Human neutrophil elastase mediates fibrinolysis shutdown through competitive degradation of plasminogen and generation of angiostatin

Abstract

BACKGROUND A subset of trauma patients undergo fibrinolysis shutdown rather than pathologic hyperfibrinolysis, contributing to organ failure. The molecular basis for fibrinolysis shutdown in trauma is incompletely understood. Elastase released from primed/activated human neutrophils (HNE) has historically been described as fibrin(ogen)olytic. However, HNE can also degrade plasminogen (PLG) to angiostatin (ANG), retaining the kringle domains but not the proteolytic function, and could thereby compete for generation of active plasmin by tissue plasminogen activator (tPA). We hypothesized that HNE can drive fibrinolysis shutdown rather than fibrinolysis. METHODS Turbidometry was performed using light scatter (λ = 620 nm) in a purified fibrinogen + PLG system and in healthy citrate plasma clotted with Ca2+/thrombin ± tPA, ±HNE, and ±ANG to evaluate HNE effects on fibrinolysis, quantified by time to transition midpoint (Tm). ΔTmfrom control is reported as percent of control ±95% CI. Purified HNE coincubated with PLG or tPA was analyzed by western blot to identify cleavage products. Exogenous HNE was mixed ex vivo with healthy volunteer blood (n = 7) and used in TEG ± tPA to evaluate effects on fibrinolysis. RESULTS HNE did not cause measurable fibrinolysis on fibrin clots, clotted plasma, or whole blood as assessed by turbidometry or TEG in the absence of tPA. Upon tPA treatment, all three methods of evaluating fibrinolysis showed delays and decreases in fibrinolysis caused by HNE relative to control: fibrin clot turbidometry ΔTm= 110.7% (CI 105.0-116.5%), clotted citrate plasma (n = 6 healthy volunteers) ΔTm= 126.1% (CI 110.4-141.8%), and whole blood native TEG (n = 7 healthy volunteers) with ΔLY30 = 28% (p = 0.043). Western blot analysis of HNE-PLG co-incubation confirmed that HNE generates angiostatin K1-3, and plasma turbidity assays treated with angiostatin K1-3 delayed fibrinolysis. CONCLUSION HNE degrades PLG and generates angiostatin K1-3, which predominates over HNE cleavage of fibrin(ogen). These findings suggest that neutrophil release of elastase may underlie trauma-induced fibrinolytic shutdown.