Alfimeprase: pharmacology of a novel fibrinolytic metalloproteinase for thrombolysis

Alfimeprase is a recombinantly produced, truncated form of fibrolase, a known directly fibrinolytic zinc metalloproteinase that was first isolated from the venom of the southern copperhead snake (Agkistrodon contortrix contortrix). Both fibrolase and alfimeprase have been shown to have direct proteolytic activity against the fibrinogen Aalpha chain. In vivo pharmacology studies have shown that thrombolysis with alfimeprase is up to 6 times more rapid than with plasminogen activators. Alfimeprase can be bound and neutralized by serum alpha(2)-macroglobulin, a prevalent mammalian protease inhibitor which is capable of forming a macromolecular complex with alfimeprase. As a result, systemic bleeding complications have been greatly reduced due to the inhibitory effects of alpha(2)-macroglobulin. This article reviews the biochemical in vitro and in vivo characteristics of this novel acting thrombolytic.     

Detecting APC-resistant factor V: a functional method without plasma dilution.

A method for detecting activated protein C (APC)-resistant factor V, especially factor V Leiden, is described, which uses reagents containing two unfractionated snake venoms. The procedure can be used for testing plasma samples from patients receiving oral anticoagulant therapy, heparin therapy and patients with lupus anticoagulant, and does not require the use of factor-V-deficient plasma. The sample plasma is first incubated with dilute venom from Agkistrodon contortrix contortrix (Southern Copperhead) which activates the endogenous protein C and then a dilute Russell's viper venom time test is performed. In individuals with APC-resistant factor V, especially factor V Leiden, a marginal prolongation of dilute Russell's viper venom time was noted [1.14 +/- 0.14 s (n = 16)]. Non-carriers were easily discriminated in each patient group, with a prolongation of 2.69 +/- 0.30 s for normal blood donors (n = 127), 2.61 +/- 0.38 s for patients taking oral anticoagulants (n = 102), 2.41 +/- 0.45 s for patients taking heparin (n = 96), and 2.38 +/- 0.41 s for patients with lupus anticoagulant (n = 22). Patients taking oral anticoagulants with moderate prolongation (between 1.5- and 2.0-fold) may have low levels of functional protein C and this might additionally indicate a subgroup of such patients at higher than normal thrombotic risk.     

Diagnostic uses of snake venom

Snake venom toxins are invaluable for the assay of coagulation factors and for the study of haemostasis generally. Thrombin-like enzymes (SVTLE) are used for fibrinogen and fibrinogen breakdown product assays as well as detecting dysfibrinogenaemias. Since SVTLE are not inhibited by heparin, they can be used for assaying antithrombin III in samples containing heparin. Snake venom prothrombin activators are utilised in prothrombin assays, whilst Russell's viper venom (RVV) can be used to assay clotting factors V, VII, X and lupus anticoagulants (LA). Activators from the taipan, Australian brown snake and saw-scaled viper have also been used to assay LA. Protein C (PC) and activated PC (APC) resistance can be measured by means of RVV, Protac (from Southern copperhead snake venom) and STA-Staclot (from Crotalus viridis helleri) whilst von Willebrand factor can be studied with Botrocetin (Bothrops jararaca). Finally, snake venom C-type lectins and metalloproteinase disintegrins are being used to study platelet glycoprotein receptors and show great potential for use in the routine coagulation laboratory.     

Effect of snake venom of Agkistrodon halys on atherosclerosis and blood characteristics in Japanese quail

Extracts of snake venom have been widely used for the treatment of vascular thrombotic diseases, yet the therapeutic mechanism is not clear. The effect of snake venom fractions on atherosclerosis in Japanese quail was studied. The venom of Agkistrodon halys was fractionated by DEAE-cellulose chromatography and the pooled protein fractions that resulted were injected intravenously into the quail with aortic atherosclerosis induced by dietary cholesterol. After 7 weeks of injections on every other day, the quail were killed, blood clotting times and serum cholesterol levels were determined, and aortic atherosclerosis and fatty liver were scored. The results showed that while no regression of atherosclerosis was observed, the lowering of serum cholesterol, prolonged blood clotting time and reduced fatty liver were significantly affected by the injection of one of the pooled protein fractions. This venom fraction contained two major protein components, one of which had arginine esterase activity. From this study we conclude that snake venom has little effect on the regression of atherosclerosis, but it prolongs blood clotting and lowers serum cholesterol.     

The reduced sensitivity of the ProC Global test in protein S deficient subjects reflects a reduction in the associated thrombotic risk.

To investigate simultaneously a defect affecting the protein C/protein S (PC/PS) anticoagulant pathway is possible thanks to a methodological approach (ProC(R) Global; Dade Behring) based on the activation of endogenous plasma PC by a snake venom extract. Factor V (FV) Leiden, the most frequent cause of hereditary thrombosis, is well detected by the test with sensitivity of 100% irrespective of the presence/absence of thrombosis in the subjects investigated. The test is also suited to detect PC or PS defect, but in this case the in vitro impairment of the PC/PS pathway is less pronounced particularly for PS defects (sensitivity for PC and PS defect, 85-100 and 30-90%, respectively). In this study, we hypothesized that the lower sensitivity described for PS defect, compared with those of PC and FV Leiden defects, could also be related to the clinical condition of the subject investigated (symptomatic/asymptomatic) rather than solely to the PS plasma activity/level. Therefore, we analyzed 126 subjects with single congenital defects in the PC/PS pathway: 46 subjects with PS deficiency (26 thrombotic cases and 20 asymptomatic relatives), 40 subjects with PC deficiency (25 thrombotic cases and 15 asymptomatic relatives), and 40 heterozygous FV Leiden subjects (25 thrombotic cases and 15 asymptomatic relatives). By a cut-off of normalized Agkistrodon contortix snake venom ratio of 0.84, the sensitivity in the whole group of cases (sensitivity a) was 76.1, 95.0 and 100%, respectively, for PS, PC and FV Leiden defects. The test failed to detect 11 (23.9%) among the 46 PS-deficient subjects, and all these cases except two belonged to the asymptomatic subgroup (9/20; 45%). Excluding the 20 asymptomatic relatives, the new sensitivity (sensitivity b) for the PS defect was 92.3%. The comparison of the sensitivity in the symptomatic PS cases and in the asymptomatic ones was significantly different (P = 0.010). Among the 40 PC-deficient subjects, only two (5.0%) were not detected by the test and they belonged indifferently to the two subgroups. Finally, none of the 40 FV Leiden heterozygotes were misdiagnosed by the test. These results suggest that in symptomatic PS-deficient cases the test could reflect a post-thrombotic effect and/or reveal potential unidentified prothrombotic influences assessing a prothrombotic risk condition.     

Snake venom activators of factor X: an overview

Activators of blood coagulation factor X have been described in the venom of many snake species belonging to the genus Viperidae and Crotalidae as well as from a few Elapid species. Based on the structural and functional properties of purified activating principles, factor X activators are either metalloproteases or serine proteases. The best known activator is RVV-X from Russell's viper (Daboia russelli), a metalloprotease consisting of a heavy chain containing the catalytic domain and two light chains which share homology with C-type lectins and which are thought to exert a regulatory function in the Ca(2+)-dependent activation of factor X. This activator is also one of the best examples of the use of exogenous activators in coagulation research and in addition it is used in many diagnostic research kits. In this paper, an overview is given of the structural and functional properties of snake venom factor X activators thus far described in the literature.     

Procoagulant proteins from snake venoms

Several procoagulant proteins from snake venoms have been isolated and characterized. They are either serine proteinases or metalloproteinases, which activate specific zymogens of coagulation factors and initiate the coagulation cascade. These procoagulant proteins are useful in treating various thrombotic and hemostatic conditions and contribute to our understanding of molecular details in the activation of specific coagulation factors. Recent studies have shown that the prothrombin activators with serine proteinase activity are structurally and functionally similar to mammalian coagulation factors. Their structural studies should provide us insight into prothrombinase complex formation. Here, we present an overview of snake venom procoagulant factors.     

Factor V activation and inactivation by venom proteases

Blood coagulation factor V is a single-chain glycoprotein with M(r) = 330,000 which plays an important role in the procoagulant and anticoagulant pathways. Thrombin activates factor V into factor Va, a two-chain molecule which is composed of a heavy (M(r) = 105,000) and a light chain (M(r) = 71,000/74,000). Factor Va accelerates factor Xa-catalysed prothrombin activation more than 1,000-fold and under physiological conditions the cofactor activity of factor Va in prothrombin activation is down-regulated by activated protein C. Factor V can also be activated by a wide variety of snake venoms (e.g. from Vipera species, Naja naja oxiana, Bothrops atrox) and by proteases present in the bristles of a South American caterpillar (Lonomia achelous). Some venoms, notably of Vipera lebetina turanica and Lonomia achelous, contain proteases that are able to inactivate factor V or factor Va. Venom factor V activators are excellent tools in studying the structure-function relationship of factor V(a) and they are also used in diagnostic tests for quantification of plasma factor V levels and for the screening of defects in the protein C pathway. In this review, the structural and functional properties of animal venom factor V activators and inactivators is described.     

Southern copperhead venom enhances tissue-type plasminogen activator induced fibrinolysis but does not directly lyse human plasma thrombi

In addition to degrading fibrinogen as a source of consumptive coagulopathy, purified fractions of southern copperhead (Agkistrodon contortrix contortrix; A. c. contortrix) venom has been demonstrated to enhance fibrinolysis. The goal of this investigation was to characterize the kinetic fibrinolytic profile of A. c. contortrix venom in the absence and presence of tissue-type plasminogen activator (tPA) to determine if intact venom had tPA independent fibrinolytic properties. Utilizing thrombelastographic methods, the coagulation and fibrinolytic kinetic profiles of human plasma exposed to A. c. contortrix venom (0–6 μg/ml) were determined in the absence or presence of tPA (0–100 IU/ml). Then, plasma was exposed to 0–6 μg/ml of venom without tPA added and coagulation observed for 3 h. Venom significantly prolonged the onset of coagulation, decreased the velocity of thrombus growth but did not significantly decrease clot strength. In the presence of tPA, venom significantly decreased clot strength, shortened the time of onset of fibrinolysis, decreased clot lysis time but did not significantly affect the maximum rate of lysis. Lastly, while venom exposure in the absence of tPA significantly prolonged the onset of coagulation and decreased the velocity of clot growth, venom exposure did not result in detectable fibrinolysis over the 3 h observation period. A. c. contortrix venom enhances tPA mediated fibrinolysis by degrading plasma coagulation kinetics. Intact A. c. contortrix venom does not possess sufficient fibrinolytic activity to cause fibrinolysis in human plasma at the concentration tested.     

Blood cells as targets of snake toxins

Snake venoms are mixtures of enzymes and peptides which exert toxicological effects by targeting their substrates or receptors upon envenomation. Snake venom proteins widely affect vascular system including circulating blood cells, coagulation factors, and vascular wall components. Many of the toxic proteins have multiple targets. For example, some metalloproteinase domain-containing snake venom protein cleaves not only fibrinogen but also receptors on platelets. Also, it is frequent that toxins from different snake venom protein families are capable of binding to a common target on cells. Most of the cytotoxic effects in the venom are usually results of the activities of metalloproteinase, C-type lectin, disintegrin, cysteine-rich protein, as well as phospholipase A(2). There has been a growing interest in studying the structure and function of these snake venom proteins because many of them have high structural homologies to proteins found in human. Therefore, the understanding of how these toxins interact with their targets may contribute to the discovery of novel physiological processes and the development of therapeutic agents for cardiovascular diseases. In this review, we summarize how snake toxins target blood cells with an emphasis on their effects on platelet function.     

Fibrinogen Bbeta14 Arg-->Cys: further evidence for a role in thrombosis.

A single base substitution (C-->T) in exon II of the Bbeta fibrinogen gene resulting in an Arg14-->Cys replacement was identified in a young woman with a history of recurrent thrombotic stroke. The patient's plasma showed prolongation of the thrombin and Reptilase times, and plasma fibrinogen, which was low when determined by chronometric assay (Clauss technique) was normal by clot weight. Dysfibrinogenaemia associated with the same mutation was identified in eight family members including two siblings with a history of venous and arterial thrombosis. Fibrin monomer polymerization with thrombin, Reptilase and Agkistrodon contortrix contortrix venom was defective. Polymerization studies revealed a reduced rate of polymerization compared with normal plasma, which improved on cooling from 37 degrees C to 20 degrees C. Plasma viscosity in the affected individuals was normal. Flow cytometric analysis of platelets from the proband and another affected member showed no increase in surface bound fibrinogen. Euglobulin clot lysis time was normal. The same point mutation has been described previously in individuals with thrombosis. This family adds further to the genotype-phenotype correlation of the dysfibrinogenaemias and provides strong evidence for a genuine association of fibrinogen BbetaArg14Cys with thrombosis. The mechanism underlying a causal relationship with the increased incidence of thrombosis remains obscure but a review of related dysfibrinogens suggests that the addition of a free thiol group rather than the loss of the thrombin cleavage site may be important.     

Modification of the ProC Global assay using dilution of patient plasma in factor V-depleted plasma as a screening assay for factor V Leiden mutation.

The activated protein C (APC) resistant-factor V (factor V Leiden) has emerged as the most common inherited risk factor for thrombosis in the Caucasian population. Beside DNA analysis, the laboratory diagnosis is often based on the detection of a poor anticoagulant response to exogenous APC. The ProC Global assay (Dade Behring, Marburg, Germany) is a global clotting assay, which was primarily developed to evaluate the functionality of the protein C anticoagulant pathway. It is based on the ability of endogenous APC, generated by activation of protein C by an extract from Agkistrodon contortrix contortrix venom, to prolong an activated partial thromboplastin time. It was previously found to be highly sensitive for the factor V Leiden mutation and for protein C deficiency, but only moderately sensitivity for protein S deficiency. Here, we evaluated the performance of a modification of the ProC Global assay using a 1 : 5 pre-dilution of patient plasma in factor V-depleted plasma in the screening of the factor V Leiden mutation-related APC resistance. For that purpose, we investigated selected frozen plasma samples from 341 patients with a history of venous thromboembolism. The sensitivity for the factor V Leiden mutation of the modified assay was found to be 100%, as all the carriers of that mutation (five homozygotes and 77 heterozygotes) had a decreased response to the assay, i.e. a normalized ratio below 0.80. Its specificity was also 100% since none of the other tested patients had a decreased response, i.e. isolated protein C (n = 3) or protein S deficiency (n = 50), or without any abnormality of the protein C pathway (n = 143), even those on oral anticoagulant treatment (n = 76). However, it would be preferable that each laboratory defines both its reference range and its cut-off level. Finally, even if larger-scale multicentre studies are needed before definite recommendations could be made, these results suggest that the ProC Global performed using a 1 : 5 pre-dilution of the patient plasma in factor V-depleted plasma could be validly used as a screening assay of the factor V Leiden mutation-related APC resistance in patients with a history of thrombosis.     

Genome wide association study for plasma levels of natural anticoagulant inhibitors and protein C anticoagulant pathway: the MARTHA project

Deficiencies of antithrombin (AT), protein C (PC) and protein S (PS) or an impaired PC anticoagulant pathway increase the risk of venous thrombosis (VT). By conducting a genome-wide association study (GWAS) on two independent samples of VT patients totalling 951 subjects typed for 472 173 single nucleotide polymorphisms (SNPs), we observed that common SNPs explain 21% and 27% of the genetic variance of plasma AT and PS levels, even though no SNP reached genome-wide significance. For PC, we showed that two PROCR SNPs, rs867186 (Ser219Gly) and rs6060278, additionally explained c. 20% (P = 1·19 × 10(-31)) of the variance of plasma PC levels. We also observed that c. 40% of the remaining genetic variance of PC levels could be due to yet unidentified common SNPs. The PROCR locus was also found to explain c. 8% (P < 10(-10)) of agkistrodon contortrix venom (ACV) (exploring the PC pathway) variability which was under the main control of the F5 and F2 loci that further explained about 40% and 10%, respectively. We presented here the first GWAS for plasma AT and free-PS levels and ACV in Caucasian samples. We identified three independent loci associated with ACV (F2, F5 and PROCR) and replicated two independent effects on plasma PC levels at the PROCR locus.     

Amino acid sequence of trocarin, a prothrombin activator from Tropidechis carinatus venom: its structural similarity to coagulation factor Xa.

Among snake venom procoagulant proteins, group II prothrombin activators are functionally similar to blood coagulation factor Xa. We have purified and partially characterized the enzymatic properties of trocarin, the group II prothrombin activator from the venom of the Australian elapid, Tropidechis carinatus (rough-scaled snake). Prothrombin activation by trocarin is enhanced by Ca2+, phospholipids, and factor Va, similar to that by factor Xa. However, its amidolytic activity on peptide substrate S-2222 is significantly lower. We have determined the complete amino acid sequence of trocarin. It is a 46,515-Dalton glycoprotein highly homologous to factor Xa and shares the same domain architecture. The light chain possesses an N-terminal Gla domain containing 11 gamma-carboxyglutamic acid residues, followed by two epidermal growth factor (EGF)-like domains; the heavy chain is a serine proteinase. Both chains are likely glycosylated: the light chain at Ser 52 and the heavy chain at Asn 45. Unlike other types of venom procoagulants, trocarin is the first true structural homologue of a coagulation factor. It clots snake plasma and thus may be similar, if not identical, to snake blood coagulation factor Xa. Unlike blood factor Xa, it is expressed in high quantities and in a nonhepatic tissue, making snake venom the richest source of factor Xa-like proteins. It induces cyanosis and death in mice at 1 mg/kg body weight. Thus, trocarin acts as a toxin in venom and a similar, if not identical, protein plays a critical role in hemostasis.     

Biochemical characteristics of fibrolase, a fibrinolytic protease from snake venom.

Fibrolase, a fibrinolytic enzyme isolated from Agkistrodon c. contortrix (southern copperhead) venom, solubilizes fibrin primarily by rapid hydrolysis of the alpha and beta chains. Fibrolase is also an A alpha, B beta fibrinogenase. The breakdown products of fibrin and fibrinogen following incubation with fibrolase were different from those observed with plasmin. This enzyme is a metalloprotease that was inhibited by ethylenediaminetetraacetic acid. Fibrolase was inhibited by dithiothreitol, suggesting that disulfide bonds are important for catalytic activity. It was also inhibited by alpha 2-macroglobulin, but not by the soybean or lima bean trypsin inhibitors, diisopropylfluorophosphate, or p-hydroxymercuribenzoate. Unlike thrombolytic agents such as streptokinase, fibrolase does not activate plasminogen as evidenced by the use of plasmin-specific chromogenic substrate S-2251 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Present tests for detection of snake venom: clinical applications

Immunologic tests for detection of snake venom and venom antibodies have important clinical applications. Enzyme-linked immunoassay (ELISA) and radioimmunoassay (RIA) provide adequate specificity and sensitivity. The former is much more widely used because it is inexpensive, relatively easy to perform, and uses stable reagents. Some ELISA systems will detect 0.5 ng of venom; however, a sensitivity of 10 to 100 ng is more usual. Minimum running time is 30 to 45 minutes; with longer times, greater sensitivity can be attained. Wound aspirate, serum, and urine are the most suitable materials for venom detection. ELISA has been used for clinical diagnosis of snakebite, to monitor antivenom dose, to study clinical syndromes associated with envenomation, to detect venom in forensic cases, and to evaluate first aid techniques. The indirect ELISA usually is used for detecting and titrating venom antibody. This is potentially useful in epidemiological studies of snakebite incidence, in evaluating potency and paraspecific activity of antivenoms, and in studying response to venom immunogens. Current ELISA systems involving snake venoms have low specificity, and most cannot reliably differentiate venoms of related snakes. Venom antibody detection assays are less satisfactory than those for venom; nonspecific reactions and cross-reactivity are unacceptably high. Methods for improvement of snake venom immunodiagnosis are discussed.     

Occupational injuries with captive lance-headed vipers (Bothrops moojeni): experience from a snake farm in Brazil

We studied occupational injuries with captive lance-headed vipers (Bothrops moojeni) that occurred in a snake farm in south-eastern Brazil from February 1981 to May 1999. The risk of injury, taking into account 13 cases of snake-associated injuries (12 of them snake bites) was 2.73 per 10,000 person-days of work, and 3.51 per 100,000 venom extractions. Thirteen cases of injury occurred in seven workers, whereas 18 workers were never injured, suggesting that some individuals have a higher risk of injury than others perhaps due to lack of concentration or overconfidence. Eight episodes of occupational injuries occurring in four technicians, including a case of eye injury due to splashed venom during extraction, are reported. Assessment of whether envenoming occurred was facilitated by knowledge of the snake species and size, history of recent venom extraction and snake feeding, and examination of snake venom glands. Hypersensitivity reactions (anaphylaxis and serum sickness) to antivenom are a risk particularly to those workers who were bitten more than once and medicated previously. Antivenom therefore should not be administered to these individuals unless there is clear evidence that envenoming occurred or is likely to have occurred. Hypersensitivity to the venom is also a health concern for workers from snake farms.     

Structure-activity relationships of snake toxins targeting platelet receptors, glycoprotein Ib-IX-V and glycoprotein VI

Initiation of thrombus formation involves the platelet adhesion receptors, glycoprotein (GP)Ib-IX-V and GPVI, that bind von Willebrand factor (vWF) and collagen, respectively. These interactions trigger intracellular signals leading to degranulation, elevation of cytosolic Ca2+, cytoskeletal rearrangements, and "inside-out" activation of the integrin, GPIIb-IIIa (alphaIIbbeta3) that binds von Willebrand Factor (vWF) or fibrinogen and mediates platelet aggregation. GPIbalpha (the vWF-binding subunit of GPIb-IX-V) of the leucine-rich repeat protein family and GPVI of the immunoglobulin superfamily not only have a parallel physiological function, but recent studies of these receptors have revealed structure-activity relationships amongst snake venom toxins which target them. Two families of venom proteins, the C-type lectin-like proteins and metalloproteinase-disintegrins, target GPIbalpha, GPVI and/or the collagen-binding integrin, GPIa-IIa (alpha2beta1). Members of the C-type lectin family interact specifically with GPIbalpha (echicetin, alboaggregin-B, agglucetin), GPVI (convulxin, ophioluxin) or GPIa-IIa (rhodocetin), whereas related proteins accomplish dual receptor occupancy and bind GPVI and GPIbalpha (alboaggregin-A, alboluxin), or GPIbalpha and GPIa-IIa (aggretin). These latter venom proteins mimic physiological ligands, vWF or collagen, which also recognize more than one receptor. Similarly, metalloproteinase-disintegrin family proteins target GPlbalpha (mocarhagin, crotalin), GPVI (alborhagin) or GPIa-IIa (jararhagin), resulting in inhibition or induction of platelet aggregation by proteolytically dependent or independent mechanisms. Anti-thrombotics based on snake venom GPIIb-IIIa inhibitors have been investigated clinically, however analogous proteins recognizing GPIb-IX-V or GPVI are yet to be therapeutically exploited.     

Ca2+-related regulatory function of fibrinogen.

Fibrinogen displays a regulation of considerable physiological significance by lowering the Ca2+ requirement for the conversion of the fibrin-stabilizing factor (Factor XIII) zymogen to the range of concentrations of this ion found in plasma. Fibrinogen modulates both Ca2+-dependent steps in the complex process of zymogen activation, involving the heterologous dissociation of subunits of the thrombin-modified zymogen (Factor XIII') species : formula: (see text) and the unmasking of iodoacetamide titratable sites during generation of transamidating activity : formula: (see text). It is interesting that a thrombin-independent pathway of zymogen activation : formula: (see text), which we found to operate at Ca2+ concentrations above 50 mM, is not affected by the presence of fibrinogen. Regulation by fibrinogen thus appears to be specific for controlling only the physiological pathway of zymogen conversion.