Abstract
Activated protein C (APC) reduces mortality in severe sepsis patients and exhibits beneficial effects in multiple animal injury models. APC anticoagulant activity involves inactivation of factors Va and VIIIa, whereas APC cytoprotective activities involve the endothelial protein C receptor and protease-activated receptor-1 (PAR-1). The relative importance of the anticoagulant activity of APC versus the direct cytoprotective effects of APC on cells for the in vivo benefits is unclear. To distinguish cytoprotective from the anticoagulant activities of APC, a protease domain mutant, 5A-APC (RR229/230AA and KKK191-193AAA), was made and compared with recombinant wild-type (rwt)-APC. This mutant had minimal anticoagulant activity but normal cytoprotective activities that were dependent on endothelial protein C receptor and protease-activated receptor-1. Whereas anticoagulantly active rwt-APC inhibited secondary-extended thrombin generation and concomitant thrombin-dependent activation of thrombin activable fibrinolysis inhibitor (TAFI) in plasma, secondary-extended thrombin generation and the activation of TAFI were essentially unopposed by 5A-APC due to its low anticoagulant activity. Compared with rwt-APC, 5A-APC had minimal profibrinolytic activity and preserved TAFI-mediated anti-inflammatory carboxypeptidase activities toward bradykinin and presumably toward the anaphlatoxins, C3a and C5a, which are well known pathological mediators in sepsis. Thus, genetic engineering can selectively alter the multiple activities of APC and provide APC mutants that retain the beneficial cytoprotective effects of APC while diminishing bleeding risk due to reduction in APC's anticoagulant and APC-dependent profibrinolytic activities.
Highlights
A positively charged surface on the protease domain of Activated protein C (APC) is required for normal interactions of APC with factor Va (21, 23, 36, 37)
We assumed that the thermodynamic contributions of residues in different APC exosites responsible for APC-factor Va interactions would be approximately additive
Certain positively charged residues in APC exosites that bind the factor Va substrate for cleavage at Arg-506 are not required for APC anti-apoptotic activity that depends on APC interactions with protease-activated receptor-1 (PAR-1) and EPCR
Summary
Human kidney 293 (HEK-293) cells expressing protein C were maintained in Dulbecco’s modified Eagle’s medium:Ham’s F-12 (Invitrogen) supplemented with penicillin-streptomycin-glutamine (Invitrogen), 10% fetal bovine serum (Omega), 0.6 mg/ml G418, and 10 g/ml vitamin K1. U937 human monocytes (ATCC) were grown in RPMI 1640 (Invitrogen) supplemented with penicillin-streptomycin-glutamine and 10% fetal bovine serum. The human endothelial cell line EA.hy926, provided by Dr C. Edgell (University of North Carolina, Chapel Hill, NC), was maintained in Dulbecco’s modified Eagle’s medium containing penicillin-streptomycin-glutamine, 4.5 mg/ml glucose, and 10% fetal bovine serum. All cells were maintained in a humidified atmosphere at 37 °C and 5% CO2
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