The third edition of the Handbook of Proteolytic Enzymes aims to be a comprehensive
reference work for the enzymes that cleave proteins and peptides, and contains over
800 chapters. Each chapter is organized into sections describing the name and history,
activity and specificity, structural chemistry, preparation, biological aspects, and
distinguishing features for a specific peptidase. The subject of Chapter 642 is Coagulation
Factor Xa.
Keywords
Coagulation factor, prothrombin, thrombin, proconvertin, Stuart’s factor, Prower’s
factor.
Databanks
MEROPS name: coagulation factor Xa
MEROPS classification: clan PA, subclan PA(S), family S1, subfamily S1A, peptidase
S01.216
IUBMB: EC 3.4.21.6 (BRENDA)
Tertiary structure: Available
Species distribution
: superclass Tetrapoda
Reference sequence from: Homo sapiens (UniProt: P00742)
Name and History
In the mid-1950s it was becoming evident that a coagulation factor different from
factors VII and IX was required for the activation of prothrombin to thrombin [1].
In 1957 it was found that the coagulation properties of blood samples from patients
thought to lack factor VII (then called proconvertin) could be normalized by mixing
the samples. This indicated that the disease was not a homogeneous entity. The new
factor, ‘
factor X
’, was also called
Stuart’s factor
or
Prower’s factor
. Factor X was found to require vitamin K for normal biosynthesis, and treatment of
patients with vitamin K antagonists such as dicoumarol or warfarin resulted in a decrease
in its biological activity [1]. It was first purified to homogeneity from bovine plasma
and later from human plasma [2], [3].
Activity and Specificity
Factor X is the zymogen of a serine protease. The activated enzyme (factor Xa) is
an Arg-specific serine protease related to trypsin, but has much narrower substrate
specificity. The N-terminal γ-carboxyglutamic acid (Gla) domain binds ~8–10 calcium
ions in a cooperative manner (average K
d ~ 0.5 mM) and mediates the interaction of factor X/Xa with phospholipid membranes
[4], [5], [6]. In the presence of calcium ions, factor Xa forms a phospholipid-bound
complex with a cofactor, factor Va. This ‘prothrombinase’ complex activates prothrombin
(Chapter 643) ~300 000-fold more rapidly than does free factor Xa. Studies of this
reaction have demonstrated that phospholipid reduces the K
m for prothrombin, whereas factor Va increases the V
max
[5]. In vivo, the surface of activated platelets is the main site at which the prothrombinase
complex is assembled.
Factor Va is derived from factor V by specific proteolytic cleavages mediated by thrombin
or factor Xa. Membrane-bound factor Va binds factor Xa with high affinity (K
d ~ 1 nM), due at least in part to interactions between the catalytic domain (residues
163–170 [7]) and residues 499–505 of factor Va [8], but has no measurable affinity
for the zymogen [5]. In the absence of factor Va, factor Xa binds to phospholipid
membranes with a K
d of ~30–100 nM. In these reactions, the phospholipid can be regarded as a means of
reducing the ‘dimensionality’ of the reaction, from three, to two dimensions, thereby
increasing the likelihood of a productive collision between factor Xa and membrane-bound
factor Va. Effective binding of factor X/Xa to membranes requires that phosphatidylserine
is exposed on the outer surface, as occurs in vesicles released from activated platelets:
phosphatidylserine is translocated to the exterior by phospholipid scramblase when
the platelet is activated [9]. This regulatory mechanism restricts binding of factor
X/Xa to discrete types of cells, according to their functional state.
Factor Xa activates prothrombin, a single-chain molecule, by hydrolyzing two peptide
bonds and the reaction may thus proceed via two pathways [5], [10]. In one pathway,
cleavage of the Arg320↓Ile321 bond yields an intermediate, meizothrombin, which may
then be cleaved at the Arg271↓Thr272 bond to yield fragment 1-2 and thrombin [11].
During both cleavages, prothrombin and meizothrombin bind to the same exosite [12],
and impairing the structural shift that accompanies activation of thrombin, prevents
cleavage of meizothrombin [13]. Meizothrombin is inactive against fibrinogen but readily
activates protein C (Chapter 644). In a second pathway, the initial cleavage occurs
at the Arg271↓Thr272 bond and the intermediates fragment 1–2 and prethrombin-2 are
formed. Prethrombin-2 is enzymatically inactive and is formed predominantly in the
absence of factor Va [14]. Subsequent hydrolysis of the Arg320↓Ile321 bond in prethrombin-2
yields thrombin. The Arg284↓Thr285 bond is also susceptible to cleavage by factor
Xa or thrombin and, in plasma, this site may be preferred over the Arg 271-Ile 272
bond [15]. In vitro, factor Va and/or phosphatidylserine modulate the specificity
of factor Xa so that the meizothrombin pathway predominates [14], [16]. This is generally
thought to be the most physiologically relevant activation pathway. Factor Xa can
also proteolytically activate factors V and VIII, and factor VII (Chapter 641), thus
participating in positive feedback loops that amplify the clotting process. Factor
Xa also inactivates factor VIIIa, by cleavage at Lys36 and Arg336 within the A1 subunit
[17]. Several synthetic substrates are available for factor Xa, 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). Full activity of factor Xa towards these substrates
requires the presence of both Na+ and Ca2+ ions, which modulate the catalytic domain
[18]. The subsite specificity has been examined in detail using proteome-derived peptide
libraries [19], phage display [20], and fluorescence-quenched substrates [21], revealing
a preference for Arg in P1 and Gly or Ala in P2; slight preferences for Pro in P4,
a small amino acid in P1′ and Asp in P3′; and aromatic or aliphatic residues (especially
Phe, Leu or Tyr) in P2′.
Free factor Xa in the bloodstream is rapidly inactivated by serine protease inhibitors
(serpins) such as antithrombin and α
1-protease inhibitor [22]. Cleavage of an exposed loop on the serpin by factor Xa
leads to formation of a covalently-linked complex (1:1 stoichiometry) and inhibition
of enzymatic activity. The rate of inactivation by antithrombin is increased several
thousand-fold by heparin/heparin sulfate, shorter, heparin-based, synthetic oligosaccharides,
and a sulfated galactan from the red algae Botryocladia occidentalis
[23]. Another serpin, protein Z-dependent protease inhibitor (ZPI), can inhibit factor
Xa, although it is far less effective than antithrombin, because it has an unfavorable
P1 Tyr. Residues in factor Xa essential for inhibition by ZPI have been identified
in the autolysis loop and heparin binding exosite [24]. Protein Z accelerates this
inhibition, and its interaction with ZPI requires association with the membrane, interaction
between the Gla domains in protein Z and ZPI, and Glu313 in the protein Z pseudoactive
site [25]. Factor Xa is also inhibited by non-serpin inhibitors such as tissue factor
pathway inhibitor (TFPI) and α2-macroglobulin. TFPI consists of three tandem Kunitz-type
domains, the second of which interacts with the active site of factor Xa: the complex
then associates with tissue factor–factor VIIa to form an inactive quaternary complex
[22], [26]. Heterologous protein inhibitors of factor Xa include soy bean and corn
trypsin inhibitors, as well as some proteins in snake venom [27]. Certain peptides,
both naturally occurring (e.g. tick anticoagulant peptide [28], ecotin [29] and the
hookworm NAP5 peptide) and synthetic (e.g. dansyl-Ile-Glu-Gly-Arg-CH2Cl, or DEGR,
and SEL2711) can directly inhibit factor Xa. In addition, many specific organic inhibitors
of factor Xa have been identified, including DX-9065a [30], a propanoic acid derivative,
and benzamidine-based compounds. Synthetic inhibitors have been developed as antithrombotic
agents, including biarylmethoxy isonipecotanilides [31], thiophene-anthranilamides
[32], quinoxalinones [33], and Razaxaban, Apixaban, Rivaroxaban and Darexaban, which
can be taken orally [34], [35], [36], [37]. The anticoagulant NAPc2 from the nematode
Ancylostoma caninum binds first to a novel exosite on factor Xa, and inhibits factor
VIIa only when it is complexed with tissue factor and factor Xa [38]. Membrane-bound
factor X/Xa can be inactivated by plasmin. Cleavage of the Lys435↓Ser436 bond generates
factor Xaβ and further cleavage at the Lys330↓Gly331 peptide bond generates factor
Xa33/13, which destroys clotting activity [39]. These cleavages expose plasminogen
binding sites that augment the generation of plasmin and thus seem to switch factor
X/Xa from a procoagulant to an anticoagulant role by stimulating fibrinolysis [40].
Structural Chemistry
The primary structure of human factor X has been determined by amino acid and DNA
sequencing [41], [42]. Factor X sequences from several other vertebrate species are
also known. Human factor X is synthesized as a single polypeptide of 488 amino acids,
including a 23-residue signal peptide (amino acids −40 to −18) to direct its translocation
into the lumen of the endoplasmic reticulum, and a 17-residue propeptide (amino acids
−17 to −1), which serves as a recognition site for γ-glutamyl carboxylase [4]; neither
of these is found in the mature protein. Prior to secretion, a tripeptide (Arg140-Lys141-Arg142)
is excised to yield a heterodimer linked by a disulfide bond between Cys132 and Cys302.
The molecular mass of the mature zymogen is 58.9 kDa (sedimentation equilibrium).
It consists of a light chain of 139 amino acids and a glycosylated heavy chain of
306 amino acids. When resolved by SDS-PAGE, the chains have apparent masses of ~17
and ~49 kDa. The N-terminus of the heavy chain (residues 143–194) constitutes an activation
peptide which is not found in the active enzyme. The N-terminus of the light chain
(amino acids 1–45) comprises the Gla domain, including the so-called aromatic amino
acid stack. Gla residues reside at positions 6, 7, 14, 16, 19, 20, 25, 26, 29, 32
and 39. The aromatic stack is followed by two EGF-like domains (residues 46–84 and
85–128).
The structure of factor Xa, lacking the Gla domain, has been solved by X-ray crystallography
at ~2.2 Å resolution [43], [44]. Overall, the folding of the catalytic domain is similar
to that of chymotrypsin and thrombin. It contains three disulfide bonds: Cys221-Cys237,
Cys350-Cys364 and Cys375-Cys403. His236, Asp282 and Ser379 constitute the catalytic
triad involved in hydrolyzing peptide bonds in a substrate. The primary specificity
(S1) pocket is formed by two loops (residues 373–379 and 398–403) that are joined
by a disulfide bond (Cys375-Cys403). An aspartyl residue (Asp373) sits at the base
of the S1 pocket, as is characteristic of trypsin-like proteases. The formation of
a salt bridge between Asp373 and Arg residues at the P1 position in a substrate is
an important interaction that explains the enzyme’s preference for hydrolyzing substrates
on the C-terminal side of Arg residues. The C-terminal EGF-like domain is in close
contact with the catalytic domain and, like the N-terminal one, has a fold similar
to that of other EGF-like domains. The fold is supported by three disulfide bonds
that link the Cys residues in the pattern 1–3, 2–4, 5–6. The three-dimensional structure
of the N-terminal EGF-like domain has been determined both in the presence and absence
of calcium ions [44], [45], [46]. The calcium-bound structures revealed a calcium-binding
site that is unique to EGF-like domains, with Gly47, Gln49, β-hydroxylated Asp63,
Gly64, and Leu65 identified as ligands for the calcium ion. However, β-hydroxylation
of Asp63 is not required for high affinity calcium binding. NMR-based structures showed
also that the calcium ion locks the EGF-like domain in the proper position relative
to the Gla domain.
The structure of the Gla domain linked to the N-terminal EGF-like domain has been
determined in the absence of calcium by NMR spectroscopy [47]. In contrast to a model
of the calcium-bound domain, the structure revealed that, in the absence of calcium,
Gla residues are exposed to the solvent and three hydrophobic residues near the N-terminus
are folded into the interior of the domain. Thus, binding of several calcium ions
to the Gla domain pulls the Gla residues to the interior and provides the energy required
to expose the hydrophobic side chains to the solution. Together with site-directed
mutagenesis studies, this established that the interaction between a Gla domain and
a phospholipid membrane includes associations of a hydrophobic nature.
Preparation
Factor X can be purified to homogeneity from plasma using conventional methods. An
initial precipitation based on its affinity for insoluble barium salts is usually
employed. This is often followed by ammonium sulfate precipitation and anion exchange
chromatography. These procedures are sufficient to obtain homogeneous bovine factor
X but purification of human factor X may require additional chromatography steps [2],
[3]. Immunoaffinity chromatography with monoclonal antibodies has also been utilized
with success [48], [49], [50]. Recently, recombinant factor X has been expressed in
several mammalian cell lines. However, at high expression levels, γ-carboxylation
and proteolytic processing are inefficient. Substitution of the factor X propeptide
with that from prothrombin improves γ-carboxylation [51] and chromatography on hydroxyapatite
can effectively isolate fully γ-carboxylated factor X [52]. Site-directed mutagenesis
has been employed to enhance cleavage of the propeptide [49], which can also be removed
by incubation with furin.
Biological Aspects
The factor X gene is located on chromosome 13 at position q34, adjacent to the factor
VII gene. It spans ~27 kb and has seven introns and eight exons. Exon I encodes the
signal peptide, exon II the propeptide/Gla domain, exon III the C-terminal part of
the Gla domain and the aromatic amino acid stack, exons IV and V the EGF-like domains,
exon VI the activation peptide region, and exons VII and VIII the catalytic domain
[42]. Factor X is synthesized mainly in the liver but its ~1700-nucleotide mRNA and/or
protein has been detected in several other tissues. Factor X is secreted into the
blood (normal concentration, 5–10 μg ml−1). The protein undergoes extensive post-translational
modification [53]. The signal peptide is removed by signal peptidase during translocation
into the endoplasmic reticulum, where the 11 Glu residues in the Gla domain are γ-carboxylated
by γ-glutamyl carboxylase. This is followed by proteolytic removal of the propeptide
by the subtilisin-like enzyme furin. Asp63 in the first EGF-like domain is converted
to erythro-β-hydroxyaspartic acid by a dioxygenase [54], [55]. In the activation peptide,
Thr159 and Thr171 are O-glycosylated and Asn181 and Asn191 are N-glycosylated. The
O-linked carbohydrate moieties appear to be important for factor X to be activated
efficiently. The activation peptide of bovine factor X contains a sulfate group O-esterified
to Tyr160. In the trans-Golgi apparatus, the factor X polypeptide is cleaved at the
Arg142↓Ser143 bond to yield a disulfide-bonded dimer. The three C-terminal residues
of the light chain (Arg140-Lys141-Arg142) are somehow removed either before secretion
or in the plasma.
Activation of factor X to a serine protease occurs predominantly by hydrolysis of
the Arg194↓Ile195 bond in the heavy chain, which releases a 52-residue activation
peptide to form factor Xaα. The cleavage causes the new N-terminus of the heavy-chain
to rearrange so that Ile195 can participate in formation of the substrate binding
pocket by forming a salt bridge with Asp378 [56]. This also contributes to the formation
of the Na+ and factor Va binding sites [57], and appears to cause the transition from
zymogen to active protease. A second cleavage, plasmin-mediated or autocatalytic,
at the Lys435↓Ser436 bond yields factor Xaβ [39]. The procoagulant activity of both
forms of factor Xa is similar.
Activation of factor X occurs via two principal pathways. It is activated by factor
VII/VIIa in complex with a non-enzymatic membrane-bound cofactor, tissue factor (TF).
This pathway is called the ‘extrinsic pathway’ and is responsible for the initiation
of coagulation, proceeding mainly on the surface of damaged endothelial cells and
macrophages, but probably also on activated platelets [58], [59]. Alternatively, factor
X is activated on the platelet surface by a membrane-bound ‘tenase’ complex comprising
factor IXa, its cofactor factor VIIIa, and calcium ions, which activates factor X
~ 106-fold more rapidly than factor IXa alone [5]. This ‘intrinsic pathway’ is responsible
for amplifying the coagulation process (see also Chapter 640) and its importance is
illustrated by the fact that hereditary deficiency of factors IX or VIII causes hemophilia
B and A, respectively. Thus, factor X plays a pivotal role in blood clotting at the
point of convergence of the two coagulation pathways. Accordingly, several rare mutations
in the factor X gene have been identified that give rise to bleeding tendencies of
variable severity (e.g. Chafa et al.
[60], Bereczky et al.
[61]). Theoretically, injection of factor Xa into patients with hemophilia should
bypass the intrinsic pathway and permit generation of thrombin, but this fails because
of the short half-life in plasma of factor Xa. However, mutants in which Ile16 or
Val17 are replaced have a much longer half-life because they do not form complexes
with antithrombin III or tissue factor inhibitor in hemophiliac plasma, yet still
are able to activate prothrombin and thus may be useful therapeutic agents [62], [63].
Factor X can also be activated by an alternative pathway which is initiated on the
surface of leukocytes and can trigger clotting. In this case the zymogen is bound
by the β2-integrin Mac-1 (CD11b) and activation occurs through hydrolysis of the Leu177↓Leu178
peptide bond in the activation peptide; a cleavage effected by cathepsin G, which
is secreted by stimulated leukocytes [64], [65]. Mac-1 binds factor X with high affinity
(K
d ~ 30 nM) but has no affinity for factor Xa. Enzymes present in venom from snakes
(e.g. RVV-X; [66]) (Chapter 235) and other toxic animals can also activate factor
X.
In addition to its direct involvement in blood coagulation, factor Xa interacts with
signalling receptors on the surface of many types of cells. It can thus elicit a variety
of responses, including cell activation, gene expression and mitogenesis. A factor
Xa receptor termed effector cell protease receptor-1 (EPR-1), with some structural
similarity to the light chain of factor V, has been cloned [67]. EPR-1 does not bind
factor X, whereas factor Xa forms a protease–receptor complex that induces cytokine
gene expression and the release of platelet-derived growth factor. In endothelial
cells, factor Xa appears to exert its effects by docking to EPR-1 and subsequently
cleaving and activating protease-activated receptor-2 (PAR-2) [68]. PAR-2 is a member
of a family of G protein-coupled receptors that are activated by cleavage of an N-terminal
peptide; the new N-terminus (a ‘tethered ligand’) then inserts into the body of the
receptor and activates it. There is also evidence that factor Xa can induce cell signalling
in vascular wall cells by activating PAR-2 and/or PAR-1 by a mechanism that is independent
of EPR-1 (e.g. McLean et al.
[69]). Factor Xa activates PAR-1 with the effect that epithelial-derived tumor cells
enter apoptosis [70] and breast, colon and lung cancer cell migration is inhibited
[71]. In epithelial cells, signaling is via the extracellular-signal regulated kinase
(ERK) pathway, leading to upregulation of Bim and caspase-3 activation [70]. In breast
cancer cells, the Rho/ROCK and Src/FAK/paxillin pathways are activated leading to
myosin light chain phosphorylation, LIMK1 activation, cofilin inactivation and stabilization
of actin filaments which are incompatible with cell migration [72].
Factor Xa has other physiological and pathological roles. It is expressed in bronchoalveolar
lavage fluid macrophages from mouse models of asthma, where it induces mucin production
[73]. Factor Xa mediates the attachment of adenovirus 5 to hepatocytes via the hexon
protein, and basic residues in the serine peptidase domain are essential for this
interaction [74]. In SARS coronavirus, the spike protein, which binds to host receptors,
is cleaved by factor Xa into subunits, facilitating viral infection [75].
Distinguishing Features
Factor X is a liver-synthesized zymogen of a serine protease that requires vitamin
K for normal biosynthesis. The protein has the same domain structure as coagulation
factors VII and IX, protein C (Chapter 644) and protein Z. Although all are synthesized
as single polypeptide chains, factor X and protein C are cleaved to form dimers prior
to secretion. The proteins are easily distinguished by SDS-PAGE under reducing conditions.
Monoclonal and polyclonal antibodies that can distinguish the proteins are commercially
available.