ALBERT

Description: Key Points Factor V and protein S are required for sepsis mortality reduction and suppression of inflammatory gene expression by activated protein C. The R506Q mutation (Leiden mutation) abrogates the anti-inflammatory cofactor function of factor V for activated protein C.

Description: Infection and inflammation are invariably associated with activation of the blood coagulation mechanism, secondary to the inflammation-induced expression of the coagulation initiator tissue factor (TF) on innate immune cells. By investigating the role of cell-surface receptors for coagulation factors in mouse endotoxemia, we found that the protein C receptor (ProcR; EPCR) was required for the normal in vivo and in vitro induction of lipopolysaccharide (LPS)-regulated gene expression. In cultured bone marrow–derived myeloid cells and in monocytic RAW264.7 cells, the LPS-induced expression of functionally active TF, assembly of the ternary TF-VIIa-Xa initiation complex of blood coagulation, and the EPCR-dependent activation of protease-activated receptor 2 (PAR2) by the ternary TF-VIIa-Xa complex were required for the normal LPS induction of messenger RNAs encoding the TLR3/4 signaling adaptor protein Pellino-1 and the transcription factor interferon regulatory factor 8. In response to in vivo challenge with LPS, mice lacking EPCR or PAR2 failed to fully initiate an interferon-regulated gene expression program that included the Irf8 target genes Lif, Iigp1, Gbp2, Gbp3, and Gbp6. The inflammation-induced expression of TF and crosstalk with EPCR, PAR2, and TLR4 therefore appear necessary for the normal evolution of interferon-regulated host responses.

Description: BACKGROUND: The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. The naturally occurring R506Q Leiden polymorphism in fV largely abrogates the anticoagulant functions of aPC by rendering fVa partially refractory to aPC proteolysis, but also by preventing the formation of the anticoagulant cofactor form of fV. Among patients enrolled in the placebo arm of the PROWESS sepsis trial, heterozygous fV Leiden carriers showed significantly reduced mortality 1, and a similar survival advantage of heterozygous Leiden carriers was documented in mice harboring the fV R504Q mutation (equivalent to the human R506Q mutation) that were challenged with endotoxin1, gram-positive (S.aureus), or gram-negative infection (Y.pestis)2. The objective of the current study was to examine how aPC-resistance of fV Leiden modulates responsiveness to sepsis therapy with aPC in mice. RESULTS: In murine sepsis models of S.aureus-induced septic peritonitis, aPC-resistance of endogenous fV R504Q prevents marked disease stage-specific deleterious effects associated with aPC's anticoagulant activity, but also abrogated the mortality-reducing benefits of therapy with the signaling-selective 5A-aPC variant that only exerts minimal anticoagulant activity towards activated fVa. In mice homozygous for the R504Q mutation (fVQQ mice), 5A-aPC failed to suppress inflammatory gene expression in the presence of fVR504Q. This finding was reproduced in an in vitro culture model of murine RAW cells and bone marrow-derived dendritic cells, in which thrombosis and thrombin generation play no role. Gene expression analyses and functional in vitro studies of LPS-induced inflammatory cell signaling showed that fV, as well as protein S were required for the aPC-mediated suppression of inflammatory tissue factor-PAR2 signaling3. Structure-function analyses of recombinant variants of aPC and fV showed that this anti-inflammatory cofactor function of protein S and fV involved the same structural features that underlie their accessory role for aPC's anticoagulant function, but did not involve the degradation of activated fVa or fVIIIa. CONCLUSION: These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection. This cofactor function is structurally related, but mechanistically distinct from the anticoagulant cofactor activities of protein S and fV. APC-resistance of fV thus emerges as a response modifier of the endogenous host response to infection, as well as the outcome of sepsis therapy with normal APC and signaling-selective variants thereof. REFERENCES 1. Kerlin BA, Yan SB, Isermann BH, et al. Survival advantage associated with heterozygous factor V Leiden mutation in patients with severe sepsis and in mouse endotoxemia. Blood. 2003;102(9):3085-3092. 2. Kerschen E, Hernandez I, Zogg M, Maas M, Weiler H. Survival advantage of heterozygous factor V Leiden carriers in murine sepsis. J Thromb Haemost. 2015;13(6):1073-1080. 3. Liang HP, Kerschen EJ, Hernandez I, et al. EPCR-dependent PAR2 activation by the blood coagulation initiation complex regulates LPS-triggered interferon responses in mice. Blood. 2015. Disclosures Camire: Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 3360 The Leiden polymorphism (Arg506Gln) in human blood coagulation factor V (fV) is the most prevalent genetic risk factor for venous thrombosis. We have now shown that heterozygous carriers, but not homozygous carriers, exhibit a robust survival advantage in murine models of lethal infection with gram-positive and gram-negative bacterial pathogens. FV Leiden augments the thrombin-mediated formation of activated protein C (aPC) and thereby enables the aPC-mediated inhibition of a specific component of the overall inflammatory response of myeloid immune cells. This specific, aPC-inhibited inflammatory response was mediated by the induction of tissue factor (TF) expression, assembly of the ternary TF-VIIa-Xa complex, and the EPCR-dependent activation of Protease Activated Receptor 2 (PAR2) by the ternary TF complex. The inhibition of inflammation-induced, PAR2-dependent gene expression by APC required factor V, protein S, and Protease Activated Receptor 3. This anti-inflammatory bioactivity of purified, plasma-derived or recombinant fV was not expressed by the fV Leiden variant. Thus, in heterozygous fV Leiden individuals, aPC levels are increased by fV Leiden, while wild type fV enhances aPC's beneficial actions on innate immune cells. This explains why heterozygous carriers are protected from sepsis mortality by aPC, whereas homozygous Leiden carriers do not respond to endogenous or therapeutically administered aPC. FV Leiden hence emerges as a unique example of a balancing and evolutionary selectable polymorphism, whose balanced nature is the consequence of beneficial, cooperative interactions of the variant fV Leiden allele that augments generation of aPC and the normal fV allele that is necessary for the protective effects of aPC. Disclosures: Weiler: BloodCenter of Wisconsin: Patents & Royalties.

Description: Recombinant wild type (wt) activated protein C (APC) reduces patient mortality in severe sepsis and multi-organ failure. APC can exert both anticoagulant activity and direct cytoprotective effects on cells (anti-inflammatory, anti-apoptotic, endothelial barrier stabilization, etc.). The contribution of distinct APC activities to the overall therapeutic efficacy in septic patients is unknown. Lethal mouse endotoxemia (i.p. LPS administration) and bacterial sepsis (i.p. Staphylococcus aureus) models were used to clarify mechanisms for APC’s beneficial mortality reduction effects and to distinguish the relative importance of APC anticoagulant effects vs. APC direct effects on cells. Murine rec wtAPC (APC) was administered as bolus plus i.v. infusion (over 〈 2 hr) in total doses ranging from 0.2 to 0.04 mg/kg and was given coincident with or at times up to 3 hr after challenge. Following induction of LPS-mediated septicemia in normal mice, APC markedly reduced mortality (eg., from 50% to 0–10% at LD-50 LPS doses). APC treatment did not alter the extent of circulating inflammatory cytokine levels at 3 or 24 hr after endotoxin exposure. The survival benefit conferred by wt APC infusions was abolished in mice with genetically reduced levels of endothelial protein C receptor (EPCR) (〈 10% of normal) or in mice genetically lacking protease-activated receptor-1 (PAR-1). Murine APC variants with either 3 or 5 Ala substitutions, 3K3A-APC (KKK192-194AAA) or 5A-APC (RR230/231AA + KKK192-194AAA) that had reduced anticoagulant activity (25 % and 〈 10 % of wt APC, respectively), but normal cytoprotective activities, were as effective as wt APC in reducing mortality after LPS challenge. A murine APC variant lacking proteolytic activity (active site S360A) did not enhance survival after LPS, showing a requirement for APC’s enzymatic activity. Thus, the survival-promoting efficacy of APC in this model requires the enzymatic active site of APC and the presence of two receptors, EPCR and PAR-1, that are known to mediate APC’s in vitro beneficial cytoprotective effects on cells. In a whole bacteria sepsis model, when APC was given to mice at the time of initiation of peritoneal Staphylococcus aureus-induced sepsis and again at 24 hr, wt APC surprisingly increased mortality (100% mortality vs. 50% at LD-50 bacteria dose). In contrast, when 3K3A-APC or 5A-APC variants with attenuated anticoagulant activity was given at 0 and 24 hr, they prevented mortality due to bacterial sepsis (0–10% vs. 50% mortality for saline control at LD-50 dose). This implies that APC’s anticoagulant action might impair beneficial coagulation-dependent host defense mechanisms in early stages of bacterial sepsis whereas the 5A-APC variant, with very low anticoagulant activity but normal cytoprotective activity, might provide beneficial cellular effects to help prevent death during bacterial sepsis. In summary, the full anticoagulant activity of APC is not required for protection against mortality in each of these models. These results highlight the importance of the cellular protein C pathway for APC therapy and suggest that APC variants with reduced anticoagulant action but normal potency for beneficial direct cellular effects merit further evaluation for sepsis therapy.

Description: Activated protein C (APC) reduces mortality in severe sepsis patients. APC exerts anticoagulant activities via inactivation of factors Va and VIIIa and cytoprotective activities via endothelial protein C receptor and protease-activated receptor-1. APC mutants with selectively altered and opposite activity profiles, that is, greatly reduced anticoagulant activity or greatly reduced cytoprotective activities, are compared here. Glu149Ala-APC exhibited enhanced in vitro anticoagulant and in vivo antithrombotic activity, but greatly diminished in vitro cytoprotective effects and in vivo reduction of endotoxin-induced murine mortality. Thus, residue Glu149 and the C-terminal region of APC's light chain are identified as functionally important for expression of multiple APC activities. In contrast to Glu149Ala-APC, 5A-APC (Lys191-193Ala + Arg229/230Ala) with protease domain mutations lacked in vivo antithrombotic activity, although it was potent in reducing endotoxin-induced mortality, as previously shown. These data imply that APC molecular species with potent antithrombotic activity, but without robust cytoprotective activity, are not sufficient to reduce mortality in endotoxemia, emphasizing the need for APC's cytoprotective actions, but not anticoagulant actions, to reduce endotoxin-induced mortality. Protein engineering can provide APC mutants that permit definitive mechanism of action studies for APC's multiple activities, and may also provide safer and more effective second-generation APC mutants with reduced bleeding risk.

Description: Recombinant activated protein C (APC) reduces mortality of patients with severe inflammatory disease associated with multi-organ failure. APC exerts anticoagulant, anti-inflammatory, and cytoprotective effects. The contribution of these distinct APC activities to the overall therapeutic efficacy in septic patients is unknown. The aim of the study is to delineate the mechanism underlying the protective effect of APC in mouse endotoxemia. We first establish an experimental mouse model to demonstrate that recombinant murine APC reduces 6 day mortality of mice subjected to LPS-induced endotoxemia. APC treatment did not alter the extent of inflammatory cytokine release. Recombinant human APC did not exhibit therapeutic efficacy in this model. In contrast, recombinant human and mouse APC reduced to a similar extent experimentally induced arterial thrombus formation. The therapeutic efficacy of wild type recombinant murine APC was abolished in genetically engineered mice with reduced expression of the endothelial protein C receptor (EPCR). Recombinant mutant murine APC with greatly reduced anticoagulant potency was as effective as wild type murine APC in reducing mortality of mice subjected to LPS-induced septicemia. Mice homozygous for the Leiden polymorphism in the factor V (fV) gene, which renders coagulation factor V partially resistant to the anticoagulant effect of APC secondary to blocked fV proteolysis at R504 (R506 in humans), were refractory to the therapeutic benefit conveyed by administration of recombinant wild type mouse APC. In summary, these findings provide evidence that the therapeutic efficacy of recombinant APC is predominantly based on the ability of APC to interact with the endothelial protein C receptor, and that the anticoagulant activity of APC is not sufficient for achieving protection against mortality in a mouse model of endotoxemia. On the other hand, cleavage of fV at R506 appears necessary for retaining therapeutic efficacy in carriers of the fV Leiden allele.