Purified human protein including FXII, FXIIa, PPK, PK and HK obtained from Enzyme Research Laboratories were used for the in-vitro experiments.
Fxiia
Manufactured by Enzyme Research
Sourced in United States
FXIIa is a laboratory product that functions as a clotting factor involved in the activation of the intrinsic pathway of the coagulation cascade. It plays a key role in the initiation and regulation of blood clotting mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Prekallikrein, by Enzyme Research (9 mentions)
Activated partial thromboplastin time reagent, by Diagnostica Stago (3 mentions)
Human cathepsin g, by Innovative Research (3 mentions)
Bovine trypsin, by Merck Group (3 mentions)
Bovine α chymotrypsin, by Merck Group (3 mentions)
Trypsin substrate s 2222, by Diapharma (3 mentions)
Thrombin, by Prolytix (3 mentions)
Plasmin, by Prolytix (3 mentions)
1 2 dioleoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (3 mentions)
1 2 dioleolyl sn glycero 3 phospho l serine, by Avanti Polar Lipids (3 mentions)
Ptt a micronized silica reagent, by Diagnostica Stago (2 mentions)
Bivalirudin, by Pfizer (2 mentions)
Hexadimethrine bromide polybrene, by Merck Group (2 mentions)
Poly l arginine, by Merck Group (2 mentions)
Anhydrous iron 3 chloride fecl3, by Merck Group (1 mentions)
S 2366 l pyro glu l pro l arg p nitroanilide, by Diapharma (1 mentions)
Fibrillar collagen type 1, by Chrono-Log (1 mentions)
Phosphatidylcholine phosphatidylserine pc ps vesicles, by Avanti Polar Lipids (1 mentions)
Z gly gly arg amc, by Bachem (1 mentions)
Plasmin, by Enzyme Research (1 mentions)
12 protocols using fxiia
1
In-Vitro Coagulation Protein Study
2
Aptamer Selection for Factor XI Inhibition
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The aptamer library comprised a ssDNA template with sequence 5′-GAATTCTAAT ACGACTCACT ATA-N40-GCGTCCAACA CATCG-3′ (please note spaces have been introduced every 10 nucleotides, into this and all other DNA sequences reported here). The forward (A) and reverse primers (B) were 5′-GAATTCTAAT ACGACTCACT ATA-3′ and 5′-GCGTCCAACAC ATCG-3′ respectively. These and all other oligonucleotides employed in this study were purchased from Integrated DNA Technologies (IDT, Coralville, IA). FXI, FXIa, FIX and FXIIa were bought from Enzyme Research Laboratories (South Bend, IN). Biotinylated goat anti-human Factor XI (FXI) antibody was purchased from Affinity Biologicals (Ancaster, ON). Dynabeads Biotin Binder was bought from Thermo Fisher Scientific (Waltham, MA). For thrombin generation assays (TGA), TGA substrate and TGA calibrator sets were obtained from Technothrombin GmbH (Vienna, Austria). Activated Partial Thromboplastin Time (APTT) reagent was from Diagnostica Stago (Asnieres, France). Chromogenic substrate S2366 was purchased from Instrumentation Laboratory (Lexington, MA).
3
Purification and Assay of Serine Proteases
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Human plasma serine
proteases including thrombin, FXa, FXIa, and FIXa were obtained from
Haematologic Technologies (Essex Junction, VT). FXIIa was purchased
from Enzyme Research Laboratories (South Bend, IN). The substrates
Spectrozyme TH, Spectrozyme FXa, and Spectrozyme FIXa were obtained
from Biomedica-Diagnostics (Windsor, NS Canada). Factor XIIa (Chromogenix
S-2302) and factor XIa (S-2366) substrates were obtained from Diapharma
(West Chester, OH). All enzymes and substrates were prepared in 20–50
mM TrisHCl buffer, pH 7.4, containing 100–150 mM NaCl, 0.1%
PEG8000, 2.5 mM CaCl2, and 0.02% Tween80. For FIXa buffer,
33% (v/v) ethylene glycol was also added.
proteases including thrombin, FXa, FXIa, and FIXa were obtained from
Haematologic Technologies (Essex Junction, VT). FXIIa was purchased
from Enzyme Research Laboratories (South Bend, IN). The substrates
Spectrozyme TH, Spectrozyme FXa, and Spectrozyme FIXa were obtained
from Biomedica-Diagnostics (Windsor, NS Canada). Factor XIIa (Chromogenix
S-2302) and factor XIa (S-2366) substrates were obtained from Diapharma
(West Chester, OH). All enzymes and substrates were prepared in 20–50
mM TrisHCl buffer, pH 7.4, containing 100–150 mM NaCl, 0.1%
PEG8000, 2.5 mM CaCl2, and 0.02% Tween80. For FIXa buffer,
33% (v/v) ethylene glycol was also added.
4
Thrombin and Coagulation Factor Assays
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α-thrombin, γ-thrombin, and PPACK (Phe-Pro-Arg-chloromethylketone)-thrombin were from Hematologic Technologies (Essex Junction, VT). FXIa, FXIIa, and pre-kallikrein were from Enzyme Research Laboratories (South Bend, IN). FIX (Benefix, recombinant FIX, protein-free) was from Wyeth-Pfizer (New York, NY). APTT (STA-PTT Automate) and PT (Neoplastine CI Plus) reagents were from Diagnostica Stago (Asnieres, France). S2238 (H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilinedihydrochloride), S2366 (L-pyroglutamyl-L-propyl-L-arginine-p-nitroanilide), and S2302 (H-D-L-prolyl-L-phenylanyl-L-arginine-p-nitroanilide) were obtained from Chromogenix-Diapharma (West Chester, OH). CM5 sensor chips and all reagents for surface plasmon resonance (BIAcore) experiments were from GE-Healthcare-Pharmacia (Piscataway, NJ). TFPI (human recombinant Tissue Factor Pathway Inhibitor) was from R&D Systems (Minneapolis, MN). Elastase (human neutrophil elastase) and human Cathepsin G were from Molecular Innovations. Dextran sulphate (DS) 500K was from Sigma Co. (Saint Louis, MO). Corn Trypsin Inhibitor (CTI) was from Kerafast Inc. (Boston, MA).
5
Colorimetric Assay for C1 Inhibitor and Factor XIIa
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C1 inhibitor from human plasma (C1-INH, GenWay Biotech, San Diego, USA) was diluted with phosphate buffer (20 mmol/l KH2PO4, 250 mmol/l KCl, pH 7.0) to a 5 mg/ml stock solution, aliquoted and stored at -80°C. The Technochrom® C1-INH test kit (Technoclone, Vienna, Austria) provided C1s esterase, a chromogenic substrate for C1s (C2H5CO-Lys(ϵ-Cbo)-Gly-Arg-pNA) and ready-to-use buffers (TRIS buffer A: 50 mmol/l TRIS, 257 mmol/l NaCl, pH 7.4; TRIS buffer B: 50 mmol/l TRIS, 257 mmol/l NaCl, pH 8.3). According to the product descriptions, both lyophilized enzyme and substrate were reconstituted with Ampuwa®.
Human α-Factor XIIa (FXIIa, Enzyme Research Laboratories, South Bend, USA) was diluted with sodium acetate buffer (4 mmol/l sodium acetate, 150 mmol/l NaCl, pH 5.3) to a 1 mg/ml stock solution, aliquoted and stored at -80°C.
The chromogenic substrate for FXIIa (S-2302TM, H-D-Pro-Phe-Arg-pNA • 2 HCl) was obtained from Chromogenix (Instrumentation Laboratory, Bedford, USA) and diluted with Ampuwa® to a 4 mmol/l stock solution.
Human α-Factor XIIa (FXIIa, Enzyme Research Laboratories, South Bend, USA) was diluted with sodium acetate buffer (4 mmol/l sodium acetate, 150 mmol/l NaCl, pH 5.3) to a 1 mg/ml stock solution, aliquoted and stored at -80°C.
The chromogenic substrate for FXIIa (S-2302TM, H-D-Pro-Phe-Arg-pNA • 2 HCl) was obtained from Chromogenix (Instrumentation Laboratory, Bedford, USA) and diluted with Ampuwa® to a 4 mmol/l stock solution.
6
Assay of Serine Proteases
7
Comprehensive Coagulation Cascade Assay
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Full-length (residues 1-263) recombinant tissue factor (TF) and factor (F) XIII were purchased from Haematologic Technologies (Essex Junction, VT, USA). FXIIa was purchased from Enzyme Research Laboratories (South Bend, IN, USA). 1,2-Dioleolyl-sn-Glycero-3-Phospho-L-Serine (PS) and 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (PC) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Phospholipid vesicles (PCPS) composed of 75% PC and 25% PS were prepared as described [22] , as were the TF/PCPS reagent and corn trypsin inhibitor (CTI) [23] . Antithrombin (AT) and factors II, V, VII, IX, X and XI were isolated from freshly frozen plasma [24] . Fibrinogen was prepared as described [25] . Recombinant human FVIIa was provided as a gift by Dr Lars Pedersen (Novo Nordisk, Bagsvaerd, Denmark). Recombinant full-length tissue factor pathway inhibitor-a (TFPI) was a gift from Dr Johnson (Chiron, Emeryville, CA, USA). Recombinant FVIII was a gift from Dr Lundblad (Baxter Healthcare, Duarte, CA, USA). D-Phe-Pro-Arg-CH 2 Cl (FPR-ck) [26] and a-thrombin [27] were produced in-house.
8
Intrinsic Coagulation Cascade Assay
9
Intrinsic Coagulation Pathway Assay
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Pooled normal plasma (PNP), Precision BioLogic (Dartmouth, Nova Scotia, Canada). FXII-deficient and PKdeficient plasmas, George King Biomed. Purified human plasma FXII, FXIIa, PK, PKa, and HK, Enzyme Research Laboratory. Plasma-derived FXI, Hematologic Technologies) S-2302 (H-D-prolyl-L-phenylalanyl-Larginine-p-nitroaniline), DiaPharma. PTT-A micronized silica reagent, Diagnostica Stago. Bivalirudin (Hospira). Hexadimethrine bromide (polybrene) and poly-L-arginine (Sigma-Aldrich). The kallikrein inhibitor KV999272 was previously described [29, 30] .
10
Coagulation Factors and DNA Isolation
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