OC 46.3 - Neutrophil Cathepsin G Cleaves Fibrinogen Affecting Clot Structure

Background: There is growing evidence that neutrophils contribute to the pathophysiology of thrombosis. Neutrophils are recruited to a site of injury, where they leave the vasculature and degranulate, releasing enzymes such as cathepsin G (CatG) and elastase, the latter of which has been previously reported to cleave fibrinogen at multiple sites, including several within the alphaC-domain. Because the alphaC-domain is relatively unstructured, we would expect it to be susceptible to proteolysis. The alphaC-domain plays important roles in fiber growth, mechanical stability, and susceptibility of the clot to fibrinolysis.

Aims: To evaluate the consequences of cleavage of fibrinogen by CatG for fibrin clot structure.

Methods: Purified human fibrinogen was incubated with buffer or purified neutrophil CatG. Aliquots were removed at various time points, boiled, then subjected to SDS-PAGE. Bands were submitted for N terminal sequencing. Protein was clotted with thrombin, and polymerization was monitored by assessing turbidity in a microplate reader. Fibrinolysis of these clots was triggered with plasmin. Viscoelastic properties were evaluated using thromboelastometry. FXIIIa-mediated crosslinking activity was followed with SDS-PAGE.

Results: We have identified four CatG cleavage sites within fibrinogen Aalpha, one within the N terminal fibrinopeptide A (L9) and three within the alphaC-domain (F394, F500, M517). When CatG-cleaved fibrinogen is then clotted by thrombin, polymerization is faster than that observed for control fibrinogen, and the resulting clot is markedly more turbid. CatG-cleaved fibrinogen also results in clots that are weaker (amplitude values 40% lower), and are more susceptible to plasmin-mediated fibrinolysis (time to 50% lysis decreased approximately 2 fold). FXIIIa mediated cross-linking is reduced as indicated by delayed appearance of gamma dimers and alpha polymers.

Conclusion(s): The modification of fibrinogen by CatG results in changes in clot structure and function. Release of CatG from activated neutrophils may affect clot formation with inflammation, with potential consequences for thrombosis.