Factor Xa

On this page: Products Physical Properties and In Vivo Processing Specificity, Assay and Kinetics Inhibitors Substrates Solubility and Solution Stability Applications and Clinical Implications

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

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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: 7.6-8.0^7,8

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.

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Inhibitors

Substrates

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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

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Products

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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

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