作用于血液系统的蛇毒蛋白酶的研究进展

蛇毒是蛇毒腺分泌的天然毒液,含有多种蛋白质、多肽、酶类和其它小分子物质,它们参与蛇的捕食消化和防御功能。蛇毒中含有许多种影响血液系统功能的蛋白质或酶,因其显著的生物功能和药用活性,受到广泛关注。本文就对蛇毒中影响血液系统的丝氨酸蛋白酶、金属蛋白酶、磷脂酶A2、L-氨基酸氧化酶和5'-核苷酸酶等蛋白酶的最新研究进展进行了综述。     

Structure and function of snake venom cysteine-rich secretory proteins.

Cysteine-rich secretory proteins (CRISPs) are primarily found in the epididymis of mammals and are expressed in diverse organisms. However, the functions of most CRISPs remain unknown. Recent studies reveal that CRISPs are widely distributed in snake venoms and that they inhibit smooth muscle contraction and cyclic nucleotide-gated ion channels. In this review, we discuss recent findings on several snake venom-derived CRISPs.     

Differential action of Indian BIG FOUR snake venom toxins on blood coagulation

Abstract

Snake venom toxins affect hemostasis by modulating blood coagulation factors resulting in pro/anti-coagulant status of blood. Most of the reported effects are in vitro which do not reflect in-vivo coagulation status. The specific interference of venom toxins on coagulation factor(s) in vivo can be used as a marker to identify the snake species responsible for envenomation and administration of species-specific anti-venom thereafter. The current review attempts to highlight specific alterations induced by BIG FOUR venomous snakes of India towards blood coagulation factors. Future insights in this regard will be valuable in identifying the snake species responsible for bite which in most cases is unknown.     

Structure and function of snake venom proteins affecting platelet plug formation

Many snake venom proteins have been isolated that affect platelet plug formation by interacting either with platelet integrins, membrane glycoprotein Ib (GPIb), or plasma von Willebrand factor (VWF). Among them, disintegrins purified from various snake venoms are strong inhibitors of platelet aggregation. Botrocetin and bitiscetin derived from Bothrops jararaca and Bitis arietans venom, respectively, induce VWF-dependent platelet agglutination in vitro. Several GPIb-binding proteins have also been isolated from snake venoms. In this review, we focus on the structure and function of those snake venom proteins that influence platelet plug formation. These proteins are potentially useful as reagents for the sub-diagnosis of platelet disorder or von Willebrand disease, as well as for clinical and basic research of thrombosis and hemostasis.     

Red blood cell aggregation by Trimeresurus mucrosquamatus snake venom.

T. mucrosquamatus venom induced aggregation of rabbit and human RBC's at a concentration higher than 25 μg/ml. This aggregation was inhibited by EDTA and could be reversed by Ca²⁺. α-Methylgalactoside, but not α-methyl-glucoside, competitively inhibited the venom-induced RBC aggregation. Trypsin and neuraminidase pretreatment of RBC did not potentiate this aggregating effect. It was concluded that venom-induced aggregation was due to a lectin-like binding to saccharide (galactosyl)-receptors of the RBC membrane. At a sublethal i.v. dose (1 mg/kg), the venom did not cause significant changes of rabbit RBC counts.     

Characterization of snake venom components acting on blood coagulation and platelet function.

Snake venoms can affect blood coagulation and platelet function in various ways. The physicochemical properties and the mechanisms of actions of the snake venom components affecting blood coagulation and platelet function are discussed.     

Thrombin-like snake venom proteinases

Proteinases affecting one or several physiological thrombin substrates are current components of Crotalidae and Viperidae venoms. Enzymes causing in vitro coagulation of fibrinogen without affecting other thrombin-susceptible blood constituents as well as enzymes affecting platelets, F. V, VIII and XIII with only minor action on fibrinogen have been isolated. Fibrinogen affecting proteinases may catalyze the release of either fibrinopeptide (Fp) A (e.g. ancrod, batroxobin) or Fp B (Agk. contortrix proteinase) or of both Fp A and B (B. gabonica proteinase). Some of these enzymes are inhibited by AT III-heparin complex (e.g. Agk. contortrix proteinase) some are not inhibited by either AT III-heparin or hirudin (e.g. batroxobin). The application of Fp A releasing venom proteinases into animals causes transformation of fibrinogen into fibrin I monomer which is rapidly degraded by fibrinolysis and thereby leads to a state of afibrinogenaemia. The administered enzyme is gradually bound to serum proteinase-inhibitors and inactivated. A species dependent interaction between venom enzyme, fibrinogen and serum proteinase inhibitors creates specific differences in dose response relationship. Thus, batroxobin isolated from B. moojeni (HOGE) proved to be a superior defibrinogenating agent in man, as compaired to the closely related enzyme isolated from B. atrox (L.). LD₅₀ of B. atrox venom in previously batroxobin defibrinogenated mice is not significantly different as compaired to normal animals, indicating an only minor role of batroxobin in Bothrops venom poisoning.     

Importance of snake venom metalloproteinases in cell biology: effects on platelets, inflammatory and endothelial cells

Snake venom metalloproteinases (SVMPs) are widely distributed in snake venoms and play important roles in hemostatic disorders and local tissue damage that follows snakebite. The impact of SVMPs on hemostasis has been extensively studied showing diverse effects both on soluble factors and cellular components. The action of SVMPs involves catalytic and anti-adhesive properties, as well as direct cellular activation and/or the release of endogenous bioactive components. The purpose of this review is to overview the action of SVMPs on the inhibition of platelet functions; angiogenesis, particularly inducing apoptosis of endothelial cells; and regarding the pro-inflammatory reaction that follows snakebite. We discuss the structural features of the molecules that may be involved in such activities. The versatility and availability of SVMPs make them important tools for cell biology research into the mechanisms of action of endogenous metalloproteinases, for insights into cellular-matrix interactions and for clinical investigations into the treatment of snakebites.     

Effects of divalent cations on snake venom cardiotoxin-induced hemolysis and H-deoxyglucose-6-phosphate release from human red blood cells

, and . Effects of divalent cations on snake venom cardiotoxin-induced hemolysis and ³H-deoxyglucose-6-phosphate release from human red blood cells. Toxicon 27, 1297–1305, 1989.—At a low concentration of Naja naja kaouthia cardiotoxin (3 μM) Ca²⁺, Sr²⁺ and Ba²⁺ (2 mM), had little to no effect on ³H-deoxyglucose-6-phosphate (³H-dGlu-6-p) or hemoglobin release. At higher concentrations of N. n. kaouthia cardiotoxin (≥ 10 μM), Ca²⁺ (2 mM), but not Sr²⁺ or Ba²⁺, significantly enhanced ³H-dGlu-6-p and hemoglobin release. Mn²⁺ (2 mM) almost completely inhibited ³H-dGlu-6-p release and hemolysis at both the 3 μM and 10 μM concentrations of cardiotoxin. At a fixed concentration of N. n. kaouthia cardiotoxin (3 μM), Ca²⁺ at low concentrations (0.5 mM) enhanced ³H-dGlu-6-p and hemoglobin release, but at higher concentrations caused a dose-dependent inhibition of cardiotoxin action. The cardiotoxin from N. n. kaouthia venom (3 μM) induced ³H-dGlu-6-p release and hemolysis release with similar time courses and to similar extents. ³H-dGlu-6-p release induced by cardiotoxin was greatly enhanced as the pH of the medium was increased from 7.0 to 8.5. Similarities between ³H-dGlu-6-p and hemoglobin release do not support opening of pores in the plasmalemma of all red blood cells as the mode of action of cardiotoxins, but suggests that complete lysis of a subpopulation of cells occurs. Cardiotoxins have two components of lysis, only one of which is Ca²⁺-dependent. The Ca²⁺-dependent lysis is only evident at higher cardiotoxin concentrations and is likely due to trace phospholipase A₂ contamination in the toxin fraction. Mn²⁺ is an effective antagonist of cardiotoxin action.     

Predictors of Bothrops jararaca venom allergy in snake handlers and snake venom handlers.

Since allergic sensitization to snake venom has been reported, anaphylactic reactions to snake venom might be an underestimated factor contributing to fatal snakebites, independently from the toxicity of the venom itself. However, little information is available on the determinants of such reaction. Hence, we studied a group of workers exposed to Bothrops jararaca venom (BJV), in order to clarify the factors related with snake venom allergy. The aim of this work was to investigate the prevalence and predictors of venom allergy among workers exposed to BJV and to confirm the involvement of IgE-mediated mechanisms in this condition. Workers exposed to BJV were assessed for venom allergy using questionnaires and immunological tests. The presence of BJV sensitization was determined through quantification of specific IgE. Allergens were studied using the Western blots and inhibition assays. Of the 67 workers evaluated, 7 (10.4%) presented specific IgE antibodies to BJV. Of those, 6 presented typical symptoms of an IgE-mediated allergic reaction when exposed to BJV. Venom sensitization was associated with length of employment (P=0.042), high levels of total IgE (P=0.034), atopy (P=0.051), and specific tasks, primarily the handling of dried venom (P=0.014). Our observations suggest that exposure to BJV can result in allergic sensitization in snake handlers through IgE-mediated mechanisms. The prevalence rate of this condition appears to be high among these workers, and the handling of dried venom, total IgE level above 100 kU/L, length of employment, and probably history of atopy were predictors of its occurrence.     

Effect of Cerastes vipera snake venom on blood and bone marrow cells

A. H. Mohamed, A. M. Saleh, S. Ahmed and M. El-Maghraby. Effect of Cerastes vipera snake venom on blood and bone marrow cells. Toxicon15, 35–40, 1977.—In vivo experimentsLethal doses of Cerastes vipera venom injected intraperitoneally in rabbits produced a significant initial rise of the erythrocyte count 15 min after venom injection, followed by a progressive drop. Total leucocyte and platelet counts gradually dropped. The bone marrow picture showed erythroid hyperplasia 3–4 hr after venom injection. Sublethal doses produced a small insignificant initial rise of red cell count followed by a progressive drop during the first hr. In the second hr the counts rose and gradually reached the pre-injection levels after 24 hr. An initial leucopenia and thrombocytopenia was followed by fluctuations in both counts with significant leucocytosis and increased percentage of neutrophil after 24 hr. The platelet counts reached the pre-injection values in 24 hr. The bone marrow picture revealed erythroid hyperplasia 5–6 hr after venom injection. Reticulocyte counts were above normal levels.In vitro studiesIncubation of 1 ml blood with 0·5 mg venom for 1 hr resulted in marked haemolysis as evidenced by reddish coloration of plasma, decreased rouleaux formation, microspherocytosis, disruption of leucocytes and drop of all cell counts.     

Snake venom proteins affecting platelets and their applications to anti-thrombotic research

Snake venoms are very complex mixtures of biologically active proteins and peptides that may affect hemostasis in many ways, by activating or inhibiting coagulant factors or platelets, or by disrupting endothelium. They have been classified into various families, including serine proteases, metalloproteinases, C-type lectins, disintegrins and phospholipases. The various members of a particular family act selectively on different blood coagulation factors, blood cells or tissues. Venom proteins affect platelet function in particular by binding to and blocking or clustering and activating receptors or by cleaving receptors or von Willebrand factor. They may also activate protease-activated receptors or modulate ADP release or thromboxane A(2) formation. L-amino acid oxidases activate platelets by producing H(2)O(2). Many of these purified components are valuable tools in platelet research, providing new information about receptor function and signaling.     

Effects of morin on snake venom phospholipase A2 (PLA2).

Flavonoids are potent anti-inflammatory compounds isolated from several plant extracts, and have been used experimentally against inflammatory processes. In this work, a PLA2 isolated from the Crotalus durissus cascavella venom and rat paw oedema were used as a model to study the effect of flavonoids on PLA2. We observed that a treatment of PLA2 with morin induces several modifications in the aromatic amino acids, with accompanying changes in its amino acid composition. In addition, results from circular dichroism spectroscopy and UV scanning revealed important structural modifications. Concomitantly, a considerable decrease in the enzymatic and antibacterial activities was observed, even though anti-inflammatory and neurotoxic activities were not affected. These apparent controversial results may be an indication that PLA2 possess a second pharmacological site which does not affect or depend on the enzymatic activity.     

Molecular diversity and accelerated evolution of C-type lectin-like proteins from snake venom.

A number of C-type lectin-like proteins that affect thrombosis and hemostasis by inhibiting or activating specific platelet membrane receptors or blood coagulation factors have been isolated from the venom of various snake species and characterized and more than 80 have been sequenced. Recent data on the primary sequences and 3D structures of C-type lectins and C-type lectin-like proteins from snake venoms have enabled us to analyze their molecular evolution. Statistical analysis of their cDNA sequences shows that C-type lectin-like proteins, with some exceptions, have evolved in an accelerated manner to acquire their diverse functions. Phylogenetic analysis shows that the A and B chains of C-type lectin-like proteins are clearly separated from C-type lectins and that the A and B chains are further divided into a group of platelet receptor-binding proteins and a group of coagulation factor-binding proteins. Elucidation of the tertiary structures of several C-type lectin-like proteins led to the discovery of a unique domain-swapping interaction between heterodimeric subunits, which creates a concave surface for ligand binding.     

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.