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.

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.

Coronary atherosclerosis represents the major cause of death in Western societies. As atherosclerosis typically progresses over years without giving rise to clinical symptoms, biomarkers are urgently needed to identify patients at risk. Over the past decade, evidence has accumulated suggesting cross-talk between the diseased vasculature and cells of the innate immune system. We therefore employed proteomics to search for biomarkers associated with severe atherosclerotic coronary lumen stenosis in circulating leukocytes. In a two-phase approach, we first performed in-depth quantitative profiling of the granulocyte proteome on a small pooled cohort of patients suffering from chronic (sub)total coronary occlusion and matched control patients using stable isotope peptide labeling, two-dimensional LC-MS/MS and data-dependent decision tree fragmentation. Over 3000 proteins were quantified, among which 57 candidate biomarker proteins remained after stringent filtering. The most promising biomarker candidates were subsequently verified in the individual samples of the discovery cohort using label-free, single-run LC-MS/MS analysis, as well as in an independent verification cohort of 25 patients with total coronary occlusion (CTO) and 19 matched controls. Our data reveal bactericidal/permeability-increasing protein (BPI) as a promising biomarker for severe atherosclerotic coronary stenosis, being down-regulated in circulating granulocytes of CTO patients.