Factor Xa

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Activity and Specificity

Factor X is the zymogen of serine proteases. The activated enzyme (Factor Xa) is an Arg-specific serine protease related to trypsin, but with a much narrower substrate specificity. The N-terminal γ-carboxyglutamic acid (Gla) domain binds approximately 8-10 calcium ions in a cooperative manner and mediates the interaction of factor X/Xa with phospholipid membranes. Factor Xa and cofactor factor Va form a phospholipid-bound complexes in the presence of calcium ions. This "prothrombinase" complex activates prothrombin about 300,000-folds more rapidly free factor Xa.

Membrane-bound factor Va binds to factor Xa with high affinity. In the absence of factor Va, factor Xa binds to phospholipid membranes with a Kd of approximately 30–100 nM. In these reactions, phospholipids can be considered as a means of reducing the three-dimensional reaction dimension to two-dimensional, thereby increasing the possibility of collisions between factor Xa and membrane-bound factor Va. Effective binding of factor X/Xa to membrane requires phosphatidylserine to be exposed on the outer surface, as occurs in vesicles released from activated platelets: when platelets are activated, phosphatidylserine is transferred to the exterior by phospholipid scramblase.

Factor Xa activates prothrombin by hydrolyzing two peptide bonds, so the reaction can proceed via two pathways. In one pathway, cleavage of the Arg320↓Ile321 bond yields an intermediate, meizothrombin, which can then be cleaved at the Arg271↓Thr272 bond to yield fragments 1-2 and thrombin. During both cleavage processes, prothrombin and methylthrombin bind to the same site and impair the structural transition, preventing thrombin cleavage. In vitro, factor Va and/or phosphatidylserine can modulate the specificity of factor Xa, leading to the predominance of the methylthrombin pathway, which is generally considered the most physiologically relevant activation pathway. Factor Xa also proteolytically activates factors V and VIII, and factor VII, thereby participating in a positive feedback loop that amplifies the clotting process.

Factor Xa can also inactivate factor VIIIa by cleavage at Lys36 and Arg336 within the A1 subunit. Including methoxycarbonyl-cyclohexylglycyl-Gly-Arg↓NHPhNO₂(Spectrozyme Xa), methanesulfonyl-d-Leu-Gly-Arg↓NHPhNO₂ (CBS 31.39), and Bz-Ile-Glu-(piperidine amide)-Gly-Arg↓NHPhNO₂ (S2337) can be used for factor Xa. Full activity of factor Xa on these substrates requires the presence of both Na⁺ and Ca²⁺ ions, which are responsible for regulating its catalytic domain. Subsite specificity has been studied in detail through methods such as proteome-derived peptide libraries, phage display, and fluorescence-quenched substrates, revealing a preference for Arg in P1 and Gly or Ala in P2; slight preferences for Pro in P4; and aromatic or aliphatic residues (especially Phe, Leu or Tyr) in P2′.

Figure 1. Schematic representation of crystal structure of the catalytic domain of Factor Xa (Levigne, S.; et al. 2007)

Biological Aspects

In addition to being directly involved in blood coagulation, factor Xa also interacts with a variety of cell surface signaling receptors. Therefore, it is closely related to various cellular activities such as cell activation, gene expression and mitosis. A factor Xa receptor termed effector cell protease receptor-1 (EPR-1) has been cloned, which shares some structural similarities with the light chain of factor V.

EPR-1 does not bind factor X, but binds to factor Xa to form a protease-receptor complex responsible for inducing cytokine gene expression and platelet-derived growth factor release. In endothelial cells, factor Xa appears to exert its effects by docking to EPR-1, followed by cleavage and activation of protease-activated receptor 2 (PAR-2). PAR-2 is a member of the G protein-coupled receptor family that is activated by cleavage of an N-terminal peptide. There is also evidence that factor Xa can activate PAR-2 and/or PAR-1 through a mechanism independent of EPR-1, thereby inducing cell signaling in vascular wall cells. Factor Xa activates PAR-1, enabling epithelial-derived tumor cells to enter apoptosis and inhibit their migration. Factor Xa also has other physiological and pathological roles. It is expressed in bronchoalveolar lavage fluid macrophages from mouse models of asthma and is responsible for inducing mucin production. Factor Xa mediates the attachment of adenovirus 5 to hepatocytes through the hexon protein, and basic residues in the serine peptidase domain are critical for this interaction. In SARS coronavirus, the spike protein bound to the host receptor is cleaved into subunits by factor Xa, thereby facilitating viral infection.