Convulxin

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Convulxin is a snake venom protein isolated from the Crotalus durissus terrificus snake. It functions as a platelet activator that binds to the glycoprotein VI receptor on the surface of platelets, thereby inducing platelet aggregation.

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Lab products found in correlation

Thrombin, by Merck Group (2 mentions) Botrocetin, by Merck Group (1 mentions) Cd62p antibody, by BD (1 mentions) Cd9 apc, by Abcam (1 mentions) Cd9 pe, by Abcam (1 mentions) Thrombin, by Cayman Chemical (1 mentions) Facs lysing solution, by BD (1 mentions) Lsrii cytometer, by BD (1 mentions) Horseradish peroxidase hrp conjugated donkey anti rabbit immunoglobulin g igg, by Cytiva (1 mentions) Sheep anti mouse igg, by Cytiva (1 mentions) Fura 2 am, by Thermo Fisher Scientific (1 mentions) U46619, by Merck Group (1 mentions) Fitc phalloidin, by Merck Group (1 mentions) Enhanced chemiluminescence western blotting detection reagent, by Cytiva (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Heparin, by Merck Group (1 mentions) Fibrinogen, by Merck Group (1 mentions) Anti p38 mapk, by Cell Signaling Technology (1 mentions) Hybond, by Cytiva (1 mentions) Accuri c6 flow cytometer, by BD (1 mentions)

Spelling variants of this product

Convulxin (CVX) (2 citations)

9 protocols using convulxin

Platelet Activation and Aggregation Assays

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For flow cytometry activation experiments, washed platelets were stimulated with thrombin (#13188, Cayman Chemicals), 2-meADP (#1624, Tocris), and convulxin (#sc-202554, Santa Cruz) for 10 min. and then CD62P antibody (#553774, BD Biosciences) and the activated GPIIb/IIIa antibody JON/A (#M023-2, Emfret) were added. Washed platelets were activated with 0.5 μg/mL botrocetin (#V5625, Sigma) and 10 μg/mL human von Willebrand factor (#HCVWF-0190, Haematologic Technologies Inc.). For platelet aggregation, whole mouse blood was centrifuged for 15 minutes at 1000 rpm, platelet rich plasma (PRP) was incubated in a 1:100 dilution of CD9-APC or CD9-PE (Abcam) for 15 minutes after samples were washed and resuspended in plasma to a concentration of 50 × 10⁶ platelets/mL. Labeled platelets were mixed 1:1 and agonist-stimulated at 37°C while shaking similar to published by others [21 (link)].

Modjeski K.L., Ture S.K., Field D.J., Cameron S.J, & Morrell C.N. (2016). Glutamate Receptor Interacting Protein 1 Mediates Platelet Adhesion and Thrombus Formation. PLoS ONE, 11(9), e0160638.

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Platelet Activation Surface Receptor Expression

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Expression of the platelet activation surface receptors glycoprotein IIb/IIIa, P-selectin, and CD63 was measured by flow cytometry. Healthy donor whole blood was collected in sodium citrate and treated with EPI (50 ng/ml) or control (HT buffer only) for either 3 min or 30 min of incubation. Blood was then diluted (1:50) with HT buffer, added to tubes with or without convulxin (5 ng/ml, Santa Cruz Biotechnology) and antibodies, and incubated at 37°C for 10 min. Allophycocyanin-conjugated anti-CD41 (clone HIP-8) was used to distinguish platelets, fluorescein isothiocyanate conjugated anti-CD62p (clone AK4) to distinguish platelets expressing surface P-selectin, anti-CD41/CD61 (clone PAC-1) to distinguish platelets with glycoprotein IIb/IIIa complex, and anti-CD63 (clone H5C6) to stain platelets expressing surface CD63. Staining with fluorophore and antibody isotype matched controls was performed to determine positive staining. All antibodies and immunoglobulin isotype controls were purchased from Biolegend. Stained samples were treated with FACS Lysing Solution at a ratio of 2:1 (Becton Dickinson) for 10 min at room temperature prior to storage at 4°C for no more than 24 h. Data were collected with a BD LSRII cytometer (Becton Dickinson) and analyzed using FlowJo (FlowJo, LLC).

Wang Z., Gergely J, & Tao T. (1992). Characterization of the Ca(2+)-triggered conformational transition in troponin C.. Proceedings of the National Academy of Sciences of the United States of America, 89(24), 11814-11817.

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Platelet Activation Signaling Pathway

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Thrombin, U46619, heparin, collagen, fibrinogen, FITC-phalloidin and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). Fura-2AM was purchased from Molecular Probes (Eugene, OR, USA). An anti-phospho-p38 mitogen-activated protein kinase (MAPK) Ser¹⁸² monoclonal antibody (mAb) and convulxin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p38 MAPK, anti-phospho-c-Jun N-terminal kinase (JNK) (Thr¹⁸³/Tyr¹⁸⁵), anti-phospho-(Ser) protein kinase C (PKC) substrate (pleckstrin; p-p47), and anti-JNK polyclonal antibodies (pAbs) were purchased from Cell Signaling (Beverly, MA, USA). Anti-phospho-protein kinase B (Akt) (Ser⁴⁷³) and anti-Akt mAbs were purchased from Biovision (Mountain View, CA, USA). Fluorescein isothiocyanate (FITC)-labeled anti-GPVI (JAQ1) mAb was obtained from Emfret Analytics (Würzburg, Germany). Hybond-P polyvinylidene fluoride (PVDF) membranes, an enhanced chemiluminescence Western blotting detection reagent, and antibodies, namely horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG), and sheep anti-mouse IgG, were purchased from Amersham (Buckinghamshire, UK). A fluorescein isothiocyanate (FITC) anti-human CD42P (P-selectin) mAb was obtained from BioLegend (San Diego, CA, USA).

Yang C.H., Hsia C.W., Jayakumar T., Sheu J.R., Hsia C.H., Khamrang T., Chen Y.J., Manubolu M, & Chang Y. (2018). Structure–Activity Relationship Study of Newly Synthesized Iridium-III Complexes as Potential Series for Treating Thrombotic Diseases. International Journal of Molecular Sciences, 19(11), 3641.

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Platelet Activation Assay by Flow Cytometry

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A 96-well V-bottom polypropylene plate (Thermo Fisher Scientific) was blocked with 1% BSA [w/v] in PBS, and 3×10⁵ platelets in PBS with 1% BSA were added to each assay well. Activation assays were adapted from the work of Nylander and Lindahl (32 (link)). Platelets were incubated with AF647 fluorescently labeled fibrinogen (Thermo Fisher Scientific) for 30 minutes and subsequently activated with agonist for 15 minutes using 30 μM ADP (Chrono-log), 0.1U/mL thrombin (Chrono-log), 5 mg/mL convulxin (Santa Cruz), or 1% BSA in PBS as a negative control. Following platelet activation, platelets were analyzed with an Accuri C6 flow cytometer (BD Biosciences).

Kanack A.J., Aoki K., Tiemeyer M, & Dahms N.M. (2021). Platelet and myeloid cell phenotypes in a rat model of Fabry disease. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 35(8), e21818.

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Platelet Mitochondrial Function Assay

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Washed platelets were incubated in DMEM with 5mM glucose, 1mM pyruvate, and 2mM glutamate for 30 minutes. Platelets were then stained with annexin V-APC, and 100nM TMRM in the presence or absence of 1U/mL thrombin (Sigma Aldrich, St. Louis, MO) plus 700ng/mL convulxin (Santa Cruz, Dallas TX) or 1μM Ionomycin (Sigma Aldrich, St. Louis, MO) for 15 minutes. Samples were then diluted 1:20 in PBS and immediately analyzed using flow cytometry.

Fidler T.P., Rowley J.W., Araujo C., Boudreau L.H., Marti A., Souvenir R., Dale K., Boilard E., Weyrich A.S, & Abel E.D. (2017). Superoxide Dismutase 2 is dispensable for platelet function. Thrombosis and haemostasis, 117(10), 1859-1867.

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Platelet Activation Reagents and Protocols

Cited 1 time

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Adenosine diphosphate (ADP) and phorbol myristate acetate were obtained from Sigma (St. Louis, MO, USA). Ristocetin and arachidonic acid (Ara) were from Chrono-Log Corp. (Haverton, PA, USA). 9,11 dideoxy 9α,11α methanoepoxy prostaglandin F2α (U46619) was purchased from Cayman Chemicals (Ann Arbor, MI, USA); thrombin receptor-activating peptide (TRAP) was from EMD Biosciences (La Jolla, CA, USA). Thrombin and PPACK (D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone) were from Haematologic Technologies (Essex Junction, VT, USA). Convulxin was from Santa Cruz Biotechnology, Inc, (Santa Cruz, CA, USA) and GPVI-His from R&D Systems (Minneapolis, MN, USA). Soluble human collagen type I, III, IV, V and VI were also from R&D Systems. Synthesis and preparation of collagen derived peptides were as described in [11] (link).

Chagas A.C., McPhie P., San H., Narum D., Reiter K., Tokomasu F., Brayner F.A., Alves L.C., Ribeiro J.M, & Calvo E. (2014). Simplagrin, a Platelet Aggregation Inhibitor from Simulium nigrimanum Salivary Glands Specifically Binds to the Von Willebrand Factor Receptor in Collagen and Inhibits Carotid Thrombus Formation In Vivo. PLoS Neglected Tropical Diseases, 8(6), e2947.

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Quantifying Platelet-Neutrophil Aggregation

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Whole-blood samples from seven to 10-day-old mice were obtained by cardiac puncture into EDTA coated microtubes (Sarstedt). To measure platelet–neutrophil aggregates (PNAs), we diluted whole blood 1:10 with M-199 serum-free media (12–117F; Lonza) containing 100 U/mL heparin.^{25 ,26} Neutrophils were labeled with Ly6G BV510 (Biolegend) and platelets were labeled with APC anti-mouse CD41 (BD Biosciences) for 15 min at 37 °C. For platelet activation studies, staining was performed in the presence of 50 ng/mL of convulxin (Santa Cruz). Samples were fixed with FACS lysis buffer, centrifuged at 500 x g for 10 min and resuspended in PBS before analysis.

Denorme F., Rustad J.L., Portier I., Crandell J.L., de Araujo C.V., Cody M.J., Campbell R.A, & Yost C.C. (2022). Neutrophil extracellular trap inhibition improves survival in neonatal mouse infectious peritonitis. Pediatric Research, 93(4), 862-869.

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Platelet Activation Assay Protocol

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Adenosine 5′- diphosphate (ADP), collagen and prostaglandin E1 (PGE-1) were obtained from Sigma-Aldrich (St. Louis, Missouri/MO, U.S.A). Convulxin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Sodium chloride (p.a.) was obtained from Arquimed (Santiago, Chile). Antiphospho (S660)-PKC-β2 and antiphospho (Tyr753)-PLC-γ2 antibodies were obtained from Santa Cruz (Biotechnology, CA, USA). Anti γ-tubulin monoclonal antibody (4D11) was obtained from Thermo Scientific (Thermo Scientific, Pierce, Rockford, IL, USA). Antibodies (anti-CD62P-PE, anti-CD61-FITC, anti-GPIIb/IIIa-FITC PAC-1 and anti-CD61-PE) were obtained from BD Pharmingen (BD Biosciences, San Diego, CA, USA).

Fuentes M., Sepúlveda C., Alarcón M., Palomo I, & Fuentes E. (2017). Buddleja globosa (matico) prevents collagen-induced platelet activation by decreasing phospholipase C-gamma 2 and protein kinase C phosphorylation signaling. Journal of Traditional and Complementary Medicine, 8(1), 66-71.

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Platelets PS Exposure, Mitochondria Analysis

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To detect phosphatidylserine positive (PS+) platelets, washed platelets were resuspended in Medium 199 (12–117F; Lonza) containing calcium and stimulated for 15 minutes with thrombin (1.0U/mL, final, ThermoFisher) and convulxin (250 ng/mL, final, Santa Cruz) in the presence of FITC-labelled Annexin V and anti-human CD41 APC. In some experiments, platelets were stimulated with A23187 (50 μM, final, Sigma Aldrich) in the presence of FITC-labelled Annexin V and anti-human CD41 APC. To measure mitochondrial membrane potential and mitochondrial reactive oxygen species, washed platelets were resuspended in M199 in the presence of mitotracker green (200 nM, final) and tetramethylrhodamine methyl ester (TMRM; 60 nM, final) or MitoSox Red (200 nM, final) for 40 minutes at 37^oC in the presence or absence of thrombin and convulxin. Samples were immediately run on a Beckman Coulter Cytoflex. TMRM and MitoSox Red MFI were normalized to MitoTracker Green MFI. For TMRM, subsequently the fold change relative to baseline was calculated.

Denorme F., Manne B.K., Portier I., Petrey A.C., Middleton E.A., Kile B.T., Rondina M.T, & Campbell R.A. (2020). COVID‐19 patients exhibit reduced procoagulant platelet responses. Journal of Thrombosis and Haemostasis, 18(11), 3067-3073.

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