APC stripped bare

Comment on Mosnier et al, page 1740

APC has anticoagulant and cytoprotective functions. For the first time, these are selectively modulated by targeted APC exosite mutation. This approach offers the prospect of improved treatment for sepsis.

Many of the proteinases generated during hemostasis have highly specific roles, each cleaving only a single protein substrate. Other proteinases have several roles involving multiple substrates. An essential requirement for proteolytic cleavage is recognition of the substrate by the proteinase. At a minimum, recognition must involve occupancy of the active site of the proteinase by the scissile bond of the substrate. But some substrates require wider interactions with the proteinase to enable the scissile bond to be positioned effectively and efficiently.FIG1 

An example of a proteinase with more than 1 substrate is activated protein C (APC). APC has well-established anticoagulant activities inactivating factor Va and factor VIIIa. The importance of its cleavage of factor Va at residue Arg506 is illustrated by the role of the factor V Leiden mutation (Arg506Gln) as a risk factor for venous thrombosis. It has been shown recently that cleavage inactivation of factor Va depends not only on the active site of APC but also on basic residues including Lys191, Lys192, Lys193, Arg229, and Arg230.^1,2  These basic residues lie in loops forming a positively charged surface, or exosite, on APC, which helps position the active site over the factor Va residue Arg506.

APC also participates in a nonanticoagulant reaction, cleavage of protease-activated receptor-1 (PAR-1) on the surface of endothelial cells.³  This reaction requires the endothelial cell protein C receptor (EPCR) and results in an antiapoptotic/neuroprotective cell phenotype.⁴  In this issue of Blood, Mosnier and colleagues demonstrate that while mutation of the above basic residues of APC dramatically reduces anticoagulant activity, it does not alter proteolysis of PAR-1 and consequent cytoprotective functions. Proximity of APC to its PAR-1 cleavage site therefore is not facilitated by its exosite but solely by its engagement with EPCR.

What is the importance of these findings? First, they illustrate how proteinases can use different mechanisms to achieve specificity. They show that substrate specificity of APC can be exquisitely controlled by fine-tuning its molecular recognition mechanisms. The mutation of the basic residues in surface loops of APC only alters interaction with factor Va and does not alter its active site and those substrates that do not require broader contact for their proteolysis. Second, they have potentially important implications for the future treatment of severe sepsis. While numerous diverse approaches to therapy of severe sepsis, including those based on bacterial toxins, cytokine function, and cell signaling pathways, have produced encouraging results in animal models, all have failed to be clinically effective.⁵  In contrast, intervention in the downstream consequences of sepsis using recombinant anticoagulant proteins has been more promising in humans. Of the 3 natural anticoagulants, antithrombin, tissue factor pathway inhibitor, and activated protein C, however, only the latter has been sufficiently decisive in terms of mortality in large clinical trials to obtain regulatory approval for routine clinical use. This has focused interest on the unique aspects of APC function, their cytoprotective effects, and their possible role in protection against sepsis. This interest has been heightened because, despite APC's beneficial effect upon mortality, bleeding remains a concern in APC therapy and may be a factor in slow uptake of this therapy. The paper by Mosnier et al provides a rationale for preparation and evaluation of restricted-specificity APC preparations with enhanced cytoprotective-to-anticoagulant activity ratios. Such products will have to undergo extensive efficacy and safety testing but offer the potential of an improved therapy based upon a solitary success in this difficult area.