Microgel morphology was characterized in situ using cryoSEM with a JEOL 7600F. Microgels suspended in water were diluted to approximately 10 μg/mL and then prepared for imaging by flash freezing in liquid nitrogen under vacuum. Samples were fractured, etched, and sputter coated with gold for visualization. For a comparison to native platelets, blood was acquired from the New York Blood Center (New York City, NY) and platelets were isolated by centrifugation at 150 g’s for 15 min with no deceleration. A second spin at 900 g for 5 min was then performed and then isolated platelets were washed and resuspended in Tyrode’s albumin buffer containing 0.35% human serum albumin (Fisher Scientific, Hampton, NH) [29 ]. Platelets were activated by the addition of 0.25 U/mL human α-thrombin (Enzyme Research Laboratories, South Bend, IN) immediately before imaging. At least 5 fields were imaged at 50000X magnification per condition.
Human α thrombin
Manufactured by Enzyme Research
Sourced in United States, United Kingdom, India
Human α-thrombin is a serine protease enzyme involved in the blood coagulation cascade. It plays a central role in the conversion of fibrinogen to fibrin, a key step in blood clot formation.
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Lab products found in correlation
Human fibrinogen, by Enzyme Research (18 mentions)
Uv 1280, by Shimadzu (15 mentions)
Diethylaminoethyl deae dextran, by Merck Group (9 mentions)
Plasmid maxi kit, by Qiagen (9 mentions)
Bovine serum albumin (bsa), by Merck Group (9 mentions)
Murine thrombin, by Enzyme Research (9 mentions)
Anti phospho ser3 cofilin1 antibody 3311s, by Cell Signaling Technology (6 mentions)
β actin sc 47778, by Santa Cruz Biotechnology (6 mentions)
Axiovert 200m, by Zeiss (6 mentions)
Trypsin edta, by Merck Group (3 mentions)
Poly 1 c, by Merck Group (3 mentions)
Thermomax microplate reader, by Molecular Devices (3 mentions)
Prism software, by GraphPad (3 mentions)
S 2238, by Diapharma (3 mentions)
U73122, by Merck Group (3 mentions)
Sepharose 2b, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Pd98059, by Merck Group (3 mentions)
Adenosine diphosphate (adp), by Chrono-Log (3 mentions)
Bay61 3606, by Merck Group (3 mentions)
29 protocols using human α thrombin
1
Cryogenic Microscopy of Microgels
2
Isolation and Stimulation of Cardiac Cell Types
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Hearts of wild-type and ΔPAR1 embryos (embryonic day [E]14.5) were isolated and incubated overnight in 0.25% trypsin/EDTA (Sigma-Aldrich). Cell suspensions were plated for 90 min to allow adherence of the CFs5 (link),16 (link),41 (link). Non-adherent meCMs were isolated by adherence overnight. MEFs were derived from wild-type embryos (E14.5) as described41 (link). In addition, rat neonatal (rn) CFs and CMs were isolated from 1 day old rat pups as described2 (link). The addition of 0.1 mM BrdU to the CM culture maintenance medium prevented the proliferation of any contaminating CFs. Equal distribution of both sexes within each litter was assumed. Cells were stimulated with Poly I:C (25 µg/mL, γ-radiated; Sigma-Aldrich), thrombin (IIa, 5–10 nM, human α-thrombin, Enzyme Research Laboratories, South Bend, IN, USA) or PAR1 agonist peptide (PAR1 AP) (TFLLR, 200 µM, Abgent, San Diego, CA, USA) in serum-free media (SFM)5 (link),30 (link),41 (link).
3
Fibrin Polymerization Turbidity Assay
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Turbidity curves of fibrin polymerization were recorded at 350 nm using a UV-1280 (Shimadzu, Tokyo, Japan). Recordings were performed as described previously [19 (link)]. The final concentration in a 20 mM N-[2-hydroxyethyl] piperazine-N’-[2-ethansulfonic acid] pH 7.4, 0.12 M NaCl buffer was as follows: Human α-thrombin (Enzyme Research Laboratories, South Bend, MA, USA): 0.05 U/mL, fibrinogen: 0.18 mg/mL, and CaCl2: 1 mM. Three parameters: Lag time, Vmax, and ΔAbs30min, were obtained from the turbidity curves as described previously [19 (link)]. Reactions were performed in triplicate experiments for each sample.
4
Thrombin Inhibition Assay with Tick Salivary Glands
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All reactions were performed at 30 °C. Kinetic assays were performed using human α-thrombin (Enzyme Research Laboratories) and chromogenic substrate S-2238 (Diapharma) using a Thermomax micro plate reader (Molecular Devices). Reactions were performed in 100 μl of TBS buffer (10 mM Tris, 0.15 M NaCl, pH 7.4) containing 0.01% Tween-20. XC-43 (0–3.2 nM) or 0 to 15 pairs of X.cheopis salivary glands were incubated with human α-thrombin (0.2 nM) at 30 °C for 5 min followed by addition of S-2238 (50, 100, 200, 400, and 600 μM). Reactions were followed for 30 min. The inhibitory constants were determined by fitting the nonlinear regression model according to the Morrison’s equation (43 (link)) using the GraphPad Prism software (GraphPad Software, Inc).
5
Platelet Activation Pathway Modulation
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Prostaglandin I2 (PGI2) sodium salt, Sepharose 2B, BSA, DMSO, and U73122 were purchased from Sigma-Aldrich (St Louis, MO). Human α-thrombin was from Enzyme Research Laboratories (South Bend, IN) and ADP from Chrono-log (Havertown, PA). PE-conjugated anti–P-selectin antibody was obtained from Becton Dickinson (San Jose, CA) and mouse anti-CD36 (clone FA6-152) was purchased from Beckman Coulter (Brea, CA). PP2, PP3, PD98059, and BAY61-3606 were obtained from EMD Millipore (Billerica, MA). PLPC (1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine) was from Avanti Polar Lipids (Alabaster, AL). KODA-PC (9-keto-12-oxo-10-dodecenoic acid ester of 2-lyso-phosphocholine) was synthesized as described previously [23] . PLPC and KODA-PC were resuspended in Tyrode's buffer (without BSA and glucose) to prepare a 500 µM stock solution just before the incubation with platelets as described previously [5] (link).
6
Quantification of Fibrinogen and Thrombin Interactions
7
Thrombin-Induced Signaling Pathway Analysis
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Human α-thrombin was purchased from Enzyme Research Laboratories (South Bend, IN). Anti-ATG7, anti-phospho-(Ser536)-RelA/p65 (3033S), anti-phospho-(Ser3)-Cofilin1 antibody (3311S), and anti-Cofilin1 (clone D3F9, 5175S) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Antibodies to RelA/p65 (SC-8008), IκBα (SC-371), and β-actin (SC-47778) were from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-TBP antibody was purchased from Abcam (AB33168, Cambridge, MA). Plasmid maxi kit was from QIAGEN Inc. (Valencia, CA) and diethylaminoethyl (DEAE)-dextran was obtained from Sigma-Aldrich Chemical Company (St. Louis, MO). All other materials were obtained from Thermo Fisher Scientific (Waltham, MA).
8
Characterization of A549 cells and signaling pathways
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Commercially-available A549 cells were characterised and sourced from ATCC (LGC Standards, UK). Human α-thrombin was purchased from Enzyme Research Laboratories, UK. Transforming growth factor β-1 (TGFβ-1) was purchased from R&D Biosystems, UK. PAR-1 agonist peptide TFLLR-NH2 was purchased from Bachem AG, Switzerland. Hirudin was purchased from Sigma, UK. MEK inhibitor, UO126, and ALK5 inhibitor, SB431542 were purchased from Calbiochem, UK. PAR-1 ATAP2 detection antibody was purchased from Santa Cruz Biotechnology, USA. Total Smad2 and Smad3, and phosphorylated Smad2 and Smad3, ERK and phosphorylation ERK antibodies were purchased from Cell Signalling Technologies, USA. PAR-1 inhibitor, RWJ58259, was synthesised in-house by the Department of Chemistry, UCL. Mithramycin A was purchased from VWR International, UK and WP631 was purchased from Insight Biotechnology, UK.
9
Recombinant Fibrinogen Polymerization Catalysis
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Fibrin polymerization of recombinant γT277R and γY278H fibrinogens catalyzed by human α-thrombin (Enzyme Research Laboratories, South Bend, MA, USA) was performed as described previously [46 (link)]. Briefly, fibrinogen (90 µL at 0.20 mg/mL) in 20 mM HBS buffer was mixed with thrombin (10 µL at 0.5 U/mL) at room temperature. The turbidity change at 350 nm was followed using a UVmini-1280 spectrophotometer (Shimadzu, Tokyo, Japan), and reactions were performed in triplicate for each sample. For reaction parameters, the lag time and maximum slope were obtained from turbidity curves. Wild-type and γR275C fibrinogens were used to compare these functions.
10
Antibody Characterization for Cell Signaling
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