Tissue factor and factor VIIa--hemostasis and beyond

Initiation of the coagulation cascade via exposure of active tissue factor (TF) to blood and formation of the factor VIIa/TF complex is essential for hemostasis and is an initial procoagulant signal in thrombosis. As of early 2012, over 20,000 articles listed on PubMed describe advances in the understanding of TF biology in the settings of hemostasis and thrombosis, as well as in signaling events in cancer, sickle cell anemia, hyperlipidemia, and a broad spectrum of inflammatory disorders. It is both inspiring and humbling, then, to consider not only what has been learned about TF regulation, but also the number of questions still remaining about its role in physiology. This supplement reviews both well-accepted and currently-controversial topics in coagulation and factor VIIa/TF biology, with particular foci on non-hemostatic roles of TF, innovative approaches for the treatment of hemophilia, and novel in vivo models of bleeding and thrombosis.     

Macrophage colony-stimulating factor in nerve growth factor preparations

Following a report that nerve growth factor preparations have granulocyte-colony-stimulating activity, we investigated the presence of colony-stimulating factors in 7s mouse submaxillary nerve growth factor and its subunits. Macrophage colonies were formed in mouse bone marrow cultures after exposure to preparations of 7s nerve growth factor, the gamma subunit, and, to a small extent, the alpha subunit; the beta subunit, which is responsible for the nerve growth function, did not stimulate colony growth. Furthermore, the esteropeptidase activity of the gamma subunit was not detected in preparations of macrophage colony-stimulating factor purified from the giant cell tumor (GCT) cell line. Immunoprecipitation of radiolabeled gamma subunit with a polyclonal antibody to L-cell macrophage colony-stimulating factor showed a protein band that could represent the gamma subunit of nerve growth factor. Separation of the macrophage activity from the esteropeptidase activity of the gamma subunit was accomplished on the basis of molecular size. Thus, macrophage colony-stimulating factor was a contaminant of nerve growth factor produced by the mouse submaxillary gland and copurified with the gamma subunit.     

Urinary procoagulant behaves as tissue factor by promoting factor VIIa-catalyzed activation of factor X

We purified the urinary procoagulant from frozen human urine by introducing phenyl-Sepharose hydrophobic chromatography. By this method, the apoprotein of the procoagulant and the lipid-like substance were separately recovered. Upon reassociation with the lipid-like substance or exogenous crude phospholipids, the apoprotein accelerated factor VIIa-catalyzed activation of factor X, probably by forming a stoichiometric complex with the catalytic enzyme. Thus the procoagulant was confirmed to be a tissue factor by its mode of participation in the blood coagulation mechanism.     

Nerve growth factor and the neurotrophic factor hypothesis

The discovery of nerve growth factor (NGF) over 40 years ago led to the formulation of the “Neurotrophic Factor Hypothesis”. This hypothesis states that developing neurons compete with each other for a limited supply of a neurotrophic factor (NTF) provided by the target tissue. Successful competitors survive; unsuccessful ones die. Subsequent research on NTFs has shown that NTF expression and actions are considerably more complex and diverse than initially predicted. Even for NGF, different regulatory patterns are seen for different neuronal populations. As would be predicted by the “Neurotrophic Factor Hypothesis”, NGF levels critically regulate basal forebrain cholinergic neuron size and neurochemical differentiation. In contrast, the level of trkA, the NGF receptor, regulates these properties in caudate-putamen cholinergic neurons. Understanding NTF regulation and actions on neurons has led to their use in clinical trials of human neurological diseases. NTFs may emerge as important therapies to prevent neuronal dysfunction and death.     

Dispersity of human factor VIII--Von Willebrand factor.

Human factor VIII — Von Willebrand factor purified by gel chromatography in the presence of dextran has been studied with large pore polyacrylamide gel electrophoresis designed for analysis of highmolecular-weight proteins. It was demonstrated that purified factor VIII-Von Willebrand factor is not a single homogeneous entity, but consists of a series of immunologically related polymers. The polydispersity of factor VIII-Von Willebrand factor was confirmed by an immunochemical variant of the reversal-electrophoresis spreading test. With this technique it could be visualized that also factor VIII in plasma and cryoprecipitate is polydisperse in nature. As a consequence, changes in the polymer distribution of factor VIII-Von Willebrand factor can be an alternative explanation for results of dissociation experiments and for abnormalities in various diseases as demonstrated by cross-immunoelectrophoresis.     

The isolation of prothrombin, factor IX and factor X from human factor IX concentrates

A relatively simple and reproducible procedure is described for the isolation of functionally and electrophoretically homogeneous prothrombin, factor IX and factor X from clinical Factor IX Concentrates. The procedure involves ammonium sulphate fractionation; then chromatography on DEAE-Sephadex A50, dextran sulphate-Sepharose and heparin-Sepharose. High recovery of all three procoagulants was obtained: 32% for factor IX; 32% for factor X; 29% for prothrombin.     

Activation of factor IX by factor XIa--a spectrophotometric assay for factor IX in human plasma

The activation of Factor IX by partially purified Factor XIa was followed by active site titration, gelelectrophoresis and by a spectrophotometric assay. The assay is based on the finding that the rate of Factor X activation in the presence of phospholipid and Ca2+ is linear in time and proportional to the amount of Factor IXa present and can be determined with the chromogenic substrate S2222. Conditions were found that allowed complete activation of Factor IX in human plasma by Factor XIa. The amount of Factor IXa present in the plasma sample can be determined with the spectrophotometric assay and is proportional with the amount of plasma present. In plasma from patients receiving vitamin-K antagonists reduced Factor IX activity is found with the spectrophotometric assay and the new assay method may be useful in monitoring oral anticoagulant therapy.     

Model of a ternary complex between activated factor VII,tissue factor and factor IX

Upon binding to tissue factor, FVIIa triggers coagulation by activating vitamin K-dependent zymogens, factor IX (FIX) and factor X (FX). To understand recognition mechanisms in the initiation step of the coagulation cascade, we present a three-dimensional model of the ternary complex between FVIIa:TF:FIX. This model was built using a full-space search algorithm in combination with computational graphics. With the known crystallographic complex FVIIa:TF kept fixed, the FIX docking was performed first with FIX Gla-EGF1 domains, followed by the FIX protease/EGF2 domains. Because the FIXa crystal structure lacks electron density for the Gla domain, we constructed a chimeric FIX molecule that contains the Gla-EGF1 domains of FVIIa and the EGF2-protease domains of FIXa. The FVIIa:TF:FIX complex has been extensively challenged against experimental data including site-directed mutagenesis, inhibitory peptide data, haemophilia B database mutations, inhibitor antibodies and a novel exosite binding inhibitor peptide. This FVIIa:TF:FIX complex provides a powerful tool to study the regulation of FVIIa production and presents new avenues for developing therapeutic inhibitory compounds of FVIIa:TF:substrate complex.     

Functional properties of factor VIIa/tissue factor formed with purified tissue factor and with tissue factor expressed on cultured endothelial cells

Factor VIIa (F. VIIa)/tissue factor (TF) function was examined using purified human TF reconstituted into mixed phospholipid vesicles and TF expressed on cultured human umbilical vein endothelial cells (HUVEC) treated with thrombin. In reaction mixtures containing either type of TF, F. VIIa, 10 nM, either 3H-factor X or 3H-factor IX, 88 nM, and Ca2+, 5 mM, F. VIIa/TF activated factor X (F. X) several fold faster than it activated factor IX (F. IX). Adding heparin, 1 U/ml, increased rates of activation of both substrates and F. X remained the preferred substrate. Adding plasma at concentrations of 5% or above inhibited factor VIIa/TF catalytic activity. Inhibition was shown to require F. Xa as a cofactor, was prevented by antibodies to extrinsic pathway inhibitor (EPI), and was reversible by decalcification. Thus, with factor VIIa/TF formed with both types of TF, EPI appeared responsible for inhibition induced by plasma. Our data indicate that functional properties of factor VIIa/TF as delineated in reaction mixtures made with purified TF reconstituted into mixed phospholipid vesicles also hold for factor VIIa/TF activity on the surface of cultured HUVEC.     

Current status on tissue factor activation of factor VIIa

Free factor VIIa displays a zymogen-like behavior with low intrinsic activity. Formation of a complex between factor VIIa and tissue factor is necessary to enhance the procoagulant activity of factor VIIa, not only by providing membrane localization, substrate exosites and positioning the active site at an appropriate distance above the surface but also by allosteric enhancement of the enzymatic activity, and this event signals initiation of blood coagulation. The interaction is of high affinity and all the domains are engaged at the interface. The crosstalk between the protease domain of factor VIIa, in particular residue Met-306, and the N-terminal domain of tissue factor provides the starting point for the allosteric activation of factor VIIa. The pathway(s) of conformational transitions in factor VIIa ensuing tissue factor binding has not been entirely mapped. The present paper is a brief compilation of our current knowledge of the allosteric mechanism by which tissue factor induces and stabilizes the active conformation of factor VIIa.     

Heterogeneity of factor IX in therapeutic factor IX concentrates

Rabbit antibody to human factor IX was used to investigate the factor IX antigenic content and electrophoretic mobility of commercial products as well as experimental “activated products”. Rocket immunoelectrophoresis of all concentrates showed a 1.2–3 fold increased antigenic content/unit factor IX clotting activity when compared to plasma. Two dimensional crossed immunoelectrophoresis of standard Factor IX concentrates produced a single sharp peak whether electrophoresed in Ca²⁺ or EDTA containing buffer. “Activated” concentrates produced a dome shaped precipitin arc. The addition of plasma to standard factor IX concentrates yielded a marked shoulder only when the electrophoresis was run in EDTA. This effect could not be reproduced by the addition of antithrombin III (AT-III). The addition of plasma to some “activated” products revealed an even more pronounced heterogeneity whether in Ca²⁺ or EDTA and the addition of AT-III in the same products produced a second precipitin peak. These results indicate that at least three forms of factor IX exist in Factor IX concentrates. The absence of detectable AT-III reacting material in the standard concentrates is $\text{a priori}$ evidence of the absence of major amounts of IXa, whereas the presence of AT-III reacting material in the “activated” concentrates is evidence of biologically active material.     

Cellular activation of factor IX (christmas factor)

We have demonstrated Factor IX activation by sonicated polymorphonuclear leukocytes (PMNs). This activation reaction required the disrupted leukocytes, calcium chloride, and a small amount of normal human plasma. The requirement for normal plasma was met by plasma deficient in all of the known coagulation factors, and thus the substance present in normal plasma which facilitates this reaction was not identified. The fact that factor XI deficient plasma supported the reaction as well as normal plasma implied that factor XIa was not involved in this activation. Strontium ions could not substitute for calcium ions in the activation reaction, also implying that factor XIa was not involved. This factor IX activating principle in leukocytes could provide a mechanism for by-passing the contact factors of blood coagulation, thus providing an explanation for the discrepancy in clinical severity between deficiencies of the contact factors on the one hand and hemophilias A and B on the other.     

Assembly of the intrinsic factor X activating complex--interactions between factor IXa, factor VIIIa and phospholipid

The activation of blood coagulation factor X by factor IXa is strongly stimulated by the non-enzymatic cofactors phospholipid, Ca2+ and activated factor VIII. In this paper we present a method by which we were able to determine binding affinities of factor IXa for phospholipids (either in the absence or presence of factor VIIIa) from kinetic measurements of factor X activation. It is shown that rates of factor X activation in the presence of phospholipids can be saturated with an excess factor VIIIa at limiting amounts of factor IXa and vice versa. Our data indicate that the enzymatic unit in the intrinsic factor X activator is a 1:1 stoichiometrical complex of factor IXa and factor VIIIa bound to phospholipid. Titrations with factor IXa at fixed concentrations of phospholipid and factor X show that the apparent dissociation constant of factor IXa for phospholipid is lowered from 10(-6) M to 10(-8) M by the presence of factor VIIIa. We conclude, that in analogy with the role of factor Va in prothrombin activation, phospholipid-bound factor VIIIa functions as a high-affinity binding site ("receptor") for factor IXa in the intrinsic factor X activating complex. Therefore, factor VIIIa increases the observed Vmax of factor X activation by 1) enhancing the kcat of the reaction and 2) increasing the amount of phospholipid-bound factor IXa that participates in factor X activation.     

Heterogeneity of factor IX BM. Difference of cleavage sites by factor XIa and Ca2+ in factor IX Kashihara, factor IX Nagoya and factor IX Niigata

Abnormal factor IX was isolated from the plasma of a patient with hemophilia B Kashihara and two patients with hemophilia BM. The F.IX was purified to homogeneity by using monoclonal anti-F.IX-Sephrose, heparin-Sepharose and DEAE-Sephadex A-50 affinity chromatography successively. The isolated proteins have the same molecular weight and the same mobility on crossed immunoelectrophoresis as normal F.IX. The limited proteolysis of purified proteins was induced by F.XIa/Ca2+ or by RVV-X/Ca2+. A time course study showed that F.IX Nagoya seemed to be cleaved by neither F.XIa nor RVV-X, F.IX Kashihara was cleaved partially by F.XIa but not by RVV-X, and that F.IX Niigata was cleaved completely at the rate similar to normal F.IX, though the resultant product of F.IX Niigata did not show any F.IXa activity. These results favored the view that hemophilia B+ or BM is a heterogeneous disorder.     

Protease nexin-2/Amyloid beta-protein precursor regulates factor VIIa and the factor VIIa-tissue factor complex

Protease nexin-2/amyloid beta-protein precursor (PN-2/AbetaPP) and its Kunitz protease inhibitory (KPI) domain were characterized as inhibitors of factor VIIa (FVIIa) and factor VIIa-tissue factor complex (FVIIa-TF). PN-2/AbetaPP and KPI domain inhibited FVIIa with an apparent K(i) of 1.1+/-0.2x 10(-7) M and 1.5+/-0.1x10(-7) M, respectively. When soluble tissue factor (TF(1-219)) was present, there was increased FVIIa inhibition by PN-2/AbetaPP or KPI domain (K(i)=7.8+/-0.3x10(-8) M and 6.8+/-0.6x10(-8) M, respectively). When relipidated tissue factor (TF(1-243)) was present, the K(i) of FVIIa inhibition by PN-2/AbetaPP increased 4.7-fold further. PN-2/AbetaPP complexed with FVIIa, as shown on gel filtration and solid phase binding assay. The apparent second-order rate constant of inhibition of FVIIa by PN-2/AbetaPP in the absence of TF(1-219) was less than that of the FVIIa-TF(1-219) complex. Antithrombin in the absence of TF(1-219) also had a lower apparent second-order rate constant of inhibition than in its presence. In a mixture that included FVIIa, relipidated TF(1-243) and factor X, PN-2/AbetaPP or KPI domain had an IC(50) at 65 and 250 nM, respectively; antithrombin and heparin (1 U/mL) had an IC(50) of 12.8 nM. These data indicate that tissue factor promoted the inhibition of FVIIa by PN-2/AbetaPP or KPI domain, but antithrombin was a better inhibitor of soluble FVIIa-TF in extrinsic tenase.     

Protein disulfide isomerase has no stimulatory chaperone effect on factor X activation by factor VIIa-soluble tissue factor

Introduction

It was recently reported that protein disulfide isomerase (PDI) stimulates factor X (FX) activation by factor VIIa (FVIIa) bound to soluble tissue factor (sTF) in a purified system and that PDI may be responsible for activating cellular tissue factor (TF) and switching it between its roles in blood coagulation and cellular signalling. This study further investigates the former effect of PDI.

Method

FX activations by FVIIa-sTF_1-219 were carried out in the presence of different forms of PDI, with annexin V or detergent present in the system and using various forms of FVIIa and FX. In addition, FVIIa-lipidated TF was used as the FX activator.

Results

Recombinant human PDI did not influence FX activation by FVIIa-sTF_1-219, whereas PDI purified from bovine liver enhanced the activation rate in a dose-dependent manner. The inclusion of annexin V or detergent abolished the stimulatory effect. Removal of the phospholipd-interactive γ-carboxyglutamic acid (Gla)-containing domain from either FVIIa or FX obliterated the bovine PDI-induced enhancement of FX activation, as did the introduction of F4A or L8A mutation in FVIIa. The presence of 25 nM bovine PDI lowered the apparent K_m for FX from far above 10 μM to 1-2 μM. No PDI effect was seen when FVIIa-lipidated TF was the FX activator.

Conclusions

FX activation is insensitive to PDI per se and a phospholipid contaminant in the bovine PDI preparation acts stimulatory when sTF, but not lipidated TF, is the cofactor. Strong support is provided by the lacking effect of bovine PDI after removal or modification of the Gla domain in either FVIIa or FX as well as by the effects of annexin V and detergents and the decreased K_m value.     

Thrombomodulin-dependent effect of factor VLeiden mutation on factor XIII activation

Introduction

Factor V Leiden mutation (FV_Leiden) is associated with impaired down-regulation of activated FV procoagulant activity and loss of FV anticoagulant function that result in an increased risk of venous thromboembolism. As the downstream effects of FV_Leiden on clot formation and fibrinolyis have only partially been revealed, we investigated its effect on the activation of factor XIII (FXIII) and the cross-linking of fibrin.

Methods

In the plasma samples of fifteen healthy individuals with known FV genotypes coagulation was initiated by recombinant human tissue factor and phospholipids with or without recombinant human thrombomodulin (rhTM). FV deficient plasma supplemented with purified wild type FV or FV_Leiden were also investigated. Clots were recovered and analyzed by SDS-PAGE and quantitative densitometric evaluation of Western blots.

Results

rhTM considerably delayed the activation of FXIII in the plasma from FV wild type individuals. This effect of rhTM was significantly impaired in the plasma from FV_Leiden carriers. The results were confirmed in experiments with FV deficient plasma supplemented by FV prepared from wild type individuals or FV_Leiden homozygotes. Fibrin γ-chain dimerization was also considerably delayed by rhTM in plasma samples from individuals without Leiden mutation, but not in plasma samples from FV_Leiden heterozygotes or homozygotes. The difference between heterozygotes and homozygotes was not statistically significant.

Conclusion

The highly diminished delaying effect of TM on FXIII activation and on the cross-linking of fibrin in FV_Leiden carriers might represent a novel mechanism contributing to the increased thrombosis risk of these individuals.