GPIb is involved in platelet aggregation induced by mucetin, a snake C-type lectin protein from Chinese habu (Trimeresurus mucrosquamatus) venom

Mucetin (Trimeresurus mucrosquamatus venom activator, TMVA) is a potent platelet activator purified from Chinese habu (Trimeresurus mucrosquamatus) venom. It belongs to the snake venom heterodimeric C-type lectin family and exists in several multimeric forms. We now show that binding to platelet glycoprotein (GP) Ib is involved in mucetin-induced platelet aggregation. Antibodies against GPIb as well as the GPIb-blocking C-type lectin echicetin inhibited mucetin-induced platelet aggregation. Binding of GPIb was confirmed by affinity chromatography and Western blotting. Antibodies against GPVI inhibited convulxin- but not mucetin-induced aggregation. Signalling by mucetin involved rapid tyrosine phosphorylation of a number of proteins including Syk, Src, LAT and PLC gamma 2. Mucetin-induced phosphorylation of the Fc gamma chain of platelet was greatly promoted by inhibition of alpha(IIb)beta(3) by the peptidomimetic EMD 132338, suggesting that phosphatases downstream of alpha(IIb)beta(3) activation are involved in dephosphorylation of Fc gamma. Unlike other multimeric snake C-type lectins that act via GPIb and only agglutinate platelets, mucetin activates alpha(IIb)beta(3). Inhibition of alpha(IIb)beta(3) strongly reduced the aggregation response to mucetin, indicating that activation of alpha(IIb)beta(3) and binding of fibrinogen are involved in mucetin-induced platelet aggregation. Apyrase and aspirin also inhibit platelet aggregation induced by mucetin, suggesting that ADP and thromboxane A2 are involved in autocrine feedback. Sequence and structural comparison with closely related members of this protein family point to features that may be responsible for the functional differences

The C-terminus of the gamma2 chain but not of the beta3 chain of laminin-332 is indirectly but indispensably necessary for integrin-mediated cell reactions

Using a recombinant mini-laminin-332, we showed that truncation of the three C-terminal amino acids of the gamma2 chain, but not of the C-terminal amino acid of the beta3 chain, completely abolished alpha3beta1 integrin binding and its cellular functions, such as attachment and spreading. However, a synthetic peptide mimicking the gamma2 chain C-terminus did not interfere with alpha3beta1 integrin binding or cell adhesion and spreading on laminin-332 as measured by protein interaction assays and electric cell-substrate impedance sensing. Nor was the soluble peptide able to restore the loss of integrin-mediated cell adhesiveness to mini-laminin-332 after deletion of the gamma2 chain C-terminus. These findings spoke against the hypothesis that the gamma2 chain C-terminus of laminin-332 is a part of the alpha3beta1 integrin interaction site. In addition, structural studies with electron microscopy showed that truncation of the gamma2 chain C-terminus opened up the compact supradomain structure of LG1-3 domains. Thus, by inducing or stabilizing an integrin binding-competent conformation or array of the LG1-3 domains, the gamma2 chain C-terminus plays an indirect but essential role in laminin-332 recognition by alpha3beta1 integrin and, hence, its cellular functions

The crystal structure of the platelet activator aggretin reveals a novel (alphabeta)2 dimeric structure

Aggretin is a C-type lectin purified from Calloselasma rhodostoma snake venom. It is a potent activator of platelets, resulting in a collagen-like response by binding and clustering platelet receptor CLEC-2. We present here the crystal structure of aggretin at 1.7 A which reveals a unique tetrameric quaternary structure. The two alphabeta heterodimers are arranged through 2-fold rotational symmetry, resulting in an antiparallel side-by-side arrangement. Aggretin thus presents two ligand binding sites on one surface and can therefore cluster ligands in a manner reminiscent of convulxin and flavocetin. To examine the molecular basis of the interaction with CLEC-2, we used a molecular modeling approach of docking the aggretin alphabeta structure with the CLEC-2 N-terminal domain (CLEC-2N). This model positions the CLEC-2N structure face down in the "saddle"-shaped binding site which lies between the aggretin alpha and beta lectin-like domains. A 2-fold rotation of this complex to generate the aggretin tetramer reveals dimer contacts for CLEC-2N which bring the N- and C-termini into the proximity of each other, and a series of contacts involving two interlocking beta-strands close to the N-terminus are described. A comparison with homologous lectin-like domains from the immunoreceptor family reveals a similar but not identical dimerization mode, suggesting this structure may represent the clustered form of CLEC-2 capable of signaling across the platelet membrane

Cytotoxicity and inhibition of platelet aggregation caused by an l-amino acid oxidase from Bothrops leucurus venom

AbstractBackgroundMultifunctional l-amino acid oxidases (LAAOs) occur widely in snake venoms.MethodsThe l-AAO from Bothrops leucurus (Bl-LAAO) venom was purified using a combination of molecular exclusion and ion-exchange chromatographies. We report some biochemical features of Bl-LAAO associated with its effect on platelet function and its cytotoxicity.ResultsBl-LAAO is a 60kDa monomeric glycoprotein. Its N-terminal sequence shows high homology to other members of the snake-venom LAAO family. Bl-LAAO catalyzes oxidative deamination of l-amino acids with the generation of H2O2. The best substrates were: l-Met, l-Norleu, l-Leu, l-Phe and l-Trp. The effects of snake venom LAAOs in hemostasis, especially their action on platelet function remain largely unknown. Bl-LAAO dose-dependently inhibited platelet aggregation of both human PRP and washed platelets. Moreover, the purified enzyme exhibited a killing effect in vitro against Leishmania sp., promastigotes, with a very low EC50 of 0.07μM. Furthermore, the cytotoxicity of Bl-LAAO was observed in the stomach cancer MKN-45, adeno carcinoma HUTU, colorectal RKO and human fibroblast LL-24 cell lines. The enzyme released enough H2O2 in culture medium to induce apoptosis in cells in a dose- and time-dependent manner. The biological effects were inhibited by catalase.ConclusionBl-LAAO, a major component of B. leucurus venom, is a cytotoxin acting primarily via the generation of high amounts of H2O2 which kill the cells.General significanceThese results allow us to consider the use of LAAOs as anticancer agents, as tools in biochemical studies to investigate cellular processes, and to obtain a better understanding of the envenomation mechanism