required for store-operated Ca(2+) entry, the main mechanism for Ca(2+) influx in platelets. Hence, we have investigated the role of the SNAP-25 and VAMPs in thrombin-induced platelet aggregation. Platelet stimulation with thrombin or selective activation of thrombin receptors PAR-1, PAR-4 or GPIb-IX-V results in platelet aggregation that, except for GPIb-IX-V receptor, requires Ca(2+) entry for full activation. Depletion of the intracellular Ca(2+) stores using pharmacological tools was unable to induce aggregation except when cytosolic Ca(2+) concentration reached a critical level (around 1.5 microM). Electrotransjection of cells with anti-SNAP-25 antibody reduced thrombin-evoked platelet aggregation, while electrotransjection of anti-VAMP-1, -2 and -3 antibody had no effect. These findings support a role for SNAP-25 but not VAMP-1, -2 and -3 in platelet aggregation, which is likely mediated by the regulation of Ca(2+) mobilization in human platelets.