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

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 convulxininduced 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 timedependent tyrosine phosphorylation of platelet proteins, and binding studies with 125 I-convulxin. Convulxin induces signal transduction in part like collagen, involving the time-dependent tyrosine phosphorylation of Fc receptor ␥ chain, phospholipase C␥2, p72 SYK , c-Cbl, and p36 -38. However, unlike collagen, pp125 FAK 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 p72 SYK , showed differential effects on collagen and convulxinstimulated signaling. These results suggest that convulxin uses the p62/GPVI but not the ␣ 2 ␤ 1 part of the collagen signaling pathways to activate platelets. Occupation and clustering of p62/GPVI may activate Src family kinases phosphorylating Fc receptor ␥ chain and, by a mechanism previously described in T-and B-cells, activate p72 SYK that is critical for downstream activation of platelets.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 ...

Variation in Human Platelet Glycoprotein VI Content Modulates Glycoprotein VI–Specific Prothrombinase Activity

“…7,8 Platelets from all the donors (nϭ4) showed a similar rate and extent of aggregation in response to CVX (10 ng/mL), without an effect of GPVI content (data not shown). Similarly, we did not observe a statistically significant difference in platelet adhesion to either CVX or CRP (nϭ4, data not shown).…”

“…Convulxin was purified from Crotalus durissus terrificus venom (Miami Serpentarium Laboratories) by the method of Polgar et al, 7 with minor modifications. Briefly, 250 mg of lyophilized venom was dissolved in 8 mL of 300 mmol/L NaCl and 100 mmol/L ammonium formate at pH 3.5 (buffer A) containing the following protease inhibitors: Pefabloc-SC (1 g/mL, Boehringer-Mannheim), N-␣-Tosyl-L-lysine chloromethyl ketone (50 g/mL), Tosyl-Lphenylalanine chloromethyl ketone (100 g/mL), benzamidine (10 mmol/L), leupeptin (4 mol/L), aprotinin (1 g/mL), EDTA (5 mmol/L), and 0.05% NaN 3 .…”

“…In contrast, in patients with von Willebrand's disease type 1 and borderline to normal ristocetin cofactor activity, collagen receptor density correlates inversely with closure time in a high-shear stress system (platelet function analyzer-100). 6 Recently, Polgar et al 7 reported that convulxin (CVX) bound to GPIV and induced platelet activation. 7 Jandrot-Perrus et al 8 reported that purified CVX induced platelet aggregation at levels between 15 and 35 pmol/L, but they noted a significant heterogeneity in platelet sensitivity to CVX among normal individuals.…”

“…6 Recently, Polgar et al 7 reported that convulxin (CVX) bound to GPIV and induced platelet activation. 7 Jandrot-Perrus et al 8 reported that purified CVX induced platelet aggregation at levels between 15 and 35 pmol/L, but they noted a significant heterogeneity in platelet sensitivity to CVX among normal individuals. In the present study, we quantified platelet GPVI content by a ligand blotting assay with the use of CVX and found significant variation among 23 normal subjects.…”

Antibody against platelet membrane glycoprotein VI in a patient with systemic lupus erythematosus

“…The F(abЈ) 2 fragment of the antibody from the ITP patient induced platelet aggregation but its monovalent Fab fragment rather inhibited the platelet aggregation induced by collagen [6]. Tyrosine phosphorylation of platelet proteins, especially Syk and c-Src, was induced by F(abЈ) 2 fragment [20] and the same tyrosine phosphorylation pattern was induced when platelets were activated by GPVI-specific agonists, collagen, collagenrelated peptide, and convulxin [21][22][23]. These results suggest that the cross-1inking or clustering of GPVI would induce the signal transduction via tyrosine phosphorylation and activate platelets.…”