Stroke therapy and diagnosis

• Cui, Xueyan; Stavik, Benedicte; Thiede, Bernd; Sandset, Per Morten & Kanse, Sandip (2022). FSAP Protects against Histone-Mediated Increase in Endothelial Permeability In Vitro. International Journal of Molecular Sciences. ISSN 1661-6596. 23(22). doi: 10.3390/ijms232213706. Full text in Research Archive Show summary

  Factor-VII-activating protease (FSAP) is involved in the regulation of hemostasis and inflammation. Extracellular histones play a role in inflammation and the conversion of latent pro-FSAP into active FSAP. FSAP has been shown to regulate endothelial permeability, but the mechanisms are not clear. Here, we have investigated the effects of FSAP on endothelial permeability in vitro. A mixture of histones from calf thymus stimulated permeability, and the wild-type (WT) serine protease domain (SPD) of FSAP blocked this effect. WT–SPD–FSAP did not influence permeability on its own, nor that stimulated by thrombin or vascular endothelial growth factor (VEGF)-A165. Histones induced a large-scale rearrangement of the junction proteins VE-cadherin and zona occludens-1 from a clear junctional distribution to a diffuse pattern. The presence of WT–SPD–FSAP inhibited these changes. Permeability changes by histones were blocked by both TLR-2 and TLR4 blocking antibodies. Histones upregulated the expression of TLR-2, but not TLR-4, in HUVEC cells, and WT–SPD–FSAP abolished the upregulation of TLR-2 expression. An inactive variant, Marburg I (MI)–SPD–FSAP, did not have any of these effects. The inhibition of histone-mediated permeability may be an important function of FSAP with relevance to sepsis, trauma, and stroke and the need to be investigated further in in vivo experiments.

• Berge-Seidl, Sebastian; Nielsen, Nis Valentin; Alfonso, Armando A. Rodriguez; Etscheid, Michael; Kandanur, Sai Priya Sarma & Haug, Bengt Erik [Show all 15 contributors for this article] (2022). Identification of a phage display-derived peptide interacting with the N-terminal region of factor VII activating protease (FSAP) enables characterization of zymogen activation. ACS Chemical Biology. ISSN 1554-8929. 17(9), p. 2631–2642. doi: 10.1021/acschembio.2c00538. Full text in Research Archive Show summary

  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.

• Etscheid, Michael; Hanschmann, Kay Martin; Sandset, Per Morten & Kanse, Sandip (2022). Development of a Factor VII Activating Protease (FSAP) generation assay and its application in studying FSAP in venous thrombosis. Thrombosis Research. ISSN 0049-3848. 220, p. 24–34. doi: 10.1016/j.thromres.2022.09.027. Full text in Research Archive Show summary

  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.

• Cui, Xueyan; Tjønnfjord, Geir Erland; Kanse, Sandip; Dahm, Anders Erik A; Iversen, Nina & Myklebust, Christiane Filion [Show all 12 contributors for this article] (2021). Tissue factor pathway inhibitor upregulates CXCR7 expression and enhances CXCL12-mediated migration in chronic lymphocytic leukemia. Scientific Reports. 11. doi: 10.1038/s41598-021-84695-8. Full text in Research Archive Show summary

  Abstract The infiltration of chronic lymphocytic leukemia (CLL) cells into lymphoid organs correlates with disease severity. CXCL12 is a key chemotactic factor for the trafficking of CLL. Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor and plays a role in CXCL12-mediated hematopoietic stem cell homing. We aim to explore the role of TFPI in CXCL12-mediated migration of CLL cells. In this study, plasma TFPI concentrations were measured by ELISA. CLL cells were isolated from patients and used for trans-endothelial migration (TEM) assays. Quantitative RT-PCR and Western blotting were used to detect the expression of CXCR7, CXCR4 and β-catenin. Immunofluorescence and co-immunoprecipitation was used to detect the binding of TFPI and glypican-3 (GPC3). We found that plasma TFPI levels in CLL patients were higher than in healthy controls, particularly in the patients with advanced disease. TFPI enhanced CXCL12-mediated TEM of CLL cells by increasing the expression of the CXCL12 receptor CXCR7, but not of the CXCL12 receptor CXCR4. The effect of TFPI on TEM was abolished by the CXCR7 inhibitor, CCX771, while the CXCR4 inhibitor AMD3100 strongly increased TEM. TFPI co-localized with GPC3 on the cell surface. An antibody to GPC3, HS20, decreased CXCR7 expression and abolished the effect of TFPI on TEM. TFPI activated β-catenin and the Wnt/β-catenin inhibitor IWP4 repressed the effect of TFPI on CXCR7 expression and TEM. We conclude that TFPI may contribute to organ infiltration in CLL patients.

• Artunc, Ferruh; Bohnert, Bernhard; Schneider, j; Staudner, Tobias; Sure, Florian & Ilyaskin, a [Show all 15 contributors for this article] (2021). Proteolytic activation of the epithelial sodium channel (ENaC) by factor VII activating protease (FSAP) and its relevance for sodium retention in nephrotic mice. Pflügers Archiv: European Journal of Physiology. ISSN 0031-6768. doi: 10.1007/s00424-021-02639-7. Full text in Research Archive Show summary

  Abstract 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.

• Gramstad, Olav Rogde; Kandanur, Sai Priya Sarma; Etscheid, Michael; Nielsen, Erik Waage & Kanse, Sandip (2021). Factor VII activating protease (FSAP) is not essential in the pathophysiology of angioedema in patients with C1 inhibitor deficiency. Molecular Immunology. ISSN 0161-5890. 142, p. 95–104. doi: 10.1016/j.molimm.2021.11.019. Full text in Research Archive Show summary

  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.<p> <p>Objective: To investigate the interaction between FSAP and C1 inhibitor (C1Inh) and evaluate the potential role of FSAP in HAE with C1Inh deficiency.<p> <p>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.<p> <p>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.<p> <p>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.

• Uhl, B; Mittmann, Laura A.; Dominik, Julian; Hennel, Roman; Smiljanov, Bojan & Haring, Florian [Show all 23 contributors for this article] (2021). uPA-PAI-1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils. EMBO Molecular Medicine. ISSN 1757-4676. 13. doi: 10.15252/emmm.202013110. Full text in Research Archive Show summary

  High intratumoral levels of urokinase-type plasminogen activator (uPA)-plasminogen activator inhibitor-1 (PAI-1) heteromers predict impaired survival and treatment response in early breast cancer. The pathogenetic role of this protein complex remains obscure. Here, we demonstrate that heteromerization of uPA and PAI-1 multiplies the potential of the single proteins to attract pro-tumorigenic neutrophils. To this end, tumor-released uPA-PAI-1 utilizes very low-density lipoprotein receptor and mitogen-activated protein kinases to initiate a pro-inflammatory program in perivascular macrophages. This enforces neutrophil trafficking to cancerous lesions and skews these immune cells toward a pro-tumorigenic phenotype, thus supporting tumor growth and metastasis. Blockade of uPA-PAI-1 heteromerization by a novel small-molecule inhibitor interfered with these events and effectively prevented tumor progression. Our findings identify a therapeutically targetable, hitherto unknown interplay between hemostasis and innate immunity that drives breast cancer progression. As a personalized immunotherapeutic strategy, blockade of uPA-PAI-1 heteromerization might be particularly beneficial for patients with highly aggressive uPA-PAI-1high tumors.

• Kara, Emrah; Nielsen, Nis Valentin; Eggertsdottir, Bergrun; Thiede, Bernd; Kanse, Sandip & Løset, Geir Åge (2020). Design and characterization of a new pVII combinatorial phage display peptide library for protease substrate mining using Factor VII activating protease (FSAP) as model. ChemBioChem. ISSN 1439-4227. 21(13), p. 1875–1884. doi: 10.1002/cbic.201900705. Full text in Research Archive Show summary

  We describe a novel, easy and efficient combinatorial phage display peptide substrate‐mining method to map the substrate specificity of proteases. The peptide library is displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, in an unmodified state. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti‐FLAG mAbs and its independency of any post‐translational modification. This library was tested with Factor‐VII activating protease (WT‐FSAP) and its single‐nucleotide polymorphism variant Marburg‐I (MI)‐FSAP. The WT‐FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI‐FSAP as a loss‐of‐function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified. Indeed one of these peptides was cleaved as free peptide, thus suggesting a broader range of WT‐FSAP substrates than previously anticipated.

• Zuchtriegel, G.; Uhl, B; Ramsauer, Michaela; Dominik, Julian; Mittmann, L & Canis, Martin [Show all 10 contributors for this article] (2020). Vitronectin stabilizes intravascular adhesion of neutrophils by coordinating beta2 integrin clustering. Haematologica. ISSN 0390-6078. doi: 10.3324/haematol.2019.226241. Full text in Research Archive Show summary

  The recruitment of neutrophils from the microvasculature to the site of injury or infection represents a key event in the inflammatory response. Vitronectin (VN) is a multifunctional macromolecule abundantly present in blood and extracellular matrix. The role of this glycoprotein in the extravasation process of circulating neutrophils remains elusive. Employing advanced in vivo/ex vivo imaging techniques in different mouse models as well as in vitro methods, we uncovered a previously unrecognized function of VN in the transition of dynamic to static intravascular interactions of neutrophils with microvascular endothelial cells. These distinct properties of VN require the heteromerization of this glycoprotein with plasminogen activator inhibitor-1 (PAI-1) on the activated venular endothelium and subsequent interactions of this protein complex with the scavenger receptor low-density lipoprotein receptor-related protein-1 (LRP-1) on intravascularly adhering neutrophils. This induces p38 mitogen-activated protein kinases (MAPK)-dependent intracellular signaling events which, in turn, regulates the proper clustering of the beta2 integrin lymphocyte function associated antigen-1 (LFA-1) on the surface of these immune cells. As a consequence of this molecular interplay, neutrophils become able to stabilize their adhesion to the microvascular endothelium and, subsequently, to extravasate to the perivascular tissue. Hence, endothelial- bound VN-PAI-1 heteromers stabilize intravascular adhesion of neutrophils by coordinating beta2 integrin clustering on the surface of these immune cells, thereby effectively controlling neutrophil trafficking to inflamed tissue. Targeting this protein complex might be beneficial for the prevention and treatment of inflammatory pathologies.

• Parahuleva, Mariana S; Worsch, M; Euler, Gerhild; Choukeir, Maryana; Mardini, Amar & Parviz, Behnoush [Show all 11 contributors for this article] (2020). Factor VII Activating Protease Expression in Human Platelets and Accumulation in Symptomatic Carotid Plaque. Journal of the American Heart Association (JAHA). 9. doi: 10.1161/JAHA.120.016445. Full text in Research Archive Show summary

  Background Factor VII activating protease (FSAP) is of interest as a marker for vascular inflammation and plaque destabilization. The aim of this study was to analyze the expression profile of FSAP in endarterectomy specimens that were taken from patients with asymptomatic and symptomatic carotid atherosclerotic plaques and to compare them with circulating FSAP levels. Methods and Results Plasma FSAP concentration, activity, and mRNA expression were measured in endarterectomy specimens and in monocytes and platelets. Plaque and plasma FSAP levels were higher in symptomatic patients (n=10) than in asymptomatic patients (n=14). Stronger FSAP immunostaining was observed in advanced symptomatic lesions, in intraplaque hemorrhage‐related structures, and in lipid‐rich areas within the necrotic core. FSAP was also colocalized with monocytes and macrophages (CD11b/CD68‐positive cells) and platelets (CD41‐positive cells) of the plaques. Moreover, human platelets expressed FSAP in vitro, at both the mRNA and protein levels. Expression is stimulated by thrombin receptor‐activating peptide and ADP and reduced by acetylsalicylic acid. Conclusions Plasma FSAP levels were significantly increased in patients with symptomatic carotid stenosis and thus may be involved in plaque development This plaque‐associated FSAP may be produced by platelets or macrophages or may be taken up from the circulation. To establish FSAP’s utility as a circulating or plaque biomarker in patients with symptomatic carotid atherosclerotic plaques, further studies are needed.

• Byskov, Kristina; Etscheid, Michael & Kanse, Sandip (2020). Cellular effects of factor VII activating protease (FSAP). Thrombosis Research. ISSN 0049-3848. 188, p. 74–78. doi: 10.1016/j.thromres.2020.02.010. Full text in Research Archive Show summary

  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.

• Ueslue, Ouezguer; Herold, Joerg & Kanse, Sandip (2019). VEGF-A-Cleavage by FSAP and Inhibition of Neo-Vascularization. Cells. doi: 10.3390/cells8111396. Full text in Research Archive Show summary

  Alternative splicing leads to the secretion of multiple forms of vascular endothelial growth factor-A (VEGF-A) that differ in their activity profiles with respect to neovascularization. FSAP (factor VII activating protease) is the zymogen form of a plasma protease that is activated (FSAPa) upon tissue injury via the release of histones. The purpose of the study was to determine if FSAPa regulates VEGF-A activity in vitro and in vivo. FSAP bound to VEGF165, but not VEGF121, and VEGF165 was cleaved in its neuropilin/proteoglycan binding domain. VEGF165 cleavage did not alter its binding to VEGF receptors but diminished its binding to neuropilin. The stimulatory effects of VEGF165 on endothelial cell proliferation, migration, and signal transduction were not altered by FSAP. Similarly, proliferation of VEGF receptor-expressing BAF3 cells, in response to VEGF165, was not modulated by FSAP. In the mouse matrigel model of angiogenesis, FSAP decreased the ability of VEGF165, basic fibroblast growth factor (bFGF), and their combination, to induce neovascularization. Lack of endogenous FSAP in mice did not influence neovascularization. Thus, FSAP inhibited VEGF165-mediated angiogenesis in the matrigel model in vivo, where VEGF’s interaction with the matrix and its diffusion are important.

• Nielsen, Nis Valentin; Roedel, Elfie; Manna, Dipankar; Etscheid, Michael; Morth, Jens Preben & Kanse, Sandip (2019). Characterization of the enzymatic activity of the serine protease domain of Factor VII activating protease (FSAP). Scientific Reports. 9(1). doi: 10.1038/s41598-019-55531-x. Full text in Research Archive Show summary

  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.

• Byskov, Kristina; Gall, Sylvain M. le; Thiede, Bernd; Camerer, Eric & Kanse, Sandip (2019). Protease activated receptors (PAR)‐1 and ‐2 mediate cellular effects of factor VII activating protease (FSAP). The FASEB Journal. ISSN 0892-6638. 34, p. 1079–1090. doi: 10.1096/fj.201801986RR. Full text in Research Archive Show summary

  Factor VII activating protease (FSAP) is a circulating serine protease implicated in thrombosis, atherosclerosis, stroke, and cancer. Using an overexpression strategy, we have systematically investigated the role of protease activated receptors (PAR)‐1, ‐2, ‐3, and ‐4 on FSAP‐mediated signaling in HEK293T and A549 cells. Cleavage of PAR‐reporter constructs and MAPK phosphorylation was used to monitor receptor activation. FSAP cleaved PAR‐2 and to a lesser degree PAR‐1, but not PAR‐3 or PAR‐4 in both cell types. Robust MAPK activation in response to FSAP was observed after PAR‐2, but not PAR‐1 overexpression in HEK293T. Recombinant serine protease domain of wild type FSAP, but not the Marburg I isoform of FSAP, could reproduce the effects of plasma purified FSAP. Canonical cleavage of both PARs was suggested by mass spectrometric analysis of synthetic peptide substrates from the N‐terminus of PARs and site directed mutagenesis studies. Surprisingly, knockdown of endogenous PAR‐1, but not PAR‐2, prevented the apoptosis‐inhibitory effect of FSAP, suggesting that PAR1 is nevertheless a direct or indirect target in some cell types. This molecular characterization of PAR‐1 and ‐2 as cellular receptors of FSAP will help to define the actions of FSAP in the context of cancer and vascular biology.

• Bohnert, Bernhard; Daiminger, Sophie; Woern, Matthias; Sure, Florian; Staudner, Tobias & Ilyaskin, Alexandr [Show all 14 contributors for this article] (2019). Urokinase-type Plasminogen Activator (uPA) Is Not Essential for Epithelial Sodium Channel (ENaC)-mediated Sodium Retention in Experimental Nephrotic Syndrome. Acta Physiologica. ISSN 1748-1708. 227(4). doi: 10.1111/apha.13286. Full text in Research Archive
• Rut, Wioletta; Nielsen, Nis Valentin; Czarna, Justyna; Poreba, Marcin; Kanse, Sandip & Drag, Marcin (2019). Fluorescent Activity-Based Probe for the Selective Detection of Factor VII Activating Protease (FSAP) in Human Plasma. Thrombosis Research. ISSN 0049-3848. 182, p. 124–132. doi: 10.1016/j.thromres.2019.08.016. Full text in Research Archive Show summary

  The zymogen form of circulating Factor VII activating protease (FSAP) is activated by histones that are released as a consequence of tissue damage or excessive inflammation. This is likely to have consequences in a number of disease conditions such as stroke, atherosclerosis, liver fibrosis, thrombosis and cancer. To investigate the existence, as well as the concentration of active FSAP (FSAPa) in complex biological systems an active site probe is needed. We used Hybrid Combinatorial Substrate Library (HyCoSuL) to screen for natural and unnatural amino acids that specifically bind to P4-P2 pockets of FSAPa. This information was used to designing a fluorogenic substrate (Ac-Pro-DTyr-Lys-Arg-ACC) as well as an irreversible, fluorogenic activity-based probe Cy5-6-Ahx-Pro-DTyr-Lys-ArgP(OPh)2. In normal human plasma the probe showed very low non-specific reactivity with some plasma proteins but upon activation of pro-FSAP with histones, strong labelling of FSAPa was observed. This labelling could be inhibited by aprotinin and was not found in the plasma of a subject that was homozygous for a polymorphism, which leads to loss of activity, or in plasma that was depleted of FSAP by antibodies. This 2nd generation substrate exhibited 6-fold higher catalytic efficiency than the 1st generation substrate and a much higher selectivity for FSAPa over other plasma proteases. This substrate and probe can be useful to detect and localize FSAPa in normal and pathological tissue and plasma to gain more insight into its functions.

• Dahm, Anders Erik A; Jacobsen, Eva-Marie; Wik, Hilde Skuterud; Jacobsen, Anne Flem; Mollnes, Tom Eirik & Kanse, Sandip [Show all 7 contributors for this article] (2019). Elevated complement C3 and C4 levels are associated with postnatal pregnancy-related venous thrombosis. Thrombosis and Haemostasis. ISSN 0340-6245. 119(9), p. 1481–1488. doi: 10.1055/s-0039-1692426. Full text in Research Archive Show summary

  High levels of complement C3 are associated with venous thrombosis (VT) in the general population. We investigated if high C3 and C4 levels were associated with pregnancy-related VT. We undertook the Norwegian VIP study, a case–control study of VT in pregnancy or within 3 months postpartum (cases, n = 313) and women without pregnancy-related VT (controls, n = 353). Determinants of C3 and C4 in the control women were investigated with linear regression and the odds ratio (OR) for pregnancy-related VT was calculated with logistic regression. We found that levels of C3 and C4 were associated with body mass index (BMI), C-reactive protein (CRP); with the coagulation factors (F) fibrinogen, FVIII, and FIX; and with the coagulation inhibitors antithrombin, protein C, protein S, and tissue factor pathway inhibitor. These associations were influenced by CRP levels. The crude OR for pregnancy-related VT was 1.8 (95% confidence interval [CI], 1.1–3.0) for C3 above the 90th percentile and 2.0 (95% CI, 1.2–3.2) for C4 above the 90th percentile. Stratification in antenatal and postnatal VT showed that C3 and C4 were only associated with postnatal VT with an OR for high C3 of 3.0 (95% CI, 1.8–5.0), and for high C4 of 2.6 (95% CI, 1.5–4.6). Adjustment for high FIX and BMI reduced the ORs. We conclude that the association between postnatal VT and C3 and C4 suggests that there is clinically relevant crosstalk between the complement and the coagulation system.

• Subramaniam, Saravanan; Kanse, Sandip; Kothari, Hema; Reinhardt, Christoph & Craig, Fletcher (2018). Post-transcriptional, post-translational and pharmacological regulation of tissue factor pathway inhibitor. Blood Coagulation and Fibrinolysis. ISSN 0957-5235. 29(8), p. 668–682. doi: 10.1097/MBC.0000000000000775. Full text in Research Archive
• Cole, John W.; Kanse, Sandip; Authors, Other & Mitchell, Braxton (2018). Genetics of the thrombomodulin-endothelial cell protein C receptor system and the risk of early-onset ischemic stroke. PLOS ONE. doi: 10.1371/journal.pone.0206554. Full text in Research Archive Show summary

  Background and purpose Polymorphisms in coagulation genes have been associated with early-onset ischemic stroke. Here we pursue an a priori hypothesis that genetic variation in the endothelial-based receptors of the thrombomodulin−protein C system (THBD and PROCR) may similarly be associated with early-onset ischemic stroke. We explored this hypothesis utilizing a multi-stage design of discovery and replication. Methods Discovery was performed in the Genetics-of-Early-Onset Stroke (GEOS) Study, a biracial population-based case-control study of ischemic stroke among men and women aged 15–49 including 829 cases of first ischemic stroke (42.2% African-American) and 850 age-comparable stroke-free controls (38.1% African-American). Twenty-four single-nucleotide-polymorphisms (SNPs) in THBD and 22 SNPs in PROCR were evaluated. Following LD pruning (r2≥0.8), we advanced uncorrelated SNPs forward for association analyses. Associated SNPs were evaluated for replication in an early-onset ischemic stroke population (onset-age<60 years) consisting of 3676 cases and 21118 non-stroke controls from 6 case–control studies. Lastly, we determined if the replicated SNPs also associated with older-onset ischemic stroke in the METASTROKE data-base. Results Among GEOS Caucasians, PROCR rs9574, which was in strong LD with 8 other SNPs, and one additional independent SNP rs2069951, were significantly associated with ischemic stroke (rs9574, OR = 1.33, p = 0.003; rs2069951, OR = 1.80, p = 0.006) using an additive-model adjusting for age, gender and population-structure. Adjusting for risk factors did not change the associations; however, associations were strengthened among those without risk factors. PROCR rs9574 also associated with early-onset ischemic stroke in the replication sample (OR = 1.08, p = 0.015), but not older-onset stroke. There were no PROCR associations in African-Americans, nor were there any THBD associations in either ethnicity. Conclusion PROCR polymorphisms are associated with early-onset ischemic stroke in Caucasians.

• Olsson, Maja; Stanne, Tara M.; Pedersén, Annie; Lorentzen, Erik; Kara, Emrah & Martinez-Palaciàn, Adoraciòn [Show all 14 contributors for this article] (2018). Genome-wide analysis of genetic determinants of circulating factor VII-activating protease (FSAP) activity. Journal of Thrombosis and Haemostasis. ISSN 1538-7933. 16(10), p. 2024–2034. doi: 10.1111/jth.14258. Full text in Research Archive Show summary

  Background Factor VII‐activating protease (FSAP) has roles in both coagulation and fibrinolysis. Recent data indicate its involvement in several other processes, such as vascular remodeling and inflammation. Plasma FSAP activity is highly variable among healthy individuals and, apart from the low‐frequency missense variant Marburg‐I (rs7080536) in the FSAP‐encoding gene HABP2, determinants of this variation are unclear. Objectives To identify novel genetic variants within and outside of the HABP2 locus that influence circulating FSAP activity. Patients/Methods We performed an exploratory genome‐wide association study (GWAS) on plasma FSAP activity amongst 3230 Swedish subjects. Directly genotyped rare variants were also analyzed with gene‐based tests. Using GWAS, we confirmed the strong association between the Marburg‐I variant and FSAP activity. HABP2 was also significant in the gene‐based analysis, and remained significant after exclusion of Marburg‐I carriers. This was attributable to a rare HABP2 stop variant (rs41292628). Carriers of this stop variant showed a similar reduction in FSAP activity as Marburg‐I carriers, and this finding was replicated. A secondary genome‐wide significant locus was identified at a 5p15 locus (rs35510613), and this finding requires future replication. This common variant is located upstream of ADCY2, which encodes a protein catalyzing the formation of cAMP. Results and Conclusions This study verified the Marburg‐I variant to be a strong regulator of FSAP activity, and identified an HABP2 stop variant with a similar impact on FSAP activity. A novel locus near ADCY2 was identified as a potential additional regulator of FSAP activity.

• Praetner, M; Zuchtriegel, G; Holzer, M; Uhl, B; Schaubaecher, J & Mittmann, L [Show all 16 contributors for this article] (2018). Plasminogen Activator Inhibitor-1 Promotes Neutrophil Infiltration and Tissue Injury on Ischemia-Reperfusion. Arteriosclerosis, Thrombosis and Vascular Biology. ISSN 1079-5642. 38(4), p. 829–842. doi: 10.1161/ATVBAHA.117.309760. Full text in Research Archive
• Etscheid, Michael; Subramaniam, Saravanan; Lochnit, Guenter; Zabczyk, Michał; Undas, Anetta & Lang, Irene M. [Show all 8 contributors for this article] (2018). Altered structure and function of fibrinogen after cleavage by Factor VII Activating Protease (FSAP). Biochimica et Biophysica Acta. ISSN 0006-3002. 1864(10), p. 3397–3406. doi: 10.1016/j.bbadis.2018.07.030. Full text in Research Archive
• Sperling, C; Maitz, MF; Grasso, Simona; Werner, C & Kanse, Sandip (2017). A Positively Charged Surface Triggers Coagulation Activation Through Factor VII Activating Protease (FSAP). ACS Applied Materials & Interfaces. ISSN 1944-8244. 9(46), p. 40107–40116. doi: 10.1021/acsami.7b14281. Full text in Research Archive Show summary

  Contact between biomedical materials and blood often initiates undesirable pro-coagulant and pro-inflammatory processes. On negatively charged materials, blood coagulation is known to be triggered through autoactivation of Factor XII, while activation on cationic surfaces follows a distinct and so far enigmatic mechanism. Because Factor VII activating protease (FSAP) is known to be activated on positively and on negatively charged macromolecules in plasma, we have investigated its interaction with charged biomaterials and its consequences for coagulation. Several activation processes in blood and plasma were characterized after contact with material surfaces with varied charge. FSAP was found to be exclusively activated by the positively charged surfaces polyethylenimine (PEI) and poly-l-lysine (PLL), not by the negatively charged glass or self-assembled monolayer with carboxyl group termination (SAM-COOH), as well as uncharged (Teflon AF) surfaces. Whole blood incubation on PEI showed that this activation was concomitant with coagulation as determined by thrombin and fibrin formation, which was high for glass (F1+2, 138 nM) and PEI (F1+2, 44 nM) but low for Teflon AF (F1+2, 3.3 nM) and SAM COOH (F1+2, 5.8 nM). Contact phase inhibitor diminished coagulation to background levels for all surfaces except PEI (F1+2: ^PEI 43 to 25 nM; glass, 58 to 1.5 nM) indicating that coagulation activation is not dependent on FXII activation on the PEI surface. A decisive role of endogenous FSAP for coagulation however was confirmed with the use of FSAP inhibitory antibodies which showed no influence on Teflon AF, glass and SAM COOH but diminished F1+2 on PEI to less than 50%. We propose that FSAP activation could be a novel mechanism of surface-driven coagulation. An inhibition of this protease might improve hemocompatibility of cationic surfaces and therefore facilitate the application of polycationic surfaces in blood. This research has been published in ACS Applied Materials and Interfaces. © 2017 American Chemical Society

• Grasso, Simona; Neumann, A; Lang, IM; Etscheid, Michael; Köckritz-Blickwede, M von & Kanse, Sandip (2017). Interaction of factor VII activating protease (FSAP) with neutrophil extracellular traps (NETs). Thrombosis Research. ISSN 0049-3848. 161, p. 36–42. doi: 10.1016/j.thromres.2017.11.012. Full text in Research Archive Show summary

  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.

• Reiterer-Farhan, Veronika; Figueras‐Puig, Cristina; Guerroue,, Francois Le; Confalonieri, Stefano; Vecchi, Manuela & Jalapothu, Dasaradha [Show all 11 contributors for this article] (2017). The pseudophosphatase STYX targets the F‐box of FBXW7 and inhibits SCFFBXW7 function. EMBO Journal. ISSN 0261-4189. 36(3), p. 260–273. doi: 10.15252/embj.201694795. Full text in Research Archive
• Byskov, Kristina; Boettger, Thomas; Ruehle, Paul F.; Nielsen, Nis Valentin; Etscheid, Michael & Kanse, Sandip (2017). Factor VII activating protease (FSAP) regulates the expression of inflammatory genes in vascular smooth muscle and endothelial cells. Atherosclerosis. ISSN 0021-9150. 265, p. 133–139. doi: 10.1016/j.atherosclerosis.2017.08.029. Full text in Research Archive Show summary

  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.

• Kara, Emrah; Manna, Dipankar; Løset, Geir Åge; Schneider, E L; Craik, C S & Kanse, Sandip (2017). Analysis of the substrate specificity of Factor VII activating protease (FSAP) and design of specific and sensitive peptide substrates. Thrombosis and Haemostasis. ISSN 0340-6245. doi: 10.1160/TH17-02-0081. Full text in Research Archive Show summary

  Factor VII (FVII) activating protease (FSAP) is a circulating serine protease that is likely to be involved in a number of disease conditions such as stroke, atherosclerosis, liver fibrosis, thrombosis and cancer. To date, no systematic information is available about the substrate specificity of FSAP. Applying phage display and positional scanning substrate combinatorial library (PS-SCL) approaches we have characterised the specificity of FSAP towards small peptides. Results were evaluated in the context of known protein substrates as well as molecular modelling of the peptides in the active site of FSAP. The representative FSAP-cleaved sequence obtained from the phage display method was Val-Leu-Lys-Arg-Ser (P4-P1’). The sequence X-Lys/Arg-Nle-Lys/Arg (P4-P1) was derived from the PS-SCL method. These results show a predilection for cleavage at a cluster of basic amino acids on the nonprime side. Quenched fluorescent substrate (Ala-Lys-Nle-Arg-AMC) (amino methyl coumarin) and (Ala-Leu-Lys-Arg-AMC) had a higher selectivity for FSAP compared to other proteases from the hemostasis system. These substrates could be used to measure FSAP activity in a complex biological system such as plasma. In histonetreated plasma there was a specific activation of pro-FSAP as validated by the use of an FSAP inhibitory antibody, corn trypsin inhibitor to inhibit Factor XIIa and hirudin to inhibit thrombin, which may account for some of the haemostasis-related effects of histones. These results will aid the development of further selective FSAP activity probes as well as specific inhibitors that will help to increase the understanding of the functions of FSAP in vivo. This research has been published in Thrombosis and Haemostasis. © 2017 Schattauer

• Serrano, Sandra Espada; Stavik, Benedicte; Holm, Sverre; Sagen, Ellen Lund; Bjerkeli, Vigdis & Skjelland, Mona [Show all 12 contributors for this article] (2017). Tissue factor pathway inhibitor attenuates ER stress-induced inflammation in human M2-polarized macrophages. Biochemical and Biophysical Research Communications - BBRC. ISSN 0006-291X. 491(2), p. 442–448. doi: 10.1016/j.bbrc.2017.07.070. Full text in Research Archive Show summary

  Endoplasmic reticulum (ER) stress has been shown to play a key role during the initiation and clinical progression of the cardiovascular diseases, such as atherosclerosis. We have recently shown that expression of tissue factor pathway inhibitor (TFPI) in human monocyte-derived macrophages (MDMs) was induced by cholesterol crystals (CC). In the present study we aimed to determine the role of TFPI under ER stress conditions using human MDMs. qRT-PCR and immunohistochemistry analysis were performed to determine the presence of the ER stress marker CCAAT/enhancer binding protein homologous protein (CHOP) and TFPI in human carotid plaque material and also in human MDMs polarized into pro-inflammatory M1 or anti-inflammatory M2 populations. CHOP mRNA levels were upregulated in the plaques compared to healthy vessels, and CHOP protein was localized in the same area as TFPI in the plaques. Both CHOP and TFPI mRNA levels were upregulated after CC treatment, especially in the M2 phenotype, and the ER stress inhibitor 4-phenylbutyric acid (PBA) reversed this effect. Furthermore, CC treatment increased the levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-8, which for TNF-α and IL-8 was inhibited by PBA, and reduced the levels of the anti-inflammatory cytokine IL-10 in M2-polarized macrophages. Knockdown of TFPI prior to CC treatment exacerbated TNF-α and IL-6 levels, but reduced IL-8 and IL-10 levels. Our results show that CC induce TFPI and cytokine expression in M2-polarized macrophages through activation of the ER stress pathway and that TFPI has a protective effect against TNF-α and IL-6 mediated inflammation. These mechanisms may have implications for the pathogenesis of atherosclerosis.

• Herold, Joerg; Nowak, Steven; Kostin, Sawa; Daniel, Jan-Marcus; Francke, Alexander & Subramaniam, Saravanan [Show all 8 contributors for this article] (2017). Factor VII activating protease (FSAP) influences vascular remodeling in the mouse hind limb ischemia model. American journal of translational research. 9(6), p. 3084–3095. Full text in Research Archive
• Leiting, Silke; Seidl, Sebastian; Martinez-Palaciàn, Adoraciòn; Muhl, Lars & Kanse, Sandip (2016). Transforming growth factor-β (TGF-β) inhibits the expression of factor VII-activating protease (FSAP) in hepatocytes. Journal of Biological Chemistry. ISSN 0021-9258. 291(40), p. 21020–21028. doi: 10.1074/jbc.M116.744631. Full text in Research Archive

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• Bohnert, Bernhard; Kanse, Sandip; Haerteis, Silke; Korbmacher, Christoph & Artunc, Ferruh (2019). Rebuttal to Editorial: Sodium Retention by uPA in Nephrotic Syndrome? Acta Physiologica. ISSN 1748-1708. doi: 10.1111/apha.13427. Full text in Research Archive
• Cui, Xueyan; Tjønnfjord, Geir Erland; Kanse, Sandip; Iversen, Nina; Dahm, Anders Erik A & Myklebust, Christiane Filion [Show all 11 contributors for this article] (2019). Tissue factor pathway inhibitor enhances transendothelial migration of chronic lymphocytic leukemia cells trhough CXCL12/CXCR7 chemotaxis. Research and Practice in Thrombosis and Haemostasis (RPTH). 3. Full text in Research Archive

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