Plasma components are required for platelet activation by the toxin of Loxosceles reclusa

We have used a partially purified toxin from the venom of the brown recluse spider, Loxosceles reclusa, to study its effects on human platelets isolated from plasma proteins. This toxin, which produced skin necrosis in rabbits, contained sphingomyelinase D activity. The toxin induced platelet aggregation and secretion of [3H]serotonin in human plasma but not in buffer or in human neonate plasma. Ca2+ was required for the interaction of toxin, platelets, and plasma factor(s). The addition of C-reactive protein restored aggregation and serotonin release of platelets incubated in human neonate plasma. The ADP-degrading enzyme, apyrase, and the non-steroidal, anti-inflammatory drug, indomethacin, inhibited platelet aggregation, suggesting that ADP secreted from platelet storage granules and indomethacin-sensitive pathway(s) are involve in the toxin-induced human platelet activation (aggregation and serotonin release). Generation of platelet activating factor (PAF) from the platelet by brown recluse toxin is not likely since the PAF receptor antagonist, BN 52021, did not inhibit platelet aggregation induced by the brown recluse toxin.     

The hemostatic derangement produced by T-2 toxin in guinea pigs

T-2 toxin produced significant coagulation abnormalities when administered parenterally to Hartley strain guinea pigs. The animals developed depressed activity of all coagulation factors except fibrinogen. Platelet aggregation in whole blood was depressed in response to ADP and collagen. The animals also exhibited an initial rise followed by a fall in hematocrit level, leukocytosis, and a decrease in platelet count. These changes were detectable within hours of toxin administration, reached a maximum at 24 hr, and returned to normal over the next 2 days. Pretreatment of animals with vitamin K1 had no effect on the activity of coagulation factors. The activated partial thromboplastin time of dilutions of plasma from animals given T-2 toxin with plasma from control animals revealed a pattern which pointed to a deficiency of coagulation factors as the principal cause of prolonged clotting times in treated animals. The presence of a weak circulating anticoagulant could not be ruled out. The addition of T-2 to plasma and blood of normal animals in a concentration of 1 microgram/ml had no effect on clotting times or platelet aggregation.     

Purification and characterization of a toxin from brown recluse spider (Loxosceles reclusa) venom gland extracts

A Toxic protein has been purified from brown recluse spider (Loxosceles reclusa) venom gland extracts. Cation-exchange chromatography at pH 4.0 was used as a high-yield method to purify the toxin to apparent homogeneity, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Activities associated with the toxin included lethality to mice, induction of necrosis in rabbits, calcium-dependent hemolysis of human erythrocytes and a decrease in the calcium-induced coagulation time of human plasma. All four activities were destroyed when the toxin was subjected to chemical modification of disulfide bonds. The toxin had an apparent mol. wt of 31,000 in the nonreduced conformation and 37,000 in the disulfide-modified form, as determined by sodium dodecyl sulfate gel electrophoresis. Iodination of the toxin did not destroy any of the four associated activities. Discontinuous polyacrylamide gel electrophoresis of the purified toxin revealed three apparent components. Collection and assay of these components showed that only two of these retained their activities, and the total recovery of activity was less than 40%.     

Purification and preliminary characterization of a plasma kallikrein inhibitor isolated from sea hares Aplysia dactylomela Rang, 1828.

An inhibitor active against pancreatic trypsin was found in the crude extract from the sea hares Aplysia dactylomelaRang, 1828. A stronger inhibitory activity against human plasma kallikrein was detectable after treating this extract at 60 degrees C, for 30 min. The plasma kallikrein inhibitor (AdKI) purification was achieved by acetone fractionation (80%) v/v, ion-exchange chromatography on Mono Q column and gel filtration chromatography on Superdex 75 column (FPLC system). By the latter a molecular mass of 2900 Da was estimated. The purified inhibitor strongly inhibits human plasma kallikrein with a K(i) value of 2.2 x 10(-10)M, while human plasmin and pancreatic trypsin were inhibited with K(i) values of 1.8 x 10(-9) and 4.7 x 10(-9)M, respectively. Chymotrypsin, pancreatic elastase, pancreatic kallikrein and thrombin are not inhibited. The effect of AdKI on plasma kallikrein was confirmed by the prolongation of activated partial thromboplastin time, using a clotting time assay. The inhibitor did not affect prothrombin time or thrombin time. AdKi is a more specific inhibitor than other serine proteinase inhibitors from marine invertebrates.     

In vitro observations on antithrombotic action of urinastatin

The inhibiting action in vitro of urinastatin on blood coagulation was studied for the purpose of therapeutic application against thrombotic disorders, and the following results were obtained: 1) Partial thromboplastin time of normal human plasma was prolonged dose-dependently by the addition of urinastatin to the reaction mixture, but prothrombin time was little inhibited by the addition of urinastatin. Thrombin time was also prolonged with urinastatin dose-dependently. 2) Using chromogenic synthetic peptide substrates, the amydolytic activities of XIIa, plasma kallikrein and Xa activated with RVV were inhibited by the addition of urinastatin to the reaction mixtures. 3) Activated partial thromboplastin time of normal plasma was prolonged by the addition of urinastatin or heparin, and simultaneous application of both urinastatin and heparin to the reaction mixture resulted in an additional inhibitory effect on APTT. Therefore, it was assumed that the different molecular structures of the clotting factors were concerned with the inhibitory actions of urinastatin and antithrombin III. Furthermore, urinastatin was indicated to have an important role in antithrombotic remedy, since it has no inhibitory action against protein C. 4) In the comparison with purified human plasmin and plasma plasmin activated with streptokinase, the strong inhibitory action of urinastatin on purified plasmin was demonstrated, but the inhibitory action of urinastatin was decreased markedly in plasma. Therefore, it is suggested that plasma may contain an inhibitory factor against the action of urinastatin.     

Coagulant and anticoagulant activities of Bothrops lanceolatus (Fer de lance) venom.

Bothrops lanceolatus venom contains caseinolytic, phospholipase, esterase and haemorrhagic activities. We have investigated the coagulant and anticoagulant actions of B. lanceolatus venom on human citrated plasma and on purified plasma components. Although B. lanceolatus venom up to 50 microg/ml was unable to clot citrated plasma, at concentrations > or = 5 microg/ml the venom dose-dependently clotted purified human fibrinogen, indicating the presence of a thrombin-like enzyme. Human plasma (final concentration > or = 12.5%) dose-dependently inhibited the venom-induced fibrinogen clotting. This finding suggested that endogenous plasma protease inhibitors can affect the venom's action on fibrinogen. To investigate this possibility, B. lanceolatus venom was incubated with different plasma protease inhibitors and the activity on fibrinogen tested. alpha(2)-Macroglobulin and alpha(1)-antitrypsin did not interfere with the coagulant activity of the venom whereas the antithrombin-III/heparin complex partially inhibited this activity. A non-toxic, acidic phospholipase A(2) purified from B. lanceolatus venom prolonged the activated partial thromboplastin time in human plasma from 39.7+/-0.5 s (control with saline) to 60.2+/-0.9 s with 50 microg of PLA(2) (p<0.001), suggesting an anticoagulant activity associated with this enzyme. This anticoagulant activity may account for some of the effects of the venom on blood coagulation.     

A model for venom-induced consumptive coagulopathy in snake bite

Many snake venoms contain procoagulant toxins that activate the coagulation cascade and cause venom-induced consumptive coagulopathy (VICC). We developed a semi-mechanistic model of the clotting cascade in order to explore the effects of the procoagulant toxin from taipan venom on this system as well as the effects of antivenom. Simulations of the time course in the change of clotting factors were compared to data collected from taipan envenomed patients. The model accurately predicted the observed concentration of clotting factors over time following taipan envenomation. Investigations from the model indicated that the upper limit of the half-life of the procoagulant toxin was 1h. Simulations from the model also suggest that antivenom for Australasian elapids has negligible effect on reducing the recovery time of the coagulation profile unless administered almost immediately after envenomation. The model has generality to be expanded to describe the effects of other venoms and drugs on the clotting cascade.     

The effects of the purified thrombin-like enzyme and anticoagulant principle of Trimeresurus gramineus venom on blood coagulation in vivo

Chaoho Ouyang and Fun-Yun Yang. The effects of the purified thrombin-like enzyme and anticoagulant principle of Trimeresurus gramineus venom on blood coagulation in vivo. Toxicon14, 197–201, 1976.—The effects of the purified thrombin-like enzyme and anticoagulant principle of Trimeresurus gramineus venom on in vivo blood coagulation of rabbits were studied. The thrombin-like enzyme caused a marked prolongation of whole blood coagulation time and one-stage plasma prothrombin time and a marked decrease of the fibrinogen concentration, while no significant change in the two-stage plasma prothrombin level was detected. It is concluded that the retardation of blood clotting by the thrombin-like enzyme was chiefly due to the decrease of plasma fibrinogen level. The anticoagulant principle caused a marked, but transient prolongation of whole blood coagulation time and one-stage plasma prothrombin time with no significant change in the two-stage plasma prothrombin level or plasma fibrinogen level. Combining these results with our previous in vitro findings, it is concluded that the retardation of blood clotting by the anticoagulant principle might be due to the interference in the interaction between prothrombin and its activation factors.     

The effects of the purified thrombin-like and anticoagulant principles of Agkistrodon acutus venom on blood coagulation in vivo.

The effects of the purified thrombin-like and anticoagulant principles of the snake venom of Agkistrodon acutus on blood coagulation of rabbits in vivo were studied. The thrombin-like principle caused a marked prolongations of whole blood coagulation time and one-stage plasma prothrombin time and a marked decrease of the fibrinogen concentration, while no significant change in the two-stage plasma prothrombin level was detected. It is concluded that the retardation of blood clotting by the thrombin-like principle was chiefly due to the decrease of plasma fibrinogen level. The anticoagulant principle caused a marked, but transient prolongation of whole blood coagulation time and one-stage plasma prothrombin time with no significant change in the two-stage plasma prothrombin level or plasma fibrinogen level. Combining these results with our previous in vitro findings, it is concluded that the retardation of blood clotting by the anticoagulant principle might be due to the interference in the interaction between prothrombin and its activation factors.     

Purification and characterization of anticoagulant protein from the Tabanus,Tabanus bivittatus

Tabanus anticoagulant protein (TAP) was isolated from the whole body of the tabanus, Tabanus bivittatus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and ion exchange chromatography on DEAE Sephadex gel). The purified TAP, with a molecular weight of 65 kDa, was assessed to be homogeneous by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 7.9 was determined by isoelectric focusing. The internal amino acid sequence of the purified protein was composed of Ser-Leu-Asn-Asn-Gln-Phe-Ala-Ser-Phe-Ile-Asp-Lys-Val-Arg. The protein was activated by Cu2+ and Zn2+, and the optimal conditions were found to be at pH 3-6 and 40-70 degrees C. Standard coagulation screen assays were used to determine thrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. TAP considerably prolonged human plasma clotting time, especially activated partial thromboplastin time in a dose-dependent manner; it showed potent and specific antithrombin activity in the chromogenic substrate assay. Specific anti-factor Xa activity in TAP was not detected. Overall, this result suggested that TAP has significant anticoagulant activity on blood coagulation system.     

Cytotoxicity and effects of T2-toxin on plasma proteines involved in coagulation,fibrinolysis and kallikrein-kinin system

The activity of both the coagulation and fibrinolytic systems was markedly depressed 24 h after a sublethal dose of T-2 toxin. T-2 toxin was active as an anticoagulant at low doses, which did not affect the basal state of the animals. The kallikrein-kinin system was also affected by depletion of the prekallikrein, which indicates increased bradykinin levels in plasma. At the same time there was an increased activity of some clinically relevant enzymes in serum, indicating tissue injuries caused by T-2 toxin. All effects observed in this study reached their maximum within 24 h after administration, which corresponds to the time animals usually die when receiving a lethal dose. T-2 toxin does not, however, seem to affect the protease enzymes by reduced protein synthesis, because of early onset of the effects, nor does it act as a trigger itself. The effect of T-2 toxin on plasma protease enzymes is probably secondary to cytotoxic effects in the vascular endothelium.     

The effect of the purified anticoagulant principle of Agkistrodon acutus venom on blood coagulation

The purified anticoagulant principle of Agkistrodon acutus venom had marked anticoagulant action when tested on whole blood clotting time, calcium clotting time and plasma prothrombin time. It did not destroy fibrinogen, induce fibrinolysis, inactivate thrombin nor interfere with the interaction between thrombin and fibrinogen. However a marked inhibition of prothrombin activation was not due to the destruction of prothrombin or its activation factors, but due to an interference in the interaction between prothrombin and its activation factors because of the reversible binding of these factors with the anticoagulant principle of the venom.     

国产冻干人凝血酶原复合物质量标准的研究

目的通过对国产人凝血酶原复合物 (PCC)质量的研究 ,建立与国际接规的质量标准。方法用人凝血因子Ⅱ、Ⅶ、Ⅸ、Ⅹ (FⅡ、FⅦ、FⅨ、FⅩ )浓制剂国家标准品 ,采用一期法测定国内用不同原料制备的PCC中FⅡ、FⅦ、FⅨ、FⅩ的效价 ;用正常人血浆作标准 ,用凝固法检测PCC的总效价及PCC中的肝素。结果用血浆作原料提纯制备的PCC ,FⅣ的效价能达到 1 0IU/ml以上 ,FⅦ效价最低 ;用组分Ⅲ作原料 ,FⅣ活性损失很大 ,只有 2 / 7批次能达到 1 0IU/ml;而FⅦ效价最高。无论用何种原料 ,FⅡ和FⅩ活性均能保持在一定水平。结论从血浆中直接提纯制备PCC ,能有效地保持FⅣ活性 ,各项指标易达到国外同品种的质量标准     

Effects of Heloderma suspectum venom on blood coagulation

The effect on blood coagulation of venom from the Gila monster Heloderma suspectum was studied in vivo and in vitro. Lethal and sublethal doses of venom failed to alter the clotting and prothrombin times of rabbit and cat blood in vivo. Venom mixed with freshly drawn blood from rabbits and cats had no effect on the clotting time. When venom was incubated with human, rabbit, and cat plasma for periods longer than 5 min, prothrombin time was prolonged. Placing venom in a boiling water bath for 15 min did not alter this.     

Comparative study on the effect of staphylococcal alpha-toxin on erythrocytes and thrombocytes

The effect of staphylococcal alpha-toxin on the release of haemoglobin from rat erythrocytes, and serotonine from rat thrombocytes, has been measured and expressed in ₅₀ values. The Hb depletion is dose-dependent and appears after a latency period. Higher doses produce complete haemolysis. Diluted and full plasma decreases the toxin potency in this respect. 5-HT release from thrombocytes starts immediately after toxin administration and is dose-dependent. However, even after 60 min of incubation with plasma, high toxin doses release only about 50 per cent of 5-HT. Diluted plasma somewhat enhances the releasing effect.     

The anti-coagulant properties of an extract from the venom sac of the oriental hornet

An extract from the venom sac of the oriental hornet inhibits the in vitro formation of thromboplastin and thrombin by human plasma and inactivates already formed tissue thromboplastin. The extract has a strong fibrinogenolytic and fibrinolytic activity when tested on purified human fibrinogen, but almost no lytic activity when tested on human plasma. The fibrinolysis is inhibited by soybean trypsin inhibitor as well as by human serum, presumably due to its anti-trypsin activity. Intravenous administration of the sac extract to dogs causes decreased clottability of the whole blood, or the recalcified plasma, and depresses prothrombin consumption and thromboplastin generation. There is a significant decline of the platelet count but a rather mild decrease of fibrinogen. All parameters tend to revert to normal after 2–3 hr. Intraperitoneal envenomation causes severe heparinemia with prolonged clotting, effects which can be reversed by the addition of protamin sulfate. The anticoagulant properties of the venom are abolished by heating at acid or alkaline pH and neutralized by anti-venom immune serum.     

Metabolism of substance P and bradykinin by human neutrophils.

The catabolism of substance P and bradykinin, two peptides involved in inflammation, by human neutrophils was investigated. Substance P was cleaved by unstimulated neutrophils, but the rate of hydrolysis increased greatly (about 4-fold) when the cells were lysed by freezing and thawing or stimulated to release with fMet-Leu-Phe and cytochalasin B. The enzyme responsible for cleaving substance P was cathepsin G, hydrolyzing the Phe7-Phe8 bond. Neutral endopeptidase 24.11 (enkephalinase) became the main inactivating enzyme only when neutrophil cytoplasts (containing plasma membrane but no subcellular particles) or washed plasma membrane enriched high speed sediments were tested. Subcellular fractionation showed the highest substance P degrading activity to be in the granules. Purified cathepsin G readily cleaved substance P with a Km of 1.13 MK, a kcat of 6.35 sec-1 and a kcat/Km of 5639 M-1 sec-1, similar to kinetic constants previously reported for the best peptide substrates of cathepsin G. Despite the high Km, purified cathepsin G did hydrolyze SP at a much lower substrate concentration (down to 1 nM) as determined by radioimmunoassay. Bradykinin was also hydrolyzed by intact neutrophils but, in contrast, was not inactivated by cathepsin G, but by neutral endopeptidase at the Pro7-Phe8 bond. The inactivation of bradykinin by intact neutrophils was decreased by phorbol 12-myristate 13-acetate, probably due to down-regulation by endocytosis of the neutral endopeptidase on the plasma membrane. Thus, both bradykinin and substance P are inactivated by human neutrophils, although by different enzymes. In spite of the less favorable kinetics in vitro than with neutral endopeptidase, cathepsin G is the main inactivator of substance P in neutrophils. This may be due to the estimated 300 to 3600-fold higher concentration of cathepsin G in neutrophils than that of the neutral endopeptidase.     

Anticoagulant properties of a red tide toxin

The effect of toxins from the Florida red tide organism Gymnodinium breve Davis (gymnodin) and from the chrysomonad Prymnesium parvum Carter (prymnesim) on the coagulation time of human blood plasma is described. Prymnesim was inactive at a concentration of 4 mg per 0·05 ml aliquot, though the toxin is known to be a potent hemolysin. Gymnodin inhibited the clotting process at a concentration of 0·05 mg per 0·05 ml aliquot. Other anticoagulating agents were studied. Activities were compared in terms of the amounts of anticoagulant required to double the prothrombin time. On this basis, gymnodin had $\text{1}\text{6}$ the activity of carrageenan and $\text{1}\text{70}$ the activity of heparin. The anticoagulant activity of gymnodin appears to be the first such activity reported for a toxin from a red tide organism. Available evidence indicated the anticoagulant properties of gymnodin were not responsible for the ichthyotoxicity.     

Non-steroidal anti-inflammatory drugs: effects on a GTP binding protein within the neutrophil plasma membrane

Sodium salicylate and other non-steroidal anti-inflammatory drugs (NSAIDs) inhibit neutrophil functions via unknown mechanisms. To examine their site of action in the neutrophil we have studied discrete events within the plasma membrane which depend upon the normal function of a GTP binding protein (G protein). We demonstrated that sodium salicylate and piroxicam inhibit neutrophil activation in response to stimuli which require signal transduction via a G protein (e.g. formyl-methionine-leucine-phenylalanine) but have no effect on stimuli which do not (e.g. phorbol myristate acetate, ionomycin). NSAIDs blocked the ADP-ribosylation of the pertussis toxin substrate in human neutrophils. This effect was associated with the capacity of NSAIDs to block pertussis toxin-dependent inhibition of neutrophil functions. Finally, NSAIDs inhibited the binding of GTP gamma S, a stable analog of GTP, to purified neutrophil membrane preparations. The data indicate that salicylate and other NSAIDs interact with a G protein in the neutrophil plasmalemma and thereby uncouple post-receptor signaling events.     

The effect of choleragen on adenyl cyclase activity and glycogen content in tissues of the rat

Injection (iv.) of purified cholera toxin (choleragen) into rats markedly activated basal adenyl cyclase activity in liver, slightly activated it in thyroid gland, kidney and spleen, while no activation was observed in heart, skeletal muscle, mediastinal fat or hypophysis. The changes in carbohydrate metabolism corresponded to the alterations in adenyl cyclase activity: depletion of liver glycogen with an increase in plasma glucose, no change in skeletal muscle glycogen, with only a slight increase in plasma lactic acid and a slight increase in heart glycogen content. The reason for the selective response of some tissues to choleragen treatment remains unknown; however, it is clear that cholera toxin effect is not confined to an epinephrine receptor, as was recently suggested.The mechanism of action of cholera toxin on adenyl cyclase from liver showed close similarities to the mechanism found in studies with mucosal epithelial cells from the small intestine. After a few hr of in vivo exposure to choleragen (10 μg per rat) the basal activity of adenyl cyclase was increased many fold, while sodium fluoride stimulation and hormone responsiveness were substantially unchanged. Addition of guanosine triphosphate to the assay system increased the basal adenyl cyclase activity in liver, both from the control and cholera toxin treated group.