Effect of blood loss during caesarean section on coagulation parameters.

INTRODUCTION: Venous thrombosis is the leading cause of pregnancy-related maternal morbidity and mortality. The thrombosis risk is increased by caesarean section and blood loss, though underlying mechanisms of these prothrombotic changes remain unknown. MATERIALS AND METHODS: This prospective study recruited 50 pregnant women at term undergoing elective caesarean section at University Hospital Magdeburg, Germany. Blood loss during surgery was correlated with the changes in total protein S, full length TFPI (TFPIfl), prothrombin, the endogenous thrombin potential (ETP) and resistance to activated protein C (APCsr) determined via calibrated automated thrombography. RESULTS: Mean blood loss was 506 ml (95%CI: 456 to 557 ml). Total protein S was 0.63 (95%CI: 0.60 to 0.67) U/ml preoperatively, decreased by 14.8% after caesarean section and almost normalised five days later. TFPIfl was 0.47 (95%CI: 0.41 to 0.53) U/ml before, remained unchanged immediately after and increased by 11.5% five days after surgery. Prothormbin was 1.10 (95%CI: 1.03 to 1.16) U/ml preoperatively, reduced by 10.4% immediately after and increased again five days after caesarean section, exceeding the preoperative values by 4.4% (-0.7 to 9.6). The ETP decreased by 3.9%, whereas the APCsr increased by 37.0% immediately after caesarean section. The changes in total protein S, prothrombin, thrombin generation and APC resistance showed a trend to be more pronounced in the subgroups with higher blood loss. DISCUSSION: Moderate blood loss during caesarean section hardly reduces thrombin generation but aggravates pregnancy-induced APC resistance and combined deficiency of TFPI and protein S, which can account for the increased thrombosis risk in early puerperium.

Design and synthesis of a multivalent catch-and-release assay to measure circulating FXIa.

BACKGROUND: Decreased blood coagulation factor (F)XIa levels have been shown to protect from thrombosis without bleeding side effects, but less is known on effects of increased FXIa levels. Studies are hampered by lack of a reliable and robust method for FXIa quantification in blood. We aim to develop a new assay employing a unique multivalent catch-and-release system. The system selectively isolates and protects homodimeric FXIa from plasma and releases free FXIa allowing subsequent quantification. METHODS: A dynamic multivalent construct was synthesized by complexing four identical FXIa inhibitors from the snake Bungarus Fasxiatus to avidin through desthiobiotin-PEG-linkers, allowing dissociation of FXIa by excess biotin. PEG-linker lengths were optimised for FXIa inhibitory activity and analysed by Michaelis-Menten kinetics. Finally, the catch-and-release assay was validated in buffer and plasma model systems. RESULTS: Monovalent and multivalent inhibitor constructs were successfully obtained by total chemical synthesis. Multimerisation of Fasxiator resulted in a 30-fold increase in affinity for FXIa from 1.6 nM to 0.05 nM. With use of this system, FXIa could be quantified down to a concentration of 7 pM in buffer and 20 pM in plasma. CONCLUSION: In this proof-of-concept study, we have shown that the catch-and-release approach is a promising technique to quantify FXIa in plasma or buffer. By binding FXIa to the multivalent construct directly after blood drawing, FXIa is hypothesized to be inaccessible for serpin inhibition or auto inactivation. This results in a close reflection of actual circulating FXIa levels at the moment of blood drawing.

Protein S is a cofactor for tissue factor pathway inhibitor.

Protein S is a vitamin K-dependent protein that acts as a cofactor of the anticoagulant protein APC. However, protein S also exhibits anticoagulant activity in the absence of APC. Thrombin generation experiments in normal plasma and in plasma deficient in tissue factor pathway inhibitor (TFPI) and/or protein S demonstrated that protein S stimulates the inhibition of TF by TFPI. Kinetic analysis in model systems containing purified proteins showed that protein S enhances the formation of the binary FXa:TFPI complex by reducing the Ki of TFPI from approximately 4 nM to approximately 0.5 nM. Enhancement of inhibitory activity of TFPI by protein S is only observed with full-length TFPI and in the presence of a negatively charged phospholipid surface. The Ki decrease brings the TFPI concentration necessary for FXa:TFPI complex formation within range of the plasma TFPI concentration which increases FXa:TFPI complex formation and accelerates feedback inhibition of the TF pathway by enhancing the formation of the quaternary TFPI:FXa:TF:FVIIa complex. Thus, protein S is not only a cofactor of APC, but also of TFPI. A reduced TFPI cofactor activity may contribute to the increased risk of venous thrombosis in protein-S deficient individuals. Using calibrated automated thrombography we have developed two assays that enable quantification of the functional activity of the TFPI/protein S system in plasma. These assays show that the activity of the TFPI/protein S system is greatly impaired in oral contraceptive users.

Post-translational modifications regulate matrix Gla protein function: importance for inhibition of vascular smooth muscle cell calcification.

BACKGROUND: Matrix Gla protein (MGP) is a small vitamin K-dependent protein containing five gamma-carboxyglutamic acid (Gla) residues that are believed to be important in binding Ca(2+), calcium crystals and bone morphogenetic protein. In addition, MGP contains phosphorylated serine residues that may further regulate its activity. In vivo, MGP has been shown to be a potent inhibitor of vascular calcification; however, the precise molecular mechanism underlying the function of MGP is not yet fully understood. METHODS AND RESULTS: We investigated the effects of MGP in human vascular smooth muscle cell (VSMC) monolayers that undergo calcification after exposure to an increase in Ca(2+) concentration. Increased calcium salt deposition was found in cells treated with the vitamin K antagonist warfarin as compared to controls, whereas cells treated with vitamin K(1) showed decreased calcification as compared to controls. With conformation-specific antibodies, it was confirmed that warfarin treatment of VSMCs resulted in uncarboxylated (Gla-deficient) MGP. To specifically test the effects of MGP on VSMC calcification, we used full-length synthetic MGP and MGP-derived peptides representing various domains in MGP. Full length MGP, the gamma-carboxylated motif (Gla) (amino acids 35-54) and the phosphorylated serine motif (amino acids 3-15) inhibited calcification. Furthermore, we showed that the peptides were not taken up by VSMCs but bound to the cell surface and to vesicle-like structures. CONCLUSIONS: These data demonstrate that both gamma-glutamyl carboxylation and serine phosphorylation of MGP contribute to its function as a calcification inhibitor and that MGP may inhibit calcification via binding to VSMC-derived vesicles.

New functional assays to selectively quantify the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S in plasma.

Essentials Protein S is a cofactor of activated protein C (APC) and tissue factor pathway inhibitor (TFPI). There are no assays to quantify separate APC and TFPI cofactor activities of protein S in plasma. We developed assays to measure the APC- and TFPI-cofactor activities of protein S in plasma. The assays were sensitive to protein S deficiency, and not affected by the Factor V Leiden mutation. SUMMARY: Background Protein S plays an important role in the down-regulation of coagulation as cofactor for activated protein C (APC) and tissue factor pathway inhibitor (TFPI). Aim To develop functional assays to quantify the APC- and TFPI-cofactor activities of protein S in plasma. Methods APC- and TFPI-cofactor activities of protein S in plasma were measured using calibrated automated thrombography in protein S-depleted plasma supplemented with a small amount of sample plasma either in the presence of anti-TFPI antibodies and APC (APC-cofactor activity) or at excess full-length TFPI without APC (TFPI-cofactor activity). Total and free protein S levels in plasma were measured by ELISAs. Results Average APC-cofactor activities of protein S were 113%, 108% and 89% in plasma from normal individuals (n = 15), FV Leiden heterozygotes (n = 14) and FV Leiden homozygotes (n = 7), respectively, whereas the average APC-cofactor activity of protein S in plasma from heterozygous protein S-deficient individuals (n = 21) was significantly lower (55%). Similar trends were observed for the TFPI-cofactor activity of protein S, with averages of 109%, 115% and 124% in plasma from individuals with normal protein S levels and different FV Leiden genotypes, and 64% in plasma from protein S-deficient patients. APC-cofactor activities of protein S correlated significantly with free and total protein S antigen levels, whereas TFPI-cofactor activities correlated less with protein S antigen levels. Conclusion We have developed functional protein S assays that measure both the APC- and TFPI-cofactor activities of protein S in plasma, which are hardly if at all affected by the FV Leiden mutation.

The C-terminus of tissue factor pathway inhibitor-α inhibits factor V activation by protecting the Arg1545 cleavage site.

Essentials The C-terminus of tissue factor pathway inhibitor (TFPIα) binds to the B-domain of factor V (FV). The functional consequences of this interaction were investigated in plasma and model systems. The TFPIα C-terminus inhibited thrombin generation in plasma, but not in the presence of FVa. The TFPIα C-terminus inhibited FV activation by preventing cleavage at Arg1545 . SUMMARY: Background Factor V (FV) is a carrier and a cofactor of the anticoagulant protein tissue factor pathway inhibitor-α (TFPIα), whose basic C-terminus binds to an acidic region in the B-domain of FV. Proteolysis of FV at Arg709 , Arg1018 and Arg1545 by activated FX (FXa) or thrombin removes the B-domain, and converts FV into a procoagulant cofactor (activated FV [FVa]) of FXa in the prothrombinase complex. However, retention of the acidic region in partially activated FV makes prothrombinase activity susceptible to inhibition by TFPIα. Objective/Methods To investigate the effect of the TFPIα C-terminal peptide (TFPIα C-term) on thrombin generation in plasma and on FV activation in model systems. Results TFPIα C-term inhibited tissue factor-initiated and FXa-initiated thrombin generation in a dose-dependent manner. Failure to inhibit thrombin generation in FV-depleted plasma reconstituted with FVa indicated that the peptide effect was mediated by the acidic region of FV, and was localized at the level of FV activation and/or prothrombinase. In model systems, TFPIα C-term inhibited both FV activation and prothrombinase activity. Western blot analysis showed that the peptide impaired cleavage at Arg1545 by both thrombin and FXa. The inhibition was stronger for FV-short, which binds TFPIα with higher affinity. Similar results were obtained with full-length TFPIα. Conclusions Cleavage of FV at Arg1545 , which abolishes the anticoagulant properties of FV and commits FV to the procoagulant pathway, is inhibited by binding of the TFPIα C-terminus to the FV acidic region. Possible targets of this new anticoagulant function of TFPIα are low-abundance FV(a) species retaining the acidic region.

Tissue factor-independent inhibition of thrombin generation by tissue factor pathway inhibitor-α.

BACKGROUND: Tissue factor pathway inhibitor-α (TFPIα) inhibits factor Xa by forming a binary TFPI-FXa complex in a reaction that is stimulated by protein S. TF-FVIIa forms a quaternary complex with TFPIα and FXa, which shuts off the initiation of coagulation via the extrinsic pathway. AIM: To investigate whether direct inhibition of FXa by TFPIα independently of TF plays a role in downregulating coagulation. METHODS: Inhibition of FXa by TFPIα in plasma was determined by measuring thrombin generation triggered with FXa, the FX activator from Russell's viper venom (RVV-X), FXIa, or FIXa. TF-independent anticoagulant activities of TFPIα and its cofactor, protein S, were quantified: (i) after neutralization of TFPIα and protein S with anti-TFPI or anti-protein S antibodies; and (ii) in TFPI-depleted or protein S-depleted plasmas supplemented with varying amounts of TFPIα or protein S. RESULTS: Both anti-TFPI and anti-protein S antibodies enhanced thrombin generation in plasma triggered with RVV-X, FXa, FIXa, or FXIa. Anti-TFPI and anti-protein S antibodies decreased the lag time and increased the peak height of thrombin generation to the same extent, indicating that inhibition of FXa by TFPIα requires the presence of protein S. TFPIα and protein S titrations in TFPI-depleted or protein S-depleted plasma in which thrombin formation was initiated with triggers other than TF also revealed TF-independent anticoagulant activity of TFPIα, which was completely dependent on the presence of protein S. CONCLUSION: Direct inhibition of FXa by TFPIα contributes to the downregulation of coagulation.

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.

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.

Importance of the alpha 3-fragment of complement C4 for the binding with C4b-binding protein.

The human regulatory complement component C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the complement system. C4BP functions as a cofactor to factor 1 in the degradation of C4b and accelerates the decay rate of the C4b2a complex. Previously, we have demonstrated that monoclonal antibodies (C4-2 and 9) directed against the alpha'-chain of C4b inhibit the binding of C4b to C4BP. In order to identify the structural domain of C4b that binds C4BP, proteolytic fragments of C4 were generated with trypsin and Staphylococcus aureus V8 protease. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and amino acid sequence analysis of the proteolytic fragments reactive with the anti-C4 mAb's revealed that the residues Ala738-Arg826 of the alpha 3-fragment of C4b are important for the interaction with C4BP.

The region Ser333-Arg356 of the alpha-chain of human C4b-binding protein is involved in the binding of complement C4b.

Human C4b-binding protein (C4BP) functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. In this study we describe a monoclonal antibody directed against the alpha-chain of C4BP that inhibits the binding of C4b to C4BP. In order to identify the structural domain of the alpha-chain of C4BP that interacts with C4b, tryptic fragments of C4BP were generated. Amino acid sequence analysis of the fragments revealed that the residues Ser333-Arg356 of the alpha-chain of C4BP contain the epitope of this antibody, and as a consequence, that this part of the alpha-chain of C4BP is likely to be involved in the interaction with C4b.

Direct anticoagulant activity of protein S-C4b binding protein complex in Heerlen heterozygotes and normals.

BACKGROUND: Plasma protein S normally circulates free (40%) or complexed with C4b-binding protein (PS-C4BP); only free protein S is a cofactor for activated protein C during factor (F) Va inactivation. Protein S-Heerlen lacks a carbohydrate group, leading to low plasma free protein S levels, but normal levels of PS-C4BP. OBJECTIVES: Because protein S-Heerlen is not associated with thrombosis, we investigated whether PS-C4BP is directly anticoagulant in plasma and whether PS-Heerlen-C4BP has enhanced direct anticoagulant activity. METHODS: An assay for protein S direct activity was applied to Heerlen-heterozygous plasmas. Free and complexed protein S were repeatedly isolated from normal and Heerlen-heterozygous plasmas and tested for direct anticoagulant activity in prothrombinase assays and in plasma. RESULTS: Heerlen-heterozygous plasmas were deficient in free and total protein S antigen but had normal to high protein S direct anticoagulant activity. Purified Heerlen-heterozygous PS-C4BP was 7-fold more potent than normal PS-C4BP in inhibiting full prothrombinase activity, and 22-fold more potent in inhibiting prothrombin activation in the absence of FVa; it also specifically prolonged plasma clotting times 14-fold more than normal PS-C4BP. Heerlen-heterozygous PS-C4BP did not compete for limiting phospholipids any better than normal PS-C4BP. However, ligand blots and surface plasmon resonance studies showed that Heerlen-heterozygous PS-C4BP bound more avidly to FXa than did normal PS-C4BP (apparent Kd = 4.3 nm vs. 82 nm). CONCLUSIONS: Plasma-derived PS-C4BP has direct anticoagulant activity in plasma and in purified systems. Enhanced direct activity of PS-Heerlen-C4BP may compensate for low free protein S levels and low cofactor activity in individuals with protein S-Heerlen.

Characteristics of recombinant W501S mutated human gamma-glutamyl carboxylase.

A mutation (W501S) in the vitamin K-dependent gamma-glutamyl carboxylase (VKC) that leads to a congenital bleeding disorder was recently discovered in two patients. To characterize the enzyme defect, recombinant VKC-W501S was expressed in and purified from insect cells. The major effect of the mutation appears to be to decrease the affinity of the carboxylase for the propeptide of its substrates. This observation agrees with recent data that place part of the propeptide binding site within residues 495-513 of VKC. Additionally, we demonstrate that the affinity between descarboxy osteocalcin (d-OC) and VKC remains unaffected by the W501S mutation. This confirms earlier data that the high-affinity site for d-OC is not located on the propeptide binding domain of VKC. Two properties of the enzyme suggest an explanation for the observation that vitamin K supplementation ameliorates the effects of the mutation: (i) since full carboxylation requires the propeptide to remain bound to the enzyme sufficiently long for full carboxylation, a reduced affinity can cause its premature release before carboxylation is complete; (ii) propeptide binding results in a decrease of the KM for vitamin K hydroquinone in wild-type, but not in mutant carboxylase, resulting in increased vitamin K requirement of affected subjects.

Identification of different forms of human C4b-binding protein lacking beta-chain and protein S binding ability.

Human C4b-binding protein (C4BP) is a multimeric regulatory component of the complement system that circulates in plasma either as a free protein or in a noncovalent complex with the vitamin K-dependent protein S. The major form of C4BP is composed of seven identical alpha-chains (70 kDa) and one beta-chain (45 kDa). C4BP was purified from human plasma after barium citrate adsorption using anti-C4BP monoclonal antibody affinity chromatography. C4BP-high and low Mr forms were both obtained from the barium citrate precipitate and the supernatant. C4BP-high and low forms from the barium citrate precipitate were separated by sodium dodecylsulfate polyacrylamide slab gel electrophoresis and extracted with Triton X-100. Both forms contained the beta-chain as was demonstrated on sodium dodecylsulfate polyacrylamide slab gel electrophoresis under reduced conditions after silver-staining and with Western-blotting using monoclonal antibodies specific for the beta-chain. The C4BP-high and low forms demonstrated similar protein S binding affinity (KA: 3.18 x 10(8) and 3.21 x 10(8) M-1, respectively) in a C4BP-protein S binding assay and a protein S ligand blot using a peroxidase-conjugated monoclonal anti-protein S antibody. The barium citrate supernatant contained two forms of C4BP-high and one form of C4BP-low. One form of C4BP-high did contain the beta-chain and was capable of protein S binding (KA: 4.35 x 10(8) M-1). The two other forms of C4BP lacked the beta-chain and were unable to bind protein S.

In vitro studies of antiphospholipid antibodies and its cofactor, beta 2-glycoprotein I, show negligible effects on endothelial cell mediated protein C activation.

The effect of sera and purified IgG isolated from plasma of 46 patients with systemic lupus erythematosus (SLE) and 9 healthy donors on the endothelial cell (EC) mediated protein C activation was investigated. Out of the 46 SLE sera used, 19 were antiphospholipid antibodies (aPL) positive. From 12 patients IgG was isolated, of which 6 contained aPL. EC were first incubated with IgG (7 mg/ml) or serum (1:1 diluted) for 1 h and then tested for their ability to promote protein C activation by thrombin, with the cells either in a monolayer or in a suspension. The normal range (mean of control values +/- 2 SD) of protein C activation was 80-120%. In contrast to others, we could not detect an inhibition of protein C activation by any of the patient IgG's or sera. The recently described cofactor for binding of antiphospholipid antibodies to phospholipids, beta 2-glycoprotein I, was purified and added to the purified IgG's. A combination of these two components did not inhibit the EC mediated protein C activation by thrombin. This study suggests that the inhibition of the protein C activation, mediated by EC, is not a general mechanism by which aPL related thrombosis can be explained.

Activated protein C resistance: molecular mechanisms.

Activated protein C (APC) resistance is usually associated with a single DNA mutation predicting replacement of Arg506 by Gln in factor V (FV). Studies using synthetic peptides suggest that FV residues 493-506 provide factor Xa (FXa) and protein S binding sites. Biochemical studies were performed to test the hypothesis that the Arg506Gln FV mutation causes APC resistance and to define the nature of the resistance of Gln506-FVa to APC. Purified Gln506-FV conveyed APC resistance to FV-deficient plasma in APTT and FXa-1-stage assays. Purified Gln506-FVa, generated either by thrombin or by FXa, was resistant to APC. Nonetheless, Gln506-FVa was not completely resistant to APC since it was inactivated by APC approximately 10-fold slower than normal Arg506-FVa, probably due to cleavage at Arg306. This reduced but significant susceptibility of Gln506-FVa to APC inactivation may help explain why APC resistance, especially for heterozygotes, is a relatively moderate risk factor for venous thrombosis. Cardiolipin promotes APC anticoagulant activity better than FXa coagulant activity, and antibodies from some antiphospholipid antibody syndrome patients downregulate APC activity. Thus, acquired APC resistance may contribute to pathogenesis of thrombosis in the antiphospholipid antibody syndrome.

Construction and characterization of thrombin-resistant variants of recombinant human protein S.

Protein S is a vitamin K-dependent plasma protein that functions as a cofactor of activated protein C (APC) in the inactivation of coagulation factors Va and VIIIa. Protein S, migrates as a doublet on reduced SDS polyacrylamide gel electrophoresis. This heterogeneity in molecular weight has been explained by limited proteolysis of protein S. Human protein S contains at Arg-49, Arg-60 and Arg-70 three potential cleavage sites. Whether cleavage occurs at all three sites is not known. To study the role of these arginine residues in human protein S, we have replaced them by leucine or isoleucine. All seven possible variants were constructed: three variants with single mutations (R49L, R60L, R70I), three variants with double mutations (R49L/R60L, R60L/R70I, R49L/R70I) and one variant with a triple mutation (R49L/R60L/R70I). On reduced SDS polyacrylamide gels the single and double variants migrate as a doublet just like the wild type protein S. The triple variant migrates as a single band at a molecular weight corresponding to the upper band of the doublet. The upper band of the single and double variants but not of the triple variant could be converted into the lower band by thrombin treatment. All variants showed cofactor activity to APC in a clotting assay. After thrombin treatment, this cofactor activity was abolished for the single (R49L, R60L, R70I) and double variants (R49L/R60L, R60L/R70I, R49L/R70I), while the triple variant (R49L/R60L/R70I) tested at several concentrations, retained its cofactor activity completely, suggesting resistance to thrombin. This shows that thrombin can cleave at all three arginine sites and that cleavage at each of these sites results in the loss of APC cofactor activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of tissue factor:factor VIIa-catalyzed factor IX and factor X activation by TFPI and TFPI constructs.

BACKGROUND: TFPI is a Kunitz-type protease inhibitor that downregulates the extrinsic coagulation pathway by inhibiting factor Xa (FXa) and FVIIa. All three Kunitz domains (KD1, KD2, and KD3) and protein S are required for optimal inhibition of FXa and FVIIa. There is limited information on Kunitz domain requirements of the inhibition of TF:FVIIa-catalyzed FIX and FX activation by TFPI. AIM: To investigate the role of the Kunitz domains of TFPI and protein S in the inhibition of FX and FIX activation. METHODS: Inhibition of TF:FVIIa-catalyzed FX and FIX activation by full-length TFPI (TFPIFL ) and TFPI constructs was quantified from progress curves of FXa and FIXa generation measured with chromogenic substrates. RESULTS AND CONCLUSIONS: TFPIFL inhibited TF:FVIIa-catalyzed FIX activation with a Ki of 16.7 nmol L(-1) . Protein S reduced the Ki to 1.0 nmol L(-1) . TFPI1-150 and KD1-KD2 had 10-fold higher Ki values and were not stimulated by protein S. Single Kunitz domains were poor inhibitors of TF:FVIIa-catalyzed FIX activation (Ki >800 nm). FX activation was measured at limiting FVIIa and excess TF or vice versa. At both conditions, TFPIFL , TFPI1-150 , and KD1-KD2 showed similar inhibition of FX activation. However, at low phospholipid concentrations, TFPIFL was ~ 15-fold more active than TFPI1-150 or KD1-KD2. Apparently, excess phospholipids act as a kind of sink for TFPIFL , limiting its availability for TF:FVIIa inhibition. Preformed FXa:TFPIFL/1-150 complexes rapidly and stoichiometrically inhibited FIX and FX activation by TF:FVIIa, indicating that binary TFPI:FXa complex formation is the limiting step in TF:FVIIa inhibition. Protein S also enhanced inhibition of TF:FVIIa-catalyzed FX activation by TFPI.

Direct inhibition of factor VIIa by TFPI and TFPI constructs.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a multi-Kunitz domain protease inhibitor that down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa. OBJECTIVES: To investigate the role of the three Kunitz domains (KDs) of TFPI in FVIIa inhibition using full-length TFPI (TFPIfl ) and truncated TFPI constructs. METHODS: Inhibition of FVIIa with/without relipidated tissue factor (TF) or soluble TF (sTF) by TFPIfl /TFPI constructs was quantified with a FVIIa-specific chromogenic substrate. RESULTS AND CONCLUSIONS: TFPIfl inhibited TF-FVIIa via a monophasic reaction, which is rather slow at low TFPIfl concentrations (t½  ≈ 5 min at 2 nm TFPI) and has a Ki of 4.6 nm. In the presence of sTF and without TF, TFPIfl was a poor FVIIa inhibitor, with Ki values of 122 nm and 1118 nm, respectively. This indicates that phospholipids and TF significantly contribute to FVIIa inhibition by TFPIfl . TFPI constructs without the KD3-c-terminus (TFPI1-150 and KD1-KD2) were 7-10-fold less effective than TFPIfl in inhibiting TF-FVIIa and sTF-FVIIa, indicating that the KD3-C-terminus significantly contributes to direct inhibition of FVIIa by TFPI. Compared with KD1-KD2, KD1 was a poor TF-FVIIa inhibitor (Ki =434 nm), which shows that the KD2 domain of TFPI also contributes to FVIIa inhibition. Protein S stimulated TF-FVIIa inhibition by TFPIfl (Ki =0.7 nm). In the presence of FXa, a tight quaternary TF-FVIIa-TFPI-FXa complex is formed with TFPIfl , TFPI1-150 and KD1-KD2, with Ki values of < 0.15 nm, 0.5 nm and 0.8 nm, respectively, indicating the KD3-C-terminus is not a prerequisite for quaternary complex formation. Phospholipids and the Gla-domain of FXa are required for quaternary complex formation.