Enzyme Explorer

Factor Xa

On this page:

 


Physical Properties and In vivo processing
Factor Xa is a serine endoproteinase and a member of the S1 peptidase family. Factor Xa plays a critical role in the coagulation cascade (824 K PDF) by catalyzing the proteolytic conversion of prothrombin to active thrombin. Factor Xa’s prothrombin conversion activity is greatly enhanced in vivo when complexed with Factor V, calcium ions and phospholipids on the activated platelet surface.

The zymogen form, Factor X, is activated in vivo by two different pathways. The intrinsic pathway utilizes a catalytic complex composed of factor IXa, factor VIII, phospholipids and calcium ions. The extrinsic pathway utilizes a complex of factor VII and tissue factor. The factor X zymogen is a 55 KDa glycoprotein with a light and heavy chain joined by a single disulfide.2


Activation of factor X involves the hydrolysis of a 9.5 KDa peptide from the amino terminus of the heavy chain of the zymogen. The resulting activated factor Xaα has a MW of 45.3 KDa. Further hydrolysis of a small glycopeptide from the carboxy-terminus of the heavy chain yields factor Xaβ, which has equal coagulant activity. One disulfide bond also links the heavy and light chains of factor Xa.2,3


Back to top

Specificity, Assay and Kinetics
Factor Xa catalyzes the hydrolysis of the Arg-Thr and then Arg-Ile bonds in prothrombin to yield active thrombin. The fairly strict recognition sequence is Ile-Glu (or Asp)-Gly-Arg--X.4,5, 6
It may sometimes cleave at other basic residues, depending on the conformation of the target protein. Factor Xa will not cleave if a proline residue follows the arginine of the recognition sequence.

pH Optimum: 7.6-8.07,8

Temperature Optimum: 37 °C6

Assay
Unit definition: One unit of activated Factor X will liberate 1.0 µmole of p-nitroanilide from N-benzoyl-L-isoleucyl-L-glutamyl-L-glycyl-L-arginine-p-nitroaniline per minute at pH 8.3 at 37 °C.

The reaction buffer is 50 mM Tris, pH 8.3, containing 5 mM calcium chloride and 0.2 mM sodium chloride.

Back to top

Inhibitors

Product #  Product Name Add to Cart
A8849 α2-Antiplasmin from human plasma lyophilized powder, ≥5 inhibitor U/mg protein
A6397 Antithrombin III from rat plasma lyophilized powder, >200 units/mg protein (E1%/280 = 6.5)
A2221 Antithrombin III from human plasma lyophilized powder, ≥95% (SDS-PAGE)
A9141 Antithrombin III from bovine plasma lyophilized powder, 200-400 units/mg protein (E1%/280 = 6.5)
D0879 Diisopropylfluorophosphate
D7910 3,4-Dichloroisocoumarin
E1398 Ecotin recombinant, expressed in Escherichia coli, ≥90% (SDS-PAGE), essentially salt-free, lyophilized powder, ≥2000 units/mg protein

Substrates

Product #  Product Name Add to Cart
B7020 N-Benzoyl-Ile-Glu-Gly-Arg p-nitroanilide acetate salt
B9760 Boc-Ile-Glu-Gly-Arg-7-amido-4-methylcoumarin hydrochloride
F3301 Factor Xa chromogenic substrate solid
51015 4-Nitrophenyl 4-guanidinobenzoate hydrochloride BioChemika, ≥95.0% (AT)

Back to top

Solubility and Solution Stability Factor Xa (Product No F9302) is supplied as a 50% aqueous glycerol solution. It can be further diluted to no more than 100 µg/ml in HEPES, PBS or TBS (at a pH of 7.4) or with 40 mM citrate buffer, pH 6.8. Dilution to a lower concentration may result in loss of protein due to adsorption. The enzyme diluent in Sigma's assay procedure for activity is 40 mM citric acid, pH 6.8 containing 0.5% sucrose.

Applications and Clinical Implications

  • Inherited factor X deficiency is an extremely rare disease. However, acquired decreased levels of factor X can be the result of several factors including amyloidosis, vitamin K deficiency and administration of oral anticoagulants. Factor X deficiency results in prolonged prothrombin and partial thromboplastin times.
  • Fusion proteins are commonly expressed with a factor Xa cleavable Ile-Glu (or Asp)-Gly-Arg--X sequence. Typically 1mg of fusion protein can be incubated with 10 µg of factor Xa for 2.5 hours at 37 °C.10,11

Back to top

Products

Product #  Product Name Add to Cart
F9302 Factor X Activated (Xa) from bovine plasma

Related Products

Product #  Product Name Add to Cart
PRKF Factor Xa Removal Kit
F5132 Factor II from human plasma

Back to top

References

  1. IUBMB Enzyme Nomenclature: http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/6.html
  2. Fujikawa, K. and Davie, E.W., Methods Enzymol., XLV, 89-95 (1976)
  3. Jesty, J and Nemerson, y., Methods Enzymol., XLV, 95-107 (1976)
  4. Walz, D.A., et al., Proc. Natl, Acad. Sci., USA, 74, 1969-1972 (1977)
  5. Butkowski, R.J., et al., J. Biol. Chem., 252, 4942 (1977)
  6. Nagai, K. and Thogerson, H.C., Methods Enzymol., 153, 461481 (1987)
  7. Srivastava, A., et al., J. Biol. Chem., 277 1855-1863 (2002)
  8.  
  9. Sperl, S., et al., Biol. Chem., 381, 312-329 (2000)
  10. Ludeman, J.P., et al., Int. J. Biochem. Cell Biol., 35, 221-225 (2003)
  11. Gardella, T.J., et al., J. Biol. Chem., 265, 15854-15859 (1990)
  12. La Vallie, E.R., and McCoy, J.M., "Enzymatic Cleavage of Fusion Proteins with Factor Xa" in Current Protocols in Molecular Biology, 2, Supplement 28, Ausubel, F.M., ed., (John Wiley & Sons, New York, 1994) p.16.4.6-16.4.7

Back to top