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Glycoprotein (GP)Ib that binds von Willebrand factor (vWF) and glycoprotein (GP)VI, that binds collagen play a significant role in platelet activation and aggregation, and are potential targets for antithrombotic treatment. They are targeted by snake venom proteinases. The effect of a such proteinase, mutalysin-II, on platelet aggregation was examined using washed human platelets and platelet-rich plasma. Its proteolytic activity on vWF, on its binding partner GPIbα, and on GPVI was analyzed by SDS-PAGE, and immunodetection with the corresponding antibodies after blotting. Dose- and time-dependently, mutalysin-II inhibits aggregation of washed platelets induced by vWF plus ristocetin and by convulxin, but with no significant effect on platelet-rich-plasma. Furthermore, mutalysin-II cleaves vWF into low molecular mass multimers of vWF and a rvWF-A1 domain to realease a ∼27-kDa fragment detectable by SDS-PAGE and blotting with mouse anti-rvWF-A1-domain IgG. Moreover, GPVI was cut by mutalysin-II into a soluble ∼55-kDa ectodomain and a fragment of ∼35-kDa. Thus, mutalysin-II inhibits vWF-induced platelet aggregation via cleavage of bound vWF-A1, and its receptor GPIbα. The additional cleavage of, GPVI, blocks collagen-induced platelets. Our data highlight mutalysin-II as an interesting platelet-directed tool targeting vWF-GPIbα binding and particularly GPVI. Thus, it might be suited for antithrombotic therapy as its combined inactivation of two receptors does not significantly compromise hemostasis, but shows high efficacy and safety. Studies are needed to further develop and demonstrate its potential benefits.

Rationale Hematoma expansion (HE) aggravates brain injury after intracerebral hemorrhage (ICH) and hypertension is a key contributor to HE. Plasma kallikrein (PK) is involved in hemorrhagic transformation in ischemic stroke mice. This study was conducted to explore the role of PK in HE in hypertensive ICH. Methods Hypertension was achieved by continuous infusion of angiotensin II (Ang II) with an osmotic pump in C57BL/6 mice. ICH was achieved by stereotactic intrastriatal injection of blood. PK-specific antibody and platelet glycoprotein VI (GPVI) agonists were administered to intervene in hematoma expansion. The hematoma volume was indicated by the erythrocyte components hemoglobin and carbonic anhydrase-1 in the ipsilateral brain hemisphere. Results Ang II-induced hypertensive mice showed enhanced hematoma expansion and worsened neurologic deficits after ICH modeling. Moreover, intrastriatal injection of blood from Ang II-treated mice into normal mice increased the area of secondary hemorrhage more than blood from untreated mice. Mechanistically, elevated PK was found in Ang II-infused mice whereas, inhibition of PK and administration of the GPVI agonist convulxin decreased hematoma expansion and improved neurologic deficits after ICH. Conclusions These findings suggest that PK inhibition and GPVI agonist treatment might serve as potential methods to intervene in HE after ICH.

Background and aims: Platelets can release extracellular vesicles (EVs) upon stimulation with various agonists. Interestingly, platelets from patients with Glanzmann thrombasthenia have reduced EV release. These platelets lack functional alpha(IIb)beta(3) integrins, indicating that alpha(IIb)beta(3) integrin is critical in vesicle release. Integrin activation is central in platelet function and is associated with e.g. adhesion, aggregation and cytoskeletal rearrangement. However, while platelet activation pathways are widely known, the mechanisms underlying EV release remain uncharacterized. We investigated the role of integrin alpha(IIb)beta(3), phosphatidyl serine (PS) exposure, cytoskeletal rearrangement and their associated signalling pathways in EV release.Methods: EVs were isolated from activated platelets. Platelet activation status was measured by multicolour flow cytometry. A panel of pharmacologic inhibitors was used to interfere in specific signalling pathways. EV release was quantified enzymatically based on membrane PS content and nanoparticle tracking analysis. In addition, real-time visualization of EV shedding with confocal microscopy and EVs with Cryo-TEM imaging was performed.Results: Platelet activation with convulxin resulted in higher EV release than with activation by thrombin. Kinetic measurements indicated that EV release followed the pattern of alpha(IIb)beta(3) integrin activation and subsequent closure paralleled by PS exposure. Prevention of alpha(IIb)beta(3) activation with the inhibitor tirofiban dramatically suppressed EV release. Similar results were obtained using a alpha(IIb)beta(3)-deficient platelets from patients with Glanzmann thrombasthenia. Inhibition of actin cytoskeleton rearrangement decreased EV release, whereas inhibition of individual signalling targets upstream of cytoskeletal rearrangement showed no such effects.Conclusion: Platelet EV release requires three main events: integrin activation and closure, PS exposure, and cytoskeletal rearrangement.

Factor Xa (FXa) has been reported to activate platelet via interaction with glycoprotein (GP) VI but the underlying mechanism has not been fully elucidated. We investigated if Nox2-derived oxidative stress is implicated in FXa-induced platelet aggregation (PA), and the effect of a FXa inhibitor, namely rivaroxaban, with or without aspirin (ASA), on PA. We performed an in vitro study measuring convulxin-induced PA, thromboxane (Tx) B2 and isoprostanes biosynthesis, soluble Nox2-dp (sNox2-dp), a marker of Nox2 activation, soluble GPVI (sGPVI) and PLA2 activation in platelets from healthy subjects (n = 5) added with and without a Nox2 inhibitor. The same variables were also examined in platelets treated with rivaroxaban (15–60 ng/ml), combined or less with ASA (25 µM). Convulxin-stimulated platelets increased sGPVI, sNox2-dp, H2O2, eicosanoid biosynthesis and PLA2 phosphorylation, which were all inhibited by a Nox2 inhibitor. Rivaroxaban alone significantly reduced PA, sGPVI, TxB2 and isoprostanes biosynthesis, concomitantly with Syk, sNox2-dp and PLA2 activation in a dose-dependent fashion; a significant effect was achieved with 30 ng/ml rivaroxaban. Docking simulation analysis showed that rivaroxaban interacts with GPVI. In platelets co-incubated with ASA, rivaroxaban amplified the ASA antiplatelet effect, which was achieved with 30 ng/ml and prevalently attributable to Nox2 inhibition and impaired isoprostane biosynthesis. Here we show that rivaroxaban, at concentrations achievable in human circulation, inhibits PA via GPVI interaction and eventually Nox2-mediated isoprostanes biosynthesis and amplifies the ASA antiplatelet effect.

Victims of Bothrops jararaca snakebites manifest bleedings, blood incoagulability, platelet dysfunction, and thrombocytopenia, and the latter has been directly implicated in the genesis of hemorrhagic diathesis. We addressed herein the direct effects of B. jararaca venom (BjV) on ex vivo platelet aggregation and granule secretion in washed human and mouse platelets. BjV directly aggregated platelets, but the extent of platelet aggregation was lower in human than mouse platelets. On the other hand, BjV (24.4 μg/mL) and thrombin (0.1 U/mL) induced a similar extent of ATP and platelet factor 4 (PF4) secretion in both species. BjV-induced platelet aggregation was independent of the platelet dense body content, as in pearl mouse (Ap3b1−/−) platelets, whose dense bodies are deficient in adenine nucleotides and serotonin, the extent of platelet aggregation was superior to that induced in BALB/c or C57BL/6 mice. BjV-induced β-hexosaminidase secretion in human platelets was less intense than that evoked by thrombin, and the contrary was observed in mouse platelets. Irreversible inactivation of platelet cyclooxygenase 1 by acetylsalicylic acid did not reduce BjV-induced platelet aggregation. BjV exerted no cytotoxic activity in human and mouse platelets, as evaluated by lactate dehydrogenase loss. Eptifibatide, which inhibits the binding of fibrinogen to platelet glycoprotein complex GPIIb-IIIa, differently blocked BjV-induced platelet aggregation in mice and humans. BjV-induced platelet aggregation did not depend on snake venom serine proteinases nor metalloproteinases in mice, whilst serine proteinases were rather important for platelet aggregation in humans. Our results show that BjV induces direct activation, aggregation, and secretion in human and mouse platelets, but it exerts diverse responses in them, which should be considered in comparative studies to understand pathophysiological events during Bothrops envenomation.

Platelets are the smallest blood cells, and their activation (platelet cohesion or aggregation) at sites of vascular injury is essential for thrombus formation. Since the use of antiplatelet therapy is an unsolved problem, there are now focused and innovative efforts to develop novel antiplatelet compounds. In this context, we assessed the antiplatelet effect of an acylhydroquinone series, synthesized by Fries rearrangement under microwave irradiation, evaluating the effect of diverse acyl chain lengths, their chlorinated derivatives, and their dimethylated derivatives both in the aromatic ring and also the effect of the introduction of a bromine atom at the terminus of the acyl chain. Findings from a primary screening of cytotoxic activity on platelets by lactate dehydrogenase assay identified 19 non-toxic compounds from the 27 acylhydroquinones evaluated. A large number of them showed IC50 values less than 10 µM acting against specific pathways of platelet aggregation. The highest activity was obtained with compound 38, it exhibited sub-micromolar IC50 of 0.98 ± 0.40, 1.10 ± 0.26, 3.98 ± 0.46, 6.79 ± 3.02 and 42.01 ± 3.48 µM against convulxin-, collagen-, TRAP-6-, PMA- and arachidonic acid-induced platelet aggregation, respectively. It also inhibited P-selectin and granulophysin expression. We demonstrated that the antiplatelet mechanism of compound 38 was through a decrease in a central target in human platelet activation as in mitochondrial function, and this could modulate a lower response of platelets to activating agonists. The results of this study show that the chemical space around ortho-carbonyl hydroquinone moiety is a rich source of biologically active compounds, signaling that the acylhydroquinone scaffold has a promising role in antiplatelet drug research.

International audience; Type III collagen binding protein (TIIICBP) was previously described as a platelet membrane protein that recognizes the KOGEOGPK peptide sequence within type III collagen. In order to better characterize this protein, we performed different approaches including mass spectrometry sequencing and functional experiments. This study leads to identify high biochemical and functional similarities between TIIICBP and kindlin-3, a member of a family of focal adhesion proteins. Indeed, mass spectrometry surveys indicated that TIIICBP contains several peptides identical to kindlin-3, covering 41% of the amino acid sequence. Polyclonal antibodies raised against a kindlin-3 specific N-terminal sequence, recognized and immunoprecipitated TIIICBP from platelet lysates. Electron microscopy and flow cytometry experiments showed that kindlin-3, as well as TIIICBP, were present associated to platelet membrane and a trans-location of cytosolic kindlin-3 to the platelet membrane was observed after platelet activation. Similarly to anti-TIIICBP antibodies and the KOGEOGPK peptide, anti-kindlin-3 antibodies inhibited platelet interactions with type III collagen under flow conditions and slowed down platelet aggregation induced by glycoprotein VI agonists; e.g. collagen-related peptides and convulxin. In addition, the anti-kindlin-3 antibody inhibited platelet aggregation induced by low e but not high e doses of ADP or thrombin which depends on a IIb b 3 integrin function. In conclusion, our results show that the peptides identified by mass spectrometry from purified TIIICBP correspond to the kindlin-3 protein and demonstrate biochemical and functional similarities between TIIICBP and kindlin-3, strengthening a key role for TIIICBP/kindlin-3 in platelet interactions with collagen by cooperating with glycoprotein VI activation and integrin clustering in focal adhesion complexes.

Summary Background: Macrophages are major immune cells and play an important role in modulating homeostasis and the immune defense mechanism. In inflammatory responses to the infection of pathogens, macrophages are activated, producing various inflammatory mediators. Snake venom C-type lectin proteins (snaclecs) have diverse targets, including platelet GPVI, GPIb, integrin α2β1 or CLEC-2 expressed in platelets, endothelial cells or myeloid cells. Methods: In this study, murine macrophages (RAW 264.7 cells) and human monocytes (THP-1) were treated with different snaclecs, including aggretin, gramicetin, trowaglerix and convulxin, in the absence or presence of LPS for 24 h. Results: The production of cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in supernatants was measured by ELISA. Aggretin increased the production of TNF-α and IL-6 in both RAW264.7 and THP-1 cells; however, the other snaclecs did not. Aggretin induced extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) tyrosine phosphorylation of RAW264.7 cells. Pretreatments with inhibitor of ERK, JNK, p38 or NF-κB abolished cytokine release caused by aggretin. Aggretin bound to THP-1 cells in a concentration-dependent manner and it displaced the CLEC-2 mAb binding to THP-1 cells and the immobilized aggretin selectively bound to CLEC-2 of both platelets and THP-1 cell lysates. Furthermore, aggretin elevated the plasma level of IL-6 in ICR mice as it was administered intramuscularly. Conclusion: These results indicate that aggretin may induce cytokine TNF-α/IL-6 release via interacting with CLEC-2 receptor and the subsequent MAPK and NF-κB activation in monocytes/macrophages.

Crotalus durissus rattlesnakes are responsible for the most lethal cases of snakebites in Brazil. Crotalus durissus collilineatus subspecies is related to a great number of accidents in Southeast and Central West regions, but few studies on its venom composition have been carried out to date. In an attempt to describe the transcriptional profile of the C. durissus collilineatus venom gland, we generated a cDNA library and the sequences obtained could be identified by similarity searches on existing databases. Out of 673 expressed sequence tags (ESTs) 489 produced readable sequences comprising 201 singletons and 47 clusters of two or more ESTs. One hundred and fifty reads (60.5%) produced significant hits to known sequences. The results showed a predominance of toxin-coding ESTs instead of transcripts coding for proteins involved in all cellular functions. The most frequent toxin was crotoxin, comprising 88% of toxin-coding sequences. Crotoxin B, a basic phospholipase A2 (PLA2) subunit of crotoxin, was represented in more variable forms comparing to the non-enzymatic subunit (crotoxin A), and most sequences coding this molecule were identified as CB1 isoform from Crotalus durissus terrificus venom. Four percent of toxin-related sequences in this study were identified as growth factors, comprising five sequences for vascular endothelial growth factor (VEGF) and one for nerve growth factor (NGF) that showed 100% of identity with C. durissus terrificus NGF. We also identified two clusters for metalloprotease from PII class comprising 3% of the toxins, and two for serine proteases, including gyroxin (2.5%). The remaining 2.5% of toxin-coding ESTs represent singletons identified as homologue sequences to cardiotoxin, convulxin, angiotensin-converting enzyme inhibitor and C-type natriuretic peptide, Ohanin, crotamin and PLA2 inhibitor. These results allowed the identification of the most common classes of toxins in C. durissus collilineatus snake venom, also showing some unknown classes for this subspecies and even for C. durissus species, such as cardiotoxins and VEGF.

Summary. Background and objectives: Injuries to the vessel wall and subsequent exposure of the matrix of the subendothelial layer resulted in thrombus formation. Platelet glycoprotein (GP) Ib and VI play a crucial role in matrix-induced activation and aggregation of platelets. Methods and results: In the present study, we reported that the GPIb-cleaving snake venom metalloproteinase (SVMP), kistomin, inhibited collagen-induced platelet aggregation. Moreover, kistomin inhibited platelet aggregation induced by convulxin (CVX, a GPVI agonist) and a GPVI-specific antibody in a concentration and time-dependent manner. Kistomin treatment decreased platelet GPVI but not integrin α2β1 and αIIbβ3, accompanied with the formation of GPVI cleavage fragments, as determined by flow cytometric and Western blot analyses. In addition, intact platelet GPVI and recombinant GPVI were digested by kistomin to release 25- and 35-kDa fragments, suggesting that kistomin cleaved GPVI near the mucin-like region. We designed four synthetic peptides ranging from Leu180 to Asn249 as the substrates for kistomin and found that kistomin cleaved these synthetic peptides at FSE205/A206TA and NKV218/F219TT, as analyzed by MALDI-TOF-MS. In addition, GPVI-specific antibody-induced tyrosine kinase phosphorylation in platelets was reduced after kistomin pretreatment, and platelet adhesion to collagen but not to fibrinogen was attenuated by kistomin. Conclusions: We provided here the first evidence that a P-I snake venom metalloproteinase, kistomin, inhibits the interaction between collagen and platelet GPVI through its proteolytic activity on GPVI, thus providing an alternative strategy for developing new anti-thrombotic agents.

Platelets play a key role in hemostasis and changes in redox balance are known to alter platelet activation and aggregation. Interestingly, activation of platelets leads to production of reactive oxygen species (ROS), but the role(s) of these ROS remain unclear. Using flow cytometry and chemiluminescence, agonist-induced ROS generation was found to be spatially distinct with stimulation through the major collagen receptor GPVI inducing only intraplatelet ROS while thrombin induced production of extracellular ROS. Platelet activation by either the GPVI-selective agonist convulxin or thrombin was differentially regulated by ROS generation. Thus, surface expression of CD62P, CD40L, or activated integrin alphaIIbbeta3 was abrogated by pharmacologic antioxidants but externalization of phosphatidylserine was not inhibited. Furthermore, extracellular antioxidants SOD/catalase markedly inhibited thrombin-, but not convulxin-, induced CD62P expression and alphaIIbbeta3 activation. The data suggest that ROS selectively regulate biochemical steps in platelet activation and that distinct source(s) of ROS and discrete redox-sensitive pathway(s) may control platelet activation in response to GPVI or thrombin stimulation. Thus, targeting ROS with site-specific antioxidants may differentially regulate platelet activation via thrombin or collagen.

In the present study, we characterized OXSI-2 ([3-(1-Methyl-1H–indol –3-yl-methylene)-2–oxo-2, 3–dihydro-1H- indole–5-sulfonamide], a putative inhibitor of Syk, and determined its specificity and selectivity in platelets. We found that OXSI-2 completely abolished convulxin-induced platelet functional responses. In order to determine whether OXSI-2 inhibited Src family kinase-mediated platelet responses, we evaluated its effect on Src family kinase (SFK)-mediated signaling events in platelets, viz. Lyn-mediated phosphorylation of Y352 on Syk, LAT- Y191 phosphorylation by Syk, and protease-activated receptor (PAR)-mediated phosphorylation of ERK. In the present work, we report that convulxin mediated Syk tyrosine 352 phosphorylation is not inhibited by OXSI-2, whereas piceatannol and PP2 abolished it. Syk-mediated Y191 LAT phosphorylation is abolished by all the three inhibitors. AYPGKF-induced phosphorylation of ERK was marginally inhibited by OXSI-2, whereas treatment with PP2 and piceatannol completely abolished it. However, PAR mediated thromboxane generation (an event mediated by ERK) was potentiated by OXSI-2 whereas PP2 and piceatannol brought thromboxane to basal levels. Protein kinase C (PKC) inhibitors are known to potentiate PAR-mediated thromboxane generation in platelets. In contrast, OXSI-2, unlike PKC inhibitors, did not inhibit secretion. Therefore, we conclude that OXSI-2 is not a Syk-selective inhibitor in platelets because of its unexplained non-specific effects.

Background: Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcR-gamma-chain, which has an immunoreceptor tyrosine-containing activation motif (ITAM). Objective: To determine why GPVI-FcR gamma signals poorly, or not at all, in response to collagen in hematopoietic cell lines, despite robust responses to the GPVI-reactive snake venom toxin convulxin. Methods and results: Using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay, a sensitive readout for sustained ITAM signaling, we demonstrate collagen-induced GPVI-FcR gamma signaling in hematopoietic cell lines. This is accompanied by relatively weak but sustained protein tyrosine phosphorylation, in contrast to the stronger but transient response to convulxin. Sustained signaling by collagen is also observed in platelets and is necessary for the maintenance of spreading on collagen. Finally, in cell lines, the inhibitory collagen receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), which is not expressed on platelets but is present on most hematopoietic cells, inhibits GPVI responses to collagen but not convulxin. Conclusion: The inability of previous studies to readily detect GPVI collagen signaling in cell lines is probably because of the weak but sustained nature of the signal and the presence of the inhibitory collagen receptor LAIR-1. In platelets, we propose that GPVI-FcR gamma has evolved to transmit sustained signals in order to maintain spreading over several hours, as well as facilitating rapid activation through release of feedback agonists and integrin activation. The establishment of a cell line NFAT assay will facilitate the molecular dissection of GPVI signaling and the identification of GPVI antagonists in drug discovery.

Thrombus formation in hemostasis or thrombotic disease is initiated by adhesion of circulating platelets to damaged blood vessel walls. Exposed subendothelial collagen interacting with platelet glycoprotein (GP) VI leads to platelet activation and integrin alpha(IIb)beta(3)-mediated aggregation. We previously showed that ligand binding to GPVI also induces metalloproteinase-dependent shedding, generating an approximately 55-kDa soluble ectodomain fragment and an approximately 10-kDa membrane-associated remnant. Here, treatment of platelets with collagen or the GPVI-targeting rattlesnake toxin convulxin also induces rapid (10-30 s) formation of a high molecular weight GPVI complex (GPVIc) under nonreducing conditions, as detected by immunoblotting with anti-GPVI antibodies. The appearance of an approximately 20-kDa remnant detectable using a polyclonal antibody against the GPVI cytoplasmic tail under nonreducing, but not reducing, conditions after ectodomain shedding and nonreduced/reduced two-dimensional SDS-polyacrylamide gel analysis of biotinylated platelets confirmed that that GPVIc was a homodimer. Formation of disulfide-linked GPVIc was prolonged in the presence of metalloproteinase inhibitor GM6001 and was independent of GPVI signaling because it was unaffected by inhibitors of Src kinases, Syk, or phosphoinositide 3-kinase. To identify the thiol involved in disulfide bond formation, wild-type or mutant GPVI, where two available sulfhydryls (Cys-274 and Cys-338) were individually mutated to serine, was expressed in rat basophilic leukemia cells. Dimerization of wild-type and C274S GPVI, but not the C338S mutant, was observed after treating cells with convulxin. We conclude that (i) a subpopulation of GPVI forms a constitutive dimer on the platelet surface, facilitating rapid disulfide cross-linking, (ii) convulxin or other GPVI agonists induce disulfide-linked GPVI dimerization independent of GPVI signaling, and (iii) the penultimate residue of the GPVI cytoplasmic tail, Cys-338, mediates disulfide-dependent dimer formation.

Previous studies have shown that anticoagulants alter platelet reactivity and the apparent effects of antiplatelet agents. This study was conducted to identify the impact of methods of preparation of blood samples on an assay of platelet function and the effects of antiplatelet agents. The activation of platelets was identified with the use of flow cytometry in response to thrombin (1 and 10 nmol/L), adenosine diphosphate (0.2 and 1 micromol/L), platelet activating factor (1 nmol/L), and convulxin (1 and 10 ng/ml). Antiplatelet effects were assessed after the addition in vitro of tirofiban (50 ng/ml) and cangrelor (10 nmol/L). Results were compared in whole blood and platelet-rich plasma (PRP) anticoagulated with corn trypsin inhibitor (32 microg/ml, a specific inhibitor of factor XIIa). The fraction of young platelets was quantified with thiazole orange, which identifies ribonucleic acid. The activation of platelets was consistently less in PRP compared with whole blood. Activation in PRP was 23 +/- 15% that in whole blood for thrombin, 65 +/- 26% for adenosine diphosphate, 40 +/- 20% for platelet activating factor, and 49 +/- 25% for convulxin (p0.01 for each comparison). The fraction of young platelets in PRP was 39 +/- 11% that in whole blood (p0.001). The effects of antiplatelet agents varied with agonist and antiplatelet agent but were generally greater in PRP compared with whole blood (p0.05). In conclusion, platelet reactivity is lower and the effects of antiplatelet agents are greater and potentially misleading in PRP compared with whole blood. The accuracy of platelet function testing may be improved by performance in whole blood.

Lectins are a structurally and functionally diverse group of proteins from different sources, capable to recognize and bind specifically carbohydrates. Several snake venoms contain calcium-dependent true lectins (SVLs) that recognize galactose. Herein, in order to enlighten some of the structure–function relationships of snake venom lectins (SVLs), we constructed theoretical models for 10 SVLs based on the Crotalus atrox lectin (CaL), the only SVL crystal structure available, and compared with other animal and plant lectins, and C-type lectin-like proteins (CLPs) that do not bind carbohydrates. Although these are theoretical structures, we could identify some SVL features, including: (i) a singular intrachain disulfide bond (Cys38–Cys133) that is not present in CLPs; (ii) a significant reorientation (39–41 A) of the 80's loop position that folds back to the globular domain, assists the carbohydrate recognition domain (CRD), and orients the dimer formation, even in BfL-1 and BfL-2, which did not present the Cys86 interchain; (iii) a CRD presenting a negative and concave surface that allows the interaction with the specific saccharide hydroxyl groups and calcium ion; (iv) the role of water molecules in some interchain interactions, similar to other animal and plant lectins; and (v) the inability of forming oligomers in contrast to CaL and some CLPs, such as convulxin.

Glycoprotein (GP) VI, a key receptor for collagen-induced platelet activation, recently emerged as a major target for developing new antithrombotics. However, little is known about its functional domains, which is a disadvantage for the rational development of antagonists. Our aim was to identify the structures determining GPVI specificity. GPVI presents homologies with members of the Ig superfamily (in particular with FcalphaRI) whose extracellular parts present two domains, D1 and D2 linked by a hinge interdomain. To identify the respective role of these domains in GPVI, we have substituted D1 and D2 by their FcalphaRI homologue in a soluble GPVI fusion protein (GPVI-Fc) and have modified the linker motif by mutagenesis. Proteins were tested for their binding to ligands and antibodies specific for GPVI and FcalphaRI. We demonstrate for the first time that D2 plays a specific and significant role in GPVI binding to collagen and that the hinge interdomain is critical for the binding to convulxin. Furthermore, binding to CRP requires elements of D1 and of the linker motif. Our results indicate that GPVI is unique amongst the receptors of its family as it uses different structural domains to interact with several agonists and provide evidence that different sites on GPVI constitute targets to develop antagonists of GPVI.

Summary. Background: Convulxin (CVX), a C-type lectin from the venom of Crotalus durissusterrificus, is a potent activator of human platelets, binding predominantly to glycoprotein (GP)VI. Native CVX is an octamer composed of four αβ-heterodimers [(αβ)4]. Two different native sequences have been reported, one bearing lysine (K), the other glutamic acid (E), at β chain residue 89, but the physiological relevance of this difference is unknown. Objective: We used the Drosophila S2 system to express recombinant CVX (rCVX) heterodimers (αβ) and site-directed mutagenesis to evaluate the influence of multimer size and the substitution βK89E on CVX function. Methods: By flow cytometry, native CVX and both recombinant forms bind to human platelets in whole blood. By surface plasmon resonance (BIAcore, Piscataway, NJ, USA), the calculated equilibrium dissociation constants (KD) were: rCVX αβ89K, 11.3 × 10−8 m; rCVX αβ89E, 9 × 10−8 m; and native CVX, 2.8 × 10−8 m. Results: Thus, the affinities of the two rCVX forms for human, recombinant GPVI are essentially the same, but the relative affinity of native CVX is about 3-fold higher. The minimum concentration of native CVX that induces maximal human platelet aggregation (70 pm) is roughly 400-fold lower than that of either rCVX (29 nm). Conclusions: These results are consistent with the hypothesis that the ability of the native CVX octamer to cluster mobile GPVI molecules within the platelet membrane may be the single most important factor that contributes to the efficiency with which CVX is able to induce platelet activation.

Platelet glycoprotein VI (GPVI) is now considered to be a major player in platelet–collagen adhesive interactions leading to thrombus formation. GPVI blockade, or its depletion, has been shown in mice to result in complete protection against arterial thrombosis, without significant prolongation of bleeding time. GPVI may therefore represent a useful antithrombotic target. In order to reaffirm the role of GPVI in platelet–collagen interactions, we developed GPVInull mice by targeted disruption methodology. GPVInull mice platelets failed to respond to a high dose of fibrillar collagen, or convulxin, a GPVI agonist, but showed a normal response to other agonists such as ADP, PMA and arachidonic acid. We report, for the first time, that a proportion of GPVInull mice is protected against lethal thromboembolism, induced by the infusion of a mixture of collagen and epinephrine. Greater than 55% of GPVInull mice survived the challenge, whereas the maximal survival from the other genotypes was 17% (n = 18 per genotype). Washed platelets obtained from GPVInull mice showed > 90% reduction in adhesion to fibrillar collagen under static conditions. Platelet adhesion to collagen under dynamic conditions using a high shear rate (2600 s− 1) was dramatically reduced using blood from GPVInull mice, while platelets from wild-type and heterozygous animals showed a similar amount of adhesion. Animals from each genotype had essentially similar tail bleeding time, suggesting that a complete deficiency of GPVI, at least in mice, does not result in an enhanced bleeding tendency. These observations clearly establish that blockade of GPVI may attenuate platelet–collagen interactions without adversely affecting the bleeding time.

Gab2, a recently identified docking protein, contains a pleckstrin homology domain and potential binding sites for SH2 and SH3 domain-containing proteins. Gab2 has been shown to support growth, differentiation, and function in a number of haematopoietic cells, although its role in platelets remains to be determined. Here we report that cross-linking of the collagen receptor GPVI by the snake venom toxin convulxin stimulates tyrosine phosphorylation of Gab2. Furthermore, platelet aggregation induced by submaximal concentrations of convulxin is attenuated in the absence of Gab2, although recovery is seen with higher concentrations of the toxin. Consistent with this, tyrosine phosphorylation of Fc receptor γ-chain, Syk, Btk, and phospholipase Cγ2 by convulxin is reduced in the absence of Gab2. In comparison, the G protein-coupled receptor agonist, thrombin, does not induce phosphorylation of Gab2 and aggregation is unaltered in the absence of the toxin. These findings provide evidence for a functional role of Gab2 in supporting platelet activation by GPVI.

Recent studies have implied that GPIb-IX-V as well as functioning as an adhesion receptor may also induce signaling to mediate binding of platelets to damaged vessel wall to prevent bleeding. Reorganization of the cytoskeleton and redistribution of platelet structural proteins and signaling molecules are thought to be important in this early activation process, though the molecular mechanisms remain to be fully defined. In this study, we have used mucetin, a snake venom lectin protein that activates platelets via GPIb, to study the redistribution of GPIb in platelets. In unstimulated platelets, a minor portion of GPIb localized to Triton-insoluble cytoskeleton fractions (TIC). This portion increased considerably after platelet activation by mucetin. We also find increased contents of the FcRgamma chain in TIC. Anti-GPIb antibodies, mocarhagin or cytochalasin D completely inhibited the cytoskeletal translocation. In addition, BAPTA-AM, a cytoplasmic calcium chelator, strongly inhibited this process. On the other hand, inhibitors of alphaIIbbeta3, PLCgamma, PKC, tyrosine kinases, ADP receptor, PI3-kinase or EDTA are effective in preventing GPIb relocation in convulxin- but not in mucetin-activated platelets. We propose that cytoskeletal translocation of GPIb is upstream of alphaIIbbeta3 activation and cross-linking of GPIb is sufficient to induce this event in mucetin-activated platelets.

Novel, cell-based assays, based on bioluminescence resonance energy transfer, have been developed for FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling at receptor-proximal steps. In a stable transfectant of the HEK-293 cell line expressing human FcepsilonRIalpha, FcepsilonRIbeta, and FcRgamma-GFP2 and Syk(1-265)-Rluc fusion proteins, FcepsilonRI cross-linking markedly increased BRET2 ratios, which are the ratios of GFP2 emission to Rluc emission. These ratios reflect the FcRgamma-GFP2-Syk(1-265)-Rluc interaction in living cells. The signals are specifically inhibited by the Src-family kinase inhibitor PP2. Separately, in transient transfectants expressing GPVI, FcRgamma-GFP2, and Syk(1-265)-Rluc, the GPVI-specific ligand convulxin induced a two-fold increase in the BRET2 ratio and this increase was also inhibited by PP2. Finally, a differential assay was developed which permits the measurement of FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling in the same cell. These assays provide useful methods for monitoring FcRgamma-Syk interaction in real time in living cells and may contribute to the understanding of signal regulation through FcRgamma-containing receptors.

We analyzed the intracellular action of sphingosine 1-phosphate (Sph-1-P), formed from sphingosine (Sph) by sphingosine kinase (SPHK), in platelets. When sphingosine kinase activity was inhibited by N,N-dimethylsphingosine (DMS), Ca2+ mobilization induced by convulxin, an agonist of the collagen receptor glycoprotein VI (GPVI), was moderately but specifically abolished; that induced via G protein-coupled receptors was not affected. Under the same conditions, however, tyrosine phosphorylation of Syk and phospholipase Cgamma2, which is essential for the GPVI-mediated signaling, was not inhibited. Sphingosine kinase activity of the platelet membrane fraction increased specifically upon stimulation with convulxin or collagen. Our results suggest that intracellular sphingosine 1-phosphate is related to Ca2+ mobilization in GPVI-mediated signaling pathways.

Glycoprotein VI (GPVI) has a crucial role in platelet responses to collagen. Still, little is known about its interaction with its ligands. In binding assays using soluble or cell-expressed human GPVI, we observed that (i) collagen, and the GPVI-specific ligands collagen-related peptides (CRP) and convulxin, competed with one another for the binding to GPVI and (ii) monoclonal antibodies directed against the extracellular part of the human receptor displayed selective inhibitory properties on GPVI interaction with its ligands. Monoclonal antibody 9E18 strongly reduced the binding of GPVI to collagen/CRP, 3F8 inhibited its interaction with convulxin, whereas 9O12 prevented all three interactions. These observations suggest that ligand-binding sites are distinct, exhibiting specific features but at the same time also sharing some common residues participating in the recognition of these ligands. The epitope of 9O12 was mapped by phage display, along with molecular modeling of human GPVI, which allowed the identification of residues within GPVI potentially involved in ligand recognition. Site-directed mutagenesis revealed that valine 34 and leucine 36 are critical for GPVI interaction with collagen and CRP. The loop might thus be part of a collagen/CRP-binding site.

Summary. The activation of the small GTPase Rap2B in resting and agonist-stimulated human platelets was investigated. Both thrombin, that stimulates heterotrimeric G-protein-coupled receptors, and the GPVI ligand convulxin, that activates a tyrosine-kinase based signaling pathway, were able to induced the rapid and sustained binding of GTP to Rap2B. Similarly, a number of other agonists tested, previously known to activate the highly related protein Rap1B, were also able to stimulate Rap2B. In contrast, platelet antagonists that increase the intracellular concentration of cAMP did not signal to Rap2B. Thrombin- and convulxin-induced activation of Rap2B was not dependent on thromboxane A2, did not require the interaction of the protein with the cytoskeleton, and was not regulated by integrin αIIbβ3-dependent outside-in signaling. When secreted ADP was neutralized, activation of Rap2B induced by thrombin, but not by convulxin, was significantly reduced. ADP itself was found to induce the rapid and sustained binding of GTP to Rap2B, and this effect was predominantly mediated by stimulation of the Gi-coupled P2Y12 receptor. Activation of Rap2B promoted by both thrombin and convulxin was regulated by intracellular Ca2+, while protein kinase C was found to be involved in convulxin- but not in thrombin-induced activation of Rap2B. Moreover, Rap2B activation induced by thrombin, but not by convulxin, was totally dependent on phosphatidylinositol 3-kinase activity. These results demonstrate that the small GTPase Rap2B is involved in platelet activation, and outline some important differences between the regulation of highly related GTPases Rap2B and Rap1B in human platelets.

Glycoprotein (GP) VI is a platelet membrane protein with a molecular weight of 62 kDa that was identified as a physiological collagen receptor from studies of patients deficient in this protein. GPVI-deficient platelets lacked specifically collagen-induced aggregation and the ability to form thrombi on a collagen surface under flow conditions, suggesting that GPVI makes an indispensable contribution to collagen-induced platelet activation. On the platelet surface, GPVI is present as a complex with the Fc receptor (FcR) gamma-chain, probably composed of two GPVI molecules and one FcR gamma-chain dimer. GPVI must form such a dimeric complex to exhibit high affinity binding to collagen. The GPVI-induced activation mechanism is initiated by tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of the FcR gamma-chain, and then this signal is transduced to many related proteins, mainly by tyrosine phosphorylation. GPVI is widely recognized as a requisite factor for the formation of platelet aggregates on a collagen surface under blood flow. However, individuals with GPVI-deficient or null platelets do not exhibit any strong bleeding tendency. Analyzing this apparent dichotomy should provide us with a more precise understanding of the mechanism of thrombus formation.

Convulxin (CVX), a C-type lectin, isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, causes cardiovascular and respiratory disturbances and is a potent platelet activator which binds to platelet glycoprotein GPVI. The structure of CVX has been solved at 2.4A resolution to a crystallographic residual of 18.6% (R(free)=26.4%). CVX is a disulfide linked heterodimer consisting of homologous alpha and beta chains. The heterodimers are additionally linked by disulfide bridges to form cyclic alpha(4)beta(4)heterotetramers. These domains exhibit significant homology to the carbohydrate-binding domains of C-type lectins, to the factor IX-binding protein (IX-bp), and to flavocetin-A (Fl-A) but sequence and structural differences are observed in both the domains in the putative Ca(2+)and carbohydrate binding regions.

Convulxin (CVX), a C-type snake protein from Crotalus durissus terrificus venom, is the quintessential agonist for studies of the collagen receptor, glycoprotein VI (GPVI) and its role in platelet adhesion to collagens. In this study, CVX, purified from venom, behaves as expected, i.e. it binds to platelet GPVI and recombinant human GPVI, induces platelet aggregation and platelet prothrombinase activity, and binds uniquely to GPVI in ligand blots of SDS-denatured proteins. Nonetheless, we find that CVX has a dual specificity for both GPVI and native but not denatured human GPIb alpha. First, CVX binds to human GPIb alpha expressed on the surface of CHO cells. Second, CVX binds weakly to murine platelet GPIb alpha but more strongly to human platelet GPIb alpha, as evidenced by comparative binding to wild-type, GPVI(-/-), FcR gamma (-/-), and human GPIb transgenic mice. Third, the binding of CVX to human GPIb alpha is inhibited by soluble, recombinant human GPVI. Fourth, CVX binding to GPIb alpha is disrupted by phenylalanine substitutions at GPIb alpha tyrosine-276, tyrosine-278, and tyrosine-279, which also disrupts von Willebrand factor and alpha-thrombin binding to GPIb alpha. Fifth, CVX binding to GPIb alpha on Chinese hamster ovary cell transfectants is inhibited by function-blocking murine monoclonal anti-GPIb alpha antibodies. Lastly, CVX fails to bind to denatured GPIb alpha in detergent extracts of platelets. Three separate preparations of CVX (two purified by the authors; one obtained commercially) produced equivalent results. These results indicate that CVX exhibits dual specificity for both native GPIb alpha and GPVI. Furthermore, the binding site on GPIb alpha for CVX may be close to that for von Willebrand factor. Therefore, a contribution of GPIb alpha to CVX-induced platelet responses needs to be carefully re-evaluated.

Antibodies raised against denatured and native forms of bothrojaracin were used to analyze the immunological similarities compared to the structural and biological features of five C-type lectin proteins from snake venom (bothrojaracin, botrocetin, Factor IX/X binding protein (FIX/Xbp), convulxin and Bothrops jararaca lectin). Anti-denatured-bothrojaracin antibodies, which recognize mainly linear epitopes, cross-reacted with botrocetin, FIX/Xbp and convulxin, as expected for homologous proteins. On the other hand, anti-native-bothrojaracin antibodies, which mostly interact with conformational epitopes, exhibited a higher degree of selectivity. These results show that differences exist at the surface of these proteins and that they should be related to their different biological activities, while they share a common and similar scaffold.

Glycoprotein VI (GPVI) is a platelet-specific glycoprotein that has been indicated to react with collagen and activate platelets. Its structure was recently identified by cDNA cloning (Clemetson, J. M., Polgar, J., Magnenat, E., Wells, T. N., and Clemetson, K. J. (1999) J. Biol. Chem. 274, 29019–29024). However, the mechanism of the interaction between collagen and GPVI has not been analyzed in detail because both collagen and GPVI are insoluble molecules. In this study, we expressed the extracellular domain of GPVI as soluble forms as follows: the monomeric form (GPVIex) and the dimeric form of GPVI fused with the human immunoglobulin Fc domain (GPVI-Fc2). Purified GPVIex strongly inhibited convulxin (Cvx)-induced platelet aggregation but only weakly inhibited that induced by collagen-related peptide. However, only GPVI-Fc2, and not GPVIex, inhibited collagen-induced platelet aggregation. The dimeric form of GPVI exhibits high affinity for collagen, as concluded from measurements of GPVI binding to immobilized collagen by both the enzyme-linked immunosorbent assay and surface plasmon resonance methods. GPVI-Fc2 bound to the surface of immobilized collagen with a dissociation constant (K D) of 5.76 × 10−7 m, but the binding of GPVIex was too weak to allow estimation of this parameter. Cvx did not inhibit the binding of dimeric GPVI to collagen, indicating that the binding site of GPVI to collagen was different from that to Cvx. Taken together, our data indicate that the high affinity binding site for collagen is composed from two chains of GPVI. Furthermore, they suggest that the binding sites for Cvx are different from the collagen-binding sites and do not need to be formed by two GPVI molecules. Because dimeric GPVI is the only form that shows high affinity to fibrous collagen, our results indicate that GPVI would be present as a dimeric form on the platelet. Moreover, surface plasmon resonance indicated that there is no detectable interaction between soluble collagen and GPVI, supporting our previous observation that GPVI only reacts with fibrous collagen.

TMVA, a novel C-type lectin-like protein that induces platelet aggregation in a dose-dependent manner, was purified from the venom of Trimeresurus mucrosquamatus. It consists of two subunits, alpha (15536 Da) and beta (14873 Da). The mature amino acid sequences of the alpha (135 amino acids) and beta subunits (123 amino acids) were deduced from cloned cDNAs. Both of the sequences show great similarity to C-type lectin-like venom proteins, including a carbohydrate recognition domain. The cysteine residues of TMVA are conserved at positions corresponding to those of flavocetin-A and convulxin, including the additional Cys135 in the alpha subunit and Cys3 in the beta subunit. SDS-PAGE, mass spectrometry analysis and amino acid sequence showed that native TMVA exists as two convertible multimers of (alpha beta)(2) and (alpha beta)(4) with molecular weights of 63680 and 128518 Da, respectively. The (alpha beta)(2) complex is stabilized by an interchain disulfide bridge between the two alpha beta-heterodimers, whereas the stabilization of the (alpha beta)(4) complex seems to involve non-covalent interactions between the (alpha beta)(2) complexes.

Envenomation by Crotalus durissus terrificus leads to coagulation disorders, myotoxicity, neurotoxicity and acute renal failure (ARF). The most serious systemic change and primary cause of death is ARF. In this work, we used RP-HPLC to isolate crotoxin, convulxin and gyroxin from venom of the related subspecies Crotalus durissus cascavella and investigated the effects of these toxins on renal function in the isolated rat kidneys perfused with Krebs–Henseleit solution containing 6% of bovine serum albumin. The parameters studied included perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), percent of sodium tubular transport (%TNa + ), percent of potassium tubular transport (%TK + ) and percent of chloride tubular transport (%TCl − ). Crotoxin (5 μg/ml) increased the PP, RVR, GFR, UF and decreased %TNa + , %TK + and %TCl − , with gyroxin (5 μg/ml) the GFR remained stable during the 120 min of perfusion, whereas PP and RVR increased significantly and the %TNa + , %TK + and %TCl − decreased significantly. Convulxin (5 μg/ml) had no effect on renal function. Crotoxin caused alterations in all renal parameters. Gyroxin produced a minor effect compared to crotoxin. These results indicated that crotoxin is the main componenet responsible for acute nephrotoxicity caused by C. d. cascavella venom.

Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake C-type lectin family and is thought to activate platelets by binding to platelet glycoprotein alpha(2)beta(1). We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to alpha(2)beta(1). Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor gamma chain (Fcgamma)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fcgamma pathway. Platelets from Fcgamma-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including alpha(2)beta(1) or GPIb for the lack of the Fcgamma pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72(SYK), p125(FAK), and PLCgamma2, whereas, in comparison with collagen and convulxin, the Fcgamma subunit neither is phosphorylated nor coprecipitates with p72(SYK). This supports an independent, GPIb- and integrin-based pathway for activation of p72(SYK) not involving the Fcgamma receptor.

Platelet endothelial cell adhesion molecule-1 (CD31) is a 130-kDa glycoprotein receptor present on the surface of platelets, neutrophils, monocytes, certain T-lymphocytes, and vascular endothelial cells. CD31 is involved in adhesion and signal transduction and is implicated in the regulation of a number of cellular processes. These include transendothelial migration of leukocytes, integrin regulation, and T-cell function, although its function in platelets remains unclear. In this study, we demonstrate the ability of the platelet agonists collagen, convulxin, and thrombin to induce tyrosine phosphorylation of CD31. Furthermore, we show that this event is independent of platelet aggregation and secretion and is accompanied by an increase in surface expression of CD31. A kinase capable of phosphorylating CD31 was detected in CD31 immunoprecipitates, and its activity was increased following activation of platelets. CD31 tyrosine phosphorylation was reduced or abolished by the Src family kinase inhibitor PP2, suggesting a role for these enzymes. In accordance with this, each of the Src family members expressed in platelets, namely Fyn, Lyn, Src, Yes, and Hck, was shown to co-immunoprecipitate with CD31. The involvement of Src family kinases in this process was confirmed through the study of mouse platelets deficient in Fyn.

Rhodnius prolixus aggregation inhibitor 1 (RPAI-1), a 19-kDa protein isolated from the salivary gland of R. prolixus, was purified by strong cation exchange and reverse-phase high performance liquid chromatographies. Based on 49 amino-terminal amino acid sequences of RPAI-1, primers were produced to generate probes to screen an R. prolixus salivary gland cDNA library. A phage containing the full-length clone of RPAI-1 codes for a mature protein of 155 amino acids. RPAI-1 shows sequence homology to triabin and pallidipin, lipocalins from Triatoma pallidipennis. The cDNA sequence was cloned in Pet17B Escherichia coli expression vector, producing an active peptide. RPAI-1 inhibits human platelet-rich plasma aggregation triggered by low concentrations of ADP, collagen, arachidonic acid, thromboxane A(2) mimetics (U46619), and very low doses of thrombin and convulxin. Here we show that ADP is the target of RPAI-1 since (i) RPAI-1 inhibits ADP-dependent large aggregation formation and secretion triggered by U46619, without affecting Ca(2+) increase and shape change; (ii) ADP restored the inhibition of U46619-induced platelet aggregation by RPAI-1, (iii) PGE(1)-induced increase of cAMP (which is antagonized by U46619 in an ADP-dependent manner) was restored by RPAI-1, (iv) RPAI-1 inhibits low concentrations of ADP-mediated responses of indomethacin-treated platelets, and (v) RPAI-1 binds to ADP, as assessed by large zone chromatography. RPAI-1 affects neither integrin alpha(2)beta(1)- nor glycoprotein VI-mediated platelet responses. We conclude that RPAI-1 is the first lipocalin described that inhibits platelet aggregation by a novel mechanism, binding to ADP.

Convulxin (Cvx) isolated from Crotalus durissus terrificus venom selectively binds with a high affinity to platelets and induces platelet aggregation by a mechanism that resembles that induced by collagen. Taking advantage that P65 has been recently cloned and expressed as a recombinant soluble protein (rec-P65), we examined the role of this non-integrin collagen receptor in platelet activation induced by Cvx. Rec-P65 blocked platelet adhesion to collagen-coated surfaces and inhibited platelet aggregation and ATP secretion induced by type I collagen. On the other hand, rec-P65 did not inhibit platelet aggregation and ATP secretion induced by Cvx, and it did not affect platelet adhesion to Cvx. In addition, ligand-blotting indicated that the Cvx binding to the collagen receptor GPVI was preserved in the presence of rec-P65. These observations indicate that P65 does not play a significant role in platelet activation by Cvx; in contrast, platelet response to collagen involves multiple receptors.

Platelet activation by collagen depends principally on two receptors, alpha(2)beta(1) integrin (GPIa-IIa) and GPVI. During this activation, the nonreceptor protein tyrosine kinase pp72(syk) is rapidly phosphorylated, but the precise contribution of alpha(2)beta(1) integrin and GPVI to signaling for this phosphorylation is not clear. We have recently found that proteolysis of platelet alpha(2)beta(1) integrin by the snake venom metalloproteinase, jararhagin, results in inhibition of collagen-induced platelet aggregation and pp72(syk) phosphorylation. In order to verify whether the treatment of platelets with jararhagin had any effect on GPVI signaling, in this study we stimulated platelets treated with either jararhagin or anti-alpha(2)beta(1) antibody with two GPVI agonists, an antibody to GPVI and convulxin. Platelet shape change and phosphorylation of pp72(syk) by both GPVI agonists was preserved, as was the structure and function of GPVI shown by (125)I-labeled convulxin binding to immunoprecipitated GPVI from jararhagin-treated platelets. In contrast, defective platelet aggregation in response to GPVI agonists occurred in both jararhagin-treated and alpha(2)beta(1)-blocked platelets. This apparent cosignaling role of alpha(2)beta(1) integrin for platelet aggregation suggests the possibility of a topographical association of this integrin with GPVI. We found that both platelet alpha(2)beta(1) integrin and GPVI coimmunoprecipitated with alpha(IIb)beta(3) integrin. Since platelet aggregation requires activation of alpha(IIb)beta(3) integrin, defective aggregation in the absence of alpha(2)beta(1) suggests that this receptor may provide a signaling link between GPVI and alpha(IIb)beta(3). Our study therefore demonstrates that platelet signaling leading to pp72(syk) phosphorylation initiated with GPVI engagement by either convulxin or GPVI antibody does not depend on alpha(2)beta(1) integrin. However, alpha(IIb)beta(3) integrin may, in this model, require functional alpha(2)beta(1) integrin for its activation.

We have cloned the platelet collagen receptor glycoprotein (GP) VI from a human bone marrow cDNA library using rapid amplification of cDNA ends with platelet mRNA to complete the 5' end sequence. GPVI was isolated from platelets using affinity chromatography on the snake C-type lectin, convulxin, as a critical step. Internal peptide sequences were obtained, and degenerate primers were designed to amplify a fragment of the GPVI cDNA, which was then used as a probe to screen the library. Purified GPVI, as well as Fab fragments of polyclonal antibodies made against the receptor, inhibited collagen-induced platelet aggregation. The GPVI receptor cDNA has an open reading frame of 1017 base pairs coding for a protein of 339 amino acids including a putative 23-amino acid signal sequence and a 19-amino acid transmembrane domain between residues 247 and 265. GPVI belongs to the immunoglobulin superfamily, and its sequence is closely related to FcalphaR and to the natural killer receptors. Its extracellular chain has two Ig-C2-like domains formed by disulfide bridges. An arginine residue is found in position 3 of the transmembrane portion, which should permit association with Fcgamma and its immunoreceptor tyrosine-based activation motif via a salt bridge. With 51 amino acids, the cytoplasmic tail is relatively long and shows little homology to the C-terminal part of the other family members. The ability of the cloned GPVI cDNA to code for a functional platelet collagen receptor was demonstrated in the megakaryocytic cell line Dami. Dami cells transfected with GPVI cDNA mobilized intracellular Ca(2+) in response to collagen, unlike the nontransfected or mock transfected Dami cells, which do not respond to collagen.

The collapse of phospholipid asymmetry across membrane bilayers mediates the physiologic processes of cell activation and apoptosis [1,2], whereas a failure to regulate transbilayer lipid movement is associated with a variety of pathologic conditions [3]. The terms ‘flippase’, ‘floppase’, and ‘scramblase’ refer to membrane transporter activities responsible for inward, outward, and bidirectional lipid movement; however, the biologic basis and regulation of these activities is not yet fully understood [4,5]. Scott syndrome (OMIM: 262890), a rare hemostatic defect characterized by an inability of stimulated platelets to externalize phosphatidylserine (PS), provides a unique tool to study cell membrane lipid handling. Heterozygosity for a novel missense mutation in the lipid translocator, adenosine triphosphate binding cassette 1 (ABCA1), was recently described in a Scott syndrome patient [6]. A paucity of cases, however, hinders genetic and platelet function studies to further define how this mutant protein contributes to expression of the Scott platelet phenotype. Spontaneous mutations of ABCA1 and targeted disruption of the murine gene produce the clinical phenotype Tangier disease (OMIM: 2054000) primarily characterized by a lack of plasma high-density lipoprotein (HDL) cholesterol and accumulation of lipid within reticuloendothelial cells [7,8]. To further clarify a role for ABCA1 in stimulated platelet PS movement, we looked for evidence of ABCA1 deficiency in platelets from Scott syndrome German shepherd dogs (GSD) [9] and performed linkage analyses to test for association of the canine Scott platelet phenotype with genetic markers within and flanking the ABCA1 locus. Canine Scott platelets display the hallmarks of their human counterpart e.g. normal aggregation, but a failure to generate prothrombinase activity and a lack of PS externalization and microparticle release in response to physiologic (thrombin plus collagen) or pharmacologic (ionophore) agonists [10]. The presence of ABCA1 protein in Scott GSD platelets was confirmed in Western blots of platelet lysates [11] (Fig. 1A) probed with an affinity-purified rabbit polyclonal antibody [ABCA1 antibody (ab14146), Abcam] that recognizes a peptide sequence of human ABCA1 with 100% homology to dog, mouse, and rat ABCA1. The free cholesterol content of Scott (n = 5) and control dog (n = 8) platelets were similar, with mean values of 14.3 μg cholesterol per 108 platelets and 12.2 μg cholesterol per 108 platelets, respectively. As Tangier platelets demonstrate impaired collagen-induced aggregation [12], we compared the aggregation of Scott and control dog platelets post-stimulation with collagen (12 μg per mL) and throughout a dosage range of the GPVI ligand, convulxin [CVX (0–30 nM)]. We found no significant difference (P = 0.13) in mean maximal collagen-induced aggregation of Scott GSD platelet-rich plasma (n = 5; 75% aggregation) vs. control dogs (n = 8; 80% aggregation), and the CVX response of Scott and control dog platelets was essentially the same (Fig. 1B). Additional manifestations of platelet dysfunction in Tangier patients include decreased reactivity to low dose thrombin and defective granule formation and release [7,12,13]. We therefore evaluated Scott GSD platelets in flow cytometry experiments to assess various granule-dependent responses. We found no significant differences (P > 0.1) between Scott GSD (n = 5) and control dogs (n = 8) in mean percentages of CD62P positive platelets (denoting P-selectin expression) post-thrombin (0.5 U per mL) stimulation (Scott GSD = 70.3%; control = 62.5%) or CVX (10 nM) stimulation (Scott GSD = 53%; control = 48.8%), mean mepacrine uptake (denoting dense granule content [14]) (Scott GSD = 68.8% positive; control = 75%) and mean CD61 positive leukocytes (denoting platelet-leukocyte aggregate formation [15]) post-thrombin (2 U per mL) stimulation (Scott GSD = 12.2%; control = 10.2%). In contrast, the mean percentage Annexin V positive platelets (denoting PS externalization) in response to combined simulation with thrombin (0.5 U per mL) plus CVX (10 nM) was significantly reduced (P < 0.0001) for Scott platelets (Scott GSD = 5.6%; control = 41%). Although species differences in platelet physiology limit direct comparisons, the pattern of activation response for Scott GSD differs from human and murine Tangier platelets, indicating that the ABCA1 protein within Scott GSD platelets is functional. Fig. 1 Platelet expression of ABCA1, aggregation response to convulxin stimulation, and ABCA1 linkage analyses in Scott German shepherd dogs (GSD). (A) Western blot analyses of ABCA1 in platelet lysates. Platelet proteins were solubilized in Laemelli buffer, ... Phenotyping of the study pedigree (Fig. 1C) for linkage analyses was based on residual serum prothrombin [9,16]. Abnormal platelet function of affected GSD (n = 22) in this screening test was confirmed by flow cytometric findings of diminished PS externalization and microvesiculation post-calcium ionophore. The GSD were genotyped for an intragenic canine ABCA1 (Gene ID: 481651) microsatellite and 10 additional markers [17] spanning CFA11 (chromosomal location of canine ABCA1) at 5–10 cM intervals. The power of these analyses to identify a disease gene was assessed by simulating 40 000 replicate data sets using the SLINK function of LINKAGE[18]; conditional on the observed pedigree, autosomal recessive inheritance, and variation of recombination rate between marker and disease locus (θ) along a gradient from 0 to 0.1. These simulations predicted approximately 80% power to identify a disease locus at cutoff LOD = 2, for markers within θ = 0.05 from the disease locus. We found that two point and multipoint linkage analyses, calculated using SUPERLINK v. 1.6 (http://bioinfo.cs.technion.ac.il/superlink/), failed to demonstrate an association between canine Scott syndrome and the ABCA1 locus [LOD

Convulxin (Cvx) isolated from Crotalus durissus terrificus venom, induces platelet aggregation, phospholipase C (PLC) activation, and tyrosyl-phosphorylation (PTP) of multiple proteins, including PLCgamma2 by a mechanism independent of integrin alphaIIb beta3. However, PTP induced by Cvx is followed by a dephosphorylation step in a platelet aggregation-dependent manner. Here we show that increasing intraplatelet content of cAMP with forskolin is associated with the inhibition of Cvx-induced platelet aggregation, ATP secretion, and inositol-phosphates production. However, the early onset of Cvx-induced PTP is not sensitive to cAMP (including PLCgamma2), and it also occurs in the presence of integrin alphaIIb beta3-antagonist (RGDS peptide, RGDS) or inhibitors of actin polymerization (cytochalasin D, CD) and tyrosine-phosphatases (phenylarsine oxide, PAO). However, forskolin, RGDS, and CD prevented the dephosphorylation step together with inhibition of platelet aggregation, whereas in the presence of phenylarsine oxide (PAO) the dephosphorylation step was replaced by an increase in the number and intensity of tyrosyl-phosphorylated proteins. Our data provide evidence to conclude that (i) cAMP inhibits platelet aggregation at a downstream site to PLCgamma2 tyrosyl-phosphorylation; (ii) Cvx-induced PTP is independent on integrin alphaIIb beta3 engagement, actin polymerization, and tyrosine-phosphatases activation; (iii) integrin alphaIIb beta3 mediates the dephosphorylation step in a platelet aggregation-dependent manner; and (iv) Cvx and collagen stimulate platelets by a similar signal transduction pathway.

1Convulxin (Cvx) is a well-characterized platelet aggregating glycoprotein isolated from Crotalus durissus terrificus and C. d. cascavella venoms. In the present report we show that Cvx induces tyrosine phosphorylation of human platelet proteins, including phospholipase C-gamma 2 (PLC gamma 2), and also stimulates [3H]arachidonic acid ([3H]AA) mobilization, pleckstrin phosphorylation, and an increase in the cytosolic Ca2+ concentration ([Ca2+]in) due to both Ca2+ entry and internal Ca2+ mobilization. Staurosporine, a potent protein kinase inhibitor, and genistein, a specific inhibitor of protein tyrosine kinases (PTK), were used to evaluate the role of protein tyrosine phosphorylation (PTP) in the signal transduction evoked by Cvx. Staurosporine and genistein inhibited in a dose-dependent manner platelet aggregation induced by Cvx. Both inhibitors significantly blocked to near basal levels breakdown of phosphatidylinositol 4,5-bisphosphate from [myo-2-3H]inositol-labeled platelets and the production of [3H]AA metabolites from [3H]AA-labeled platelets after challenge with Cvx. Cvx provokes an increase in [Ca2+]in in Fura-2-loaded platelets that was abolished by concentrations of staurosporine which also inhibited Cvx-induced platelet aggregation. In addition, Cvx stimulates a rapid increase in tyrosine phosphorylation of human platelets proteins with molecular masses of 40, 72/74, 78/80, 105, 120, and 145 kDa, followed by dephosphorylation. Furthermore, Cvx stimulates a rapid tyrosyl phosphorylation of a 145-kDa molecular mass protein that was identified as PLC gamma 2. PTP induced by Cvx was not inhibited when platelets were stimulated in the presence of indomethacin, apyrase, EDTA, or RGDS peptide. These results indicate that PTP is chronologically proximal to Cvx binding to platelets, and is independent of aggregation or fibrinogen binding to the integrin alpha IIb beta 3. On the other hand, the dephosphorylation step is inhibited by RGDS peptide or EDTA, suggesting that integrin alpha IIb beta 3 is envolved in this step. The profile obtained with Cvx resembles that obtained in platelets adherent to an immobilized ligand, such as immobilized collagen, in which PTP is independent on integrin alpha IIb beta 3. Thus, we suggest that Cvx is an example of a protein with adhesion molecule-like properties; i.e., it is an adhesin. In conclusion, our results show that Cvx induces multiple signaling pathways in platelets via a PTK-dependent pathway involving PLC gamma 2 tyrosyl phosphorylation, with the subsequent platelet responses. Cvx is unique among platelet soluble agonists because under test tube stirring conditions it induces a PTP profile independently of integrin alpha IIb beta 3.

We analyzed the interaction of convulxin (Cvx), a 72-kDa protein isolated from the venom of Crotalus durissus terrificus, with human platelets. Cvx is a potent platelet agonist that induces an increase in the intracellular Ca2+ concentration ([Ca2+]i), granule exocytosis and aggregation. 125I-Labeled Cvx binds specifically and rapidly to platelets at binding sites of high and moderate affinity. Platelets adhere to immobilized Cvx in a time-dependent but cation-independent manner. Platelet exocytosis and aggregation induced by Cvx were inhibited by an anti-integrin alpha2beta1 monoclonal antibody (6F1) and by the Fab fragments of a polyclonal anti-glycoprotein VI (GPVI) antibody. Both the adhesion of platelets to Cvx and the Cvx-induced increase in [Ca2+]i were inhibited by anti-GPVI Fab fragments but not by 6F1. Ligand blotting assay showed that 125I-Cvx binds to a 57-kDa platelet protein with an electrophoretic mobility identical to that of GPVI. In addition, we observed the following: (i) 125I-Cvx binds to GPVI immunoprecipitated by the anti-GPVI antibody from a platelet lysate, and (ii) Cvx inhibits the binding of anti-GPVI IgG to GPVI. Taken together, these results demonstrate that GPVI behaves as a Cvx receptor and that the alpha2beta1 integrin appears to be involved in the later stages of Cvx-induced platelet activation, i.e. exocytosis and aggregation.

The effects of convulxin, isolated from the venom of Crotalus durissus terrificus, on the localization and distribution of wheat germ agglutinin (WGA) binding sites on platelet surfaces have been investigated at an ultrastructural level. A post-embedding cytochemical technique using WGA-gold complexes was used and the quantitative intensity of WGA-labeling on the surface membrane of platelets after convulxin stimulation was determined. In the presence of Ca2+, convulxin induced platelet release and aggregation, while in its absence, the platelets formed pseudopodia and showed release reaction, but without aggregation. After treatment with convulxin, WGA-labeling on the surface membrane decreased compared with intact washed (control) platelets. In the presence of Ca2+, clusters of gold label were often found on the surface membrane. However, the WGA-labeling intensity of the membrane of the open canalicular system increased significantly compared with that of platelets stimulated by convulxin in the absence of Ca2+. Direct morphological evidence demonstrates qualitative and quantitative alterations of WGA-labeling on the surface membrane of platelets, after convulxin stimulation. The possibility is considered that WGA-binding glycoproteins on the surface membrane are involved in the aggregation response after convulxin stimulation.

Convulxin is a high molecular weight toxin isolated from rattlesnake ( Crotalus durissus terrificus, Crotalus durissus collilineatus, Crotalus durissus cascavella ) venom. When administered i.v. it reduces the number of circulating platelets and leukocytes of anesthetized rabbits in a dose dependent manner. Small doses induce reversible platelet aggregation, and larger doses cause lysis. The doses of convulxin that cause platelet lysis also evoke cardiovascular and respiratory disturbances in anesthetized animals. These effects were also observed when platelets which were preincubated with convulxin in vitro were administered to animals which had received antivenom serum. This indicates that convulxin may act by releasing substances contained in the platelets.

It was concluded in a previous paper that the high Mr platelet-activating glycoprotein isolated earlier from the venom of Crotalus durissus cascavella has an hexameric structure of the alpha 3 beta 3 type involving two distinct subunits. Data reported here demonstrate that these two subunits are separable from each other by ion exchange chromatography under denaturating conditions, have similar Mrs (alpha = 12,540 et beta = 13,770) and exist in a one to one ratio within the native molecule. Carbohydrate analysis indicated that they are both similarly glycosylated to a small extent. They have slightly different amino-acid compositions, a common N-terminal sequence up to the fifth residue and similar extinction coefficients at 280 nm. The native molecule has a calculated Mr of 78,930. Additional data demonstrated that convulxin from the venom of Crotalus durissus terrificus is the same platelet-activating agent as the presently described platelet-activating glycoprotein (PAG) from the venom of Crotalus durissus cascavella.

A very potent platelet-aggregating glycoprotein, convulxin, was purified from the venom of Crotalus durissus cascavella by gel filtration on Sephadex G75 and by adsorption to Sepharose 4B gel. The apparent molecular weights of the native protein were 78400 and 60000 daltons determined by SDS electrophoresis and by gel filtration under denaturating conditions, respectively. Under the same conditions, the apparent molecular weights of the reduced protein were 13000 and 12000 respectively. These discrepancies are due to the presence of a carbohydrate moiety in the molecule. Analysis for carbohydrates showed the presence of around 4, 8 % sugars. Convulxin is built up of closely similar subunits linked by disulfide brides and is devoid of free sulhydryl groups.