Platelet Activation and Signal Transduction by Convulxin, a C-type Lectin from Crotalus durissus terrificus (Tropical Rattlesnake) Venom via the p62/GPVI Collagen Receptor*

(Received for publication, January 9, 1997)

János Polgár Dagger , Jeannine M. Clemetson Dagger , Beate E. Kehrel §, Markus Wiedemann Dagger , Edith M. Magnenat , Timothy N. C. Wells and Kenneth J. Clemetson Dagger par

From the Dagger  Theodor Kocher Institute, University of Berne, Freiestrasse 1, CH-3012 Berne, Switzerland, § Experiment, Hämostaseforschung, Innere Medizin A, Universitätskliniken Münster, Domagkstrasse 3,  D-48149 Münster, Federal Republic of Germany, and  Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development S.A., 14, Chemin des Aulx, CH-1228 Plan-les-Ouates, Geneva, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Convulxin, a powerful platelet activator, was isolated from Crotalus durissus terrificus venom, and 20 amino acid N-terminal sequences of both subunits were determined. These indicated that convulxin belongs to the heterodimeric C-type lectin family. Neither antibodies against GPIb nor echicetin had any effect on convulxin-induced platelet aggregation showing that, in contrast to other venom C-type lectins acting on platelets, GPIb is not involved in convulxin-induced platelet activation. In addition, partially reduced/denatured convulxin only affects collagen-induced platelet aggregation. The mechanism of convulxin-induced platelet activation was examined by platelet aggregation, detection of time-dependent tyrosine phosphorylation of platelet proteins, and binding studies with 125I-convulxin. Convulxin induces signal transduction in part like collagen, involving the time-dependent tyrosine phosphorylation of Fc receptor gamma  chain, phospholipase Cgamma 2, p72SYK, c-Cbl, and p36-38. However, unlike collagen, pp125FAK and some other bands are not tyrosine-phosphorylated. Convulxin binds to a glycosylated 62-kDa membrane component in platelet lysate and to p62/GPVI immunoprecipitated by human anti-p62/GPVI antibodies. Convulxin subunits inhibit both aggregation and tyrosine phosphorylation in response to collagen. Piceatannol, a tyrosine kinase inhibitor with some specificity for p72SYK, showed differential effects on collagen and convulxin-stimulated signaling. These results suggest that convulxin uses the p62/GPVI but not the alpha 2beta 1 part of the collagen signaling pathways to activate platelets. Occupation and clustering of p62/GPVI may activate Src family kinases phosphorylating Fc receptor gamma  chain and, by a mechanism previously described in T- and B-cells, activate p72SYK that is critical for downstream activation of platelets.


INTRODUCTION

A large number of C-type lectins from snake venoms have been described over the last few years with effects on hemostasis. While most of these inhibit the function of the coagulation factors and platelet components that they bind to, a few activate platelets by direct or indirect effects. So far all of these have been shown to affect the von Willebrand factor (vWf)1-platelet GPIb-V-IX axis. They include botrocetin (1) and bitiscetin (2) that bind to and change the conformation of vWf so that it can bind to GPIb and thus activate platelets and alboaggregin B (3) that activates platelets directly by binding to, and presumably clustering, GPIb. A further snake peptide from the venom of some Crotalus species has subunits with a molecular mass similar to the C-type lectins, is a strong activator of platelet phospholipase C, and has been termed convulxin (4-8). We have isolated a similar, possibly identical, molecule from Crotalus durissus terrificus venom and show that it belongs to the heterodimeric, C-type lectin family. It activates platelets not via GPIb but through the p62/GPVI component of the platelet collagen receptor, probably by a clustering effect, and induces signals similar to a set of those induced by collagen in platelets.

Several platelet membrane glycoproteins have been implicated as collagen receptors, in particular GPIa-IIa (the alpha 2beta 1 integrin) (9-12), CD36 (13), and p62/GPVI (14-16). Since patients platelets lacking alpha 2beta 1 do not adhere to or aggregate to collagen (9, 10), and antibodies against alpha 2beta 1 block platelet adhesion to immobilized collagen (17-19), there is general agreement about the pivotal role of alpha 2beta 1 in the adhesion of platelets to collagen fibers. Furthermore, it was suggested that platelet adhesion to collagen through the alpha 2beta 1 integrin induces rapid tyrosine phosphorylation of pp125FAK; thus, the alpha 2beta 1 integrin plays a more direct role in early events of collagen-induced platelet activation, other than mediating the initial adhesion to collagen (20, 21). Many of the platelet responses, induced by collagen, can be induced by synthetic, triple helical, collagen-like peptides based upon typical collagen sequences incorporating multiple glycine-proline-hydroxyproline repeats (22-24). These responses are independent of the alpha 2beta 1 receptor, which shows that receptor(s) other than alpha 2beta 1 are also involved in the collagen-induced events. A platelet-activating antibody, anti-p62 IgG, found in a patient with autoimmune thrombocytopenia with a selective deficiency in collagen-induced platelet aggregation (25), immunoprecipitated a 62-kDa protein from normal platelets that was later described as GPVI (14). All four p62/GPVI-deficient patients, reported so far (14-16, 25), showed defective platelet responses only to collagen despite the normal expression of alpha 2beta 1. Although CD36, which has been found to be associated with Src family kinases (26), also may play some role, the fact that patients lacking CD36 adhere normally and aggregate to collagen (27-30) shows that CD36 is less critical in collagen-induced platelet activation than alpha 2beta 1 and p62/GPVI. A characteristic of platelet activation by collagen compared via G-protein-linked receptors, such as that for thrombin, is the early engagement of a wide range of tyrosine kinases, phosphatases, and their substrates. Collagen stimulation of platelets results in the phosphorylation of numerous proteins (reviewed in Ref. 31). Recent reports include collagen-induced tyrosine phosphorylation of phospholipase Cgamma 2 (PLCgamma 2) providing a mechanism for further platelet activation by increasing cytosolic Ca2+ and activating protein kinase C (32, 33) and of the Fc receptor gamma  chain (Fcgamma ) providing a mechanism for activation of the major tyrosine kinase p72SYK (24). Because there are at least two collagen receptors on platelets, but the relative contribution of each to collagen-induced platelet activation has not yet been determined, it is of interest to find platelet agonists that mimic the effect of collagen by acting through only one of the collagen receptors.

Here we show that convulxin, a powerful platelet agonist purified from C. durissus terrificus venom, belongs to the C-type lectin family, induces platelet activation/aggregation predominantly through one of the collagen receptors, p62/GPVI, and identify some of the signaling molecules involved.


EXPERIMENTAL PROCEDURES

Materials

Lyophilized C. durissus terrificus venom, Protein A-Sepharose, peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies, bovine serum albumin, wheat germ agglutinin (WGA), and Triton X-114 were from Sigma. Sepharose 4B was from Pharmacia Fine Chemicals (Uppsala, Sweden). Iloprost was a kind gift from Schering AG (Zürich, Switzerland). Na125I was from Amersham Corp. (Zürich, Switzerland), IODO-GEN and the SuperSignal chemiluminescence detection systems were from Pierce. Autoradiography (Fuji RX) films were from Fujifilm (Dielsdorf AG, Switzerland). Methylated type I calf skin collagen was a kind gift from Dr. J. Rauterberg. Piceatannol was from Boehringer Mannheim (Germany). Anti-phosphotyrosine mAb (4G10) was from Upstate Biotechnology Inc. (Lake Placid, NY); anti-p72SYK (4D10) mAb, anti-c-Cbl (C-15), and anti-pp125FAK (A-17) polyclonal antibodies and Grb2-(54-164)-AC were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-Fcgamma RII (IV.3) mAb was from Medarex Inc. (Annandale, NJ); AP-1 anti-GPIb mAb was a kind gift from Dr. T. J. Kunicki; SZ-2 anti-GPIb mAb was a kind gift from Dr. C. Ruan; Ib-23 anti-GPIb mAb and Ro44-9883, a GPIIb-IIIa inhibitor, were kind gifts from Dr. B. Steiner; PTA-1 mAb was a kind gift from Prof. G. Burns; human anti-GPVI polyclonal antibody was a kind gift from Prof. M. Okuma; anti-GP60 (a putative laminin receptor) mAb was a kind gift from Dr. S. Sentoso; mAb 6F1 against alpha 2beta 1 was a kind gift from Prof. B. Coller; polyclonal antibody against Fcepsilon RIgamma was a kind gift from Prof. J.-P. Kinet. Echicetin was purified from lyophilized Echis carinatus sochureki venom (Latoxan, Rosans, France) as described previously (34). PVDF membranes were PolyScreen from DuPont NEN. Octyl-N-methylglucamide was from Oxyl Chemie (Bobingen, Germany).

Purification of Convulxin

Lyophilized C. durissus terrificus venom was dissolved at 250 mg/8 ml in 300 mM NaCl, 100 mM ammonium formate, pH 3.5 (buffer A). Insoluble components were removed by centrifugation, and the supernatant was loaded on a Fractogel EMD BioSEC 650 (S) column (16 × 1200 mm; Merck, Darmstadt, Germany) equilibrated with buffer A. At 0.5 ml/min flow rate, 5-ml fractions were collected. Fractions were analyzed by SDS-PAGE/silver staining under nonreducing and reducing conditions and assayed for their ability to induce platelet aggregation. The pH of 100-µl aliquots was adjusted to 7.4 by adding 10 µl of 1 M Tris, pH 8.5, before the aggregation studies. Fractions that induced platelet aggregation and showed strong 85-kDa (nonreduced) and 14- and 16-kDa (reduced) bands on analysis by SDS-PAGE/silver staining were pooled (15 ml), dialyzed against diluted HCl, pH 3.5, dissolved in 3 ml of buffer A, and rechromatographed under the same conditions as above. Fractions corresponding to the main peak, high purity convulxin, were concentrated to about 20% of their original volume on a Speed-Vac. If a sample became opalescent during this step, a small volume of 0.1% trifluoroacetic acid was added to redissolve the protein. After dialysis against first, 150 mM NaCl, 50 mM phosphate, pH 5.0, second, the same buffer at pH 6.5, and finally, the same buffer or 150 mM NaCl, 50 mM Tris/HCl, pH 7.4, the convulxin was stored at 4 °C until used.

Purification and Sequence Analysis of Convulxin Subunits

To 1 volume of 300 µg/ml convulxin in 6 M guanidine HCl, 0.1 M Tris, pH 8.0, was added to 1/20 volume 440 mM DTT followed by incubation at 45 °C for 30 min. 1/20 volume of 1 M 4-vinylpyridine was added, and after 1 h incubation at room temperature the sample was diluted with 2 volumes of 0.2% trifluoroacetic acid. Modified convulxin subunits were isolated by reverse phase HPLC on a wide pore C4 column (4.6 × 250 mm; J. T. Baker Inc.) using an acetonitrile gradient (0.1% trifluoroacetic acid). N-terminal sequencing of S-pyridylethylated alpha  and beta  subunits of convulxin was performed on an Applied Biosystem model 477 A pulsed-liquid-phase protein sequencer with model 120 A on-line phenylthiohydantoin amino acid analyzer.

Preparation of Partially Reduced/Denatured Convulxin

Partially reduced/denatured convulxin was prepared in the same way as S-pyridylethylated alpha  and beta  subunits of convulxin, omitting 4-vinylpyridine so that the free -SH groups were not chemically modified in these samples. The protein was isolated, free from denaturing and reducing agents, by reverse phase HPLC, freeze-dried, and dissolved in 0.05% trifluoroacetic acid. This treatment resulted in three main fractions corresponding to the two subunits and a fraction that contained both subunits. The ratio of these three fractions varied between preparations.

Labeling of Convulxin with 125I

Convulxin was labeled by the IODO-GEN procedure (35). Convulxin, 80 µg, and 800 µCi of Na125I were added to a glass tube that had been coated with 150 µg of IODO-GEN. The reaction mixture was incubated for 30 min at room temperature with gentle agitation. The reaction was stopped by adding 30 mg/ml (final) KI and labeled convulxin was separated from free Na125I on a Bio Gel P-6 DG column (0.9 × 16 cm, Bio-Rad) using 5 mg/ml bovine serum albumin, 300 mM NaCl, 50 mM Tris/HCl, pH 7.4. The specific activity of labeled convulxin was between 0.5 and 1.2 × 109 cpm/mg.

Computer Analysis of the Sequence Data

Computer analysis of the sequence data was performed on a VAX/VMS system using the suite of programs from the University of Wisconsin Genetics Computer Group.

Protein Determination

Protein determination was performed by the BCA protein assay (Pierce) with bovine serum albumin as a standard.

SDS-PAGE/Silver Staining

SDS-PAGE was performed according to Laemmli (36), and the gels were silver-stained by the method of Morrissey (37).

Preparation of Washed Platelets, Platelet Aggregation, and Immunoprecipitations

Human platelets were isolated from buffy coats, less than 20 h after blood collection, obtained from the Central Laboratory of the Swiss Red Cross Blood Transfusion Service. To one buffy coat was added 30 ml of 100 mM citrate, pH 6.5. Platelet-rich plasma and the platelet pellet was isolated by successive centrifugation steps. Platelets were resuspended in 113 mM NaCl, 4.3 mM K2HPO4, 4.3 mM Na2HPO4, 24.4 mM NaH2PO4, 5.5 mM glucose, pH 6.5 (buffer B) and centrifuged at 250 × g for 5 min. The platelet-rich supernatant was centrifuged at 1000 × g for 10 min, and platelets were washed with buffer B once more. Washed platelets were resuspended in 20 mM Hepes, 140 mM NaCl, 4 mM KCl, 5.5 mM glucose, pH 7.4 (buffer C), and the platelet count was adjusted to 5 × 108 platelets/ml by dilution with buffer C. Samples were kept at room temperature until used for aggregation studies. Platelet aggregation was monitored by light transmission in an aggregometer (Lumitec, France) with continuous stirring at 1100 rpm at 37 °C. Platelets were preincubated in buffer containing 2 mM CaCl2 at 37 °C for 2 min before starting the measurement by adding the samples for analysis. For immunoprecipitation, aliquots (700 µl, 5 × 108 platelets/ml) of control, resting as well as activated platelets were solubilized in phosphate-buffered saline containing 1.2% Triton X-100 with 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 2 mM N-ethylmaleimide (NEM), 2 mM benzamidine, and 2 mM sodium orthovanadate. After centrifugation, platelet lysates, precleared with Protein A-Sepharose, were stirred for 2 h with specific antibodies before adding 20 µl of Protein A-Sepharose followed by 6-8 h incubation.

Triton X-114 Phase Separation and Wheat Germ Agglutinin Affinity Chromatography

Human platelets were isolated from 10 buffy coats as described previously (38). The platelet count was adjusted to 1 × 109 platelets/ml with 20 mM Tris/HCl, pH 7.4, 10 mM EDTA, 154 mM NaCl (TENA buffer), and Triton X-114 phase separation was performed as described previously (39). The Triton phase was diluted 10 times with 2 mM sodium orthovanadate, 2 mM NEM, 4 mM EDTA, 20 mM NaCl, 10 mM Tris/HCl, pH 7.4 (buffer D), and centrifuged at 10,000 × g for 30 min at 4 °C. The supernatant was applied to a column of WGA-Sepharose 4B (2.6 × 15 cm, 1 mg of WGA/ml of Sepharose 4B) equilibrated with buffer D. The column was washed intensively with buffer D containing 0.1% octyl-N-methylglucamide. The bound material was eluted with 2.5% N-acetylglucosamine in buffer D containing 0.5% octyl-N-methylglucamide. Fractions containing glycoproteins were pooled, concentrated 10-fold by ultrafiltration, dialyzed against water, and freeze-dried. The sample was dissolved in 4 ml of phosphate-buffered saline, pH 7.2, containing 2 mM sodium orthovanadate, 2 mM NEM, 4 mM EDTA. 750-µl aliquots of this preparation were used for immunoprecipitations.

Identification of 125I-Labeled Convulxin-binding Proteins in Immunoprecipitated Platelet Proteins by an Affinity Blotting Method

Aliquots (750 µl) of platelet glycoprotein samples (see above) were incubated at 4 °C for 3 h with 5 µl of anti-PTA1, anti-p62/GPVI, and anti-GP60 antibodies followed by incubation with 5 mg of Protein A-Sepharose at 4 °C overnight. The Protein A-Sepharose samples were washed four times by centrifugation in ice-cold phosphate-buffered saline, pH 7.2, containing 2 mM sodium orthovanadate, 2 mM NEM, and 4 mM EDTA. The samples were boiled in 40 µl of 2% SDS, 50 mM Tris/HCl, pH 8.0, for 5 min and centrifuged, and the supernatants were electrophoresed on a 7-17% gradient SDS-polyacrylamide gel. Proteins were transferred electrophoretically to PVDF membrane, and convulxin-binding proteins were detected using 125I-labeled convulxin. The membranes were incubated with 1 µg/ml labeled convulxin in 2% bovine serum albumin, 300 mM NaCl, 50 mM Tris/HCl, pH 7.4, for 3 h followed by washing 7 times with the same buffer for 30 min. The membranes were rinsed with water, dried, and radioactive labeling detected by autoradiography using a PhosphorImager (Molecular Dynamics).


RESULTS

Convulxin Is a Heterodimeric C-type Lectin

Convulxin was purified from lyophilized C. durissus terrificus venom by a two-step gel filtration method (Fig. 1). The final product showed an 85-kDa broad band under nonreducing conditions and two bands, 14 and 16-kDa, under reducing conditions when analyzed by SDS-PAGE/silver staining (see inset in Fig. 1B). Treatment with DTT alone under nondenaturing conditions was not sufficient to separate the subunits by reverse phase HPLC. However, S-pyridylethylation allowed a separation of the subunits by reverse phase HPLC. Fractionation of reduced S-pyridylethylated convulxin subunits is shown in Fig. 2. Peaks in the 3-10-min interval represent non-protein peaks originated from the reagents used for reduction and S-pyridylethylation. Fractions 1 and 2 contained homogenous single chain proteins with apparent Mr of 16 and 14, respectively, on analysis by SDS-PAGE/silver staining (see inset of Fig. 2). Following the nomenclature of Marlas (7), we called the 14-kDa protein (fraction 2) the alpha subunit and the 16-kDa protein (fraction 1) the beta  subunit. N-terminal sequence analysis gave the 20 amino acid sequence GFCCPSHWSXYDRYCYKVFK for the alpha  subunit and GLHCPSDWYYYDQHCYRIFN for the beta  subunit. Computer analysis of the sequences showed that convulxin is a heterodimeric C-type lectin with strong sequence similarity to other venom C-type lectins (Fig. 3).


Fig. 1. Purification of convulxin from C. durissus terrificus venom. Typical chromatograms of gel filtration chromatography on Fractogel EMD BioSEC 650 (S) column. A, separation of crude venom; bar indicates pooled, active fractions. B, rechromatography of pooled, active fractions from A. The inset in B shows convulxin bands analyzed by SDS-PAGE/silver staining. N, nonreducing conditions; R, reducing conditions. mAU, milliabsorbance units at 280 nm. (For details see "Experimental Procedures.")
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Fig. 2. Fractionation of reduced and S-pyridylethylated convulxin on reverse phase HPLC column. 1, convulxin alpha  subunit; 2, convulxin beta  subunit; 3, probably a differently glycosylated form of the convulxin beta  subunit (not sequenced). Note that peaks in the 3-10-min interval originated from the reagents used for reduction and S-pyridylethylation. mAU, milliabsorbance units at 216 nm; %B, percentage of 80% acetonitrile. The inset shows fractions 1-3 analyzed by SDS-PAGE/silver staining. (For details see "Experimental Procedures.")
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Fig. 3. Sequence comparison of alpha  and beta  subunits of heterodimeric venom C-type lectins that act on platelets. Cvx alpha  and Cvx beta , alpha  and beta  subunits of C. durissus terrificus convulxin (present report). Echi alpha , alpha  subunit of E. carinatus echicetin; Echi beta , beta  subunit of E. carinatus echicetin (46); GPIb-BP alpha  and GPIb-BP beta , alpha  and beta  subunits of Bothrops jararaca GPIb-binding protein (47); Albo alpha  and Albo beta , alpha  and beta  subunits of Trimeresurus albolabris alboaggregin-B (55); Botro alpha  and Botro beta , alpha  and beta  subunits of B. jararaca two-chain botrocetin (1). Positions with amino acids conserved in at least 8 of the 10 sequences are shown in bold. Venom C-type lectins that act on platelets through GPIb are grouped separately in this sequence comparison.
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Convulxin Does Not Act through GPIb or alpha 2beta 1

Convulxin is a powerful platelet agonist and, at concentrations as low as 3-10 ng/ml, induced maximal aggregation of isolated human platelets (data not shown). The strong sequence similarity to C-type lectins that bind to GPIb suggested that GPIb might be involved in platelet activation induced by convulxin. However, neither antibodies against GPIb (Ib-23, SZ-2, and AP-2) nor echicetin, a snake venom C-type lectin that binds to GPIb and inhibits platelet agonists acting through GPIb (34), had any effect on convulxin-induced platelet aggregation (data not shown). In addition, partially reduced/denatured convulxin had no effect on ristocetin-vWf-induced platelet agglutination (see below). These data show that GPIb is not involved in convulxin-induced platelet activation. A mAb against alpha 2beta 1 (6F1), which inhibits collagen-induced platelet activation, had little effect on convulxin-induced platelet aggregation (data not shown).

Partially Reduced/Denatured Convulxin Only Affects Collagen-induced Platelet Aggregation

Neither of the reduced, S-pyridylethylated convulxin subunits activated platelets (data not shown), suggesting that the platelet activation effect of convulxin requires its native conformation rather than a linear sequence in its subunits. Neither incubation of convulxin with up to 50 mM DTT at 37 °C for 30 min nor freezing and thawing the convulxin sample in the presence of 50 mM DTT decreased the ability of convulxin to aggregate platelets. After reduction of convulxin under denaturing conditions, in the presence of 6 M guanidine HCl, reverse phase HPLC gave three main peaks corresponding to the two subunits and a fraction that contained both subunits. The ratio of these peaks varied between preparations (n = 10, data not shown). None of these partially reduced/denatured convulxin subunits induced platelet aggregation or signal transduction. Seven out of ten preparations, containing either alpha , beta , or both subunits, had no effect on platelet aggregation induced by various agonists. However, the others inhibited convulxin-induced aggregation as well as collagen-induced platelet aggregation in a concentration-dependent manner, whereas they had no effect on platelet aggregation induced by thrombin or ristocetin-vWf. These data show that convulxin and its two subunits bind to a common receptor that is involved in collagen activation of platelets.

Convulxin Binds to p62/GPVI

The fact that the N-terminal sequences of convulxin are rich in tyrosine suggested that it might be readily labeled by 125I without affecting its biological activity, and this proved to be the case. The platelet membrane protein binding 125I-labeled convulxin was identified. It is a 62-kDa hydrophobic, platelet membrane glycoprotein (Fig. 4, lane 1) present in the Triton phase after Triton X-114 phase separation of platelet membrane proteins that binds to WGA (Fig. 4, lane 2). 125I-Labeled convulxin binds strongly only to the 62-kDa protein immunoprecipitated with anti-p62/GPVI antiserum (Fig. 4, lane 6) and not to other platelet membrane glycoproteins with similar molecular masses and physicochemical properties immunoprecipitated by specific antibodies (Fig. 4, lane 4, anti-PTA-1 mAb and lane 5, anti-GP60 laminin receptor mAb). This argues convincingly that the p62/GPVI collagen receptor is the binding site for convulxin on platelets.


Fig. 4. Identification of platelet glycoproteins that bind 125I-labeled convulxin. Samples were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with 125I-labeled convulxin, washed, and analyzed by autoradiography (see "Experimental Procedures"). Lane 1, platelet lysate (2% SDS), 50 µl, 1 × 109 platelets/ml; lane 2, WGA-bound fraction of Triton X-114 phase of platelet glycoproteins, 10 µl; lane 3, empty lane; lane 4, control sample for immunoprecipitates. Protein A-Sepharose was treated as samples 5-7 but without antibody; lane 5, immunoprecipitates with anti-PTA-1 antibody; lane 6, immunoprecipitates with anti-p62/GPVI antibody; lane 7, immunoprecipitates with an anti-GP60 putative laminin receptor antibody. Note that the slightly faster migration of the labeled band in lane 2 is due to the presence of Triton X-114 in this sample.
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Tyrosine Phosphorylation Signal Transduction by Convulxin Compared with Collagen

Fig. 5 shows a time range of tyrosine phosphorylation for platelets activated by 5 mg/ml collagen compared with that with 30 ng/ml convulxin. These relative amounts were chosen because they gave comparable rates of platelet aggregation as estimated by the slope of the aggregometer curve. Such aggregation slopes are often used as the basis for comparison of activation rates. With convulxin several proteins are phosphorylated more rapidly but also more intensely than with collagen. These include Fcgamma , p36-38, p72SYK, PI3K, c-Cbl, and PLCgamma 2. These tyrosine phosphorylations induced by convulxin were not inhibited by raising the adenylate cyclase level by adding Iloprost (final concentration 0.15 nM) to the platelet suspension 5 min before the agonist (data not shown). These components were identified by immunoprecipitation with specific antibodies or, in the case of p36-38, by precipitation with a GST-Grb2 SH2 fusion protein. Some of these data are shown in Fig. 6. In Fig. 6A the first three lanes show the tyrosine phosphorylation pattern at 30 s of resting platelets, platelets activated by collagen, and platelets activated by convulxin, respectively, and the following three lanes show the phosphotyrosine proteins binding to GST-Grb2 SH2 domain from these platelet preparations. This clearly identifies the tyrosine-phosphorylated p36-38 band present in the platelets as a major Grb2 SH2-binding protein that is much more tyrosine-phosphorylated in convulxin-activated platelets and is distinct from the immunoprecipitated Fcgamma RIIA receptor that has a higher molecular mass as shown in the five lanes on the right. Thus p36-38 is perhaps the human equivalent of, or at least related to, the Lnk adaptor protein (40). Fcgamma RIIA also shows raised levels of tyrosine phosphorylation after platelet activation by collagen or convulxin but much less than p72SYK, Fcgamma , and c-Cbl. Similarly, in Fig. 6B the upper bands show the level of tyrosine phosphorylation in immunoprecipitates from resting, collagen-, and convulxin-activated platelets, with p72SYK, Fcgamma , and c-Cbl antibodies, respectively, and the lower bands show the same blots after stripping and restaining with the corresponding antibodies to show that equal amounts of each component were immunoprecipitated from each platelet preparation. p72SYK, Fcgamma , and c-Cbl are all tyrosine-phosphorylated after platelet activation by collagen compared with virtually nil or very low levels in resting platelets but are even more dramatically tyrosine-phosphorylated after platelet activation by convulxin. Fig. 6C shows that whereas convulxin is a powerful activator of pp125FAK, this was delayed compared with collagen and was completely inhibited in the presence of 1 µM Ro44-9883, a specific, efficient inhibitor of fibrinogen binding to GPIIb-IIIa (41), indicating that the activation of pp125FAK by convulxin is via activation of GPIIb-IIIa and release and binding of fibrinogen rather than via alpha 2beta 1 that is an early event in platelet activation by collagen and is not prevented by GPIIb-IIIa inhibitors. In some cases, however, the collagen induced a more rapid and stronger earlier phosphorylation than convulxin. This was the case with a band at 125 kDa (Fig. 5), identified as p125FAK in Fig. 6C, and with so far unidentified bands at about 32 and 28 kDa (Fig. 5). As well as p36-38, GST-Grb2 SH2 fusion protein precipitated an additional band at about 30 kDa from a lysate of collagen-activated platelets but not from convulxin-activated platelets (Fig. 6A).


Fig. 5. Time dependence of tyrosine phosphorylation in proteins from platelets activated by collagen or convulxin. Washed platelets (700 µl) were stirred at 1100 rpm at 37 °C. After addition of 3 µg of collagen or 30 ng of convulxin, aliquots were removed at the times indicated and dissolved in SDS buffer containing inhibitors. After separation by SDS-PAGE gel electrophoresis (7-20% acrylamide gradient) and transfer to PVDF membranes, the proteins were incubated with the anti-phosphotyrosine antibody 4G10 before detection by a peroxidase-linked second antibody and chemiluminescence.
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Fig. 6. Identification of bands with increased tyrosine phosphorylation in platelets after activation by collagen or convulxin. A, control platelets or platelet suspension activated by collagen (coll) or convulxin (con) were lysed in Triton X-100 buffer containing inhibitors. Aliquots were incubated with GST-Grb2 SH2 agarose conjugate or immunoprecipitated with anti-Fcgamma RIIA antibodies. The proteins eluted from the GST-Grb2 SH2 agarose and the immunoprecipitates were separated by SDS-PAGE gel electrophoresis (7-20% acrylamide gradient). After transfer, the proteins were incubated with 4G10 anti-phosphotyrosine antibody and detected by a peroxidase-linked second antibody and chemiluminescence. B, control platelets or platelets suspension activated by collagen or convulxin for 30 s were lysed in Triton X-100 buffer containing inhibitors. Aliquots were immunoprecipitated with antibodies against p72SYK, Fcgamma , or c-Cbl. After SDS-PAGE and Western blotting, the proteins were detected with 4G10 anti-phosphotyrosine antibody. The membranes were stripped and treated with anti-p72SYK, anti-Fcgamma , or anti-c-Cbl antibodies. C, control platelets or platelet suspension were activated by convulxin in the presence of 1 µM Ro44-9883, a GPIIb/IIIa inhibitor, and by convulxin or collagen. Aliquots were immunoprecipitated with antibodies against pp125FAK. After SDS-PAGE and transfer to PVDF membrane, the proteins were detected with 4G10 anti-phosphotyrosine antibody. The membranes were stripped and treated with anti-pp125FAK antibody.
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Partially Reduced/Denatured Convulxin Subunits Inhibit Aggregation and Tyrosine Phosphorylation Signal Transduction by Collagen

Partially reduced/denatured convulxin subunits inhibit collagen-induced platelet aggregation in a dose-dependent way. Fig. 7A shows the aggregation response to collagen in the presence and absence of an intermediate dose of a typical preparation of subunit(s). In the presence of the subunit(s) the aggregation is rapid at first but quickly reaches a maximum and reverses, leading to platelet disaggregation. Fig. 7B shows the time range tyrosine phosphorylation profile. Both subunits gave similar effects. Compared with the collagen control, in the presence of the subunits the tyrosine phosphorylation is slower, much less intense, of shorter duration, and those bands that do show clear but brief increases included those corresponding to pp125FAK and the 32- and 28-kDa groups. Tyrosine phosphorylation of bands corresponding to p36-38 and to Fcgamma were strongly inhibited and did not increase during the aggregation phase.


Fig. 7. Aggregation response and time dependence of tyrosine phosphorylation in proteins from platelets activated by collagen or by collagen in the presence of convulxin subunits. Washed platelets (700 µl) were stirred at 1100 rpm at 37 °C in the presence or absence of 200 ng of convulxin subunits. After 90 s, 1 µg of collagen was added. A, aggregation curves. B, aliquots were removed at the times indicated and dissolved in SDS buffer containing inhibitors. After separation by SDS-PAGE gel electrophoresis (7-20% acrylamide gradient) and transfer to PVDF membranes, the proteins were incubated with the anti-phosphotyrosine antibody 4G10 before detection by a peroxidase-linked second antibody and chemiluminescence.
[View Larger Version of this Image (45K GIF file)]

Effects of Piceatannol on Signaling through Tyrosine Phosphorylation by Convulxin Compared with Collagen

As previously reported (42) piceatannol inhibits both platelet aggregation and tyrosine phosphorylation induced by collagen. In the case of convulxin, however, the aggregation was not completely inhibited at doses of convulxin giving an equivalent aggregation rate, whereas that of collagen was completely inhibited. In this experiment lower amounts of the agonists were used to be able to inhibit convulxin-induced aggregation at relevant doses of piceatannol. With convulxin a brief reversible aggregation similar to, but weaker than, that shown in Fig. 7A for the convulxin subunits was observed. In addition, unlike with collagen, the tyrosine phosphorylation in general was not inhibited and, compared with control, convulxin- activated platelets lasted longer (Fig. 8). Those proteins with prolonged tyrosine phosphorylation included p36-38, Fcgamma , and several unidentified bands including two at about 80 and 180 kDa. On the other hand, tyrosine phosphorylation of some bands was inhibited by piceatannol in convulxin-activated platelets (Fig. 8B). These included unidentified bands at 85 and 120 kDa. Tyrosine phosphorylation of p72SYK lasted longer in collagen than in convulxin-activated platelets, peaking in the latter between 10 and 30 s. Tyrosine phosphorylation of p72SYK was clearly inhibited by piceatannol in collagen-activated platelets. In convulxin-activated platelets it appeared inhibited at 10 s but less so at 30 s, otherwise the time course was not affected.


Fig. 8. Time dependence of tyrosine phosphorylation in proteins from platelets activated by collagen and convulxin in the presence and absence of piceatannol. Washed platelets (700 µl) were stirred at 1100 rpm at 37 °C for 7 min in the presence of 25 µM piceatannol. After addition of 1 µg of collagen or 10 ng of convulxin, aliquots were removed at the times indicated and dissolved in SDS buffer containing inhibitors. After separation by SDS-PAGE gel electrophoresis (7-20% acrylamide gradient) and transfer to PVDF membranes, the proteins were incubated with the anti-phosphotyrosine antibody 4G10 before detection by a peroxidase-linked second antibody and chemiluminescence.
[View Larger Version of this Image (57K GIF file)]


DISCUSSION

The venom from several Crotalus species contains a heterodimeric protein that is a powerful platelet activator. We isolated, separated, and determined the N-terminal sequence, 20 amino acids, of the alpha  and beta  subunits of this protein purified from C. durissus terrificus venom and show that this protein belongs to the C-type lectin family, with sequences most similar to alboaggregin B. The previously published very short N-terminal sequences of the protein identified and defined as convulxin in the venom of Crotalus durissus cascavella, GFRPD for both subunits (7), are not identical to the sequences we found. Thus, while it is likely to be the same protein and would appear to have similar properties, at the moment we cannot exclude that it is a closely related protein since many snake venoms do contain such families of proteins, and the relative amounts may vary depending on the place where the snake comes from and the major prey in that area (43). In the absence of information to the contrary we shall use the name convulxin here. Because so far the only C-type lectins that activate platelets do so via the vWf-GPIb axis, we first of all checked if this was true for convulxin, but the platelet activation was inhibited neither by specific anti-GPIb antibodies that inhibit platelet-activation by vWf-ristocetin nor by echicetin, another C-type lectin from E. carinatus venom demonstrated to bind to GPIb and to inhibit platelet activation (34).

Earlier results showing that platelet activation by both collagen and convulxin was antagonized by methylation inhibitors that did not interfere with other platelet agonists, such as thrombin, ADP, or Ca2+ ionophore A 23187 (44, 45), and specific crossed desensitization between convulxin- and collagen-activated platelets (5), suggested that convulxin and collagen might share common receptors. The mechanism of platelet activation by collagen is still controversial. At least three molecules have been implicated as receptors, GPIa-IIa (the integrin alpha 2beta 1) (9-12), CD36 (13), and p62/GPVI (14-16). Accumulated evidence points to a major role of alpha 2beta 1 in adhesion, whereas p62/GPVI may be more important in activation, shape change, and secretion. To find out whether convulxin receptors might be involved, we prepared purified subunits of convulxin under conditions where we expected at least some retention of activity and examined their effect on collagen-induced platelet activation. There has been some controversy about the functionality of reduced, separated subunits of snake venom C-type lectins. For example, among those recognizing GPIb, echicetin beta  was shown to retain activity and to inhibit thrombin and vWf activation of platelets (46), whereas the isolated subunits of GPIb-binding protein were inactive (47). An explanation of these differences might be that the active preparations were probably only partly reduced and could, at least partly, refold to an active conformation, whereas the inactive preparations had been efficiently reduced and alkylated, so that refolding and disulfide bridge formation were effectively prevented. Thus it is essential not to block the free -SH groups to allow reformation of disulfide bridges after separation of the subunits. Partially reduced/denatured samples containing alpha , beta , or both subunits of convulxin did not induce platelet aggregation or tyrosine phosphorylation (Fig. 7), but they did inhibit platelet aggregation induced by collagen while having no effect on platelet aggregation induced by thrombin or vWf-ristocetin, strongly implicating the collagen receptor(s) in convulxin-induced platelet activation. However, a mAb to alpha 2beta 1 that blocks collagen-induced platelet aggregation had only very weak effects on convulxin-induced aggregation. A remaining candidate for the convulxin receptor is p62/GPVI. 125I-Labeled convulxin bound to a 62-kDa membrane glycoprotein and to a similar sized molecule in the material immunoprecipitated by anti-p62/GPVI antibodies known to activate platelets by a collagen-like mechanism (48) but not to material immunoprecipitated by antibodies recognizing other platelet membrane glycoproteins of similar molecular mass and physicochemical properties (anti-PTA-1 or anti-GP60 putative laminin receptor antibodies). Thus, the evidence supports a model where both subunits of convulxin bind to the p62/GPVI collagen receptor. This heterodimeric binding to the same protein together with the fact that convulxin exists as disulfide-linked hexamers, alpha 3beta 3, which are able to form even larger structural units up to 150 Å in diameter (6, 7), immediately suggests an activation mechanism based on cross-linking and clustering p62/GPVI. It has been suggested many times that collagen with its repetitive structure and many available binding sites may also work by such a mechanism. In addition, signal transduction by convulxin shows many similarities to that induced by collagen and, as recently shown, by collagen-like peptides (23). Important molecules identified in collagen-induced signaling include the gamma  subunit of Fcepsilon RI, p72SYK, PLCgamma 2, and a p36-38 molecule (24). In platelets activated by convulxin these are all rapidly tyrosine-phosphorylated as well suggesting that, as with collagen itself, it is the clustering of p62/GPVI that has a major role in activating these pathways. Studies with collagen, collagen-like peptides, and WGA have all shown that Fcgamma and PLCgamma 2 are rapidly phosphorylated/activated, and it has been suggested that p62/GPVI may be tightly associated with Fcgamma and activates Src family kinases by a mechanism previously described in T- and B-cells (reviewed in Ref. 49) leading to activation of p72SYK that is critical for downstream engagement of the enzymes involved in further platelet stimulation (24). The results described above clearly indicate that convulxin interacts with platelets predominantly via the p62/GPVI receptor, whereas collagen uses additional receptors including GPIa/IIa (alpha 2beta 1) and possibly CD36. It might therefore be expected that the signal transduction resulting from engagement of these receptors would show related differences. Overall the pattern of tyrosine phosphorylation in platelets activated by convulxin compared with collagen did show considerable similarity indicating that they do share common pathways. However, there were also marked differences in the strength and timing of the signals. A problem in comparing such signals is choosing an appropriate concentration of reagent to use in each case. In this case we decided to use amounts that give comparable rates of aggregation as reflected by the slope of their aggregation curves. The rate of aggregation reflects the final confluence of signaling pathways in activation of platelets. In the convulxin-activated platelets Fcgamma , p36-38, p72SYK, PI3K, c-Cbl, and PLCgamma 2 show a more rapid and more intense phosphorylation compared with platelets activated with collagen. Logically these components should then be linked to the p62/GPVI receptor pathway. On the other hand, bands at 125, 32, and 28 kDa were less tyrosine-phosphorylated than those in the corresponding controls activated by collagen and should therefore be related to other receptors such as alpha 2beta 1. In collagen-activated platelets the 125-kDa band was shown to be pp125FAK, and it was also found to be strongly tyrosine-phosphorylated later in convulxin-activated platelets (Fig. 6). However, since this could be prevented by a specific GPIIb-IIIa inhibitor, it is clearly related to release of fibrinogen and binding to activated GPIIb-IIIa in convulxin-activated platelets and not to binding to alpha 2beta 1 as with collagen. It was also of interest to note that when platelet aggregation to collagen was blocked by convulxin subunits the tyrosine phosphorylation of the pp125FAK, 32-, and 28-kDa bands was markedly less affected suggesting that, in this case, the bulk of the signal transduction occurs via alpha 2beta 1 but that it was not adequate to maintain the platelets in an activated state and they therefore disaggregated. This distinctive use by convulxin of the p62/GPVI pathway but not the alpha 2beta 1 is also supported by the lack of effect of anti-alpha 2beta 1 antibodies on convulxin-induced platelet aggregation as well as the strong parallels with the effects of the collagen-like peptides and the anti-p62/GPVI antibodies on platelets. Activation of platelets with collagen-like peptides that are also thought not to use the alpha 2beta 1 receptor gave a very similar tyrosine phosphorylation pattern to that obtained with convulxin (23, 24). The collagen-like peptides also gave enhanced and rapid tyrosine phosphorylation of bands in the positions of p36-38, p72SYK, PI3K, c-Cbl, and PLCgamma 2, whereas bands at 125, 32, and 28 kDa were also less tyrosine-phosphorylated than those in the corresponding controls activated by collagen (23).

Recently, it was demonstrated that the tyrosine kinase inhibitor, piceatannol, which shows specificity for p72SYK (50), was an efficient inhibitor of collagen-induced platelet aggregation and, indeed, inhibited tyrosine phosphorylation of signal transduction components (42). Surprisingly, with convulxin as agonist it showed some dramatically different effects causing rather a prolonged phosphorylation of several components including p36-38. This would indicate that in convulxin-activated platelets p72SYK has a major role in activation of tyrosine phosphatases, whereas in collagen-activated platelets there are alternative pathways for activation of these phosphatases via other receptors (51) or, alternatively, that piceatannol can also inhibit directly a phosphatase activated by the p62/GPVI pathway, whereas the other pathways activate additional non-inhibitable phosphatases. It might also indicate that p72SYK is not involved in phosphorylating p36-38 in convulxin-activated platelets. It was also recently shown that anti-alpha 2beta 1 antibodies block tyrosine phosphorylation of both p72SYK and PLCgamma 2 by collagen (42). On the other hand, cross-linking anti-alpha 2beta 1 antibodies activated platelets and increased phosphorylation of both p72SYK and PLCgamma 2. However, this involved the Fcgamma RIIA receptor, and F(ab)2 fragments were not stimulatory. There is thus good evidence linking p62/GPVI with Fcgamma and p72SYK in a common pathway that resembles that of the T- and B-cell receptors. Both p36-38-like molecules and c-Cbl have been closely implicated in these latter receptor mechanisms (52, 53).

The fact that agents such as convulxin or the collagen-like peptides are able to activate platelets efficiently and in a very similar way to collagen raises the question why alpha 2beta 1 and CD36 are necessary in physiological situations. The main evidence for a role for these other receptors comes from inhibitory studies with specific antibodies. The CD36 negative platelets respond relatively normally to collagen (27-30), and it was never possible to check if the alpha 2beta 1-deficient platelets (9, 10) had normal levels of p62/GPVI nor was signal transduction investigated. However, since collagen contains p62/GPVI binding sites, supported by the poor response to collagen of p62/GPVI-deficient platelets, as well as the inhibitory effect of the convulxin subunits on collagen-induced activation and the characteristics of the platelet response to collagen-like peptides, why are the alpha 2beta 1 receptors necessary? There are several plausible answers. The alpha 2beta 1 receptors are certainly necessary for platelet adhesion (19). In addition, the p62/GPVI-binding sites on collagen may be cryptic and require the binding of the alpha 2beta 1 receptor to expose them correctly to the p62/GPVI receptor. This is supported by evidence that the alpha 2beta 1 receptor has less critical requirements for binding collagen than does p62/GPVI, which needs intact tertiary and quaternary structures (22). Alternatively, since both receptors appear capable of mediating signals, the activating potential may depend upon an adequate matrix of platelet binding sites of both types being present on collagen to ensure that the receptors are clustered and brought into sufficient proximity for activation mechanisms to occur. If either receptor (or binding site) is blocked then an adequate clustering is not attained and activation is prevented. This would explain why ligands, containing one type of binding site presented in multimeric form in a more closely packed arrangement, such as in convulxin or the collagen-like peptides, are such potent activators of platelets. It will be of considerable interest to see if collagen-like peptides containing multimeric forms of the alpha 2beta 1-binding motif from collagen can also activate platelets directly or whether this motif is only effective in conjunction with p62/GPVI- or Fcgamma RIIA-binding motifs. It was also recently shown, in one of the patients with p62/GPVI-deficient platelets, that while collagen did not activate p72SYK it did activate c-Src kinase normally (54), implying that this occurs via other receptors, presumably alpha 2beta 1. The importance of alpha 2beta 1 may also be associated with its primary role in adhesion and with the necessity to modulate the extremely powerful responses induced via p62/GPVI alone. Damaged subendothelial tissue probably exposes sites on collagen for both types of receptor. It is important that platelet activation after binding to collagen should be limited in extent and should be responsive to feedback mechanisms, both positive and negative; hence, under physiological conditions a two-receptor mechanism provides the required flexibility.

The finding that convulxin binds to and acts predominantly, if not exclusively, via p62/GPVI adds it to anti-p62/GPVI antibodies and collagen-like peptides as p62/GPVI-specific reagents. The unique structure and properties of convulxin offer considerable opportunities for the isolation and characterization, as well as for further exploration of the signaling responses of this important receptor.


FOOTNOTES

*   This work was supported in part by Grant 31-42336.94 (to K. J. C.) from the Swiss National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence should be addressed: Theodor Kocher Institute, University of Berne, Freiestrasse 1, CH-3012 Berne, Switzerland. Tel.: 41 31 631 41 48; Fax: 41 31 631 37 99; E-mail: clemetson{at}tki.unibe.ch.
1   The abbreviations used are: vWf, von Willebrand factor; GP, glycoprotein; PLCgamma 2, phospholipase Cgamma 2; GST, glutathione S-transferase; Grb2, growth factor receptor binding protein 2; SH2, Src homology region 2; WGA, wheat germ agglutinin; Fcgamma , Fc receptor gamma  chain; mAb, monoclonal antibody; NEM, N-ethylmaleimide; HPLC, high performance liquid chromatography; PVDF, polyvinylidene difluoride; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.

ACKNOWLEDGEMENTS

We thank Corinne Birbaum for technical assistance. We are grateful to the Central Laboratory of the Swiss Red Cross Blood Transfusion Service, Berne, for the supply of buffy coats.


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