Z-dependent Protease Inhibitor Research Articles

La protéine Z : une protéine régulatrice de la coagulation au niveau artériel ?

Protein Z (PZ) is a vitamin K dependent factor identified in human plasma in 1984 whose physiological function was poorly understood. It was recently shown that protein Z is implicated in the down-regulation of coagulation by forming a complex with a plasma proteinase inhibitor called protein Z-dependent protease inhibitor (ZPI) which inhibits activated factor Xa on phospholipid surfaces. In the absence of an additional challenge, the disruption of PZ gene in mice is asymptomatic, but the association with the factor VLeiden mutation leads to a near complete mortality during the neonatal period with microvascular thrombosis. Unexpectedly, in humans, a relationship between protein Z deficiency and ischemic strokes, was firstly evidenced, but not confirmed by all the epidemiological study. Additional studies suggest that protein Z deficiency could be also a risk factor for acute coronary syndromes, early fetal losses, and increased the arterial risk in antiphospholipid syndrome. This review analyzes the different studies so far published and discusses the different results obtained in order to understand whether or not protein Z deficiency could be considered as an arterial ischemic risk factor.

Heritability of plasma concentrations of clotting factors and measures of a prethrombotic state in a protein C‐deficient family

Earlier studies found strong support for a genetic basis for regulation of coagulation factor levels and measures of a prethrombotic state (d-dimer, prothrombin fragment 1.2). Estimation of how much of the variation in the levels of coagulation factors and measures of a prethrombotic state, including measures of protein C activation and inactivation, could be attributed to heritability and household effect. Blood samples were collected from 330 members of a large kindred of French-Canadian origin with type I protein C deficiency. Heritability and common household effect were estimated for plasma concentrations of prothrombin, factor (F)V, factor VIII, factor (F)IX, fibrinogen, von Willebrand factor (VWF), antithrombin, protein C, protein S, protein Z, protein Z-dependent protease inhibitor (ZPI), fibrinopeptide A (FPA), protein C activation peptide (PCP), activated protein C-protein C inhibitor complex (APC-PCI), activated protein C-alpha1-antitrypsin complex (APC-alpha1AT), prothrombin fragment 1.2 (F1.2) and d-dimer, using the variance component method in sequential oligo-genic linkage analysis routines (SOLAR). The highest heritability was found for measures of thrombin activity (PCP and FPA). High estimates were also found for prothrombin, FV, FIX, protein C, protein Z, ZPI, APC-PCI and APC-alpha1AT. An important influence of shared household effect on phenotypic variation was found for VWF, antithrombin, protein S and F1.2. We found strong evidence for the heritability of single coagulation factors and measures of a prethrombotic state. Hemostatic markers with statistically significant heritability constitute potential targets for the identification of novel genes involved in the control of quantitative trait loci.

Autoimmune antiphospholipid antibodies impair the inhibition of activated factor X by protein Z/protein Z-dependent protease inhibitor

The hemostatic process is tightly regulated by several antithrombotic mechanisms. Among them, protein Z (PZ)-dependent protease inhibitor (ZPI) potently inhibits factor (F)Xa in a manner dependent on calcium ions, phospholipids and PZ. Autoimmune antiphospholipid antibodies (aPL) are mainly directed against phospholipid-binding plasma proteins such as beta2-glycoprotein I (beta2GPI) and prothrombin, and are known to interfere with phospholipid-dependent hemostatic pathways. In this study, we investigated whether purified aPL are able to interfere with inhibition of FXa by PZ/ZPI. beta2GPI modestly delayed the FXa inactivation by PZ/ZPI and most isolated aPL-IgGs were found to further increase the inhibitory potential of beta2GPI on PZ/ZPI activity. Without beta2GPI, the PZ/ZPI activity was unaffected by the addition of aPL-IgG. As PZ deficiency is hypothesized to lead to a prothrombotic state, we performed a case-control study to measure plasma levels of PZ and ZPI in 66 patients with autoimmune aPL and 152 normal controls. The prevalence of low PZ levels (below the 5th percentile of controls) was significantly greater in the 37 patients with definite antiphospholipid syndrome (APS) (24.3%) but not in the 29 aPL patients not fulfilling the criteria for APS (10.3%) compared with the normal group (4.6%, P < 0.001 vs. APS). ZPI antigen levels were similar in patients with aPL and normal controls. Concomitant PZ deficiency increased by approximately sevenfold the risk of arterial thrombosis in aPL patients. Taken together, these data suggest that the PZ/ZPI system is commonly impaired in aPL patients thus probably increasing the thrombotic risk.

Low Protein Z Levels and Risk of Ischemic Stroke: Differences by Diabetic Status and Gender

ABSTRACT Protein Z was recently shown to act as an essential cofactor for protein Z-dependent protease inhibitor, a potent downregulator of coagulation Factor Xa. Thus, deficiency of protein Z is hypothesized to lead to a prothrombotic state, but two publications reported opposing results for the relationship of protein Z levels with ischemic stroke in young European subjects (mean age 33–40 years). We performed a study of stroke in a different ethnic population of greater mean age (57 years) to further clarify this issue. An ELISA was developed to measure protein Z antigen in 154 patients with ischemic stroke and in 206 controls in a largely Hispanic population. Low plasma protein Z values were significantly associated with ischemic stroke except in diabetic subjects and females. The mean protein Z value was significantly lower in stroke cases than in controls for nondiabetic subjects [1.78 ± 0.77 (S.D.) versus 2.28 ± 0.88 μg/ml, P < 0.0001] and for males (1.90 ± 0.90 versus 2.42 ± 0.99 μg/ml, P = 0.0004). Stroke risk was higher in subjects with protein Z levels at or below the fifteenth percentile of controls (≤1.46 μg/ml). The odds ratios were: 2.6 for all subjects (95% C.I. = 1.5–4.3); 3.8 for nondiabetic subjects (95% C.I. = 2.2–6.8); and 3.6 for males (95% C.I. = 2.0–6.4). This study also revealed high protein Z levels associated with high triglyceride levels in controls ( P = 0.015). Protein Z is suggested to be a physiologic downregulator of blood coagulation and low protein Z levels are associated with increased risk of ischemic stroke, particularly in males and in the absence of diabetes.

Protein Z influences the prothrombotic phenotype in Factor V Leiden patients

Protein Z enhances the inhibition of factor Xa by protein Z-dependent protease inhibitor (ZPI). Thus, diminution of protein Z should induce prothrombotic tendency due to lowered cofactor activity for ZPI. In Factor V Leiden mice, prothrombotic tendency of severe diminution or lack of protein Z was demonstrated. We here present first studies in humans, indicating that diminution of protein Z in factor V Leiden patients aggravates thromboembolic risk.

Mouse Protein Z-Dependent Protease Inhibitor cDNA

Protein Z-dependent protease inhibitor (ZPI) is plasma proteinase inhibitor in the serpin superfamily that produces rapid inhibition of factor Xa in the presence of phospholipids, Ca++ and protein Z (PZ). Mouse ZPI cDNA was isolated and cloned from mouse liver RNA using RT-PCR. The cDNA contains 100 nucleotides 5' of a translation initiation codon and an open reading frame of 1344 nucleotides followed by a 163 nucleotide 3' untranslated sequence with a poly (A) tail. The cDNA predicts a signal peptide containing 21 amino acids and a mature protein of 427 residues with 8 potential sites for N-linked glycosylation. The oligonucleotide and predicted amino acid sequences of mouse ZPI are 72% and 81% homologous with those of human ZPI. Like human ZPI, mouse ZPI contains tyrosine-serine (P1-P1') at its reactive center in contrast to the rat molecule which contains tyrosine-cysteine. By Northern analysis, mouse ZPI mRNA is 1.6 kb in size and, similar to both human and rat, it is detectable in liver, but not in heart, brain, spleen, lung, kidney, skeletal muscle or testes.

Protein Z-Dependent Regulation of Coagulation

Protein Z (PZ) is a 62 kDa vitamin K-dependent plasma protein that serves as a cofactor for the inhibition of factor Xa by protein Z-dependent protease inhibitor (ZPI). ZPI is a recently identified 72 kDa member of the serpin superfamily of proteinase inhibitors that contains a tyrosine at its reactive center. PZ circulates in plasma in a complex with ZPI. Inhibition of factor Xa by ZPI in the presence of phospholipids and Ca++ is enhanced 1000-fold by PZ, but ZPI also inhibits factor XIa in a process that does not require PZ, phospholipids or Ca++. ZPI activity is consumed during coagulation through proteolysis mediated by factor Xa with PZ and factor Xla. Concomitant PZ deficiency dramatically increases the severity of the prothrombotic phenotype of factor VLeiden mice. Studies to determine the potential roles of PZ and ZPI deficiency in human thrombosis are in progress.

Protein Z Circulates in Plasma in a Complex with Protein Z-Dependent Protease Inhibitor

Protein Z (PZ) is a vitamin K-dependent plasma protein that forms a Ca++-dependent complex with factor Xa at phospholipid surfaces. This interaction between PZ and factor Xa enhances by >1,000-fold the inhibition of factor Xa by the serpin called protein Z-dependent protease inhibitor (ZPI). These experiments show that PZ also binds ZPI in a process that does not require Ca++ or phospholipids. In pooled normal plasma, which contains excess ZPI relative to PZ, all the PZ appears to be bound in a complex with ZPI. The binding of PZ to ZPI reduces the rate and extent of factor XIa inhibition produced by ZPI. During the course of these studies, it was noted that a PZ purification procedure, that included NaSCN (2.0 M) elution of PZ from an immunoaffinity column, produced aggregated, inactive forms of PZ.

Characterization of the protein Z–dependent protease inhibitor

Protein Z-dependent protease inhibitor (ZPI) is a 72-kd member of the serpin superfamily of proteinase inhibitors that produces rapid inhibition of factor Xa in the presence of protein Z (PZ), procoagulant phospholipids, and Ca++ (t1/2 less than 10 seconds). The rate of factor Xa inhibition by ZPI is reduced more than 1000-fold in the absence of PZ. The factor Xa–ZPI complex is not stable to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, but is detectable by alkaline–polyacrylamide gel electrophoresis. The combination of PZ and ZPI dramatically delays the initiation and reduces the ultimate rate of thrombin generation in mixtures containing prothrombin, factor V, phospholipids, and Ca++. In similar mixtures containing factor Va, however, PZ and ZPI do not inhibit thrombin generation. Thus, the major effect of PZ and ZPI is to dampen the coagulation response prior to the formation of the prothrombinase complex. Besides factor Xa, ZPI also inhibits factor XIa in the absence of PZ, phospholipids, and Ca++. Heparin (0.2 U/mL) enhances the rate (t1/2 = 25 seconds vs 50 seconds) and the extent (99% vs 93% at 30 minutes) of factor XIa inhibition by ZPI. During its inhibitory interaction with factor Xa and factor XIa, ZPI is proteolytically cleaved with the release of a 4.2-kd peptide. The N-terminal amino acid sequence of this peptide (SMPPVIKVDRPF) establishes Y387 as the P1 residue at the reactive center of ZPI. ZPI activity is consumed during the in vitro coagulation of plasma through a proteolytic process that involves the actions of factor Xa with PZ and factor XIa.

Characterization of the protein Z–dependent protease inhibitor

AbstractProtein Z-dependent protease inhibitor (ZPI) is a 72-kd member of the serpin superfamily of proteinase inhibitors that produces rapid inhibition of factor Xa in the presence of protein Z (PZ), procoagulant phospholipids, and Ca++ (t1/2 less than 10 seconds). The rate of factor Xa inhibition by ZPI is reduced more than 1000-fold in the absence of PZ. The factor Xa–ZPI complex is not stable to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, but is detectable by alkaline–polyacrylamide gel electrophoresis. The combination of PZ and ZPI dramatically delays the initiation and reduces the ultimate rate of thrombin generation in mixtures containing prothrombin, factor V, phospholipids, and Ca++. In similar mixtures containing factor Va, however, PZ and ZPI do not inhibit thrombin generation. Thus, the major effect of PZ and ZPI is to dampen the coagulation response prior to the formation of the prothrombinase complex. Besides factor Xa, ZPI also inhibits factor XIa in the absence of PZ, phospholipids, and Ca++. Heparin (0.2 U/mL) enhances the rate (t1/2 = 25 seconds vs 50 seconds) and the extent (99% vs 93% at 30 minutes) of factor XIa inhibition by ZPI. During its inhibitory interaction with factor Xa and factor XIa, ZPI is proteolytically cleaved with the release of a 4.2-kd peptide. The N-terminal amino acid sequence of this peptide (SMPPVIKVDRPF) establishes Y387 as the P1 residue at the reactive center of ZPI. ZPI activity is consumed during the in vitro coagulation of plasma through a proteolytic process that involves the actions of factor Xa with PZ and factor XIa.