Characterization and cDNA cloning of aminopeptidase A from the venom of Gloydius blomhoffi brevicaudus

The aminopeptidase activities of snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis, Bothrops jararaca and Crotalus atrox were investigated. Aminopeptidase A (APA), aminopeptidase B and aminopeptidase N activities were present in all snake venoms. The strongest APA activity was found in venom from G. blomhoffi brevicaudus. The susceptibility to metallopeptidase inhibitors and the pH optimum of the partially purified enzyme from G. blomhoffi brevicaudus venom were similar to those of known APAs from mammals. A G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences in known APAs. Molecular cloning of APA from G. blomhoffi brevicaudus venom predicted that it was a type II integral membrane protein containing 958 amino acid residues with 17 potential N-linked glycosylation sites. It possessed a His-Glu-Xaa-Xaa-His-(Xaa)₁₈-Glu zinc binding motif that allowed the classification of this protein as a member of the M1 family of zinc-metallopeptidases, or gluzincins. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian APA sequences. This is the first study to report the primary structure of APA from a reptile.     

Molecular cloning of albolatin, a novel snake venom metalloprotease from green pit viper (Trimeresurus albolabris), and expression of its disintegrin domain

Disintegrins are snake venom-derived, RGD- or KGD-containing peptides that can inhibit integrin-mediated platelet aggregation and cell–matix interactions. The aim of this study is to analyze the full-length cDNA sequence of a snake venom metalloprotease (SVMP) from green pit viper (Trimeresurus albolabris) venom and characterize functions of its disintegrin domain on human platelets. From the primary cDNA library of venom glands, a partial sequence of a novel SVMP (Albolatin) was obtained. Using the 5′-RACE, the 2040 bp full-length sequence of albolatin mRNA was derived. The deduced amino acid sequence revealed a type P-II SVMP of 484 amino acid residues comprising a signal region, pro-peptide, inactive metalloprotease domain and a disintegrin domain. It showed 85% amino acid identical to Trimeresurus jerdonii jerdonitin and 81% to Gloydius halys agkistin. Sequence alignment revealed that all cysteines were conserved except for an extra cysteine in the protease domain of albolatin. The disintegrin domain of albolatin, which comprised 76 amino acids with a KGDW sequence, was expressed in Pichia pastoris with the yield of 3.3 mg/L of culture medium. The molecular weights were 11 kDa in reduced and 22 kDa in non-reduced states indicating a homodimer. It can inhibit collagen-induced platelet aggregation with IC₅₀ of 976 nM and, therefore, should be investigated for a potential to be a novel therapeutic agent.     

Active fragments of the antihemorrhagic protein HSF from serum of habu (Trimeresurus flavoviridis).

Certain snakes have antihemorrhagic proteins in their sera. Habu serum factor (HSF), an antihemorrhagic protein isolated from the serum of the Japanese habu snake (Trimeresurus flavoviridis) is composed of two cystatin-like domains (D1 and D2) and a His-rich domain, and it inhibits several snake venom metalloproteinases (SVMPs). The activity of HSF can be abolished by trinitrophenylation of Lys residues with 2,4,6-trinitrobenzene sulphonic acid. Upon complex formation of HSF with SVMP, however, the loss of its inhibitory activity by the chemical modification was suppressed, and Lys(15), Lys(41), and Lys(103) residues in HSF were not trinitrophenylated. In order to identify the domain that is critical to the inhibitory activity on SVMPs, native HSF was digested with papain followed by cleavage with cyanogen bromide, yielding a low-molecular mass fragment that was composed of two peptide chains (residues 5-89 and 312-317) linked by a disulfide bond. This fragment inhibited several SVMPs and showed significant antihemorrhagic activity. This indicates that the N-terminal half of D1 is indispensable for the antihemorrhagic activity of HSF. Furthermore, a three-dimensional model of two cystatin-like domains constructed by the homology modeling has indicated that three Lys residues (15, 41, and 103) are exposed to the same surface of HSF molecule.     

Purification, primary structures and evolution of coagulant proteases from Deinagkistrodon actus venom

Deinagkistrodon (formerly Agkistrodon) actus (Taiwan) snake venom was found to contain at least seven closely related coagulant proteases. One of them, named actibin, was purified to homogeneity by means of four chromatographic steps. Actibin acted on fibrinogen to form fibrin clots with extremely high specific activity of 1630 NIH units/mg and preferentially released fibrinopeptide A. Actibin was an acidic glycoprotein (pI 3.4) with molecular weight of 41,000, which was reduced to 28,800 after deglycosylation with N-glycanase. The k_cat/K_m values of actibin for hydrolysis of tosyl-l-arginine methyl ester and benzoyl-l-arginine p-nitroanilide were one-third to a half those for thrombin, reflecting a high potency of actibin in fibrinogen clotting. The amidase activities of actibin and its family proteases were inhibited by 3,4-dichloroisocoumarin, a serine protease inhibitor, indicating that actibin and its family proteases are serine proteases. Four cDNAs, named DaP1 and DaP7-DaP9, encoding D. actus coagulant proteases were cloned. All cDNAs contain an open reading frame of 780 bp coding for 260 amino acid residues, including a signal peptide of 24 amino acid residues. Their amino acid sequences predicted are highly homologous to one another with one to five amino acid substitutions. When four D. actus protease cDNAs were compared with the cDNAs coding for Trimeresurus flavoviridis and T. gramineus venom serine proteases, accelerated evolution was clearly observed. Similarity of the nucleotide sequences of four D. actus protease cDNAs with no synonymous and one to five nonsynonymous substitutions seems not to be in direct conformity with accelerated evolution. This possibly suggests that they have evolved to a similar direction to enhance their clotting activity rather than to produce other physiological activities.     

Purification and characterization of a coagulant enzyme, okinaxobin II, from Trimeresurs okinavensis (himehabu snake) venom which releases fibrinopeptides A and B

A coagulant enzyme, okinaxobin I, which was purified from Trimeresurus okinavensis (himehabu snake) venom, releases specifically fibrinopeptide B from fibrinogen to form fibrin clots. In the present study, its isozyme denoted as okinaxobin II has been purified to homogeneity from the same venom by chromatographies on Sephadex G-100, CM-Toyopearl 650M, and FPLC Mono-Q columns. Differently from okinaxobin I, okinaxobin II specifically cleaved fibrinopeptides A and B from fibrinogen similarly as found for -thrombin. The enzyme acted on fibrinogen with specific activity of 42 NIH units/mg at optimum pH of 8.0. Okinaxobin II was a monomeric glycoprotein with a mol. wt of 37,500 on SDS-PAGE, which was reduced to 29,500 after treatment with N-glycanase. Okinaxobin II was much more basic (pI=8.1) than okinaxobin I (pI=5.4). The N-terminal sequence was highly similar to those of okinazobin I and some other snake venom coagulant enzymes such as flavoxobin (Trimeresurus flavoviridis), batroxobin (Bothrops atrox and Bothrops moojeni), and catroxobin (Crotalus atrox). Okinaxobin II hydrolyzed tosyl-L-arginine methly ester and benzoyl-L-arginine p-nitroanilide. The esterase activity was strongly inhibited by diisopropylfluorophosphate and to a lesser extent by tosyl-L-lysine chloromethly ketone, indicating that the enzyme is a serine protease like -thrombin. In terms of amino acid composition, okinaxobin II was similar to okinaxobin I and dissimilar to -thrombin.     

Molecular cloning of serine proteases from elapid snake venoms

Serine proteases are widely distributed in viperid snake venoms, but rare in elapid snake venoms. Previously, we have identified a fibrinogenolytic enzyme termed OhS1 from the venom of Ophiophagus hannah. The results indicated that OhS1 might be a serine protease, but there was no structural evidence previously. In the present study, the primary structure of OhS1 was determined by protein sequencing, in combination with RT-PCR and 5′-RACE methods. OhS1 precursor is composed of an 18-amino acid signal peptide, a 6-amino acid putative activation peptide and 236-amino acid mature protein. OhS1 homologues from Naja atra and Bungarus multicinctus were also cloned and reported. These elapid venom serine proteases exhibited 60% sequence identity with serine proteases from the snake venoms of the Viperidae and Colubridae family. Phylogenetic analysis indicated that snake venom serine protease might have a common ancestor.     

Primary structure of a thrombin-like serine protease, kangshuanmei, from the venom of Agkistrodon halys brevicaudus stejneger.

The complete amino acid sequence of the thrombin-like serine protease, named kangshuanmei, isolated from the venom of a Chinese snake Agkistrodon halys brevicaudus stejneger, was determined by Edman degradation. The serine protease was composed of 236 amino acid residues and conserved the catalytic triad as His43, Asp88 and Ser182. The protease had four sites of asparagine-linked glycosylation at 81, 99, 148 and 229, and contained fucose, N-acetylglucosamin, galactose, mannose and N-acetylneuraminic acid. The amino acid sequence exhibited considerable similarities with other thrombin-like proteases isolated from the snake venoms of the Viperidae family. However, the enzymatic characteristics of kangshuanmei distinct from that of thrombin and the other protease from the venom of Viperidae family may be derived from the structural difference of the sequence in the functional regions, especially corresponding to thrombin exosite 1, 2 and hydrophobic pocket.     

Bhalternin: Functional and structural characterization of a new thrombin-like enzyme from Bothrops alternatus snake venom

A serine protease from Bothrops alternatus snake venom was isolated using DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose column chromatography. The purified enzyme, named Bhalternin, ran as a single protein band on analytical polyacrylamide gel electrophoresis (SDS-PAGE) and showed molecular weights of 31,500 and 27,000 under reducing and non-reducing conditions, respectively. Its complete cDNA was obtained by RT-PCR and the 708 bp codified for a mature protein of 236 amino acid residues. The multiple alignment of its deduced amino acid sequence showed a structural similarly with other serine proteases from snake venoms. Bhalternin was proteolytically active against bovine fibrinogen and albumin as substrates. When Bhalternin and bovine fibrinogen were incubated at 37 °C, at a ratio of 1:100 (w/w), the enzyme cleaved preferentially the Aα-chain, apparently not degrading the Bβ and γ-chains. Stability tests showed that the intervals of optimum temperature and pH for the fibrinogenolytic activity were 30-40 °C and 7.0-8.0, respectively. Also, the inhibitory effects of benzamidine on the fibrinogenolytic activity of Bhalternin indicate that it is a serine protease. This enzyme caused morphological alterations in heart, liver, lung and muscle of mice and it was found to cause blood clotting in vitro and defibrinogenation when intraperitoneally administered to mice, suggesting it to be a thrombin-like enzyme. Therefore, Bhaltenin may be of interest as a therapeutic agent in the treatment and prevention of thrombotic disorders.     

Expression and characterization of a recombinant fibrinogenolytic serine protease from green pit viper (Trimeresurus albolabris) venom

Viper venom serine proteases (SPs) display several effects on hemostatic system. Molecular cloning showed that Trimeresurus albolabris venom comprised a mixture of five SPs with thrombin-like (2), fibrinogenase (2) and plasminogen-activating (1) activities. Because only few fibrinogenolytic SP sequences were reported, we decided to express albofibrase, a novel fibrinogenase from T. albolabris using Pichia pastoris system. The recombinant active form of enzyme was 30 kDa including 2.2 kDa of glycosylation. Albofibrase showed an fibrinogenase activity. In addition, a plasminogen activating and clotting effect were detectable. Albofibrase prolonged APTT and PT in a time-dependent manner. The effect was neutralized by pre-incubation with equine antivenom to T. albolabris. Therefore, the protein is potentially useful as a new anticoagulant as the antidote is clinically available. Sequence analysis compared with other snake venom fibrinogenases and SPs could not find any unique residues responsible for their various effects. Structure–function relationship should be further studied using mutagenesis in order to explore the mechanisms of venom protease functional diversity.     

Antihemorrhagin in the blood serum of king cobra (Ophiophagus hannah): purification and characterization

King cobra (Ophiophagus hannah) serum was found to possess antihemorrhagic activity against king cobra hemorrhagin. The activity was stronger than that in commercial king cobra antivenom. An antihemorrhagin has been purified by ion exchange chromatography, affinity chromatography and gel filtration with a 22-fold purification and an overall yield of 12% of the total antihemorrhagic activity contained in crude serum. The purified antihemorrhagin was homogeneous in disc-PAGE and SDS–PAGE. Its apparent molecular weight determined by SDS–PAGE was 120 kDa. The antihemorrhagin was also active against other hemorrhagic snake venoms obtained in Thailand and Japan such as Calloselasma rhodostoma, Trimeresurus albolabris, Trimeresurus macrops and Trimeresurus flavoviridis (Japanese Habu). It inhibited the proteolytic activity of king cobra venom. It is an acid- and thermolabile protein and does not form precipitin lines against king cobra venom.     

Immunological studies on Egyptian polyvalent antivenins

Polyvalent antivenin was prepared against the Egyptian snake venoms Naja haje, N. nigricollis, Cerastes cerastes and C. vipera, and tested by neutralization and immunodiffusion tests. The antivenin was of high potency against the Egyptian snake venoms, especially C. cerastes and C. vipera, followed by Echis carinatus, E. coloratus, N. haje, N. nigricollis and Walterinnesia aegyptia. The titre against the African and Indian cobra venoms N. nivea, N. haje (Ethiopian) and N. naja was lower, while poor or no effect was shown against Bitis arietans, B. gabonica and Trimeresurus flavoviridis venoms. The polyvalent serum prepared in horses not previously immunized, was comparable to sera obtained by using horses, which were previously immunized against a single venom. However, the latter sera, still maintained their higher titre against the original sensitizing venom. Comparison of polyvalent and monovalent N. nigricollis antivenins, revealed that while the polyvalent serum was of better effectiveness against the Egyptian viper venoms especially cerastes venoms, more protection was provided by the monovalent serum against the naja venoms.     

Citrate inhibition of snake venom proteases

Thirty snake venoms had a citrate content of 2.3 to 12.9%, dry basis, by an aconitase–isocitric dehydrogenase coupled enzyme assay. This is a venom concentration range of approximately 30 to 150 mM citrate assuming 25% venom solids content. Inhibition of snake venom protease activity by the addition of exogenous citrate was obtained using azure blue hide powder and azocasein as substrates. Protease inhibitions of 7.5% for Crotalus atrox venom to 78% for Bothrops picadoi venom were observed with citrate. Complete inhibition of snake venom protease activity by citrate was not observed. Bothrops asper (Pacifico) venom showed a 41% protease inhibition by citrate with azocasein as the substrate and 46% inhibition of Bothrops asper (Alantico) venom protease with azure blue hide power as a substrate. Trypsin was not inhibited in this system. Citrate may inhibit some venom protease activity by forming a complex with the zinc of zinc-dependent enzymes.     

Cloning of cDNAs encoding C-type lectins from Elapidae snakes Bungarus fasciatus and Bungarus multicinctus.

A number of C-type lectins with various biological activities have been purified and characterized from Viperidae snake venoms. In contrast, only a few reports could be found in literature concerning the C-type lectins in Elapidae snake venoms. Based on the published cDNA sequences of C-type lectins from Viperidae snake venoms, oligonucleotide primers were designed and used to screen the cDNA libraries made from the venom glands of Bungarus fasciatus and Bungarus multicinctus. This allowed the cloning of three full length cDNAs encoding C-type lectins. The encoded proteins, named BFL-1, BFL-2 and BML, exhibit high degrees of sequence identities with Viperidae snake venom saccharide-binding lectins (around 60% with Trimeresurus stejnegeri venom lectin, Crotalus atrox venom lectin and Agkistrodon piscivorus venom lectin). They show much less identities with other venom C-type lectin-like proteins (around 30% with the platelet glycoprotein Ib-binding protein from Agkistrodon blomhoffi venom and the factor IX/X-binding protein from Trimeresurus flavoviridis venom). The cDNAs revealed that the precursors contain potential signal peptides characterized by a hydrophobic core. To our knowledge, these are the first cDNA cloning of group VII C-type lectins (Drickamer K. 1993. Prog. Nucleic Acid Res. Mol. Biol. 45, 207–232) from Elapidae snake venom glands.     

Characterization of a thrombin-like serine protease, Kangshuanmei, isolated from the venom of a Chinese snake, Agkistrodon halys brevicaudus stejneger

An enzyme, referred to as Kangshuanmei, was isolated from the venom of the Chinese snake Agkistrodon halys brevicaudus stejneger by gel filtration chromatography followed by affinity chromatography. Kangshuanmei is composed of a single polypeptide chain with a molecular weight of approximately 34,000, estimated by SDS-PAGE. The enzyme hydrolyzed both benzoyl-arginine ethyl ester and H-D-Phe-Pip-Arg-p-nitroanilide, specific substrates for thrombin. The protease activity of Kangshuanmei was inhibited by 4-(2-aminoethyl)-benzensulfonyl fluoride, but was not affected by EDTA. The enzyme acted on human fibrinogen to form a fibrin clot and released three fragments. These fragments were shown to be fibrinopeptide A, fibrinopeptide B, and the Bbeta1-42 peptide of fibrinogen, respectively. These results indicate that Kangshuanmei is a thrombin-like serine protease with coagulant activity. However, the enzyme did not induce activation of blood coagulation factor XIII, unlike thrombin. Moreover, antithrombin-III, the specific thrombin inhibitor in plasma, had no inhibitory effect on the thrombin-like amidolytic activity of Kangshuanmei. The N-terminal amino acid sequence of the enzyme up to 50 residues was determined by a peptide sequencer. The N-terminal sequence of Kangshuanmei was highly homologous to most thrombin-like serine proteases from the venom of the snakes of the crotalidae family.     

Immediate active movement following Bitis arietans or Naja mossambica venom injection diminishes or prevents necrosis in mice

Tourniquet use, which localises necrotising snake venom to the bite site, is suspected to exacerbate local necrosis. In view of this, the effect of immediate active movement on the outcome of necrotising snake venom injection was investigated in mice. A minimal macroscopic necrotic dose of Bitis arietans venom (2.5 μg) was administered subcutaneously into the dorsum of the right hind feet of 40 mice of which 20 were previously anaesthetised. The 20 non-anaesthetised mice swam for 10 min. All mice were euthanased on day 4. A further 40 mice underwent an identical experiment using 20 μg Naja mossambica venom and were euthanased on day 6. The areas of resulting macroscopic necrosis were measured in the B. arietans venom injected mice, while the largest diameter of macroscopic necrosis was determined in the N. mossambica venom injected mice. Immediate active movement (swimming) significantly prevented or reduced the area of necrosis in comparison to non-ambulant mice.     

The efficacy of two antivenoms against the in vitro myotoxic effects of black snake (Pseudechis) venoms in the chick biventer cervicis nerve-muscle preparation

Neurotoxicity is rarely seen following human systemic envenoming by Australasian black snakes (genus Pseudechis) with myotoxicity being the most prominent feature following bites by some species. This study investigated the in vitro myotoxicity of venoms from seven species of Australasian Pseudechis and determined the efficacy of CSL black and tiger snake antivenoms.

All Pseudechis venoms (10 μg/ml) significantly inhibited direct twitches of the chick biventer cervicis nerve-muscle preparation (p<0.05, one-way ANOVA). Prior addition of black snake antivenom (5 U/ml) prevented the inhibitory effects of all Pseudechis venoms (p<0.05, one-way ANOVA), except Pseudechis butleri. Prior addition of tiger snake antivenom (5 U/ml) prevented the venom-induced reduction in direct twitches to Notechis scutatus venom and all Pseudechis venoms (p<0.05, one-way ANOVA), with the exception of Pseudechis australis and Pseudechis colletti venoms. Black or tiger snake antivenom (5 U/ml) added 1 h after the addition of venom inhibited further reduction of direct twitches by N. scutatus and most Pseudechis venoms, but did not significantly restore twitch height. PLA₂ activity was found in all venoms with the following rank order: Pseudechis porphyriacus>P. australis>Pseudechis papuanus>P. butleri>Pseudechis guttatus≥Pseudechis pailsii>P. colletti>N. scutatus.

The results of the present study suggest that Australasian Pseudechis venoms possess variable myotoxic activity. The ability of black or tiger snake antivenom to prevent or inhibit further venom-induced effects varied across the genus.     

Insights into the loss of muscle mass following B. jararacussu venom in mice

Bothrops jararacussu snake venom produces myonecrosis and nerve degeneration. In this work, we investigated whether nerve lesions or impaired muscle regeneration contributed to the permanent loss of muscle mass, a long-term sequela of envenoming. The right soleus muscle of adult male mice was injected with B. jararacussu venom (80 μg) while the left muscle received only saline (control). The mice were killed after 2 and 3 months and the muscles were removed and processed for examination by transmission electron microscopy and light microscopy. The nerve fibers, Schwann cells and neuromuscular junctions had regenerated in venom-treated muscle. The total number of muscle fibers was significantly lower (p<0.05) than in the control (617±48 versus 1235±97, respectively; mean±SEM, n=10). These results show that the loss of muscle mass was most likely related to a decrease in the ability of the muscle to regenerate rather than to nerve lesions.     

Properties and cDNA cloning of antihemorrhagic factors in sera of Chinese and Japanese mamushi (Gloydius blomhoffi).

An antihemorrhagic protein has been isolated from the serum of Chinese mamushi (Gloydius blomhoffi brevicaudus) by using a combination of ethanol precipitation and a reverse-phase high-performance liquid chromatography (HPLC) on a C8 column. This protein-designated Chinese mamushi serum factor (cMSF)-suppressed mamushi venom-induced hemorrhage in a dose-dependent manner. It had no effect on trypsin, chymotrypsin, thermolysin, and papain but inhibited the proteinase activities of several snake venom metalloproteinases (SVMPs) including hemorrhagic enzymes isolated from the venoms of mamushi and habu (Trimeresurus flavoviridis). A similar protein (Japanese MSF, jMSF) with antihemorrhagic activity has also been purified from the sera of Japanese mamushi (G. blomhoffi). The N-terminal 70 and 51 residues of the intact cMSF and jMSF were directly analyzed; a similarity between the sequences of two MSFs to that of antihemorrhagic protein (HSF) from habu serum was noticed. To obtain the complete amino acid sequences of MSFs, cDNAs encoding these proteins were cloned from the liver mRNA of Chinese and Japanese vipers based on their N-terminal amino acid sequences. The mature forms of both MSFs consisted of 305 amino acids with a 19-residue signal sequence, and a unique 17-residue deletion was detected in their His-rich domains.     

Pharmacokinetics of thrombin-like enzyme from venom of Agkistrodon halys ussuriensis Emelianov determined by ELISA in the rat.

A thrombin-like enzyme (TLE) was separated and purified from the venom of a northeast Chinese snake Agkistrodon halys ussuriensis Emelianov. Experiments were performed in rats to determine the pharmacokinetic parameters following an intravenous (i.v.) or a subcutaneous (s.c.) injection of the thrombin-like enzyme. The plasma levels of TLE were estimated by enzyme-linked immunosorbent assay. The method exhibited high reproducibility and accuracy in correlating optical densities with TLE concentrations (0.2–30 ng ml⁻¹, r=0.99). The plasma concentration-time course after i.v. administration of 50 μg kg⁻¹ TLE was well fitted by a two-compartment open model. The half-life of the -phase was 18.0±3.2 min, and that of the β-phase 3.9±0.7 h. The apparent volume of distribution was 1.8±0.5 l kg⁻¹, and clearance was 5.4±0.5 ml min⁻¹ kg⁻¹. When the TLE was injected s.c. at a dose of 0.75 mg kg⁻¹, the changes in plasma concentration were best described by a two-compartment model with a first-order absorption. The maximal plasma level of 51±2.7 ng ml⁻¹ was reached at 5.2±0.5 h. The absorption rate constant was 0.3±0.03 h⁻¹. The area under the plasma concentration-time curve (AUC) was 2.8±0.8 μg h⁻¹ ml⁻¹.     

Snake fetuin: Isolation and structural analysis of new fetuin family proteins from the sera of venomous snakes

Novel proteins were isolated from the sera of Chinese Mamushi (Gloydius blomhoffi brevicaudus) and Habu (Trimeresurus flavoviridis). The primary structures of these proteins were determined by protein sequencing, and the nucleotide sequences were established by cDNA cloning from the liver mRNAs. They belonged to the fetuin family having a double-headed cystatin-like domain and a His-rich domain, akin to HSF, an antihemorrhagic factor isolated from Habu serum. They showed no antihemorrhagic activity and were designated HSF-like proteins (HLPs). Mamushi serum contained two different HLPs termed HLP-A and HLP-B. Both HLP-A and Habu HLP had a unique 17-residue deletion in their His-rich domains. HLP-B comprised two glycosylated polypeptide chains and inhibited the precipitation of calcium phosphate as potently as does bovine fetuin. HLP-B was hence identified as a snake fetuin. The phylogenetic analysis of the fetuin family of proteins showed that antihemorrhagins and HLPs have evolved from this snake fetuin.