[Effect of Yunnan-cobra venom factor in overcoming acute humoral rejection after allograft cardiac transplantation in presensitized recipients: experiment with rats].

OBJECTIVE: To investigate the effect of Yunnan-cobra venom factor (Y-CVF) in overcoming acute humoral rejection after allograft cardiac transplantation in presensitized recipients. METHODS: Fifteen Lewis rats received the transplantation of full-thickness skin graft of BN rats three times so as to be presensitized. Fifteen pairs of Lewis rat, as recipients of heart, and BN rat, as heart donors, were randomly divided into 2 groups: experimental group (n = 8), and control group (n = 7). The Lewis rats in the experimental group received heart transplantation of the heart of the BN rat 7 approximately 10 days after the third skin transplantation, and were injected with Y-CVF 80 microg/kg 24 hours before the heart transplantation. The Lewis rat in the control group received only the heart transplantation without Y-CVF injection. Blood samples were collected from all rats before pre-sensitization and 7 days after the third skin transplantation so as to determine the titer of anti-BN rat lymphocyte antibody. 0 and 24 hours, and 6 and 8 days after Y-CVF injection blood samples were collected from the Lewis rats to determine the total complement activity with the complement activity before Y-CVF injection defined as 100%. The survival time of the transplanted heart was observed. After the transplanted hearts stopped to beat, they were resected and underwent HE staining and microscopy. Immunohistochemistry was used to examine the deposition of IgG and complement 3 (C3). RESULTS: The titer of anti-BN rat lymphocyte antibody was 0 before the pre-sensitization, and increased to 1:1028 - 1:2056 7 days after the third skin pre-sensitization. The serum total complement activity of the Lewis rats decreased to 0 twenty-four hours after the Y-CVF injection, recovered to 2.01% - 15.41% 6 days after, and returned to the normal level (89.61% - 109.46%) 8 days after. The mean survival time of the transplanted hearts of the control group was 12.71 +/- 13.94 hours (with a range of 1.5 - 15 hours), significantly shorter than that of the experimental group (99.50 +/- 38.72 hours, with a range of 23 - 153 hours, P < 0.01). Pathological examination showed that the acute humoral rejection in the control group was mainly complement-dependent antibody-mediated humoral rejection characterized by intravascular thrombosis, and that in the experimental group was mainly cellular rejection, characterized by extensive infiltration of mononuclear cells. Immunohistochemistry showed that remarkable IgG deposition was seen in the cardiac muscle cells and vascular endothelial cells in both groups; however, C3 deposition in vascular endothelial cells could be seen only in the control group. CONCLUSION: Administration of CVF is effective in overcoming the acute humoral rejection after allograft cardiac transplantation in presensitized recipients.

Characterization and molecular cloning of dabocetin, a potent antiplatelet C-type lectin-like protein from Daboia russellii siamensis venom.

A novel C-type lectin-like protein, dabocetin, was purified from Daboia russellii siamensis venom. On SDS-polyacrylamide gel electrophoresis, it showed a single band with an apparent molecular weight of 28 kDa and two distinct bands with the apparent molecular weights of 15.0 kDa and 14.5 kDa under non-reducing and reducing conditions, respectively. cDNA clones containing the coding sequences for dabocetin alpha and beta subunits were isolated and sequenced. The deduced protein sequences of both subunits were confirmed by N-terminal amino acid sequencing and trypsin-digested peptide mass fingerprinting. Dabocetin did not induce platelet aggregation in platelet-rich plasma. It also had little effect on the platelet aggregation induced by ADP, TMVA or stejnulxin. Whereas, dabocetin inhibited ristocetin-induced platelet agglutination in platelet-rich plasma in a dose-dependent manner with an IC50 value of 0.35 microM. Flow cytometry analysis showed that dabocetin significantly inhibited mAb SZ2 binding to platelet membrane glycoprotein Ib alpha, indicating that platelet membrane glycoprotein Ib is involved in the inhibitory effect of dabocetin on ristocetin-induced platelet agglutination.

cDNA cloning and functional expression of jerdostatin, a novel RTS-disintegrin from Trimeresurus jerdonii and a specific antagonist of the alpha1beta1 integrin.

Jerdostatin represents a novel RTS-containing short disintegrin cloned by reverse transcriptase-PCR from the venom gland mRNA of the Chinese Jerdons pit viper Trimeresurus jerdonii. The jerdostatins precursor cDNA contained a 333-bp open reading frame encoding a signal peptide, a pre-peptide, and a 43-amino acid disintegrin domain, whose amino acid sequence displayed 80% identity with that of the KTS-disintegrins obtustatin and viperistatin. The jerdostatin cDNA structure represents the first complete open reading frame of a short disintegrin and points to the emergence of jerdostatin from a short-coding gene. The different residues between jerdostatin and obtustatin/viperistatin are segregated within the integrin-recognition loop and the C-terminal tail. Native jerdostatin (r-jerdostatin-R21) and a R21K mutant (r-jerdostatin-K21) were produced in Escherichia coli. In each case, two conformers were isolated. One-dimensional (1)H NMR showed that conformers 1 and 2 of r-jerdostatin-R21 represent, respectively, well folded and unfolded proteins. The two conformers of the wild-type and the R21K mutant inhibited the adhesion of alpha(1)-K562 cells to collagen IV with IC(50) values of 180 and 703 nm, respectively. The IC(50) values of conformers 2 of r-jerdostatin-R21 and r-jerdostatin-K21 were, respectively, 5.95 and 12.5 microm. Neither r-jerdostatin-R21 nor r-jerdostatin-K21 showed inhibitory activity toward other integrins, including alpha(IIb)beta(3), alpha(v)beta(3), alpha(2)beta(1), alpha(5)beta(1), alpha(4)beta(1), alpha(6)beta(1), and alpha(9)beta(1) up to a concentration of 24 mum. Although the RTS motif appears to be more potent than KTS inhibiting the alpha(1)beta(1) integrin, r-jerdostatin-R21 is less active than the KTS-disintegrins, strongly suggesting that substitutions outside the integrin-binding motif and/or C-terminal proteolytic processing are responsible for the decreased inhibitory activity.

Molecular characterization of a weak fibrinogen-clotting enzyme from Trimeresurus jerdonii venom.

A fibrinogen-clotting enzyme designed as jerdonobin-II was isolated from the venom of Trimeresurus jerdonii. It differed in molecular weight and N-terminal sequence with the previously isolated jerdonobin, a thrombin-like enzyme from the same venom. The enzyme consists of a single polypeptide chain with molecular weights of 30,000 and 32,000 under non-reducing and reducing conditions, respectively. Jerdonobin-II showed weak fibrinogen clotting activity and its activity unit on fibrinogen was calculated to be less than one unit using human thrombin as standard. The precursor protein sequence of jerodonobin-II was deduced from cloned cDNA sequence. The sequence shows high similarity (identity=89%) to TSV-PA, a specific plasminogen activator from venom of T. stejnegeri. Despite of the sequence similarity, jerdonobin-II was found devoid of plasminogen activating effect. Sequence alignment analysis suggested that the replacement of Lys239 in TSV-PA to Gln239 in jerdonobin-II might play an important role on their plasminogen activating activity difference.

A novel disintegrin, jerdonatin, inhibits platelet aggregation and sperm-egg binding.

A novel disintegrin, jerdonatin, was purified to homogeneity from Trimeresurus jerdonii venom by gel filtration and reversed-phase high-pressure liquid chromatography. We isolated the cDNA encoding jerdonatin from the snake venom gland. Jerdonatin cDNA precursor encoded pre-peptide, metalloprotease and disintegrin domain. Jerdonatin is composed of 72 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonatin was determined to be 8011 Da by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Jerdonatin inhibited ADP- and collagen-induced human platelet aggregation with IC50 of 123 and 135 nM, respectively. We also investigated the effect of jerdonatin on the binding of B6D2F1 hybrid mice spermatozoa to mice zona-free eggs and their subsequent fusion. Jerdonatin significantly inhibited sperm-egg binding in a concentration-dependent manner, but had no effect on the fusion of sperm-egg. These results indicate that integrins on the egg play a role in mammalian fertilization.

A novel high molecular weight metalloproteinase cleaves fragment F1 of activated human prothrombin.

A hemorrhagic proteinase, jerdohagin, was purified from Trimeresurus jerdonii venom by gel filtration and ion-exchange chromatographies. It was a single chain polypeptide with an apparent molecular weight of 96 kDa as estimated by SDS-PAGE under the non-reducing and reducing conditions. Internal peptide sequencing indicated that it consisted of metalloproteinase, disintegrin-like and cysteine-rich domains and belonged to the class III snake venom metalloproteinases (class P-III SVMPs). Like other typical metalloproteinases, hemorrhagic activities of jerdohagin were completely inhibited by EDTA, but not by PMSF. Jerdohagin preferentially degraded alpha-chain of human fibrinogen. Interestingly, jerdohagin did not activate human prothrombin, whereas it cleaved human prothrombin and fragment F1 of activated human prothrombin.

Purification, cloning and biological characterization of a novel disintegrin from Trimeresurus jerdonii venom.

A novel disintegrin, jerdonin, was purified from the Trimeresurus jerdonii venom by means of gel filtration and reverse phase high pressure liquid chromatography. Its coding cDNA was also isolated from the venom gland. The jerdonin coding cDNA is part of a precursor composed of proprotein, metalloproteinase, and disintegrin domains. From the deduced amino acid sequence, jerdonin is composed of 71 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonin was determined to be 7483Da by matrix-assisted laser desorption ionization time of flight mass spectrometry. Jerdonin inhibited ADP- and collagen-induced human platelet aggregation with IC(50) of 220 and 240 nM, respectively. In vivo, jerdonin inhibited the growth of subcutaneously inoculated B16 solid tumor in C57BL/6 mice and improved the survival time of the tumor-bearing mice.

Purification, characterization and primary structure of a chymotrypsin inhibitor from Naja atra venom.

A chymotrypsin inhibitor, designated NA-CI, was isolated from the venom of the Chinese cobra Naja atra by three-step chromatography. It inhibited bovine alpha-chymotrypsin with a Ki of 25 nM. The molecular mass of NA-CI was determined to be 6403.8 Da by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) analysis. The complete amino acid sequence was determined after digestion of S-carboxymethylated inhibitor with Staphylococcus aureus V8 protease and porcine trypsin. NA-CI was a single polypeptide chain composed of 57 amino acid residues. The main contact site with the protease (P1) has a Phe, showing the specificity of the inhibitor. NA-CI shared great similarity with the chymotrypsin inhibitor from Naja naja venom (identities=89.5%) and other snake venom protease inhibitors.

TMVA, a novel GPIb-binding protein, significantly prevents platelet microthrombi formation and prolongs discordant cardiac xenograft survival.

In xenotransplantation, donor endothelium is the first target of immunological attack. Activation of the endothelial cell by preformed natural antibodies leads to platelet binding via the interaction of the glycoprotein (GP) Ib and von Willebrand factor (vWF). TMVA is a novel GPIb-binding protein purified from the venom of Trimeresurus mucrosquamatus. In this study, the inhibitory effect of TMVA on platelet aggregation in rats and the effect on discordant guinea pig-to-rat cardiac xenograft survival were investigated. Three doses (8, 20 or 40 microg/kg) of TMVA were infused intravenously to 30 rats respectively. Platelet aggregation rate was assayed 0.5, 12, and 24 h after TMVA administration. Wister rats underwent guinea pig cardiac cervical heterotopic transplantation using single dosing of TMVA (20 microg/kg, i.v., 0.5 h before reperfusion). Additionally, levels of TXB(2) and 6-keto-PGF(1alpha) within rejected graft tissues were determined by radioimmunoassay. Treatment with TMVA at a dose of 20 or 40 microg/kg resulted in complete inhibition of platelet aggregation 0.5 h after TMVA administration. Rats receiving guinea pig cardiac xenografts after TMVA therapy had significantly prolonged xenograft survival. Histologic and immunopathologic analysis of cardiac xenografts in TMVA treatment group showed no intragraft platelet microthrombi formation and fibrin deposition. Additionally, the ratio of 6-keto-PGF(1alpha) to TXB(2) in TMVA treatment group was significantly higher than those in control group. We conclude that the use of this novel GPIb-binding protein was very effective in preventing platelet microthrombi formation and fibrin deposition in a guinea pig-to-rat model and resulted in prolongation of xenograft survival. The increased ratio of PGI(2)/TXA(2) in TMVA treatment group may protect xenografts from the endothelial cell activation and contribute to the prolongation of xenograft survival.

A new protein structure of P-II class snake venom metalloproteinases: it comprises metalloproteinase and disintegrin domains.

A new metalloproteinase-disintegrin, named Jerdonitin, was purified from Trimeresurus jerdonii venom with a molecular weight of 36 kDa on SDS-PAGE. It dose-dependently inhibited ADP-induced human platelet aggregation with IC(50) of 120nM. cDNA cloning and sequencing revealed that Jerdonitin belonged to the class II of snake venom metalloproteinases (SVMPs) (P-II class). Different from other P-II class SVMPs, metalloproteinase and disintegrin domains of its natural protein were not separated, confirmed by internal peptide sequencing. Compared to other P-II class SVMPs, Jerdonitin has two additional cysteines (Cys219 and Cys238) located in the spacer domain and disintegrin domain, respectively. They probably form a disulfide bond and therefore the metalloproteinase and disintegrin domains cannot be separated by posttranslationally processing. In summary, comparison of the amino acid sequences of Jerdonitin with those of other P-II class SVMPs by sequence alignment and phylogenetic analysis, in conjunction with natural protein structure data, suggested that it was a new type of P-II class SVMPs.

Purification and characterization of alpha-mucrofibrase, a novel serine protease with alpha-fibrinogenase activity from the venom of Chinese Habu (Trimeresurus mucrosquamatus).

A novel fibrinogenolytic protease, named alpha-mucrofibrase, was purified from the venom of Chinese Habu (Trimeresurus mucrosquamatus) by DEAE-Sephadex A-50 ion-exchange chromatography and Sephadex G-100 (super fine) gel filtration alpha-Mucrofibrase is a single-chain polypeptide of approximately 29 kDa. It is stable even at 95 degrees C, and the most susceptible hydrolysis substrate is S-2302. It cleaved primarily the Aalpha chain of fibrinogen followed by the Bbeta chain, while the gamma chain was partially affected. N-terminal sequence of this fibrinogenolytic enzyme has great homology with those of other snake venom serine proteases. The esterase activity of alpha-mucrofibrase is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by metal chelator (EDTA), suggesting this fibrinogenase belongs to the venom serine protease family.

Purification and cloning of a novel C-type lectin-like protein with platelet aggregation activity from Trimeresurus mucrosquamatus venom.

TMVA, a novel C-type lectin-like protein that induces platelet aggregation in a dose-dependent manner, was purified from the venom of Trimeresurus mucrosquamatus. It consists of two subunits, alpha (15536 Da) and beta (14873 Da). The mature amino acid sequences of the alpha (135 amino acids) and beta subunits (123 amino acids) were deduced from cloned cDNAs. Both of the sequences show great similarity to C-type lectin-like venom proteins, including a carbohydrate recognition domain. The cysteine residues of TMVA are conserved at positions corresponding to those of flavocetin-A and convulxin, including the additional Cys135 in the alpha subunit and Cys3 in the beta subunit. SDS-PAGE, mass spectrometry analysis and amino acid sequence showed that native TMVA exists as two convertible multimers of (alpha beta)(2) and (alpha beta)(4) with molecular weights of 63680 and 128518 Da, respectively. The (alpha beta)(2) complex is stabilized by an interchain disulfide bridge between the two alpha beta-heterodimers, whereas the stabilization of the (alpha beta)(4) complex seems to involve non-covalent interactions between the (alpha beta)(2) complexes.

A Highly Active Anticomplement Factor from the Venom of Naja kaouthia.

A highly active anticomplement factor (cobra venom factor) from the venom of Naja kaouthia in South Yunnan, China was isolated by sequential column chromatography (SP-Sephadex-C-25, Q Sepharose HP and Sephadex G-150). It displays strong anticomplement activities in vitro and in vivo, and has anticomplement activity of 1 515 u per mg. The purified anticomplement factor was homogeneous on SDS-PAGE with a molecular weight of 149 kD. It is composed of three polypeptide chains which are connected by disulfide bonds. The molecular weights of the three chains were determined to be 65.4 kD (alpha-chain), 52.1 kD (beta-chain), and 35.5 kD (gamma-chain). The gamma-chain displayed size heterogeneity, and two bands could be clearly detected in gels. All polypeptide chains were stained with periodate-Schiff reagent, suggesting the presence of carbohydrate. Neutral sugar and sialicacid were determined to be 1.78% and 0.38%, respectively. The isoelectric point of this anticomplement factor was 6.2. Its amino acid composition was analyzed and N-terminal amino acid sequence of each polypeptide chain was determined.