About 0.25 nM FVIIa (NovoSeven; Novo Nordisk) was preincubated with 5 nM TF (Dade Innovin; Siemens) in HN-BSA buffer (20 mM Hepes, 140 mM NaCl, 5 mg/ml BSA, pH 7.4) supplemented with 3 mM CaCl2 for 15 min at 37 °C to allow the formation of the TF–FVIIa complex. After incubating the complex with 0 to 20 μM tick protein for another 15 min at 37 °C, the residual FVIIa activity was probed by adding 0.5 mM Spectrozyme FVIIa (ImmBioMed) and following the absorbance at 405 nm in a plate reader. 0.1 nM human FXa (Enzyme Research Laboratories) in HN-BSA buffer was incubated with 0 to 20 μM tick protein for 15 min at 37 °C. Residual FXa activity was probed by adding 120 μM Biophen CS-11 (65) (Aniara) and following the absorbance at 405 nm.
Human fxa
Manufactured by Enzyme Research
Sourced in United States
Human FXa is a laboratory reagent that contains the activated form of coagulation factor X (FXa). FXa is a serine protease that plays a crucial role in the blood clotting cascade. This product is intended for use in various in vitro research applications, such as studying the mechanisms of blood coagulation and the development of anticoagulant therapies.
Automatically generated - may contain errors
Lab products found in correlation
Prothrombin, by Enzyme Research (18 mentions)
Thrombin, by Enzyme Research (9 mentions)
Human prothrombin, by Enzyme Research (9 mentions)
Spectrozyme th, by Sekisui (6 mentions)
Spectrozyme fxa, by Sekisui (6 mentions)
Dade innovin, by Siemens (3 mentions)
Novoseven, by Novo Nordisk (3 mentions)
Heparan sulfate from bovine kidney, by Merck Group (3 mentions)
Human plasmin, by Merck Group (3 mentions)
Human thrombin, by Merck Group (3 mentions)
Human leukocyte cathepsin g, by Merck Group (3 mentions)
Imac sepharose 6 fast flow, by GE Healthcare (3 mentions)
α chymotrypsin from bovine pancreas, by Merck Group (3 mentions)
Trypsin, by Merck Group (3 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (3 mentions)
Goat anti mouse irdye 680, by LI COR (3 mentions)
Irdye 800cw goat anti rabbit, by LI COR (3 mentions)
Enoxaparin, by Sandoz (3 mentions)
S 2765, by Diapharma (3 mentions)
Thrombin, by Prolytix (3 mentions)
6 protocols using human fxa
1
Inhibition of FVIIa and FXa by Tick Proteins
2
Characterization of Lamprey AGTR1 Receptor
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Human thrombin (≥2 800 NIH U/ml), human plasmin, human leukocyte cathepsin G, trypsin and α-chymotrypsin from bovine pancreas, affinity-isolated anti-hemagglutinin (HA) antibodies from rabbits and heparan sulfate from bovine kidney were purchased from Sigma-Aldrich. Human FXa (176 IU/mg) was from Enzyme Research Laboratories. Horse anti-mouse IgG and Q5 high-fidelity DNA polymerase (2 000 U/ml) were from New England BioLabs. Horseradish peroxidase-linked anti-rabbit antibodies from donkey and IMAC Sepharose 6 Fast Flow were purchased from GE Healthcare. The FXa substrate S-2222 was from Chromogenix. PCR primers (Table S1
3
Coagulation Factors Protocol Synthesis
4
Anticoagulant Reagents and Enzyme Characterization
5
Chromogenic Assay for Factor Xa Activity
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Assays were based on a previously published method. Briefly, human FXa (Enzyme Research Laboratories) was diluted to 50 U ml−1 with PBS. The chromogenic substrate S-2765 (Diapharma) was diluted to 1 mg ml−1 in water. For in vitro studies, fondaparinux and 12-mer oligosaccharides were dissolved in PBS at various concentrations (0–131 nM). 16 μl of sample was incubated with 60 μl of 35 μl ml−1 antithrombin (Cutter Biologics) for 2 min at room temperature. Next, 100 μl of FXa was added and incubated for 4 min at room temperature. 30 μl of S-2765 substrate was added and the absorbance of the reaction mixture was measured at 405 nm continuously for 5 min. PBS serves as a control sample. The maximum slope for each sample was convert to percent FXa activity by dividing by the maximum slope for the control sample.
For ex vivo studies, mouse plasma collected after the 6 h reperfusion period and assayed the same as described above.
For ex vivo studies, mouse plasma collected after the 6 h reperfusion period and assayed the same as described above.
6
Quantification of Activated Factor V
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FV variants were diluted to ~20 pM in assay buffer (25 mM HEPES [pH 7.4], 175 mM NaCl, 5 mg mL À1 bovine serum albumin, 3 mM CaCl 2 ), and activated with thrombin (2.5 nM; Haematologic Technologies, River Road, VT, USA) for 10 min at 37 °C. FVa was quantified in a prothrombinase-based assay containing human FXa (5 nM; Enzyme Research Laboratories, South Bend, IN, USA), human prothrombin (1 lM, purified in-house) and phospholipid vesicles (20 lM DOPS/DOPC, 10/ 90 mol mol À1 ) in the presence of 2.6 mM CaCl 2 . After 2 min, aliquots were removed from the prothrombinase mixture and quenched in ice-cold EDTA-containing buffer. The generated thrombin was quantified with chromogenic substrate S-2238 (Pepscan, Lelystad, The Netherlands). The assay was calibrated by use of a 1 : 1000 dilution of pooled normal plasma.
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