Biomedical subjects

T M Hackeng

Publications and source records attributed to T M Hackeng.

At least 19 recordsLinked to original sources

PubMed2001

Purified protein S contains multimeric forms with increased APC-independent anticoagulant activity.

Protein S, the cofactor of activated protein C (APC), also expresses anticoagulant activity independent of APC by directly inhibiting prothrombin activation via interactions with factor Xa, factor Va, and phospholipids. In different studies, however, large variations in APC-independent anticoagulant activities have been reported for protein S. The investigation presented here shows that within purified protein S preparations different forms of protein S are present, of which a hitherto unrecognized form (<5% of total protein S) binds with high affinity to phospholipid bilayers (K(d) < 1 nM). The remaining protein S (>95%) has a low affinity (K(d) = 250 nM) for phospholipids. Using their different affinities for phospholipids, separation of the forms of protein S was achieved. Native polyacrylamide gel electrophoresis demonstrated that the form of protein S that binds to phospholipids with low affinity migrated as a single band, whereas the high-affinity protein S exhibited several bands that migrated with reduced mobility. Size-exclusion chromatography revealed that the slower-migrating bands represented multimeric forms of protein S. Multimeric protein S (<5% of total protein S) appeared to have a 100-fold higher APC-independent anticoagulant activity than the abundant form of protein S. Comparison of purified protein S preparations that exhibited a 4-fold difference in APC-independent anticoagulant activity showed that the ability to inhibit prothrombin activation correlated with the content of multimeric protein S. Multimeric protein S could not be identified in normal human plasma, and it is therefore unlikely that this form of protein S contributes to the APC-independent anticoagulant activity of protein S that is observed in plasma.

Adsorption↗

PubMed2001

Total chemical synthesis of human matrix Gla protein.

Human matrix Gla protein (MGP) is a vitamin K-dependent extracellular matrix protein that binds Ca2+ ions and that is involved in the prevention of vascular calcification. MGP is a 10.6-kD protein (84 amino acids) containing five gamma-carboxyglutamic acid (Gla) residues and one disulfide bond. Studies of the mechanism by which MGP prevents calcification of the arterial media are hampered by the low solubility of the protein (<10 microg/mL). Because of solubility problems, processing of a recombinantly expressed MGP-fusion protein chimera to obtain MGP was unsuccessful. Here we describe the total chemical synthesis of MGP by tBoc solid-phase peptide synthesis (SPPS) and native chemical ligation. Peptide Tyr1-Ala53 was synthesized on a derivatized resin yielding a C-terminal thioester group. Peptide Cys54-Lys84 was synthesized on Lys-PAM resin yielding a C-terminal carboxylic acid. Subsequent native chemical ligation of the two peptides resulted in the formation of a native peptide bond between Ala53 and Cys54. Folding of the 1-84-polypeptide chain in 3 M guanidine (pH 8) resulted in a decrease of molecular mass from 10,605 to 10,603 (ESI-MS), representing the loss of two protons because of the formation of the Cys54-Cys60 internal disulfide bond. Like native MGP, synthetic MGP had the same low solubility when brought into aqueous buffer solutions with physiological salt concentrations, confirming its native like structure. However, the solubility of MGP markedly increased in borate buffer at pH 7.4 in the absence of sodium chloride. Ca2+-binding to MGP was confirmed by analytical HPLC, on which the retention time of MGP was reduced in the presence of CaCl2. Circular dichroism studies revealed a sharp increase in alpha-helicity at 0.2 mM CaCl2 that may explain the Ca2+-dependent shift in high-pressure liquid chromatography (HPLC)-retention time of MGP. In conclusion, facile and efficient chemical synthesis in combination with native chemical ligation yielded MGP preparations that can aid in unraveling the mechanism by which MGP prevents vascular calcification.

Blotting, Western↗

PubMed2000

Chemical synthesis and spontaneous folding of a multidomain protein: anticoagulant microprotein S.

Because of recent high-yield native ligation techniques, chemical synthesis of larger multidomain bioactive proteins is rapidly coming within reach. Here we describe the total chemical synthesis of a designed "microprotein S," comprising the gamma-carboxyglutamic acid-rich module, the thrombin-sensitive module, and the first epidermal growth factor-like module of human plasma protein S (residues 1-116). Synthetic microprotein S expressed anticoagulant cofactor activity for activated protein C in the down-regulation of blood coagulation, and the anticoagulant activity of microprotein S was not neutralized by C4b-binding protein, a natural inhibitor of native protein S in plasma. The correct folding of this complex multidomain protein was enhanced compared with individual modules because the gamma-carboxyglutamic acid-rich module and the thrombin-sensitive module markedly facilitated correct folding of the first epidermal growth factor-like module compared with folding of the first epidermal growth factor-like module alone. These results demonstrate that total chemical synthesis of proteins offers an effective way to generate multidomain biologically active proteins.

1-Carboxyglutamic Acid↗

PubMed2000

Conformational changes in activated protein C caused by binding of the first epidermal growth factor-like module of protein S.

The first epidermal growth factor-like module of human plasma protein S (EGF1, residues 76-116) was chemically synthesized and tested for its ability to inhibit the anticoagulant cofactor activity of protein S for the anticoagulant protease, activated protein C (APC). EGF1 completely inhibited the stimulation of APC activity by protein S in plasma coagulation assays, with 50% inhibition at approx. 1 microM+ EGF1, suggesting direct binding of EGF1 to APC. To investigate a direct interaction between EGF1 and APC, fluorescence resonance energy transfer (FRET) experiments were employed. APC labelled in the active site with fluorescein as the donor, and phospholipid vesicles containing octadecylrhodamine as the acceptor, showed that EGF1 association with APC caused an increase in energy transfer consistent with a relocation of the active site of APC from 94 A (9.4 nm) to 85 A above the phospholipid surface (assuming kappa(2)=2/3). An identical increase in energy transfer between the APC active site-bound fluorescein and phospholipid-bound rhodamine was obtained upon association of protein S or protein S-C4b-binding protein complex with APC. The latter suggests the presence of a ternary complex of protein S-C4b-binding protein with APC on the phospholipid surface. To confirm a direct interaction of EGF1 with APC, rhodamine was covalently attached to the alpha-N-terminus of EGF1, and binding of the labelled EGF1 to APC was directly demonstrated using FRET. The data suggested a separation between the active site of APC and the N-terminus of EGF1 of 76 A (kappa(2)=2/3), placing the APC-bound protein S-EGF1 close to, but above, the phospholipid surface and near the two EGF domains of APC. Thus we provide direct evidence for binding of protein S-EGF1 to APC and show that it induces a conformational change in APC.

Amino Acid Sequence↗

PubMed2000

Inactivation of active thrombin-activable fibrinolysis inhibitor takes place by a process that involves conformational instability rather than proteolytic cleavage.

Thrombin-activable fibrinolysis inhibitor (TAFI) is present in the circulation as an inactive zymogen. Thrombin converts TAFI to a carboxypeptidase B-like enzyme (TAFIa) by cleaving at Arg(92) in a process accelerated by the cofactor, thrombomodulin. TAFIa attenuates fibrinolysis. TAFIa can be inactivated by both proteolysis by thrombin and spontaneous temperature-dependent loss of activity. The identity of the thrombin cleavage site responsible for loss of TAFIa activity was suggested to be Arg(330), but site-directed mutagenesis of this residue did not prevent inactivation of TAFIa by thrombin. In this study we followed TAFI activation and TAFIa inactivation by thrombin/thrombomodulin in time and characterized the cleavage pattern of TAFI using matrix-assisted laser desorption ionization mass spectrometry. Mass matching of the fragments revealed that TAFIa was cleaved at Arg(302). Studies of a mutant R302Q-TAFI confirmed identification of this thrombin cleavage site and, furthermore, suggested that inactivation of TAFIa is based on its conformational instability rather than proteolytic cleavage at Arg(302).

Carboxypeptidase B2↗

PubMed2000

Cardiolipin is a normal component of human plasma lipoproteins.

Anticardiolipin (anti-CL) antibodies, diagnostic for antiphospholipid antibody syndrome, are associated with increased risks of venous and arterial thrombosis. Because CL selectively enhances activated protein C/protein S-dependent anticoagulant activities in purified systems and because CL is not known to be a normal plasma component, we searched for CL in plasma. Plasma lipid extracts [chloroform/methanol (2:1, vol/vol)] were subjected to analyses by using TLC, analytical HPLC, and MS. A plasma lipid component was purified that was indistinguishable from reference CL (M:1448). When CL in 40 fasting plasma lipid extracts (20 males, 20 females) was quantitated by using HPLC, CL (mean +/- SD) was 14.9 +/- 3.7 microgram/ml (range 9.1 to 24.2) and CL was not correlated with phosphatidylserine (3.8 +/- 1.7 microgram/ml), phosphatidylethanolamine (64 +/- 20 microgram/ml), or choline-containing phospholipid (1,580 +/- 280 microgram/ml). Based on studies of fasting blood donors, CL (>/=94%) was recovered in very low density, low density, and high density lipoproteins (11 +/- 5.3%, 67 +/- 11.0%, and 17 +/- 10%, respectively), showing that the majority of plasma CL (67%) is in low density lipoprotein. Analysis of relative phospholipid contents of lipoproteins indicated that high density lipoprotein is selectively enriched in CL and phosphatidylethanolamine. These results shows that CL is a normal plasma component and suggest that the epitopes of antiphospholipid antibodies could include CL or oxidized CL in lipoproteins or in complexes with plasma proteins (e. g., beta(2)-glycoprotein I, prothrombin, protein C, or protein S) or with platelet or endothelial surface proteins.

Antibodies, Antiphospholipid↗

PubMed2000

Cardiolipin enhances protein C pathway anticoagulant activity.

The anticoagulant activity of activated protein C (APC) was studied using factor Xa-1-stage assays of both the procoagulant and anticoagulant activities of phospholipid vesicles containing phosphatidylserine or cardiolipin as active phospholipids. In the absence of APC, phosphatidylserine vesicles showed higher procoagulant activity than cardiolipin vesicles whereas cardiolipin vesicles supported APC-dependent anticoagulant activity better than phosphatidylserine vesicles. Enhancement of APC anticoagulant activity in plasma by cardiolipin was markedly stimulated by the APC cofactor protein S. In purified reaction mixtures, cardiolipin in phospholipid vesicles dose-dependently enhanced APC anticoagulant activity. This effect of cardiolipin was partially dependent on protein S, and immunoblotting studies showed that cardiolipin enhanced the APC-mediated cleavage of the factor Va heavy chain at Arg506 and Arg306. In solid-phase binding assays, increasing amounts of cardiolipin in multicomponent phospholipid vesicles increased the affinity for protein S and to a lesser extent APC. These data are consistent with the hypothesis that cardiolipin stimulates the anticoagulant protein C pathway by increasing the affinity of phospholipid surfaces for protein S:APC and by enhancing inactivation of factor Va by APC due to cleavages at Arg506 and Arg306 in factor Va. Based on this, it is further hypothesized that anti-cardiolipin or anti-oxidized cardiolipin antibodies may be thrombogenic because they inhibit phospholipid-dependent expression of the anticoagulant protein C pathway.

Blood Coagulation↗

PubMed1999

Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology.

The total chemical synthesis of proteins has great potential for increasing our understanding of the molecular basis of protein function. The introduction of native chemical ligation techniques to join unprotected peptides next to a cysteine residue has greatly facilitated the synthesis of proteins of moderate size. Here, we describe a straightforward methodology that has enabled us to rapidly analyze the compatibility of the native chemical ligation strategy for X-Cys ligation sites, where X is any of the 20 naturally occurring amino acids. The simplified methodology avoids the necessity of specific amino acid thioester linkers or alkylation of C-terminal thioacid peptides. Experiments using matrix-assisted laser-desorption ionization MS analysis of combinatorial ligations of LYRAX-C-terminal thioester peptides to the peptide CRANK show that all 20 amino acids are suitable for ligation, with Val, Ile, and Pro representing less favorable choices because of slow ligation rates. To illustrate the method's utility, two 124-aa proteins were manually synthesized by using a three-step, four-piece ligation to yield a fully active human secretory phospholipase A(2) and a catalytically inactive analog. The combination of flexibility in design with general access because of simplified methodology broadens the applicability and versatility of chemical protein synthesis.

Amino Acid Sequence↗

PubMed1998

Inhibition of prothrombinase by human secretory phospholipase A2 involves binding to factor Xa.

Human group II secretory phospholipase A2 (hsPLA2) exhibits significant anticoagulant activity that does not require its enzymatic activity. We examined which coagulation factor was targeted by hsPLA2 and analyzed which region of the protein may be involved in this inhibition. Prothrombin time coagulation assays indicated that hsPLA2 did not inhibit activated factor V (FVa) activity, whereas activated factor X (FXa) one-stage coagulation assays suggested that FXa was inhibited. The inhibitory effect of hsPLA2 on prothrombinase activity of FXa, FV, phospholipids, and Ca2+ complex was markedly enhanced upon preincubation of hsPLA2 with FXa but not with FV. Prothrombinase activity was also strongly inhibited by hsPLA2 in the absence of PL. High concentrations of FVa in the prothrombinase generation assay reversed the inhibitory effect of hsPLA2. By using isothermal titration calorimetry, we demonstrated that hsPLA2 binds to FXa in solution with a 1:1 stoichiometry and a Kd of 230 nM. By using surface plasmon resonance we determined the rate constants, kon and koff, of the FXa/hsPLA2 interaction and analyzed the Ca2+ effect on these constants. When preincubated with FXa, synthetic peptides comprising residues 51-74 and 51-62 of hsPLA2 inhibited prothrombinase assays, providing evidence that this part of the molecule, which shares similarities with a region of FVa that binds to FXa, is likely involved in the anticoagulant interaction of hsPLA2 with FXa. In conclusion, we propose that residues 51-62 of hsPLA2 bind to FXa at a FVa-binding site and that hsPLA2 decreases the prothrombinase generation by preventing FXa.FVa complex formation.

Amino Acid Sequence↗

PubMed1998

Binding site for blood coagulation factor Xa involving residues 311-325 in factor Va.

Factor Va inactivation by activated protein C is associated with cleavages at Arg306, Arg506, and Arg679 with Arg306 cleavage causing the major activity loss. To study functional roles of the Arg306 region, overlapping 15-mer peptides representing the sequence of factor Va residues 271-345 were synthesized and screened for anticoagulant activities. The peptide containing residues 311-325 (VP311) noncompetitively inhibited prothrombin activation by factor Xa, but only in the presence of factor Va. Fluorescence studies showed that VP311 bound to fluorescence-labeled 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg factor Xa in solution with a Kd of 70 microM. Diisopropylphosphoryl factor Xa and factor Xa but not factor VII/VIIa or prothrombin bound to immobilized VP311 with relatively high affinity. These results support the hypothesis that residues 311-325, which are positioned between the A1 and A2 domains of factor Va and likely exposed to solvent, contribute to the binding of factor Xa by factor Va. Based on this hypothesis, it is suggested that cleavage by activated protein C at Arg306 in factor Va not only severs the covalent connection between the A1 and A2 domains but also disrupts the environment and structure of residues 311-325, thereby down-regulating the binding of factor Xa to factor Va.

Amino Acid Sequence↗

PubMed1998

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-tetramethyluron ium 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 beta-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% beta-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.

Amino Acid Sequence↗

PubMed1997

A factor IX-deficient mouse model for hemophilia B gene therapy.

We have generated a mouse where the clotting factor IX (FIX) gene has been disrupted by homologous recombination. The FIX nullizygous (-/-) mouse was devoid of factor IX antigen in plasma. Consistent with the bleeding disorder, the factor IX coagulant activities for wild-type (+/+), heterozygous (+/-), and homozygous (-/-) mice were 92%, 53%, and <5%, respectively, in activated partial thromboplastin time assays. Plasma factor IX activity in the deficient mice (-/-) was restored by introducing wild-type murine FIX gene via adenoviral vectors. Thus, these factor IX-deficient mice provide a useful animal model for gene therapy studies of hemophilia B.

Adenoviridae↗

PubMed1997

Total chemical synthesis of enzymatically active human type II secretory phospholipase A2.

Human group II secretory phospholipase A2 (sPLA2) is an enzyme found in the alpha granules of platelets and at inflammatory sites. Although its physiological function is unclear, sPLA2 can inhibit blood coagulation reactions independent of its lipolytic action. To study the molecular basis of PLA2 activities, we developed a total chemical synthesis of sPLA2 by chemical ligation of large unprotected peptides. The synthetic segments PLA2-(1-58)-alphaCOSCH2COOH and PLA2-(59-124) were prepared by stepwise solid-phase peptide synthesis and ligated to yield a peptide bond between Gly58 and Cys59. The 124-residue polypeptide product (mass: 13,920 +/- 2 Da) was folded to yield one major product (mass: 13,905 +/- 1 Da), the loss of 15 +/- 3 Da reflecting the formation of seven disulfide bonds. Circular dichroism studies of synthetic sPLA2 showed alpha-helix, beta-structure, and random coil contents consistent with those found in the crystal structure of sPLA2. Synthetic sPLA2 had kcat and Km values identical to those of recombinant sPLA2 for hydrolysis of 1,2-bis(heptanoylthio)-phosphatidylcholine. Synthetic sPLA2, like recombinant sPLA2, inhibited thrombin generation from prothrombinase complex (factors Xa, V, II, Ca2+, and phospholipids). In the absence of phospholipids, both synthetic and recombinant sPLA2 inhibited by 70% prothrombin activation by factors Xa, Va, and Ca2+. Thus, synthetic sPLA2 is a phospholipid-independent anticoagulant like recombinant or natural sPLA2. This study demonstrates that chemical synthesis of sPLA2 yields a fully active native-like enzyme and offers a straightforward tool to provide sPLA2 analogs for structure-activity studies of anticoagulant, lipolytic, or inflammatory activities.

Amino Acid Sequence↗

PubMed1997

The carbohydrate moiety of factor V modulates inactivation by activated protein C.

An important risk factor for thrombosis is the polymorphism R506Q in factor V that causes resistance of factor Va to proteolytic inactivation by activated protein C (APC). To study the potential influence of the carbohydrate moieties of factor Va on its inactivation by APC, factor V was subjected to mild deglycosylation (neuraminidase plus N-glycanase) under nondenaturing conditions. The APC resistance ratio values (ratio of activated partial thromboplastin time [APTT] clotting times with and without APC) of the treated factor V were increased (2.4 to 3.4) as measured in APTT assays. O-glycanase treatment of factor V did not change the APC resistance ratio. The procoagulant activity of factor V as well as its activation by thrombin was not affected by mild deglycosylation. Treatment of factor V with neuraminidase and N-glycanase mainly altered the electrophoretic mobility of the factor Va heavy chain, whereas treatment with O-glycanase changed the mobility of the connecting region. This suggests that the removal of the N-linked carbohydrates from the heavy chain of factor Va, which is the substrate for APC, is responsible for the increase in susceptibility to inactivation by APC. Thus, variability in carbohydrate could account for some of the known variability in APC resistance ratios, including the presence of borderline or low APC resistance ratios among patients who lack the R506Q mutation.

Amidohydrolases↗

PubMed1996

Protein C activation on endothelial cells by prothrombin activation products generated in situ: meizothrombin is a better protein C activator than alpha-thrombin.

The conversion of protein C into activated protein C (APC) by the thrombin-thrombomodulin complex on the surface of endothelial cells initiates an essential negative feedback reaction on blood coagulation. APC, together with its non-enzymic cofactor protein S, inactivates factors Va and VIIIa, the non-enzymic protein cofactors of the prothrombinase and intrinsic tenase complex, by proteolytic degradation. In this study we report that prothrombin activation products, generated by the prothrombinase complex on the surface of quiescent endothelial cells, are able to activate protein C. Subsequent inactivation of factor Va by the APC that was formed decreased the rate of prothrombin activation, thus demonstrating in vitro the negative feedback loop on coagulation factor activation. The anticoagulant feedback reaction of APC on the prothrombinase complex was stimulated 3-4-fold by the addition of protein S but not by thrombin-cleaved protein S or by protein S complexed with C4b-binding protein. Stimulation of endothelial cells with 50 pM tumour necrosis factor (TNF) or 500 pM interleukin 1 (IL-1) resulted in a 70% decrease in activation of protein C by exogenously added alpha-thrombin, which seemed to be due to down-regulation of thrombomodulin activity on the surface of endothelial cells. However, when prothrombin activation products generated in situ were allowed to activate protein C, stimulation of endothelial cells with TNF and IL-1 resulted in only a 25% decrease in activation of protein C. Stimulation with TNF or IL-1 did not affect the ability of endothelial cells to support prothrombinase activity. We investigated whether the differences in extent of protein C activation by exogenously added alpha-thrombin and by prothrombin activation products generated in situ were due to meizothrombin formed during prothrombin activation. Previous reports from our groups revealed that meizothrombin is generated as a transient intermediate during prothrombin activation on phospholipid vesicles and endothelial cells. Here we show that meizothrombin is at least a 6-fold better activator of protein C on the surface of endothelial cells than is alpha-thrombin. These results demonstrate that meizothrombin, formed during the initial phase of prothrombin activation, efficiently down-regulates both its own formation and that of thrombin.

Cells, Cultured↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1995

Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII.

Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic factor X activation via a specific interaction with factor VIII. In the presence of endothelial cells, the intrinsic activation of factor X was inhibited by protein S with an IC50 value of 0.28 +/- 0.04 mumol/L corresponding to the plasma concentration of protein S. This inhibitory effect was even more pronounced when the intrinsic factor X activation was studied in the presence of activated platelets (IC50 = 0.15 +/- 0.02 mumol/L). When a nonlimiting concentration of phospholipid vesicles was used, the plasma concentration of protein S (300 nmol/L) inhibited the intrinsic factor X activation by 40%. Thrombin-cleaved protein S inhibited the endothelial cell-mediated factor X activation with an IC50 similar to that of native protein S (0.26 +/- 0.02 mumol/L). Protein S in complex with C4b-binding protein inhibited the endothelial cell-mediated factor X activation more potently than protein S alone (IC50 = 0.19 +/- 0.03 mumol/L). Using thrombin activated factor VIII, IC50 values of 0.53 +/- 0.09 mumol/L and 0.46 +/- 0.10 mumol/L were found for native protein S and thrombin-cleaved protein S, respectively. The possible interactions of protein S with factor IXa, phospholipids, and factor VIII were investigated. The enzymatic activity of factor IXa was not affected by protein S, and interaction of protein S with the phospholipid surface could not fully explain the inhibitory effect of protein S on the factor X activation. Using a solid-phase binding assay, we showed a specific, saturable, and reversible binding of protein S to factor VIII with a high affinity. The concentration of protein S where half-maximal binding was reached (B1/2max) was 0.41 +/- 0.06 mumol/L. A similar affinity was found for the interaction of thrombin-cleaved protein S with factor VIII (B1/2max = 0.40 +/- 0.04 mumol/L). The affinity of the complex protein S with C4B-binding protein appeared to be five times higher (B1/2max = 0.07 +/- 0.03 mumol/L). Because the affinities of the interaction of the different forms of protein S with factor VIII correspond to the IC50 values observed for the intrinsic factor X activating complex, the interaction of protein S with factor VIII may explain the inhibitory effect of protein S on the intrinsic factor X activating complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Anticoagulants↗

PubMed1995

Increased prothrombin activation in protein S-deficient plasma under flow conditions on endothelial cell matrix: an independent anticoagulant function of protein S in plasma.

Protein S is a vitamin K-dependent nonenzymatic coagulation factor involved in the regulation of activated protein C (aPC). In this study, we report an aPC-independent anticoagulant function of protein S in plasma under flow conditions. Plasma, anticoagulated with low-molecular-weight heparin allowing tissue factor-dependent prothrombin activation, was perfused at a wall shear rate of 100 s-1 over tissue factor containing matrices of stimulated endothelial cells placed in a perfusion chamber. Fractions were collected in time at the outlet and prothrombin activation was determined by measuring the activation fragment F1+2 of prothrombin. In normal plasma, a time-dependent prothrombin activation was detected by the generation of fragment1+2. Prothrombin activation had ceased after 12 minutes perfusion, independent of the amount of tissue factor present in the matrix. Depletion of protein S from plasma or inhibition of protein S in plasma by monoclonal antibodies induced a 5- to 25-fold increase of prothrombin activation on the procoagulant endothelial cell matrix. A prolonged prothrombin activation was detected in protein S-depleted plasma up to 20 minutes after onset of the thrombin generation. The increased prothrombin activation in protein S-depleted plasma could not be explained by the absence of the cofactor function of protein S for aPC because depletion of protein C from plasma did not result in increased prothrombin activation. These data provide further evidence for a strong anticoagulant function of protein S in plasma independent from activated protein C.

Antibodies, Monoclonal↗