Activity Of Activated Protein C Research Articles

Reduction Of Histone H1 Cytotoxicity By Activated Protein C and Its Exosite Variants

In vivo and in vitro data in murine and baboon sepsis models have shown pathogenic effects of extracellular histones H3 and H4 in the circulation. The plasma serine protease, activated protein C (APC), is protective in these models by proteolysis of histones H3 and H4, and APC remarkably reduces mortality in these models. Histones H3 and H4 are also pathogenic in lung, liver, and kidney injury models. The function of extracellular histone H1, the linker histone between nucleosomes, has not been well investigated. Here we test the hypothesis that H1 can exert cytotoxic activity on lung epithelial cells and endothelial cells, and that H1 can affect endothelial cell barrier. Survival of lung epithelial (NCI H460) and endothelial (EA.hy926) cells was measured with an LDH (lactate dehydrogenase) release cytotoxicity assay in which LDH release follows loss of cell membrane integrity. Histone H1 (calf thymus-derived lysine-rich histone fraction) exhibited significant cytotoxicity on both lung epithelial and endothelial cells. Compared to other histone preparations (e.g., recombinant H2A, H2B, H3, and H4), H1 showed similar cytotoxicity on endothelial cells, whereas, on epithelial cells, H1 was more cytotoxic than other histones. Pre-incubation of all histones with plasma-derived human APC blunted their cytotoxic effects. Immunoblot analysis demonstrated site-specific, limited proteolysis of H1 and of other histones by APC, suggesting that APC protected the cells by cleaving H1 and other histones. When TransEndothelial Resistance (TER) measurements were performed on endothelial cells with the iCelligence system, the results showed that H1 histones exerted complex effects on cells, including a significant reduction in transcellular impedance observed at 10-20 hr post-treatment, implying disruption of the endothelium by H1. Studies showed that APC protected endothelial cells from this H1-induced decrease in TER. For example, the normalized cell index value for endothelial cells in TER assays was reduced by 10%, 16%, and 30% following treatment with 100, 200, and 400 µg/mL histone H1, respectively, and APC potently blunted this H1-induced reduction of the normalized cell index in TER assays. These experiments show that histone H1 has potent cytotoxic effects on endothelial and epithelial cells, as previously observed by others for histones H3 and H4, and that APC can prevent these effects. Future work should identify cell surface receptors and signaling mechanisms for H1 cytotoxic effects that may involve toll-like receptors. Our findings also suggest that histone H1 could be targeted for therapeutic purposes. Identification of the surface residues of APC that interact with H1 or other histones may help provide APC mutants defective in histone cleavage that could be used for in vivo proof of concept studies. Exosites around the active site of APC can directly interact with its substrates and influence APC activity by determining APC's affinity for substrates. A recombinant APC variant with Lys191-193 mutated to Ala (3K3A-APC) appeared to have enhanced cytoprotective effects in H1-mediated cytotoxicity assays whereas a variant of APC with residues Glu330 and Glu333 mutated to Ala (E330A/E333A-APC) showed reduced protection against H1-mediated cytotoxicity. Immunoblot analysis of reaction mixtures showed reduced cleavage of H1 by the E330A/E333A-APC mutant compared to normal APC, suggesting that residues Glu330 and Glu333 contribute to affinity for highly positively charged histones like H1. In summary, data show that histone H1 exerts cytotoxic effects on endothelial and epithelial cells and that a negatively charged exosite on APC, which includes Glu330 and Glu333, contributes to APC's ability to proteolyze H1 and reduce its cytotoxicity. Disclosures:No relevant conflicts of interest to declare.

Production of extracellular proteinases—protein C activators of blood plasma—by the micromycete Aspergillus ochraceus during submerged and solid-state fermentation

The conditions for the submerged and solid-state cultivation of the micromycete Aspergillus ochraceus VKM F-4104D, producing extracellular proteinases that activate protein C in human blood plasma, were optimized. It is shown that the protein C-activating activity of the micromycete in a solid-state culture was 1.5-3.5 times higher than in a submerged culture (as calculated per milliliter of culture medium). Among the extracellular proteins secreted by A. ochraceus VKM F-4104D during submerged and solid-state cultivation, a protein C-activating proteinase with a pI of 6.0-6.3 was identified.

Antibody SPC-54 provides acute in vivo blockage of the murine protein C system

Multiple protective effects of pharmacological activated protein C (APC) are reported in several organ pathologies. To help evaluate the endogenous murine PC system, we characterized a rat monoclonal anti-mouse PC antibody, SPC-54, which inhibited the amidolytic and anticoagulant activities of murine APC by>95%. SPC-54 blocked active site titration of purified APC using the active site titrant, biotinylated FPR-chloromethylketone, showing that SPC-54 blocks access to APC's active site to inhibit all enzymatic activity. A single injection of SPC-54 (10mg/kg) neutralized circulating PC in mice for at least 7days, and immunoblotting and immuno-precipitation with protein G-agarose confirmed that SPC-54 in vivo was bound to PC in plasma. Pre-infusion of SPC-54 in tissue factor-induced murine acute thromboembolism experiments caused a major decrease in mean survival time compared to controls (7min vs. 42.5min, P=0.0016). SPC-54 decreased lung perfusion in this model by 54% when monitored by vascular perfusion methodologies using infrared fluorescence of Evans blue dye. In LD50 endotoxemia murine models, SPC-54 infused at 7hr after endotoxin administration increased mortality from 42% to 100% (P<0.001). In summary, monoclonal antibody SPC-54 ablates in vitro and in vivo APC protective functions and enzymatic activity. The ability of SPC-54 to block the endogenous PC/APC system provides a powerful tool to understand better the role of the endogenous PC system in murine injury models and in cell bioassays and also to neutralize the enzymatic activities of murine APC in any assay system.

Expression and functional characterisation of natural R147W and K150del variants of protein C in the Chinese population

Protein C is a vitamin K-dependent serine protease zymogen in plasma which upon activation to activated protein C (APC) by thrombin down-regulates the clotting cascade by limited proteolysis of the procoagulant cofactors Va and VIIIa. In addition to its anticoagulant activity, APC also exhibits potent cytoprotective and anti-inflammatory activities. While the anticoagulant activity of APC is enhanced by the cofactor function of protein S on membrane phospholipids, the cytoprotective intracellular signalling activity of APC requires complex formation with endothelial protein C receptor (EPCR) expressed on the vascular endothelium. Two natural variants of APC [Arg-147 to Trp substitution (R147W) and Lys-150 deletion (K150del)] have been identified in the Chinese population as hotspot mutants occurring with high frequencies of 27.8% and 13.9%, respectively, among 36 protein C-deficient subjects. The affected individuals exhibit variable thrombotic tendencies. To understand the underlying cause of the thrombotic phenotype in these patients, we expressed these two protein C variants in mammalian cells and characterised their anticoagulant and anti-inflammatory properties using established in vitro and cellular assays. Our results suggest that both R147W and K150del variants have normal amidolytic and proteolytic activities in the absence of cofactors. However, the R147W mutant exhibits ~3 times lower affinity for binding to EPCR and the K150del variant has ~2-3-fold impaired anticoagulant activity in the presence of protein S. These results provide some insight into the possible pathogenic mechanism of protein C deficiency in Chinese patients carrying these mutations.

Unmasking Asian thrombophilia: is APC dysfunction the real culprit?

See also Tang L, Lu X, Yu JM, Wang QY, Yang R, Guo T, Mei H, Hu Y. PROC c.574_576del polymorphism: a common genetic risk factor for venous thrombosis in the Chinese population. This issue, pp 2019–26.

A Novel Heparin-dependent Inhibitor of Activated Protein C That Potentiates Consumptive Coagulopathy in Russell's Viper Envenomation

The activation of coagulation factors V and X by Russell's viper venom (RVV) has been implicated in the development of consumptive coagulopathies in severely envenomed patients. However, factor Va is prone to inactivation by activated protein C (APC), an important serine protease that negatively regulates blood coagulation. It is therefore hypothesized that APC may be down-regulated by some of the venom components. In this study, we managed to isolate a potent Kunitz-type APC inhibitor, named DrKIn-I. Using chromogenic substrate, DrKIn-I dose-dependently inhibited the activity of APC. Heparin potentiated the inhibition and reduced the IC(50) of DrKIn-I by 25-fold. DrKIn-I, together with heparin, also protected factor Va from APC-mediated inactivation. Using surface plasmon resonance, DrKIn-I exhibited fast binding kinetics with APC (association rate constant = 1.7 × 10(7) M(-1) s(-1)). Direct binding assays and kinetic studies revealed that this inhibition (K(i) = 53 pM) is due to the tight binding interactions of DrKIn-I with both heparin and APC. DrKIn-I also effectively reversed the anticoagulant activity of APC and completely restored the thrombin generation in APC-containing plasma. Furthermore, although the injection of either DrKIn-I or RVV-X (the venom factor X-activator) into ICR mice did not significantly deplete the plasma fibrinogen concentration, co-administration of DrKIn-I with RVV-X resulted in complete fibrinogen consumption and the deposition of fibrin thrombi in the glomerular capillaries. Our results provide new insights into the pathogenesis of RVV-induced coagulopathies and indicate that DrKIn-I is a novel APC inhibitor that is associated with potentially fatal thrombotic complications in Russell's viper envenomation.

The Multiple Layers of Signaling Selectivity at Protease-Activated Receptors

The Protease-Activated Receptors (PARs) are G-protein-coupled receptors (GPCRs) characterized by a unique mechanism of activation. They carry built in their extended N-terminal structure their own activating agonist, in the form of a cryptic tethered ligand, unmasked by an irreversible proteolytic cleavage. Besides, PARs display several other particular properties, that converge and create interacting and intertwined layers of molecular processes regulating receptor's selective signaling with important biological and pharmacological consequences. These include the operation of multiple proteases, co-factors and protease inhibitors expressed in many types of cells and tissues, creating a dynamic balance between activators and inhibitors of PAR function in a tissue specific way. Membrane microdomain compartmentalization and allosteric modulation through intermolecular interactions between PARs adds further complexity to the receptor signaling and desensitization. Furthermore, molecular components interacting with thrombin and PARs take on new roles. In particular, activated protein C (APC) forms a significant negative feedback loop for thrombin with anticoagulant properties. In addition, APC exerts anti-inflammatory and direct neuroprotective effects in vivo and in vitro. This has informed the pharmacological dissection of anticoagulant from the anti-inflammatory and neuroprotective actions of APC and the generation of engineered APC mutations with diminished risk of serious bleeding, while preserving the cytoprotective effects of APC on cells. Even more important, these advances have made possible a paradigm shift, away from a "neurocentric" and towards a "vasculo-neuronal-inflammatory model of action", which supports novel pharmacological strategies targeting multiple disease mechanisms.

Activated protein C promotes protease-activated receptor-1 cytoprotective signaling through β-arrestin and dishevelled-2 scaffolds

Protease-activated receptor-1 (PAR1) is a guanine nucleotide-binding (G) protein-coupled receptor that elicits cellular responses to coagulant and anticoagulant proteases. Activation of PAR1 by the coagulant protease thrombin results in Ras homolog gene family member A (RhoA) activation, disassembly of adherens junctions, and disruption of the endothelial barrier. In contrast, activation of PAR1 with the anticoagulant protease activated protein C (APC) results in activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and endothelial barrier protection. We previously showed that APC cytoprotective signaling requires the compartmentalization of PAR1 in caveolar microdomains. However, the mechanism by which APC-activated PAR1 promotes cytoprotective signaling in human endothelial cells remains poorly understood. Here we show that APC-activated PAR1 cytoprotective signaling is mediated by β-arrestin recruitment and activation of the dishevelled-2 (Dvl-2) scaffold and not by G protein α inhibiting activity polypeptide 2 (Gα(i)) signaling. In human endothelial cells, PAR1 and β-arrestins form a preassembled complex and cosegregate in caveolin-1-enriched fractions. Remarkably, we found that depletion of β-arrestin expression by RNA interference resulted in the loss of APC-induced Rac1 activation but not of thrombin-stimulated RhoA signaling. APC also failed to protect against thrombin-induced endothelial barrier permeability in cells deficient in β-arrestin expression. We further demonstrate that APC activation of PAR1 results in β-arrestin-dependent recruitment of Dvl-2, which is critical for Rac1 signaling and endothelial barrier protection but not for thrombin-induced RhoA signaling. Our findings identify a role for β-arrestin and Dvl-2 scaffolds in APC-activated PAR1 cytoprotective signaling in human endothelial cells.

Protein S Regulates Factor IXa in the Absence and Presence of Factor VIIIa Independently of Activated Protein C

Abstract 1197Coagulation is a finely tuned process. During thrombin formation, several anticoagulant reactions are initiated to prevent systematic activation of coagulation, and impairment of anticoagulant activity causes an increased risk of venous thrombosis. One such anticoagulant factor is protein S, deficiencies of which have been linked to venous and arterial thrombosis. While protein S has been studied for over three decades, the precise role this protein plays in attenuating the hemostatic response is far from clear.Protein S is a vitamin K-dependent plasma protein that functions in feedback regulation of thrombin generation. Protein S was initially identified as a cofactor for activated protein C (APC) but later it was observed that there is only a 3–10 fold increase in APC activity in the presence of protein S. Plasma coagulation assays in the absence of APC suggest that protein S may have other anticoagulant role(s).We report here an anticoagulant activity of Protein S mediated by inhibition of fIXa in the absence and presence of fVIIIa independent of APC. Although an APC-independent anticoagulant activity has been reported for protein S interacting with fVIIIa, no study has shown that the inhibitory effect of protein S is mediated through its interaction with fIXa, thus making our observations novel and significant. Moreover, previous studies that reported an interaction between fVIIIa and protein S were performed with low amounts of phospholipid, a condition that produces activity measurement artifacts due to the presence of active protein S multimers.We used both ex vivo (plasma studies) and in vitro methods at high phospholipid (100–200 micro molar) concentration to determine whether and how the intrinsic pathway of blood coagulation is regulated by protein S. We obtained the following results: 1) activated partial thromboplastin time (aPTT) assays with protein S-supplemented plasma confirmed that protein S prolongs clotting time, and a normal clotting time was restored with addition of anti-protein S antibody, 2) a modified aPPT assay with fIX-deficient plasma confirmed that protein S affects fIX-initiated clotting time, 3) thrombin generation assay through fIXa/fVIIIa pathway, initiated with a limiting amount of tissue factor (TF), was regulated by protein S, 4) in vitro studies with fIXa/fVIIIa and protein S in the presence of phosphatidylserine (PS) vesicles showed ∼40% and ∼65% inhibition in the activity of fIXa in the absence and presence of fVIIIa, respectively, and 5) protein S altered only the KM for fX activation by fIXa but altered both kcat and KM for fX activation by fIXa and fVIIIa. Our findings underscore the central role of protein S in regulation of coagulation. We anticipate these results will unravel important implications for the evaluation of thrombotic risk associated with protein S-deficiency. Disclosures:No relevant conflicts of interest to declare.

1-11 CARTILAGE BIOLOGY IN OSTEOARTHRITIS: ACTIVATION OF MATRIX DEGRADING ENZYMES

While osteoarthritis (OA) is a disease involving all joint tissues, progressive cartilage erosion remains a pathognomonic feature and a major indicator of the need for replacement surgery. Through the use of genetically-modified mice, our understanding of the molecular mechanisms that drive cartilage erosion in OA has greatly improved. Factors regulating two particular pathways have emerged as being critical in modulating OA cartilage pathology: (1) chondrocyte differentiation/hypertrophy, and (2) proteolysis of key matrix proteins (aggrecan and collagen) by metalloproteinases. These two pathways may converge as chondrocyte hypertrophic differentiation is associated with upregulation of a number of significant matrix degrading enzymes (ADAMTS and MMPs). However, data from MMP-13 KO mice may suggest it is the MMP activity rather than chondrocyte hypertrophy per se, that is the critical step in OA-cartilage degradation. Despite localised pathology we found wide-spread upregulation of chondrocyte MMP1, 2, 3, 13 and 14 mRNA in OA, suggesting local proenzyme activation regulates focal cartilage breakdown. We identified the serine proteinase, activated protein C (APC) but not its zymogen (PC), as a potential regulator of MMP activation in cartilage. PC and the proteins necessary for its activation (EPCR, TM) are expressed by chondrocytes, and PC/APC is increased in areas of MMP activation in OA. In healthy cartilage, an inflammatory stimulus (IL-1, TNF) is required for APC effect. In contrast, APC alone induces aggrecan and collagen proteolysis by MMPs in OA human cartilage. The effect of APC was still augmented by IL-1 stimulation in OA cartilage, even where IL-1 itself had no significant effect on degradation. APC did not regulate expression of MMPs or TIMPs but increased activation of pro-MMP2, 9 and 13 present in OA human cartilage. We found that APC cannot directly activate recombinant pro-MMP2, 9 or 13, and therefore investigated whether it was acting through PARs. Chondrocyte PAR expression is increased in OA and by inflammatory cytokines, and PARs are implicated in regulating MMP expression and activation. We found no difference in vitro in basal or IL-1 stimulated aggrecan or collagen proteolysis, MMP activity or mRNA expression in PAR-1 or PAR-2 KO compared with wild type (WT) cartilage, suggesting neither PAR alone is critical for cartilage degradation in mice. We confirmed this in vivo, observing no change in cartilage erosion compared to WT in PAR-1 or -2 KO 8 weeks after DMM-induced OA, a time when MMP-13 activity is critical for cartilage breakdown. In WT cartilage in the presence of IL-1, APC induced aggrecan and collagen proteolysis, and pro-MMP2, 9 and 13 activation. There was no diminution in APC action in PAR-1 or -2 KO cartilage, indicating its effect was not dependent on either receptor. Surprisingly, APC administration in WT mice commencing 1 week after DMM surgery and continuing twice weekly until sacrifice (week 8) significantly reduced cartilage degradation. This chondroprotection was not associated with altered cartilage aggrecan loss nor the appearance of the MMP-cleaved aggrecan neoepiope DIPEN. Initiating APC treatment 4 weeks after DMM surgery failed to alter cartilage degradation. These in vivo results implicate the effects of APC on other tissues in regulating OA cartilage pathology, and suggest that early intervention, perhaps to modify acute joint inflammation, may be required prevent long-term cartilage degradation after joint injury. Local activation of MMPs is a critical step in cartilage degradation in OA, providing a potential therapeutic target. However, understanding the interaction between all joint tissues, and the role of inflammation in the initiation and progression of cartilage degradation in post-traumatic OA requires further investigation.

Activated protein C protects against myocardial ischemic/reperfusion injury through AMP‐activated protein kinase signaling

Activated protein C (APC) is a vitamin K-dependent plasma serine protease that down-regulates clotting and inflammatory pathways. It is known that APC exerts a cardioprotective effect by decreasing apoptosis of cardiomyocytes and inhibiting expression of inflammatory mediators after myocardial ischemia. The objective of this study was to understand the mechanism of the APC-mediated cardioprotection against ischemic injury. Cardioprotective activities of wild-type APC and two derivatives, having either dramatically reduced anticoagulant activity or lacking signaling activity, were monitored in an acute ischemia/reperfusion injury model in which the left anterior descending coronary artery (LAD) was occluded. APC reduced the myocardial infarct size by a mechanism that was largely independent of its anticoagulant activity. Thus, the non-anticoagulant APC-2Cys mutant, but not the non-signaling APC-E170A mutant, attenuated myocardial infarct size by EPCR and PAR-1-dependent mechanisms. Further studies revealed that APC acts directly on cardiomyocytes to stimulate the AMP-activated protein kinase (AMPK) signaling pathway. The activation of AMPK by APC ameliorated the post-ischemic cardiac dysfunction in isolated perfused mouse hearts. Moreover, both APC and APC-2Cys inhibited production of TNFα and IL-6 in vivo by attenuating the ischemia/reperfusion-induced JNK and NF-κB signaling pathways. APC exerts a cardioprotective function in ischemic/reperfusion injury through modulation of AMPK, NF-κB and JNK signaling pathways.

Introduction to mini‐theme issue on “protease‐activated receptor signaling”

Introduction to mini‐theme issue on “protease‐activated receptor signaling”

The occupancy of endothelial protein C receptor by its ligand modulates the par‐1 dependent signaling specificity of coagulation proteases

Several recent studies have demonstrated that the activation of protease-activated receptor 1 (PAR-1) by thrombin and activated protein C (APC) on cultured vascular endothelial cells elicits paradoxical proinflammatory and antiinflammatory responses, respectively. Noting that the protective intracellular signaling activity of APC requires the interaction of the protease with its receptor, endothelial protein C receptor (EPCR), we recently hypothesized that the occupancy of EPCR by protein C may also change the PAR-1-dependent signaling specificity of thrombin. In support of this hypothesis, we demonstrated that EPCR is associated with caveolin-1 in lipid rafts of endothelial cells and that the occupancy of EPCR by the Gla-domain of protein C/APC leads to its dissociation from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway through the coupling of PAR-1 to the pertussis toxin sensitive G(i) -protein. Thus, when EPCR is bound by protein C, a PAR-1-dependent protective signaling response in cultured endothelial cells can be mediated by either thrombin or APC. This article will briefly review the mechanism by which the occupancy of EPCR by its natural ligand modulates the PAR-1-dependent signaling specificity of coagulation proteases.

The structure-function relationship of activated protein C

Protein C is the central enzyme of the natural anticoagulant pathway and its activated form APC (activated protein C) is able to proteolyse non-active as well as active coagulation factors V and VIII. Proteolysis renders these cofactors inactive, resulting in an attenuation of thrombin formation and overall down-regulation of coagulation. Presences of the APC cofactor, protein S, thrombomodulin, endothelial protein C receptor and a phospholipid surface are important for the expression of anticoagulant APC activity. Notably, APC also has direct cytoprotective effects on cells: APC is able to protect the endothelial barrier function and expresses anti-inflammatory and anti-apoptotic activities. Exact molecular mechanisms have thus far not been completely described but it has been shown that both the protease activated receptor 1 and EPCR are essential for the cytoprotective activity of APC. Recently it was shown that also other receptors like sphingosine 1 phosphate receptor 1, Cd11b/CD18 and tyrosine kinase with immunoglobulin-like and EGF-like domains 2 are likewise important for APC signalling. Mutagenesis studies are being performed to map the various APC functions and interactions onto its 3D structure and to dissect anticoagulant and cytoprotective properties. The results of these studies have provided a wealth of structure-function information. With this review we describe the state-of-the-art of the intricate structure-function relationships of APC, a protein that harbours several important functions for the maintenance of both humoral and tissue homeostasis.

Transcriptome Analysis Revealed Unique Genes as Targets for the Anti-inflammatory Action of Activated Protein C in Human Macrophages

BackgroundActivated protein C (APC) has been introduced as a therapeutic agent for treatment of patients with severe sepsis due to its unique anticoagulant and anti-inflammatory properties in the vascular system. In this study we investigated novel targets for the anti-inflammatory action of APC in human macrophages.MethodsUsing a genome-wide approach, effects of APC on the expression profile in inflammatory activated human macrophages were analyzed.ResultsWe identified, for the first time, genes that are specifically regulated by APC under inflammatory conditions, such as chromatin binding protein 4B (CHMP4B) and p300/CBP-associated factor (PCAF), thus indicating a role of APC in the epigenetic control of gene transcription. A functional assay showed the influence of APC in the acetyltransferase/deacetylase activity of nuclear extracts from inflamed macrophages.ConclusionOur data sheds new light on APC targets in inflammation and opens new lines of investigation that may be explored in order to further elucidate its unique molecule properties.

Factor V Is an Anticoagulant Cofactor for Activated Protein C during Inactivation of Factor Va

Coagulation factor V (FV) promotes inactivation of activated factor VIII (FVIIIa) by activated protein C (APC) and protein S. Loss of this APC cofactor activity is proposed to be partially responsible for the APC resistance phenotype of FV_Leiden. However, FVIIIa loses activity rapidly due to dissociation of the A2 domain, and this may be the primary mechanism of FVIIIa inactivation. APC/protein S also readily inactivates activated FV (FVa). We therefore hypothesized that FV can function as an anticoagulant cofactor for APC/protein S in the inactivation of FVa. FV was titrated into FV-deficient plasma, and the APC sensitivity ratio (APCsr; a measure of APC activity) was measured in a clotting assay that was not sensitive to FVIII. Our results showed an increase in APCsr as the FV concentration increased, suggesting an anticoagulant function for FV in this assay. FV_Leiden showed APC resistance with an APCsr of 1.0. Therefore, under our experimental conditions, FV acted as an anticoagulant cofactor for APC in the inactivation of FVa.

The efficacy of activated protein C in murine endotoxemia is dependent on integrin CD11b.

Activated protein C (APC), the only FDA-approved biotherapeutic drug for sepsis, possesses anticoagulant, antiinflammatory, and barrier-protective activities. However, the mechanisms underlying its anti-inflammatory functions are not well defined. Here, we report that the antiinflammatory activity of APC on macrophages is dependent on integrin CD11b/CD18, but not on endothelial protein C receptor (EPCR). We showed that CD11b/CD18 bound APC within specialized membrane microdomains/lipid rafts and facilitated APC cleavage and activation of protease-activated receptor-1 (PAR1), leading to enhanced production of sphingosine-1-phosphate (S1P) and suppression of the proinflammatory response of activated macrophages. Deletion of the gamma-carboxyglutamic acid domain of APC, a region critical for its anticoagulant activity and EPCR-dependent barrier protection, had no effect on its antiinflammatory function. Genetic inactivation of CD11b, PAR1, or sphingosine kinase-1, but not EPCR, abolished the ability of APC to suppress the macrophage inflammatory response in vitro. Using an LPS-induced mouse model of lethal endotoxemia, we showed that APC administration reduced the mortality of wild-type mice, but not CD11b-deficient mice. These data establish what we believe to be a novel mechanism underlying the antiinflammatory activity of APC in the setting of endotoxemia and provide clear evidence that the antiinflammatory function of APC is distinct from its barrier-protective function and anticoagulant activities.

Hepatocyte growth factor activator (HGFA): its regulation by protein C inhibitor

Protein C inhibitor (PCI; SERPINA5) is a plasma serine protease inhibitor, and a potent inhibitor of activated protein C (APC), which plays a critical role in the anticoagulant protein C pathway. Recently, PCI was also found to form a complex with the serine protease hepatocyte growth factor activator (HGFA), inhibiting the HGFA-catalyzed activation of the single-chain hepatocyte growth factor precursor. In vivo studies using human PCI-transgenic (hPCI-Tg) mice, which mimic PCI expression in humans, showed that the regeneration rate of the liver after partial hepatectomy was significantly impaired as compared with wild-type mice. The decreased liver regeneration in hPCI-Tg mice was restored by pretreatment with antibody against human PCI. Furthermore, APC protected hepatic nonparenchymal cells from thrombin-induced inflammation in vitro, suggesting that plasma PCI may inhibit the cytoprotective action of APC on hepatic cells in hPCI-Tg mice. It was shown that the levels of HGFA-PCI are increased in plasma of patients who have been subjected to hepatectomy, as compared with complex levels in the plasma of normal individuals. Thus, PCI may play a role as a potent inhibitor of HGFA and APC in plasma and/or at the sites of tissue injury in the regulation of tissue regeneration.

Zinc ions bind to and inhibit activated protein C

Zn2+ ions were found to efficiently inhibit activated protein C (APC), suggesting a potential regulatory function for such inhibition. APC activity assays employing a chromogenic peptide substrate demonstrated that the inhibition was reversible and the apparent K I was 13 +/- 2 microM. k cat was seven fold decreased whereas K M was unaffected in the presence of 10 microM Zn2+. The inhibitory effect of Zn2+ on APC activity was also observed when factor Va was used as a substrate in an assay coupled to a prothrombinase assay. The interaction of Zn2+ with APC was accompanied by a reversible approximately 40% decrease in tryptophan fluorescence, consistent with the ion inducing a conformational change in the protein. The apparent K D was 7.4 +/- 1.5 microM and thus correlated well with the apparent K I. In the presence of physiological Ca2+ concentration the K I and K D values were three to four fold enhanced, presumably due to the Ca2+-induced conformational change affecting the conformation of the Zn2+-binding site. The inhibition mechanism was non-competitive both in the absence and presence of Ca2+. Comparisons of sequences and structures suggested several possible sites for zinc binding. The magnitude of the apparent KI in relation to the blood and platelet concentrations of Zn2+ supports a physiological role for this ion in the regulation of anticoagulant activity of APC. These findings broaden the understanding of this versatile serine protease and enable the future development of potentially more efficient anticoagulant APC variants for treatments of thrombotic diseases.

Covalent Antithrombin-Heparin Complex Catalysis of Activated Protein C Inhibition by Protein C Inhibitor.

Abstract 3167Poster Board III-107 IntroductionThrombin (IIa), when bound to thrombomodulin (TM), readily converts protein C (PC) into activated PC (APC). APC functions as an anticoagulant by inactivating activated factors V (FVa) and VIII (FVIIIa), and indirectly reduces IIa generation. Once formed, APC activity is controlled through inhibition by PC inhibitor (PCI), a reaction that is catalyzed by heparin (H). Chan et al. developed a covalent antithrombin-heparin (ATH) complex with increased anticoagulant activity compared to H. The current investigation looked at the role and mechanism by which ATH affects the inhibition of APC by PCI. MethodsDiscontinous second order rate constant inhibition assays of APC+PCI, in the presence of ATH or unfractionated H (UFH), were performed. These experiments were repeated in the presence of low molecular weight H (LMWH) or ATH (LMWATH), or a high molecular weight ATH (HMWATH). The affinity of ATH or UFH for APC or PCI was assessed using native electrophoresis and APC or PCI immobilized onto agarose beads. ResultsSecond order rate experiments revealed that, at peak values, ATH (k2=2.0 × 107 ± 1.2 × 106) was a significantly slower catalyst of APC inhibition by PCI than UFH (k2= 3.0 × 107 ± 2.0 × 106; p=0.005). The peak reaction rate value for UFH occurred at a higher catalyst concentration (300nM) compared to ATH (60nM). LMWH was a poor catalyst of APC inhibition by PCI (k2= 4.2 × 105 ± 8.2 × 104). However, both LMWATH and HMWATH had high catalytic function (k2= 4.1 × 107 ± 2.1 × 106 and k2= 2.7 × 107 ± 4.8 × 105, respectively). Although UFH and ATH were able to bind to either APC or PCI at a pH of 7.3, increasing the pH to 8.8 eliminated all binding affinity, except that of ATH for APC. ConclusionUFH functions mechanistically through a template-mediated effect by bridging APC with PCI. The catalytic function of UFH is dependent upon H chain length, as LMWH was ineffective at enhancing APC inhibition by PCI. On the other hand, ATH may act mainly through conformational APC activation. There was no H chain length dependence for ATH, as LMWATH was able to significantly catalyze PCI inhibition of APC. We speculate that ATH's AT moiety may assist interaction with APC but prevents the H component of ATH from bridging the enzyme with the inhibitor. Compared to UFH, ATH is a more potent inhibitor of IIa, but is less effective at inhibiting APC and thereby decreasing IIa generation. These results add to the growing evidence that ATH is a superior anticoagulant to UFH. DisclosuresNo relevant conflicts of interest to declare.