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

      Highlights

      • Development of a robust, simple and highly specific FSAP generation assay (FGA)
      • Carriers of the Marburg I-polymorphism can be identified by strongly delayed FSAP generation, low peak FSAP and low FSAP potential
      • Delayed FSAP generation significantly correlated with VT in post-menopausal women after excluding Marburg I carriers

      Abstract

      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.

      Graphical abstract

      Abbreviations:

      AMC (aminomethyl coumarin), AUC (area under the curve), CTI (corn trypsin inhibitor), EFP (endogenous FSAP potential), FG(A) (FSAP generation (assay)), FSAP (Factor VII Activating Protease), MI-SNP (Marburg I single nucleotide polymorphism), RFU (relative fluorescence unit), TGA (thrombin generation assay), TtPk (time to peak), VT (venous thrombosis)

      Keywords

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      References

        • Choi-Miura N.H.
        • Tobe T.
        • Sumiya J.
        • Nakano Y.
        • Sano Y.
        • Mazda T.
        • Tomita M.
        Purification and characterization of a novel hyaluronan-binding protein (PHBP) from human plasma: it has three EGF, a kringle and a serine protease domain, similar to hepatocyte growth factor activator.
        J. Biochem. 1996; 119: 1157-1165
        • Hunfeld A.
        • Etscheid M.
        • König H.
        • Seitz R.
        • Dodt J.
        Detection of a novel plasma serine protease during purification of vitamin K-dependent coagulation factors.
        FEBS Lett. 1999; 456: 290-294
        • Römisch J.
        • Feußner A.
        • Vermöhlen S.
        • Stöhr H.-A.
        A protease isolated from human plasma activating factor VII independent of tissue factor.
        Blood Coagul. Fibrinolysis. 1999; 10: 471-479
        • Römisch J.
        • Feussner A.
        • Stöhr H.-A.
        Quantification of the factor VII-and single chain plasminogen activator-activating protease in plasmas of healthy subjects.
        Blood Coagul. Fibrinolysis. 2001; 12: 375-383
        • Yamamichi S.
        • Fujiwara Y.
        • Kikuchi T.
        • Nishitani M.
        • Matsushita Y.
        • Hasumi K.
        Extracellular histone induces plasma hyaluronan-binding protein (factor VII activating protease) activation in vivo.
        Biochem. Biophys. Res. Commun. 2011; 409: 483-488
        • Römisch J.
        • Vermöhlen S.
        • Feußner A.
        • Stöhr H.-A.
        The FVII activating protease cleaves single-chain plasminogen activators.
        Haemostasis. 2000; 29: 292-299
        • Kanse S.M.
        • Declerck P.J.
        • Ruf W.
        • Broze G.
        • Etscheid M.
        Factor VII-activating protease promotes the proteolysis and inhibition of tissue factor pathway inhibitor.
        Arterioscler. Thromb. Vasc. Biol. 2012; 32: 427-433
        • Etscheid M.
        • Subramaniam S.
        • Lochnit G.
        • Zabczyk M.
        • Undas A.
        • Lang I.M.
        • Hanschmann K.-M.
        • Kanse S.M.
        Altered structure and function of fibrinogen after cleavage by factor VII activating protease (FSAP).
        Biochim. Biophys. Acta Mol. basis Dis. 1864; 2018: 3397-3406https://doi.org/10.1016/j.bbadis.2018.07.030
        • Kanse S.M.
        • Gallenmueller A.
        • Zeerleder S.
        • Stephan F.
        • Rannou O.
        • Denk S.
        • Etscheid M.
        • Lochnit G.
        • Krueger M.
        • Huber-Lang M.
        Factor VII-activating protease is activated in multiple trauma patients and generates anaphylatoxin C5a.
        J. Immunol. 2012; 188: 2858-2865https://doi.org/10.4049/jimmunol.1103029
        • Römisch J.
        • Feussner A.
        • Nerlich C.
        • Stoehr H.A.
        • Weimer T.
        The frequentMarburg I polymorphism impairs the pro-urokinase activating potency of the factor VII activating protease (FSAP).
        Blood Coagul. Fibrinolysis. 2002; 13: 433-441
        • Etscheid M.
        • Muhl L.
        • Pons D.
        • Jukema J.W.
        • König H.
        • Kanse S.M.
        The Marburg I polymorphism of factor VII activating protease is associated with low proteolytic and low pro-coagulant activity.
        Thromb. Res. 2012; 130: 935-941https://doi.org/10.1016/j.thromres.2012.07.023
        • Willeit J.
        • Kiechl S.
        • Weimer T.
        • Mair A.
        • Santer P.
        • Wiedermann C.J.
        • Roemisch J.
        Marburg I polymorphism of factor VII–activating protease: a prominent risk predictor of carotid stenosis.
        Circulation. 2003; 107: 667-670
        • Trompet S.
        • Pons D.
        • Kanse S.M.
        • de Craen A.J.
        • Ikram M.A.
        • Verschuren J.J.
        • Zwinderman A.H.
        • Doevendans P.A.
        • Tio R.A.
        • de Winter R.J.
        • Slagboom P.E.
        • Westendorp R.G.
        • Jukema J.W.
        Factor VII activating protease polymorphism (G534E) is associated with increased risk for stroke and mortality.
        Stroke Res. Treat. 2011; 2011424759
        • Ireland H.
        • Miller G.J.
        • Webb K.E.
        • Cooper J.A.
        • Humphries S.E.
        The factor VII activating protease G511E (Marburg) variant and cardiovascular risk.
        Thromb. Haemost. 2004; 92: 986-992
        • Wasmuth H.E.
        • Tag C.G.
        • van de L.E.
        • Hellerbrand C.
        • Mueller T.
        • Berg T.
        • Puhl G.
        • Neuhaus P.
        • Samuel D.
        • Trautwein C.
        • Kanse S.M.
        • Weiskirchen R.
        The Marburg I variant (G534E) of the factor VII-activating protease determines liver fibrosis in hepatitis C infection by reduced proteolysis of platelet-derived growth factor BB.
        Hepatology. 2009; 49: 775-780
        • Ahmad-Nejad P.
        • Dempfle C.E.
        • Weiss C.
        • Bugert P.
        • Borggrefe M.
        • Neumaier M.
        The G534E-polymorphism of the gene encoding the factor VII-activating protease is a risk factor for venous thrombosis and recurrent events.
        Thromb. Res. 2012; 130: 441-444
        • Hoppe B.
        • Tolou F.
        • Radtke H.
        • Kiesewetter H.
        • Dorner T.
        • Salama A.
        Marburg I polymorphism of factor VII-activating protease is associated with idiopathic venous thromboembolism.
        Blood. 2005; 105: 1549-1551
        • van Minkelen R.
        • de Visser M.C.
        • Vos H.L.
        • Bertina R.M.
        • Rosendaal F.R.
        The Marburg I polymorphism of factor VII-activating protease is not associated with venous thrombosis.
        Blood. 2005; 105: 4898
        • Franchi F.
        • Martinelli I.
        • Biguzzi E.
        • Bucciarelli P.
        • Mannucci P.M.
        Marburg I polymorphism of factor VII-activating protease and risk of venous thromboembolism.
        Blood. 2006; 107: 1731https://doi.org/10.1182/blood-2005-09-3603
        • Gulesserian T.
        • Hron G.
        • Endler G.
        • Eichinger S.
        • Wagner O.
        • Kyrle P.A.
        Marburg I polymorphism of factor VII-activating protease and risk of recurrent venous thromboembolism.
        Thromb. Haemost. 2006; 95: 65-67
        • Weisbach V.
        • Ruppel R.
        • Eckstein R.
        The Marburg I polymorphism of factor VII-activating protease and the risk of venous thromboembolism.
        Thromb. Haemost. 2007; 97: 870-872
        • Pecheniuk N.M.
        • Elias D.J.
        • Xu X.
        • Griffin J.H.
        Failure to validate association of gene polymorphisms in EPCR, PAR-1, FSAP and protein S Tokushima with venous thromboembolism among californians of european ancestry.
        Thromb. Haemost. 2008; 99: 453-455
        • Sidelmann J.J.
        • Vitzthum F.
        • Funding E.
        • Munster A.M.
        • Gram J.
        • Jespersen J.
        Factor VII-activating protease in patients with acute deep venous thrombosis.
        Thromb. Res. 2008; 122: 848-853
        • Olsson M.
        • Stanne T.M.
        • Pedersen A.
        • Lorentzen E.
        • Kara E.
        • Martinez-Palacian A.
        • Rønnow Sand N.P.
        • Jacobsen A.F.
        • Sandset P.M.
        • Sidelmann J.J.
        • Engström G.
        • Melander O.
        • Kanse S.M.
        • Jern C.
        Genome-wide analysis of genetic determinants of circulating factor VII-activating protease (FSAP) activity.
        J. Thromb. Haemost. 2018; 16: 2024-2034https://doi.org/10.1111/jth.14258
        • Joshi A.U.
        • Orset C.
        • Engelhardt B.
        • Baumgart-Vogt E.
        • Gerriets T.
        • Vivien D.
        • Kanse S.M.
        Deficiency of factor VII activating protease alters the outcome of ischemic stroke in mice.
        Eur. J. Neurosci. 2015; : 965-975https://doi.org/10.1111/ejn.12830
        • Tian D.-S.
        • Qin C.
        • Zhou L.-Q.
        • Yang S.
        • Chen M.
        • Xiao J.
        • Shang K.
        • Bosco D.B.
        • Wu L.-J.
        • Wang W.
        FSAP aggravated endothelial dysfunction and neurological deficits in acute ischemic stroke due to large vessel occlusion.
        Signal Transduct. Target. Ther. 2022; 7: 6https://doi.org/10.1038/s41392-021-00802-1
        • Parahuleva M.S.
        • Ball N.
        • Parviz B.
        • Zandt D.
        • Abdallah Y.
        • Tillmanns H.
        • Hoelschermann H.
        • Kanse S.M.
        Factor seven activating protease (FSAP) expression in human placenta and its role in trophoblast migration.
        Eur. J. Obstet. Gynecol. Reprod. Biol. 2013; 167: 34-40https://doi.org/10.1016/j.ejogrb.2012.10.035
        • Stephan F.
        • Hazelzet J.A.
        • Bulder I.
        • Boermeester M.A.
        • van Till J.O.
        • van der Poll T.
        • Wuillemin W.A.
        • Aarden L.A.
        • Zeerleder S.
        Activation of factor VII-activating protease in human inflammation: a sensor for cell death.
        Crit. Care. 2011; 15: R110
        • Stephan S.
        • Schwarz H.
        • Borchert A.
        • Bussfeld D.
        • Quak E.
        • Simshaeuser-Knaub B.
        • Teigelkamp S.
        • Behrens F.
        • Vitzthum F.
        Tests for the measurement of factor VII-activating protease (FSAP) activity and antigen levels in citrated plasma, their correlation to PCR testing, and utility for the detection of the Marburg I-polymorphism of FSAP.
        Clin. Chem. Lab. Med. 2008; 46: 1109-1116
        • Stephan S.
        • Schwarz H.
        • Haude-Barten A.
        • Sidelmann J.J.
        • Fischer B.
        • Althaus H.
        • Hahn M.
        • Kappel A.
        • Ehm M.
        • Vitzthum F.
        Direct chromogenic substrate immuno-capture activity assay for testing of factor VII-activating protease.
        Clin. Chem. Lab. Med. 2011; 49: 1199-1204https://doi.org/10.1515/CCLM.2011.199
        • Gramstad O.R.
        • Kandanur S.P.S.
        • Etscheid M.
        • Nielsen E.W.
        • Kanse S.M.
        Factor VII activating protease (FSAP) is not essential in the pathophysiology of angioedema in patients with C1 inhibitor deficiency.
        Mol. Immunol. 2022; 142: 95-104https://doi.org/10.1016/j.molimm.2021.11.019
        • Rut W.
        • Nielsen N.V.
        • Czarna J.
        • Poreba M.
        • Kanse S.M.
        • Drag M.
        Fluorescent activity-based probe for the selective detection of factor VII activating protease (FSAP) in human plasma.
        Thromb. Res. 2019; 182: 124-132https://doi.org/10.1016/j.thromres.2019.08.016
        • Kintigh J.
        • Monagle P.
        • Ignjatovic V.
        A review of commercially available thrombin generation assays.
        Res. Pract. Thromb. Haemost. 2018; 2: 42-48https://doi.org/10.1002/rth2.12048
        • Hemker H.C.
        • Giesen P.
        • Al Dieri R.
        • Regnault V.
        • de Smedt E.
        • Wagenvoord R.
        • Lecompte T.
        • Béguin S.
        Calibrated automated thrombin generation measurement in clotting plasma.
        Pathophysiol. Haemost. Thromb. 2003; 33: 4-15https://doi.org/10.1159/000071636
        • Høibraaten E.
        • Qvigstad E.
        • Arnesen H.
        • Larsen S.
        • Wickstrøm E.
        • Sandset P.M.
        Increased risk of recurrent venous thromboembolism during hormone replacement therapy–results of the randomized, double-blind, placebo-controlled estrogen in venous thromboembolism trial (EVTET).
        Thromb. Haemost. 2000; 84: 961-967
        • Eilertsen A.L.
        • Sandvik L.
        • Mowinckel M.C.
        • Andersen T.O.
        • Qvigstad E.
        • Sandset P.M.
        Differential effects of conventional and low dose oral hormone therapy (HT), tibolone, and raloxifene on coagulation and fibrinolysis.
        Thromb. Res. 2007; 120: 371-379https://doi.org/10.1016/j.thromres.2006.10.013
        • Morrison J.F.
        Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors.
        Biochim. Biophys. Acta. 1969; 185: 269-286https://doi.org/10.1016/0005-2744(69)90420-3
        • Leatherbarrow R.J.
        GraFit Version 7.
        Erithacus Software, Horley, U.K2009
        • Hanson E.
        • Kanse S.M.
        • Joshi A.
        • Jood K.
        • Nilsson S.
        • Blomstrand C.
        • Jern C.
        Plasma factor VII-activating protease antigen levels and activity are increased in ischemic stroke.
        J. Thromb. Haemost. 2012; 10: 848-856https://doi.org/10.1111/j.1538-7836.2012.04692.x
        • Tapp H.J.
        • Grundmann C.
        • Kusch M.
        • König H.
        Calibrating thrombin generation in different samples: less effort with a less efficient substrate.
        Open Atheroscler. Thromb. J. 2009; : 6-11
        • Chandler W.L.
        • Roshal M.
        Optimization of plasma fluorogenic thrombin-generation assays.
        Am. J. Clin. Pathol. 2009; 132: 169-179https://doi.org/10.1309/AJCP6AY4HTRAAJFQ
        • Marsman G.
        • von Richthofen H.
        • Bulder I.
        • Lupu F.
        • Hazelzet J.
        • Luken B.M.
        • Zeerleder S.
        DNA and factor VII-activating protease protect against the cytotoxicity of histones.
        Blood Adv. 2017; 1: 2491-2502https://doi.org/10.1182/bloodadvances.2017010959
        • Nielsen N.V.
        • Roedel E.
        • Manna D.
        • Etscheid M.
        • Morth J.P.
        • Kanse S.M.
        Characterization of the enzymatic activity of the serine protease domain of factor VII activating protease (FSAP).
        Sci. Rep. 2019; 9: 18990https://doi.org/10.1038/s41598-019-55531-x
        • Stavenuiter F.
        • Dienava-Verdoold I.
        • Boon-Spijker M.G.
        • Brinkman H.J.
        • Meijer A.B.
        • Mertens K.
        Factor seven activating protease (FSAP): does it activate factor VII?.
        J. Thromb. Haemost. 2012; 10: 859-866
        • Hemker H.C.
        • Kremers R.
        Data management in thrombin generation.
        Thromb. Res. 2013; 131: 3-11https://doi.org/10.1016/j.thromres.2012.10.011