Correlates of antithrombin, protein C, protein S, and TFPI in a healthy elderly cohort.

The majority of fatal acute myocardial infarctions occur in the elderly. Since these events are predominantly thrombotic, we studied the cross-sectional associations of the anticoagulant proteins Antithrombin, Protein C, Protein S. and Tissue Factor Pathway Inhibitor (TFPI) in a subgroup (n = 400) of the Cardiovascular Health Study (a study of healthy men and women > or = 65 years) free of clinical cardiovascular disease (CVD). We did not observe any strong age-associated trends, although Protein C was lower in older women (p < or = 0.001), and TFPI was higher in older men (p < or = 0.01). The inhibitors were highly intercorrelated, and were associated with increased levels of inflammation-sensitive proteins (e.g., fibrinogen. plasminogen), lipids (especially total and LDL-cholesterol), and coagulation factors, such as Factors VIIc, IXc, and Xc. None was associated with the procoagulant markers Prothrombin Fragment F1-2 or Fibrinopeptide A. Only TFPI was associated with subclinical atherosclerosis: ankle-arm index and internal carotid artery stenosis, p trend < or = 0.01; and carotid wall thickness, p trend < or = 0.05. In multivariate analysis the independent predictors of TFPI were levels of fibrinogen; the fibrinolytic marker plasmin-antiplasmin complex; LDL-cholesterol; and carotid wall thickness (R2 for the model = 0.35). In summary, the inhibitors did not appear to increase with age, and were predominantly associated with inflammation markers and lipids. Since markers of thrombin production do increase with age, we hypothesize that an age-related hemostatic imbalance may ensue, with associated increased thrombotic risk. Only TFPI was associated with subclinical CVD, suggesting that it may more closely reflect endothelial damage.

Interaction of factor IXa with factor VIIIa. Effects of protease domain Ca2+ binding site, proteolysis in the autolysis loop, phospholipid, and factor X.

We previously identified a high affinity Ca2+ binding site in the protease domain of factor IXa involving Glu235 (Glu70 in chymotrypsinogen numbering; hereafter, the numbers in brackets refer to the chymotrypsin equivalents) and Glu245[80] as putative ligands. To delineate the function of this Ca2+ binding site, we expressed IXwild type (IXWT), IXE235K, and IXE245V in 293 kidney cells and compared their properties with those of factor IX isolated from normal plasma (IXNP); each protein had the same Mr and gamma-carboxyglutamic acid content. Activation of each factor IX protein by factor VIIa.Ca2+.tissue factor was normal as analyzed by sodium dodecyl sulfate-gel electrophoresis. The coagulant activity of IXaWT was approximately 93%, of IXaE235K was approximately 27%, and of IXaE245V was approximately 4% compared with that of IXaNP. In contrast, activation by factor XIa.Ca2+ led to proteolysis at Arg318-Ser319[150-151] in the protease domain autolysis loop of IXaE245V with a concomitant loss of coagulant activity; this proteolysis was moderate in IXaE235K and minimal in IXaWT or IXaNP. Interaction of each activated mutant with an active site probe, p-aminobenzamidine, was also examined; the Kd of interaction in the absence and presence (in parentheses) of Ca2+ was: IXaNP or IXaWT 230 microM (78 microM), IXaE235K 150 microM (145 microM), IXaE245V 225 microM (240 microM), and autolysis loop cleaved IXaE245V 330 microM (350 microM). Next, we evaluated the apparent Kd (Kd,app) of interaction of each activated mutant with factor VIIIa. We first investigated the EC50 of interaction of IXaNP as well as of IXaWT with factor VIIIa in the presence and absence of phospholipid (PL) and varying concentrations of factor X. At each factor X concentration and constant factor VIIIa, EC50 was the free IXaNP or IXaWT concentration that yielded a half-maximal rate of factor Xa generation. EC50 values for IXaNP and IXaWT were similar and are as follows: PL-minus/X-minus (extrapolated), 2.8 microM; PL-minus/X-saturating, 0.25 microM; PLplus/X-minus, 1.6 nM; and PL-plus/X-saturating, 0.09 nM. Further, Kd,app of binding of active site-blocked factor IXa to factor VIIIa was calculated from its ability to inhibit IXaWT in the Tenase assay. Kd,app values in the absence and presence (in parentheses) of PL were: IXaNP or IXaWT, 0. 19 microM (0.07 nM); IXaE235K, 0.68 microM (0.26 nM); IXaE245V, 2.5 microM (1.35 nM); and autolysis loop-cleaved IXaE245V, 15.6 microM (14.3 nM). We conclude that (a) PL increases the apparent affinity of factor IXa for factor VIIIa approximately 2,000-fold, and the substrate, factor X, increases this affinity approximately 10-15-fold; (b) the protease domain Ca2+ binding site increases this affinity approximately 15-fold, and lysine at position 235 only partly substitutes for Ca2+; (c) Ca2+ binding to the protease domain increases the S1 reactivity approximately 3-fold and prevents proteolysis in the autolysis loop; and (d) proteolysis in the autolysis loop leads to a loss of catalytic efficiency with retention of S1 binding site and a further approximately 8-fold reduction in affinity of factor IXa for factor VIIIa.

Interaction of calcium with native and decarboxylated human factor X. Effect of proteolysis in the autolysis loop on catalytic efficiency and factor Va binding.

Human factor X is a two-chain, 58-kDa, vitamin K-dependent blood coagulation zymogen. The light chain of factor X consists of an NH2-terminal gamma-carboxyglutamic acid (Gla) domain, followed by a few helical hydrophobic residues and the two epidermal growth factor-like domains, whereas the heavy chain contains the serine protease domain. In this study, native factor X was found to contain three classes of Ca2+-binding sites: two high affinity (Kd 100 +/- 30 microM), four intermediate affinity (Kd 450 +/- 70 microM), and five to six low affinity (Kd 2 +/- 0.2 mM). Decarboxylated factor X in which the Gla residues were converted to Glu retained the two high affinity sites (Kd 140 +/- 20 microM). In contrast, factor X lacking the Gla domain as well as a part of the helical hydrophobic residues (des-44-X) retained only one high affinity Ca2+-binding site (Kd 130 +/- 20 microM). Moreover, a synthetic peptide composed of residues 238-277 (58-97 in chymotrypsinogen numbering) from the protease domain of factor X bound one Ca2+ with high affinity (Kd 150 +/- 20 microM). From competitive inhibition assays for binding of active site-blocked factor Xa to factor Va in the prothrombinase complex, the Kd for peptide-Va interaction was calculated to be approximately 10 microM as compared with 30 pM for factor Xa and approximately 1.5 microM for decarboxylated factor Xa. A peptide containing residues 238-262(58-82) bound Ca2+ with reduced affinity (Kd approximately 600 microM) and did not inhibit Xa:Va interaction. In contrast, a peptide containing residues 253-277(73-97) inhibited Xa:Va interaction (Kd approximately 10 microM) but did not bind Ca2+. In additional studies, Ca2+ increased the amidolytic activity of native and des-44-Xa toward a tetrapeptide substrate (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide) by approximately 1.6-fold. The half-maximal increase was observed at approximately 150 microM Ca2+ and the effect was primarily on the kcat. Ca2+ also significantly protected cleavage at Arg-332-Gln-333(150-151) in the protease domain autolysis loop. Des-44-Xa in which the autolysis loop was cleaved possessed

High affinity Ca(2+)-binding site in the serine protease domain of human factor VIIa and its role in tissue factor binding and development of catalytic activity.

Factor VIIa, in the presence of Ca2+ and tissue factor (TF), initiates the extrinsic pathway of blood coagulation. The light chain (amino acids 1-152) of factor VIIa consists of an N-terminal gamma-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor-like domains, whereas the heavy chain (amino acids 153-406) contains the serine protease domain. In this study, both recombinant factor VIIa (rVIIa) and factor VIIa lacking the Gla domain were found to contain two high-affinity (Kd approximately 150 microM) Ca2+ binding sites. The rVIIa also contained approximately 6-7 low-affinity (Kd approximately 1 mM) Ca(2+)-binding sites. By analogy to other serine proteases, one of the two high affinity Ca(2+)-binding sites in factor VIIa may be formed involving Glu-210 and Glu-220 of the protease domain. In support of this, a synthetic peptide composed of residues 206-242 of factor VIIa bound one Ca2+ with Kd approximately 230 microM; however, Ca2+ binding was observed only in Tris buffer (pH 7.5) containing 1 M NaCl and not in buffer containing 0.1 M NaCl. In both low or high salt +/- Ca2+, the peptide existed as a monomer as determined by sedimentation equilibrium measurements and had no detectable secondary structure as determined by CD measurements. This indicates that subtle changes undetectable by CD may occur in the conformation of the peptide that favor calcium binding in high salt. In the presence of recombinant TF and 5 mM Ca2+, the peptide inhibited the amidolytic activity of rVIIa toward the synthetic substrate, S-2288. The concentration of the peptide required for half-maximal inhibition was approximately 5-fold higher in the low salt buffer than that in the high salt buffer. From direct binding and competitive inhibition assays of active site-blocked 125I-rVIIa binding to TF, the Kd for peptide-TF interaction was calculated to be approximately 15 microM in the high salt and approximately 55 microM in the low salt buffer containing 5 mM Ca2+. Moreover, as inferred from S-2288 hydrolysis, the Kd for VIIa.TF interaction was approximately 1.5 microM in the absence of Ca2+, and, as inferred from factor X activation studies, it was approximately 10 pM in the presence of Ca2+. Thus, Ca2+ decreases the functional Kd of VIIa.TF interaction approximately 150,000-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Tissue factor pathway inhibitor and von Willebrand factor antigen levels in adult respiratory distress syndrome and in a primate model of sepsis.

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein primarily synthesized by the endothelium. A major fraction (approximately 85%) of TFPI remains associated with the endothelium, whereas a small fraction (approximately 15%) is secreted into the blood. In our attempts to search for a marker(s) of endothelial injury in the setting of adult respiratory distress syndrome (ARDS), we retrospectively measured plasma TFPI levels in patients at risk for and with ARDS caused by several etiologic factors. Plasma von Willebrand factor antigen (vWF-Ag), another endothelial-specific protein, was also measured in these patients. The mean plasma TFPI levels were slightly elevated (approximately 1.3-fold), whereas vWF-Ag levels were significantly elevated (approximately 3-fold) in the at-risk group as compared with those in the normal subjects. Both the TFPI (approximately 1.8-fold) and the vWF-Ag (approximately 4-fold) levels were further elevated in the ARDS group. Moreover, the sequential plasma samples from patients with ARDS had progressively increased levels of vWF-Ag and TFPI up to Days 4 and 8, respectively. Neither plasma vWF-Ag nor TFPI levels correlated with mortality in the at-risk group or the ARDS group. TFPI levels were also measured in bronchoalveolar lavage fluids (BALF). The levels (ng/ml) were: normal subjects, 0.05 +/- 0.02 SE; at-risk group, 0.35 +/- 0.16 SE; ARDS group, 0.99 +/- 0.28 SE. Thus, the BALF TFPI levels were increased approximately 7-fold in the at-risk group and approximately 20-fold in the ARDS group relative to the value in the normal subjects. These findings indicate increased local synthesis of TFPI in the alveolar space both in the at-risk patients and in those with ARDS. In additional studies in a primate model of sepsis, lethal doses (LD100) of E. coli administered to baboons resulted in a progressive increase in TFPI levels (approximately 2-fold at 6 h), whereas sublethal doses caused only minimal increase (approximately 1.2-fold). The vWF-Ag levels were elevated approximately 5-fold after infusion of LD100 concentrations of E. coli at 6 h and 4-fold after infusion of sublethal concentrations of E. coli at 24 h. Autopsies on animals in the LD100 group revealed pulmonary congestion, leukocyte infiltration, edema, and hemorrhage, all suggestive of acute lung injury. Thus, in the setting of acute lung injury plasma vWF-Ag appears to be considerably increased prior to significant damage to the endothelium, whereas increased plasma TFPI occurs only after severe injury.(ABSTRACT TRUNCATED AT 400 WORDS)

Hemophilia B caused by five different nondeletion mutations in the protease domain of factor IX.

Factor IX is a multidomain protein and is the proenzyme of a serine protease, factor IXa, essential for hemostasis. In this report, we describe the molecular basis of hemophilia B (deficiency of factor IX activity) in five patients who have neither deletions nor rearrangements of the factor IX gene. By enzymatic amplification and sequencing of all exons and promoter regions, the following causative mutation in the protease domain of factor IX was identified in each patient: IXSchmallenberg: nucleotide 31,215G----T, Ser365Ile; IXVarel: nucleotide 31,214A----G, Ser365Gly; IXMechtal: nucleotide 31,211G----C, Asp364His; IXDreihacken: nucleotide 30,864G----A, Arg248Gln; and IXMonschau: nucleotide 30,855A----T, Glu245Val. In IXVarel, nucleotide 31,213T was also replaced by C, which results in a silent mutation (GAT----GAC) at Asp-364. Thus, this patient has a double base-pair substitution of TA to CG at nucleotides 31,213 and 31,214 but only a single amino acid change of Ser-365 to Gly. This patient also developed an antibody to factor IX during replacement therapy, which suggests that deletion of the factor IX gene is not necessary for development of the antibody in hemophilia B patients. The levels of plasma factor IX antigen in the patients ranged from 40% to 100% except for IXDreihacken (Arg248Gln), in which case it was approximately 4% of normal. The Ser365Gly and Ser365Ile mutants are nonfunctional because of lack of the active site serine residue. Mutant Asp364His is inactive because it cannot form the hydrogen bond between the carboxylate group of Asp-364 and the alpha-amino group of Val-181 generated after activation. As observed in other homologous serine proteases, this hydrogen bond is essential for maintaining the correct active site conformation in normal factor IXa (IXaN). Purified Arg248Gln had approximately 41% and Glu245Val had approximately 17% of the activity of normal factor IX (IXN) in a partial thromboplastin time (aPTT) assay. In immunodot blot experiments, the isolated Glu245Val mutant did and the Arg248Gln mutant did not bind to an anti-IXN monoclonal antibody that has been shown previously to inhibit the interaction of factor VIIIa with factor IXaN. We have recently shown that a high-affinity calcium binding site exists in the protease domain of IXN; among the proposed Ca(2+)-binding ligands is the carboxyl group of Glu-245. Further, a part of the epitope for the above antibody was shown to be contained in the 231 to 265 residue segment of factor IX.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibody-probed conformational transitions in the protease domain of human factor IX upon calcium binding and zymogen activation: putative high-affinity Ca(2+)-binding site in the protease domain.

The Fab fragment of a monoclonal antibody (mAb) reactive to the N-terminal half (residues 180-310) of the protease domain of human factor IX has been previously shown to inhibit the binding of factor IXa to its cofactor, factor VIIIa. These data suggested that this segment of factor IXa may participate in binding to factor VIIIa. We now report that the binding rate (kon) of the mAb is 3-fold higher in the presence of Ca2+ than in its absence for both factors IX and IXa; the half-maximal effect was observed at approximately 300 microM Ca2+. Furthermore, the off rate (koff) of the mAb is 10-fold higher for factor IXa than for factor IX with or without Ca2+. Moreover, like the kon for mAb binding, the incorporation of dansyl-Glu-Gly-Arg chloromethyl ketone (dEGR-CK) into factor IXa was approximately 3 times faster in the presence of Ca2+ than in its absence. Since steric factors govern the kon and the strength of noncovalent interactions governs the koff, the data indicate that the region of factor IX at residues 180-310 undergoes two separate conformational changes before expression of its biologic activity: one upon Ca2+ binding and the other upon zymogen activation. Furthermore, the dEGF-CK incorporation data suggest that both conformational changes also affect the active site residues. Analyses of the known three-dimensional structures of serine proteases indicate that in human factor IX a high-affinity Ca(2+)-binding site may be formed by the carboxyl groups of glutamates 235 and 245 and by the main chain carbonyl oxygens of residues 237 and 240. In support of this conclusion, a synthetic peptide including residues 231-265 was shown to bind Ca2+ with a Kd of approximately 500 microM. This peptide also bound to the mAb, although with approximately 500-fold reduced affinity. Moreover, like factor IX, the peptide bound to the mAb more strongly (approximately 3-fold) in the presence of Ca2+ than in its absence. Thus, it appears that a part of the epitope for the mAb described above is contained in the proposed Ca(2+)-binding site in the protease domain of human factor IX. This proposed site is analogous to the Ca(2+)-binding site in trypsin and elastase, and it may be involved in binding factor IXa to factor VIIIa.