Development of a Factor VII Activating Protease (FSAP) generation assay and its application in studying FSAP in venous thrombosis.

Human genetic studies based on the Marburg I polymorphism in the factor VII activating protease (FSAP) encoding gene, analysis of FSAP activity in plasma and biochemical characterization of FSAP substrates indicate a possible causal link between FSAP activity and venous thrombosis. We hypothesized that a direct standardized assay to measure FSAP activity in plasma could provide convincing arguments for or against such a potential link. Using Ac-Pro-DTyr-Lys-Arg-AMC as a highly specific and sensitive substrate, histones as a trigger to activate pro-FSAP and plasma-purified active FSAP as a calibrator, we have developed a fluorogenic kinetic assay that reveals the FSAP generating potential in human plasma in real time. This assay is similar to the thrombin generation assay and allows analysis of lag phase, time to peak and velocity, as well as peak FSAP and the endogenous FSAP potential (EFP) of plasma samples. Carriers of the Marburg I polymorphism showed clearly delayed FSAP generation and lower peak FSAP and EFP level. There were no significant differences in all FSAP activity parameters between plasma from patients with a history of venous thrombosis and controls. When excluding Marburg I carriers, which were evenly distributed between groups, delayed FSAP generation significantly correlated with venous thrombosis in postmenopausal women. The novel FSAP activity assay is robust and easy to perform and will be a useful tool for analyzing plasma FSAP activity, also, in other pathophysiological conditions.

Identification of a Phage Display-Derived Peptide Interacting with the N-Terminal Region of Factor VII Activating Protease (FSAP) Enables Characterization of Zymogen Activation.

Factor VII Activating protease (FSAP) has a protective effect in diverse disease conditions as inferred from studies in FSAP-/- mice and humans deficient in FSAP activity due to single-nucleotide polymorphism. The zymogen form of FSAP in plasma is activated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear whether this activation mechanism is specific and amenable to manipulation. Using a phage display approach, we have identified a Cys-constrained 11 amino acid peptide, NNKC9/41, that activates pro-FSAP in plasma. The synthetic linear peptide has a propensity to cyclize through the terminal Cys groups, of which the antiparallel cyclic dimer, but not the monocyclic peptide, is the active component. Other commonly found zymogens in the plasma, related to the hemostasis system, were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30-35) in an intrinsically disordered stretch in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41, and this was reversed by MA-FSAP-38C7, demonstrating the utility of this peptide. Peptide NNKC9/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP, elucidate its biological role, and provide a starting point for the pharmacological manipulation of FSAP activity.

Factor VII activating protease (FSAP) inhibits the outcome of ischemic stroke in mouse models.

The outcome of ischemic stroke can be improved by further refinements of thrombolysis and reperfusion strategies. Factor VII activating protease (FSAP) is a circulating serine protease that could be important in this context. Its levels are raised in patients as well as mice after stroke and a single nucleotide polymorphism (SNP) in the coding sequence, which results in an inactive enzyme, is linked to an increased risk of stroke. In vitro, FSAP cleaves fibrinogen to promote fibrinolysis, activates protease-activated receptors, and decreases the cellular cytotoxicity of histones. Based on these facts, we hypothesized that FSAP can be used as a treatment for ischemic stroke. A combination of tissue plasminogen activator (tPA), a thrombolytic drug, and recombinant serine protease domain of FSAP (FSAP-SPD) improved regional cerebral perfusion and neurological outcome and reduced infarct size in a mouse model of thromboembolic stroke. FSAP-SPD also improved stroke outcomes and diminished the negative consequences of co-treatment with tPA in the transient middle cerebral artery occlusion model of stroke without altering cerebral perfusion. The inactive MI-isoform of FSAP had no impact in either model. FSAP enhanced the lysis of blood clots in vitro, but in the tail transection model of hemostasis, FSAP-SPD treatment provoked a faster clotting time indicating that it also has pro-coagulant actions. Thus, apart from enhancing thrombolysis, FSAP has multiple effects on stroke progression and represents a promising novel therapeutic strategy in the treatment of ischemic stroke.

Factor VII activating protease (FSAP) is not essential in the pathophysiology of angioedema in patients with C1 inhibitor deficiency.

BACKGROUND: Excessive bradykinin (BK) generation from high molecular weight kininogen (HK) by plasma kallikrein (PK) due to lack of protease inhibition is central to the pathophysiology of hereditary angioedema (HAE). Inadequate protease inhibition may contribute to HAE through a number of plasma proteases including factor VII activating protease (FSAP) that can also cleave HK. OBJECTIVE: To investigate the interaction between FSAP and C1 inhibitor (C1Inh) and evaluate the potential role of FSAP in HAE with C1Inh deficiency. MATERIALS AND METHODS: Plasma samples from 20 persons with HAE types 1 or 2 in remission were studied and compared to healthy controls. We measured and compared antigenic FSAP levels, spontaneous FSAP activity, FSAP generation potential, activation of plasma pre-kallikrein (PPK) by FSAP, and the formation of FSAP-C1Inh and FSAP-alpha2-antiplasmin (FSAP-α2AP) complexes. Furthermore, we measured HK cleavage and PK activation after activation of endogenous pro-FSAP and after addition of exogenous FSAP. RESULTS: In plasma from HAE patients, there is increased basal FSAP activity compared to healthy volunteers. HAE plasma exhibits decreased formation of FSAP-C1Inh complexes and increased formation of FSAP-α2AP complexes in histone-activated plasma. Although exogenous FSAP can cleave HK in plasma, this was not seen when endogenous plasma pro-FSAP was activated with histones in either group. PK was also not activated by FSAP in plasma. CONCLUSION: In this study, we established that FSAP activity is increased and the pattern of FSAP-inhibitor complexes is altered in HAE patients. However, we did not find evidence suggesting that FSAP contributes directly to HAE attacks.

Proteolytic activation of the epithelial sodium channel (ENaC) by factor VII activating protease (FSAP) and its relevance for sodium retention in nephrotic mice.

Proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases is thought to contribute to renal sodium retention in nephrotic syndrome. However, the identity of the responsible proteases remains elusive. This study evaluated factor VII activating protease (FSAP) as a candidate in this context. We analyzed FSAP in the urine of patients with nephrotic syndrome and nephrotic mice and investigated its ability to activate human ENaC expressed in Xenopus laevis oocytes. Moreover, we studied sodium retention in FSAP-deficient mice (Habp2-/-) with experimental nephrotic syndrome induced by doxorubicin. In urine samples from nephrotic humans, high concentrations of FSAP were detected both as zymogen and in its active state. Recombinant serine protease domain of FSAP stimulated ENaC-mediated whole-cell currents in a time- and concentration-dependent manner. Mutating the putative prostasin cleavage site in γ-ENaC (γRKRK178AAAA) prevented channel stimulation by the serine protease domain of FSAP. In a mouse model for nephrotic syndrome, active FSAP was present in nephrotic urine of Habp2+/+ but not of Habp2-/- mice. However, Habp2-/- mice were not protected from sodium retention compared to nephrotic Habp2+/+ mice. Western blot analysis revealed that in nephrotic Habp2-/- mice, proteolytic cleavage of α- and γ-ENaC was similar to that in nephrotic Habp2+/+ animals. In conclusion, active FSAP is excreted in the urine of nephrotic patients and mice and activates ENaC in vitro involving the putative prostasin cleavage site of γ-ENaC. However, endogenous FSAP is not essential for sodium retention in nephrotic mice.

Cellular effects of factor VII activating protease (FSAP).

Factor VII activating protease (FSAP) is a circulating serine protease of broad specificity that is likely to be involved in many pathophysiological processes. The activation of the circulating zymogen form of FSAP by histones, released from damaged cells, underlines its roles in regulating host responses to tissue damage and inflammation. Some of the direct cellular effects of FSAP are mediated through protease-activated receptors (PARs). Knock-down of each one of the four PARs in endothelial cells indicated that PAR-1 and -3 are involved in regulating endothelial permeability in response to FSAP. Overexpression of PARs in cell lines led to the conclusion that PAR-2 and -1 were the main receptors for FSAP. Studies with synthetic peptides and receptor mutants demonstrate that FSAP cleaves PAR-1 and -2 at their canonical cleavage site. However, PAR-1 is not activated by FSAP in all cells, which may be related to other, as yet, undefined factors. Inhibition of apoptosis by FSAP is mediated through PAR-1 and was observed in neurons, astrocytes and A549 cells. FSAP also mediates cellular effects by modulating the activity of growth factors, generation of bradykinin, C5a and C3a generation or histone inactivation. These cellular effects need to be further investigated at the in vivo level.

Characterization of the enzymatic activity of the serine protease domain of Factor VII activating protease (FSAP).

Factor VII (FVII) activating protease (FSAP) is a circulating serine protease. Human genetic studies, based on the Marburg I (MI) (Gly221Glu, chymotrypsin numbering system) polymorphism, implicate FSAP in the pathogenesis of many diseases. Here, we describe the molecular and functional changes caused by the Gly221Glu substitution in the 220 loop using recombinant proteins expressed in E. coli. The serine protease domain (SPD) of wild type (WT) FSAP displayed auto-catalytic activation whereas the MI isoform displayed very low autocatalytic activation and low proteolytic activity against the chromogenic substrate S-2288, Factor VII, tissue factor pathway inhibitor as well as pro-urokinase. Introduction of a thermolysin cleavage site in the activation position (Arg15Gln) led to cleavage of both WT- and MI-SPD and the resulting WT-SPD, but not the MI-SPD, was active. Mutating the Gly221 position to Asp, Gln and Leu led to a loss of activity whereas the Ala substitution was partially active. These results suggest a disturbance of the active site, or non-accessibility of the substrate to the active site in MI-SPD. With respect to regulation with metal ions, calcium, more than sodium, increased the enzymatic activity of WT-SPD. Thus, we describe a novel method for the production of recombinant FSAP-SPD to understand the role of the MI-single nucleotide polymorphism (SNP) in the regulation of its activity.

Altered structure and function of fibrinogen after cleavage by Factor VII Activating Protease (FSAP).

Factor VII Activating Protease (FSAP) is a plasma protease affecting both coagulation and fibrinolysis. Although a role in hemostasis is still unclear, the identification of additional physiologic substrates will help to elucidate its role in this context. FSAP has been reported to cleave fibrinogen, but the functional consequences of this are not known. We have therefore undertaken this study to determine the implications of this cleavage for fibrin-clot formation and its lysis. Treatment of human fibrinogen with FSAP released an N-terminal peptide from the Bß chain (Bß1-53) and subsequently the fibrinopeptide B; within the Aα chain a partial truncation of the αC-region by multiple cleavages was seen. The truncated fibrinogen showed a delayed thrombin-catalyzed polymerization and formed fibrin clots of reduced turbidity, indicative of thinner fibrin fibers. Confocal laser scanning and scanning electron microscopy of these clots revealed a less coarse fibrin network with thinner fibers and a smaller pore size. A lower pore size was also seen in permeability studies. Unexpectedly, FSAP-treated fibrinogen or plasma exhibited a significantly faster tPA-driven lysis, which correlated exclusively with cleavage of fibrinogen and not with activation of plasminogen activators. Similar observations were also made in plasma after activation of endogenous zymogen FSAP, but not in plasma of carrier of the rare Marburg I single nucleotide polymorphism. In conclusion, altering fibrin clot properties by fibrinogenolysis is a novel function of FSAP in the vasculature, which facilitates clot lysis and may in vivo contribute to reduced fibrin deposition during thrombosis.

Interaction of factor VII activating protease (FSAP) with neutrophil extracellular traps (NETs).

The circulating zymogen form of Factor VII activating protease (FSAP) can be activated by histones and nucleosomes in vivo. These cell-death-associated nuclear factors are also actively extruded into the extracellular space by neutrophils through a process called neutrophil extracellular trap (NET) formation (NETosis). NETs are thought to be involved in host defense, inflammation as well as thrombosis. We have investigated the bidirectional interactions of FSAP and NETs. Phorbol ester-mediated NET formation was marginally stimulated by FSAP. Plasma-derived FSAP as well as exogenous FSAP bound to NETs. There was co-localization of FSAP and NETs in coronary thrombi from patients with acute myocardial infarction. Contrary to our expectations no activation of pro-FSAP by NETs was evident. However, after disintegration of NETs with DNase, a robust activation of pro-FSAP, due to release of histones from nucleosomes, was detected. The released histones were in turn degraded by FSAP. Histone cytotoxicity towards endothelial cells was neutralized by FSAP more potently than by activated protein C (APC). One more consequence of histone degradation was a decrease in nucleosome release from apoptotic neutrophils. Taken together, NETs bind to FSAP, but do not activate pro-FSAP unless histones are released from NETs by DNAse. This activation of FSAP is likely to be important in diminishing the cytotoxic effect of histones, thus limiting the damaging effect of NETosis.

Factor VII activating protease (FSAP) regulates the expression of inflammatory genes in vascular smooth muscle and endothelial cells.

BACKGROUND AND AIMS: The factor VII activating protease (FSAP) knockout mice have a bigger neointima after vascular injury and a larger infarct volume after stroke. The Marburg I (MI) single nucleotide polymorphism (SNP) in the FSAP-encoding gene is associated with an increased risk of stroke and carotid stenosis in humans. We hypothesize that the regulation of gene expression by FSAP in vascular cells accounts for its vasculo-regulatory properties. METHODS: Vascular smooth muscle cells (VSMC) and endothelial cells (EC) were stimulated with FSAP and a microarray-based expression analysis was performed. Selected genes were further investigated by qPCR. Receptor- and pathway-inhibitors were used to elucidate the mechanisms involved. RESULTS: Pathways significantly activated by FSAP include those related to inflammation, apoptosis and cell growth in VSMC and inflammation in EC. The key upregulated genes in VSMC were AREG, PTGS2 and IL6; and in EC these were SELE, VCAM1, and IL8. Secretion of IL6 in VSMC and IL8 in EC was also stimulated by FSAP. Recombinant wild type protease domain of FSAP, but not the MI-isoform, could recapitulate most of these effects. In VSMC, but not EC, gene expression by FSAP was impaired by PAR1 (protease-activated receptor1) receptor antagonists. In VSMC, FSAP-induced expression of AREG and IL6 was blocked by cAMP and MAPK pathway inhibitors indicating that multiple signalling pathways are likely to be involved. CONCLUSIONS: The stimulation of inflammation- and proliferative/apoptosis-related genes in VSMC and EC provides a comprehensive basis for understanding the role of FSAP in vascular diseases.

Defective thrombus formation in mice lacking endogenous factor VII activating protease (FSAP).

Factor VII (FVII) activating protease (FSAP) is a circulating protease with a putative function in blood coagulation and fibrinolysis. Genetic epidemiological studies have implied a role for FSAP in carotid stenosis, stroke and thrombosis. To date, no in vivo evidence is available to support these claims. We have, for the first time, used FSAP-/- mice to define its role in thrombosis and haemostasis in vivo and to characterise the molecular mechanisms involved. FeCl3-induced arterial thrombosis in carotid and mesenteric artery revealed that the occlusion time was significantly increased in FSAP-/- mice (p< 0.01) and that some FSAP-/- mice did not occlude at all. FSAP-/- mice were protected from lethal pulmonary thromboembolism induced by collagen/ epinephrine infusion (p< 0.01). Although no spontaneous bleeding was evident, in the tail bleeding assay a re-bleeding pattern was observed in FSAP-/- mice. To explain these observations at a mechanistic level we then determined how haemostasis factors and putative FSAP substrates were altered in FSAP-/- mice. Tissue factor pathway inhibitor (TFPI) levels were higher in FSAP-/- mice compared to WT mice whereas FVIIa levels were unchanged. Other coagulation factors as well as markers of platelet activation and function revealed no significant differences between WT and FSAP-/- mice. This phenotype of FSAP-/- mice could be reversed by application of exogenous FSAP. In conclusion, a lack of endogenous FSAP impaired the formation of stable, occlusive thrombi in mice. The underlying in vivo effect of FSAP is more likely to be related to the modulation of TFPI rather than FVIIa.

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.

Factor VII-activating protease promotes the proteolysis and inhibition of tissue factor pathway inhibitor.

OBJECTIVE: Factor VII-activating protease (FSAP) activates both factor VII and pro-urokinase and inhibits platelet-derived growth factor-BB, thus regulating hemostasis- and remodeling-associated processes in the vasculature. A genetic variant of FSAP (Marburg I polymorphism) results in low enzymatic activity and is associated with an enhanced risk of carotid stenosis and stroke. We postulate that there are additional substrates for FSAP that will help to explain its role in vascular biology and have searched for such a substrate. METHODS AND RESULTS: Using screening procedures to determine the influence of FSAP on various hemostasis-related processes on endothelial cells, we discovered that FSAP inhibited tissue factor pathway inhibitor (TFPI), a major anticoagulant secreted by these cells. Proteolytic degradation of TFPI by FSAP could also be demonstrated by Western blotting, and the exact cleavage sites were determined by N-terminal sequencing. The Marburg I variant of FSAP had a diminished ability to inhibit TFPI. A monoclonal antibody to FSAP that specifically inhibited FSAP binding to TFPI reversed the inhibitory effect of FSAP on TFPI. CONCLUSIONS: The identification of TFPI as a sensitive substrate for FSAP increases our understanding of its role in regulating hemostasis and proliferative remodeling events in the vasculature.

The hyaluronic acid-binding protease: a novel vascular and inflammatory mediator?

A serine protease in human plasma termed hyaluronan-binding protease HABP is structurally related to plasminogen-activators, coagulation FXII and hepathocyte growth factor activator. This protease has coagulation and fibrinolysis-related activities, although a physiologic role in haemostasis still requires confirmation. In more recent years accumulating information became available supporting the hypothesis that HABP plays also a significant role in the regulation of cells in the vasculature and in the perivascular environment. On the one hand HABP generates bradykinin or bFGF on the surface of human umbilical vein endothelial cells (HUVECs), triggering intracellular signalling via the bradykinin receptor 2 and FGFR-1. Other data indicate that beside endothelial cells also vascular smooth muscle cells are a target for HABP. As major mechanism of cell regulation a high affinity of HABP to growth factors with the subsequent proteolytic cleavage and inactivation has been identified. The current knowledge of the physiologic and clinical relevance of HABP as a vascular and possibly inflammatory mediator is summarized in this review.

Induction of intracellular signalling in human endothelial cells by the hyaluronan-binding protease involves two distinct pathways.

Recently a novel plasma serine protease with high affinity to hyaluronic acid and glycosaminoglycans, such as heparin and heparan sulfate, has been described and termed hyaluronan-binding protease (HABP). HABP cleaves kininogen in vitro, releasing the vasoactive peptide bradykinin, and activates plasminogen activators, suggesting a vascular cell-directed physiological function of this novel plasma protease. Here we show that HABP stimulates human umbilical vein endothelial cells (HUVECs) by activating two distinct cell-surface receptors. On the one hand, HABP releases bradykinin from cell surface-bound or soluble kininogen and triggers a bradykinin B2-receptor-dependent mobilisation of intracellular Ca2+. On the other hand, HABP activates the p44/42-dependent MAPK (ERK1/2) signalling cascade independent of the B2-receptor, but involving the fibroblast growth factor receptor-1 and basic fibroblast growth factor. This signalling pathway leads to phosphorylation of the kinases Raf, MEK1/2 and ERK1/2. The extracellular activity of HABP also affects the gene expression level through phosphorylation of two transcription factors, the cAMP-responsive element binding protein CREB and the proto-oncogene c-Myc. Our results indicate a proangiogenic potential of HABP, which, in combination with a profibrinolytic activity, directs the physiological function of this plasma protease to processes in which clot lysis, cell motility and neovascularisation are pivotal processes, e.g., in wound healing, tissue repair and tumour progression.

Inhibition of bFGF/EGF-dependent endothelial cell proliferation by the hyaluronan-binding protease from human plasma.

Recently we identified a plasma serine protease with a high affinity to glycosaminoglycans like heparin or hyaluronic acid, termed hyaluronan-binding protease (HABP). Since glycosaminoglycans are found on cell surfaces and in the extracellular matrix a physiological role of this plasma protease in a pericellular environment was postulated. Here we studied the influence of HABP on the regulation of endothelial cell growth. We found that HABP efficiently prevented the basic fibroblast growth factor/epidermal growth factor (bFGF/EGF)-dependent proliferation of human umbilical vein endothelial cells. Proteolytic cleavage of adhesion molecules was found to be involved, but was not solely responsible for the anti-proliferative activity. Pre-treatment of growth factor-supplemented cell culture medium with HABP indicated that no direct contact between the active protease and cells was required for growth inhibition. In vitro studies revealed a growth factor-directed activity of HABP, resulting in complexation and partial hydrolysis and, thus, inactivation of basic fibroblast growth factor, a potent mitogen for endothelial cells. Heparin and heparan sulfate fully protected bFGF from complexation and cleavage by HABP, although these glycosaminoglycans are known to enhance the proteolytic activity of HABP. This finding suggested that free circulating bFGF rather than bFGF bound to heparan sulfate proteoglycans would be a physiologic substrate. In conclusion, down-regulation of bFGF-dependent endothelial cell growth represents an important mechanism through which HABP could control cell growth in physiologic or pathologic processes like angiogenesis, wound healing or tumor development.

The hyaluronan-binding serine protease from human plasma cleaves HMW and LMW kininogen and releases bradykinin.

The influence of the hyaluronan-binding protease (PHBSP), a plasma enzyme with FVII- and pro-urokinase-activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The pro-cofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the N-terminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5H and D6H are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the pro-cofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogen-cleaving and bradykinin-releasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system.