Role of the N-terminal Epidermal Growth Factor-like Domain of Factor X/Xa

Kittur, Farooqahmed S.; Manithody, Chandrashekhara; Rezaie, Alireza R.

doi:10.1074/jbc.m402302200

Cited by 12 publications

(13 citation statements)

References 43 publications

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“…Thus, the EGF-1 domain of fXa may function as a spacer to maintain the active site pocket of the protease far above the membrane surface. This has been evidenced by the observation that the catalytic efficiency of fXades-EGF-1 toward prothrombin in the prothrombinase complex has been markedly impaired (26). The normal reactivity of the EGF-1 deletion mutant of fXa with the ZPI-PZ complex was surprising, and suggests that the cofactor-inhibitor complex has sufficient flexibility to interact with the mutant protease on PC/PS vesicles.…”

Section: Discussionmentioning

confidence: 94%

Identification of Factor Xa Residues Critical for Interaction with Protein Z-dependent Protease Inhibitor

Rezaie

Manithody

Yang

2005

Journal of Biological Chemistry

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Protein Z-dependent protease inhibitor (ZPI) is a plasma serpin, which can rapidly inactivate factor Xa (fXa) in the presence of protein Z (PZ), negatively charged phospholipids, and Ca 2؉ . To investigate the mechanism by which ZPI inactivates fXa, we expressed the serpin in mammalian cells and characterized its reactivity with both wild-type and selected mutants of fXa that 1) contained substitutions in the autolysis loop and the heparin binding exosite, 2) lacked the first EGF-like domain (fXa-des-EGF-1), or 3) contained the Gla domain of protein C (fXa/PC-Gla). Inhibition studies in both the presence and absence of PZ revealed that Arg-143, Lys-147, and Arg-154 of the autolysis loop and Lys-96, Lys-169, and Lys-236 of the heparin binding exosite are required for recognition of ZPI, with Arg-143 being essential for the interaction. Similar studies with fXa-des-EGF-1 and fXa/PC-Gla suggested that protein-protein interaction with either the Gla or the EGF-1 domain may not play a dominant role in the PZ-dependent recognition of fXa by the serpin on phospholipid vesicles. Further studies showed that an inactive Ser-195 to Ala mutant of fXa effectively competes with wild-type fXa for binding to the non-serpin inhibitors tissue factor pathway inhibitor and recombinant tick anticoagulant peptide, but does not compete for binding to ZPI. This suggests that the catalytic residue of fXa is required for interaction with ZPI.Factor Xa (fXa) 2 is a vitamin K-dependent serine protease that is responsible for generation of thrombin from prothrombin in the coagulation cascade (1-4). It circulates in plasma as a light and heavy chain molecule held together by a disulfide bond (5). The N-terminal light chain contains the non-catalytic ␥-carboxyglutamic acid (Gla) and two epidermal growth factor-like (EGF) domains, while the C-terminal heavy chain contains the trypsin-like catalytic domain of the molecule (2, 5). The proteolytic activity of fXa in plasma is regulated by three physiological inhibitors, tissue factor pathway inhibitor (TFPI) (6), antithrombin (AT) (7,8), and protein Z (PZ)-dependent protease inhibitor (ZPI) (9 -12). TFPI is a multidomain Kunitz-type inhibitor that regulates the coagulation cascade by the fXa-dependent inhibition of the factor VIIa-tissue factor complex during the initial stages of the clotting cascade (6). It functions by binding to the active site pocket of fXa by the second Kunitz domain, and thereafter binding tightly to the active site of the factor VIIa-tissue factor complex via the first Kunitz domain, thereby rendering both proteases inactive in a quaternary complex (6). AT, on the other hand, is a serine protease inhibitor (serpin) that regulates the proteolytic activity of fXa and other coagulation proteases by covalently binding to the active site through an exposed reactive center loop, and undergoing a conformational change, which leads to entrapment of the target protease in inactive and SDS-stable complex with the serpin (13-15). ZPI is also a serpin, and there is some evidence that...

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Section: Discussionmentioning

confidence: 94%

Identification of Factor Xa Residues Critical for Interaction with Protein Z-dependent Protease Inhibitor

Rezaie

Manithody

Yang

2005

Journal of Biological Chemistry

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“…The expression, purification, and characterization of recombinant ZPI in HEK-293 cells has been described previously (15). The expression and purification of fXa lacking the EGF1 domain (fXadesEGF1) or both Gla and EGF1 domains (E2-fXa) has been described (16). The activated protein C mutants in which either the Gla domain or both the Gla and the first EGF-like domains of the protease were replaced with the corresponding regions of fXa (APC-fX/Gla and APC-fX/Gla/EGF1) were expressed in the same expression system as described (17).…”

Section: Methodsmentioning

confidence: 99%

Protein Z-dependent Protease Inhibitor Binds to the C-terminal Domain of Protein Z

Rezaie

Bae

Manithody

et al. 2008

Journal of Biological Chemistry

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Protein Z (PZ) is a multidomain vitamin K-dependent plasma protein that functions as a cofactor to promote the inactivation of factor Xa (fXa) by PZ-dependent protease inhibitor (ZPI) by three orders of magnitude. To understand the mechanism by which PZ improves the reactivity of fXa with ZPI, we expressed wild-type PZ, PZ lacking the ␥-carboxyglutamic acid domain (GD-PZ), and a chimeric PZ mutant in which both Gla and EGFlike domains of the molecule were substituted with identical domains of fXa. The ZPI binding and the cofactor function of the PZ derivatives were characterized in both binding and kinetic assays. The binding assay indicated that all PZ derivatives interact with ZPI with a similar dissociation constant (K D ) of ϳ7 nM. However, the apparent K D for the chimeric PZ-mediated ZPI inhibition of fXa was elevated 6-fold on PC/PS vesicles and its capacity to function as a cofactor to accelerate the ZPI inhibition of fXa was also decreased 6-fold. The cofactor activity of GD-PZ was dramatically impaired; however, the deletion mutant exhibited a normal cofactor function in solution. A chimeric activated protein C mutant containing the Gla domain of fXa was susceptible to inhibition by ZPI in the presence of PZ. These results suggest that: (i) the ZPI interactive site of PZ is located within the C-terminal domain of the cofactor and (ii) a specific interaction between the Gla domains of PZ and fXa contributes ϳ6-fold to the acceleration of the ZPI inhibition of fXa on phospholipid membranes. Protein Z (PZ)2 is a vitamin K-dependent coagulation glycoprotein, which has a genetic organization that is identical to that of factor Xa (fXa) and other similar vitamin K-dependent coagulation factors (1, 2). Thus, PZ has an N-terminal ␥-carboxyglutamic acid (Gla) domain that is followed by two epidermal growth factor (EGF)-like domains (light chain homologue) and a C-terminal pseudo catalytic domain (heavy chain homologue) (1, 2). Unlike fXa and other vitamin K-dependent coagulation proteases, two of the catalytic triad residues of the C-terminal domain have not been conserved in the homologous regions of PZ (1, 2). Thus, PZ has no catalytic function, but instead it binds to PZ-dependent protease inhibitor (ZPI) with a high affinity, thereby promoting the ZPI inhibition of fXa in the presence of PC/PS vesicles and Ca 2ϩ by approximately three orders of magnitude (3-5). ZPI is a 72-kDa serpin with a plasma concentration of 2.6 -2.9 g/ml that appears to interact with the active site pocket of fXa by a covalent mechanism similar to that observed with antithrombin and most other inhibitory serpins (4 -6). However, unlike antithrombin, which can inhibit all coagulation proteases, ZPI is a specific inhibitor of fXa and factor XIa (4, 7, 8), though recent results have indicated that it may also inhibit factor IXa (9). ZPI may play a critical role in the regulation of the coagulation cascade because its deficiency is associated with venous thromboembolic diseases (10, 11). ZPI by itself is a poor inhibitor of fXa, unless i...

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“…A structural role for the EGF1 domain can be inferred from the observation that the isolated EGF1 domain and Gla domain bind Ca 2+ with at least 10-fold lower affinity than when they are covalently linked, suggesting that the domain-domain interaction is required for the proper folding and function of fXa on the membrane surface [9,10]. Recent results have excluded any interaction between either Gla or EGF1 of fXa with fVa in the prothrombinase complex [11,12]. However, the EGF1 domain of fXa may serve an essential spacer function between the Gla domain and the serine protease domain to maintain the active site at an appropriate height above the membrane surface in order to cleave the membrane-bound substrate [12].…”

Section: Introductionmentioning

confidence: 99%

FRET studies with Factor X mutants provide insight into the topography of the membrane-bound Factor X/Xa

Qureshi

Yang

Yegneswaran

et al. 2007

Biochemical Journal

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FRET (fluorescence resonance energy transfer) studies have shown that the vitamin K-dependent coagulation proteases bind to membrane surfaces perpendicularly, positioning their active sites above the membrane surfaces. To investigate whether EGF (epidermal growth factor) domains of these proteases play a spacer function in this model of the membrane interaction, we used FRET to measure the distance between the donor fluorescein dye in the active sites of Fl-FPR (fluorescein-D-Phe-Pro-Arg-chloromethane)-inhibited fXa (activated Factor Xa) and its N-terminal EGF deletion mutant (fXa-desEGF1), and the acceptor OR (octadecylrhodamine) dye incorporated into phospholipid vesicles composed of 80% phosphatidylcholine and 20% phosphatidylserine. The average distance of closest approach (L) between fluorescein in the active site and OR at the vesicle surface was determined to be 56+/-1 A (1 A=0.1 nm) and 63+/-1 A for fXa-desEGF1 compared with 72+/-2 A and 75+/-1 A for fXa, in the absence and presence of fVa (activated Factor V) respectively, assuming kappa2=2/3. In comparison, an L value of 95+/-6 A was obtained for a S195C mutant of fXa in the absence of fVa in which fluorescein was attached directly to Cys(195) of fXa. These results suggest that (i) EGF1 plays a spacer function in holding the active site of fXa above the membrane surface, (ii) the average distance between fluorescein attached to Fl-FPR in the active site of fXa and OR at the vesicle surface may not reflect the actual distance of the active-site residue relative to the membrane surface, and (iii) fVa alters the orientation and/or the height of residue 195 above the membrane surface.

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Role of the N-terminal Epidermal Growth Factor-like Domain of Factor X/Xa

Cited by 12 publications

References 43 publications

Identification of Factor Xa Residues Critical for Interaction with Protein Z-dependent Protease Inhibitor

Identification of Factor Xa Residues Critical for Interaction with Protein Z-dependent Protease Inhibitor

Protein Z-dependent Protease Inhibitor Binds to the C-terminal Domain of Protein Z

FRET studies with Factor X mutants provide insight into the topography of the membrane-bound Factor X/Xa

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