Interaction of viper venom serine peptidases with thrombin receptors on human platelets

The serine peptidases, thrombocytin and PA-BJ, isolated from the venom of Bothrops atrox and Bothrops jararaca, respectively, induce platelet aggregation and granule secretion without clotting fibrinogen. The specific platelet aggregation activity of each enzyme was about 15 times lower than that of thrombin. This activity was blocked by monoclonal antibodies recognizing protease activated receptor 1 (PAR1) and by heparin, but not by hirudin nor thrombomodulin. Both enzymes induced calcium mobilization in platelets and desensitized platelets to the action of thrombin and the SFLLRN peptide. We compared the effect of thrombin, PA-BJ, and thrombocytin on the degradation of the soluble N-terminal domain of the PAR1 receptor. The major cleavage site by thrombin and both viper enzymes was Arg41-Ser42. In addition, a rapid cleavage of the peptide bond at Arg46-Asn47 by the viper enzymes was observed, resulting in the inactivation of the tethered ligand. PA-BJ and thrombocytin both cleaved at 41-42 and 46-47 peptide bonds, and fragment 42-103 disappeared rapidly. Both viper enzymes caused calcium mobilization in fibroblasts transfected with PAR4 and desensitized these cells to the thrombin action. In conclusion, both PAR1 and PAR4 mediate the effect of viper venom serine peptidases on platelets.     

蛇毒丝氨酸蛋白酶多样性──底物专一性免疫化学及序列比较研究

本文报道烙铁头（Ｔｒｉｍｅｒｅｓｕｒｕｓｍｕｃｒｏｓｑｕａｍａｔｕｓ）蛇毒纤维蛋白原溶酶（ＴＭＶＦｇ），眼镜王蛇（Ｏｐｈｉｏｐｈａｇｕｓｈａｎｎａｈ）蛇毒纤维蛋白原溶酶（ｏｈＳ１），竹叶青（Ｔｒｉｍｅｒｅｓｕｒｕｓｓｔｅｊｎｅｇｅｒｉ）蛇毒专一纤溶酶原激活剂（ｓｖ－ｐＡ）对５种小分子多肽底物的底物专一性，及这些蛇毒丝氨酸蛋白酶对各种凝血因子（第Ｘ因子、凝血酶原、纤溶酶原、蛋白Ｃ）的作用，并和其它蛇毒丝氨酸蛋白酶如矛头蝮（Ｂｏｔｈｒｏｐｓａｔｒｏｘ）蛇毒凝血酶样酶（Ｂａｔｒｏｘｏｂｉｎ）、铜头蝮（Ａｇｋｉｓｔｒｏｄｏｎｃｏｎｔｏｒｔｒｉｘｃｏｎｔｏｒｔｒｉｘ）蛇毒蛋白Ｃ激活剂ＡＣＣ－Ｃ、蝰蛇（Ｖｉｐｅｒａｒｕｓｓｅｌｌｉ）毒第Ⅴ因子激活剂ＲＶＶ－Ｖ进行比较研究。通过酶标偶联免疫反应研究了抗ｓｖ－ＰＡ抗体与各种丝氨酸蛋白酶的免疫交叉反应，并对蛇毒丝氨酸蛋白酶及相应功能的哺乳动物蛋白酶进行了序列比较分析。从底物专一性多样性及已知序列结构分化上对这一类蛇毒丝氨酸蛋白酶的结构与功能进行了探讨和研究。     

The isolation of a component of the venom of Trimeresurus okinavensis that causes the aggregation of blood platelets

1. A substance has been isolated from the venom of the Okinawan pit viper (Trimeresurus okinavensis) that causes the aggregation of human blood platelets. The reaction resembles that between platelets and thrombin or connective tissue, in that the platelets release ADP. 2. The purified venom fraction is a protein-polysaccharide, and after Pronase digestion had mol.wt. 4x10(6), containing 10% peptide, 85% neutral sugars and 5% hexosamines. It is distinct from the proteolytic, esterolytic and coagulant materials also present in the venom, and has not been shown to possess any enzymic activity. How it reacts with platelets is not yet known.     

Purification and Functional Characterisation of Rhinocerase,a Novel Serine Protease from the Venom of Bitis gabonica rhinoceros

Background

Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders.

Methodology/Principal Findings

In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36kDa, which reduces to 31kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading α and β chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate.

Conclusions/Significance

A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes.     

Purification and characterization of a Kunitz-type trypsin inhibitor from Leaf-nosed viper venom.

A Kunitz-type trypsin inhibitor was purified from Leaf-nosed viper venom and the primary structure determined by peptide analysis. In relation to other trypsin inhibitors, the protein has an extended C-terminal segment and a distinct pattern of residue alterations at the functionally important contact sites with proteases.     

Functional phenotyping of human plasma using a 361-fluorogenic substrate biosensing microarray

A microarray presenting glycerol nanodroplets of fluorogenic peptide substrates was used as a biosensor for the detection of multiple enzyme activities within human plasma. Using 10 different plasma proteases (kallikrein, factor XIIa, factor XIa, factor IXa, factor VIIa, factor Xa, thrombin, activated protein C, uPA and plasmin) and a 361-compound fluorogenic substrate library (Ac-Ala-P3-P2-Arg-coumarin for P = all amino acids except Cys), a database was created for deconvoluting the relative activity of each individual enzyme signal in human plasma treated with various activators (calcium, kaolin, or uPA). Three separate deconvolution protocols were tested: searching for "optimal" sensing substrate sequences for a set of 5 enzymes and using these substrates to detect protease signals in plasma; ranking the "optimal" sensing substrates for 10 proteases using local error minimization, resulting in a set of substrates which were bundled via weighted averaging into a super-pixel that had biosensing properties not obtainable by any individual fluorogenic substrate; and treating each 361-element map measured for each plasma preparation as a weighted sum of the 10 maps obtained for the 10 purified enzymes using a global error minimization. The similarity of the results from these latter two protocols indicated that a small subset of <90 substrates contained the majority of biochemical information. The results were consistent with the state of the coagulation cascade expected when treated with the given activators. This method may allow development of future biosensors using minimal and non-specific markers. These substrates can be applied to real-time diagnostic biosensing of complex protease mixtures.     

The factor V-activating enzyme (RVV-V) from Russell's viper venom. Identification of isoproteins RVV-V alpha, -V beta, and -V gamma and their complete amino acid sequences

The complete amino acid sequences of two isoproteins of the factor V-activating enzyme (RVV-V) isolated from Vipera russelli (Russell's viper) venom were determined by sequencing S-pyridylethylated derivatives of the proteins and their peptide fragments generated by either chemical (cyanogen bromide and 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine) or enzymatic (trypsin, alpha-chymotrypsin, and lysyl endopeptidase) cleavages. Both enzymes, designated RVV-V alpha and RVV-V gamma, consist of 236 amino acid residues and have a N-linked oligosaccharide chain at Asn229. The six amino acid substitutions between RVV-V alpha and -V gamma are: Thr22(alpha)-Ala22(gamma), Gly29(alpha)-Ala29(gamma), Gln191(alpha)-Glu191(gamma), Ile192(alpha)-Met192(gamma), Gln193(alpha)-His193(gamma), and Asn224(alpha)-Ser224(gamma). The molecular weights were calculated as 26,182 for RVV-V alpha and 26,167 for RVV-V gamma. The sequences of the RVV-V isoproteins exhibited 62% identity with that of batroxobin, a thrombin-like enzyme present in Bothrops atrox venom, and 33% identity with that of human thrombin B chain. The most interesting difference between the structures of RVV-V and other trypsin-type serine proteases is that the conservative Ser214-Trp215-Gly216 sequence (chymotrypsinogen numbering), considered as the site of antiparallel beta-sheet formation between the protein substrate and most serine proteases, has been replaced by the corresponding sequence Ala-Gly-Gly.     

竹叶青蛇毒凝血酶样酶氨基酸序列报道

蛇毒凝血酶样酶可作为蛋白酶结构与功能研究的良好模型,并已广泛用于各种血栓疾病的诊断和治疗,因而测定其一级结构具有重要意义．利用逆转录与聚合酶链反应相结合的ＲＴ－ＰＣＲ法,扩增出竹叶青蛇毒凝血酶样酶（ＴＳＶ－ＴＥＬ１）的ｃＤＮＡ;将扩增的ｃＤＮＡ片段克隆入ｐＧＥＭ－Ｔ载体中,经末端终止法测定核苷酸序列,推导出竹叶青蛇毒凝血酶样酶的全序列．竹叶青蛇毒凝血酶样酶由２３４个氨基酸组成并含有１个位于Ａｓｎ２０的Ｎ－型糖基结合位点．竹叶青蛇毒凝血酶样酶序列与其它蛇种来源凝血酶样酶具有较大相似性,其与黄绿烙铁头蛇毒凝血酶样酶序列相似度为８４％,与美洲矛头蝮蛇毒凝血酶样酶序列相似度为６８％,而与牛凝血酶Ｂ链序列相似度仅为２５％．     

Mechanism by which heparin proteoglycan modulates mast cell chymase activity.

Chymases are highly basic chymotrypsin-like serine proteases expressed exclusively by mast cells. Large amounts of chymases complexed with heparin proteoglycan (PG) are released in vivo during mast cell activation. The tight binding of chymase to heparin PG results in increased activity of the protease toward certain substrates, e.g., thrombin and MeO-Suc-Arg-Pro-Tyr-pNA (S-2586). In this study, the mechanism by which heparin PG modulates chymase activity was investigated, using thrombin and various chromogenic peptide substrates as model substrates. Incubation of thrombin with oligonucleotides that block the heparin-binding site of thrombin abolished the stimulatory effect of heparin PG on thrombin inactivation. Further, thrombin mutants with defects in their heparin-binding regions were less efficiently inactivated by chymase-heparin PG than wild type thrombin. These findings suggest a model for chymase stimulation where heparin PG may promote the chymase-catalyzed cleavage of heparin-binding substrates by simultaneously binding to both chymase and substrate. Experiments in which various chromogenic peptide substrates were utilized showed that heparin PG enhanced the activity of chymase toward positively charged peptide substrates such as S-2586, whereas the cleavage of uncharged substrates was not affected by the presence of heparin PG. On the basis of the latter findings, an alternative stimulation mechanism is discussed where heparin PG may stimulate chymase activity by blocking positively charged regions in chymase, thereby reducing the level of electrostatic repulsion between chymase and positively charged substrates.     

Primary structure of a protein C activator from Agkistrodon contortrix contortrix venom

The amino acid sequence of a protease, protein C activator, from Agkistrodon contortrix contortrix venom was determined. Peptide fragments obtained by chemical or enzymatic cleavage of the S-carboxymethylated protein were purified by gel filtration and reverse-phase high-performance liquid chromatography. The present study demonstrates that protein C activator from A. contortrix contortrix venom is a trypsin-type serine protease that is composed of 231 residues with a molecular weight of 25,095 for the polypeptide portion of the molecule. By analogy to the mammalian serine proteases, the catalytic triad in venom protein C activator consists of His-40, Asp-85, and Ser-177. The protein also contains three N-linked glycosylation sites at Asn-21, Asn-78, and Asn-129. The amino acid sequence of protein C activator exhibits a high degree of sequence identity with other snake venom proteases: 73% with batroxobin, 68% with flavoxobin, and 55% with Russell's viper venom factor V activator.     

Blood coagulation induced by the venom of Bothrops atrox. 1. Identification, purification, and properties of a prothrombin activator

In this paper, we show that the procoagulant action of Bothrops atrox venom is due in part to a protein component that activates prothrombin. The venom prothrombin activator was purified by ion-exchange chromatography and gel filtration. It was separated from a protease by affinity chromatography in a p-aminobenzamidine-CH-Sepharose column. It is a protein of about Mr 70,000, consisting of a single polypeptide chain. We have studied the kinetics of activation of prothrombin under different experimental conditions. The prothrombin activator from B. atrox venom is insensitive to reagents of serine and thiol proteases but is inactivated by ion chelators and by various divalent ions. These results suggest that it is a metalloenzyme. The prothrombin activator from B. atrox venom is inactive on the chromogenic substrates S-2337 and S-2238, and it is selective for prothrombin since it does not act on other blood coagulation factors such as fibrinogen and factor X. We have also studied the pattern of peptide cleavages produced in the human prothrombin molecule during the activation by the activator from B. atrox venom and compared it to that obtained with ecarin, a prothrombin activator from Echis carinatus venom. In the presence of thrombin inhibitors, e.g., hirudin, we found that the activators from B. atrox venom and ecarin act in a similar, or identical, manner by producing a thrombin intermediate, meizothrombin. In the absence of thrombin inhibitors, several peptides are generated, and alpha-thrombin is produced as a consequence of meizothrombin action.     

Isolation of a proteinase with plasminogen-activating activity from Lachesis muta muta (bushmaster) snake venom

A plasminogen activator enzyme (LV-PA) from Lachesis muta muta venom was purified to homogeneity using gel filtration and anion exchange chromatography. SDS-PAGE under reducing conditions showed a single protein band with an Mr of 33,000 Da. It is an acidic glycoprotein which activates plasminogen to plasmin indirectly, functioning via prior formation of a molecular complex, known as plasminogen activator. The purified preparation catalyzes the hydrolysis of several p-nitroanilide peptide substrates containing Lys at the scissile bond. In contrast, no hydrolysis was detected on the synthetic substrates TAME and BAPNA, which contain arginine. By the use of the plasmin-specific chromogenic substrate Tos-Gly-Pro-Lys-pNA, the preparation had a plasmin-like activity of 0.68 U/mg, which was 35.8-fold higher than that of the crude venom from which it was prepared. In vitro, fibrin hydrolysis using LV-PA as plasminogen activator displayed more similarity with the effect produced by streptokinase (SK). SDS-PAGE (10%) analysis showed a 115-kDa complex formation after incubation of plasminogen with either LV-PA or SK. At a molar ratio of 50:1 (fibrinogen:enzyme), the preparation exhibited weakly fibrinogenolytic activity. However, LV-PA is distinguished from thrombin in that it does not clot fibrinogen. After incubation of LV-PA with platelet-rich plasma, the enzyme (2 microM) showed no effect on platelet aggregation induced by ADP, epinephrine, or collagen. Comparison of the N-terminal sequence of LV-PA with other snake venom plasminogen activators revealed that LV-PA exhibits a high degree of sequence identity with the TsVPA from Trimeresurus stejnegeri (90%) and with the Haly-PA from Agkistrodon halys (85%). LV-PA also has homology with other snake venom serine proteinases such as the thrombin-like/gyroxin analogue (38%) from bushmaster venom and with other coagulation serine proteases. The proteinase was readily inhibited by treatment with p-nitrophenyl p-guanidinebenzoate, p-aminobenzamidine, and phenylmethanesulfonyl fluoride but was not affected by metal chelators.     

Characterization of kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom

1. A kallikrein-like enzyme from the venom of Crotalus ruber ruber (red rattlesnake) had been isolated and characterized by Mori and Sugihara. The enzyme was active upon the kallikrein substrates, Pro-Phe-Arg-MCA and z-Phe-Arg-MCA, and slightly hydrolyzed Boc-Val-Leu-Lys-MCA, and Boc-Phe-Ser-Arg-MCA. 2. Unlike thrombin, the newly isolated kallikrein-like enzyme did not cause formation of a fibrin clot when fibrinogen was mixed with the enzyme. 3. The B beta chain of fibrinogen was first split and A alpha chain was cleaved later. Pancreatic kallikrein hydrolyzed only the A alpha chain without affecting the B beta chain. 4. The kallikrein-like enzyme produced kallidin (Lys-bradykinin) by splitting the Met-Lys bond instead of producing bradykinin. 5. The kallikrein analog JSI-450 (Ac-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gln-Val-Ser-NH2) was also cleaved at the site of the Arg-Ser bond. 6. Its NH2-terminal amino acid sequence (Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-Arg-Pro-Phe-Leu-Val-Ala-Leu-Tyr- Asp-Ser-) is homologous to the rat pancreatic kallikrein and other snake venom proteases.     

Degradation of coagulation proteins by an enzyme from Malayan pit viper (Akistrodon rhodostoma) venom

Three hydrolases from the crude venom of the Malayan pit viper (Akistrodon rhodostoma) can be differentiated. The first, which we designate ARH alpha, is the well-known fibrinogenolytic enzyme ancrod. The second, ARH beta, which has not been described previously, is identified by its electrophoretic mobility after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), by its ability to hydrolyze H-D-phenylalanyl-L-piperyl-L-arginyl-rho-nitroanilide, and by inhibition of its activity by diisopropyl phosphorofluoridate. The third, ARH gamma, also previously not described, has been purified by using gel permeation and ion-exchange chromatography and preparative PAGE. Chemical, electrophoretic, and hydrodynamic data indicate that it is a single-chain, nonglobular glycoprotein with a molecular weight of 25,600. ARH gamma catalyzes the degradation of several plasma vitamin K dependent coagulation factors, including factor IX, factor X, prothrombin, and protein C. The products are electrophoretically similar to factor IXa beta, factor Xa, thrombin, and activated protein C, respectively. However, these products contain little or no enzymatic activity. ARH gamma-degraded factor IX, factor X, prothrombin, and protein C can be subsequently activated by factor XIa, Russell's viper venom X coagulant protein, crude taipan snake venom, and thrombin, respectively. The N-terminal sequence of the peptides resulting from the ARH gamma digest of porcine factor IX shows that at least three bonds are hydrolyzed: (1) at position 152, seven residues from the Arg145-Ala146 factor XIa cleavage site; (2) at position 167 within the factor IX activation peptide; and (3) at position 177, three residues from the Arg180-Val181 factor XIa cleavage site. The degradation of factor IX by ARH gamma is not affected by several serine protease inhibitors. ARH gamma catalyzes the degradation of both the heavy and light chains of porcine factor VIII which results in the inability of thrombin to activate factor VIII. ARH gamma also catalyzes the degradation of porcine antithrombin III which abolishes its ability to inhibit thrombin. These findings may have relevance to studies of hemostatic derangements following envenomation by this snake. Additionally, several novel coagulation factor derivatives have been generated for structure-function studies.     

Characterization of cerastobin, a thrombin-like enzyme from the venom of Cerastes vipera (Sahara sand viper)

Cerastobin, a thrombin-like enzyme, was isolated from the venom of Cerastes vipera (Sahara sand viper) in homogeneous form. Cerastobin had a molecular weight of 38,000 with 348 amino acid residues. It had an isoelectric point of 7.7 (a pH optimum of 7.9 and a temperature optimum of 45 degrees C). Cerastobin hydrolyzed arginine-containing synthetic substrates such as TAME, BAME, and BAEE, but BAPNA was not hydrolyzed. Cerastobin had thrombin-like activity, producing fibrin from fibrinogen and also hydrolyzing chromogenic substrates for thrombin such as 2AcOH.H-D-CHG-But-Arg-pNA (CBS 34.47) and H-D-Phe-Pip-Arg-pNA (S-2238). It showed kallikrein-like activity and hydrolyzed kallikrein substrates 2AcOH.H-D-Phe-Gly-Arg-pNA (CBS 33.27) and H-D-Pro-Phe-Arg-pNA (S-2302). It produced bradykinin from bradykininogen, as uterus contraction was observed. A serine inhibitor, DFP, exerted a pronounced inhibitory effect, suggesting that cerastobin is a serine-type protease. The sequence of 37 residues from the amino-terminal end was investigated. The amino-terminal amino acid was valine as it is in most other thrombin-like enzymes. The amino acid sequence of cerastobin was similar to that of thrombin in some residues and had some homology with that of kallikrein. However, cerastobin showed a high degree of homology to thrombin-like enzymes isolated from various snake venoms. Factor X was partially degraded by cerastobin. It was also found that antithrombin III was degraded by the enzyme. The alpha and beta chains of fibrin monomer were preferentially hydrolyzed by cerastobin, but the gamma chain was quite resistant.     

Isolation and characterization of an apoptotic and platelet aggregation inhibiting L-amino acid oxidase from Vipera berus berus (common viper) venom

An L-amino acid oxidase was isolated from the venom of the common viper Vipera berus berus by a three-step procedure combining gel filtration, ion exchange and hydrophobic chromatography. The enzyme is a non-covalently bound homodimer with a monomeric molecular mass of 57.7 kDa. The N-terminal amino acid sequence and the internal peptide sequences show close structural homology with other snake venom L-amino acid oxidases. The purified protein catalyzed oxidative desamination of L-amino acids, the most specific substrate is L-Phe. The best substrates among the studied 20 amino acids were: L-Met, L-Leu, L-Phe, L-Ile, L-Arg and L-His. Five amino acids, L-Ser, L-Pro, Gly, L-Thr and L-Cys, were not oxidized. The enzyme inhibited ADP-induced platelet aggregation dose-dependently with an IC50 of 0.07 microM. The effect was neutralized by catalase. V. berus berus LAAO induced apoptosis in cultured HeLa and K562 cells as shown by DNA fragmentation gel pattern. The induction of apoptosis was inhibited by catalase.     

On the modeling of snake venom serine proteinase interactions with benzamidine-based thrombin inhibitors

Pit viper venoms contain a number of serine proteinases that exhibit one or more thrombin-like activities on fibrinogen and platelets, this being the case for the kinin-releasing and fibrinogen-clotting KN-BJ from the venom of Bothrops jararaca. A three-dimensional structural model of the KN-BJ2 serine proteinase was built by homology modeling using the snake venom plasminogen activator TSV-PA as a major template and porcine kallikrein as additional structural support. A set of intrinsic buried waters was included in the model and its behavior under dynamic conditions was molecular dynamics simulated, revealing a most interesting similarity pattern to kallikrein. The benzamidine-based thrombin inhibitors alpha-NAPAP, 3-TAPAP, and 4-TAPAP were docked into the refined model, allowing for a more insightful functional characterization of the enzyme and a better understanding of the reported comparatively low affinity of KN-BJ2 toward those inhibitors.     

Detection of Venom after Antivenom Is Not Associated with Persistent Coagulopathy in a Prospective Cohort of Russell's Viper (Daboia russelii) Envenomings

Background

Venom recurrence or persistence in the circulation after antivenom treatment has been documented many times in viper envenoming. However, it has not been associated with clinical recurrence for many snakes, including Russell''s viper (Daboia spp.). We compare the recovery of coagulopathy to the recurrence or persistence of venom in patients with Russell''s viper envenoming.

Methodology/Principal Findings

The study included patients with Russell''s viper (D. russelii) envenoming presenting over a 30 month period who had Russell''s viper venom detected by enzyme immunoassay. Demographics, information on the snake bite, and clinical effects were collected for all patients. All patients had serum collected for venom specific enzyme immunoassay and citrate plasma to measure fibrinogen levels and prothrombin time (international normalised ratio; INR). Patients with venom recurrence/persistence were compared to those with no detectable recurrence of venom. There were 55 patients with confirmed Russell''s viper envenoming and coagulopathy with low fibrinogen concentrations: 31 with venom recurrence/persistence, and 24 with no venom detected post-antivenom. Fibrinogen concentrations increased and INR decreased after antivenom in both the recurrence and non-recurrence patients. Clinical features, laboratory parameters, antivenom dose and length of hospital were similar for both groups. Pre-antivenom venom concentrations were higher in patients with venom recurrence/persistence with a median venom concentration of 385 ng/mL (16–1521 ng/mL) compared to 128 ng/mL (14–1492 ng/mL; p = 0.008).

Conclusion

Recurrence of Russell''s viper venom was not associated with a recurrence of coagulopathy and length of hospital stay. Further work is required to determine if the detection of venom recurrence is due to the venom specific enzyme immunoassay detecting both venom-antivenom complexes as well as free venom.     

Platelet stimulation by thrombin and other proteases.

The mechanism of stimulation of platelets by thrombin and other proteases was studied by following kinetics of secretion of Ca2+ or ATP. The progress-time curves of secretion were analyzed for rate and total amount released. The reaction of thrombin was perturbed by addition of hydroxylamine or a competitive inhibitor and by variation of pH and it was compared with the reactions of other proteases. Trypsin and papain, with specificities for arginyl residues, induced secretion with a time course that was nearly identical with that induced by thrombin when saturating levels of enzyme were used. At low levels of enzyme, trypsin and papain gave extended lags in the progress-time curves. Higher concentrations of trypsin and papain were required for saturation of the measured parameters. Human plasmin (lysly specificity) and bovine chymotrypsin (aromatic amino acid specificity) failed to induce platelet secretion. Active site inhibited thrombin was also ineffective. Both yield and kinetics depended on pH, with the pH profile for each enzyme similar to its profile for hydrolysis of synthetic substrates. Studies at low pH also showed that the early part of the reaction undergoes a change in rate-determining step from enzyme dependent at low enzyme to enzyme indepdenent at high enzyme. Hydroxylamine, a nucleophile that would be expected to accelerate hydrolytic reactions, actually decreased both the rate of initial reactions and yield. A competitive inhibitor of thrombin also decreased both rate and yield; a calculated inhibition constant was in agreement with the value for a synthetic substrate, suggesting that the interaction of thrombin with platelets is analogous to reaction with substrates. A modification of our previous model is proposed in order to accommodate the results described here and to reaoncile the apparent contradictions that enzyme was found not to turn over in the reaction (Detwiler, T. C., and Feinman, R. D. (1973), Biochemistry 12, 282), that catalytic activity is required (Davey, M. G., and Luscher, E. F. (1967), Nature (London) 216, 875; this paper), and that the reaction is characterized by an apparent equilibrium binding (Tollefsen, D. M., Feagler J. R., and Majerus, P. W. (1974), J. Biol. Chem. 249, 2646). The essential feature is a reversible catalytic step with no dissociation of enzyme from product. This is followed by irreversible, thrombin-independent platelet processes leading to secretion, with yield dependent on the equilibrium concentration of the thrombin product. The model thus has aspects of catalysis, stoichiometry, and an agonist-receptor equilibrium.