Factor VII-activating Protease Research Articles

Ozone exposure and prothrombosis: Mechanistic insights from a randomized controlled exposure trial

Epidemiological studies have associated ozone exposure with cardiovascular diseases, but the molecular mechanisms were not elucidated. We performed an untargeted serum proteomic analysis in a randomized, crossover, controlled exposure trial. We recruited 32 healthy young adults and asked them to receive filtered air and 200-ppb ozone exposures for 2 h in a random order before serum collection. Linear mixed-effect models were used to identify differentially expressed proteins (DEPs) between the two exposures and Gene Ontology enrichment and ingenuity pathway analysis were performed to determine their biological function. A total of 56 DEPs were identified. For example, acute ozone exposure increased coagulation factor X and factor VII-activating protease by 20.96% and 28.35%, respectively. Whereas, protein Z, protein Z-dependent protease inhibitor, and plasminogen decreased by 13.62%, 33.54%, and 10.47%, respectively. We also observed a 42.32% decrease in paraoxonase 3 and evident changes in four apolipoproteins. Additionally, we found 18.21% and 95.82% increases in L-selectin and β2-microglobulin, respectively, and significant changes in three complements. DEPs and enriched pathways suggest that short-term ozone exposure may promote coagulation, suppress fibrinolysis, disrupt lipoprotein metabolism, activate immune responses, and affect the complement system. These findings provide additional insights into the mechanisms linking acute ozone exposure to thrombosis.

Diagnostic and prognostic value of HABP2 as a novel biomarker for endometrial cancer.

BackgroundEndometrial cancer is the fifth most common malignant disorder in women, with its incidence increasing. A biomarker with diagnostic and prognostic value remains to be found. The HABP2 protein, or Factor VII-activating protease, encodes a hyaluronic acid-binding protein.MethodsPatient data including clinical characteristics and RNAseq information of HABP2 was obtained from The Cancer Genome Atlas (TCGA), and analyzed by R statistic packages. A total of 370 women with endometrial cancer were enrolled in the study. To study the diagnostic value of HABP2 in patients with endometrial cancer, receiver operating characteristic (ROC) curves were plotted by the pROC package. To study the prognostic value of HABP2 in patients with endometrial cancer, the survival package in R was used and the Cox model was established.ResultsHABP2 expression was lower in endometrial cancer compared with normal endometrial tissues. HABP2 showed moderate diagnostic value for endometrial cancer, with HBP2 expression associated with vital status, histologic grade, and residual tumor. HABP2 was an independent prognostic factor, with low HABP2 expression indicating a better overall survival.ConclusionsHABP2 has diagnostic and prognostic value and maybe a novel biomarker for endometrial cancer.

Association between FSAP 1601G>A polymorphism and venous thromboembolism risk: A meta-analysis.

Previous several studies have shown that factor VII-activating protease (FSAP) gene 1601G>A polymorphism is related to the occurrence of venous thromboembolism, but the results are inconsistent and controversial. Therefore, we conducted a meta-analysis to explore the association between FSAP 1601G>A polymorphism and venous thromboembolism susceptibility. We managed a systematic literature search through Pubmed, Embase, Web of Science, China National Knowledge Infrastructure, and WanFang databases to collect research data related to FSAP gene 1601G>A polymorphism and susceptibility to venous thromboembolism published before May 2019. Data analysis was performed through Revman 5.3 and Stata 12.0 software, the pooled odd ratios and 95% confidence intervals were calculated. Additionally, the sensitivity analysis and publication bias assessment were also performed. A total of seven case-control studies were included and evaluated, including 2411 venous thromboembolism cases and 2850 controls. The meta-analysis results revealed that the FSAP 1601G>A mutation is associated with venous thromboembolism risk, and statistically significance was observed under three genetic comparison models (A: G, odds ratio: 1.33, 95% confidence interval: 1.07-1.66; GA: GG, odds ratio: 1.34, 95% confidence interval: 1.06-1.68; and GA + AA: GG, odds ratio: 1.33, 95% confidence interval: 1.06-1.66). This study demonstrated that the FSAP 1601G>A polymorphism may be associated with venous thromboembolism susceptibility.

Expression of the Marburg I Single Nucleotide Polymorphism (MI-SNP) and the Marburg II Single Nucleotide Polymorphism (MII-SNP) of the Factor VII-Activating Protease (FSAP) Gene and Risk of Coronary Artery Disease (CAD): A Pilot Study in a Single Population.

BackgroundFactor VII-activating protease (FSAP) has a role in vascular inflammation and may have a role coronary artery disease (CAD). The aim of this study was to investigate the association between two naturally occurring single nucleotide polymorphisms (SNPs) in the FSAP gene and the risk of coronary artery disease (CAD).Material/MethodsOf 733 patients, 173 patients had symptoms of angina, and 560 patients had CAD confirmed by coronary angiography. All patients were genotyped for SNPs of the FSAP gene, Marburg I (MI-SNP) and Marburg II (MII-SNP), using 5′ exonuclease TaqMan assays. Logistic regression analysis was used to evaluate the association between two gene polymorphisms, metabolic and other cardiovascular risk factors in patients with CAD.ResultsThe presence of MI-SNP and MII-SNP FSAP gene polymorphisms were not associated with the presence of CAD. However, the MII-SNP polymorphism was significantly associated with a reduced risk of developing CAD (OR=0.422; 95% CI, 0.194–0.920; P=0.035); the MI-SNP polymorphism was associated with absence of hyperlipoproteinemia (OR=0.601; 95% CI, 0.344–1.051; P=0.074). There was no significant association between expression of the MI-SNP and MII-SNP FSAP gene polymorphisms and the incidence of myocardial infarction, or of a history of diabetes mellitus, arterial hypertension, obesity, or smoking.ConclusionsThe MI-SNP and MII-SNP FSAP gene polymorphisms were not predictive or prognostic biomarkers for CAD or its main risk factors. However, the presence of the MII-SNP polymorphism was associated with a reduced risk of developing CAD.

Factor VII-activating protease: sex-related association with coronary artery calcification.

: Factor VII-activating protease (FSAP) may regulate development of cardiovascular disease (CVD). We evaluated sex differences in FSAP measures and examined the association between FSAP and coronary artery calcification (CAC) in a middle-aged population. Participants were randomly selected citizens aged 50 or 60 without CVD, diabetes mellitus, Marburg I polymorphism, or hormone replacement therapy (HRT). FSAP protein concentration (total FSAP), FSAP urokinase-activating capacity (FSAP GP), and FSAP GP/total FSAP (specific FSAP activity) were measured. Cardiac computed tomography (CT) determined the Agatston score, dividing the study population in three groups: (1) Agatston score = 0 U, (2) Agatston score = 1-99 U, or (3) Agatston score more than 99 U. A total of 134 women and 116 men were included. Total FSAP, FSAP GP, and specific FSAP activity were independently higher in women (97.4%, 81.1%, 0.84, respectively) compared with men (87.5%, 68.7%, 0.79, respectively) (P < 0.001). In women, total FSAP was significantly different between (3) Agatston score (111.5%) and (1) Agatston score (95.4%), respectively, (2) Agatston score (96.8%), (P < 0.05). Also, the specific activity of FSAP was significantly different between (3) Agatston score (0.77) and (1) Agatston score (0.85), respectively, (2) Agatston score (0.86) (P < 0.05). No difference in FSAP measures was observed in men. FSAP measures are higher in women compared with age-matched men. The extent of CAC in women is positively associated with total FSAP, but negatively associated with the specific activity of FSAP suggesting that FSAP may play a role in the evolution of CVD in women.

Factor VII‐activating protease deficiency promotes neointima formation by enhancing leukocyte accumulation

Background Factor VII-activating protease (FSAP) is a multifunctional circulating plasma serine protease involved in thrombosis and vascular remodeling processes. The Marburg I single-nucleotide polymorphism (MI-SNP) in the FSAP-coding gene is characterized by low proteolytic activity, and is associated with increased rates of stroke and carotid stenosis in humans. Objectives To determine whether neointima formation after vascular injury is increased in FSAP-/- mice. Methods and Results The neointimal lesion size and the proliferation of vascular smooth muscle cells (VSMCs) were significantly enhanced in FSAP-/- mice as compared with C57BL/6 control mice after wire-induced injury of the femoral artery. Accumulation of leukocytes and macrophages was increased within the lesions of FSAP-/- mice at day 3 and day 14. Quantitative zymography demonstrated enhanced activity of gelatinases/matrix metalloproteinase (MMP)-2 and MMP-9 within the neointimal lesions of FSAP-/- mice, and immunohistochemistry showed particular costaining of MMP-9 with accumulating leukocytes. Using intravital microscopy, we observed that FSAP deficiency promoted the intravascular adherence and the subsequent transmigration of leukocytes in vivo in response to chemokine ligand 2 (CCL2). CCL2 expression was increased in FSAP-/- monocytes but not in the vessel wall. There was no difference in the expression of platelet-derived growth factor (PDGF-BB). Conclusions FSAP deficiency causes an increase in CCL2 expression and CCL2-mediated infiltration of leukocytes into the injured vessel, thereby promoting SMC proliferation and migration by the activation of leukocyte-derived gelatinases. These results provide a possible explanation for the observed association of the loss-of-function MI-SNP with vascular proliferative diseases.

Transforming Growth Factor-β (TGF-β) Inhibits the Expression of Factor VII-activating Protease (FSAP) in Hepatocytes

Deletion of the Habp2 gene encoding Factor VII-activating protease (FSAP) increases liver fibrosis in mice. A single nucleotide polymorphism (G534E) in HABP2 leads to lower enzymatic activity and is associated with enhanced liver fibrosis in humans. Liver fibrosis is associated with a decrease in FSAP expression but, to date, nothing is known about how this might be regulated. Primary mouse hepatocytes or the hepatocyte cell line, AML12, were treated with different factors, and expression of FSAP was determined. Of the various regulatory factors tested, only transforming growth factor-β (TGF-β) demonstrated a concentration- and time-dependent inhibition of FSAP expression at the mRNA and protein level. The TGF-β-Type I receptor (ALK-5) antagonist SB431542 and Smad2 siRNA, but neither SIS3, which inhibits SMAD3, nor siRNA against Smad3 could block this effect. Various regions of the HABP2 promoter region were cloned into reporter constructs, and the promoter activity was determined. Accordingly, the promoter activity, which could phenocopy changes in Habp2 mRNA in response to TGF-β, was found to be located in the 177-bp region upstream of the transcription start site, and this region did not contain any SMAD binding sites. Mutation analysis of the promoter and chromatin immunoprecipitation assays were performed to identify an important role for the ATF3 binding element. Thus, TGF-β is the most likely mediator responsible for the decrease in FSAP expression in liver fibrosis.

FSAP-mediated nucleosome release from late apoptotic cells is inhibited by autoantibodies present in SLE.

Inefficient clearance of apoptotic cells and the subsequent exposure of the immune system to nuclear contents are crucially involved in the pathogenesis of systemic lupus erythematosus (SLE). Factor VII-activating protease (FSAP) is activated in serum upon contact with dead cells, and releases nucleosomes from late apoptotic cells into the extracellular environment. We investigated whether FSAP-mediated nucleosome release from late apoptotic cells is affected in SLE patients. Nucleosome release in sera of 27 SLE patients and 30 healthy controls was investigated by incubating late apoptotic Jurkat cells with serum and analyzing the remaining DNA content by flow cytometry. We found that nucleosome release in sera of SLE patients with high disease activity was significantly decreased when compared with that in SLE sera obtained during low disease activity or from healthy individuals. Upon removal of IgG/IgM antibodies from SLE sera, nucleosome release was restored. Similarly, monoclonal antinuclear antibodies inhibited nucleosome release in healthy donor serum or by plasma-purified FSAP. This inhibition was lost when Fab fragments were used, suggesting that antigen cross-linking is involved. In conclusion, FSAP-mediated nucleosome release from late apoptotic cells is greatly impaired in SLE patient sera, possibly hampering the clearance of these cells and thereby propagating inflammation.

Factor VII-Activating Protease Is a Regulator of Histone Cytotoxicity

Background: During sepsis, ischemia-reperfusion injury and sterile inflammation, levels of circulating histones and nucleosomes have been found to correlate with disease severity. Although the pathophysiological effects of circulating nucleosomes are not fully understood, free histones have been described to mediate cytotoxicity and contribute to disease severity. Several plasma proteins have been identified that modulate these cytotoxic effects by neutralizing or degrading histones. We have previously described that the plasma serine protease Factor VII-activating protease (FSAP) is activated upon contact with late apoptotic or necrotic cells and induces release of nucleosomes from these cells. During this release, histone H1 is cleaved. In addition, it has been described that FSAP is (auto)activated upon contact with histones and subsequently degrades histone H3.Aims: To investigate the regulation of histone-induced cytotoxicity by FSAP. Moreover, we aimed to compare differences in the cytotoxicity of histones and nucleosomes (histone+DNA) and potential differences in their proteolysis by FSAP.Methods: To determine FSAP activation and proteolysis of histones in serum we incubated normal donor or FSAP-deficient serum with 100ug/ml calf thymus histones. Upon its activation FSAP is rapidly bound by plasma inhibitors and we measured complexes of FSAP and its plasma inhibitor alpha 2-antiplasmin as an indirect readout for FSAP activation. To detect FSAP mediated proteolysis of histones we performed immunoblotting for histone H3. Results were corroborated using plasma-purified or recombinant FSAP. To determine histone-mediated cytotoxicity, we measured LDH levels after overnight incubation of HEK293 or ECRF cells with 100ug/ml calf thymus histone that had undergone a pre-incubation step in the presence or absence of healthy donor serum, FSAP-deficient serum or FSAP-depleted serum. Moreover, we tested the cytotoxicity of purified nucleosomes that were pre-incubated in the presence or absence of benzonase to degrade DNA.Results: EndogenousFSAP in serum was activated upon contact with histones, and histone proteolysis was observed. Similarly, histones were proteolyzed upon incubation using active plasma-purified or pre-activated recombinant FSAP, whereas no histone proteolysis was observed using recombinant inactive FSAP. These results indicate that FSAP is responsible for the observed histone degradation. FSAP-mediated histone degradation was able to protect HEK293 and ECRF cells against histone-induced cytotoxicity, showing that the resultant histone breakdown products had lost their cytotoxic effects. Recombinant inactive FSAP did not affect histone-induced cytotoxicity, indicating that the catalytic activity of FSAP is required for the observed protective effect of FSAP on cytotoxicity. When FSAP-deficient or FSAP-depleted serum was used, this led to greatly diminished histone proteolysis and reduced histone-induced cytotoxicity. Using our assay, we found that nucleosomes did not show cytotoxic effects unless they had been pre-incubated with benzonase to release histones. Notably, whereas intact nucleosomes were resistant against degradation by FSAP, FSAP protected against the cytotoxicity induced by histones released from these nucleosomes.Conclusion: FSAP in serum is activated by histones and subsequently proteolyses histones ,resulting in protection of cells from histone-induced cytotoxicity. Histones in the form of nucleosomes did not induce cytotoxicity, however after DNA removal the histones regained cytotoxicity. FSAP efficiently protected against the cytotoxicity of the unbound histones. We predict that the form in which histones are released into the blood is highly relevant in the context of human health and disease. We speculate that when endogenous DNases in serum degrade the DNA in nucleosomes, or alternatively, when highly cytotoxic histones are inadvertently released from dead cells in their free form rather than in the form of nucleosomes, FSAP mediates their efficient degradation to protect against the damaging effects of free histones. Neutralization of histones by FSAP may be an effective therapeutic approach to treat systemic inflammation. DisclosuresZeerleder:ViroPharma: Other: Receives an unrestricted grant from Viropharma.

Diabetes‐independent increase of factor VII‐activating protease activation in patients with Gram‐negative sepsis (melioidosis)

BackgroundThe plasma protease factor VII-activating protease (FSAP) can release nucleosomes from late apoptotic cells. Nucleosomes are markers of cell death, and extracellular cell-free DNA has been suggested to play an important role in inflammation and has been demonstrated to correlate with severity and outcome in sepsis patients.ObjectiveTo investigate FSAP activation in patients suffering from Burkholderia pseudomallei infection (melioidosis), an important cause of Gram-negative sepsis in Southeast Asia. As diabetes mellitus (DM) is the most important risk factor for both melioidosis and sepsis, we were also able to examine the role of DM in FSAP activation in this cohort of patients.MethodsIn a prospective observational study, complexes of FSAP with α2-antiplasmin (AP) were assayed in 44 patients with melioidosis, 34 of whom were classified as diabetic. Eighty-two healthy subjects served as controls (52 with DM and 30 without).ResultsFSAP–AP complex levels were markedly elevated in patients as compared with controls. The FSAP level increased by 16.82 AU mL−1 in patients with melioidosis after adjustment for the effect of DM in the regression model. As expected, FSAP activation was correlated with nucleosome release (slope = 0.74). No difference in FSAP activation on admission was seen between survivors and non-survivors, but the extent of FSAP activation correlated with stage of the disease; repeated testing during convalescence showed a return towards normal values (day 0 vs. day 28, 4.16 AU mL−1, 95% confidence interval [CI] 1.42–12.22).ConclusionPatients with Gram-negative sepsis caused by B. pseudomallei have abundant FSAP activation, which significantly correlates with stage of disease. The presence of DM, however, does not influence the extent of FSAP activation.

Cooperation of Factor VII–Activating Protease and Serum DNase I in the Release of Nucleosomes From Necrotic Cells

Removal of dead cells is essential in the maintenance of tissue homeostasis, and efficient removal prevents exposure of intracellular content to the immune system, which could lead to autoimmunity. The plasma protease factor VII-activating protease (FSAP) can release nucleosomes from late apoptotic cells. FSAP circulates as an inactive single-chain protein, which is activated upon contact with either apoptotic cells or necrotic cells. The purpose of this study was to investigate the role of FSAP in the release of nucleosomes from necrotic cells. Necrotic Jurkat cells were incubated with serum, purified 2-chain FSAP, and/or DNase I. Nucleosome release was analyzed by flow cytometry, and agarose gel electrophoresis was performed to detect DNA breakdown. Incubation with serum released nucleosomes from necrotic cells. Incubation with FSAP-deficient serum or serum in which FSAP was inhibited by a blocking antibody was unable to release nucleosomes from necrotic cells, confirming that FSAP is indeed the essential serum factor in this process. Together with serum DNase I, FSAP induced the release of DNA from the cells, the appearance of nucleosomes in the supernatant, and the fragmentation of chromatin into eventually mononucleosomes. FSAP and DNase I are the essential serum factors that cooperate in necrotic cell DNA degradation and nucleosome release. We propose that this mechanism may be important in the removal of potential autoantigens.

Complexes of factor VII-activating protease with plasminogen activator inhibitor-1 in human sepsis

Complexes of factor VII-activating protease with plasminogen activator inhibitor-1 in human sepsis -

The Factor VII-activating Protease (FSAP) Enhances the Activity of Bone Morphogenetic Protein-2 (BMP-2)

Factor VII-activating protease (FSAP) is a circulating protease involved in the pathogenesis of atherosclerosis, calcification, and fibrotic processes. To understand how FSAP controls the balance of local growth factors, we have investigated its effect on the regulation of bone morphogenetic proteins (BMPs). BMP-2 is produced as a large pro-form and secreted as a mature heparin-binding growth factor after intracellular processing by pro-protein convertases (PCs). In this study, we discovered that FSAP enhances the biological activity of mature BMP-2 as well as its pro-form, as shown by osteogenic differentiation of C2C12 myoblasts. These findings were complemented by knockdown of FSAP in hepatocytes, which revealed BMP-2 processing by endogenous FSAP. N-terminal sequencing indicated that pro-BMP-2 was cleaved by FSAP at the canonical PC cleavage site, giving rise to mature BMP-2 (Arg(282)↓Gln(283)), as well as in the N-terminal heparin binding region of mature BMP-2, generating a truncated mature BMP-2 peptide (Arg(289)↓Lys(290)). Similarly, mature BMP-2 was also cleaved to a truncated peptide within its N-terminal region (Arg(289)↓Lys(290)). Plasmin exhibited a similar activity, but it was weaker compared with FSAP. Thrombin, Factor VIIa, Factor Xa, and activated protein C were not effective. These results were further supported by the observation that the mutation of the heparin binding region of BMP-2 inhibited the processing by FSAP but not by PC. Thus, the proteolysis and activation of pro-BMP-2 and mature BMP-2 by FSAP can regulate cell differentiation and calcification in vasculature and may explain why polymorphisms in the gene encoding for FSAP are related to vascular diseases.

Tissue factor pathway inhibitor is an inhibitor of factor VII‐activating protease

Factor VII-activating protease (FSAP) is a serine protease that circulates in plasma in its inactive single-chain form and can be activated upon contact with dead cells. When activated by apoptotic cells, FSAP leads to the release of nucleosomes. The serpins C1-inhibitor and α(2) -antiplasmin are reported to be the major inhibitors of FSAP. However, regulation of FSAP activity by Kunitz-type inhibitors is not well studied. To compare the inhibition of FSAP activity and FSAP-induced nucleosome release from apoptotic cells by tissue factor pathway inhibitor (TFPI) with that of C1-inhibitor and α(2) -antiplasmin. Apoptotic cells were incubated with plasma or FSAP in presence of the inhibitor, and nucleosome release was analyzed with flow cytometry. Monoclonal antibodies against TFPI and altered forms of TFPI were used to investigate which domains of TFPI contribute to FSAP inhibition. We show that TFPI abrogates FSAP activity and nucleosome release from apoptotic cells. TFPI is a much more efficient inhibitor than C1-inhibitor or α(2) -antiplasmin. The active site of K2 is required for inhibition of FSAP. A direct binding interaction between FSAP and the C-terminal domain of TFPI is also required for efficient inhibition. Inhibition of FSAP-induced nucleosome release by recombinant TFPI might, in part, explain the anti-inflammatory effects of recombinant TFPI infusion observed in animal and human sepsis.

Plasma factor VII‐activating protease antigen levels and activity are increased in ischemic stroke

Factor VII-activating protease (FSAP) is a recently discovered plasma protease with a role in the regulation of hemostasis and vascular remodeling processes. Higher levels and activity of FSAP have been reported in patients with deep vein thrombosis, but there are no data on plasma FSAP in ischemic stroke (IS). To investigate whether FSAP antigen levels and activity are associated with IS and/or etiologic subtypes of IS. To assess the potential association between FSAP and IS, plasma FSAP antigen levels and activity were measured in 600 consecutive IS patients and 600 population-based controls from the case-control study the Sahlgrenska Academy Study on Ischemic Stroke (SAHLSIS). Blood sampling was performed in the acute phase and 3 months after the index stroke. FSAP was also investigated at the genetic level by genotyping of 33 single-nucleotide polymorphisms. Increased FSAP antigen level and activity, at both time-points, were independently associated with IS. Subtype analysis revealed similar associations for both FSAP measures, at both time-points, in all main IS subtypes. FSAP genotypes showed association with both FSAP plasma measurements, but not with IS. Increased plasma FSAP antigen levels and activity were associated with IS and all main etiologic subtypes, suggesting a possible role for FSAP in the pathophysiology of IS, irrespective of the underlying etiology.

Factor VII-Activating Protease Is Activated in Multiple Trauma Patients and Generates Anaphylatoxin C5a

Severe tissue injury results in early activation of serine protease systems including the coagulation and complement cascade. In this context, little is known about factor VII-activating protease (FSAP), which is activated by substances released from damaged cells such as histones and nucleosomes. Therefore, we have measured FSAP activation in trauma patients and have identified novel FSAP substrates in human plasma. Mass spectrometry-based methods were used to identify FSAP binding proteins in plasma. Anaphylatoxin generation was measured by ELISA, Western blotting, protein sequencing, and chemotaxis assays. Plasma samples from trauma patients were analyzed for FSAP Ag and activity, nucleosomes, C5a, and C3a. Among others, we found complement components C3 and C5 in FSAP coimmunoprecipitates. C3 and C5 were cleaved by FSAP in a dose- and time-dependent manner generating functional C3a and C5a anaphylatoxins. Activation of endogenous FSAP in plasma led to increased C5a generation, but this was not the case in plasma of a homozygous carrier of Marburg I single nucleotide polymorphism with lower FSAP activity. In multiple trauma patients there was a large increase in circulating FSAP activity and nucleosomes immediately after the injury. A high correlation between FSAP activity and C5a was found. These data suggest that activation of FSAP by tissue injury triggers anaphylatoxin generation and thereby modulates the posttraumatic inflammatory response in vivo. A strong link between C5a, nucleosomes, and FSAP activity indicates that this new principle might be important in the regulation of inflammation.

The G534E-polymorphism of the gene encoding the Factor VII-activating protease is a risk factor for venous thrombosis and recurrent events

IntroductionA single nucleotide polymorphism of the factor VII activating protease (FSAP), FSAP Marburg I (rs7080536) has been identified as a risk factor for venous thrombosis, but its clinical role has so far been controversial in part due to small cohort sizes. The aim of the present case-control study was to elucidate the impact of the FSAP Marburg I polymorphism (FSAP-MI) on the development of venous thromboembolic disease (VTE) with other known sequence variations, including Factor V Leiden (rs6025) and Factor II G20210A (rs1799963). Materials and MethodsThe study included 891 patients (312 male and 579 female) with a history of deep venous thrombosis (DVT) and/or pulmonary embolism (PE) and 1283 healthy blood donors with no history of venous thromboembolic disease. ResultsWe found that besides to the well-established aforementioned sequence variations of FV and Prothrombin, the FSAP Marburg I (FSAP-MI) polymorphism was significantly associated with the development of DVTs (1.65 (1.16-2.34) OR (95% CI)) and recurrent thromboembolic events (DVT and PE) (2.13 (1.35-3.36) OR (95% CI)). Comparing patients displaying one or more events FSAP-MI was still associated with the development of recurrent thromboembolic events (1.64 (1- 2.69) OR (95% CI)). ConclusionsWe conclude that FSAP Marburg-I genotyping may be used to determine the risk for thromboembolic disorders in patients with suspected thrombophilia and known DVT or PE.

The effect of factor VII activating protease treatment on proliferation and collagen synthesis of cultured human pulmonary fibroblasts

To examine the influence of factor VII-activating protease (FSAP) treatment on cell proliferation and collagen synthesis in human pulmonary fibroblasts (HPF). Cultured HPF were treated with heparin (10 mg/L), platelet-derived growth factor-BB (PDGF-BB, 20 μg/L), FSAP (12 mg/L), aprotinin (10 mg/L) separately and in different mixture. The proliferation of the HPF was determined by 5-bromodeoxyuridine (BrDU) incorporation. The collagen III mRNA was determined by real-time polymerase chain reaction (PCR), and its protein expression, as well as the phosphorylation of p42 / p44 mitogen activated protein kinase (MAPK) were both determined using Western blotting. PDGF BB significantly (all P < 0.05) induced cell proliferation (1.23 ± 0.06 vs. 1.01 ± 0.01 without heparin, 1.24 ± 0.04 vs. 0.98 ± 0.01 with heparin), collagen III mRNA transcription (1.79 ± 0.02 vs. 1.00 ± 0.00 without aprotinin, 1.57 ± 0.01 vs. 1.00 ± 0.00 with aprotinin), and collagen III protein expression (0.54 ± 0.26 vs. 0.17 ± 0.05 without aprotinin, 0.59 ± 0.31 vs. 0.24 ± 0.15 with aprotinin) in HPF. PDGF BB also significantly (both P < 0.05) induced p42 / p44 MAPK phosphorylation in HPF (0.89 ± 0.24 vs. 0.51 ± 0.17 without aprotinin, 0.97 ± 0.41 vs. 0.37 ± 0.05 with aprotinin). In the presence of heparin FSAP significantly (all P < 0.05) inhibited the PDGF BB induced HPF proliferation (0.92 ± 0.23 vs 1.19 ± 0.11); collagen III mRNA transcription (0.61 ± 0.02 vs. 1.79 ± 0.02) and collagen III protein expression (0.15 ± 0.12 vs. 0.54 ± 0.26). FSAP also inhibited PDGF induced p42 / p44 MAPK phosphorylation in HPF (0.57 ± 0.16 vs. 0.89 ± 0.24) significantly (P < 0.05). The presence of aprotinin lead to a reversal of the inhibitory effect of FSAP on collagen III mRNA transcription (2.37 ± 0.21 vs.0.61 ± 0.02), collagen III protein expression (0.55 ± 0.24 vs. 0.15 ± 0.12) and p42 / p44 MAPK phosphorylation (0.86 ± 0.41 vs. 0.57 ± 0.16, all P < 0.05). FSAP could inhibit the PDGF-BB induced proliferation and collagen III synthesis in HPF in vitro through the suppression of p42 / p44 MAPK phosphorylation.

Plasma factor VII-activating protease is increased by oral contraceptives and induces factor VII activation in-vivo

Oral contraceptive (OC) use influences the hemostatic system significantly and is a risk factor for development of cardiovascular disease. Factor VII-activating protease (FSAP) has potential effects on hemostasis. The 1601GA genotype of the 1601 G/A polymorphism in the FSAP gene expresses a FSAP alloenzyme with reduced pro-fibrinolytic activity. Presently, we address whether OC use and OC formulation affect FSAP measures in human blood. Healthy women (n = 588) were allocated to six cycles of OCs with estrogen contents of 20 μg (n = 158), 30 μg (n = 284), 35 μg (n = 79) or 50 μg (n = 67) combined with various progestins. FSAP genotypes, FSAP and factor VII (FVII) plasma measures were assessed at baseline and after 6 cycles of OC. The 1601GA genotype was present in 49 (8.3%) of the women and was associated with significantly reduced levels of FSAP (P ≤ 0.001). OC use increased FSAP antigen by 25% and FSAP activity by 59% (P < 0.001). The FSAP increase was comparable in the seven different OC treatment groups (P > 0.05). The relative increase in FSAP activity was significantly higher in women carrying the 1601GG genotype (63%) than in women carrying 1601GA genotype (50%) (P = 0.01) and was associated with an increased activation of FVII. In conclusion: OC use increases the plasma measures of FSAP. The increase in FSAP is comparable in the seven OC-groups studied but is more significant in women carrying the 1601GG genotype than in women with the 1601GA genotype and results in increased activation of FVII suggesting that FSAP-induced activation of FVII takes place in-vivo and not only in-vitro as hitherto described.

Direct chromogenic substrate immuno-capture activity assay for testing of factor VII-activating protease

The Marburg I (MRI) single nucleotide polymorphism (SNP) of the factor VII-activating protease (FSAP) gene has been associated with thrombophilia and atherosclerotic disease. PCR is used to detect the SNP. Also, the specific FSAP activity to cleave single-chain urokinase-type plasminogen activator (scu-PA) serves as a surrogate for PCR testing. Development of further assays is indicated in order to increase testing opportunities for future studies. A direct chromogenic substrate immuno-capture activity assay for FSAP (FSAP dcs activity assay) was established. Performance characteristics of the FSAP dcs activity assay were compared to the FSAP scu-PA activity assay. The FSAP dcs activity assay detects FSAP activity from 25% to 150% of the norm. Total CVs ranged from 6% to 10% for FSAP wild type samples and 9%-18% for MRI samples. Correlation between the FSAP dcs and scu-PA activity assays was low (R=0.7). The FSAP dcs activity determined the presence of the MRI FSAP alloenzyme with a diagnostic sensitivity and specificity of 100% [95% confidence interval (CI): 89.6%-100%] and 96.2% (95% CI: 93.2%-97.4%), respectively, whereas the specific FSAP dcs activity increased specificity to 99.0% (95% CI: 97.2%-99.6%). The specific FSAP dcs activity represents a reliable method for the detection of the FSAP MRI alloenzyme. Due to the limited correlation between the FSAP dcs and scu-PA activity assays, these different measurands may exhibit different utility in research and clinical applications. Thus, the FSAP dcs activity assay can represent a valuable complement or alternative for FSAP testing in future studies.