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

The influence of the hyaluronanbinding 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 procofactor 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 Nterminus 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 D5(H) and D6(H) 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

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

publishedVersio

Untersuchungen zur Thrombogenität von therapeutischen Immunglobulinen

Eine Forschergruppe aus der Abteilung Hämatologie des Paul-Ehrlich-Instituts (PEI) arbeitet seit Jahren intensiv an der Entwicklung von Methoden zum Nachweis möglicher Thrombogenität in Blutprodukten. Eines der Ziele besteht in der Entwicklung eines universellen Thrombin-Generierungstests, der es ermöglicht, im Labor ein erhöhtes Thromboserisiko durch aktivierte Gerinnungsfaktoren in diesen Produkten zu erkennen. Die Bedeutung eines solchen Tests für die Arzneimittelsicherheit wurde im August letzten Jahres deutlich, als gehäuft thromboembolische Komplikationen nach Gabe eines intravenösen Immunglobulinpräparates gemeldet wurden. Durch Untersuchungen des betroffenen Präparates mit Hilfe bereits etablierter sowie neuartiger Tests durch Hersteller, PEI und andere Prüflaboratorien konnte die Ursache für die thromboembolischen Komplikationen identifiziert werden. Der Hersteller führte Änderungen im Herstellungsprozess ein, wodurch eine erhöhte Thrombogenität des Produktes vermieden werden kann

The Marburg I polymorphism of factor VII activating protease is associated with low proteolytic and low pro-coagulant activity

Cardiolog

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

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

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