Human Protein Z.

Broze, George J.; Miletich, Joseph P.

doi:10.1172/jci111317

Cited by 140 publications

(99 citation statements)

References 34 publications

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“…An unusual feature of this serpin is that the P1 bait residue is a tyrosine, which is unexpected based on the arginine-specific target proteases of ZPI and the fact that most other serpins that inhibit coagulation proteases contain a P1 arginine residue (6). The serpin circulates as a tight stoichiometric complex with protein Z (8), a vitamin K-dependent protein whose domain structure resembles that of factor Xa in consisting of a Gla domain, two epidermal growth factor domains, and a protease domain, but with the protease domain lacking catalytic function (9). Inhibition of free factor Xa in the absence of protein Z is about 1000-fold slower than inhibition of membrane-bound factor Xa by ZPI-protein Z complex (7).…”

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confidence: 99%

Kinetic Characterization of the Protein Z-dependent Protease Inhibitor Reaction with Blood Coagulation Factor Xa

Huang

Swanson

Broze

et al. 2008

Journal of Biological Chemistry

109

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Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulation factors Xa (FXa) and XIa. Here we show that ZPI and its cofactor, protein Z (PZ), inhibit procoagulant membrane-bound factor Xa by the branched pathway acyl-intermediate trapping mechanism used by other serpins, but with significant variations of this mechanism that are unique to ZPI. Rapid kinetic analyses showed that the reaction proceeded by the initial assembly of a membraneassociated PZ-ZPI-FXa Michaelis complex (K M 53 ؎ 5 nM) followed by conversion to a stable ZPI-FXa complex (k lim 1.2 ؎ 0.1 s ؊1 ). Cofactor premixing experiments together with independent kinetic analyses of ZPI-PZ and factor Xa-PZ-membrane complex formation suggested that assembly of the Michaelis complex through either ZPI-PZ-lipid or factor Xa-PZ-lipid intermediates was rate-limiting. Reaction stoichiometry analyses and native PAGE showed that for every factor Xa molecule inhibited by ZPI, two serpin molecules were cleaved. Native PAGE and immunoblotting showed that PZ dissociated from ZPI once ZPI forms a stable complex with FXa, and kinetic analyses confirmed that PZ acted catalytically to accelerate the membrane-dependent ZPI-factor Xa reaction. The ZPI-FXa complex was only transiently stable and dissociated with a rate constant that showed a bell-shaped pH dependence indicative of participation of factor Xa active-site residues. The complex was detectable by SDS-PAGE when denatured at low pH, consistent with it being a kinetically trapped covalent acyl-intermediate. Together our findings show that ZPI functions like other serpins to regulate the activity of FXa but in a manner uniquely dependent on protein Z, procoagulant membranes, and pH.Activation of factor X is a focal point for regulation of the blood coagulation cascade. Formation of factor Xa through the extrinsic pathway and its amplification through the intrinsic pathway are regulated by the Kunitz inhibitor, tissue factor pathway inhibitor, and by the serpin, antithrombin, and its cofactor, heparin (1, 2). Whereas tissue factor pathway inhibitor regulates the activity of factor Xa bound in a physiologic complex with membrane-bound tissue factor and factor VIIa, antithrombin and heparin poorly inhibit factor Xa bound in procoagulant membrane-associated physiologic complexes and instead appear to target factor Xa that escapes from its membrane sites of action (3, 4). Broze and co-workers (5, 6) have identified another serpin, protein Z-dependent protease inhibitor or ZPI, that specifically inhibits factor Xa bound to procoagulant membranes, suggesting a potential means for dynamic regulation of factor Xa bound in procoagulant complexes such as prothrombinase and factor Xase. ZPI 2 is a member of the serpin superfamily of protein protease inhibitors and is unusual in requiring protein Z, anionic phospholipids, and calcium as cofactors to rapidly inhibit factor Xa (6). Factor XIa is also inhibited by ZPI,...

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mentioning

confidence: 99%

Kinetic Characterization of the Protein Z-dependent Protease Inhibitor Reaction with Blood Coagulation Factor Xa

Huang

Swanson

Broze

et al. 2008

Journal of Biological Chemistry

109

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show abstract

“…Protein Z is a vitamin K-dependent plasma glycoprotein that presents structural similarities with some coagulation factors such as factors VII, IX, X and protein C [1].…”

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confidence: 99%

Protein Z is not synthesised by human umbilical vein endothelial cells

Cesari

Sofi

Evangelisti

et al. 2006

Thrombosis Research

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“…Activated FX (FXa) can generate a tiny amount of thrombin from prothrombin in an extremely inefficient reaction (2). Tissue factor pathway inhibitor (TFPI) binds to TF-FVIIa-FXa to limit the production of FXa and FIXa by TF-FVIIa (3,4). Nevertheless, once produced, thrombin and the initially formed FXa activate small quantities of factor V (FV) to FVa and factor VIII (FVIII) to FVIIIa (5)(6)(7)(8).…”

mentioning

confidence: 99%

Role of the Gla and First Epidermal Growth Factor-like Domains of Factor X in the Prothrombinase and Tissue Factor-Factor VIIa Complexes

Thiec

Cherel

Olivier

2003

Journal of Biological Chemistry

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Factor X (FX) has high structure homology with other proteins of blood coagulation such as factor IX (FIX) and factor VII (FVII). These proteins present at their aminoterminal extremity a ␥-carboxyglutamic acid containing domain (Gla domain), followed by two epidermal growth factor-like (EGF1 and EGF2) domains, an activation peptide, and a serine protease domain. After vascular damage, the tissue factor-FVIIa (TF-FVIIa) complex activates both FX and FIX. FXa interacts stoichiometrically with tissue pathway inhibitor (TFPI), regulating TF-FVIIa activity by forming the TF-FVIIa-TFPI-FXa quaternary complex. Conversely, FXa boosts coagulation by its association with its cofactor, factor Va (FVa). To investigate the contribution of the Gla and EGF1 domains of FX in these complexes, FX chimeras were produced in which FIX Gla and EGF1 domains substituted the corresponding domains of FX. The affinity of the two chimeras, FX/FIX(Gla) and FX/FIX(EGF1), for the TF-FVIIa complex was markedly reduced compared with that of wild-type-FX (wt-FX) independently of the presence of phospholipids. Furthermore, the association rate constants of preformed FX/FIX(Gla)-TFPI and FX/FIX(EGF1)-TFPI complexes with TF-FVIIa were, respectively, 10-and 5-fold slower than that of wt-FXa-TFPI complex. Finally, the apparent affinity of FVa was 2-fold higher for the chimeras than for wt-FX in the presence of phospholipids and equal in their absence. These data demonstrate that FX Gla and EGF1 domains contain residues, which interact with TF-FVIIa exosites contributing to the formation of the TF-FVIIa-FX and TF-FVIIa-TFPI-FXa complexes. On the opposite, FXa Gla and EGF1 domains are not directly involved in FVa binding.The blood coagulation cascade consists of a series of enzymatic conversions driven by the formation of complexes between serine proteases and cell membrane-bound cofactors. Human factor X (FX) 1 is one of the serine protease zymogens playing a central role in coagulation processes leading to the formation of a fibrin clot. This is illustrated by the behavior of FX as a substrate or as an enzyme in three essential blood coagulation complexes. First, FX is a natural substrate, as well as factor IX (FIX), of the tissue factor-factor VIIa (TF-FVIIa) complex (1) considered as the initial enzyme complex in the cascade following vascular damage. FX activation by TF-FVIIa results from specific cleavage and release of a 52-residue activation peptide. Activated FX (FXa) can generate a tiny amount of thrombin from prothrombin in an extremely inefficient reaction (2). Tissue factor pathway inhibitor (TFPI) binds to TF-FVIIa-FXa to limit the production of FXa and FIXa by TF-FVIIa (3, 4). Nevertheless, once produced, thrombin and the initially formed FXa activate small quantities of factor V (FV) to FVa and factor VIII (FVIII) to FVIIIa (5-8). The activation of these two cofactors leads to the formation of two other essential procoagulant complexes, both involving FX, at the surface of procoagulant phospholipids in the presence of calcium ions (9),...

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Human Protein Z.

Cited by 140 publications

References 34 publications

Kinetic Characterization of the Protein Z-dependent Protease Inhibitor Reaction with Blood Coagulation Factor Xa

Kinetic Characterization of the Protein Z-dependent Protease Inhibitor Reaction with Blood Coagulation Factor Xa

Protein Z is not synthesised by human umbilical vein endothelial cells

Role of the Gla and First Epidermal Growth Factor-like Domains of Factor X in the Prothrombinase and Tissue Factor-Factor VIIa Complexes

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