ISOLATION AND PROPERTIES OF A BLOOD-COAGULATION FACTOR-X ACTIVATOR FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.

AN ACTIVATOR OF BLOOD-COAGULATION FACTOR-X FROM THE VENOM OF BUNGARUS-FASCIATUS

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mel. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.

Effects of Pallas' viper (Agkistrodon halys pallas) venom on blood coagulation and characterization of a prothrombin activator

The action of Pallas' viper (Agkistrodon halys pallas) venom on blood coagulation was examined in vitro and a strong anticoagulant effect was observed. This action was abolished after treatment with a specific inhibitor of phospholipase A(2) activity (p-bromophenacyl bromide), revealing a procoagulant action in low concentrations of treated venom (around 1 mu g/ml). The effect of the venom an haemostasis was further characterized by measuring its ability to activate purified blood coagulation factors. It is concluded that A. halys pallas venom contains prothrombin activation activity. A prothrombin activator (aharin) was purified from the venom by Sephadex G-75 gel filtration and ion-exchange chromatography on a Mono-Q column. It consisted of a single polypeptide chain, with a mol. wt of 63,000. Purified aharin possessed no amidolytic activity on chromogenic substrates. It did not act on other blood coagulation factors, such as factor X and plasminogen, nor did it cleave or clot purified fibrinogen. The prothrombin activation activity of aharin was readily inhibited by ethylenediamine tetracetic acid (a metal chelator), but specific serine protease inhibitors such as diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride had no effect on it. These observations suggest that, like those prothrombin activators from Echis carinatus and Bothrops atrox venoms, the prothrombin activator from A. halys pallas venom is a metalloproteinase. (C) 1998 Elsevier Science Ltd. All rights reserved.

Cloning and characterization of a blood coagulation factor IX-binding protein from the venom of Trimeresurus stejnegeri

A blood coagulation factor IX-binding protein (TSV-FIX-BP) was isolated from the snake venom of Trimeresurus stejnegeri. On SDS-polyacrylamide gel electrophoresis, TSV-FIX-BP showed a single band with an apparent molecular weight of 23,000 under non-reducing conditions. and two distinct bands with apparent molecular weights of 14,800 and 14,000 under reducing conditions. cDNA clones containing the coding sequences of TSV-FIX-BP were isolated and sequenced to determine the structure of the precusors of TSV-FIX-BP subunits. The deduced amino acid sequences of two subunits of TSV-FIX-BP were confirmed by N-terminal protein sequencing and trypsin-digested peptide mass fingerprinting. TSV-FIX-BP was a nonenzymatic C-type lectin-like anti-coagulant. The anti-coagulant activity of TSV-FIX-BP was mainly caused by its dose dependent interaction with blood coagulation factor IX but not with blood coagulation factor X. (C) 2003 Elsevier Science Ltd. All rights reserved.

Plant and marine derived (n-3) polyunsaturated fatty acids do not affect blood coagulation and fibrinolytic factors in moderately hyperlipidemic humans

Dietary alpha-linolenic acid (ALA) can be converted to long-chain (n-3) PUFA in humans and may potentially reproduce the beneficial effects of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids on risk factors for coronary heart disease (CHID). This study compared the effects of increased intakes of ALA with those of dietary EPA and DHA on blood coagulation and fibrinolytic factors in fasting subjects. A placebo-controlled, parallel study was conducted in 150 moderately hyperlipidemic subjects, age 25-72 y. Subjects were randomly assigned to one of five interventions and consumed a total intake of 0.8 or 1.7g/d EPA+DHA, 4.5 or 9.5g/d ALA or control (linoleic acid; LA) for 6 mo. Fatty acids were incorporated into 25 g of fat spread, which replaced the subject's normal spread and three capsules. Long-term supplementation with either dietary EPA+DHA or estimated biologically equivalent amounts of ALA did not affect factors VIIa, VIIc, VIIag, XIIa, XIIag, fibrinogen concentrations, plasminogen activator inhibitor-1 or tissue plasminogen activator activity compared with the control. (n-3) PUFA of plant or marine origin do not differ from one another or from LA in their effect on a range of blood coagulation and fibrinolytic factors.

Effects of Compounds from Passiflora edulis Sims f. flavicarpa Juice on Blood Coagulation and on Proteolytic Enzymes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CrataBL, a lectin and Factor Xa inhibitor, plays a role in blood coagulation and impairs thrombus formation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

[Proteins influencing the blood coagulation]

This review describes some natural proteins, which can be employed, either as factor concentrates derived from human plasma or as recombinant drug, to modulate the coagulation system. I will address some biochemical characteristics and the physiological role of von Willebrand factor, the coagulation factors of the extrinsic and intrinsic pathways, and the physiological anticoagulant protein C. In addition, I will detail the pharmacological compounds, which are available for influencing or substituting the coagulation proteins: desmopressin (DDAVP), single coagulation factor concentrates, prothrombin complex concentrates, and protein C concentrate. In particular, I will address some treatment topics of general medical interest, such as the treatment of massive bleeding, the correction of the coagulopathy induced by vitamin K-antagonists in patients with cerebral haemorrhage, and of the coagulopathy of meningococcemia. Finally, I will describe some properties and practical clinical applications of the recombinant anticoagulans lepirudin and bivalirudin, which are derived from hirudin, the natural anticoagulant of the medical leech.

Altered blood coagulation in patients with posttraumatic stress disorder

OBJECTIVE: Posttraumatic stress disorder (PTSD) has been associated with an increased cardiovascular risk, though the pathophysiologic mechanisms involved are elusive. A hypercoagulable state before occurrence of coronary thrombosis contributes to atherosclerosis development. We investigated whether PTSD would be associated with increased coagulation activity. METHODS: We measured resting plasma levels of clotting factor VII activity (FVII:C), FVIII:C, FXII:C, fibrinogen, and D-dimer in 14 otherwise healthy patients with PTSD and in 14 age- and gender-matched, trauma-exposed non-PTSD controls. Categorical and dimensional diagnoses of PTSD were made using the Clinician-Administered PTSD Scale (CAPS) interview. We also investigated to what extent the relationship between PTSD and coagulation measures would be confounded by demographics, cardiovascular risk factors, lifestyle variables, time since trauma, and mood. RESULTS: Coagulation factor levels did not significantly differ between patients with a categorical diagnosis of PTSD and controls while controlling for covariates. In all subjects, FVIII:C was predicted by hyperarousal severity (beta = 0.46, p = .014) independent of covariates and by overall PTSD symptom severity (beta = 0.38, p = .045); the latter association was of borderline significance when separately controlling for gender, smoking, exercise, and anxiety (p values <.07). In patients, fibrinogen was predicted by hyperarousal severity (beta = 0.70, p = .005) and by overall PTSD symptom severity (beta = 0.61, p = .020), with mood partially affecting these associations. FVII:C, fibrinogen, and D-dimer showed no independent association with PTSD symptoms. CONCLUSIONS: PTSD may elicit hypercoagulability, even at subthreshold levels, offering one psychobiological pathway by which posttraumatic stress might contribute to atherosclerosis progression and clinical cardiovascular disease.

Oral melatonin reduces blood coagulation activity: a placebo-controlled study in healthy young men

Melatonin has previously been suggested to affect hemostatic function but studies on the issue are scant. We hypothesized that, in humans, oral administration of melatonin is associated with decreased plasma levels of procoagulant hemostatic measures compared with placebo medication and that plasma melatonin concentration shows an inverse association with procoagulant measures. Forty-six healthy men (mean age 25 +/- 4 yr) were randomized, single-blinded, to either 3 mg of oral melatonin (n = 25) or placebo medication (n = 21). One hour thereafter, levels of melatonin, fibrinogen, and D-dimer as well as activities of coagulation factor VII (FVII:C) and VIII (FVIII:C) were measured in plasma. Multivariate analysis of covariance and regression analysis controlled for age, body mass index, mean arterial blood pressure, heart rate, and norepinephrine plasma level. Subjects on melatonin had significantly lower mean levels of FVIII:C (81%, 95% CI 71-92 versus 103%, 95% CI 90-119; P = 0.018) and of fibrinogen (1.92 g/L, 95% CI 1.76-2.08 versus 2.26 g/L, 95% CI 2.09-2.43; P = 0.007) than those on placebo explaining 14 and 17% of the respective variance. In all subjects, increased plasma melatonin concentration independently predicted lower levels of FVIII:C (P = 0.037) and fibrinogen (P = 0.022) explaining 9 and 11% of the respective variance. Melatonin medication and plasma concentration were not significantly associated with FVII:C and D-dimer levels. A single dose of oral melatonin was associated with lower plasma levels of procoagulant factors 60 min later. There might be a dose-response relationship between the plasma concentration of melatonin and coagulation activity.