Suppressing protein Z-dependent inhibition of factor Xa improves coagulation in hemophilia A.

Essentials Protein Z (PZ) catalyzes PZ-dependent proteinase inhibitor (ZPI) inactivation of factor (F)Xa. Gene-deletion of PZ or ZPI improves coagulation in hemophilia (FVIII knockout) mice. A PZ blocking antibody enhances thrombin generation in human hemophilia plasma. Suppression of the PZ/ZPI pathway may ameliorate the phenotype of severe hemophilia. SUMMARY: Background Hemostasis requires a balance between procoagulant and anticoagulant factors. Hemophiliacs bleed because of a procoagulant deficiency. Targeted reduction in the activity of endogenous anticoagulant pathways is currently being investigated as a means of improving hemostasis in hemophilia. Protein Z (PZ) is a cofactor that serves as a catalyst for PZ-dependent protease inhibitor (ZPI) inactivation of activated factor X at phospholipid surfaces. Objectives To evaluate the effects of PZ or ZPI gene deletion in hemophilic mice, and of blocking PZ in human hemophilic plasma. Methods A tail vein rebleeding assay (TVRB) was developed on the basis of the serial disruption of clots forming over a period of 15 min following tail vein laceration in an anesthetized mouse. Wild-type (WT)/FVIII knockout FVIIIKO, PZ knockout PZKO/FVIIIKO and ZPI knockout ZPIKO/FVIIIKO mice were evaluated in this model, and their plasmas were tested in thrombin generation assays. A mAb against PZ was evaluated in human hemophilic plasma thrombin generation assays. Results The numbers of clot disruptions (mean ± standard error of the mean) in the TVRB were: 4.0 ± 0.9 for WT/FVIIIKO mice; 23.8 ± 1.1 for WT/FVIIIKO mice supplemented with 100% FVIII; 15.2 ± 1.1 for PZKO/FVIIIKO mice; and 14.7 ± 1.2 for ZPIKO/FVIIIKO mice. Thrombin generation in PZKO/FVIIIKO and ZPIKO/FVIIIKO mouse plasmas was similar to that in FVIIIKO plasma supplemented with ~ 15% recombinant FVIII. A mAb against PZ added to human hemophilic plasma enhanced thrombin generation to an extent similar to the addition of ~ 15% FVIII. Conclusions Blockade of the PZ/ZPI system may be sufficient to ameliorate the phenotype of severe hemophilia.

Re-evaluation of mouse tissue factor pathway inhibitor and comparison of mouse and human tissue factor pathway inhibitor physiology.

Essentials Mouse models are often used to define roles of tissue factor pathway inhibitor (TFPI) in man. TFPI isoform-specific KOs reveal unexpected differences between mouse and human TFPI physiology. Mouse plasma contains 20 times more TFPI than man, derived from TFPIγ, a form not found in man. TFPIγ null mice, expressing only TFPI isoforms α and ß, may better reflect the human situation. SUMMARY: Background Mouse models can provide insight into the pathophysiology of human thrombosis and hemostasis. Tissue factor pathway inhibitor (TFPI) regulates coagulation through protein S (PS)-enhanced factor (F) Xa inhibition and FXa-dependent inhibition of FVIIa/tissue factor (TF) activity. TFPI is expressed as isoforms α and ß in man, and α, ß and γ in the mouse. Objective Assess the reliability of extending TFPI-related studies in mice to humans. Method Compare mouse and human TFPI physiology using a variety of methods. Results Mouse TFPI and human TFPI are similar in regard to: (i) the mechanisms for FVIIa/TF and FXa inhibition; (ii) TFPIα is a soluble form and TFPIß is glycosyl phosphatidyl inositol (GPI) membrane anchored; (iii) the predominant circulating form of TFPI in plasma is lipoprotein-associated; (iv) low levels of TFPIα circulate in plasma and increase following heparin treatment; and (v) TFPIα is the isoform in platelets. They differ in that: (i) mouse TFPI circulates at a ~20-fold higher concentration; (ii) mouse lines with isolated isoform deletions show this circulating mouse TFPI is derived from TFPIγ; (iii) sequences homologous to the mouse TFPIγ exon are present in many species, including man, but in primates are unfavorable for splicing; and (iv) tandem mass spectrometry (MS/MS) detects sequences for TFPI isoforms α and ß in human plasma and α and γ in mouse plasma. Conclusion To dissect the pathophysiological roles of human TFPIα and TFPIß, studies in TFPIγ null mice, expressing only α and ß, only α or only ß should better reflect the human situation.

Protein Z-dependent protease inhibitor and protein Z in peripheral arterial disease patients.

SUMMARY INTRODUCTION: Protein Z is a vitamin K-dependent protein that serves as a cofactor for the inhibition of activated factor X by the serpin protein Z-dependent protease inhibitor (ZPI). Protein Z plasma levels have been shown to be reduced in patients with peripheral arterial disease (PAD), but ZPI levels have not yet been reported. The aim of this study was to more fully assess the protein Z-ZPI system in individuals with atherosclerosis selected by the presence of symptomatic PAD. MATERIALS AND METHODS: Protein Z and ZPI levels were determined in 95 PAD patients (73 males; 22 females) [median age: 73 years (range, 50-86 years)] and in 190 controls comparable for age and gender. Protein Z was measured using a commercial immunoassay, and ZPI was measured with a homemade immunoassay and a functional assay. RESULTS: Protein Z antigen, ZPI antigen and ZPI function were found to be significantly lower in PAD patients with respect to controls [protein Z, median 72.5% (range: 3.4-123.7%) vs. 90.7% (range: 32.1-203.2%), P < 0.0001; ZPI antigen, 86.1% (range: 25.1-149.5%) vs. 93.2% (range: 48.9-171.3%), P = 0.004; ZPI function, 83.5% (range: 21.1-135.2%) vs. 97.2% (range: 50.5-175.5%), P < 0.0001]. The lowest tertiles of protein Z antigen [odds ratio (OR) 5.4, 95% confidence interval (CI) 2.2-13.5, P < 0.0001] and ZPI function (OR 2.4, 95% CI 1.1-5.5, P = 0.03) were associated with PAD on multivariate analysis after adjustment for age, gender, and traditional cardiovascular risk factors. A significant inverse relationship was also observed between protein Z and ZPI levels and the number of traditional cardiovascular risk factors and the clinical severity of disease (Fontaine stage). CONCLUSIONS: Low levels of protein Z antigen and protein Z activity are significantly associated with the occurrence and severity of atherosclerotic PAD.

Foaming dynamics in Hele-Shaw cells.

We have studied foaming dynamics in Hele-Shaw cells partially filled with a soap and water mixture. A series of upside-down flips produces an intermittent wetting of the cell and leads to foam formation. As a function of the number of flips, an increasing number of bubbles composes the foam, until saturation is observed. Statistical analysis shows that the bubble size follows a Gamma distribution. Contrary to common belief, this foaming dynamics by "shaking" creates homogeneous foam, even though the system may pass through transient heterogeneous configurations. A mechanistic interpretation is proposed and included into a theoretical model.

Comparison of cell-surface TFPIalpha and beta.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is mainly produced by endothelial cells and alternative mRNA splicing generates two forms, TFPIalpha and TFPIbeta. A portion of expressed TFPI remains associated with the cell surface through both direct (TFPIbeta) and indirect (TFPIalpha) glycosylphosphatidyl-inositol (GPT)-mediated anchorage. OBJECTIVE: Compare the structure and properties of TFPIalpha and TFPIbeta. METHODS: TFPIalpha and TFPIbeta, with protein molecular masses of 36 and 28 kDa, respectively, migrate similarly (46 kDa) on SDS-PAGE. Experiments using specific glycosidases were carried out to determine the different glycosylation pattern of the two forms. ECV304 cells, a cell line with some endothelial properties, were stimulated with IL-lbeta, LPS, and TNFalpha for up to 24 hrs and mRNA levels and protein synthesis were determined. Stable clones of ECV304 cells that express reduced levels of TFPIalpha, TFPIbeta or both were produced using a plasmid-based small-interfering RNA technique. Surface TFPI activity was determined by a two-stage chromogenic assay based on the ability of each form to inhibit FXa activation by FVIIa on cells with comparable amount of tissue factor (TF). RESULTS AND CONCLUSIONS: The deglycosylation studies show that the difference in molecular masses is due to a greater degree of sialylation in O-linked carbohydrate in TFPIbeta. The mRNA and protein levels of neither form of TFPI were affected by stimulation of cells with inflammatory stimuli. Although TFPIalpha comprises 80% of the surface-TFPI, TFPIbeta was responsible for the bulk of the cellular FVIIa/TF inhibitory activity, suggesting a potential alternative role for cell surface TFPIalpha.

Interaction of sodium dodecyl sulfate with methacrylate-PEG comb copolymers.

A series of sodium methacrylate and poly(ethylene glycol) (PEG) comb copolymers (MAA/PEG) with approximate PEG chain lengths of 7, 11, and 22 ethylene oxide units were synthesized by free radical polymerization. Their weight-average molecular mass was found to be approximately 66 000. A commercial sample of a PEG comb polymer with an acrylic backbone was also used in the studies (Sokalan HP 80). The interaction of the MAA/PEG comb polymers and pure sodium methacrylate (SPMA) with sodium dodecyl sulfate (SDS) was studied by ESR spectroscopy using 5-doxyl stearic acid (5-DSA) spin probe and by conductivity measurements. Surfactant aggregation in water occurred at SDS concentrations lower than the surfactant critical micelle concentration (cmc) and depended on the polymer concentration. The observations have been attributed to changes in the effective ionic strength of the systems due to the polymer itself, and it has been concluded that there is no interaction between the MAA/PEG comb copolymers or SPMA and SDS. This has been confirmed by the fact that the decrease in surfactant aggregation concentration is similar in magnitude to the decrease observed on adding NaCl when counterion ion condensation effects are taken into account. It is apparent that the electrostatic repulsions between the surfactant molecules and the methacrylate backbone of the MAA/PEG comb copolymers inhibit association of SDS with the PEG side chains.

The first epidermal growth factor-like domains of factor Xa and factor IXa are important for the activation of the factor VII--tissue factor complex.

During tissue factor (TF)-induced coagulation, the factor (F)VIIa-TF complex activates factor (F)X and factor (F)IX. Through positive feedback, the generated FXa and FIXa activate FVII-TF. The first epidermal growth factor-like (EGF1) domains of FX and FIX serve as important TF-recognition motifs when FVIIa-TF activates FX or FIX. Here, we investigated the role of EGF1 domains of FXa and FIXa during the activation of FVII-TF and inhibition by tissue factor pathway inhibitor (TFPI). FXaPCEGF1 (EGF1 domain of FXa replaced with that of protein C), and FXaQ49P (EGF1 domain mutant with impaired calcium-binding), and the corresponding FIXa mutants were generated, and their abilities to activate FVII-TF were compared with the wild-type (WT) enzymes. In the absence of TF, the rates of FVII activation were similar between WT enzymes and mutant FXa and FIXa proteases. In the presence of either soluble TF (sTF) or relipidated TF, each mutant of FXa or FIXa activated FVII-TF at a slower rate than the corresponding WT enzyme. Kinetics of inhibition of the amidolytic activity of WT and the mutant FXa proteases by either two-domain or full-length TFPI were similar. However, compared with the complex of TFPI-FXaWT, the abilities of the complexes of TFPI-FXa mutants to inhibit FVIIa-TF were impaired. We conclude that the EGF1 domains of FXa and FIXa are important for the activation of FVII-TF and for the formation of FVIIa-TF-FXa-TFPI complex.

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

BACKGROUND: 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). OBJECTIVES: 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. PATIENTS AND METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

The rediscovery and isolation of TFPI.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type proteinase inhibitor that produces factor (F)Xa-dependent feedback inhibition of the factor VIIa/tissue factor (FVIIa/TF) catalytic complex that is responsible for the initiation of coagulation. Since 1985, when Rapaport and colleagues reported that the lipoprotein fraction of plasma contained a FXa-dependent inhibitor of FVIIa/TF, myriad articles have established its biochemical structure, its mechanism of action, and its physiological importance. This brief personal account reviews historical studies that established the existence of the inhibitor and the events that led to its initial isolation.

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.

High titers of autoantibodies to tissue factor pathway inhibitor are associated with the antiphospholipid syndrome.

As the activity of the tissue factor pathway inhibitor (TFPI) may be impaired in patients with antiphospholipid antibodies (aPL), 162 aPL patients were evaluated for autoantibodies to recombinant TFPI (anti-TFPI) using an optimized ELISA. Anti-TFPI (>18 U mL(-1) for IgG and/or > 15 U mL-1 for IgM) were detected in 54 patients with aPL (33.3%) and in three out of 79 normal controls (3.8%, P < 0.0001). Among aPL patients, the prevalence of positive anti-TFPI was 38.3 and 28.4% in those with or without diagnosis of definite antiphospholipid syndrome (APS). Patients with definite APS had a significantly greater frequency of high titer (>50 U mL(-1)) anti-TFPI than aPL patients from the no definite APS group (18.5% vs. 6.2%, OR 3.7, P= 0.017). Most aPL recognized full-length TFPI, but not a truncated form of TFPI lacking the C-terminus of the molecule. Isolated IgGs from subjects with anti-TFPI impaired the dose-dependent inhibitory effect of TFPI on factor Xa activity in the presence, but not in the absence of phospholipid vesicles. Thus, aPL with high titer anti-TFPI limit TFPI action and are associated with the APS.

Spontaneous detachment of oil drops from solid substrates: governing factors.

We carried out experiments on detachment of oil drops from glass substrates in solutions of an anionic surfactant. The three-phase contact line shrinks spontaneously, and eventually the oil drop detaches from the substrate. Consecutive video frames of such drops are digitized, and the time dependencies of the contact radius and angle are determined. Three stages of detachment of a drop, situated above a horizontal substrate, can be distinguished. They correspond to three different driving factors: (1) the interfacial tension decrease because of surfactant adsorption, (2) the aqueous meniscus spontaneously advances owing to the penetration of water between the oil and solid phases, and (3) at sufficiently small contact radius the shape of the oil-water interface becomes unstable and the drop detaches under the action of buoyancy. Analyzing the experimental data, we identified two important characteristics of the drop-detachment process: the velocity of spontaneous advance of the contact line and the line drag coefficient. In the case of moving contact line, a dynamic Young equation must be used, which takes into account the line drag force. The latter is proportional to the velocity of contact-line motion. The experimental data agree with the latter dependence, from whose slope the line drag coefficient is determined.