Characterization of two platelet aggregation inhibitor-like polypeptides from viper venom (original) (raw)
Related papers
Biochemistry, 2003
The venoms of Viperidae snakes contain numerous serine proteinases that have been recognized to possess one or more of the essential activities of thrombin on fibrinogen and platelets. Among them, a platelet proaggregant protein, cerastocytin, has been isolated from the venom of the Tunisian viper Cerastes cerastes. Using the RACE-PCR technique, we isolated and identified the complete nucleotide sequence of a cDNA serine proteinase precursor. The recombinant protein was designated rCC-PPP (for C. cerastes platelet proaggregant protein), since its deduced amino acid sequence is more than 96% identical to the partial polypeptide sequences that have been determined for natural cerastocytin. The structure of the rCC-PPP cDNA is similar to that of snake venom serine proteinases. The expression of rCC-PPP in Escherichia coli system allowed, for the first time, the preparation and purification of an active protein from snake venom with platelet proaggregant and fibrinogenolytic activities. Purified rCC-PPP efficiently activates blood platelets at nanomolar (8 nM) concentrations, as do natural cerastocytin (5 nM) and thrombin (1 nM). It is able to clot purified fibrinogen and to hydrolyze R-chains. Thus, rCC-PPP could be therefore considered a cerastocytin isoform. By comparison with other snake venom serine proteinases, a Gly replaces the conserved Cys 42 . This implies that rCC-PPP lacks the conserved Cys 42 -Cys 58 disulfide bridge. A structural analysis performed by molecular modeling indicated that the segment of residues Tyr 67 -Arg 80 of rCC-PPP corresponds to anion-binding exosite 1 of thrombin that is involved in its capacity to induce platelet aggregation. Furthermore, the surface of the rCC-PPP molecule is characterized by a hydrophobic pocket, comprising the 90 loop (Phe 90 -Val 99 ), Tyr 172 , and Trp 215 residues, which might be involved in the fibrinogen clotting activity of rCC-PPP.
A potent platelet aggregation inhibitor purified from Agkistrodon halys (mamushi) snake venom
1983
Askivtrodon~(mamuain) snake venom. Ta~ak+on 21, 797-804, 1983 .-By means of gel filtration on Sepludex G-7S, DEAE~ephadex A-SO column chromatography and three gel filtrations on Sephadex G-75, a potent platelet aggregation inhibitor was purified from Agkistrodon lwlys snake veaam end shown to be a single peptide chain, as judged by SDS-pdyacrylamide gel ekdrophoreaia. The purified phuelet aggregation inhibitor was an acidic protein with a molecular wdght of 14,000 a~P PharP~hP~Aa activity. Its inhibitory activity on platelet aggregadon was heat stable (at %°C, 30 min) in an addic medium (pH S .S), while its phaspholipase A enzymatic activity was hart labile under the same conditions. Its inhibitory activity on platelet aggregation induced by thrombin, sodium ararhitlro+nf~~~~or ionophore A-?3187 was 13on~ompetidve and dosedepeadent with a similar m (.v 11 Etg/ml). It exerted its inhibitory acdon without pre-incubation with platelet suspension, however, its inhibitory effect could be moderately increased after longer incubation (30 min) .
Platelet aggregation inhibitors from Agkistrodon acutus snake venom
1986
aggregation inhibitors from Agkisrrodon acutus snake venom. Toxicon 24, 1099-1106, 1986 .-Among all the purified components from A. acutus venom, including ADPase, S'-nucleotidase, phospholipase A, and fibrinogenases, only the venom ADPase (SO-100 pg/ml) shows marked inhibitory action on ADP(10 NM}, collagen(10 Ng/ml)and sodium arachidonate(100 plvl}induced platelet aggregations of rabbit platelet-rich plasma. The venom S'-nucleotidase (100 ptg/ml) inhibited ADP-induced platelet aggregation by 31f4% (n=4, PG0 .05). Fibrinogenolytic enzymes (fractions I and IX, 100 Ng/ml) did not significantly inhibit platelet aggregation induced by ADP (10 pM), collagen (10 Ng/ml) or sodium arachidonate (100 NM). However, whrn the fibrinogenase (fraction IX, 100 hg/mq was preincubated with platelet-rich plasma for 30 min it inhibited collagen(20 Ng/ml} and ADP(10 pM}induced platelet aggregations by 34t9~k (n=4, P~f0.03) and 33t6ge (n=4, P<0 .05), respectively. The phospholipase A, (100 pg/ml) did not affect platelet aggregation. The venom ADPase is a single chain polypeptide with a molecular weight of 94,000. The specific ADPase activity is estimated to be 4.3 pmoles Pi/min/mg of protein. It also possesses phosphodiesterase and weak S'nucleotidase activities .
FEBS Letters, 1991
Interference with blood coagulation is one of the main causes of the pathological manifestation in snakebites by the families of Crotalidue and Viperirlrre [I]. Recently, another mechanism by which snakebitcs may interfere with hemostasis was discovered in trigramin, a naturally-occurring low molecular weight peptide from the venom of Trirneresurus gramineus [2]. This peptidc inhibits platelet aggregation by specifically and competitively inhibiting fibrinogen binding to the receptors associated with the GPIIb/GPIIIa complex in activated platelets. Having recently developed a methad to study the adhesion of platelets to collagen in the absence of platelet aggregation [3], we screened a number of crude snake venoms to determine whether any possessed proteins that inhibited platelet adhesion to collagen, a process thought to be mediated by receptors associated with the GPIa/GPIIa complex [4-63. We report here the isolation and identification of very similar 50 kDa proteins present in 4 different snake venoms which have the ability to specifically inhibit the adhesion of human platelets to collagen. 2. MA'I'ERIALS AND METWQDS Snake venoms were purchased from Sigma and reconstituted in 0.9% NaCl at 10 m&ml. The following venoms (all at a final concentration of lOpc& protein/ml
Biochemistry, 1996
Binding of the multimeric adhesive glycoprotein, von Willebrand Factor (vWF), to the platelet membrane glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion and initiates signal transduction leading to platelet activation. Recently described viper venom proteins that bind to the GP Ib R-chain and inhibit vWF binding provide novel probes for studying receptor function. We have purified a 50-kDa form of alboaggregin from the white-lipped tree viper (Trimeresurus albolabris) and two 25-kDa proteins, CHH-A and CHH-B, from the timber rattlesnake (Crotalus horridus horridus) in addition to a previously described 25-kDa alboaggregin and echicetin. Complete or partial amino acid sequencing of CHH-A, CHH-B, and 50-kDa alboaggregin and cross-reactivity of these proteins with an anti-botrocetin antiserum confirmed that they were disulfide-linked heterodimers or higher multimers of the C-type lectin protein family. These proteins, together with 25-kDa alboaggregin and echicetin, specifically bound to GP IbR within the N-terminal peptide domain, His-1-Glu-282, and inhibited vWF binding with comparable IC 50 values (∼0.2 µg/mL). However, cross-blocking studies between these structurally related proteins and anti-GP IbR monoclonal antibodies demonstrated that the venom protein binding sites were not congruent. Further, the 50-kDa alboaggregin, but not the other venom proteins, potently induced platelet activation as assessed by dense granule serotonin release or elevation of cytosolic ionized calcium. Treatment of platelets with the 50-kDa alboaggregin was associated with activation of protein kinase C and tyrosine kinase(s), resulting in a platelet protein phosphorylation profile similar to that seen on shearstress-induced vWF binding to platelets. These results suggest that the 50-kDa alboaggregin induces cytoplasmic signaling coincident with its binding to the GP Ib-IX-V complex and provides a potentially useful probe for studying the mechanism of vWF-dependent platelet activation.
Species-dependent Specificity of Platelet Aggregation Inhibitors from Snake Venom
Journal of Comparative Pathology, 1999
Echistatin, flavoridin and kistrin belong to a family of low molecular weight snake-venom proteins, termed "disintegrins" because of their ability to bind integrin receptors on the cell surfaces. Most disintegrins contain the tripeptide arginine-glycine-aspartic acid (RGD) sequence, which represents a common cell adhesion recognition site. Here we report the differing activity of echistatin, flavoridin and kistrin on adenosine 5′-diphosphate (ADP)-induced aggregation of platelets from the buffalo, dog and horse. The three disintegrins inhibited the aggregation of platelets from all three animal species at nanomolar concentrations. Echistatin was the most active of the disintegrins towards equine platelets, but flavoridin and kistrin showed a higher potency than echistatin in inhibiting aggregation of platelets from the buffalo and dog. Kistrin was 1•6-fold more effective than flavoridin in inhibiting ADPinduced aggregation of platelets from either the buffalo or dog, whereas flavoridin was 2•1-fold more active than kistrin in inhibiting aggregation of equine platelets. The species-dependent platelet sensitivity to these snakevenom proteins may reflect structural differences of the integrin receptor GP IIb/IIIa on the platelet surface in different mammalian species.
Toxicon, 1984
I Yait and C. OtrxeNa. Mechanism of action of the plateld aggregation inhibitor purified from Agkistrodon ltalys (mamuahi) snake venom. Toxirnn 22, 243-251, 1984 .-The plateld aggregation inhibitor purified from Agkistrodon lralys snake venom inhibited rabbit platelet aggregations induced by thrombin, sodium arachidonate, collagen or ionophorc A-23187. The ic was about 11 pg/ml in plateld aggregation regardless of which aggregation indticer was used. ß-Mercaptoethanol abolished both the phospholipase A enzymatic and plateld aggregation inhibitory acdvitiea of this venom inhibitor. p-Bromophenacyl bromido-tnated venom inhibitor last almost completely its phosphilipase A enzymatic activity, but retained its platelet aggregation inhibitory effect. In the presence of EGTA, the venom inhibitor still showed the same inhibitory acdvity on thrombin-, sodium ararhidonate-, collagen-or ionophorc A23187-induced platelet aggregations triggered by successive addition of Cam. The activation of platelet phospholipase A and the serotonin release reaction triggered by Cam influx wen unaffected by this venom inhibitor. It also inhibited the clot rdraction of plateld-rich plasma. It is concluded that the inhibitory effect of the venom inhibitor on plateld aggregation is indepeadmt of its phospholipase A enzymatic activity. Its mode of action is different from those of other known platelet inhibitory drugs. This venom inhibitor possibly ads on a common step subsequent to platelet shape change, leading to inhibition of platelet aggregation .
Biochemical Journal, 1995
A potent platelet aggregation inducer, aggretin, was purified from Malayan-pit-viper (Calloselasma rhodostoma) venom by ionic-exchange chromatography, gel-filtration chromatography and HPLC. It is a heterodimeric protein (29 kDa) devoid of esterase, phospholipase A and thrombin-like activity. Aggretin (> 5 nM) elicited platelet aggregation with a lag period in both human platelet-rich plasma and washed platelet suspension. EDTA (5 mM), prostaglandin E1 (1 microM) and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (‘TMB-8’; 100 microM) abolished its aggregating activity, indicating that exogenous bivalent cations and intracellular Ca2+ mobilization are essential for aggretin-induced platelet aggregation. Neomycin (4 mM) and mepacrine (50 microM) completely inhibited aggretin (33 nM)-induced aggregation; however, creatine phosphate/creatine phosphokinase (5 mM, 5 units/ml) and indomethacin (50 microM) did not significantly affect its aggregating activity. Aggretin caused ...
Monomeric and Dimeric Disintegrins: Platelet Active Agents from Viper Venom
Toxin Reviews, 2007
When the term "disintegrin" was first coined in 1990, it described a family of naturally occurring proteins with low molecular weights and highly conserved sequences, both in their cysteine arrangements and adhesive Arg-Gly-Asp (RGD) motifs. Another common characteristic was the inhibitory potential these proteins demonstrated in interacting with cell-surface integrin receptors. Measurement of the effect by disintegrins on the interaction between the platelet receptor αIIbβ3 and its ligand, fibrinogen, has become a hallmark assay for comparing the activities of members of this increasingly diverse family discovered in the past two decades. This review focuses on the inhibitory profiles, based on platelet function, of the monomeric and heterodimeric disintegrins described to date, as well as the informative contributions of disintegrin mutations in our understanding of the structure-function relationships between ligand and αIIbβ3. The challenge of naming future examples of these proteins is also addressed.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1995
A potent, proteinaceous inducer of platelet aggregation designated as IVa, has been purified to homogeneity from Cerastes cerastes venom by molecular sieve and ion exchange chromatography. It is composed of 2 subunits with total M r of 62 000 as shown by native gel chromatography and chemical cross-linking with disuccinimidyl suberate. It is not clear at the present time whether both subunits are identical gene products, however, both have identical N-terminal sequences for the first 15 amino acids. The protein has a pl above 9.6. IVa (0.1 /xg/ml) could aggregate platelets up to 80% and was inhibited by p-APMSF, leupeptin, iodoacetamide, protein kinase C inhibitor, phosphatase inhibitor, ATP and PGE~, while it was insensitive to acetylsalicylic acid, ADP scavenger system, protein kinase A inhibitor and hirudin. Protein IVa is a serine proteinase with thrombin-like activity as it hydrolysed thrombin chromogenic substrate CBS 34.47, its aggregatory activity was partially inhibited by monoclonal antibodies against GPIb and the thrombin receptor, as was the thrombin, and its ability to induce intracellular Ca 2+ release was blocked by pretreating platelets with thrombin. Unlike thrombin, the IVa protein showed very weak coagulant activity as indicated by plasma recalcification time and fibfinogen clotting time although it could hydrolyse fibrinogen a-chains.