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 PARTICIPATION OF FACTOR Xa IN PROTHROMBINASE
The participation of the serine protease factor Xa in the prothrombinase
complex is illustrated. Membrane bound factor Xa binds to membrane bound factor Va to form
the prothrombinase complex. This complex effectively converts the zymogen prothrombin (II)
to the active serine protease thrombin (IIa) by proteolytic removal of the fragment 1.2
(F1.2) portion of prothrombin.
Activation of the zymogen,
factor X, by either the intrinsic or extrinsic factor Xase complexes produces the active serine protease factor Xa (1,2). The activation of factor X requires proteolytic cleavage of the heavy chain, resulting in the release of an activation glycopeptide. The heavy chain region in factor Xa contains the serine protease catalytic domain, while the light chain, as in the zymogen, contains the membrane binding domain.
Factor Xa participates in the prothrombinase complex, which catalyzes the rapid conversion of
prothrombin to thrombin. Prothrombinase is an enzyme complex composed of factor Xa (enzyme) and
factor Va (cofactor) assembled on a cellular surface in the presence of calcium ions. Although factor Xa can independently catalyze the activation of
prothrombin, the rate at which this reaction occurs is increased nearly 300,000-fold with complete assembly of the prothrombinase complex. The clotting activity of factor Xa in vivo is terminated by either inactivation of the cofactor, factor Va, or by direct inhibition of factor Xa by inhibitors, such as
ATIII, after disassembly of the prothrombinase complex.
In recent years, molecular biologists have utilized factor Xa for site specific cleavage of fusion proteins expressed in bacteria (9-12). A factor Xa-sensitive site is incorporated between the recombinant protein of interest and peptides or proteins which facilitate purification and/or expression. The target protein is released from the expressed hybrid by cleavage with factor Xa. The factor Xa can then be easily removed by affinity chromatography.
Factor Xa is prepared by activating purified factor X with the factor X activator isolated from
Russell's viper venom. Factor Xa is purified from the activation mixture by chromatography over benzamidine-Sepharose followed by gel filtration (1,3). Several modified forms of factor Xa are also available including: A) active-site blocked factor Xa containing either the tripeptide chloromethyl ketone inhibitor EGRck, or the fluorescent inhibitor Dansyl-EGRck; and B) human Gla-domainless
β-factor
Xa. The enzyme is supplied in 50% (vol/vol) glycerol/H2O and should be stored at -20°C. Purity is determined by SDS-PAGE analysis and activity is measured in a factor Xa clotting assay and/or chromogenic substrate assay.
Properties of
Factor Xa
| Localization: |
Plasma |
| Mode of action: |
Enzyme component of the prothrombinase complex |
| Molecular weight: |
46,000 (human) (4)
45,300 (bovine) (5) |
| Extinction coefficient: |
| E |
|
= 11.6 (human) (6) |
|
|
= 12.4 (bovine) (7) |
|
| Specific activity: |
approximately 1000 units/mg |
| Structure: |
two subunits,
Mr=16,200 and 29,000 (human) (6), Mr=16,500 and 28,800 (bovine) (5),
NH2-terminal gla domain, two EGF domains |
| Percent carbohydrate: |
3.0 % (human) (8)
2.1 % (bovine) (8) |
| Post-translational modifications: |
eleven gla residues,
one
β-hydroxyaspartate |
|
Catalog
Number |
Description |
|
HCXA-0060 |
Human Factor Xa (Compliment
fluorogenic substrate(s): HTI Catalog # SN-7) |
|
HCBXA-0061 |
Human β-Factor
Xa |
|
HCXA-GD |
Human
Gla-Domainless β-Factor
Xa |
|
HCXA-EGR |
Human
Factor Xa -EGR |
|
HCXA-DEGR |
Human
Factor Xa - DEGR |
|
HCXA-BEGR |
Human
Factor Xa - BEGR (Biotinylated EGR) |
|
BCXA-1060 |
Bovine
Factor Xa |
|
BCXA-EGR |
Bovine
Factor Xa - EGR |
|
BCXA-DEGR |
Bovine
Factor Xa - DEGR |
|
MCXA-5060 |
Mouse
Factor Xa |
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References
1. Jesty, J., et al., Methods Enzymol., 45, 95 (1976).
2. Jackson, C.M., Ann. Rev. Biochem., 49, 765 (1980).
3. Krishnaswamy, S., et al., J.Biol. Chem., 262, 3291 (1987).
4. Discipio, R.G., et al., Biochemistry, 16, 5253 (1977).
5. Fujikawa, K. and Davie, E.W., Methods Enzymol., 45, 89 (1976).
6. Krishnaswamy, S., et al., J. Biol. Chem., 261, 8997 (1986).
7. Discipio, R.G., et al., Biochemistry, 16, 698 (1977).
8. Jackson, C.M., et al., Biochemistry, 7, 4506 (1968).
9. Fujikawa, K., Biochemistry, 13, 5290 (1974).
10. Nagai, K., et al., Proc. Natl. Acad. Sci. USA, 82, 7252 (1985).
11. Aurell, L., et al., Thromb. Res., 11, 595 (1984).
12. Nagai, K. and Thogersen, H., Nature, 309, 810 (1984).
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