In view of these in vitro findings we tested

In view of these in vitro findings, we tested the association between thrombin generation and complement activation in a second, independent thrombosis/thrombolysis model, and used this model to determine whether C5 is activated by thrombolytic pathways in vivo. Using wild-type mice with stable carotid artery thrombi induced by ferric chloride, we first showed that 5min after occlusion, TAT levels correlated poorly with systemic levels of C3a and C5a (r=−0·36, p=0·39 for C3a; r=−0·05, p=0·91 for C5a; n=8), consistent with our observations in the venous thrombosis model. To determine the impact of plasminogen activation in this setting, we intravenously administered the tPA analog Tenecteplase (n=8) into mice that had been challenged with arterial occlusion and measured C3a and C5a 5min after restoration of blood flow, or 30min after infusion if blood flow was not restored (Fig. 4a, b). Of these, 3 mice did not re-perfuse and 5 mice did re-perfuse. Regardless of outcome, all mice that received Tenecteplase were included in the analysis. As compared to ferric chloride-challenged mice that were not infused with Tenecteplase (n=9), Tenecteplase caused a significant (~2-fold) increase in C3a and C5a levels. Notably, this increase is in line with published studies in a small group of patients treated with recombinant tPA following acute myocardial infarction (Bennett et al., 1987). Our finding that C5 could not be cleaved by recombinant tPA (Fig. 2d, iii) makes it flunixin meglumine highly unlikely that the elevated C5a levels were attributable to Tenecteplase-mediated generation of C5. We further excluded this possibility by showing in vitro that exposure of C5 to high concentrations of tPA (up to 200nM) for 10 and 30min, did not yield measurable amounts of C5a (data not shown). Taken together, these data are consistent with a direct effect of plasmin on complement activation during thrombosis.
Discussion Thrombus formation leading to pathological vaso-occlusive events (e.g. acute coronary syndrome, stroke, deep vein thrombosis and pulmonary embolus) is a major cause of death worldwide (Mozaffarian et al., 2015; Raskob et al., 2014). Initiation, propagation, and resolution of a thrombus rely on the recruitment of platelets and inflammatory cells, and this is mediated partly by local release of the complement activation factor C5a. C5a is a potent anaphylatoxic peptide, inducing a range of pro-inflammatory and pro-thrombotic effects via its cognate G-protein-coupled receptors, C5aR and C5L2. C5a activates platelets, leukocytes and endothelial cells, upregulates expression of adhesion molecules, induces secretion of pro-inflammatory and procoagulant cytokines, promotes tissue factor expression by neutrophils and release of tissue factor-containing microparticles, induces the formation of neutrophil extracellular traps (NETs), and amplifies complement activation through positive feedback loops (Oikonomopoulou et al., 2012). Given the current findings, we propose a model in which plasmin, via liberation of C5a, contributes to leukocyte trafficking during thrombus formation, propagation and/or resolution (Fig. 5). The precise local contribution of C5a (and C3a) to thrombus formation and resolution, is difficult to ascertain, particularly since clearance of these peptides is short and likely dynamically changes in this setting. Nonetheless, with generation of C5a, terminal complement pathway complexes form which also regulate coagulation. C5b-7 induces tissue factor expression by monocytic cells (Langer et al., 2013), while the MAC induces VWF and P-selectin secretion, platelet microparticle release, and endothelial cell and platelet membrane changes that favor prothrombinase assembly and thrombin generation (Hamilton et al., 1990; Wiedmer et al., 1986; Sims et al., 1988). Since C5 activation is associated with many disease states, including acute lung injury, arthritis, sepsis (Huber-Lang et al., 2006; Kessel et al., 2014; Yan and Gao, 2012), and thrombosis (Distelmaier et al., 2009; Cheung et al., 1994), the present studies suggest that interventions at the level of plasmin may have broad clinical utility.