COnvulxin, a potent platelet-aggregating protein from Crotalus durissus terrificus venom, specifically binds to platelets

Background: Fucoidan was tested as a novel drug for hemophilia, but its effect on platelets has not been studied. Results: We show that fucoidan activates human and mouse platelets and that activation is blocked in CLEC-2 knock-out murine platelets. Conclusion: Fucoidan is a novel agonist for the CLEC-2. Significance: Understanding the effects of fucoidan on platelets can help in designing efficient drugs for hemophilia.

Section: Methodsmentioning

confidence: 99%

Fucoidan Is a Novel Platelet Agonist for the C-type Lectin-like Receptor 2 (CLEC-2)

Manne

Getz

Hughes

et al. 2013

“…Molecular biology reagents were from Invitrogen (Carlsbad, CA). Convulxin was purified as described (14).…”

Section: Methodsmentioning

confidence: 99%

Dipetalodipin, a Novel Multifunctional Salivary Lipocalin That Inhibits Platelet Aggregation, Vasoconstriction, and Angiogenesis through Unique Binding Specificity for TXA2, PGF2α, and 15(S)-HETE

Assumpção

Alvarenga

Ribeiro

et al. 2010

Self Cite

Dipetalodipin (DPTL) is an 18 kDa protein cloned from salivary glands of the triatomine Dipetalogaster maxima. DPTL belongs to the lipocalin superfamily and has strong sequence similarity to pallidipin, a salivary inhibitor of collagen-induced platelet aggregation. DPTL expressed in Escherichia coli was found to inhibit platelet aggregation by collagen, U-46619, or arachidonic acid without affecting aggregation induced by ADP, convulxin, PMA, and ristocetin. An assay based on incubation of DPTL with small molecules (e.g. prostanoids, leukotrienes, lipids, biogenic amines) followed by chromatography, mass spectrometry, and isothermal titration calorimetry showed that DPTL binds with high affinity to carbocyclic TXA 2 , TXA 2 mimetic (U-46619), TXB 2 , PGH 2 mimetic (U-51605), PGD 2, PGJ 2 , and PGF 2␣ . It also interacts with 15(S)-HETE, being the first lipocalin described to date to bind to a derivative of 15-lipoxygenase. Binding was not observed to other prostaglandins (e.g. PGE 1 , PGE 2 , 8-iso-PGF 2␣ , prostacyclin), leukotrienes (e.g,.

“…Materials-Convulxin was purified from the venom of Crotalus durissus terrificus (from Fundaçã o Ezequiel Dias (FUNED), Minas Gerais, Brazil or Latoxan, Valence, France) as previously described (19). Convulxin was labeled with 125 I using the Iodogen procedure (Pierce) and Na 125 I (Amersham Pharmacia Biotech) or was coupled to fluorescein (FITC) as previously described (7).…”

Section: Methodsmentioning

confidence: 99%

“…Proteins (40 g) were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (6,19). Membranes were incubated with polyclonal anti-GPVI IgG or with 3J24-2 or the anti-FcR ␥ chain mAbs.…”

Section: Methodsmentioning

confidence: 99%

Expression and Function of the Collagen Receptor GPVI during Megakaryocyte Maturation

Lagrue-Lak-Hal

Debili

Kingbury³

et al. 2001

In this report, the expression and function of the platelet collagen receptor glycoprotein VI (GPVI) were studied in human megakaryocytes during differentiation and maturation of mobilized blood and cord blood derived CD34 ؉ cells. By flow cytometry, using an anti-GPVI monoclonal antibody or convulxin, a GPVI-specific ligand, GPVI was detected only on CD41؉ cells including some CD41 ؉ /CD34؉ cells, suggesting expression at a stage of differentiation similar to CD41. These results were confirmed at the mRNA level using reverse transcriptionpolymerase chain reaction. GPVI expression was low during megakaryocytic differentiation but increased in the more mature megakaryocytes (CD41 high ). As in platelets, megakaryocyte GPVI associates with the Fc receptor ␥ chain (FcR␥). The FcR ␥ chain was detected at the RNA and protein level at all stages of megakaryocyte maturation preceding the expression of GPVI. The other collagen receptor, ␣ 2 ␤ 1 integrin (CD49b/CD29), had a pattern of expression similar to GPVI. Megakaryocytic GPVI was recognized as a 55-kDa protein by immunoblotting and ligand blotting, and thus it presented a slightly lower apparent molecular mass than platelet GPVI (58 kDa). Megakaryocytes began to adhere to immobilized convulxin via GPVI after only 8 -10 days of culture, at a time when megakaryocytes were maturing. At this stage of maturation, they also adhered to immobilized collagen by ␣ 2 ␤ 1 integrin-dependent and -independent mechanisms. Convulxin induced a very similar pattern of protein tyrosine phosphorylation in megakaryocytes and platelets including Syk, FcR␥, and PLC ␥2 . Our results showed that GPVI is expressed early during megakaryocytic differentiation but functionally allows megakaryocyte adherence to collagen only at late stages of differentiation when its expression increases.At sites of vascular injury, platelets adhere and are activated by contact with collagen fibers exposed with the subendothelium matrix leading to thrombus formation. Several collagen receptors are present on platelets: integrin ␣ 2 ␤ 1 (CD49b/ CD29), GPIV 1 (CD36), GPVI, p65, and a recently cloned type III collagen receptor (1, 2). The direct interaction between collagen and integrin ␣ 2 ␤ 1 adheres platelets at the collagen surface, but platelet activation and thrombus formation and attachment are subsequently mainly supported by GPVI. In addition to collagen, two GPVI-specific ligands have been described: collagen-related peptides (3) and convulxin, a snake venom protein (4). GPVI has recently been characterized as a new member of the immunoglobulin superfamily of cell-associated receptors, in which expression is restricted to the hematopoietic lineage (5-7). It is coexpressed as a noncovalent complex with the common immunoglobulin receptor ␥ (FcR␥) chain, which acts as the signaling subunit of the complex (8, 9) As a consequence, signaling pathways coupled to GPVI dimerization are identical to those coupled to other immune receptors; tyrosine phosphorylation of the FcR ␥ chain on its immunoreceptor tyrosine-...