Nonenzymatic anticoagulant activity of the mutant serine protease Ser360Ala-activated protein C mediated by factor Va.

The human plasma serine protease, activated protein C (APC), primarily exerts its anticoagulant function by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. A recombinant active site Ser 360 to Ala mutation of protein C was prepared, and the mutant protein was expressed in human 293 kidney cells and purified. The activation peptide of the mutant protein C zymogen was cleaved by a snake venom activator, Protac C, but the "activated" S360A APC did not have amidolytic activity. However, it did exhibit significant anticoagulant activity both in clotting assays and in a purified protein assay system that measured prothrombinase activity. The S360A APC was compared to plasma-derived and wild-type recombinant APC. The anticoagulant activity of the mutant, but not native APC, was resistant to diisopropyl fluorophosphate, whereas all APCs were inhibited by monoclonal antibodies against APC. In contrast to native APC, S360A APC was not inactivated by serine protease inhibitors in plasma and did not bind to the highly reactive mutant protease inhibitor M358R alpha 1 antitrypsin. Since plasma serpins provide the major mechanism for inactivating APC in vivo, this suggests that S360A APC would have a long half-life in vivo, with potential therapeutic advantages. S360A APC rapidly inhibited factor Va in a nonenzymatic manner since it apparently did not proteolyze factor Va. These data suggest that native APC may exhibit rapid nonenzymatic anticoagulant activity followed by enzymatic irreversible proteolysis of factor Va. The results of clotting assays and prothrombinase assays showed that S360A APC could not inhibit the variant Gln 506-FVa compared with normal Arg 506-FVa, suggesting that the active site of S360A APC binds to FVa at or near Arg 506.     

Interdomain engineered disulfide bond permitting elucidation of mechanisms of inactivation of coagulation factor Va by activated protein C

Procoagulant factor Va (FVa) is inactivated via limited proteolysis at three Arg residues in the A2 domain by the anticoagulant serine protease, activated protein C (APC). Cleavage by APC at Arg306 in FVa causes dissociation of the A2 domain from the heterotrimeric A1:A2:A3 structure and complete loss of procoagulant activity. To help distinguish inactivation mechanisms involving A2 domain dissociation from inactivation mechanisms involving unfavorable changes in factor Xa (FXa) affinity, we used our FVa homology model to engineer recombinant FVa mutants containing an interdomain disulfide bond (Cys609-Cys1691) between the A2 and A3 domains (A2-SS-A3 mutants) in addition to cleavage site mutations, Arg506Gln and Arg679Gln. SDS-PAGE analysis showed that the disulfide bond in A2-SS-A3 mutants prevented dissociation of the A2 domain. In the absence of A2 domain dissociation from the A1:A2:A3 trimer, APC cleavage at Arg306 alone caused a sevenfold decrease in affinity for FXa, whereas APC cleavages at Arg306, Arg506, and Arg679 caused a 70-fold decrease in affinity for FXa and a 10-fold decrease in the k(cat) of the prothrombinase complex for prothrombin without any effect on the apparent K(m) for prothrombin. Therefore, for FVa inactivation by APC, dissociation of the A2 domain may provide only a modest final step, whereas the critical events are the cleavages at Arg506 and Arg306, which effectively inactivate FVa before A2 dissociation can take place. Nonetheless, for FVa Leiden (Gln506-FVa) inactivation by APC, A2 domain dissociation may become mechanistically important, depending on the ambient FXa concentration.     

Factor Va residues 311-325 represent an activated protein C binding region

Activated protein C (APC) inactivates factor Va (fVa) by proteolytically cleaving fVa heavy chain at Arg(506), Arg(306), and Arg(679). Factor Xa (fXa) protects fVa from inactivation by APC. To test the hypothesis that fXa and APC share overlapping fVa binding sites, 15 amino acid-overlapping peptides representing the heavy chain (residues 1-709) of fVa were screened for inhibition of fVa inactivation by APC. As reported, VP311-325, a peptide comprising residues 311-325 in fVa, dose-dependently and potently inhibited fVa-dependent prothrombin activation by fXa in the absence of APC. This peptide also inhibited the inactivation of fVa by APC, suggesting that this region of fVa interacts with APC. The peptide inhibited the APC-dependent cleavage of both Arg(506) and Arg(306) because inhibition was observed with plasma-derived fVa and recombinant R506Q and RR306/679QQ fVa. VP311-325 altered the fluorescence emission of dansyl-active site-labeled APC(i) but not a dansyl-active site-labeled thrombin control, showing that the peptide binds to APC(i). This peptide also inhibited the resonance energy transfer between membrane-bound fluorescein-labeled fVa (donor) and rhodamine-active site-labeled S360C-APC (acceptor). These data suggest that peptide VP311-325 represents both an APC and fXa binding region in fVa.     

The effect of Arg306-->Ala and Arg506-->Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S.

Factor Va (fVa) is inactivated by activated protein C (APC) by cleavage of the heavy chain at Arg306, Arg506, and Arg679. Site-directed mutagenesis of human factor V cDNA was used to substitute Arg306-->Ala (rfVa306A) and Arg506-->Gln (rfVa506Q). Both the single and double mutants (rfVa306A/506Q) were constructed. The activation of these procofactors by alpha-thrombin and their inactivation by APC were assessed in coagulation assays using factor V-deficient plasma. All recombinant and wild-type proteins had similar initial cofactor activity and identical activation products (a factor Va molecule composed of light and heavy chains). Inactivation of factor Va purified from human plasma (fVaPLASMA) in HBS Ca2+ +0.5% BSA or in conditioned media by APC in the presence of phospholipid vesicles resulted in identical inactivation profiles and displayed identical cleavage patterns. Recombinant wild-type factor Va (rfVaWT) was inactivated by APC in the presence of phospholipid vesicles at an overall rate slower than fVaPLASMA. The rfVa306A and rfVa506Q mutants were each inactivated at rates slower than rfVaWT and fVaPLASMA. Following a 90-min incubation with APC, rfVa306A and rfVa506Q retain approximately 30-40% of the initial cofactor activity. The double mutant, rfVa306A/506Q, was completely resistant to cleavage and inactivation by APC retaining 100% of the initial cofactor activity following a 90-min incubation in the presence of APC. Recombinant fVaWT, rfVa306A, rfVa506Q, and rfVa306A/506Q were also used to evaluate the effect of protein S on the individual cleavage sites of the cofactor by APC. The initial rates of rfVaWT and rfVa306A inactivation in the presence of protein S were unchanged, indicating cleavage at Arg506 is not affected by protein S. The initial rate of rfVa506Q inactivation was increased, suggesting protein S slightly accelerates the cleavage at Arg306. Overall, the data demonstrate high specificity with respect to cleavage sites for APC on factor Va and demonstrate that cleavages of the cofactor at both Arg306 and Arg506 are required for efficient factor Va inactivation.     

Characterization of a thrombomodulin binding site on protein C and its comparison to an activated protein C binding site for factor Va

Activation of the anticoagulant human plasma serine protease zymogen, protein C, by a complex of thrombin and the membrane protein, thrombomodulin, generates activated protein C, a physiologic anti-thrombotic, anti-inflammatory and anti-apoptotic agent. Alanine-scanning site-directed mutagenesis of residues in five surface loops of an extensive basic surface on protein C was used to identify residues that play essential roles in its activation by the thrombin-thrombomodulin complex. Twenty-three residues in the protein C protease domain were mutated to alanine, singly, in pairs or in triple mutation combinations, and mutants were characterized for their effectiveness as substrates of the thrombin-thrombomodulin complex. Three protein C residues, K192, R229, and R230, in two loops, were identified that provided major contributions to interactions with thrombin-thrombomodulin, while six residues, S190, K191, K217, K218, W231, and R312, in four loops, appeared to provide minor contributions. These protein C residues delineated a positively charged area on the molecule's surface that largely overlapped the previously characterized factor Va binding site on activated protein C. Thus, the extensive basic surface of protein C and activated protein C provides distinctly different, though significantly overlapping, binding sites for recognition by thrombin-thrombomodulin and factor Va.     

Binding sites for blood coagulation factor Xa and protein S involving residues 493-506 in factor Va.

Inactivation due to cleavage of Factor Va (FVa) at Arg 506 by activated protein C (APC) helps to downregulate blood coagulation. To identify potential functional roles of amino acids near Arg 506, synthetic overlapping pentadecapeptides comprising FVa heavy chain residues 481-525 were tested for their ability to inhibit prothrombin activation by prothrombinase complexes [Factor Xa (FXa):FVa:phospholipids:Ca2+]. The most potent inhibition was observed for peptide VP493 (residues 493-506), with 50% inhibition at 2.5 microM. VP493 also inhibited FXa in plasma in FXa-1-stage clotting assays by 50% at 3 microM. When the C-terminal carboxamide group of VP493 was replaced by a carboxyl group, most prothrombinase inhibitory activity was lost. VP493 preincubated with FXa inhibited prothrombinase with a pattern of mixed inhibition. Homologous peptides from Factor VIII sequences did not inhibit prothrombinase. Affinity-purified antibodies to VP493 inhibited prothrombinase activity and prolonged FXa-1-stage clotting times. VP493 also blocked the ability of protein S to inhibit prothrombinase independently of APC. Immobilized VP493 bound specifically with similar affinity to both FXa and protein S (Kd approximately 40 nM), but did not measurably bind prothrombin or APC. These studies suggest that FVa residues 493-506 contribute to binding sites for both FXa and protein S, providing a rationale for the ability of protein S to negate the protective effect of FXa toward APC cleavage of FVa. Possible loss of this FVa binding site for FXa due to cleavage at Arg 506 by APC may help explain why this cleavage causes 40% decrease in FVa activity and facilitates inactivation of FVa.     

Generation of an antibody with a designed specificity difference for protein C and activated protein C

Rabbit polyclonal antibodies to a synthetic peptide, NH₂-Asp-Thr-Asn-Gln-Val-Asp-Gln-Lys-Asp-Gln-Leu-Asp-Phe-Arg-CONH₂ (APep), have been produced. This sequence is identical to that contained in the tetradecapeptide released from bovine protein C (PC) as a result of its conversion to its activated form (APC), except that Phe₁₃ replaced the normal Pro₁₃, in order to discourage cross-reactivity of antibodies to the carboxylterminal portion of APep with PC. The antibody pool obtained reacted with PC and showed virtually no cross-reactivity toward either APC or several typical plasma proteins. This general approach should serve well as a means of production of antibodies with a designed specificity capable of distinguishing between forms of the same protein that arise by release of peptide material.     

Neutral glycosphingolipid-dependent inactivation of coagulation factor Va by activated protein C and protein S.

To test whether neutral glycosphingolipids can serve as anticoagulant cofactors, the effects of incorporation of neutral glycosphingolipids into phospholipid vesicles on anticoagulant and procoagulant reactions were studied. Glucosylceramide (GlcCer), lactosylceramide (LacCer), and globotriaosylceramide (Gb(3)Cer) in vesicles containing phosphatidylserine (PS) and phosphatidylcholine (PC) dose dependently enhanced factor Va inactivation by the anticoagulant factors, activated protein C (APC) and protein S. Addition of GlcCer to PC/PS vesicles enhanced protein S-dependent APC cleavage in factor Va at Arg-506 by 13-fold, whereas PC/PS vesicles alone minimally affected protein S enhancement of this reaction. Incorporation into PC/PS vesicles of GlcCer, LacCer, or Gb(3)Cer, but not galactosylceramide or globotetraosylceramide, dose dependently prolonged factor Xa-1-stage clotting times of normal plasma in the presence of added APC without affecting baseline clotting times in the absence of APC, showing that certain neutral glycosphingolipids enhance anticoagulant but not procoagulant reactions in plasma. Thus, certain neutral glycosphingolipids (e.g. GlcCer, LacCer, and Gb(3)Cer) can enhance anticoagulant activity of APC/protein S by mechanisms that are distinctly different from those of phospholipids alone. We speculate that under some circumstances certain neutral glycosphingolipids either in lipoprotein particles or in cell membranes may help form antithrombotic microdomains that might enhance down-regulation of thrombin by APC in vivo.     

Charged residues at protein interaction interfaces: unexpected conservation and orchestrated divergence

Protein-protein interactions play an essential role in the functioning of cell. The importance of charged residues and their diverse role in protein-protein interactions have been well studied using experimental and computational methods. Often, charged residues located in protein interaction interfaces are conserved across the families of homologous proteins and protein complexes. However, on a large scale, it has been recently shown that charged residues are significantly less conserved than other residue types in protein interaction interfaces. The goal of this work is to understand the role of charged residues in the protein interaction interfaces through their conservation patterns. Here, we propose a simple approach where the structural conservation of the charged residue pairs is analyzed among the pairs of homologous binary complexes. Specifically, we determine a large set of homologous interactions using an interaction interface similarity measure and catalog the basic types of conservation patterns among the charged residue pairs. We find an unexpected conservation pattern, which we call the correlated reappearance, occurring among the pairs of homologous interfaces more frequently than the fully conserved pairs of charged residues. Furthermore, the analysis of the conservation patterns across different superkingdoms as well as structural classes of proteins has revealed that the correlated reappearance of charged residues is by far the most prevalent conservation pattern, often occurring more frequently than the unconserved charged residues. We discuss a possible role that the new conservation pattern may play in the long-range electrostatic steering effect.     

Importance of individual activated protein C cleavage site regions in coagulation factor V for factor Va inactivation and for factor Xa activation.

Activated protein C (APC) cleavage of Factor Va (FVa) at residues R506 and R306 correlates with its inactivation. APC resistance and increased thrombotic risk are due to the mutation R506Q in Factor V (FV). To study the effects of individual cleavages in FVa by APC and the importance of regions near the cleavage sites, the following recombinant (r) human FVs were prepared and purified: wild-type, Q306-rFV, Q506-rFV, and Q306Q506-rFV. All had similar time courses for thrombin activation. Q506-rFVa was cleaved by APC at R306 and was moderately resistant to APC in plasma-clotting assays and in prothrombinase assays measuring FVa residual activity, in agreement with studies of purified plasma-derived Q506-FVa. Q306-rFVa was cleaved by APC at R506 and gave a low APC-resistance ratio similar to Q506-rFVa in clotting assays, whereas unactivated Q306-rFV gave a near-normal APC-resistance ratio. When FVa residual activity was measured after long exposure to APC, Q306-rFVa was inactivated by only < or = 40% under conditions where Q506-rFVa was inactivated > 90%, supporting the hypothesis that efficient inactivation of normal FVa by APC requires cleavage at R306. In addition, the heavy chain of Q306-rFVa was cleaved at R506 much more rapidly than activity was lost, suggesting that FVa cleaved at only R506 is partially active. Under the same conditions, Q306Q506-rFVa lost no activity and was not cleaved by APC. Therefore, cleavage at either R506 or R306 appears essential for significant inactivation of FVa by APC. Modest loss of activity, probably due to cleavage at R679, was observed for the single site rFVa mutants, as evidenced by a second phase of inactivation. Q306Q506-rFVa had a low activity-to-antigen ratio of 0.50-0.77, possibly due to abnormal Factor Xa (FXa) binding. Furthermore, Q306Q506-rFV was very resistant to cleavage and activation by FXa. Q306Q506-rFV appeared to bind FXa and inhibit FXa's ability to activate normal FV. Thus, APC may downregulate FV/Va partly by impairing FXa-binding sites upon cleavage at R306 and R506. This study shows that R306 is the most important cleavage site for normal efficient inactivation of FVa by APC and supports other studies suggesting that regions near R306 and R506 provide FXa-binding sites and that FVa cleaved at only R506 retains partial activity.     

The effect of calcium (II) on the binding of anticoagulation factor I with activated coagulation factor X

Anticoagulation factor I (ACF I) from the venom of Agkistrodon acutus forms a 1:1 complex with activated coagulation factor X (FXa) in a Ca²⁺-dependent fashion and thereby prolongs the clotting time. In the present study, the dependence of the binding of ACF I with FXa on the concentration of Ca²⁺ ions was quantitatively analyzed by HPLC, and the result showed that the maximal binding of ACF I to FXa occurred at concentration of Ca²⁺ ions of about 1 mM. The binding of Ca²⁺ ions to ACF I was investigated by equilibrium dialysis and two Ca²⁺-binding sites with different affinities were identified. At pH 7.6, the apparent association constants K₁ and K₂ for these two sites were (1.8 ± 0.5) × 10⁵ and (2.7 ± 0.6) × 10⁴ M^–1 (mean ± SE, n = 4), respectively. It was evident from the observation of Ca²⁺-induced changes in the intrinsic fluorescence of ACF I that ACF I underwent a conformational change upon binding of Ca²⁺ ions. The occupation of both Ca²⁺-binding sites in ACF I required a concentration of Ca²⁺ ions of about 1 mM, which is equal to the effective concentration of Ca²⁺ ions required both for maximal binding of ACF I to FXa and for the maximal enhancement of emission fluorescence of ACF I. It could be deduced from these results that the occupation of both Ca²⁺-binding sites in ACF I with Ca²⁺ ions and subsequent conformational rearrangement might be essential for the binding of ACF I to FXa.     

Autolysis loop restricts the specificity of activated protein C: analysis by FRET and functional assays

We previously demonstrated that the substitution of the autolysis loop (residues 143-154 in chymotrypsin numbering) of APC with the corresponding loop of trypsin (APC-Tryp 143-154) has no influence on the proteolytic activity of the protease toward fVa, however, this substitution increases the reactivity of APC with plasma inhibitors so that the mutant exhibits no anticoagulant activity in plasma. To further investigate the role of the autolysis loop in APC and determine whether this loop is a target for modulation by protein S, we evaluated the activity of APC-Tryp 143-154 toward fVa and several plasma inhibitors both in the absence and presence of protein S. Furthermore, we evaluated the active-site topography of APC-Tryp 143-154 by determining the average distance of the closest approach (L) between a fluorescein dye tethered to a tripeptide inhibitor, attached to the active-site of APC-Tryp 143-154, and octadecylrhodamine dyes incorporated into PCPS vesicles both in the absence and presence of protein S. The activity of APC-Tryp 143-154 toward fVa was identical to that of wild-type APC both in the presence and absence of protein S. However, the reactivity of APC-Tryp 143-154 with plasma inhibitors was preferentially improved independent of protein S. The FRET analysis revealed a dramatic change in the active-site topography of APC both in the absence and presence of protein S. Anisotropy measurements revealed that the fluorescein dye has a remarkable degree of rotational freedom in the active-site of APC-Tryp 143-154. These results suggest that the autolysis loop of APC may not be a target for modulation by protein S. This loop, however, plays a critical role in restricting both the specificity and spatial environment of the active-site groove of APC.     

Chemical synthesis of human protein S thrombin-sensitive module and first epidermal growth factor module

Human plasma protein S is a nonenzymatic cofactor for activated protein C (APC) in the inactivation of coagulation factors Va and VIIIa, and helps to provide an essential negative feedback on blood coagulation. Previous indirect evidence suggested that the thrombin-sensitive region (TSR:residues 47–75, 1 disulfide) and the first epidermal growth factorlike region (EGF1: residues 76–116, 3 disulfides) of protein S may be functionally important for expression of its APC cofactor activity. To study the functional importance of these modules directly, access to the isolated TSR and EGF1 modules would be preferred. Recombinant expression of protein S intact TSR and correctly folded EGF1 has not been possible. Here we describe the synthesis of both TSR and EGF1 modules by stepwise solid phase peptide synthesis using the in situ neutralization/2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate activation procedure for tert-butoxycarbonyl chemistry. For the TSR, correct intramodular disulfide bonding was confirmed. To overcome folding difficulties with the EGF1, a two-step oxidation procedure was used in which the cysteines involved in the middle, crossing, disulfide bond (Cys⁸⁵-Cys¹⁰²) remained protected with acetamidomethyl (Acm) groups after hydrogen fluoride treatment of the peptide resin. Selective formation of the first two disulfide bonds (Cys⁸⁰-Cys⁹³ and Cys¹⁰⁴-Cys¹¹³) was followed by release of the Acm groups and subsequent formation of the third disulfide bond (Cys⁸⁵-Cys¹⁰²). CD studies revealed 54% of β-sheet/turn in the EGF1 that is characteristic for EGF modules. Deuterium exchange studies suggested a very tightly packed core in EGF1 that is not accessible to the bulk solvent, likely a result from the compact structure caused by its three disulfide bonds. The 30% β-sheet structure observed in the TSR involved amide protons that could be readily exchanged by deuterons, likely reflecting a more flexible structure of the TSR loop in contrast to the rigid structure of EGF1. The establishment of synthetic access to the TSR and EGF1 of protein S provides a versatile tool to study interactions of these modules with the blood coagulation components of the anticoagulant plasma protein C pathway. © 1998 John Wiley & Sons, Inc. Biopoly 46: 53–63, 1998     

Identification of surface epitopes of human coagulation factor Va that are important for interaction with activated protein C and heparin

Inactivation of factor Va (FVa) by activated protein C (APC) is a key reaction in the down-regulation of thrombin formation. FVa inactivation by APC is correlated with a loss of FXa cofactor activity as a result of three proteolytic cleavages in the FVa heavy chain at Arg306, Arg506, and Arg679. Recently, we have shown that heparin specifically inhibits the APC-mediated cleavage at Arg506 and stimulates cleavage at Arg306. Three-dimensional molecular models of APC docked at the Arg306 and Arg506 cleavage sites in FVa have identified several FVa amino acids that may be important for FVa inactivation by APC in the absence and presence of heparin. Mutagenesis of Lys320, Arg321, and Arg400 to Ala resulted in an increased inactivation rate by APC at Arg306, which indicates the importance of these residues in the FVa-APC interaction. No heparin-mediated stimulation of Arg306 cleavage was observed for these mutants, and stimulation by protein S was similar to that of wild type FVa. With this, we have now demonstrated that a cluster of basic residues in FVa comprising Lys320, Arg321, and Arg400 is required for the heparin-mediated stimulation of cleavage at Arg306 by APC. Furthermore, mutations that were introduced near the Arg506 cleavage site had a significant but modest effect on the rate of APC-catalyzed FVa inactivation, suggesting an extended interaction surface between the FVa Arg506 site and APC.     

Mutating the charged residues in the binding pocket of cellular retinoic acid-binding protein simultaneously reduces its binding affinity to retinoic acid and increases its thermostability.

Three-dimensional modeling of the complex between retinoic acid-binding protein (CRABP) and retinoic acid suggests that binding of the ligand is mediated by interaction between the carboxyl group of retinoic acid and two charged amino acids (Arg-111 and Arg-131) whose side chains project into the barrel of the protein. To assess the contribution of these amino acids to protein-ligand interaction, amino acid substitutions were made by oligonucleotide-directed, site-specific mutagenesis. The wild-type and mutant proteins were expressed in E. coli and subsequently purified. Like wild-type CRABP, the mutant proteins are composed mainly of beta-strands as determined by circular dichroism in the presence and absence of ligand, and thus presumably are folded into the same compact barrel structure as the wild-type protein. Mutants in which Arg-111 and Arg-131 are replaced by glutamine bind retinoic acid with significantly lower affinity than the wild-type protein, arguing that these two residues indeed interact with the ligand. The mutant proteins are more resistant to thermal denaturation than wild-type CRABP in the absence of retinoic acid, but they are not as thermostable as the CRABP-retinoic acid complex. These data suggest a model for CRABP-retinoic acid interaction in which the repulsive forces between the positively-charged arginine residues provide conformational flexibility to the native protein for retinoic acid to enter the binding pocket. Elimination of the positively-charged pair of amino acids produces a protein that is more thermostable than wild-type CRABP but less effective at ligand-binding.     

Structure and evolution of the human genes encoding protein C and coagulation factor IX

Human protein C is a vitamin K-dependent plasma protein that serves as a feedback down-regulator of the coagulation cascade by specifically degrading the protein cofactors VIIIa and Va. The protein C precursor consists of the following domains: leader peptide, "gla" region, two epidermal growth factor segments, and the activation peptide/serine protease. Comparison of amino acid sequences reveals that protein C and factor IX are homologous. A comparison of the genes for protein C and factor IX shows that all seven of the introns within the protein coding regions are in identical positions and correspond to protein structure-function domain boundries. However, the base compositions of the two genes (coding and noncoding regions) are remarkably different: approximately 60% guanine + cytosine (G + C) for protein C versus approximately 40% G + C for factor IX. One possible explanation for this phenomenon is that the factor IX gene (located on the X chromosome) has undergone extensive deoxycytosine methylation and subsequent spontaneous deamination mutagenesis, resulting in a net C to thymine (and G to adenine) transition. This would suggest that the protein C gene may represent a more primitive form of the gene duplication precursor.     

Identification of FAKTS as a novel 14-3-3-associated nuclear protein

Through bioinformatics and experimental approaches, we have assigned the first biochemical property to a predicted protein product in the human genome as a new 14-3-3 binding protein. 14-3-3 client proteins represent a diverse group of regulatory molecules that often function as signaling integrators in response to various environmental cues and include proteins such as Bad and Foxo. Using 14-3-3 as a probe in a yeast two-hybrid screen, we identified a novel 14-3-3 binding protein with unknown function, initially designated as clone 546. Confocal microscopy revealed that clone 546 localized to the nucleus of mammalian cells. Additional studies show that the gene encoding clone 546 is expressed in many human tissues, including the thymus, as well as a number of cancer cell lines. The interaction of clone 546 with 14-3-3 was confirmed in mammalian cells. Interestingly, this interaction was markedly enhanced by the expression of activated Akt/PKB, suggesting a phosphorylation dependent event. Mutational analysis was carried out to identify Ser479 as the predominant residue that mediates the clone 546/14-3-3 association. Phosphorylation of Ser479 by AKT/PKB further supports a critical role for Akt/PKB in regulation of the clone 546/14-3-3 interaction. On the basis of these findings, we named this undefined protein FAKTS: Fourteen-three-three associated AKT Substrate.     

Endothelial protein C receptor is expressed in rat cortical and hippocampal neurons and is necessary for protective effect of activated protein C at glutamate excitotoxicity

Activated protein C (APC) is an anticoagulant and anti-inflammatory factor that acts via endothelial protein C receptor (EPCR). Interestingly, APC also exhibits neuroprotective activities. In the present study, we demonstrate for the first time expression of EPCR, the receptor for APC, in rat cortical and hippocampal neurons. Moreover, exposing the neurons to glutamate excitotoxicity we studied the functional consequence of the expression of EPCR. By cytotoxicity assay we showed that EPCR was necessary for the APC-mediated protective effect in both neuronal cell types in culture. The effect of APC was abrogated in the presence of blocking EPCR antibodies. Analysis of neuronal death by cell labelling with dyes which allow distinguishing living and dead cells confirmed that the anti-apoptotic effect of APC was dependent on both EPCR and protease-activated receptor-1. Thus, we suggest that binding of APC to EPCR on neurons and subsequent activation of protease-activated receptor-1 by the complex of APC-EPCR promotes survival mechanisms after exposure of neurons to damaging factors.     

Identification of a binding site for blood coagulation factor Xa on the heavy chain of factor Va. Amino acid residues 323-331 of factor V represent an interactive site for activated factor X

We have recently shown that amino acid region 307-348 of factor Va heavy chain (42 amino acids, N42R) is critical for cofactor activity and may contain a binding site for factor Xa and/or prothrombin [(2001) J. Biol. Chem. 276, 18614-18623]. To ascertain the importance of this region for factor Va cofactor activity, we have synthesized eight overlapping peptides (10 amino acid each) spanning amino acid region 307-351 of the heavy chain of factor Va and tested them for inhibition of prothrombinase activity. The peptides were also tested for the inhibition of the binding of factor Va to membrane-bound active site fluorescent labeled Glu-Gly-Arg human factor Xa ([OG488]-EGR-hXa). Factor Va binds specifically to membrane-bound [OG488]-EGR-hXa (10nM) with half-maximum saturation reached at approximately 6 nM. N42R was also found to interact with [OG488]-EGR-hXa with half-maximal saturation observed at approximately 230 nM peptide. N42R was found to inhibit prothrombinase activity with an IC50 of approximately 250 nM. A nonapeptide containing amino acid region 323-331 of factor Va (AP4') was found to be a potent inhibitor of prothrombinase. Kinetic analyses revealed that AP4' is a noncompetitive inhibitor of prothrombinase with respect to prothrombin, with a K(i) of 5.7 microM. Thus, the peptide interferes with the factor Va-factor Xa interaction. Displacement experiments revealed that the nonapeptide inhibits the direct interaction of factor Va with [OG488]-EGR-hXa (IC50 approximately 7.5 microM). The nonapeptide was also found to bind directly to [OG488]-EGR-hXa and to increase the catalytic efficiency of factor Xa toward prothrombin in the absence of factor Va. In contrast, a peptadecapeptide from N42R encompassing amino acid region 337-351 of factor Va (P15H) had no effect on either prothrombinase activity or the ability of the cofactor to interact with [OG488]-EGR-hXa. Our data demonstrate that amino acid sequence 323-331 of factor Va heavy chain contains a binding site for factor Xa.     

Anti‐inflammatory effects of activated protein C on human dendritic cells

Activated protein C (APC) has an anticoagulant action and plays an important role in blood coagulation homeostasis. In addition to its anticoagulant action, APC is known to have cytoprotective effects, such as anti‐apoptotic action and endothelial barrier protection, on vascular endothelial cells and monocytes. However, the effects of APC on DCs have not been clarified. To investigate the effects of APC on human DCs, monocytes were isolated from peripheral blood and DC differentiation induced with LPS. APC significantly inhibited the production of inflammatory cytokines TNF‐α and IL‐6 during differentiation of immature DCs to mature DCs, but did not inhibit the production of IL‐12 and anti‐inflammatory cytokine IL‐10. Interestingly, treatment with 5 μg/mL, but not 25 μg/mL, of APC significantly enhanced production of IL‐10. In addition, protein C, which is the zymogen of APC, did not affect production of these cytokines. On the other hand, flow cytometric analysis of DC's surface molecules indicated that APC does not significantly affect expression of CD83, a marker of mDC differentiation, and the co‐stimulatory molecules CD40, CD80 and CD86. These results suggest that APC has anti‐inflammatory effects on human DCs and may be effective against some inflammatory diseases in which the pathogenesis involves TNF‐α and/or IL‐6 production.