From studies with mouse models Huber Lang et al

From studies with mouse models (Huber-Lang et al., 2006; Hoth et al., 2014; Khan et al., 2013; Auger et al., 2012; Zecher et al., 2014; Borkowska et al., 2014), several groups have concluded that thrombin is the major coagulation enzyme that generates C5a under pathologic conditions. This role for thrombin was supported by observations that thrombin generates C5 in vitro (Huber-Lang et al., 2006), and inhibition of thrombin dampens severity of disease and reduces C5a levels in murine models of disease (Huber-Lang et al., 2006; Hoth et al., 2014; Khan et al., 2013; Auger et al., 2012; Zecher et al., 2014; Borkowska et al., 2014). How do we reconcile these findings with the fact that the residues flanking the R751 C5 convertase cleavage site necessary to generate C5a lack similarity to thrombin cleavage sites in all other classic thrombin substrates (e.g., protein C, PAR1, fibrinogen, factor V, factor VIII) (Krisinger et al., 2012), and that thrombin is an inefficient cutter of C5 at that site (Krisinger et al., 2012)? That thrombin participates in C5a generation during coagulation is not challenged by the present findings. However, this reaction likely does not occur via direct C5 cleavage. Indeed, in our experiments, with thrombin concentrations that more closely approximate the dynamics of thrombin generation in plasma and blood (Brummel et al., 2002; Dielis et al., 2008), C5a could not be measured. Moreover, the C5T product that is generated by thrombin-mediated cleavage of C5 at the thrombin-sensitive R947 site (Krisinger et al., 2012), does not exhibit C5a-like chemotactic/migration properties in vitro (data not shown). The present studies are consistent with the concept that thrombin contributes to C5a generation, but indirectly via plasmin-mediated events. Thrombin is fundamentally important for the initiation of fibrinolysis since it generates fibrin, an important cofactor for tPA-mediated plasmin generation. Thrombin further amplifies plasmin generation by inducing endothelial secretion of tPA and rxr receptor of urokinase-type plasminogen activator (van den Eijnden-Schrauwen et al., 1995). Given the relative kinetics of thrombin versus plasmin in generating C5a, it is unlikely that thrombin, alone, generates C5a. Rather, our data support the premise that, in combination with C5 convertase, plasmin (and/or downstream proteolytic effectors of plasmin) is the major mediator, and that the amount of thrombin only affects C5a levels insofar as thrombin affects the kinetics of fibrinolysis/plasmin generation. The caveat to this premise is that very high thrombin concentrations that may transiently accumulate during thrombus formation (Brummel-Ziedins et al., 2005) could cleave C5 at R751 to generate C5a (Krisinger et al., 2012). Interestingly, reports of a role for thrombin in generating C5a come from studies with mice lacking C3 (Huber-Lang et al., 2006; Khan et al., 2013; Auger et al., 2012; Borkowska et al., 2014). These mice, for unexplained reasons, have elevated levels of prothrombin (Huber-Lang et al., 2006) which can result in increased generation of thrombin following activation of coagulation (Aleman et al., 2013; Kyrle et al., 1998). It is reasonable to consider that the high thrombin levels reached in C3-deficient mice might contribute to C5a generation. However, with excess thrombin and fibrin deposition, plasmin generation would also be markedly increased, and plasmin would be significantly favored over thrombin in generating C5a from C5. Indeed, this apparent paradox of heightened plasmin generation with a larger thrombus is in line with our observation that IVC clot weights strongly correlated with C5a levels. By interfering with deposition of fibrin, a critical cofactor for plasmin generation, thrombin inhibition would reduce C5a generation. Overall, thrombin is important in C5a generation, but indirectly, via enhanced production of plasmin. These findings are consistent with, and indeed, extend previous in vitro studies in which high concentrations of plasmin cleaved C5 (Amara et al., 2010). Attempts to measure murine levels of plasma plasmin–antiplasmin complexes were confounded by a lack of reliable assays. However, in vivo gain-of-function studies using pharmacologic-intervention (Tenecteplase infusion), allow us to conclude that plasmin plays a physiologically relevant role in the generation of C5a.