The accelerating effect of fibrinogen and early fibrinogen degradation products on erythrocyte sedimentation.

The fibrinogen molecule has a number of biological activities and some of these are shared by the larger degradation products. This study was carried out to investigate the erythrocyte sedimentation-accelerating property of fibrinogen and how this property might be modified by proteolytic digestion with plasmin. The sedimentation rate of washed human red cells suspended in saline, was found to be directly proportional to the concentration of added purified human fibrinogen, down to 250 mg%, below which there was no difference from saline controls. Plasmic digestion of fibrinogen yielding fragment X, did not reduce the accelerating affect on erythrocyte sedimentation, indicating that the intact carboxyl terminal end of the A alpha chain is unnecessary for this phenomenon. Further digestion to fragment Y reduced the effect slightly but digestion to fragments D and E abolished the accelerating effect completely.

Aprotinin reduces blood loss after cardiopulmonary bypass by direct inhibition of plasmin.

The effectiveness and mechanism of aprotinin reduced bleeding after cardiopulmonary bypass surgery was studied in a double blind randomised study of 106 patients undergoing valve replacement surgery. Aprotinin therapy was associated with significant reduction in perioperative bleeding and postoperative blood transfusion requirements. Although initially tissue plasminogen activator (t-PA) activity was lower in the aprotinin than placebo group, as surgery proceeded this difference was reversed due to less plasminogen activator inhibitor-1 release in the aprotinin group. This indicates that aprotinin-mediated suppression of fibrinolysis as demonstrated by reduced D-dimer concentration was not related to t-PA. Furthermore, similar perioperative reduction of plasminogen levels in aprotinin and placebo groups indicated a similar degree of conversion of plasminogen to plasmin. However, less plasmin bound with alpha 2-antiplasmin in the plasma in the aprotinin group as it was already complexed with aprotinin where it remained protected from the natural inhibitor on the intact fibrin surface. The reduced fibrinolytic activity of the aprotinin group was thus brought about by the complexing of aprotinin with the plasmin which was bound to the fibrin surface.

Exercise-induced fibrinolysis--fact or fiction?

The effect of strenuous exercise on the fibrinolytic and coagulation mechanisms was examined in six healthy male subjects. Five min bicycle exercise at a work-rate of 800 to 1200 kpm. min-1 produced an abrupt increase in plasma plasminogen activator levels which disappeared after 90 min. However, there was no change in early or late fibrin degradation products nor was there a change in fibrinopeptide A levels or beta-thromboglobulin levels after exercise although activated partial thromboplastin times were significantly shortened. It is concluded that strenuous exercise does not produce any real increase in fibrinogen-fibrin conversion nor any real increase in the breakdown of these proteins. The role of exercise-induced release of plasminogen activator remains unclear, but probably helps to maintain plasma levels in a discontinuous manner concurrently with the continuous low-level secretion from the vascular wall. The shortening of partial thromboplastin time may be due to the raised levels of plasminogen activator changing the activation state of other coagulation factors.

The rapid fibrin plate - a method for plasminogen activator assay.

Most of the technical problems associated with the fibrin plate method have been overcome in recent years with the exception of the long incubation period. This study was carried out to investigate plasminogen-enrichment as a means of shortening this period. Fibrin plates made up to contain 2.0 casein units of added plasminogen each, were opaque, firm, did not lyse spontaneously and yielded biometrically-valid parallel-line assays for streptokinase and urokinase after a 3 hour incubation period. Urokinase assays were more accurate than those for streptokinase probably because of the latter's shallow dose-response curve. Plasminogen-enrichment appears therefore to be a convenient way of producing a "rapid" fibrin plate requiring incubation for 3 hours compared with the usual 16 to 20 hours.

The effect of pentosan polysulphate (SP54) on the fibrinolytic enzyme system--a human volunteer and experimental animal study.

Pentosan polysulphate causes an increase in plasminogen activator activity in plasma both after oral ingestion and after subcutaneous injection. The effect is greatest after 3 h and has disappeared by 6 h. Repeat doses by mouth over 5 days elicit a similar response. The recorded increase in activity is due largely to the release of tissue-type plasminogen activator (tPA) from the endothelium according to the antigen assay although there could be a small contribution from Factor XII-related "intrinsic" fibrinolysis induced in vitro. SP54 enhances activity ex vivo by a non-specific surface effect, and this phenomenon may contribute the increased levels of activity seen in vitro. Administration of SP54 to animals elicits a similar increase in activator activity, the intramuscular route being slightly more effective. Results with an inferior vena cava thrombosis model in the rat suggest that pentosan polysulphate may induce a thrombolytic effect.

A comparison of pentosan polysulphate and heparin. II: Effects of subcutaneous injection.

A comparison has been made between the effects of pentosan polysulphate (SP54) and mucosal heparin following subcutaneous injection in man. Unlike heparin, pentosan polysulphate has relatively little effect in vivo as measured by anti-factor Xa clotting assay and none by an anti-Xa amidolytic assay (S-2222). However, pentosan polysulphate is at least as potent as heparin on a weight basis in producing activation of lipoprotein lipase, shortening of the euglobulin clot lysis time and impairing the generation of factor Xa. Our data indicate that pentosan polysulphate has more marked effects in vivo than in vitro, that the action of the drug on clotting is mediated mainly via an At III-independent pathway, and that its effects are not confined to the coagulation system.

Sites of action of Mojave toxin isolated from the venom of the Mojave rattlesnake.

1 Mojave toxin isolated from the venom of the Mojave rattlesnake (Crotalus scutulatus scutulatus) produced an irreversible blockade of the contractile response of the mouse hemidiaphragm to stimulation of the phrenic nerve in vitro, at concentrations of 0.16 to 20 mug/ml; the response to direct stimulation was not affected over a testing period of several hours.2 Mojave toxin (1 to 4 mug/g) was injected into the tail vein of mice and the intoxicated hemidiaphragm preparation was removed either for testing the contractile response or for intracellular recording.3 In fully intoxicated hemidiaphragms the contractile response to indirect stimulation was either small and transient or absent, whilst the response to direct stimulation was well maintained.4 Intracellular recording showed that resting membrane potentials of the muscle fibres were within the normal range. Endplates were difficult to locate but miniature endplate potentials (m.e.p.ps) were recorded at sites at which neurally evoked responses either could not be detected or did not exceed 2 mV which corresponds to transmitter release of a few quanta only.5 The mean frequency of m.e.p.ps at fully intoxicated endplates was not significantly different from controls but potassium depolarization produced only a small increase in m.e.p.p. frequency relative to the control response. A 50 Hz tetanus had no effect on m.e.p.p. frequency.6 When a sub-lethal dose (3 mug) of Mojave toxin was injected into one hindlimb of mice and the tissues examined at 72 h, there was histological evidence of myonecrosis.7 The isolated perfused heart of the rat was exposed to recycled Mojave toxin (50 and 100 mug/ml) but showed no change in rate or force of ventricular contraction.8 Post-mortem examination of intoxicated mice showed a frequent incidence of localized areas of interstitial and intra-alveolar haemorrhage in the lungs. Other organs including skin and muscle were not affected.9 Mojave toxin showed antigenic similarities to crotoxin, the lethal neurotoxin in the venom of the South American rattlesnake, as determined by the ability of antiserum raised against crotoxin to neutralize Mojave toxin.10 With systemic Mojave intoxication of rapid onset, the cause of death was respiratory paralysis. However, the toxin acts at multiple sites at differing rates of action. With a slower rate of intoxication, impaired respiration may act synergistically with cardiovascular changes to produce circulatory failure. The desirability of using an antivenin with a high titre against Mojave toxin is indicated.

Preoperative platelet dysfunction increases the benefit of aprotinin in cardiopulmonary bypass.

BACKGROUND: This study was designed to determine the benefit of aprotinin therapy in reducing bleeding during and after cardiopulmonary bypass in patients with preoperative platelet dysfunction. Platelet function involvement in the mechanism by which aprotinin acts was also investigated. METHODS: In a double-blind, randomized study, patients received high-dose aprotinin (n = 54) or placebo (n = 52). Whole blood aggregation was measured preoperatively. Platelet function and activation in both groups were assessed intraoperatively and postoperatively at five times. RESULTS: Aprotinin significantly reduced perioperative bleeding and postoperative blood transfusion. Placebo-treated patients with reduced preoperative platelet aggregation bled more postoperatively, but aprotinin reduced the bleeding in patients with normal or reduced platelet function to similar levels. Any cardiopulmonary bypass-induced changes in platelet aggregation, platelet activation as measured by P-selectin expression, and von Willebrand factor antigen and function were similar in aprotinin-treated and placebo-treated groups. CONCLUSIONS: The mechanism by which aprotinin reduced bleeding was independent of any effect on platelet function. However, aprotinin produced a greater reduction in bleeding among patients whose condition was hemostatically compromised by preoperative platelet dysfunction.

Heparin and a low molecular weight fraction enhances thrombolysis and by this pathway exercises a protective effect against thrombosis.

In human volunteers unfractionated heparin and a low molecular weight fraction of heparin (LMWH) caused an increase in plasma plasminogen activator (PA) which peaked at 3 hours after subcutaneous injection. Using a perfused isolated rabbit ear model the enhancement of PA activity was confirmed and was related to the anti-Xa activity of both products infused. Using a modified rabbit Wessler model for thrombus formation it was found that, when using doses of heparin and LMWH sufficient to give a 100% antithrombotic effect, antifibrinolytic drugs (eg. epsilon-ACA and aprotinin), negated this protective effect. It is concluded that the effect of heparin and LMWH on haemostasis is mediated in part through the enhancement which these drugs have on fibrinolysis, the latter being arguably a major defence against fibrin formation during thrombosis.

Accidental envenoming by a Gaboon viper (Bitis gabonica): the haemostatic disturbances observed and investigation of in vitro haemostatic properties of whole venom.

We report the successful treatment of envenoming by the Gaboon viper (Bitis gabonica) and include results of in vitro investigations of the haemostatic properties of the whole venom. The patient was admitted to casualty soon after the bite with chest tightness, dizziness, nausea and swelling at the site of the bite and was treated immediately with polyspecific antivenom, hydrocortisone, chlorpheniramine and antibiotics. Results of haemostatic investigations were essentially normal on admission but on day 3 the thrombin time became prolonged and was associated with significant hypofibrinogenaemia and elevated D-dimers. Factors V and VIII, antithrombin III and protein C levels and platelet number were not significantly reduced. The haemostatic disturbances persisted for more than 24 h despite treatment with blood products (16 units of cryoprecipitate, 2 units of fresh frozen plasma and 6 units of platelet concentrate). Resolution of the abnormalities occurred only after administration of a further dose of antivenom. The period of hypofibrinogenaemia occurred at a time when venom antigen was undetectable in plasma by enzyme-linked immunosorbent assay. Studies in vitro with whole venom and a panel of amidolytic substrates commonly employed for measurement of haemostatic proteins revealed significant activity of venom with substrates sensitive to kallikrein and plasmin. The venom inhibited washed platelet aggregation induced by collagen, thrombin, arachidonic acid and the calcium ionophore A23187 in a dose-dependent manner.

The Gaboon viper (Bitis gabonica): its biology, venom components and toxinology.

The Gaboon viper has acquired an impressive reputation which is at least partly unfounded. This handsome animal with such striking features is undoubtedly docile which accounts for the very low incidence of bite amongst humans. There are only six detailed clinical reports on the effect of bite and these are summarized in the review. The viper does indeed produce prodigious amounts of venom, but the toxicity, weight for weight, is rather low compared to other poisonous snakes. Venom extractions have been carried out on four snakes over a 13-year-period and the effects of this venom have been studied in a variety of experimental animals. Systemic envenomation is characterized by immediate abrupt hypotension, subsequent cardiac damage and dyspnoea. The individual venom components responsible for these effects have not been isolated but it seems likely that the two enzymes which have been studied extensively (phospholipase A2 and the thrombin-like enzyme, gabonase) do not contribute significantly to lethality. We propose three principal activities which give rise to the major signs of systemic envenomation. Haemorrhagin; causing widespread damage to microvasculature which leads to the pulmonary oedema and hence dyspnoea, and locally causes blistering. Cardiotoxin; a long-acting material causing cardiac muscle damage, arrhythmia and ultimately cardiac failure. Peripheral vasodilator; a short acting effect, operating either locally via bradykinin formation and/or unknown peptides or centrally on the vasomotor centre.

Comparison of the physiological effects in rabbits of gaboon viper (Bitis gabonica) venoms from different sources.

The cardiovascular, respiratory and metabolic effects of B. gabonica venoms obtained from specimens originating from Ghana, Togo, Nigeria, Uganda and Tanzania were examined in anaesthetized rabbits. Intravenous injection of all venoms (0.125-2.0 mg/kg) induced hypotension. Nigeria venom was the least potent in this respect. Following doses of all venoms there was a brief bradycardia and a transient increase in respiratory rate and depth. At high doses (greater than or equal to 1.0 mg/kg), all venoms induced severe ST depression and T wave inversion. In addition, Togo venom, and to a lesser extent Tanzania and Ghana venoms, were potent in inducing extrasystoles. None of the venoms produced any significant changes in haematocrit, plasma proteins or arterial blood gas and pH levels. All venoms increased blood glucose and lactate levels by 1.3-2.1 fold and 2.2-4.0 fold respectively while the respiratory quotient remained unchanged. Togo venom was significantly (P less than 0.05) more lethal than the other venoms. The pattern of haemorrhage observed at post-mortem was the same for all venoms with the heart, ureters, adrenals, kidneys, lungs, stomach and intestines being the most affected. When combined on a subspecies basis, the results suggest that there are no significant differences in the physiological effects of venoms representing B. g. rhinoceros (West African gaboon viper) and B. g. gabonica (East African gaboon viper).

Preliminary studies of the effects of a Peruvian snake Bothrops pictus (jergon of the coast) venom upon fibrinogen.

Bothrops pictus (jergon of the coast) venom has a coagulant effect in vitro on both canine fibrinogen and on human and canine plasma, with a greater affinity for canine plasma. In vivo a single dose of venom produced partial defibrinogenation in conscious dogs, plasma fibrinogen being reduced to about 60% of control values after 6 hr.

Isolation and partial characterization of a prothrombin-activating enzyme from the venom of the Australian rough-scaled snake (Tropidechis carinatus).

A prothrombin activator from the venom of Tropidechis carinatus has been isolated by means of gel filtration and benzamidine-based affinity chromatography, a novel use of the latter technique. Two bands possessing prothrombinase activity were obtained from the affinity chromatography procedure and designated A1 and A2. The bulk of the enzyme activity was recovered in peak A2 which represented 27-31% of the starting activity and a 14-16-fold purification. The venom contained, in total, around 5% by weight of the two isoforms of the prothrombin activator. The two fractions were electrophoretically similar on polyacrylamide electrophoresis, migrating with a mol. wt of 64,500 under native conditions and as a single band of 41,500 under reducing conditions. The prothrombinase was dependent on factor Va, phospholipid and calcium ions for its activity and is, thus, a member of the type II class of prothrombinases requiring such co-factors. The enzyme did not possess any phospholipase activity nor did it cleave the substrates N-alpha-benzoyl-L-arginine-p-nitroanilide (BAPNA), N-benzoyl-L-tyrosine ethyl ester (BTEE), azocollagen or azocasein, indicating a lack of amidolytic, esterolytic and broad-spectrum protease activity.

Isolation and characterisation of two haemorrhagic proteins (HTa and HTb) from the venom of Bitis gabonica (Gaboon viper).

Two distinct haemorrhagic proteinases, HTa and HTb, were isolated from the venom of Bitis gabonica by gel filtration and ion-exchange chromatography with native mol. wts of 180,000 and 111,000, respectively. After reduction with dithiothreitol, smaller mol. wts of 77,600 and 69,200 were recorded for HTa and HTb, suggesting that under native conditions the haemorrhagins exist as dimeric molecules. Both toxins possessed caseinolytic and collagenase activity although HTa was 15-36 times more potent than HTb with respect to collagenase activity. No zinc could be detected in the toxins; however, dialysis against ethylenediamine tetracetic acid (EDTA) reduced caseinolytic activity, suggesting the dependence of the latter on other metal ions. HTa and HTb had a marked effect on the intrinsic cascade coagulation mechanism (factors IX, XI and XII) but no effect on the final common coagulation pathway (factor X and prothrombin). Light and electron microscopical studies demonstrated that both HTa and HTb caused organ-specific lesions, with the lungs, diaphragm and body wall muscle being most affected. HTa caused widespread haemorrhage whilst HTb caused discrete focal lesions near the site of injection and elsewhere. However, both toxins appeared to cause capillary rupture by the separation of cells from one another and both caused cell detachment and cell death of bovine endothelial cells cultured in vitro, consonant with the massive disruption of capillaries seen in vivo.

The effect of Bitis gabonica (Gaboon viper) snake venom on external iliac and mesenteric arterial circulation in the dog.

The effects of Gaboon viper (Bitis gabonica) venom on external iliac and mesenteric arterial blood flow and resistance were investigated in eight anaesthetized, close-chest dogs. Venom doses in the range 0.125-0.5 mg/kg produced a profound fall in external iliac and mesenteric arterial resistance, which recovered to control values after 30 min. After a third dose of venom, the mean arterial blood pressure failed to recover and the animals died after a period of severe hypotension. External iliac arterial blood flow rose concomitantly with the fall in external iliac resistance and decreased to a value significantly below control after 30 min. Paradoxically, mesenteric blood flow fell during the period of vasodilation. The results suggest that widespread vasodilation of muscle vascular beds (of which the external iliac circulation is representative) leads to shunting of blood away from the less-dilated mesenteric circulation. Venom-induced peritoneal haemorrhage caused a fall in blood volume and increase in viscosity. These undoubtedly contributed to the severe haemodynamic deterioration of the preparations after the third injection of the venom.

The effects of Gaboon viper (Bitis gabonica) venom on the electrical and mechanical activity of the guinea-pig myocardium.

The effects of Bitis gabonica venom were tested on guinea-pig heart, using both Langendorff preparations and isolated atrial strips or papillary muscles. In the self-paced whole heart, a single passage of 50 micrograms of venom per ml produced in sequence: irregularities of the A-V conduction and decrease of the contractile strength, progressive failure to relax and systolic arrest of the heart. Pretreatment with atropine reduced but did not abolish these effects. Venom recycled through the heart was effective at a much lower dose. The relationship between resting membrane potential and [K+]o was unaffected by envenomation, suggesting that the action of the venom cannot be ascribed to a loss of ionic selectivity of the cell membrane. The peak amplitude of action potentials declined in papillary muscle exposed to venom at physiological [K+]o, while in atrial cells it was affected only at higher [K+]o. Maximum upstroke rate of the action potential vs. resting potential at different [K+]o gave a sigmoid relationship, characterized by a higher upper asymptote as compared to controls, and by a shift of the curve towards more negative voltage values. A marked shortening of the action potential duration, paralleled by a decrease in time to peak tension, was recorded as well. 'Slow' action potentials, elicited in 20 mM K+ solution, were completely abolished within 10 min of perfusion with venom. These results are consistent with the hypothesis that the venom interacts with both transmembrane Ca2+ inflow and Ca2+ binding at the external side of the cell membrane. A transient positive inotropic effect induced by the venom was observed in papillary muscle and in atropinized atrium. This effect was abolished by previous administration of reserpine to the animal or by addition of propranolol to the perfusing solution, suggesting a venom-induced release of both adrenergic and cholinergic transmitters from nerve endings within the cardiac tissue.

Isolation and characterization of a fibrinogen-clotting enzyme from venom of the snake, Lachesis muta muta (Peruvian bushmaster).

A fibrinogen-clotting enzyme from the venom of the Peruvian bushmaster snake was purified to homogeneity by gel filtration on Sephadex G-100 followed by DEAE-cellulose ion-exchange chromatography using a linear ionic strength gradient with NaCl. The specific activity of the enzyme was 866 NIH U/mg, representing a 55-fold purification, with a recovery of 45%. The amino acid composition was Asx30, Thr14, Ser15, Glx33, Pro23, Gly22, Ala15, Val22, Cys18, Met3, Ile18, Leu23, Tyr2, Phe13, His8, Lys11, Arg11. The total carbohydrate content was 13.4%, comprised of 3.4% hexose, 8.7% hexosamine and 1.3% sialic acid. The enzyme was active against the synthetic amide substrate alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and against the ester substrates alpha-N-benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME). Kinetic parameters for TAME esterolysis were: Vmax, 135 mumoles/min/mg and Km, 2.5 x 10(-4) M. The pH optimum was 8.0. Vmax for BAPNA amidolysis was 0.363 mumoles/min/mg and Km, 7.5 x 10(-5) M. Enzyme activity was reduced by diethylpyrocarbonate and by photo-oxidation, suggesting that the enzyme is a serine protease with a histidine residue involved in the active site. The enzyme released fibrinopeptide A rapidly from purified human fibrinogen and fibrinopeptide B more slowly. Factor XIII was not activated and the clotting activity was not inhibited by heparin. A dose of 50 micrograms/kg brought about defibrinogenation in anaesthetized rats but rabbits were unaffected. A dose of 80 micrograms/kg defibrinogenated conscious rats after 5 hr. There were no hypotensive or haemorrhagic effects.

Acid-base, plasma lactate and glucose changes in the rabbit following administration of Gaboon viper (Bitis gabonica) venom.

The acid-base and metabolic effects of Bitis gabonica venom administered intravenously to the anaesthetised rabbit were studied. Doubling doses of venom from 0.125 mg/kg to 1.0 mg/kg were used. Venom caused progressive and significant increases in plasma glucose and plasma lactate levels although oxygen consumption only became significantly lower after the fourth dose. Standard base excess (SBE) became significantly more negative after the third dose of venom and the fall in pH became significant at the same point. The results indicate that venom induces a metabolic acidosis in the rabbit and because the acidosis occurs in the absence of any fall in arterial PO2, it cannot be considered a consequence of impaired pulmonary ventilation. The reduction in oxygen uptake is likely to occur at a cellular level with a shift from aerobic to anaerobic metabolism hence the increase in plasma lactate levels. However, the magnitude of the acidosis is unlikely to be the principal cause of death under experimental conditions.

The mechanical and electrical effects of rhinoceros viper (Bitis nasicornis) venom on the isolated perfused guinea pig heart and atrial preparations.

The mechanical and electrical effects of the venom of Bitis nasicornis were studied on the guinea-pig Langendorff and left atrial myocardium preparations. While Langendorff preparations were treated with individual doses of 0.1, 0.6 and 1.4 mg, isolated left atria were treated using concentrations of 2.0, 20 and 200 micrograms/ml of venom in the perfusion solution. In the Langendorff preparation, transient increases in left ventricular systolic pressure (LVSP) and heart rate (HR) were seen after 0.1 mg of venom. When 0.6 mg of venom was given, the increases were followed by decreases, while 1.4 mg doses simply induced decreases in LVSP and HR. After both 0.6 and 1.4 mg doses the decreases were accompanied by increases in left ventricular diastolic pressure. In addition to these mechanical effects, transient increases in HR with atrio-ventricular blocks, ventricular extrasystoles and tachycardia were observed after each dose. In the left atrium the 2 micrograms/ml venom concentration produced an increase, followed by a decrease, in the maximum tension developed, which was only seen to decrease with higher concentrations of 20 and 200 micrograms/ml of venom. A dose dependent significant reduction in the action potential duration was observed for the doses of 0.6 and 1.4 mg in the ventricle and for all three concentrations in the atrium.