Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function

The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits APC anticoagulant activity. In this study, we investigate the mechanisms by which sEPCR modulates APC function. Soluble EPCR inhibited the inactivation of factor Va by APC only in the presence of phospholipid vesicles. By using flow cytometric analysis in the presence of 3 mM CaCl(2) and 0. 6 mM MgCl(2), sEPCR inhibited the binding of protein C and APC to phospholipid vesicles (K(i) = 40 +/- 7 and 33 +/- 4 nM, respectively). Without MgCl(2), the K(i) values increased approximately 4-fold. Double label flow cytometric analysis using fluorescein-APC and Texas Red-sEPCR indicated that the APC.sEPCR complex does not interact with phospholipid vesicles. By using surface plasmon resonance, we found that sEPCR also inhibited binding of protein C to phospholipid in a dose-dependent fashion (K(i) = 32 nM). To explore the possibility that sEPCR evokes structural changes in APC, fluorescence spectroscopy studies were performed to monitor sEPCR/Fl-APC interactions. sEPCR binds saturably to Fl-APC (K(d) = 27 +/- 13 nM) with a maximum decrease in Fl-APC fluorescence of 10.8 +/- 0.6%. sEPCR also stimulated the amidolytic activity of APC toward synthetic substrates. We conclude that sEPCR binding to APC blocks phospholipid interaction and alters the active site of APC.     

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.     

Multifunctional specificity of the protein C/activated protein C Gla domain

Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla domain is implicated in both interactions. We sought to identify how the protein C Gla domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla domain. We hypothesized that the unique protein C/APC Gla domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla domains.     

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.     

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.     

Identification of a sequence of human activated protein C (residues 390-404) essential for its anticoagulant activity.

Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. To identify the regions on the surface that mediate anticoagulant activity, 26 synthetic peptides were prepared representing 90% of the human protein C heavy chain primary structure and tested for their ability to inhibit APC anticoagulant activity. Peptide-(390-404) specifically inhibited APC activity in activated partial thromboplastin time and Xa-1-stage coagulation assays in normal, in protein S-depleted and Factor VIII-deficient plasma with 50% inhibition at 5 microM peptide. Polyclonal antibodies raised against this peptide and immunoaffinity-purified on a protein C-Sepharose column inhibited APC anticoagulant activity in activated partial thromboplastin time and Xa-1-stage assays in normal, protein S-depleted, and Factor VIII-deficient plasma with half-maximal inhibition at 30 nM anti-(390-404) antibody. Neither the peptide-(390-404) nor the anti-(390-404) antibodies inhibited APC amidolytic activity or the reaction of APC with recombinant [Arg358] alpha 1-antitrypsin. Furthermore, in a purified system, peptide-(390-404) inhibited APC-catalyzed inactivation of Factor Va in the presence as well as in the absence of phospholipids with 50% inhibition at 4 microM peptide. These data suggest that the region containing residues 390-404 in APC is essential for anticoagulant activity and is available to interact with antibodies or with other proteins such as the macromolecular substrates Factors Va or VIIIa.     

Functional properties and active-site topographies of factor X Gla- and prothrombin Gla-domain chimeras of activated protein C

Substitution of the Gla-domain of activated protein C (APC) with the Gla-domain of prothrombin (APC-PTGla) improves the anticoagulant activity of APC independent of protein S. Previous FRET studies showed that this substitution alters the active-site topography of this mutant, rendering it identical to the active site of the APC-protein S complex. In this study, we characterized the functional properties and the active-site topography of another APC chimera containing the Gla-domain of factor X (APC-FXGla). We discovered that the anticoagulant activity of this mutant was similarly improved independent of protein S. The average distance of the closest approach (L) between the donor dye fluorescein attached to the active site of APC derivatives and the acceptor dye octadecylrhodamine incorporated into PC/PS vesicles was determined to be 99 A for APC and 84-86 A for both APC-PTGla and APC-FXGla. Protein S minimally influenced the L values of the APC chimeras, however, it lowered this value to 87 A for wild-type APC. Further studies revealed that neither chimera elicits a protective signaling response in the TNF-alpha-activated endothelial cells. These results suggest that unique structural features within the Gla-domain of APC enable the protease to interact with endothelial protein C receptor in the antiinflammatory pathway, while the same features also cause an inherently lower specific activity for APC in the anticoagulant pathway. This adaptation has made APC a cofactor-dependent protease, requiring the cofactor function of protein S for its optimal anticoagulant function, which appears to involve the alteration of the active-site topography of APC above the membrane surface.     

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.     

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.     

Binding of calcium to anticoagulant protein S: role of the fourth EGF module

Protein S is an anticoagulant protein containing a Gla (enclosing gamma-carboxyglutamic acids) module, a TSR (thrombin sensitive region) module, four EGF (epidermal growth factor)-like modules, and a SHBG (sex hormone binding globulin)-like region. Protein S is a cofactor to activated protein C (APC) in the degradation of coagulation factors Va and VIIIa but also has APC-independent activities. The function of the fourth EGF module (EGF4) in protein S has so far not been clear. We have now investigated this module through studies of recombinant wild-type protein S and a naturally occurring mutant (Asn217Ser). The mutant has essentially normal APC anticoagulant activity and a previously reported secretion defect. In the wild-type protein, Asn217 is normally beta-hydroxylated. The binding of calcium to wild-type protein S is characterized by four high-affinity binding sites with K(D) values ranging from 10(-)(7) to 10(-)(9) M. Three of these binding sites are located in EGF modules. Using surface plasmon resonance, competition with a calcium chelator, and antibody-based methods, we found that one high-affinity binding site for calcium was lost in protein S Asn217Ser but that the mutation also affected the calcium-dependent conformation of EGF1. We conclude that binding of calcium to EGF4 of protein S, involving Asn217, is important for the maintenance of the structure of protein S. Also, the abolition of binding of calcium to EGF4, related to Asn217, impairs both the structure and function of EGF1.     

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.     

Recognition of anionic phospholipid membranes by an antihemostatic protein from a blood-feeding insect

The saliva of blood-feeding insects contains a variety of molecules having antihemostatic activity. Here, we describe nitrophorin 7 (NP7), a salivary protein that binds with high affinity to anionic phospholipid membranes. The protein is apparently targeted to the negatively charged surfaces of activated platelets and other cells, where it can serve as a vasodilator, antihistamine, platelet aggregation inhibitor, and anticoagulant. As with other members of the nitrophorin group, NP7 reversibly binds a molecule of NO and binds histamine with high affinity. The protein differs from other nitrophorins in that it binds to membranes containing phosphatidylserine. Sedimentation and surface plasmon resonance experiments, revealed two classes of phospholipid-binding sites having K(d) values of 4.8 and 755 nM. NP7 inhibits prothrombin activation by blocking phospholipid binding sites for the prothrombinase complex on the surfaces of vesicles and activated platelets. As a NO complex, NP7 inhibits collagen and ADP-induced platelet aggregation and induces disaggregation of ADP-stimulated platelets by an NO-mediated mechanism. Molecular modeling of NP7 revealed a putative, positively charged membrane interaction surface comprised mainly of a helix lying outside of the lipocalin beta-barrel structure.     

Characterization of bothrojaracin interaction with human prothrombin

Bothrojaracin (BJC) is a 27-kD snake venom protein from Bothrops jararaca that has been characterized as a potent thrombin inhibitor. BJC binds to exosites I and II, with a dissociation constant of 0.7 nM, and influences but does not block the proteinase catalytic site. BJC also binds prothrombin through an interaction that has not been characterized. In the present work we characterize the interaction of BJC with prothrombin quantitatively for the first time, and identify the BJC binding site on human prothrombin. Gel filtration chromatography demonstrated calcium-independent, 1:1 complex formation between fluorescein-labeled BJC ([5F]BJC) and prothrombin, whereas no interactions were observed with activation fragments 1 or 2 of prothrombin. Isothermal titration calorimetry showed that binding of BJC to prothrombin is endothermic, with a dissociation constant of 76 +/- 32 nM. The exosite I-specific ligand, hirudin(54-65) (Hir(54-65) (SO(3)(-)), displaced competitively [5F]BJC from prothrombin. Titration of the fluorescent hirudin(54-65) derivative, [5F]Hir(54-65)(SO(3)(-)), with human prothrombin showed a dissociation constant of 7.0 +/- 0.2 microM, indicating a approximately 100-fold lower binding affinity than that exhibited by BJC. Both ligands, however, displayed a similar, approximately 100-fold increase in affinity for exosite I when prothrombin was activated to thrombin. BJC efficiently displaced [5F]Hir(54-65)(SO(3)(-)) from complexes formed with thrombin or prothrombin with dissociation constants of 0.7 +/- 0.9 nM and 11 +/- 80 nM, respectively, indicating that BJC and Hir(54-65)(SO(3)(-)) compete for the same exosite on these molecules. The results indicate that BJC is a potent and specific probe of the partially exposed anion-binding exosite (proexosite I) of human prothrombin.     

The anticoagulant protein C pathway

The anticoagulant protein C system regulates the activity of coagulation factors VIIIa and Va, cofactors in the activation of factor X and prothrombin, respectively. Protein C is activated on endothelium by the thrombin-thrombomodulin-EPCR (endothelial protein C receptor) complex. Activated protein C (APC)-mediated cleavages of factors VIIIa and Va occur on negatively charged phospholipid membranes and involve protein cofactors, protein S and factor V. APC also has anti-inflammatory and anti-apoptotic activities that involve binding of APC to EPCR and cleavage of PAR-1 (protease-activated receptor-1). Genetic defects affecting the protein C system are the most common risk factors of venous thrombosis. The protein C system contains multi-domain proteins, the molecular recognition of which will be reviewed.     

Intracellular adenosine 3',5'-phosphate binding proteins in Dictyostelium discoideum: partial purification and characterization in aggregation competent cells

Three adenosine 3',5'-phosphate (cAMP) binding proteins were separated and partially purified from cytoplasmic extracts of Dictyostelium discoideum cells developed to aggregation competence. Two species, A and B, representing respectively 50% and 20% of the total activity, bind cAMP with very rapid kinetics and high specificity. Species A (Kd = 7.5 nM) is a monomeric protein of 36 000 daltons with a sedimentation coefficient of 2.3 S. Species B, which binds cAMP with positive cooperativity, also displays a high affinity for the ligand (Kd = 3.2 nM). This protein is present in the extracts as an equilibrium between monomeric, dimeric, and tetrameric forms with respective sedimentation coefficients of 2.4, 4.5, and 6.5 S; binding of cAMP to the monomer induces the appearance of the multimeric forms. A third cAMP binding protein (species C, Kd - 9.5 nM) was characterized as a larger protein (Mr 190 000, sedimentation coefficient of 9.2 S) which also binds adenosine and adenosyl derivatives. Species C represents 30% of the activity in the extracts and resemble the "adenosine analogue binding proteins" described in mammalian cells. The relevance of the properties of these proteins to the developmental process of D. discoideum amoebas is discussed.     

Alu-family repeat binding protein from HeLa cells which interacts with regulatory region of SV40 virus genome

Using a gel retardation assay the protein which binds selectively to the Alu-family repeat (AFR) has been identified and partially purified from HeLa cell nuclear extract. The protein (AFR-binding protein, ABP) forms multiple discrete complexes with AFR even in the presence of 200 to 2000-fold excess of non-specific (E. coli) DNA. The most stable complex has a relative mobility in 4% polyacrylamide gel (as compared to the free Alu-fragment) of 0.54. Heterogeneity of protein-DNA bands seen in the polyacrylamide gel suggests that ABP is able to form multimeric complexes with AFR. Competition experiments show that ABP do not interact with the RNA polymerase III promoter and with the TGGCA-sequence, but a high affinity binding site for ABP was found within a 660 bp restriction fragment containing the SV40 virus promoter and replication origin.     

Thermodynamic linkage between the S1 site,the Na+ site,and the Ca2+ site in the protease domain of human activated protein C (APC). Sodium ion in the APC crystal structure is coordinated to four carbonyl groups from two separate loops

The serine protease domain of activated protein C (APC) contains a Na+ and a Ca2+ site. However, the number and identity of the APC residues that coordinate to Na+ is not precisely known. Further, the functional link between the Na+ and the Ca2+ site is insufficiently defined, and their linkage to the substrate S1 site has not been studied. Here, we systematically investigate the functional significance of these two cation sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na+ binds to the substrate-occupied APC with K(d) values of approximately 24 mm in the absence and approximately 6 mm in the presence of Ca2+. Sodium-occupied APC has approximately 100-fold increased catalytic efficiency ( approximately 4-fold decrease in K(m) and approximately 25-fold increase in k(cat)) in hydrolyzing S-2288 (H-d-Ile-Pro-Arg-p-nitroanilide) and Ca2+ further increases this k(cat) slightly ( approximately 1.2-fold). Ca2+ binds to the protease domain of APC with K(d) values of approximately 438 microm in the absence and approximately 105 microm in the presence of Na+. Ca2+ binding to the protease domain of APC does not affect K(m) but increases the k(cat) approximately 10-fold, and Na+ further increases this k(cat) approximately 3-fold and decreases the K(m) value approximately 3.7-fold. In agreement with the K(m) data, sodium-occupied APC has approximately 4-fold increased affinity in binding to p-aminobenzamidine (S1 probe). Crystallographically, the Ca2+ site in APC is similar to that in trypsin, and the Na+ site is similar to that in factor Xa but not thrombin. Collectively, the Na+ site is thermodynamically linked to the S1 site as well as to the protease domain Ca2+ site, whereas the Ca2+ site is only linked to the Na+ site. The significance of these findings is that under physiologic conditions, most of the APC will exist in Na2+-APC-Ca2+ form, which has 110-fold increased proteolytic activity.     

Notecarin D binds human factor V and factor Va with high affinity in the absence of membranes

Notecarin D (NotD) is a prothrombin (ProT) activator in the venom of the tiger snake, Notechis scutatus, and a factor Xa (FXa) homolog. NotD binds specifically to the FXa binding site expressed on factor V (FV) upon activation to factor Va (FVa) by thrombin. NotD active site-labeled with 5-fluorescein ([5F]FFR-NotD) binds FV and FVa with remarkably high affinity in the absence of phospholipids (K(D) 12 and ≤ 0.01 nm, respectively). In the presence of membranes, the affinity of [5F]FFR-NotD for FVa is similar, but increased ～55-fold for FV. Binding of FXa active site-labeled with Oregon Green to FV and FVa in the presence of phospholipids is ～5,000- and ～80-fold weaker than [5F]FFR-NotD, respectively. NotD reports FVa and not FV binding by a 3-fold increase in tripeptide substrate hydrolysis, demonstrating allosteric regulation by FVa. The NotD·FVa·membrane complex activates ProT with K(m)((app)) similar to prothrombinase, and ～85-fold weaker without membranes. Active site-blocked NotD exhibits potent anticoagulant activity in plasma thrombin generation assays, representing inhibition of productive prothrombinase assembly and possible disruption of FXa inhibition by the tissue factor pathway inhibitor. The results show that high affinity binding of NotD to FVa is membrane-independent, unlike the strict membrane dependence of FXa for high affinity FVa binding.     

Interaction between protein S and complement C4b-binding protein (C4BP). Affinity studies using chimeras containing c4bp beta-chain short consensus repeats.

Human C4b-binding protein (C4BP) is a regulator of the complement system and plays an important role in the regulation of the anticoagulant protein C pathway. C4BP can bind anticoagulant protein S, resulting in a decreased cofactor function of protein S for activated protein C. C4BP is a multimeric protein containing several identical alpha-chains and a single beta-chain (C4BPbeta), each chain being composed of short consensus repeats (SCRs). Previous studies have localized the protein S binding site to the NH2-terminal SCR (SCR-1) of C4BPbeta. To further localize the protein S binding site, we constructed chimeras containing C4BPbeta SCR-1, SCR-2, SCR-3, SCR-1+2, SCR-1+3, and SCR-2+3 fused to tissue-type plasminogen activator. Binding assays of protein S with these chimeras indicated that SCR-2 contributes to the interaction of protein S with SCR-1, since the affinity of protein S for SCR-1+2 was up to 5-fold higher compared with SCR-1 and SCR-1+3. Using an assay that measures protein S cofactor activity, we showed that cofactor activity was decreased due to binding to constructs that contain SCR-1. SCR-1+2 inhibited more potently than SCR-1 and SCR-1+3. SCR-3 had no additional effect on SCR-1, and therefore the effect of SCR-2 was specific. In conclusion, beta-chain SCR-2 contributes to the interaction of C4BP with protein S.     

The effect of membrane composition on the hemostatic balance

The phospholipid composition requirements for optimal prothrombin activation and factor Va inactivation by activated protein C (APC) anticoagulant were examined. Vesicles composed of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) supported factor Va inactivation relatively well. However, optimal factor Va inactivation still required relatively high concentrations of phosphatidylserine (PS). In addition, at a fixed concentration of phospholipid, PS, and APC, vesicles devoid of PE never attained a rate of factor Va inactivation achievable with vesicles containing PE. Polyunsaturation of any vesicle component also contributed significantly to APC inactivation of factor Va. Thus, PE makes an important contribution to factor Va inactivation that cannot be mimicked by PS. In the absence of polyunsaturation in the other membrane constituents, this contribution was dependent upon the presence of both the PE headgroup per se and unsaturation of the 1,2 fatty acids. Although PE did not affect prothrombin activation rates at optimal PS concentrations, PE reduced the requirement for PS approximately 10-fold. The Km(app) for prothrombin and the Kd(app) for factor Xa-factor Va decreased as a function of increasing PS concentration, reaching optimal values at 10-15% PS in the absence of PE but only 1% PS in the presence of PE. Fatty acid polyunsaturation had minimal effects. A lupus anticoagulant immunoglobulin was more inhibitory to both prothrombinase and factor Va inactivation in the presence of PE. The degree of inhibition of APC was significantly greater and much more dependent on the phospholipid composition than that of prothrombinase. Thus, subtle changes in the phospholipid composition of cells may control procoagulant and anticoagulant reactions differentially under both normal and pathological conditions.