Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity.

To evaluate the contributions of individual gamma-carboxyglutamic acid (gla) residues to the overall Ca(2+)-dependent anticoagulant activity of activated human protein C (APC), we used recombinant (r) DNA technology to generate protein C (PC) variants in which each of the gla precursor glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) was separately altered to aspartic acid (D). In one case, a gla26V mutation ([gla26V]r-PC) was constructed because a patient with this particular substitution in coagulation factor IX had been previously identified. Two additional r-PC mutants were generated, viz, an r-PC variant containing a substitution at arginine (R) 15 ([R15]r-PC), because this particular R residue is conserved in all gla-containing blood coagulation proteins, as well as a variant r-PC with substitution of an E at position 32 ([F31L, Q32E]r-PC), because gla residues are found in other proteins at this sequence location. This latter protein did undergo gamma-carboxylation at the newly inserted E32 position. For each of the 11 recombinant variants, a subpopulation of PC molecules that were gamma-carboxylated at all nonmutated gla-precursor E residues has been purified by anion exchange chromatography and, where necessary, affinity chromatography on an antihuman PC column. The r-PC muteins were converted to their respective r-APC forms and assayed for their amidolytic activities and Ca(2+)-dependent anticoagulant properties. While no significant differences were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic assays, lack of a single gla residue at any of the following locations, viz, 7, 16, 20, or 26, led to virtual complete disappearance of the Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as compared with its wt counterpart. On the other hand, single eliminations of any of the gla residues located at positions 6, 14, or 19 of r-APC resulted in variant recombinant molecules with substantial anticoagulant activity (80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25 and 29 resulted in r-APC variants with significant but low (24% and 9% of wtr-APC, respectively) levels of anticoagulant activity. The variant, [R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC, while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC, resulted in a recombinant enzyme with an anticoagulant activity equivalent to that of wtr-APC.     

Gla domain-mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding

Protein C is a member of the vitamin K- dependent protein family. Proteins in this family have similar gamma-carboxyglutamic acid (Gla)-rich domains, but their affinities for negatively charged phospholipid membranes vary more than 1000-fold. We have shown that it is possible to enhance anticoagulant activity and membrane affinity of protein C by selective mutagenesis of the Gla domain. In this study, 3 new mutants, Q10G11N12 (QGN), S23E32D33Y44 (SEDY), and Q10G11N12S23E32D33Y44 (QGNSEDY), were created. In plasma-based coagulation assays, the activated form of QGNSEDY (QGNSEDY-APC) demonstrated approximately 20-fold higher anticoagulant activity than wild-type activated protein C (WT APC), while QGN-APC and SEDY-APC did not. Both normal activated factor V (FVa) and FVa Leiden (Arg506Gln) were degraded much more efficiently by QGNSEDY-APC than by WT APC in the presence as well as in the absence of protein S. Binding of protein C variants to negatively charged phospholipid membranes was investigated using light scattering and the BIAcore technique. QGNSEDY demonstrated 3- to 7-fold enhanced binding as compared with WT protein C, suggesting the membrane affinity to be influenced by several residues located at different parts of the Gla domain. The anticoagulant activity as well as phospholipid binding ability was only enhanced when multiple regions of the Gla domain were modified. The results provide insights into the molecular mechanisms that are involved in determining the binding affinity of the interaction between Gla domains and phospholipid membranes. The unique properties of QGNSEDY-APC suggest this APC variant possibly to have greater therapeutic potential than WT APC.     

Inhibition of activated protein C anticoagulant activity by prothrombin

In this study, we test the hypothesis that prothrombin levels may modulate activated protein C (APC) anticoagulant activity. Prothrombin in purified systems or plasma dramatically inhibited the ability of APC to inactivate factor Va and to anticoagulate plasma. This was not due solely to competition for binding to the membrane surface, as prothrombin also inhibited factor Va inactivation by APC in the absence of a membrane surface. Compared with normal factor Va, inactivation of factor Va Leiden by APC was much less sensitive to prothrombin inhibition. This may account for the observation that the Leiden mutation has less of an effect on plasma-based clotting assays than would be predicted from the purified system. Reduction of protein C levels to 20% of normal constitutes a significant risk of thrombosis, yet these levels are observed in neonates and patients on oral anticoagulant therapy. In both situations, the correspondingly low prothrombin levels would result in an increased effectiveness of the remaining functional APC of approximately 5-fold. Thus, while the protein C activation system is impaired by the reduction in protein C levels, the APC that is formed is a more effective anticoagulant, allowing protein C levels to be reduced without significant thrombotic risk. In situations where prothrombin is high and protein C levels are low, as in early stages of oral anticoagulant therapy, the reduction in protein C would result only in impaired function of the anticoagulant system, possibly explaining the tendency for warfarin-induced skin necrosis.     

Inhibition of activated protein C and its cofactor protein S by antiphospholipid antibodies

Five unrelated Japanese beta-thalassaemia genes, from one homozygote and four heterozygotes, have been systematically characterized using DNA polymorphism analysis, polymerase chain reaction, dot-blot hybridization and direct sequencing of amplified genomic DNA. Four different molecular defects were observed on three different beta-globin gene frameworks. One of these, the A----G mutation in the TATA box, a previously described mutation, was detected by dot-blot hybridization in one homozygote and one heterozygote with the beta-globin gene of framework 2. The second mutation is a C----T substitution at position 654 of IVS-2, the mutation commonly found in Chinese, which was associated with the framework 1 gene. Another two mutations, both associated with framework 3 genes, are novel ones; an amber mutation in codon 90 (GAG to TAG) and a frameshift (+G) insertion in codon 54, both of which cause a beta 0-thalassaemia phenotype by premature termination of the beta-globin chain synthesis.     

Monocytes, but not endothelial cells, downregulate the anticoagulant activity of activated protein C

Activated protein C (APC) is a natural anticoagulant and inhibits thrombin generation by degrading factors Va and VIIIa. We evaluated the ability of APC to inhibit blood coagulation triggered by lipopolysaccharide (LPS)-stimulated [tissue factor (TF)-expressing] human mononuclear cells (MNCs) or umbilical vein endothelial cells (HUVECs). Using a plasma recalcification assay, we found that APC (up to 53.3 nmol/l final concentration) had a poor anticoagulant effect in the presence of LPS-stimulated MNCs, whereas it caused a marked prolongation of clotting time in the presence of LPS-stimulated HUVECs. A poor response to APC was also observed when platelet-free MNCs, monocyte-enriched preparations or the monocytoid cell line U937 were tested. Using a TF-independent (FXa-induced) thrombin generation assay, we demonstrated that both LPS-stimulated and unstimulated MNCs negated the inhibitory activity of APC. Direct determination of FVa activity indicated that MNCs were less efficient than HUVECs in promoting FVa inactivation by APC. Together, our results suggest that MNCs, at variance with HUVECs, protect factor Va from inactivation by APC, probably through the expression of a membrane component not present on endothelial cells. These strengthen the importance of monocytes in fibrin deposition associated with pathological conditions characterized by monocyte recruitment and activation.     

The helical domain of intestinal fatty acid binding protein is critical for collisional transfer of fatty acids to phospholipid membranes

Fatty acid binding proteins (FABPs) exhibit a β-barrel topology, comprising 10 antiparallel β-sheets capped by two short α-helical segments. Previous studies suggested that fatty acid transfer from several FABPs occurs during interaction between the protein and the acceptor membrane, and that the helical domain of the FABPs plays an important role in this process. In this study, we employed a helix-less variant of intestinal FABP (IFABP-HL) and examined the rate and mechanism of transfer of fluorescent anthroyloxy fatty acids (AOFA) from this protein to model membranes in comparison to the wild type (wIFABP). In marked contrast to wIFABP, IFABP-HL does not show significant modification of the AOFA transfer rate as a function of either the concentration or the composition of the acceptor membranes. These results suggest that the transfer of fatty acids from IFABP-HL occurs by an aqueous diffusion-mediated process, i.e., in the absence of the helical domain, effective collisional transfer of fatty acids to membranes does not occur. Binding of wIFABP and IFABP-HL to membranes was directly analyzed by using a cytochrome c competition assay, and it was shown that IFABP-HL was 80% less efficient in preventing cytochrome c from binding to membranes than the native IFABP. Collectively, these results indicate that the α-helical region of IFABP is involved in membrane interactions and thus plays a critical role in the collisional mechanism of fatty acid transfer from IFABP to phospholipid membranes.     

The cofactor role of protein S in the acceleration of whole blood clot lysis by activated protein C in vitro

The effect of purified human activated protein C (APC) and protein S on fibrinolysis was studied by using an in vitro blood clot lysis technique. Blood clots were formed from citrated blood (supplemented with 125I-fibrinogen) by adding thrombin and Ca2+-ions; lysis of the clots was achieved by adding tissue-type plasminogen activator. The release of labeled fibrin degradation products from the clots into the supernatant was followed in time. We clearly demonstrated that APC accelerates whole blood clot lysis in vitro. The effect of APC was completely quenched by antiprotein C IgG, pretreatment of APC with diisopropylfluorophosphate, and preincubation of the blood with antiprotein S IgG. This demonstrates that both the active site of APC and the presence of the cofactor, protein S, are essential for the expression of the profibrinolytic properties. At present, the substrate of APC involved in the regulation of fibrinolysis is not yet known. Analysis of the radiolabeled fibrin degradation products demonstrated that APC had no effect on the fibrin cross-linking capacity of factor XIII.     

Protein S is a cofactor for activated protein C neutralization of an inhibitor of plasminogen activation released from platelets

Platelets stimulated with thrombin release an inhibitor of plasminogen activator (PAI), which has been shown previously to be neutralized by activated protein C (APC). The requirements for optimal neutralization of PAI activity were investigated. The releasate of gel-filtered human platelets stimulated with thrombin served as a source of PAI. When 6 X 10(8) platelets/mL were incubated with thrombin (1 IU/mL), the releasate contained 18 to 26 ng/mL PAI as determined by incubation of the releasate with urokinase and measurement of residual urokinase activity on plasminogen (S2251). Preincubation of PAI with up to 4 micrograms/mL APC for two hours yielded less than 20% neutralization of PAI activity. In the presence of protein S, phospholipid, and Ca2+, neutralization of PAI activity was time-dependent with 50% neutralization occurring in two hours with 1 microgram/mL APC. The cofactor effects of protein S and phospholipid were concentration- dependent with half-maximal acceleration at approximately 3 micrograms/mL protein S and 10 micrograms/mL phospholipid when the experiments were performed at 1 microgram/mL APC. Diisopropylfluorophosphate-inactivated APC, gla-domainless APC, and thrombin-cleaved protein S had no effect on PAI activity, indicating requirement for preservation of the APC active site and of the Ca2+ binding ability of both APC and protein S. These results suggest coordinate binding of APC and protein S onto phospholipid membrane as a prerequisite for optimal expression of PAI neutralized by APC.     

Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C.

Although patients with thromboembolic disease frequently have family histories of thrombosis, well-defined defects such as inherited deficiencies of anticoagulant proteins are found only in a minority of cases. Based on the hypothesis that a poor anticoagulant response to activated protein C (APC) would predispose to thrombosis, a set of new coagulation assays was developed that measure the anticoagulant response in plasma to APC. A middle-aged man with a history of multiple thrombotic events was identified. The addition of APC to his plasma did not result in a normal anticoagulant response as measured by prolongation of clotting time in an activated partial thromboplastin time (APTT) assay. Four of the proband's relatives had medical histories of multiple thrombotic events, and they and several other family members responded poorly to APC in the APTT-based assay. Subnormal anticoagulant responses to APC were also found in factor IXa- and Xa-based assays. Several possible mechanisms for the observed phenomenon were ruled out, such as functional protein S deficiency, a protein C-inhibitory antibody, or a fast-acting protease inhibitor against APC. Moreover, restriction fragment-length polymorphism analysis excluded possible linkage of the underlying molecular defect to factor VIII and von Willebrand factor genes. We now describe a previously unrecognized mechanism for familial thromboembolic disease that is characterized by poor anticoagulant response to APC. This would appear to be explained best by a hypothesized inherited deficiency of a previously unrecognized cofactor to APC. As we have identified two additional, unrelated cases with thrombosis and inherited poor anticoagulant response to APC, this may constitute an important cause for familial thrombophilia.     

Enhancement of vitamin-K-dependent protein function by modification of the gamma-carboxyglutamic acid domain: studies of protein C and factor VII

Vitamin-K-dependent proteins are found in both the pro- and anti-coagulation cascades, and their use in coagulation therapies is expanding rapidly. The vitamin-K-dependent, gamma-carboxyglutamic acid (Gla)-containing regions of proteins in this family are homologous and are responsible for membrane association. Site-directed mutations that enhance the membrane affinity of protein C, an anticoagulant, and of factor VII, a procoagulant, have been identified. These protein C and Factor VII mutants show enhanced activity in many assays, offering opportunities to study the role of membrane in blood clotting reactions and proteins that may have greater therapeutic value.     

Conservative mutations in the C2 domains of factor VIII and factor V alter phospholipid binding and cofactor activity

Factor VIII and factor V share structural homology and bind to phospholipid membranes via tandem, lectin-like C domains. Their respective C2 domains bind via 2 pairs of hydrophobic amino acids and an amphipathic cluster. In contrast, the factor V-like, homologous subunit (Pt-FV) of a prothrombin activator from Pseudonaja textilis venom is reported to function without membrane binding. We hypothesized that the distinct membrane-interactive amino acids of these proteins contribute to the differing membrane-dependent properties. We prepared mutants in which the C2 domain hydrophobic amino acid pairs were changed to the homologous residues of the other protein and a factor V mutant with 5 amino acids changed to those from Pt-FV (FV(MTTS/Y)). Factor VIII mutants were active on additional membrane sites and had altered apparent affinities for factor X. Some factor V mutants, including FV(MTTS/Y), had increased membrane interaction and apparent membrane-independent activity that was the result of phospholipid retained during purification. Phospholipid-free FV(MTTS/Y) showed increased activity, particularly a 10-fold increase in activity on membranes lacking phosphatidylserine. The reduced phosphatidylserine requirement correlated to increased activity on resting and stimulated platelets. We hypothesize that altered membrane binding contributes to toxicity of Pt-FV.     

The protein C ω-loop substitution Asn2Ile is associated with reduced protein C anticoagulant activity

We report a kindred with heritable protein C (PC) deficiency in which two siblings with severe thrombosis showed a composite type I and IIb PC deficiency phenotype, identified using commercial PC assays (proband: PC antigen 42 u/dl, amidolytic activity 40 u/dl, anticoagulant activity 9 u/dl). The independent PROC nucleotide variations c.669C>A (predictive of Ser181Arg) and c.131C>T (predictive of Asn2Ile) segregated with the type I and type IIb PC deficiency phenotypes respectively, but co-segregated in the siblings with severe thrombosis. Soluble thrombomodulin (sTM)-mediated inhibition of plasma thrombin generation from an individual with PC-Asn2Ile was lower (endogenous thrombin potential (ETP) 56 ± 1% that of ETP determined without sTM) than control plasma (ETP 15 ± 2%) indicating reduced PC anticoagulant activity. Recombinant APC-Asn2Ile exhibited normal amidolytic activity but impaired anticoagulant activity. Protein S (PS)-dependent anticoagulant activity of recombinant APC-Asn2Ile and binding of recombinant APC-Asn2Ile to endothelial protein C receptor (EPCR) were reduced compared to recombinant wild-type APC. Asn2 lies within the ω-loop of the PC/APC Gla domain and this region is critical for calcium-induced folding and subsequent interactions with anionic phospholipids, EPCR and PS. The disruption of these interactions in this naturally-occurring PC variant highlights their collective importance in mediating APC anticoagulant activity in vivo.     

Inherited resistance to activated protein C is corrected by anticoagulant cofactor activity found to be a property of factor V.

Recently, our laboratory described a defect inanticoagulant response to activated protein C (APC). This response, APCresistance, was shown to be inherited and associated with familialthrombophilia. As other possible mechanisms were excluded, APC resistance washypothesized to be due to deficiency of a previously unrecognized cofactor ofAPC. The aim of the present study was to isolate and characterize this factor.Plasma from an individual with pronounced inherited APC resistance was used astest plasma in a biological assay which monitored APC cofactor activity duringits isolation from normal plasma. A purification procedure was devised thatyielded a protein which was shown to be identical to coagulation factor V. Itproved impossible to separate the APC cofactor activity from factor V, even byaffinity chromatography using a monoclonal antibody against factor V. Theaffinity-purified factor V corrected the poor anticoagulant response to APC ofAPC-resistant plasma in a dose-dependent manner. Because the APC-resistantplasma contained normal levels of factor V procoagulant activity, the resultsindicated APC resistance to be due to a selective defect in the anticoagulantfunction of factor V. The present results show factor V not only to expressprocoagulant properties after its activation by thrombin but also to play animportant part in the anticoagulant system as cofactor to APC.     

Deletion or replacement of the second EGF-like domain of protein S results in loss of APC cofactor activity

Human protein S (PS), a cofactor of anticoagulant-activated protein C (APC), is a modular protein containing 4 epidermal growth factor (EGF)-like domains. EGF1 appears to mediate PS interaction with APC, but the roles of EGFs 2, 3, and 4 are less clear. We synthesized PS variants lacking single EGF domains (EGF2, 3, or 4) and assessed their APC cofactor activity in a factor Va inactivation assay. The variant lacking EGF2 (variant 134) showed the most dramatic loss of activity (approximately 10% of recombinant wild-type PS activity). Replacement of EGF2 by an additional EGF3 (variant 1334) resulted in a comparable loss of activity, suggesting that the loss of a specific rather than "spacer" function of EGF2 was responsible. We confirmed that the variant 134 had a functional gamma-carboxyglutamic acid (Gla) domain and that EGF1 was correctly folded. This is the first clear evidence that EGF2 is required for the expression of PS activity.     

Regulation of factor VIIIa by human activated protein C and protein S: inactivation of cofactor in the intrinsic factor Xase

Factor VIIIa is a trimer of A1, A2, and A3-C1-C2 subunits. Inactivation of the cofactor by human activated protein C (APC) results from preferential cleavage at Arg336 within the A1 subunit, followed by cleavage at Arg562 bisecting the A2 subunit. In the presence of human protein S, the rate of APC-dependent factor VIIIa inactivation increased several-fold and correlated with an increased rate of cleavage at Arg562. (Active site-modified) factor IXa, blocked cleavage at the A2 site. However, APC-catalyzed inactivation of factor VIIIa proceeded at a similar rate independent of factor IXa, consistent with the location of the preferential cleavage site within the A1 subunit. Addition of protein S failed to increase the rate of cleavage at the A2 site when factor IXa was present. In the presence of factor X, cofactor inactivation was inhibited, due to a reduced rate of cleavage at Arg336. However, inclusion of protein S restored near original rates of factor VIIIa inactivation and cleavage at the A1 site, thus overcoming the factor X-dependent protective effect. These results suggest that in the human system, protein S stimulates APC-catalyzed factor VIIIa inactivation by facilitating cleavage of A2 subunit (an effect retarded in the presence of factor IXa), as well as abrogating protective interactions of the cofactor with factor X. (Blood. 2000;95:1714-1720)     

Protein S negates the activated protein C inhibitory activity of plasma.

The inactivation of factor Va by the natural anticoagulant, activated protein C (APC) is subject to a number of regulatory mechanisms. This report examines the efficacy of APC in plasma and evaluates the role of the APC cofactor protein S in this milieu. The ability of protein S to enhance the anticoagulant effects of activated protein C was demonstrated using a factor Xa recalcification time of Al(OH)3 adsorbed plasma. At a set concentration of APC, increasing concentrations of protein S resulted in a linear and saturable potentiation of the activity of APC. This result was not reflected in a purified component assay, where the extent of factor Va inactivation by APC was only marginally augmented by protein S. The efficacy of the cofactor was not affected by variations in the concentration of factor Va. Similarly, increasing the protein S:APC molar ratio of 200:1 resulted in only a trivial enhancement of APC activity. To directly examine the proteolysis of factor Va by APC in plasma, a novel assay system containing Al(OH)3 adsorbed, factor V deficient plasma supplemented with purified human factor Va was developed. The addition of APC in varying concentrations to this system consistently yielded factor Va inactivation rates inferior to those seen in a purified component assay. This finding is consistent with the presence of APC inhibitory activity in plasma. When protein S was added to the plasma system, factor Va inactivation by APC was restored to a similar level to that observed in the purified system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acquired activated protein C resistance is associated with the co-existence of anti-prothrombin antibodies and lupus anticoagulant activity in patients with systemic lupus erythematosus

Venous thromboembolism (VTE) is one of the common manifestations in the anti-phospholipid (aPL) syndrome. We examined the levels of IgG antibodies (Abs) to beta2-glycoprotein I (beta2-GP I) and prothrombin, lupus anticoagulant (LA) activity, activated protein C resistance (APC-R), and factor V Leiden in 96 patients with systemic lupus erythematosus (SLE); 19 with VTE and 77 without VTE. Acquired APC-R, which was not found in any patient with the factor V Leiden mutation, was present in 33 (34.4%) out of the 96 patients with SLE. The presence of acquired APC-R was a strong risk factor for VTE. The SLE patients were divided into four groups according to the results of enzyme-linked immunosorbent assay (ELISA) and LA activity for each aPL Abs: ELISA+, LA+; ELISA+, LA-; ELISA-, LA+; and ELISA-, LA-. A significant association was observed between APC-R and the co-existence of anti-beta2-GP I Abs and LA activity or of anti-prothrombin Abs and LA activity. There was no association between APC-R and the presence of anti-beta2-GP I Abs, anti-prothrombin Abs, or LA activity alone. However, when multivariate logistical regression analysis was performed, it was clear that only the co-existence of anti-prothrombin and LA activity was a significant risk factor for APC-R. These findings indicate that the co-existence of anti-prothrombin Abs and LA activity may be an important factor in the pathogenesis of acquired APC-R in patients with SLE.     

Evaluation of protein C and protein S levels during oral anticoagulant therapy

A number of workers have examined protein C in relation to other vitamin K dependent factors during warfarin therapy and successfully identified protein C deficient patients by ratio calculation. However, protein S deficiency has not been addressed in this manner. This study compares protein C and protein S by functional and antigenic determination with procoagulant factors of similar half life (Factors VII and II) in an attempt to identify protein C and protein S deficient patients whilst on oral anticoagulant therapy. Procoagulant and anticoagulant factors were compared by linear regression in a population of normal blood donors and patients on stabilized warfarin therapy to obtain expected values for protein C and protein S dependent upon FVII and FII levels, respectively. Observed over expected values for protein C and protein S were calculated for individual patients and normal ranges derived. Comparison of similarly calculated observed over expected protein C and protein S ratios with these normal ranges successfully identified known protein C and protein S deficient patients who were taking warfarin at time of testing.     

Interaction of the protein C/protein S anticoagulant system, the endothelium and pregnancy.

Normal pregnancy is associated with significant changes in haemostasis, lipid metabolism and endothelial function. This suggests that maternal adaptation in these systems is required for successful pregnancy outcome. A number of acquired and heritable prothrombotic abnormalities are associated with complications in pregnancy. A common feature of these abnormalities is their ability to alter endothelial function or the protein C/protein S system and increase thrombin generation. In this review the normal function of the endothelium and the protein C/protein S system is detailed. The changes which characterize normal and complicated pregnancies are outlined and the evidence for the impact of heritable and acquired disorders of the protein C/protein S system on pre-eclampsia and fetal loss are discussed.