Agkistrodon Piscivorus Research Articles

Presence of heat-stable hemorrhagic toxins in snake venoms

Twenty-eight snake venoms (seven Agkistrodon venoms, six Bothrops venoms, 13 Crotalus venoms, one Sistrurus venom, and one Bitis venom) were examined for the presence of heat-stable (100°C, 5 min) hemorrhagic toxins. Both heated and unheated venoms were analyzed for their ptotein composition by SDS-PAGE, and tested for their hemorrhagic activity in vivo in mice and for their proteolytic activity on two different substrates. Heating all venoms led to the denaturation and loss of some proteins; however, most of the venoms retained a significant number of proteins. Seventeen venoms contained more than seven proteins after heating, whereas five venoms contained only one to three proteins. All but nine of the heated venoms had substantial hemorrhagic activity, and Agkistrodon piscivorus piscivorus venom had very high activity, almost four times that of the second most hemorrhagic venom from Crotalus viridis lutosus. Most venoms, heated or unheated, had activity on the two protease substrates. Using succinylated casein as the substrate, there was a wide range of activity, and heating drastically reduced the activity of all venoms except that of Crotalus ruber and Crotalus molossus molossus. With azocoll as substrate, all but two of the unheated venoms ( Crotalus adamanteus and Crotalus viridis concolor) had very high activity, whereas upon heating, all except five venoms lost essentially all of their activity. Heated venoms from snakes in the Agkistrodon genus (except for Agkistrodon blomhoffi blomhoffi, an Asian snake) retained activity on azocoll, and this activity tended to correlate better with hemorrhagic activity of the venom than did proteolytic activity on casein.

Quantification of the interaction between lysolecithin and phospholipase A 2

The rate of hydrolysis of phosphatidylcholine bilayers by phospholipase A 2 may be either enhanced or inhibited by the presence of lysolecithin depending on the experimental conditions examined. To further understand the relationship of lysolecithin to phospholipase A 2 activity, the binding of lysolecithin to phospholipase A 2 from the venom of Agkistrodon piscivorus piscivorus was examined by fluorescence spectroscopy. The tryptophan emission intensity of the enzyme was enhanced by 70% upon addition of lysolecithin. The binding isotherm for lysolecithin to the phospholipase A 2 estimated from the fluorescence change was biphasic, with a clear break in the curve occurring at the critical micelle concentration of the lysolecithin. Several observations suggested that the phospholipase A 2 was also capable of hydrolyzing the lysolecithin although at a rate far below that of phospholipid hydrolysis. These experiments were repeated using several other species of phospholipase A 2, and the results were found to be general among the enzymes except the lys-49 isozyme from A. p. piscivorus which displayed neither the dependence on the critical micelle concentration for binding nor the ability to hydrolyze lysolecithin. These results were used as the basis for a quantitative analysis of enzyme fluorescence changes that occur during the time course of phospholipid hydrolysis and of the mechanism whereby lysolecithin inhibits the hydrolysis of phosphatidylcholine bilayers by phospholipase A 2.

Additional Specimens of the Western Cottonmouth (Agkistrodon piscivorus leucostoma, Reptilia: Squamata) from Kansas

The collection of two Western Cottonmouths (Agkistrodon piscivorus leucostoma) from Cherokee County in 1991 is reported. The history of this taxon in Kansas is discussed and previous records are discredited. The collection of the latest specimens confirms the presence of this taxon in extreme east-central Cherokee County. The history of Agkistrodon piscivorus leucostoma in Kansas is a curious one. Although the taxon had been reported from the state as early as 1937 (Hall and Smith, 1947) and as recently as 1977 (Collins, 1977; Perry, 1977; Rundquist et al., 1978), no undoubtedly valid specimens have been collected from Kansas (Collins, 1982). We herein report on two specimens of A. p. leucostoma collected in 1991 that establish this species' existence in Kansas. On 14 September 1991, an adult male A. p. leucostoma (KU 218677) was taken by Shane Eckhardt at ca. 4.8 km E of the jct. of U.S. route 69 and Kansas route 160, Cherokee County, extreme southeastern Kansas. The animal was found in late afternoon at a litter-strewn parking area approximately 125 m W of the Spring River. Associated habitat is mixed hardwood deciduous forest (primarily Quercus and hickory) in the Spring River floodplain in the Cherokee Plain physiographic province (Kiichler, 1974). The specimen (KU 218677) is an adult male with an SV of 778 mm, TL 925 mm. Meristic characters are as follows: ventrals133, subcaudals-45, undivided subcaudals -24, supralabials-8-undet. (damaged), infralabials 9-10, scale row formula--23-25-21. The dorsal pattern is normal for A. p. leucostoma, as is the ventral pattern. There is damage to the right mandible and a portion of the venter in the anterior quarter of the specimen. As a consequence of news media publicity connected with KU 218677, This content downloaded from 157.55.39.208 on Fri, 14 Oct 2016 04:07:07 UTC All use subject to http://about.jstor.org/terms VOLUME 96, NUMBERS 1-2 149 another specimen found earlier was reported to us by Mr. Eckhardt. On 29 August 1991, an adult male A. p. leucostoma (KU 218780) was collected at 1.6 km S, 3.4 km E of the junction of U.S. route 69 and Kansas route 160, Cherokee County, Kansas, by Ken Outt. The animal was found near a small concrete culvert that runs under a gravel road. Associated habitat was cultivated fields to the north of the road and mixed hardwood deciduous secondary growth forest immediately to the south. While recovering this snake, Mr. Outt was bitten by it on the first phalange of his right index finger and was mildly envenomated. This second specimen is an adult male with an SV of 600 mm, TL 705 mm. Meristic characters are as follows: ventrals136, subcaudals-43, undivided subcaudals--17, supralabials--8-8, infralabials11-11, scale row formula 24-24-2 1. The dorsal pattern is somewhat more distinct than normally seen in living adult A. p. leucostoma, but this may be an artifact of preservation. The ventral pattern is normal. The specimen has sustained severe midbody trauma, apparently after having been run over by a vehicle. In addition, there are three major dislocating fractures of the vertebral column anterior to the midbody trauma.

Variability of Venom-Neutralizing Properties of Serum from Snakes of the Colubrid Genus Lampropeltis

Abstract : Venom neutralization properties and protein content of serum from 11 taxa of Lampropeltis were studied. Most serum samples contained 6.5% to 9.5% protein. Lampropeltis g. getulus and L. g. floridana serum showed the broadest spectrum of effective neutralization of venoms from 10 crotaline taxa. Lampropeltis t. triangulum, L. t. hondurensis, L. mexicana greeri, and L. alterna effectively neutralized many of the venoms assayed, but were less efficacious than the other Lampropeltis species tested. Most of the serum samples investigated had variably effective neutralization capacities for venoms with strong hemorrhagic activities (Crotalus atrox, C. adamanteus, C. v. viridis). Sera from L. g. holbrooki and L. g. floridana were particularly effective in neutralizing venoms of Agkistrodon piscivorus conanti and A. contortrix mokasen. Only L. g. getulus, L. g. floridana, and L. ruthveni sera neutralized over 100 LD of C. v. helleri venom per ml. Only four serum samples (L. g. getulus, L. g. floridana, L. calligaster, and L. t. triangulum) were effective against type A C. s. scutulatus venom (contained high concentrations of the potent neurotoxin, Mojave toxin). All Lampropeltis sera assayed had effective neutralization potential for type B C. s. scutulatus venom, which has strong hemorrhagic and proteolytic activities and lacks Mojave toxin. All serum samples assayed were ineffective against venom of the elapid Micrurus f. fulvius. Serum from Elaphe g. guttata effectively neutralized several crotaline venoms, while Rhinocheilus lecontei antonii serum had only marginal neutralization capacity for several venoms

Expression of a group II phospholipase A 2 from the venom of Agkistrodon piscivorus piscivorus in Escherichia coli: Recovery and renaturation from bacterial inclusion bodies

A synthetic gene encoding the Group 11 phospholipase A 2 (PLA 2) from the venom of Agkistrodon piscivorus piscivorus has been constructed and expressed with high efficiency in Escherichia coli. No enzymatic activity was recovered when the polypeptide contained the initiator Met residue. Replacement of an Asn residue penultimate to the initiator Met with Ser or Gly permitted removal of the initiator Met by the endogenous methionine aminopeptidase. The amino-terminal serine (N-Ser) and amino-terminal glycine PLA 2's were isolated from intracellular inclusion bodies and were renatured with 25% recovery. Automated Edman degradation confirmed the removal of the initiator Met and confirmed the sequence of the first 40 residues of N-Ser PLA 2. The recombinant proteins were purified to apparent homogeneity and showed the same specific activity as the wild-type protein. N-Ser PLA 2 demonstrated the same kinetics of activation as the wild type enzyme on large vesicles of zwitterionic lipid.

Local heat and cold application after eastern cottonmouth moccasin (Agkistrodon piscivorus) envenomation in the rat: Effect on tissue injury

We studied the effect of local application of heat or cold on the development of tissue necrosis in envenomated rats. Anesthetized animals had 6 mg/kg venom from Agkistrodon piscivorus injected subcutaneously into the lateral aspect of a hind limb. Heat or cold was applied to the site of envenomation in the experimental groups for 4–6 hr, and the injected area was examined histologically after 24 hr. Neither local treatment, with or without the presence of systemic antivenin, significantly affected the severity of tissue necrosis induced by the venom in comparison to a control group left at ambient temperature.

Calcium and magnesium dependence of phospholipase A2-catalyzed hydrolysis of phosphatidylcholine small unilamellar vesicles.

The Ca2+ requirement for lipid hydrolysis catalyzed by phospholipase A2 from Agkistrodon piscivorus piscivorus (App-D49) and porcine pancreas has been examined using small, unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC SUV). Hydrolysis was affected by product inhibition even at early times, and the extent of this inhibition depended on the concentration of divalent cations. The Ca2+ requirement for half-maximal rates of hydrolysis reflected, in part, this non-catalytic role of divalent cations. The presence of 10 mM Mg2+, a cation which does not support catalysis, reduced the Ca2+ required for half-maximal rates of hydrolysis from millimolar concentrations to 40 microM for App-D49. Since the dissociation constant of the enzyme for Ca2+ in solution is 2 mM, these results indicate a change in the interaction of the enzyme with Ca2+ under catalytic conditions. The kinetic dissociation constant of Ca2+ for the pancreatic enzyme was 20 microM which is substantially lower than the dissociation constant in solution, 0.35 mM. The similarity of apparent kinetic dissociation constants for these enzymes suggests that structurally similar features determine the affinity for Ca2+ under catalytic conditions. Evidence is presented that the affinity of phospholipase A2 for Ca2+ changes subsequent to the initial interaction of the enzyme with the substrate interface. However, the apparent Michaelis constant, KMapp, for App-D49, 0.03-0.06 mM, is independent of [Ca2+] and is about the same as the equilibrium dissociation constant for DPPC SUV, 0.14 mM. We thus suggest that KMapp is a steady-state constant.

Reversibility of the activation of soluble phospholipase A 2 on lipid bilayers: Implications for the activation mechanism

The time-courses of hydrolysis of large vesicles of dipalmitoylphosphatidylcholine were compared using four species of phospholipase A 2 ( Agkistrodon piscivorus piscivorus, Crotalus adamanteus and Naja naja venoms and porcine pancreatic). In all four cases, the hydrolysis rate suddenly increases 10 to 100-fold at the time (τ) when a specific mole fraction of reaction products has accumulated. The intrinsic fluorescence emission of the three venom enzymes also increases suddenly at time τ. Both the activation and the fluorescence change are reversible with a half-time of about 50 s for the activity and 2 to 6 s for the fluorescence. These reversal rates and the vesicle concentration dependence of τ are considered for monomer and dimer enzyme activation models. Apparently, at least three states of the enzyme exist beyond the initial unbound state: (1) inactive and bound, (2) inactive with high fluorescence and (3) active. The dimer model already contains the necessary number of states but requires that the activation rate be much lower than the reversal rate to account for the vesicle concentration dependence of τ. Success of the monomer model requires an enzyme state additional to those proposed previously. Although these results do not exclude either the monomer or dimer models conclusively, they do impose important constraints on each model.

Molecular details of the activation of soluble phospholipase A2 on lipid bilayers. Comparison of computer simulations with experimental results.

The initial rate of hydrolysis of large unilamellar vesicles of dipalmitoylphosphatidylcholine by phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus is small and elevates gradually until it suddenly increases by a factor of 10 to 1000 depending on the experimental conditions. This abrupt onset of high enzyme activity appears to be correlated to a specific mole fraction of reaction product at which point a cooperative compositional phase transition in the bilayer occurs. Five models that describe the activation process in terms of its being coupled to the putative product-induced lipid transition are presented. These models include one in which the lipid structure enhances the affinity of enzyme binding to the bilayer surface, two in which the equilibrium position between an active and an inactive form of the enzyme-substrate complex is altered, and two in which the rate of a quasi-irreversible spontaneous activation process is increased. Whether the active form of the enzyme is a monomer or dimer is also considered in the last two pairs of models. Computer simulations of time courses for the different models show how a set of four experimental observables distinguishes qualitatively among them. Comparison of the experimental behavior with the computer-simulated behavior of the observables for each model indicates that activation of phospholipase A2 on the lipid surface involves formation of an enzyme dimer which spontaneously converts to an active form. The active enzyme persists in the active state as it exchanges between vesicles. This model of activation is similar to that proposed previously for activation of porcine pancreatic phospholipase A2.

Citrate is a major component of snake venoms

Citrate has been identified as a major component of snake venoms by gas liquid chromatography and mass spectrometry. The venoms of Bothrops asper, Crotalus atrox, Crotalus viridis viridis, Crotalus adamanteus, Sistrurus miliarius barbouri, Crotalus horridus horridus, Agkistrodon contortrix mokasen, Agkistrodon contortrix contortrix and Agkistrodon piscivorus piscivorus contain citrate at concentration levels which can serve as effective buffers. Calcium, magnesium, zinc, iron, sodium and potassium salts of citrate would be constituents of venom.

Seasonal activity and road mortality of the snakes of the Pa-hay-okee wetlands of Everglades National Park, USA

The composition and activity of the snake community of the Pa-hay-okee wetlands of Everglades National Park is described. The study was conducted in February and March 1986 and from February 1987 to January 1989. A total of 1172 individuals of 16 taxa were observed, with Thamnophis sauritus, T. sirtalis, Nerodia fasciata pictiventris and Agkistrodon piscivorus representing 89·2% of the total sample. The seasonal activity of the snakes was closely related to fluctuations in the water table. Periods of greatest snake activity coincided with the periods of greatest human visitation. Seventy-three percent of all snakes observed on the park's main road were either injured or dead. Management recommendations are provided to minimize loss.

Effects of pH and calcium ion on self-association properties of two dimeric phospholipases A2.

The monomer-dimer equilibria of the dimeric phospholipases A2 from Crotalus atrox and Agkistrodon piscivorus piscivorus venoms were examined chromatographically as a function of pH and in the presence versus absence of the essential cofactor, calcium ion. At neutral pH without calcium, the subunits of both enzymes reequilibrated sufficiently slowly that dimer and monomer were separated by size exclusion chromatography. At pH 4.2 and lower, the dimers underwent rapid dissociation and reassociation, eluting as single broad peaks whose position as a function of applied protein concentration could be analyzed to determine association constants using an algorithm that estimates these values based on elution positions. Lowering the pH from 7.0 to 4.2 increased the self-association constant of the C. atrox enzyme by 1 order of magnitude and that of the A. p. piscivorus dimer by a factor of 3. Calcium ion, an essential cofactor of phospholipase A2, converted the kinetic behavior of the dimers at neutral pH from slow to virtually instantaneous on the time scale of the chromatography runs, 40 min. Calcium ion also altered the thermodynamic stability of the enzymes; the association constant of A. p. piscivorus phospholipase A2 in neutral pH buffer was reduced by approximately 2 orders of magnitude, whereas that of C. atrox was increased by a factor of 6. The structural basis for the disparate effects of calcium ion on these two acidic, dimeric venom phospholipases A2 is uncertain. This study illustrates the importance of calcium ion and pH on the solution behavior of the dimeric members of this class of enzymes.

Poisonous Snakebite in Central Texas Possible Indicators for Antivenin Treatment

Sixty-seven patients hospitalized for poisonous snakebite between 1975 and 1990 were managed by elevation, tetanus prophylaxis, intravenous fluids and antibiotics, and often by a limited excision of the bite site in the Emergency Department, with sequential laboratory studies as needed. Antivenin was used for systemic envenomation, and 23 of the 67 patients (34%) received 133 vials. Thirteen of the twenty-three patients (56%) had adverse reactions to the antivenin. Two significant observations arose. First age was an indicator. Eleven of eighteen patients 12 years or younger (61%) received antivenin, whereas 12 of 49 patients older than 12 years (24%) received antivenin (p = 0.0085, Fisher's exact test). Second species of snake was an indicator. Sixty-two snakes were identified (93%). Of 39 rattlesnake (Crotalus and Sistrurus) bites, 20 patients received antivenin (53%), but of 23 copperhead and water moccasin (Agkistrodon) bites, only three patients (12.5%) received antivenin (p = 0.0025). Antivenin may be indicated for use in systemic rattlesnake envenomation, especially in younger patients.

Myotoxin II from Bothrops asper (terciopelo) venom is a lysine-49 phospholipase A 2

A basic, dimeric myotoxic protein, myotoxin II, purified from Bothrops asper venom has a similar molecular weight and is immunologically cross-reactive with antibodies raised to previously isolated B. asper phospholipases A 2, except that it shows only 0.1% of the phospholipase activity against l-α-phosphatidylcholine in the presence of Triton X-100. Its 121 amino acid sequence, determined by automated Edman degradation, clearly identifies it as a Lys-49 phospholipase A 2. Key amino acid differences between myotoxin II and phospholipase active proteins in the Ca 2+-binding loop region, include Lys for Asp-49, Asn for Tyr-28, and Leu for Gly-32. The latter substitution has not previously been seen in Lys-49 proteins. Other substitutions near the amino terminus (Leu for Phe-5 and Gln for several different amino acids at position 11) may prove useful for identifying other Lys-49 proteins in viperid and crotalid venoms. Myotoxin II shows greater sequence identity with other Lys-49 proteins from different snake venoms ( Agkistrodon piscivorus piscivorus, Bothrops atrox, and Trimeresurus flavoviridis) than with another phospholipase A 2 active Asp-49 molecule isolated from the same B. asper venom. This work demonstrates that phospholipase activity per se, is not required in phospholipase molecules for either myotoxicity or edema inducing activities.

Neutralization of kinin-releasing enzymes of crotalid venoms by monospecific and polyspecific antivenoms

G. S. Bailey, A. Al-Joufi, S. Rawat and D. C. Smith. The neutralization of kinin-releasing enzymes of crotalid venoms by monospecific and polyspecific antivenoms. Toxicon 29, 777–781, 1991.—The amounts of kinin-releasing enzymes in the venoms of Crotalus atrox, Crotalus adamanteus, Crotalus scutulatus scutulatus and Agkistrodon piscivorus piscivorus were measured by determining the amounts of kinin released from a sheep kininogen substrate by means of a specific radioimmunoassay. Four monospecific and two commercial polyspecific antivenom IgG samples were tested for their ability to reduce the kinin-releasing activities of the four crotalid venoms measured in vitro. All of the antivenom IgG samples were able to neutralize venom kininogenase activity to varying extents. One of the commercial polyspecific antivenoms was of equal or higher potency than the corresponding monospecific antivenoms for three of the venoms tested, indicating a high degree of cross-neutralization. F(ab’) 2 and especially Fab fragments of that polyspecific antivenom IgG were also effective in reducing the kinin-releasing activities of the four crotalid venoms.

Immunological properties of the fibrinolytic enzyme (fibrolase) from southern copperhead ( Agkistrodon contortrix contortrix) venom and its purification by immunoaffinity chromatography

H. M. Chen, A. L. Guan and F. S. Markland, Jr. Immunological properties of the fibrinolytic enzyme (fibrolase) from southern copperhead ( Agkistrodon contortrix contortrix) venom and its purification by immunoaffinity chromatography. Toxicon 29, 683–694.—An antibody to the fibrinolytic enzyme in southern copperhead venom was produced by immunizing rabbits with chromatographically purified enzyme. The antibody was purified from rabbit blood by ammonium sulfate fractionation and protein-A affinity chromatography. The purified antibody reacted only with the fibrinolytic enzyme in southern copperhead venom as demonstrated by immunodiffusion and immunoelectrophoresis. Western immunoblotting revealed that several snake venoms, including Agkistrodon piscivorus conanti, Crotalus atrox, Crotalus basiliscus basiliscus, and Bothrops asper, cross-reacted with the antibody to varying degrees. However, Deinagkistrodon acutus showed no cross-reaction. Immobilized antibody has been used, in combination with molecular sieve chromatography, to purify the fibrinolytic enzyme from southern copperhead venom. In this two-step purification procedure, the enzyme was purified in good yield within two days. The specific activity of the enzyme purified by the immunoaffinity chromatography procedure is comparable with that of enzyme purified by a four-step chromatographic procedure. The mol. wt of the purified enzyme is approximately 23,000–24,000 as determined by SDS-PAGE. Interestingly, the enzyme purified by this two-step immunoaffinity chromatography procedure possesses virtually no hemorrhagic activity.

Use of flow cytometry to screen for the effects of environmental mutagens: Baseline DNA values in cottonmouth snakes

Since the late 1970's flow cytometry has been accepted as a fast and accurate technique for the quantification of nuclear DNA content (Deaven 1982). However, use of the technology has typically been confined to clinical settings due to the high cost of the instrumentation and the requirement for trained personnel. Flow cytometry recently has been applied to the study of genetic damage in wild populations. McBee and Bickham (1988) detected DNA damage in wild rodents inhabiting a dumpsite contaminated with petrochemicals, and Bickham et al. (1988) detected DNA aneuploidy in turtles found in seepage basins contaminated with radiation. Given these few studies, there is a need to establish a data base for the application of flow cytometry to environmental screening.

The crystal structure of a lysine 49 phospholipase A2 from the venom of the cottonmouth snake at 2.0-A resolution.

The crystal structure of a lysine 49 phospholipase A2 from the venom of the cottonmouth snake at 2.0-A resolution.

HPLC-based two-step purification of fibrinolytic enzymes from the venom of Agkistrodon contortrix contortrix and Agkistrodon piscivorus conanti

In investigations aimed at characterizing snake venom blood clot-dissolving enzymes, we have developed a rapid two-step high-performance chromatography method for the isolation of these fibrinolytic enzymes from the venoms of Agkistrodon contortrix contortrix and Agkistrodon piscivorus conanti. The first step consisted of hydrophobic interaction chromatography on a propyl-aspartamide column. Fractions containing the fibrinolytic activity were then concentrated and applied to a hydroxylapatite column. The resulting preparation, assessed for purity by reverse-phase chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was homogeneous. The molecular weight of both venom fibrinolytic enzymes was approximately 23,000 and amino acid analysis, immunological cross-reaction, cyanogen bromide, and tryptic digestion indicate a significant degree of structural similarity. However, the general proteolytic activity of the A. p. conanti venom enzyme was significantly lower than the corresponding activity of the A. c. contortrix venom, whereas their fibrinolytic activities were quite similar.

Binding of the snake venom-derived proteins applaggin and echistatin to the arginine-glycine-aspartic acid recognition site(s) on platelet glycoprotein IIb.IIIa complex inhibits receptor function.

In the present report we describe the platelet-binding characteristics of applaggin and echistatin, potent inhibitors of fibrinogen-dependent platelet aggregation derived from Agkistrodon piscivorus piscivorus and Echis carinatus snake venoms, respectively. Both molecules bound to unstimulated platelets in a specific and saturable manner. At saturation there were 37,100 +/- 3,150 (mean, +/- S.D.) molecules of applaggin and 27,200 +/- 2,816 molecules of echistatin bound/platelet, with dissociation constants (Kd) of 1.4 +/- 0.6 x 10(-7) M and 4.9 +/- 1.2 x 10(-7) M, respectively. Stimulation of platelets with ADP (10 microM) + epinephrine (2 microM) resulted in an increase in the number of molecules bound at saturation to 42,300 +/- 2,105 for applaggin and 32,185 +/- 3,180 for echistatin, with a Kd of 5.6 +/- 0.3 x 10(-8) M and 1.8 +/- 0.6 x 10(-7) M, respectively. The synthetic peptide (Arg)8-Gly-Asp-Val was a competitive antagonist of applaggin and echistatin binding to unstimulated platelets (Ki = 25 and 36 microM, respectively). Applaggin and echistatin inhibited the binding of fibrinogen to stimulated platelets in a dose-dependent manner, with an IC50 of 9 and 25 nM, respectively. In concert with inhibition of platelet aggregation, applaggin and echistatin inhibited platelet secretion and synthesis of thromboxane A2 induced by ADP, collagen, and human gamma-thrombin. The monclonal antibody, LJ-CP3, which inhibits the binding of Arg-Gly-Asp containing ligands to platelet GPIIb.IIIa, also inhibited applaggin binding to unstimulated platelets in a competitive manner (Ki = 4.5 microM). Thus, applaggin and echistatin bind to the platelet GPIIb.IIIa complex, and the Arg-Gly-Asp sequence plays a central role in mediating this interaction.