p75NTR Promotes Astrocyte Proliferation in Response to Cortical Stab Wound

Astrogliosis after brain trauma can have a significant impact on functional recovery. However, little is known about the mechanisms underlying astrocyte proliferation and subsequent astrogliosis. In this study, we established a cortical stab wound injury mouse model and observed dramatic astrocyte activation and nerve growth factor receptor (p75NTR) upregulation near the lesion. We also found profound alterations in the cell cycle of astrocytes near the lesion, with a switch from a mitotically quiescent (G0) phase to the G2/M and S phases. However, no changes in the level of astrocyte apoptosis were observed. Cell cycle progression to the G2/M and S phases and CDK2 protein levels in response to cortical stab wound was inhibited after p75NTR knockdown in mouse astrocytes. Conversely, p75NTR overexpression in mouse astrocytes was sufficient in promoting cell cycle progression. In conclusion, our results suggested that p75NTR upregulation in astrocytes after brain injury induces cell cycle entry by promoting CDK2 expression and promoting astrocyte proliferation. Our findings provided a better understanding of astrocytic responses after cortical stab wound injury in mice.

     

Loss of the Androgen Receptor Cofactor p44/WDR77 Induces Astrogliosis

Astrogliosis is induced by neuronal damage and is also a pathological feature of the major aging-related neurodegenerative disorders. The mechanisms that control the cascade of astrogliosis have not been well established. In a previous study, we identified a novel androgen receptor (AR)-interacting protein, p44/WDR77, that plays a critical role in the proliferation and differentiation of prostate epithelial cells. In the present study, we found that deletion of the p44/WDR77 gene caused premature death with dramatic astrogliosis in mouse brain. We further found that p44/WDR77 is expressed in astrocytes and that loss of p44/WDR77 expression in astrocytes leads to growth arrest and astrogliosis. The astrocyte activation induced by deletion of the p44/WDR77 gene was associated with upregulation of p21(Cip1) expression and NF-κB activation. Silencing p21(Cip1) or NF-κB p65 expression with short hairpin RNA (shRNA) abolished astrocyte activation and rescued the astrocyte growth inhibition induced by deletion of the p44/WDR77 gene. Our results reveal a novel role for p44/WDR77 in the control of astrocyte activation through p21(Cip1) and NF-κB signaling.     

Kallikrein 6 is a novel molecular trigger of reactive astrogliosis

Kallikrein-related peptidase 6 (KLK6) is a trypsin-like serine protease upregulated at sites of central nervous system (CNS) injury, including de novo expression by reactive astrocytes, yet its physiological actions are largely undefined. Taken with recent evidence that KLK6 activates G-protein-coupled protease-activated receptors (PARs), we hypothesized that injury-induced elevations in KLK6 contribute to the development of astrogliosis and that this occurs in a PAR-dependent fashion. Using primary murine astrocytes and the Neu7 astrocyte cell line, we show that KLK6 causes astrocytes to transform from an epitheliod to a stellate morphology and to secrete interleukin 6 (IL-6). By contrast, KLK6 reduced expression of glial fibrillary acidic protein (GFAP). The stellation-promoting activities of KLK6 were shown to be dependent on activation of the thrombin receptor, PAR1, as a PAR1-specific inhibitor, SCH79797, blocked KLK6-induced morphological changes. The ability of KLK6 to promote astrocyte stellation was also shown to be linked to activation of protein kinase C (PKC). These studies indicate that KLK6 is positioned to serve as a molecular trigger of select physiological processes involved in the development of astrogliosis and that this is likely to occur at least in part by activation of the G-protein-coupled receptor, PAR1.     

A novel role of lactosylceramide in the regulation of tumor necrosis factor alpha-mediated proliferation of rat primary astrocytes. Implications for astrogliosis following neurotrauma

The present study describes the role of glycosphingolipids in neuroinflammatory disease and investigates tumor necrosis factor alpha (TNFalpha)-induced astrogliosis following spinal cord injury. Astrogliosis is the hallmark of neuroinflammation and is characterized by proliferation of astrocytes and increased glial fibrillary acidic protein (GFAP) gene expression. In primary astrocytes, TNFalpha stimulation increased the intracellular levels of lactosylceramide (LacCer) and induced GFAP expression and astrocyte proliferation. D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCl (PDMP), a glucosylceramide synthase and LacCer synthase (GalT-2) inhibitor, inhibited astrocyte proliferation and GFAP expression, which were reversed by exogenous supplementation of LacCer but not by other glycosphingolipids. TNFalpha caused a rapid increase in the activity of GalT-2 and synthesis of LacCer. Silencing of GalT-2 gene using antisense oligonucleotides also attenuated the proliferation of astrocytes and GFAP expression. The PDMP and antisense-mediated inhibition of proliferation and GFAP expression was well correlated with decreased Ras/ERK1/2 pathway activation. Furthermore, TNFalpha-mediated astrocyte proliferation and GFAP expression was also inhibited by LY294002, a phosphatidylinositol 3-kinase inhibitor, which was reversed by exogenous LacCer. LY294002 also inhibited TNFalpha-induced GalT-2 activation and LacCer synthesis, suggesting a phosphatidylinositol 3-kinase-mediated regulation of GalT-2. In vivo, PDMP treatment attenuated chronic ERK1/2 activation and spinal cord injury (SCI)-induced astrocyte proliferation with improved functional recovery post-SCI. Therefore, the in vivo studies support the conclusions drawn from cell culture studies and provide evidence for the role of LacCer in TNFalpha-induced astrogliosis in a rat model of SCI. To our knowledge, this is the first report demonstrating the role of LacCer in the regulation of TNFalpha-induced proliferation and reactivity of primary astrocytes.     

BMP Signaling in Astrocytes Downregulates EGFR to Modulate Survival and Maturation

Astrocytes constitute a major cell population in the brain with a myriad of essential functions, yet we know remarkably little about the signaling pathways and mechanisms that direct astrocyte maturation. To explore the signals regulating astrocyte development, we prospectively purified and cultured immature postnatal rodent astrocytes. We identified fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs) as robust trophic factors for immature astrocytes. We showed that astrocytes respond directly to BMPs via phosphorylation of the smad1/5/8 pathway. In vitro, BMP signaling promoted immature astrocytes to adopt multiple characteristics of mature astrocytes, including a more process-bearing morphology, aquaporin-4 (AQP4) and S100β immunoreactivity, limited proliferation, and strong downregulation of epidermal growth factor receptor (EGFR). In vivo, activation of the smad1/5/8 pathway in astrocytes was seen during early postnatal development, but inhibition of astrocyte proliferation was not observed. These insights can aid in the further dissection of the mechanisms and pathways controlling astrocyte biology and development.     

Engineering a disulfide bond to stabilize the calcium-binding loop of activated protein C eliminates its anticoagulant but not its protective signaling properties

In addition to an anticoagulant activity, activated protein C (APC) also exhibits anti-inflammatory and cytoprotective properties. These properties may contribute to the beneficial effect of APC in treating severe sepsis patients. A higher incidence of bleeding because of its anticoagulant function has been found to be a major drawback of APC as an effective anti-inflammatory drug. In this study, we have prepared a protein C variant in which an engineered disulfide bond between two beta-sheets stabilized the functionally critical Ca2+-binding 70-80 loop of the molecule. The 70-80 loop of this mutant no longer bound Ca2+, and the activation of the mutant by thrombin was enhanced 60-80-fold independently of thrombomodulin. The anticoagulant activity of the activated protein C mutant was nearly eliminated as determined by a plasma-based clotting assay. However, the endothelial protein C receptor- and protease-activated receptor-1-dependent protective signaling properties of the mutant were minimally altered as determined by staurosporine-induced endothelial cell apoptosis, thrombin-induced endothelial cell permeability, and tumor necrosis-alpha-mediated neutrophil adhesion and migration assays. These results suggest that the mutant lost its ability to interact with the procoagulant cofactors but not with the protective signaling molecules; thus this mutant provides an important tool for in vivo studies to examine the role of anticoagulant versus anti-inflammatory function of activated protein C.     

Reciprocal Modulation of Astrocyte Stellation by Thrombin and Protease Nexin-1

When cultured astroglia are treated with agents that elevate intracellular cyclic AMP, they become process-bearing stellate cells and resemble differentiated astrocytes in vivo. Thrombin rapidly reversed the stellation induced by dibutyryl cyclic AMP, forskolin, or isoproterenol in cultured rat astrocytes; half-maximal and maximal effects occurred at 0.5 and 8 pM, respectively. The proteolytic activity of thrombin was required for stellation reversal, as thrombin derivatized at its catalytic site serine with a diisopropylphospho group was inactive. Two thrombin inhibitors, protease nexin-1 and hirudin, blocked and reversed the effect of thrombin. The stellation reversal effect of thrombin was specific, as 300-1,000-fold higher concentrations of other serine proteinases, including plasmin, urokinase, trypsin, and T cell serine proteinase-1, were ineffective. Thrombin is a mitogen for astrocytes at concentrations in excess of 30 pM. Thrombin increased both cell number and ornithine decarboxylase activity, an early marker for mitogenic stimulation, in astrocyte cultures. The lowest thrombin concentrations that completely reversed astrocyte stellation, however, did not increase ornithine decarboxylase activity. Moreover, several other mitogens for astrocytes did not reverse dibutyryl cyclic AMP-induced stellation. Thus, the stellation reversal effect of thrombin is distinct from the mitogenic response.     

PAR1 cleavage and signaling in response to activated protein C and thrombin

Activated protein C (APC), a natural anticoagulant protease, can trigger cellular responses via protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin. Whether this phenomenon contributes to the physiological effects of APC is unknown. Toward answering this question, we compared the kinetics of PAR1 cleavage on endothelial cells by APC versus thrombin. APC did cleave PAR1 on the endothelial surface, and antibodies to the endothelial protein C receptor inhibited such cleavage. Importantly, however, APC was approximately 10(4)-fold less potent than thrombin in this setting. APC and thrombin both triggered PAR1-mediated responses in endothelial cells including expression of antiapoptotic (tumor necrosis factor-alpha-induced a20 and iap-1) and chemokine (interleukin-8 (il-8) and cxcl3) genes, but again, APC was approximately 10(4)-fold less potent than thrombin. The addition of zymogen protein C to endothelial cultures did not alter the rate of PAR1 cleavage at low or high concentrations of thrombin, and PAR1 cleavage was substantial at thrombin concentrations too low to trigger detectable conversion of protein C to APC. Thus, locally generated APC did not contribute to PAR1 cleavage beyond that effected by thrombin in this system. Although consistent with reports that sufficiently high concentrations of APC can cleave and activate PAR1 in culture, our data suggest that a significant physiological role for PAR1 activation by APC is unlikely.     

Regulation of blood coagulation

The protein C anticoagulant pathway converts the coagulation signal generated by thrombin into an anticoagulant response through the activation of protein C by the thrombin-thrombomodulin (TM) complex. The activated protein C (APC) thus formed interacts with protein S to inactivate two critical coagulation cofactors, factors Va and VIIIa, thereby dampening further thrombin generation. The proposed mechanisms by which TM switches the specificity of thrombin include conformational changes in thrombin, blocking access of normal substrates to thrombin and providing a binding site for protein C. The function of protein S appears to be to alter the cleavage site preferences of APC in factor Va, probably by changing the distance of the active site of APC relative to the membrane surface. The clinical relevance of this pathway is now established through the identification of deficient individuals with severe thrombotic complications and through the analysis of families with partial deficiencies in these components and an increased thrombotic tendency. One possible reason that even partial deficiencies are a thrombotic risk is that the function of the pathway can be down-regulated by inflammatory mediators. For instance, clinical studies have shown that the extent to which protein C levels decrease in patients with septic shock is predictive of a negative outcome. Initial clinical studies suggest that supplementation with protein C may be useful in the treatment of acute inflammatory diseases such as sepsis.     

Thrombin activates mitogen-activated protein kinase in primary astrocyte cultures

Thrombin is known to evoke numerous inflammatory and proliferative responses in a wide variety of its target cells. Recent studies have demonstrated morphoregulatory and mitogenic effects of thrombin on astroglial cells (astrocytes). The present study deals with thrombin-induced activation of mitogen-activated protein (MAP) kinase in primary cultures of rat astrocytes. Treatment of serum-starved astrocytes with thrombin resulted in a rapid activation of tyrosine (Tyr) phosphorylation of a set of proteins including a prominent one with a molecular mass of 42 kDa (p42). The identity of p42 with MAP kinase was confirmed by MAP kinase-immunoreactivity of isolated [i.e., immunoprecipitated with anti-phosphotyrosine (PY) antibodies] p42 and by increased myelin basic protein (MBP) kinase activity present in MAP kinase immunoprecipitates of thrombin-treated cultures. Pertussis toxin (PTX) pretreatment failed to inhibit thrombin stimulation of p42 phosphorylation, indicating the lack of involvement of PTX sensitive G proteins in the mechanism of activation of MAP kinase by thrombin. Chronic exposure of cultures to phorbol 12-myristate 13-acetate to down-regulate PKC resulted in an attenuation of thrombin-induced p42 Tyr phosphorylation, although H-7, a known PKC inhibitor, failed to block thrombin effect. However, staurosporine, a nonspecific protein kinase inhibitor, prevented the activation of p42 phosphorylation. It is concluded that thrombin induces MAP kinase activation in astrocytes by a mechanism involving a staurosporine-sensitive pathway. © 1995 Wiley-Liss, Inc.     

Activated protein C mutant with minimal anticoagulant activity, normal cytoprotective activity, and preservation of thrombin activable fibrinolysis inhibitor-dependent cytoprotective functions

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.     

The role of the Ca2+-sensitive tyrosine kinase Pyk2 and Src in thrombin signalling in rat astrocytes

We have recently demonstrated that multiple signalling pathways are involved in thrombin-induced proliferation in rat astrocytes. Thrombin acts by protease-activated receptor-1 (PAR-1) via mitogen-activated protein kinase activity. Signalling includes both Gi/(betagamma subunits)-phosphatidylinositol 3-kinase and a Gq-phospholipase C/Ca2+/protein kinase C (PKC) pathway. In the present study, we investigated the possible protein tyrosine kinases which might be involved in thrombin signalling cascades. We found that, in astrocytes, thrombin can evoke phosphorylation of proline-rich tyrosine kinase (Pyk2) via PAR-1. This process is dependent on the increase in intracellular Ca2+ and PKC activity. Moreover, in response to thrombin stimulation Pyk2 formed a complex with Src tyrosine kinase and adapter protein growth factor receptor-bound protein 2 (Grb2), which could be coprecipitated. Furthermore, both thrombin-induced Pyk2 phosphorylation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation can be attenuated by Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. From these data we conclude that PAR-1 uses Ca2+- and PKC-dependent Pyk2 to activate Src, thereby leading to ERK1/2 activation, which predominantly recruits Grb2 in rat astrocytes.     

Thrombin Attenuates Neuronal Cell Death and Modulates Astrocyte Reactivity Induced by β-Amyloid In Vitro

Abstract: β-Amyloid protein has been implicated as a potential causative agent in the neuropathology associated with Alzheimer's disease. This possibility is supported by observations that β-amyloid induces neuronal degeneration and astrocyte reactivity in vitro by as yet undefined mechanism(s). In this report, we present data demonstrating that the pathological effects of β-amyloid on cultured cells are modulated by activation of the thrombin receptor. At concentrations between 50 and 500 n M , thrombin pretreatment significantly attenuates neurotoxicity mediated by fibrillar aggregates of β1–42 and β25–35 peptides. In cultured astrocytes, the stellate morphology induced by β1–42 and β25–35 aggregates can be prevented and reversed by thrombin exposures between 10 p M and 1 µ M . In contrast, thrombin potentiates rather than attenuates the β-amyloid-induced increased expression of basic fibroblast growth factor, suggesting that thrombin differentially modulates the effects of β-amyloid on astrocytes. Thrombin's effects on both neurons and astrocytes are mimicked by thrombin receptor-activating peptide and inhibited by two potent thrombin inhibitors, hirudin and protease nexin-1. These data provide both new insight into the signaling pathways underlying the cellular effects of β-amyloid and additional support for the role of thrombin as an important mediator of neuropathological events.     

Multiple astrocyte responses to lysophosphatidic acids

Lysophosphatidic acid (LPA) and LPA receptors are enriched in the brain. Moreover, the levels of these receptors and ligand are modulated during brain development and injury, respectively, suggesting multiple roles for LPA in the brain. In cultured astrocytes and glioma-derived cells, LPA increases intracellular calcium concentrations and causes morphological changes. LPA also induces glioma cell migration. In normal astrocytes, LPA stimulates reactive oxygen species synthesis, activation of multiple protein kinases and expression of c-fos and c-jun. It is noteworthy that LPA-induced astrocyte responses vary as a function of the specific brain region of origin of the astrocytes. This may be one factor in the finding of LPA-stimulated proliferation in some, but not all, astrocyte studies. The species and/or developmental stage also differed in many of the astrocyte proliferation analyses. Micromolar LPA is required to elicit some astrocyte responses, including the stimulation of cytokine expression and inhibition of glutamate uptake. These events could significantly impact on survival of injured neurons and micromolar LPA concentrations are likely in diverse brain pathologies. There are important aspects of astrocyte LPA responses still to be fully evaluated, including functions in development and activation, synergy between LPA and other biomediators, and astrocyte interactions with other cells.     

Participation of protease-activated receptor-1 in thrombin-induced microglial activation

Activation of microglia, the resident macrophages in the CNS, plays a significant role in neuronal death or degeneration in a broad spectrum of CNS disorders. Recent studies indicate that nanomolar concentrations of the serine protease, thrombin, can activate microglia in culture. However, in contrast to other neural cells responsive to thrombin, the participation of novel protease-activated receptors (PARs), such as the prototypic thrombin receptor PAR1, in thrombin-induced microglial activation was cast in doubt. In this report, by utilizing primary microglial cultures from PAR1 knockout (PAR1-/-) mice, application of the PAR1 active peptide TRAP-6 (SFLLRN) in comparison to a scrambled peptide (LFLNR), we have unambiguously demonstrated that murine microglia constitutively express PAR1 mRNA that is translated into fully functional protein. Activation of the microglial PAR1 induces a rapid cytosolic free [Ca2+]i increase and transient activation of both p38 and p44/42 mitogen-activated protein kinases. Moreover, although in part, this PAR1 activation directly contributes to thrombin-induced microglial proliferation. Furthermore, although not directly inducing tumor necrosis factor-alpha (TNF-alpha) release, PAR1 activation up-regulates microglial CD40 expression and potentiates CD40 ligand-induced TNF-alpha production, thus indirectly contributing to microglial activation. Taken together, these results demonstrate an essential role of PAR1 in thrombin-induced microglial activation. In addition, strategies aimed at blocking thrombin signaling through PAR1 may be therapeutically valuable for diseases associated with cerebral vascular damage and significant inflammation with microglial activation.     

Role of Gap Junction Protein Connexin43 in Astrogliosis Induced by Brain Injury

Astrogliosis is a process that involves morphological and biochemical changes associated with astrocyte activation in response to cell damage in the brain. The upregulation of intermediate filament proteins including glial fibrillary acidic protein (GFAP), nestin and vimentin are often used as indicators for astrogliosis. Although connexin43 (Cx43), a channel protein widely expressed in adult astrocytes, exhibits enhanced immunoreactivity in the peri-lesion region, its role in astrogliosis is still unclear. Here, we correlated the temporal and spatial expression of Cx43 to the activation of astrocytes and microglia in response to an acute needle stab wound in vivo. We found large numbers of microglia devoid of Cx43 in the needle wound at 3 days post injury (dpi) while reactive astrocytes expressing Cx43 were present in the peripheral zone surrounding the injury site. A redistribution of Cx43 to the needle site, corresponding to the increased presence of GFAP-positive reactive astrocytes in the region, was only apparent from 6 dpi and sustained until at least 15 dpi. Interestingly, the extent of microglial activation and subsequent astrogliosis in the brain of Cx43 knockout mice was significantly larger than those of wild type, suggesting that Cx43 expression limits the degree of microgliosis. Although Cx43 is not essential for astrogliosis and microglial activation induced by a needle injury, our results demonstrate that Cx43 is a useful marker for injury induced astrogliosis due to its enhanced expression specifically within a small region of the lesion for an extended period. As a channel protein, Cx43 is a potential in vivo diagnostic tool of asymptomatic brain injury.     

S100B content and secretion decrease in astrocytes cultured in high-glucose medium

S100B is an astrocyte calcium-binding protein that plays a regulatory role in the cytoskeleton and cell cycle. Moreover, extracellular S100B, a marker of glial activation in several conditions of brain injury, has a trophic or apoptotic effect on neurons, depending on its concentration. Hyperglycemic rats show changes in glial parameters, including S100B expression. Here, we investigated cell density, morphological and biochemical alterations in primary cortical astrocytes from rats and C6 glioma cells cultured in high-glucose medium. Astrocytes and C6 glioma cells have a reduced content of S100B and glial fibrillary acidic protein when cultured in a high-glucose environment, as well as a reduced content of glutathione and cell proliferation rate. Although these cells have been used indistinctly to study S100B secretion, we observed a contrasting profile of S100B secretion in a high-glucose medium: a decrease in primary astrocytes and an increase in C6 glioma cells. Based on the in vitro neurotrophic effects of the S100B protein, our data suggest that chronic elevated glucose levels affect astrocyte activity, reducing extracellular secretion of S100B and that this, in turn, could affect neuronal activity and survival. Such astrocyte alterations could contribute to cognitive deficit and other impairments observed in diabetic patients.     

Dissociation of activated protein C functions by elimination of protein S cofactor enhancement

Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.     

Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C

Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg⁵⁰⁶ and Arg³⁰⁶ and of factor VIIIa (FVIIIa) by cleavage at Arg³³⁶ and Arg⁵⁶². To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a K_D of 0.11 ± 0.05 nm and competed with active site-labeled Oregon Green activated coagulation factor X for binding to FVa. The binding of APC(S360A) to FVa was not affected by protein S but was inhibited by prothrombin. APC(S360A) binding to FVa was critically dependent upon the presence of Arg⁵⁰⁶ and not Arg³⁰⁶ and additionally required an active site accessible to substrates. Inhibition of FVIIIa activity by APC(S360A) was >100-fold less efficient than inhibition of FVa. Our results show that despite exosite interactions near the Arg⁵⁰⁶ cleavage site, binding of APC(S360A) to FVa is almost completely dependent on Arg⁵⁰⁶ interacting with APC(S360A) to form a nonproductive Michaelis complex. Because docking of APC to FVa and FVIIIa constitutes the first step in the inactivation of the cofactors, we hypothesize that the observed anticoagulant activity may be important for in vivo regulation of thrombin formation.     

Protective signaling by activated protein C is mechanistically linked to protein C activation on endothelial cells

Activated protein C (APC) has endothelial barrier protective effects that require binding to endothelial protein C receptor (EPCR) and cleavage of protease activated receptor-1 (PAR1) and that may play a role in the anti-inflammatory action of APC. In this study we investigated whether protein C (PC) activation by thrombin on the endothelial cell surface may be linked to efficient protective signaling. To minimize direct thrombin effects on endothelial permeability we used the anticoagulant double mutant thrombin W215A/E217A (WE). Activation of PC by WE on the endothelial cell surface generated APC with high barrier protective activity. Comparable barrier protective effects by exogenous APC required a 4-fold higher concentration of APC. To demonstrate conclusively that protective effects in the presence of WE are mediated by APC generation and not direct signaling by WE, we used a PC variant with a substitution of the active site serine with alanine (PC S360A). Barrier protective effects of a low concentration of exogenous APC were blocked by both wildtype PC and PC S360A, consistent with their expected role as competitive inhibitors for APC binding to EPCR. WE induced protective signaling only in the presence of wild type PC but not PC S360A and PAR1 cleavage was required for these protective effects. These data demonstrate that the endogenous PC activation pathway on the endothelial cell surface is mechanistically linked to PAR1-dependent autocrine barrier protective signaling by the generated APC. WE may have powerful protective effects in systemic inflammation through signaling by the endogenously generated APC.