Convulxin

Manufactured by Enzo Life Sciences

Sourced in United States

Convulxin is a protein isolated from the venom of the South American bushmaster snake (Lachesis muta). It is a potent activator of the blood platelet receptor, glycoprotein VI, and is used as a research tool to study platelet function and activation pathways.

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Lab products found in correlation

Thrombin, by Merck Group (2 mentions) Jon a pe, by Emfret Analytics (2 mentions) Gf109203x, by Enzo Life Sciences (2 mentions) Adenosine diphosphate (adp), by Merck Group (2 mentions) Tyrode s salts, by Merck Group (1 mentions) Fitc conjugated pac 1, by BD (1 mentions) Adenosine diphosphate adp, by Merck Group (1 mentions) Anti cd62p apc, by BioLegend (1 mentions) Pac 1 fitc, by BD (1 mentions) Human fibrinogen, by Merck Group (1 mentions) Atipamezol, by Pfizer (1 mentions) Prostacyclin, by Merck Group (1 mentions) Heparin sodium, by Merck Group (1 mentions) Ecl solution, by PerkinElmer (1 mentions) Igepal ca 630, by Merck Group (1 mentions) Kollagenreagent horm, by Takeda Pharmaceuticals (1 mentions) α thrombin, by Roche (1 mentions) Apyrase type 3, by Merck Group (1 mentions) U46619, by Alexis Biochemicals (1 mentions) Medetomidine, by Pfizer (1 mentions)

8 protocols using convulxin

Platelet Activation Assay by Flow Cytometry

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MKs were pelleted and resuspended in Tyrode’s Salts (Sigma) with 0.1% bovine serum albumin (BSA) containing FITC-conjugated PAC-1 (BD Biosciences), PacBlue-conjugated CD42a (eBioscience), and APC-conjugated CD42b (eBioscience) at a concentration of roughly 100,000 cells per 50 μl. Following addition of Convulxin (Enzo Biochem) or Thrombin (Sigma), cells were incubated at room temperature in the dark for 10 min. Cells were then incubated on ice for 10 min. An additional 100 μl Tyrode’s Salts containing 0.1% BSA was added, and cells were immediately analyzed by flow cytometry.

Thom C.S., Jobaliya C.D., Lorenz K., Maguire J.A., Gagne A., Gadue P., French D.L, & Voight B.F. (2020). Tropomyosin 1 genetically constrains in vitro hematopoiesis. BMC Biology, 18, 52.

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Platelet Activation Assay with Agonists

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For platelet activation, 2 × 10⁶ platelets washed in modified Tyrode’s buffer (134 mM NaCl, 0.34 mM Na₂HPO₄, 2.9 mM KCl, 12 mM NaHCO₃, 20 mM HEPES, 5 mM glucose) containing 1 mM CaCl₂ were incubated with 0.1 U/mL thrombin (Sigma-Aldrich), 25 μM adenosine diphosphate (ADP; Sigma-Aldrich), 20 ng/mL convulxin (Enzo Life Sciences), or vehicle alone in the presence of 2 μg/mL JON/A-PE (Emfret Analytics), 12 μg/mL PAC-1–FITC (BD Biosciences), or 10 μg/mL anti-CD62P–APC (BioLegend) and analyzed 10 minutes later.^{38 (link)} Platelet activation was expressed as the ratio between the mean fluorescence intensity measured after the stimulation with each agonist and the mean fluorescence intensity measured after incubation with vehicle alone.^{39 (link)}

Abbonante V., Gruppi C., Battiston M., Zulian A., Di Buduo C.A., Chrisam M., Sereni L., Laurent P.A., Semplicini C., Lombardi E., Mazzucato M., Moccia F., Petronilli V., Villa A., Bello L., Pegoraro E., Bernardi P., Braghetta P., De Marco L., Bonaldo P, & Balduini A. (2021). Ablation of collagen VI leads to the release of platelets with altered function. Blood Advances, 5(23), 5150-5163.

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Platelet activation assays using agonists

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Collagen (Kollagenreagent Horm; Nycomed), convulxin (Enzo Lifesciences) α-thrombin (Roche Diagnostics), adenosine diphosphate (ADP), sodium heparin, human fibrinogen, apyrase type III, prostacyclin (PGI₂), Igepal CA-630 (all from Sigma-Aldrich), U46619 (Alexis Biochemicals) and ECL solution (PerkinElmer) were purchased, collagen-related peptide (CRP) was generated as described [21] (link). Rhodocytin was a generous gift from Prof. Dr. J. Eble (Münster University Hospital, Germany). The anesthetic drugs medetomidine (Pfizer), midazolam (Roche Pharma AG), and fentanyl (Janssen-Cilag GmbH) and the antagonists atipamezol (Pfizer), flumazenil, and naloxon (both from Delta Select GmbH) were used according to the regulation of the local authorities. The antibody against the activated form of integrin αIIbβ3 (JON/A-PE) was from Emfret Analytics. Anti-murine CD84 monoclonal antibody JER1 [19] (link) and other antibodies were generated and modified in our laboratories as described [22] (link).

Hofmann S., Braun A., Pozgaj R., Morowski M., Vögtle T, & Nieswandt B. (2014). Mice Lacking the SLAM Family Member CD84 Display Unaltered Platelet Function in Hemostasis and Thrombosis. PLoS ONE, 9(12), e115306.

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Activation of Platelet Receptors

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All cell culture reagents were purchased from Invitrogen. Human α-thrombin was purchased from Haematological Technologies (specific activity 3200–3400 U/mg) (Essex Junction, VT). PAR4 activating peptide (AYPGKF-NH2) and PAR1 activating peptide (SFLLRN-NH2) were synthesized at PolyPeptide Laboratories (San Diego, CA). Convulxin was purchased from Enzo Life Sciences Inc. (Farmingdale, NY). Collagen, ADP, and CHRONO-LUME were purchased from Chrono-log Corporation (Havertown, PA). Protease inhibitor cocktail tablets were purchases from Roche. Rose-Bengal sodium salt, fibrinogen from human plasma, red blood cell lysing buffer, and sepharose 2B were purchased from Sigma-Aldrich. The CAN12 antibody was prepared against the human PAR4 sequence (CANDSDTLELPD) by Bethyl Laboratories as a custom synthesis using a goat as the host. The antibody was affinity purified using a CANDSDTLTLPD peptide-specific immunosorbent. For control experiments, ChromPure goat IgG, whole molecule was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). The secondary antibodies IRDYE 800CW donkey anti-mouse IgG, IRDYE 680RD donkey anti-goat IgG, and IR 680RD goat anti-rabbit IgG were purchased from LI-COR Biosciences (Lincoln, NE).

Mumaw M.M., de la Fuente M., Noble D.N, & Nieman M.T. (2014). Targeting the anionic region of human protease activated receptor 4 (PAR4) inhibits platelet aggregation and thrombosis without interfering with hemostasis. Journal of thrombosis and haemostasis : JTH, 12(8), 1331-1341.

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Platelet Aggregation Assay by LTA

Cited 1 time

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Aggregation of washed human platelets was monitored by light transmission aggregometry (LTA) using an Apact4S Plus aggregometer (DiaSys Greiner, Flacht, Germany). Platelets were stimulated by ADP (Sigma-Aldrich, Saint Louis, MO, USA), convulxin (Enzo life sciences, Lausen, Switzerland), echicetin beads (EB) (prepared as previously described [28 (link),53 (link)]), or PDBu (Sigma-Aldrich, Saint Louis, MO, USA) under stirring conditions (1000 s⁻¹) at 37 °C. Platelets were pre-treated with different effectors prior to stimulation: Src family kinase inhibitor PP2 (Abcam, Cambridge, UK), Syk inhibitors OXSI-2 (Merck, Darmstadt, Germany) and PRT-060318 (Sellckem, Houston, TX, USA), PKC pan-inhibitor GF109203X (Enzo life sciences, Lausen, Switzerland), or cAMP-elevating agent iloprost (Bayer AG, Leverkusen, Germany).

Makhoul S., Dorschel S., Gambaryan S., Walter U, & Jurk K. (2019). Feedback Regulation of Syk by Protein Kinase C in Human Platelets. International Journal of Molecular Sciences, 21(1), 176.

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Platelet Activation Signaling Pathway

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Convulxin and GF109203X were obtained from Enzo Life Sciences (Lausen, Switzerland). PRT-060318 was from Selleckchem (Houston, Texas, United States). Acalabrutinib was purchased from Abcam (Cambridge, United Kingdom). AR-C669931 (AR-C) was from the Medicines Company (Parsippany, New Jersey, United States). Total Syk (4D10), total PLCγ2 (B-10), and total Akt1 (B-1) antibodies were from Santa Cruz Biotechnology (Dallas, Texas, United States). Phospho-Syk (S297, Y352, Y525/526), total Btk (D3H5), phospho-Btk (S180, Y223, Y551), phospho-PLCγ2 (Y759, Y1217), phospho-LAT Y220, phospho-Akt (T308, S473), phospho-MEK1/2 S217/S221, phospho-Erk1/2 T202/Y204, phospho-p38 T180/Y182, total MEK1/2, α-actinin, and βactin were provided by Cell Signaling Technologies (Danvers, Massachusetts, United States). Secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse antibodies were obtained from BioRad Laboratories (Hercules, California, United States). FITC-conjugated mouse anti-human CD62P, CD63, and PAC-1 antibodies were from BD Biosciences (Heidelberg, Germany).

Zhang P., von Ungern-Sternberg S., Hastenplug L., Solari F.A., Sickmann A., Kuijpers M.J.E., Heemskerk J.W.M., Walter U, & Jurk K. (2024). Multi-phased Kinetics and Interaction of Protein Kinase Signaling in Glycoprotein VI-Induced Platelet αIIbβ3 Integrin Activation and Degranulation. Thrombosis and haemostasis.

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Platelet Activation Pathway Regulators

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Rabbit polyclonal antibodies against human TIMP1, TIMP2, TIMP3, or TIMP4, as well as AlexaFluor488-labeled anti-mouse or anti-rabbit IgG raised in goats, were purchased from Abcam (Cambridge, UK). Purified recombinant human (rh) ADAM10, ADAM17, TIMP 1, TIMP2, TIMP3 or TIMP4 were purchased from R&D Systems (Minneapolis, MI, USA). Equine Type I collagen (Horm suspension) was from Takeda (Lincz, Austria). Convulxin was purchased from Enzo Life Sciences (Framingham, USA). Collagen-related peptide (CRP) from Auspep (Tullamarine, Australia) was chemically cross-linked (CRP-xL) in-house as described. 35 (link)

Lee C.S.M., Kaur A., Montague S.J., Hicks S.M., Andrews R.K, & Gardiner E.E. (2023). Tissue inhibitors of metalloproteinases (TIMPs) modulate platelet ADAM10 activity. Platelets, 34(1).

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Isolation and Characterization of Platelet-Derived Extracellular Vesicles

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Washed platelets (2 × 10 8 /ml) were activated with different agonists (100 ng/ml convulxin, Enzo Life Sciences), 50 μM TRAP-6 (AnaSpec Inc.), or 5 nM thrombin (Haematologic Technologies) for 30 min at 37 °C. Integrin α IIb β 3 ligand binding was blocked with 10 nM tirofiban (CAS 144494-65-5, Correvio Int.). Integrin dependent outsidein signalling pathways were blocked with either 10 μM cytochalasin D (CAS 22144-77-0, Sigma), 10 μM PTPN1 inhibitor (CAS 765317-72-4, Merck), 10 μM SYK inhibitor (CAS 622387-85-3, Cayman Chemicals), 10 μM calpain inhibitor I (CAS 110044-82-1, Cayman chemicals), 1 μM ROCK (GSK429286A, selleckchem) or 10 μM Gα 13 inhibitor (made inhouse according to Shen B. et al. [28] , see below). Platelets were preincubated for 5 min at 37 °C with indicated inhibitors prior to activation, as described. Activated platelets were spun down by centrifugation at 300g for 5 min, after which the EV-containing supernatant was filtered with PK50 MiniSart sterile 0.8 μm filters (Sartorius). The filtrate was centrifuged at 20,000g for 1 h at 4 °C to pellet the EVs [22, 23, 29] . Pellets were resuspended in platelet buffer pH 7.45, snap frozen into liquid nitrogen and stored at -80 °C.

Heinzmann A.C.A., Karel M.F.A., Coenen D.M., Vajen T., Meulendijks N.M.M., Nagy M., Suylen D.P.L., Cosemans J.M.E.M., Heemskerk J.W.M., Hackeng T.M, & Koenen R.R. (2020). Complementary roles of platelet α_IIbβ₃ integrin, phosphatidylserine exposure and cytoskeletal rearrangement in the release of extracellular vesicles. Atherosclerosis, 310.

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