FVII, FVIIa, and downstream markers of extrinsic pathway activation differ by EPCR Ser219Gly variant in healthy men.

Objective: The purpose of this study was to determine the effect of a variant in EPCR (Ser219Gly), previously shown to affect EPCR shedding, on plasma FVII, FVIIa, and downstream markers of activated coagulation.

Methods And Results: Statistical analysis was undertaken in approximately 2000 healthy middle aged men (NPHSII). Higher soluble EPCR levels were confirmed for Gly allele carriers (P<0.

Relationship between markers of activated coagulation, their correlation with inflammation, and association with coronary heart disease (NPHSII).

Objective: To determine whether activation of coagulation increases in parallel with inflammation and whether coagulation activation markers (CAMs) are independently associated with coronary heart disease (CHD), in the prospective study, NPHSII.

Methods: Surveillance of 2997 men between 50 and 63 years yielded 314 first CHD events during 36507 person-years of observation. The plasma levels of activated factor XII (FXIIa), the peptides released upon activation of factor X (FXpep) and factor IX (FIXpep), activated factor VII (FVIIa), prothrombin fragment 1 + 2 (F1 + 2) and fibrinopeptide A (FpA) served as indices of activity along the coagulation pathway.

A critical evaluation of the prothrombin time for monitoring oral anticoagulant therapy.

The Quick prothrombin time is the most common clotting test performed, principally for monitoring oral anticoagulant therapy. The International Normalized Ratio (INR) for comparing patient results from prothrombin time measurements and the International Standardized Index (ISI) for achieving greater consistency of results using different thromboplastins have made it possible to compare the results of vitamin K antagonist drug therapy that was impossible before the introduction of the INR and ISI. However, INR values obtained from the same patient plasma sample using different thromboplastins are significantly different.

Epidemiological and genetic associations of activated factor XII concentration with factor VII activity, fibrinopeptide A concentration, and risk of coronary heart disease in men.

Background: The relations of plasma activated factor XII (FXIIa) concentration and a common polymorphism (C46T) of the factor XII gene with hemostatic status and risk of coronary heart disease (CHD) were examined by prospective surveillance.

Methods And Results: Genotyping for the C46T variant was performed in 2624 men 50 to 61 years of age who were free of CHD at baseline. The genotype distribution was as follows: CC, 56.

A monoclonal antibody raised against human beta-factor XIIa which also recognizes alpha-factor XIIa but not factor XII or complexes of factor XIIa with C1 esterase inhibitor.

A monoclonal antibody (mAb 2/215) against human beta-factor XIIa (beta-FXIIa), was shown by equilibrium binding studies to have a high affinity for alpha-factor XIIa (alpha-FXIIa) (Kd 1.8 nM) and beta-FXIIa (Kd 0.65 nM) but no detectable reaction with FXII zymogen or alpha-FXIIa:C1 esterase inhibitor (C1-INH) complex.

Very low activated factor VII and reduced factor VII antigen in familial abetalipoproteinaemia.

Abetalipoproteinaemia is a rare disorder of apolipoprotein B metabolism associated with extremely low plasma concentrations of triglyceride. To discover whether the general positive association between factor VII and triglyceride levels extends to this condition, 5 patients were compared with 18 controls. All patients had a triglyceride below 100 micromol/l.

Risk of coronary heart disease and activation of factor XII in middle-aged men.

Increased activity is known to be present in the extrinsic, intrinsic, and final common pathways of the hemostatic system in men at high risk of coronary heart disease (CHD), but the status of the contact system of coagulation in this condition is uncertain. Plasma levels of activated factor XII (XIIa), the initial product of contact activation, have therefore been measured by ELISA in 2464 men aged 51 to 62 years, clinically free of CHD, who were taking part in a prospective cardiovascular survey based in general medical practices. Statistically significant, independent, and positive associations of XIIa were found with serum cholesterol and triglyceride concentrations, blood pressure, body mass index, factor VII activity, plasma fibrinogen concentration, and tobacco smoking, all associated with CHD.

Elucidation of the core residues of an epitope using membrane-based combinatorial peptide libraries.

Combinatorial peptide libraries have proved to be a valuable tool for the study of the interaction of a functional protein with its ligand. Here, the epitope for a monoclonal antibody 201/9, raised against beta-factor XIIa, has been identified with a two-step approach using peptide libraries attached to a polymer (polyvinylidene difluoride) membrane. First, the octapeptide libraries with two amino acids defined at position 2 and 4, represented by the formula X-O2-X-O4-X-X-X-X, were synthesized on a sheet of polymer membrane in which X represents a mixture of all the natural -amino acids except cysteine, while O2 and O4 each represent a single amino acid.

Multiple interactive residues of recognition: elucidation of discontinuous epitopes with linear peptides.

The discontinuous epitopes of beta-factor XIIa for three mAbs were mapped by a linear peptide-based immunoblotting technique, referred to as multiple interactive residues of recognition. The Abs were incubated with a set of overlapping synthetic peptides, deduced from the cDNA sequences of beta-factor XIIa, on a polymer membrane, and the signal was amplified by an ECL assay. Several discrete sequences of the protein were recognized by each Ab.

Activation of factor VII during alimentary lipemia occurs in healthy adults and patients with congenital factor XII or factor XI deficiency, but not in patients with factor IX deficiency.

Factor VII activity (FVIIc), a risk marker for coronary heart disease, is increased during postprandial lipemia. Factor VII activation accompanies lipolysis of triglyceride-rich lipoproteins, but the nature of this association and whether it is causal remain uncertain. To explore this issue, four patients with homozygous factor XII deficiency, four with complete factor XI deficiency, six with factor IX deficiency, and their respective age- and sex-matched controls were given two isocaloric dietary regimens, one providing on average 136 g fat and the other 19 g fat.

An enzyme-linked immunosorbent assay (ELISA) for the measurement of activated factor XII (Hageman factor) in human plasma.

A direct enzyme-linked immunosorbent assay (ELISA) employing 2/215 mouse monoclonal hybridoma antibody is described. The assay is capable of detecting activated factor XII in human plasma and can be used to assess early detection of the intrinsic blood coagulation pathway. No cross reactivity with human factor XII zymogen has been found.

Factor VII-deficient substrate plasmas depleted of protein C raise the sensitivity of the factor VII bio-assay to activated factor VII: an international study.

Plasma from healthy individuals, pregnant women and patients on warfarin were distributed to 3 laboratories supporting major cardiovascular surveys (Northwick Park, Muenster and Houston) for assay of factor VII coagulant activity (VIIc) with their own bio-assays. The mean VIIc in 147 samples agreed to within 1% of standard in Northwick Park and Houston, but was 14% of standard lower in Muenster owing to its more potent standard. In samples with an increased VIIc the Northwick Park assay gave a higher result than the other assays owing to its increased responsiveness to activated factor VII (VIIa).

Mechanisms of thrombin generation during surgery and cardiopulmonary bypass.

Although in vitro studies have been invaluable in revealing the complex biochemistry of the blood coagulation system, the mechanisms involved during the in vivo response to hypercoagulable stimuli are still unclear. We have used plasma-based enzyme-linked immunosorbent assays (ELISAs) to study the mechanisms by which the coagulation system is activated in vivo during human cardiopulmonary bypass (CPB) surgery (n = 8). A novel immunoassay for factor XIIa was used to detect activation of the contact system, factor IX activation peptide (FIXAP) was used as a marker for activation of factor IX, and prothrombin fragment F1 + 2 (F1 + 2) was used as a marker for thrombin generation.

The autoactivation of factor XII in the presence of long-chain saturated fatty acids--a comparison with the potency of sulphatides and dextran sulphate.

The incubation of purified human factor XII (Hageman factor [HF]) in the presence of long-chain saturated fatty acids (FA) like stearate (C-18) or behenate (C-22) resulted in a time-dependent increase of amidolytic activity. The HF autoactivation progress curves were sigmoidal. The first order rate for the initial period was constant; this was followed by a period of decreasing rate and a plateau of zero rate.

Comparative study of reaction kinetics in membrane and agarose bead affinity systems.

Compared to conventional agarose bead affinity supports, a microporous nylon membrane exhibits greatly improved reaction kinetics as quantified in the reaction between gamma-globulin and immobilised protein A. The improvement is only observed when the solution of gamma-globulin is forced through the membrane pores. In the absence of flow in the pores, it is possible to relate approximately the rate of uptake onto either type of affinity support to independently determined diffusion coefficients.

The increased rate of activation of factor XII in late pregnancy can contribute to the increased reactivity of factor VII.

The amidolytic activity of enzymes derived from factor XII (XIIa) was 3-fold higher in plasmas collected during pregnancy than from control subjects. Factor VII coagulant activity (VIIc) and XIIa increased in both kinds of plasmas on incubation on ice for 24 h (cold activation). These increases could be attributed to the decreased potency of C1 inhibitor (C1INH).

The prothrombin activation peptide regulates synthesis of the vitamin K-dependent proteins in the rabbit.

The turnover of 125I-bovine prothrombin fragment 1 was studied in the rabbit. The t1/2 of the peptide in the intravascular compartment was 11.5 hours and this compartment accounted for between 7.

A novel method for the purification of HIV-1 p24 protein from hybrid Ty virus-like particles (Ty-VLPs).

The self-assembly properties of a protein encoded by the yeast retrotransposon Ty can be exploited to produce large amounts of recombinant, particulate fusion proteins as hybrid Ty virus-like particles (Ty-VLPs). This system has now been adapted to allow the release of the additional protein by incorporation of a protease cleavage site between the yeast carrier protein and the protein of interest. The purification of the additional protein is facilitated by exploiting the ease with which Ty-VLPs can be purified from other yeast cell components due to their particulate nature.

HIV-1 TAT "activates" presynthesized RNA in the nucleus.

Replication of the human immunodeficiency virus (HIV-1) depends upon the viral TAT protein. TAT stimulates gene expression via a target response sequence (TAR) located within the HIV-1 LTR. As TAR is located in the transcribed region it could act as a signal in either the DNA, the RNA, or both.

The activation of the contact phase of coagulation by physiologic surfaces in plasma: the effect of large negatively charged liposomal vesicles.

The endogenous, negatively charged surface that induces activation of the contact coagulation factors was investigated in plasmas taken from women in late pregnancy and control subjects of child-bearing age. The plasmas from the two groups of subjects were incubated at 4 degrees C for 24 hours either in plastic or in glass tubes and the factor VII coagulant activity (VIIc) was assayed in the treated plasmas. The activation of factor VII under these conditions involves the generation of enzymes derived from factor XII (XIIa).

The role of beta-hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX.

beta-Hydroxyaspartic acid is a post-translationally modified amino acid found in a number of plasma proteins in a domain homologous to epidermal growth factor. Its presence can be correlated with a high affinity Ca2+ binding site, with a dissociation constant of 10-100 microM. We describe a system for the expression of human coagulation factor IX in dog kidney cells in tissue culture, in which the post-translational modifications and the biochemical activity are indistinguishable from factor IX synthesized in vivo.

Structure and order of the protein and carbohydrate domains of prothrombin fragment 1.

The three-dimensional structure of prothrombin fragment 1 has been determined by X-ray crystallography at 3.8 A resolution. The fragment is composed of a number of structural units, some of which are ordered while others are disordered.

Vitamin K-dependent blood coagulation proteins form hetero-dimers.

The later stages of the blood coagulation cascade are characterized by the presence of vitamin K-dependent proteins and their involvement in membrane-bound, multi-protein converting complexes with an essential requirement for calcium ions. Specific interactions between zymogens and activating enzymes have not yet been identified. Here we describe a crystallographic study of prothrombin fragment 1 (residues 1-156 of prothrombin) which indicates that vitamin K-dependent coagulation proteins have specific association sites that allow them to form hetero-dimers.