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Thrombin

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Thrombin is a serine protease that plays a central role in the coagulation cascade. It catalyzes the conversion of fibrinogen to fibrin, which is the final step in the formation of a blood clot.

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Lab products found in correlation

Adenosine diphosphate (adp), by Chrono-Log (12 mentions) U46619, by Chrono-Log (7 mentions) Stir bars, by Chrono-Log (7 mentions) Anti β actin, by Santa Cruz Biotechnology (5 mentions) Apyrase, by Merck Group (4 mentions) Luciferin luciferase reagent, by Chrono-Log (3 mentions) Fibrinogen alexa fluor 488 conjugate, by Thermo Fisher Scientific (3 mentions) Polyvinylidene difluoride pvdf membrane, by GE Healthcare (3 mentions) Mouse monoclonal to cd62p p selectin antibody, by BioLegend (3 mentions) Fura 2 am 2 acetoxymethyl, by Thermo Fisher Scientific (3 mentions) Anti p38 mapk, by Cell Signaling Technology (3 mentions) Adenosine diphosphate (adp), by Merck Group (3 mentions) Fura 2 am, by Thermo Fisher Scientific (3 mentions) Ticagrelor, by MedChemExpress (2 mentions) Seratrodast, by MedChemExpress (2 mentions) Vorapaxar, by MedChemExpress (2 mentions) Fluo 3 am calcium indicators, by Beyotime (2 mentions) Fitc conjugated anti human cd62p p selectin, by BioLegend (2 mentions) Fitc conjugated anti human pac 1, by BioLegend (2 mentions) Pac 1 antibody, by BD (2 mentions)

48 protocols using thrombin

1

Antiplatelet Effects of 20(S)-panaxadiol

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Materials 20(S)-panaxadiol (PD, purity ≥98% by HPLC) was obtained from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). Hemocoagulase was obtained from Jinzhou Ahon Pharmaceutical Co, Ltd.
(Liaoning, China). Prothrombin (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and brinogen (FIB) kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
Luciferin/luciferase reagent and thrombin were purchased from Chrono-Log Corporation (Pennsylvania, USA). Vorapaxar, ticagrelor, and seratrodast were obtained from MedChemExpress (New Jersey, USA). Cyclic adenosine monophosphate (cAMP) ELISA kit was obtained Sino Best Biological Technology Co., Ltd. (Shanghai, China). FITC-conjugated anti-human CD62P (P-selectin) and FITC-conjugated anti-human PAC-1 were obtained BioLegend (California, USA). Fluo-3 AM Calcium indicators were purchased from Beyotime Biotechnology (Shanghai, China). Akt, p-Akt, PI3K, p-PI3K, GSK3β, p-GSK3β, and β-actin were purchased from Abcam (Cambridge, Britain).
Zhang H., Su W., Yang C., Tang X., Pan D., Zhang Y., Zhao D., Qi B., & Li X. (2021). 20(S)-Panaxadiol Enhances Hemostatic Effect on Activated Platelet by Affecting Calcium Signaling.
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2

Characterization of Medicinal XML Compound

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XML used in our research was provided by Yunnan Tengchong Pharmaceutical Corporation (Yunnan, China), in accordance with applicable Good Manufacturing Practice (GMP). We performed HPLC analysis to identify the chemical characteristics of XML samples satisfying the national standards for XML (HPLC≥99%; Figure 2). Collagen, thrombin, and arachidonic acid (AA) were purchased from Chrono-Log Corp. (Havertown, PA, USA). Antibodies against total-Syk, phospho-Syk (Tyr525/526), total-PLCγ2, phospho-PLCγ2 (Tyr759), total-Akt, phospho-Akt (Thr308), total-GSK3β, phospho-GSK3β (Ser9), total-Erk1/2, phospho-Erk1/2 (Thr202/Tyr204), total-JNK, phospho-JNK (Thr183/Tyr185), total-p38, and phospho-p38 (Thr180/Tyr182) were obtained from cell signaling (Beverly, MA, USA). CD61 and CD62P (P-selectin) antibodies and PAC-1 antibody were purchased from BD Biosciences (San Jose, CA, USA). ECL Western blotting detection reagent was obtained from Pierce Chemical Co. (Rockford, Illinois, USA). XML was dissolved in saline solution and stored at room temperature until used.
Wang H., Ye Y., Wan W., Wang L., Li R., Li L., Yang L., Yang L., Gu Y., Dong L, & Meng Z. (2019). Xinmailong Modulates Platelet Function and Inhibits Thrombus Formation via the Platelet αIIbβ3-Mediated Signaling Pathway. Frontiers in Pharmacology, 10, 923.
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3

Platelet Activation Assay Protocol

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Thrombin was purchased from Chrono-log. The anticoagulant peptide H-Gly-Pro-Arg-Pro-NH2 (cat. no. H-1998) was purchased from Bachem. Adenosine diphosphate (ADP), formyl-methionyl-leucyl phenylalanine (fMLF), HEPES, and NaCl were purchased from Sigma. KCl, MgS04, and formaldehyde were purchased from Merck. Phosphate-buffered saline (PBS; 0.14 M NaCl, 0.0027 M KCl, 0.0101 M phosphate buffer, pH 7.4; Medicago) was purchased from Saveen & Werner. eBioscience™ 1-step Fix/Lyse Solution (10X) was purchased from Invitrogen, ThermoFisher Scientific. The following fluorescently conjugated antibodies were purchased from BD Pharmingen: CD42a-FITC (clone ALMA.16), CD61-PE (clone VI-PL2), CD11b-PE-Cy™5 (clone ICRF44), and κ isotype control IgG1-PE (clone MOPC-31 C); BD Biosciences: CD62P-PE (clone AC1.2); and eBioscience™, Invitrogen, ThermoFisher Scientific: CD45-FITC (clone HI30), CD142-APC (clone HTF-1), κ isotype control IgG1-FITC (clone P3.6.2.8.1), and κ isotype control IgG1-APC (clone P3.6.2.8.1).
Chao Y., Rebetz J., Bläckberg A., Hovold G., Sunnerhagen T., Rasmussen M., Semple J.W, & Shannon O. (2021). Distinct phenotypes of platelet, monocyte, and neutrophil activation occur during the acute and convalescent phase of COVID-19. Platelets.
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4

Platelet Activation Pathway Analysis

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G-Ro was obtained from Ambo Institute (Daejon, Korea). Thrombin was obtained from Chrono-Log Corporation (Havertown, PA, USA). Anti-VASP, anti-phosphor-VASP (Ser157), anti-phosphor-VASP (Ser239), anti-rabbit IgG-HRP-horseradish peroxidase conjugate (HRP), and lysis buffer were purchased from Cell Signaling (Beverly, MA, USA). The αIIb/β3 inhibitor eptifibatide, GR 144053, and anti-β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyvinylidene difluoride membrane was purchased from GE Healthcare (Piseataway, NJ, USA). Enhanced chemiluminescence solution was purchased from GE Healthcare (Chalfont St. Giles, UK). cAMP and cyclic guanosine monophosphate (cGMP) enzyme immunoassay kits were purchased from Cayman Chemical (Ann Arbor, MI, USA). An A-kinase inhibitor Rp-8-Br-cAMPS, an A-kinase activator 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP), and a G-kinase activator 8-Br-cGMP were purchased from Sigma Chemical Corporation (St. Louis, MO, USA). Fibrinogen Alexa Fluor 488 conjugate was obtained from Invitrogen Molecular Probes (Eugene, OR, USA).
Shin J.H., Kwon H.W., Cho H.J., Rhee M.H, & Park H.J. (2015). Vasodilator-stimulated phosphoprotein-phosphorylation by ginsenoside Ro inhibits fibrinogen binding to αIIb/β3 in thrombin-induced human platelets. Journal of Ginseng Research, 40(4), 359-365.
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5

Platelet Activation by Thrombin

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Mice were treated with 2 doses of 400ng DT or PBS and platelets were purified as described above. Purified platelets (100µl aliquots) were placed in a 37°C water bath in the presence (0.1U/ml) or absence (0U/ml) of thrombin (Chronolog, Havertown, PA) for 15 minutes and prepared for flow cytometry as described [33 (link)].
Wuescher L.M., Takashima A, & Worth R.G. (2014). A novel conditional platelet depletion mouse model reveals the importance of platelets in protection against S. aureus bacteremia. Journal of thrombosis and haemostasis : JTH, 13(2), 303-313.
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6

Collagen and Thrombogenic Pathway Activation

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Collagen (type I), 9, 11-dideoxy-11α, 9α-epoxymethanoprostaglandin (U46619), and thrombin were purchased from Chrono-Log Corporation (Havertown, PA, USA). Anti-phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182), anti-phospho-c-Jun N-terminal kinase (JNK) (Thr183/Tyr185), anti-phospho-(Ser) protein kinase C (PKC) substrate, anti-JNK polyclonal antibodies (pAb), and anti-p38 MAPK and anti-Akt monoclonal antibodies (mAb) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-phospho-Akt (Ser473) pAb was purchased from Biorbyt (Cambridge, UK), and anti-pleckstrin (p47) pAb was purchased from Gene Tex (Irvine, CA, USA). Hybond-P polyvinylidene difluoride (PVDF) membranes, enhanced chemiluminescence (ECL) Western blotting detection reagent, and the analysis system were purchased from GE Healthcare Life Sciences (Buckinghamshire, UK). Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse immunoglobulin G antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
Lee T.Y., Jayakumar T., Thanasekaran P., Lin K.C., Chen H.M., Veerakumar P, & Sheu J.R. (2020). Carbon Dot Nanoparticles Exert Inhibitory Effects on Human Platelets and Reduce Mortality in Mice with Acute Pulmonary Thromboembolism. Nanomaterials, 10(7), 1254.
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7

Ginsenoside Compound Effects on Platelet Signaling

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KRG-TS was obtained from Korea Ginseng Corporation R&D Headquarters (Daejeon, Korea). Thrombin was purchased from Chrono-log Corporation (Havertown, PA, USA). The ATP assay kit was purchased from Biomedical Research Service Center (Buffalo, NY, USA). Serotonin enzyme-linked immunosorbent assay (ELISA) kit was purchased from Labor Diagnostika Nord GmbH & Co. (Nordhorn, Germany). Fura 2-acetoxymethyl (Fura 2-AM) and other reagents were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Antiphospho-IP3RI (Ser1756), antiphospho-PKA catalytic subunit (PKAc; Thr197), antirabbit immunoglobulin G (IgG)–horseradish peroxidase conjugate (HRP), and lysis buffer were obtained from Cell Signaling (Beverly, MA, USA). Polyvinylidene difluoride (PVDF) membrane was obtained from GE Healthcare (Piscataway, NJ, USA). Enhanced chemiluminescence (ECL) solution was purchased from GE Healthcare (Chalfont St, Giles, Buckinghamshire, UK). Protopanaxadiol saponins [PPDSs; G-Ra1, G-Rb1, G-Rb2, G-Rb3, G-Rc, G-Rd, G-Rg3 (20R), G-Rg3 (20S), and G-Rh2 (20R)] and protopanaxatriol saponins [PPTSs; G-Re, G-Rf, G-Rg1, G-Rg2 (20R), and G-Rh1 (20S)] were purchased from Ambo Institute (Daejeon, Korea).
Shin J.H., Kwon H.W., Cho H.J., Rhee M.H, & Park H.J. (2015). Inhibitory effects of total saponin from Korean Red Ginseng on [Ca2+]i mobilization through phosphorylation of cyclic adenosine monophosphate-dependent protein kinase catalytic subunit and inositol 1,4,5-trisphosphate receptor type I in human platelets. Journal of Ginseng Research, 39(4), 354-364.
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8

Yuzu Extract's Antiplatelet Effects

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Ethanolic yuzu extract (YE) was obtained from Konkuk University (Seoul, Republic of Korea). Briefly, yuzu were harvested in the southern region of Korea, Goheung (Republic of Korea). Whole fruits, including seeds, pulp and skin, were extracted with 70% ethanol and dried to remove the solvent, then dissolved in saline for the experiments. Hesperidin, naringin, apyrase, acetylsalicylic acid (ASA), bovine serum albumin (BSA), fura 2-AM, β-nicotinamide adenine dinucleotide (reduced form, β-NADH) and pyruvic acid were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Collagen, thrombin, adenosine diphosphate (ADP), arachidonic acid (AA), and luciferin-luciferase reagent were purchased from Chrono-Log Co. (Harvertown, PA, USA). Antibodies against phospholipase Cγ (PLCγ), phosphor-PLCγ, PI3K/protein kinase B (Akt), and phospho-Akt were purchased from Cell Signaling (Danvers, MA, USA).
Kim T.H., Kim H.M., Park S.W, & Jung Y.S. (2015). Inhibitory Effects of Yuzu and Its Components on Human Platelet Aggregation. Biomolecules & Therapeutics, 23(2), 149-155.
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9

Mulberroside C Signaling Pathway Analysis

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Mulberroside C (Figure 1) was provided by ChemFaces (Wuhan, China). Mouse monoclonal to CD62P (P-selectin) antibody was purchased from Biolegend (San Diego, CA, USA). Fura 2-AM (2-acetoxymethyl) and Alexa Fluor 488-conjugated fibrinogen were obtained from Invitrogen (Eugene, OR, USA). The BCA protein assay kit was obtained from Pierce Biotechnology (Rockford, IL, USA). The serotonin ELISA kit was provided by Labor Diagnostika Nord GmbH and Co. (Nordhorn, Germany). Collagen, U46619, and thrombin were purchased from Chrono-Log Co. (Havertown, PA, USA). The thromboxane B2 assay kit, cAMP, cGMP enzyme immunoassay kit, and ATP assay kit were obtained from Cayman Chemical (Ann Arbor, MI, USA). Cell signaling (Beverly, MA, USA) supplied antiphospho-p38MAPK, antiphospho-ERK (1/2), antiphospho-VASP (Ser157), antiphospho-VASP (Ser239), antiphospho-cPLA2 (Ser505), antiphospho-PI3K (Tyr458), antiphospho-Akt (Ser473), antiphospho-inositol-3-phosphate receptor type I (Ser1756), antiphospho-PLCγ2 (Tyr759), anti-β-actin, and antirabbit secondary antibodies.
Kwon H.W., Lee D.H., Rhee M.H, & Shin J.H. (2021). In Vitro Antiplatelet Activity of Mulberroside C through the Up-Regulation of Cyclic Nucleotide Signaling Pathways and Down-Regulation of Phosphoproteins. Genes, 12(7), 1024.
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10

Platelet Activation and Signaling Pathways

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ADP, MG132 were obtained from Sigma Aldrich (St. Louis, MO). Thrombin, Collagen, stir bars and other disposables were from Chrono-Log (Havertown, PA), and U46619 was obtained from Cayman Chemical (Ann Arbor, MI). The proteasome inhibitor bortezomib (Velcade) was generous gift from Dr. Steven Schwarze (University of Kentucky College of Medicine, Lexington, KY). FITC-conjugated Annexin V, anti—P-selectin, and PAC-1 antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Antibodies against TAK1, TAB2, CARMA1, MALT1, Bcl10, Ubiquitin, IKKα, IKKβ, IKKγ, VAMP-8, SNAP-23, Syn-11, and pSer/Thr/Tyr from Santa Cruz (Santa Cruz, CA), respectively. Other reagents were of analytical grade.
Karim Z.A., Vemana H.P, & Khasawneh F.T. (2015). MALT1-Ubiquitination Triggers Non-Genomic NF-κB/IKK Signaling upon Platelet Activation. PLoS ONE, 10(3), e0119363.
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