PB1197 - Neutrophil serine proteases cleave fibrinogen to alter fibrin clot structure

Background: Growing evidence implicates neutrophils in the pathophysiology of thrombosis. With inflammation, neutrophils are recruited to injury sites, where they release granular serine proteases (NSPs) such as cathepsin G (CatG), proteinase 3 (PR3), and elastase. We previously described cleavage of the fibrinogen alphaC-domain by CatG. PR3 has been reported to proteolyze the alphaC-domain, releasing a fragment that has utility as a marker for alpha1-antitrypsin deficiency, but any consequences for clot structure have not been described. The alphaC-domain, which plays important roles in fiber growth, mechanical stability, and susceptibility to fibrinolysis, is particularly susceptible to proteolysis due to its unstructured nature.

Aims: Evaluate the consequences of fibrinogen cleavage by NSPs on clot structure.

Methods: Purified human fibrinogen (some including Alexa-Fluor488 labelled protein) was incubated with purified neutrophil CatG, PR3 or vehicle. Timed aliquots were subjected to SDS-PAGE and N-terminal sequencing. In some experiments, proteolytic fragments were removed using concanavalin A affinity chromatography. Intact fibrinogen, PR3-cleaved fibrinogen, CatG-cleaved fibrinogen (CGFg), and CatG-cleaved fibrinogen with fragments removed (CGFg-alpha) were clotted with thrombin. Polymerization was monitored by turbidity and fibers imaged using laser-scanning confocal microscopy.

Results: We identified two alphaC-domain PR3 cleavage sites: a novel site at V397 and the reported V522 site. Similar to our observations for CGFg, thrombin-mediated polymerization of PR3-cleaved fibrinogen is faster than observed for control fibrinogen, with markedly more turbid clots. CGFg and CGFg-alpha formed clots with 91% and 44% (respectively) fewer fibers than intact fibrinogen.

Conclusion(s): Fibrinogen processing by PR3 results in changes in clot structure like those we reported for CGFg. Truncation of the alphaC-domain by either NSP results in marked changes in clot structure. In the presence of these proteolytic fragments, fibrin polymerization is markedly impaired. Release of NSPs from activated neutrophils can modulate fibrin formation and structure, with potential consequences for immunothrombosis.