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PubMed1996

Identification of the endothelial cell binding site for factor IX.

We previously demonstrated that the primary region of factor IX and IXa responsible for saturable specific binding to bovine aortic endothelial cells resides in residues 3-11 at the amino terminus of factor IX. We also demonstrated that mutations of lysine to alanine at residue 5, factor IX K5A, or valine to lysine at residue 10, factor IX V10K, resulted in a molecule unable to bind to endothelial cells. Moreover, a mutation with lysine to arginine at residue 5, factor IX K5R, resulted in a factor IX molecule with increased affinity for the endothelial cell binding site. In this paper we report that collagen IV is a strong candidate for the factor IX binding site on endothelial cells. Factor IX and factor IX K5R compete with 125I-labeled factor IX for binding to tetrameric collagen IV immobilized on microtiter plates, while factor X, factor VII, and factor IX K5A or V10K fail to compete. The Kd for wild-type factor IX binding to collagen IV in the presence of heparin was 6.8 +/- 2 nM, and the Kd for factor IX K5R was 1.1 +/- 0.2 nM, which agrees well with our previously published Kd values of 7.4 and 2.4 nM for binding of the same proteins to endothelial cells. Our working assumption is that we have identified the endothelial cell binding site and that it is collagen IV. Its physiological relevance remains to be determined.

Animals↗
PubMed1995

Coagulation factor XIa cleaves the RHDS sequence and abolishes the cell adhesive properties of the amyloid beta-protein.

Amyloid beta-protein (A beta) is the major constituent of senile plaques and cerebrovascular amyloid deposits in Alzheimer's disease and is proteolytically derived from its transmembrane parent protein the amyloid beta-protein precursor (A beta PP). Although the physiological role(s) of secreted A beta PPs are not fully understood, several potential functions have been described including the regulation of hemostatic enzymes factors XIa and IXa and a role in cell adhesion. In the present study, we investigated the proteolytic processing of A beta PP by factor XIa (FXIa). Incubation of the human glioblastoma cell line U138 stably transfected to overexpress the 695 isoform of A beta PP with FXIa (2.5-5 nM) resulted in proteolytic cleavage of secreted A beta PP. Higher concentrations of FXIa (> 25 nM) resulted in loss in cell adherence. Coincubation of FXIa with purified, recombinant Kunitz protease inhibitor domain of A beta PP blocked both the proteolytic processing of A beta PP and the loss of cell adhesion. The RHDS cell adhesion site of A beta PP resides within residues 5-8 of the A beta domain. Incubation of synthetic A beta 1-40 peptide with increasing concentrations of FXIa resulted in cleavage of A beta between Arg5 and His6 within the cell adhesion domain of the peptide. FXIa-digested A beta 1-40 or A beta PP695 lost their abilities to serve as cell adhesion substrates consistent with cleavage through this cell adhesion site. Together, these results suggest a new potential biological function for FXIa in the modulation of cell adhesion. In addition, we have shown that FXIa can proteolytically alter A beta and therefore possibly modify its physiological and perhaps pathological properties.

Amino Acid Sequence↗
PubMed1990

Anti-human factor IX monoclonal antibodies specific for calcium ion-induced conformations.

Four monoclonal antibodies have been produced, which are specific for the Ca2+ or Sr2(+)-induced conformation of human factor IX. Certain, but not all, gamma-carboxy-glutamic acid residues in factor IX are involved in the epitope expression together with the conformation stabilized by the adjacent region of Gla-domain and a disulfide bridge. All the antibodies interfered with the binding of factor IX to phospholipids and inhibited the procoagulant activity of factors IX and IXa beta.

Antibodies, Monoclonal↗
PubMed1990

[The advisability of modifying the current outline of blood coagulation].

The necessity of modifying the scheme of blood coagulation employed in the practice of clinicians is determined by the fact that the schemes offered before were short of the following important moments: 1) the whole totality of the mechanisms of factor VII activation (by factors III, XIIa, IXa, Xa, IIa, phospholipids and lipoproteins) and importance of its activation and increase of the level to reveal the risk of the development of the DIC syndrome, thromboses, sudden cardiac death; 2) formation during transformation of fibrinogen to fibrin of high molecular weight complexes of fibrin monomers (stabilized and not stabilized by factor XIIIa), whose presence in the blood plasma and serum provides important evidence for intravascular coagulation; 3) peptide splitting from prothrombin (fragments 1, 2) and fibrinogen (peptides A and B, 1-42, 15-42) the determination of which is also used for revealing activation of the blood and fibrinolytic system. Those and a number of other characteristic features are taken into account in the scheme of blood coagulation offered by the authors. A scheme of the functioning of the main anticoagulant mechanisms (antithrombin III and heparinoids; thrombomodulin, the system of proteins C and S) is also provided.

Blood Coagulation↗
PubMed1996

Factor XSt. Louis II. Identification of a glycine substitution at residue 7 and characterization of the recombinant protein.

A molecular defect in factor X (fX) results from a point mutation that causes glycine substitution for gamma-carboxylated glutamic acid at position 7. The variant (fXSt. Louis II) and wild type (fXWT) proteins were produced in a mammalian expression system and characterized. fXSt. Louis II has <1% and approximately 3% of normal clotting activity in modified prothrombin time and partial thromboplastin time assays, respectively. The rate of activation of fXSt. Louis II by factor VIIa and tissue factor is undetectable under conditions that result in complete activation of fXWT; activation by factors VIIIa and IXa is approximately 30% of normal activation. The X-activating protein from Russell's viper venom activates fXSt. Louis II completely but at a reduced rate. Thrombin generation on phoshopolipid vesicles or activated platelets is approximately 30% or approximately 5%, respectively. Membrane-dependent autolysis is markedly reduced for fXSt. Louis II. In reactions that are not surface-dependent, fXSt. Louis II is nearly identical to that of fXWT. The rate of inhibition by antithrombin is indistiguishable, as is the rate of thrombin formation in the absence of phospholipid, with or without factor Va.

Antithrombin III↗
PubMed1985

Activated protein C decreases plasminogen activator-inhibitor activity in endothelial cell-conditioned medium.

Confluent cultures of endothelial cells from human umbilical cord were used to study the effect of activated human protein C (APC) on the production of plasminogen activators, plasminogen activator-inhibitor, and factor VIII-related antigen. Addition of APC to the cells in a serum-free medium did not affect the production of tissue-type plasminogen activator (t-PA) or factor VIII-related antigen; under all measured conditions, no urokinase activity was found. However, less plasminogen activator-inhibitor activity accumulated in the conditioned medium in the presence of APC. This decrease was dose dependent and could be prevented by specific anti-protein C antibodies. No decrease was observed with the zymogen protein C or with diisopropylfluorophosphate-inactivated APC. APC also decreased the t-PA inhibitor activity in endothelial cell-conditioned medium in the absence of cells, which suggests that the effect of APC is at least partly due to a direct effect of APC on the plasminogen activator-inhibitor. High concentrations of thrombin-but not of factor Xa or IXa--had a similar effect on the t-PA inhibitor activity. The effect of APC on the plasminogen activator-inhibitor provides a new mechanism by which APC may enhance fibrinolysis. The data suggest that activation of the coagulation system may lead to a secondary increase of the fibrinolytic activity by changing the balance between plasminogen activator(s) and its (their) fast-acting inhibitor.

Animals↗
PubMedDate not supplied

A therapeutic, highly purified factor XI concentrate from human plasma.

A highly purified factor XI (FXI) concentrate was prepared from human plasma by a process comprising a filter adsorption step and chromatography on a cation exchange resin. The freeze-dried FXI, which solubilized quickly, had high specific activity (130-150 U/mg protein), high potency (approx. 100 U/mL), and excellent stability for at least 24 hours at room temperature in the liquid state. The overall recovery was about 220 U of FXI per liter of plasma. Minor protein contaminants (C1-inhibitor, fibronectin, IgG, and alpha-2-macroglobulin) were found to be between 0.13 and 0.46 mg per 1000 U of FXI. Fibrinogen and relevant coagulation factors (factors II, V, VII, IX, X, XII, XIII, and VIII/von Willebrand factor) were undetectable, as evidenced by immunologic and immunoelectrophoretic data. Components of the kinin system were present in trace amounts or were undetectable. No evidence of activated factors such as factors Xa and IXa was found. Proteolytic activity, as assessed by S-2288 chromogenic substrate, was negligible and thrombin was undetectable. A solvent-detergent treatment was included prior to chromatographic purification to enhance viral safety against lipid-enveloped viruses. In vitro and in vivo animal studies demonstrated the absence of thrombogenic, hypotensive, or toxic effects. No thrombogenic activity was found in the Wessler model in rabbits at doses of 900 to 1100 U of FXI per kg of body weight. This FXI preparation could be beneficial in substitution therapy of congenital or acquired FXI deficiency, especially as a way to avoid the use of fresh-frozen plasma.

Animals↗
PubMed1988

The saturable high affinity association of factor X to ADP-stimulated monocytes defines a novel function of the Mac-1 receptor.

Initiation of the coagulation protease cascade as it assembles on cell surfaces requires limited proteolytic activation of the zymogen factor X. Not previously suspected to be the ligand of an organizing receptor on cell surfaces, we now describe that factor X specifically associates with cells of monocyte lineage and we identify the high affinity receptor for this zymogen. Following stimulation with ADP (10 microM), or with the ionophore ionomycin (1 microM), isolated human monocytes bind 125I-factor X in a saturable fashion with a dissociation constant (Kd) of 21.8-44.9 nM. Equilibrium binding analyses indicate that the reaction is optimal at room temperature, requires Ca2+ ions, and saturates at 128,500 +/- 21,300 molecules of 125I-factor X specifically associated with the cell surface. Molar excess of unlabeled factor X inhibits and reverses the binding, whereas the homologous gamma-carboxylated coagulation proteins factors II, VII, IX, IXa, and Xa are without effect. Similarly, chelation of divalent ions immediately dissociates bound 125I-factor X. The monoblast cell line U 937 and the monocytic cell line THP-1 when stimulated with ADP or ionomycin, bind 125I-factor X with characteristics similar to monocytes. Receptor identity was explored using antibodies to the leukocyte adhesive receptors Mac-1, LFA-1, and p150.95. Monoclonal antibodies specific for the alpha subunit of Mac-1 (M 1/70, LM 2/1) or for the common beta subunit (TS 1/18, 60.3) bound equally to resting and ADP- or ionomycin-stimulated cells and also completely blocked the binding of 125I-factor X to stimulated monocytes, U 937, or THP-1 cells. To distinguish between modulatory effects of the monoclonal antibodies and direct spatial hindrance binding of 125I-factor X to Mac-1 was analyzed directly. OKM10 anti-alpha subunit of Mac-1 monoclonal antibody immunoprecipitated 125I-factor X chemically cross-linked to its receptor on stimulated cells. In addition, the complement protein fragment C3bi, which is a recognized ligand for Mac-1, competitively inhibited the association of 125I-factor X. These findings indicate that human blood monocytes and less differentiated cells of this lineage possess an inducible receptor specific for factor X; and also support the conclusion that the heterodimeric leukocyte adhesive receptor Mac-1 functions as the specific receptor structure. We suggest that the novel properties of this receptor may be of importance in the organization and regulation of certain coagulation protease cascades on the monocyte surface.

Adenosine Diphosphate↗
PubMed1995

Mathematical model of serine protease inhibition in the tissue factor pathway to thrombin.

A mathematical model has been developed to simulate the generation of thrombin by the tissue factor pathway. The model gives reasonable predictions of published experimental results without the adjustment of any parameter values. The model also accounts explicitly for the effects of serine protease inhibitors on thrombin generation. Simulations to define the optimum affinity profile of an inhibitor in this system indicate that for an inhibitor simultaneously potent against VIIa, IXa, and Xa, inhibition of thrombin generation decreases dramatically as the affinity for thrombin increases. Additional simulations show that the reason for this behavior is the sequestration of the inhibitor by small amounts of thrombin generated early in the reaction. This model is also useful for predicting the potency of compounds that inhibit thrombosis in rats. We believe that this is the first mathematical model of blood coagulation that considers the effects of exogenous inhibitors. Such a model, or extensions thereof, should be useful for evaluating targets for therapeutic intervention in the processes of blood coagulation.

Animals↗
PubMed1995

The human high mobility group (HMG)-box transcription factor TCF-1: novel isoforms due to alternative splicing and usage of a new exon IXA.

The C-terminal peptide sequences of the human lymphocyte-specific high mobility group (HMG)-box transcription factor TCF-1 are determined by alternative splice mechanisms affecting the exons VIII to X. Here we report, in addition to four splice forms described previously (TCF-1A, B, C, D), the identification of three novel transcripts designated TCF-1E, F, G. Cloning and sequencing of the novel cDNAs revealed (i) joining of the exons VIII and IX to an internal exon X splice acceptor site resulting in a new open reading frame (ORF) of 99 amino acids derived from exon X sequences, (ii) the identification of an additional functional splice acceptor site within exon X, and (iii) a new 81-nucleotide insertion between exon VIII and exon X sequences in a novel transcript form. Genomic cloning and sequence analysis of this transcribed segment of 81 basepairs revealed that it was bordered by canonical splice consensus sites and located in a distance of some 400 bp from both the exons IX and X. It was therefore termed exon IXA. Novel ORFs were generated as a consequence of these alternative splice mechanisms resulting in TCF-1 gene products with significantly different C-terminal peptide sequences, which are prone to selective protein-protein interactions or transactivating functions.

Alternative Splicing↗
PubMed1991

Activated factor IX-antithrombin III complexes in human blood: quantification by an enzyme-linked differential antibody immunoassay and determination of the in vivo half-life.

An enzyme-linked immunoassay has been developed to quantitate the activated human factor IX-antithrombin III complex (IXa-AT III). Test plasma was absorbed onto barium citrate and eluted to remove free antithrombin III (AT III). The IXa-AT III complex in the eluate was then measured by using polystyrene balls coated with immobilized rabbit anti-AT III antibody-binding fragments and anti-factor IX (anti-FIX) antibody-binding fragments labeled with beta-D-galactosidase. The assay was very sensitive, detecting a concentration as low as 0.15 fmol/assay/100 microliters plasma, which corresponds to as little as 0.002% of activated FIX in plasma. Experiments with purified FIX, IXa-alpha, AT III, IXa-AT III, normal plasma, AT III-depleted plasma, and FIX-deficient plasma demonstrated that the assay is specific. IXa-AT III generation could be detected in normal native whole blood within 2.0 minutes after venipuncture. The complex could not be detected in native whole blood from patients with severe hemophilia B even at 1 hour after venipuncture. The mean plasma level of IXa-AT III in healthy adults (n = 32, ages 23 to 54 years) was 9.67 +/- 2.86 pmol/L (mean +/- SD), whereas in eight patients with disseminated intravascular coagulation levels ranged from 11.7 to 35.0 pmol/L. The IXa-AT III titer was significantly reduced in three patients with severe factor VII deficiency (5.70 +/- 1.99 pmol/L) but was normal in three patients with severe factor XI deficiency (11.2 +/- 1.59 pmol/L). Purified IXa-AT III was cleared in vivo in rabbits with a half-life value of 30 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2000

Proteolytic processing of human coagulation factor IX by plasmin.

Previous studies have shown that thrombin generation in vivo caused a 92% decrease in factor IX (F.IX) activity and the appearance of a cleavage product after immunoblotting that comigrated with activated F.IX (F.IXa). Under these conditions, the fibrinolytic system was clearly activated, suggesting plasmin may have altered F.IX. Thus, the effect(s) of plasmin on human F.IX was determined in vitro. Plasmin (50 nM) decreased the 1-stage clotting activity of F.IX (4 microM) by 80% and the activity of F.IXa (4 microM) by 50% after 30 minutes at 37 degrees C. Plasmin hydrolysis of F.IX yields products of 45, 30, 20, and 14 kd on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2 products of 52 and 14 kd under nonreducing conditions. Plasmin-treated F.IX did not bind the active site probe, p-aminobenzamidine, or form an SDS-stable complex with antithrombin. It only marginally activated human factor X in the presence of phospholipid and activated factor VIII. Although dansyl-Glu-Gly-Arg-chloromethyl ketone inactivated-F. IXa inhibited the clotting activity of F.IXa, plasmin-treated F.IX did not. Plasmin cleaves F.IX after Lys43, Arg145, Arg180, Lys316, and Arg318, but F.IXa is not appreciably generated despite cleavage at the 2 normal activation sites (Arg145 and Arg180). Tissue plasminogen activator-catalyzed lysis of fibrin formed in human plasma results in generation of the 45- and 30-kd fragments of F.IX and decreased F.IX clotting activity. Collectively, the results suggest that plasmin is able to down-regulate coagulation by inactivating F.IX.

Amino Acid Substitution↗
PubMed2001

Surface-mediated control of blood coagulation: the role of binding site densities and platelet deposition.

A mathematical model of the extrinsic or tissue factor (TF) pathway of blood coagulation is formulated and results from a computational study of its behavior are presented. The model takes into account plasma-phase and surface-bound enzymes and zymogens, coagulation inhibitors, and activated and unactivated platelets. It includes both plasma-phase and membrane-phase reactions, and accounts for chemical and cellular transport by flow and diffusion, albeit in a simplified manner by assuming the existence of a thin, well-mixed fluid layer, near the surface, whose thickness depends on flow. There are three main conclusions from these studies. (i) The model system responds in a threshold manner to changes in the availability of particular surface binding sites; an increase in TF binding sites, as would occur with vascular injury, changes the system's production of thrombin dramatically. (ii) The model suggests that platelets adhering to and covering the subendothelium, rather than chemical inhibitors, may play the dominant role in blocking the activity of the TF:VIIa enzyme complex. This, in turn, suggests that a role of the IXa-tenase pathway for activating factor X to Xa is to continue factor Xa production after platelets have covered the TF:VIIa complexes on the subendothelium. (iii) The model gives a kinetic explanation of the reduced thrombin production in hemophilias A and B.

Binding Sites↗
PubMed1980

Activation of 125I-factor IX and 125I-factor X: effect of tissue factor and factor VII, factor Xa and thrombin.

Activation of Factor IX and Factor X was studied by adding 125I-Factor IX or 125I-Factor X to reaction mixtures and quantitating cleavage products by reduced sodium dodecylsulfate gel electrophoresis. Thrombin failed to activate Factors IX or X; Factor Xa produced insignificant amounts of cleavage products of both factors. In contrast, the reaction product of tissue factor and Factor VII cleaved large amounts of both Factor IX and Factor X in purified systems and in plasma. In incubation mixtures of plasma containing added 125I-Factor IX or 125I-Factor X, tissue factor and Ca2+ ions, the percentage of total radioactivity in the heavy chain peak of 125I-IXa and the heavy chain peak of 125I-Xa increased at a similar rate. When the tissue factor was diluted, similar curves were obtained for percent cleavage of 125I-Factor IX and percent cleavage of 125I-Factor X plotted against tissue factor concentration. These findings support the hypothesis that activation of Factor IX by the tissue factor-Factor VII reaction produce represents a physiologically significant step in normal haemostasis.

Blood Coagulation↗
PubMed1999

Epitope specificity and inactivation mechanisms of factor VIII inhibitor antibodies.

The domain specificity of anti-factor VIII (FVIII) inhibitor antibodies was determined in assays using FVIII domains generated by thrombin cleavage or expressed as recombinant polypeptides to neutralise the inhibitor. The results revealed the existence of three major types of inhibitors, and various combinations of these antibodies were found in haemophilic and autoantibody patients. Anti-A2 domain inhibitors prevent normal function of the FVIII/factor IXa (FIXa)/phospholipid complex in an unknown manner. Binding of FVIII to phospholipid and to von Willebrand factor is blocked by anti-C2 domain antibodies, and the binding of FVIII to FIXa is prevented by anti-A3 domain antibodies. A rare type of inhibitor prevents release of activated FVIII from von Willebrand factor (vWf), and another probably interferes with FVIII binding to factor X (FX) because it shares the epitope of a monoclonal antibody with this property.

Binding Sites↗
PubMed1993

[Interaction of blood and the vascular wall: hemostatic aspects].

The coagulation enzyme thrombin, a serine protease like all other coagulation factors, plays a central role in the hemostatic processes engaged after injurious events. It induces, with particular efficacy, the aggregation of blood platelets (primary hemostasis) and accounts, via splitting of fibrinogen to fibrin, for the event actually responsible for the coagulation of blood (secondary hemostasis). As is well-known, thrombin itself is generated by a cascade of activation events involving various coagulation factors (F). In this respect the "tissue factor" (TF, formerly known as thromboplastin), in combination with F VIIa, attains decisive significance, not only in the extrinsic pathway of coagulation (activation of F X-->Xa), but also in the intrinsic pathway (activation of F IX-->IXa). Under physiological circumstances, platelet aggregation and coagulation are restricted to the area of the vascular lesion, since the surrounding intact endothelium inhibits an intraluminal spreading of both processes. These "antithrombotic" features of the endothelium encompass antiaggregatory mechanisms (formation and release of prostacyclin [PGI2], adenosine, EDRF [NO], degradation of ADP and other nucleotides mediated by ecto-nucleotidases) as well as anti-coagulatory properties (formation and release of "tissue factor pathway inhibitor" [TFPI], which blocks the coagulation cascade by joining F Xa, TF and F VIIa into an inactive complex, thrombomodulin--thrombin induced activation of protein C, which, together with protein S, inactivates F Va and F VIIIa, thereby attenuating further generation of thrombin, and the heparan sulfate-enhanced activation of antithrombin III and heparin-cofactor II).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Antithrombotic actions and pharmacokinetics of heparin fractions and fragments.

During the last few years many reports on the antithrombotic actions of low molecular weight fractions (LMF) have become available and are being tested in the experimental and clinical settings. Although derived from commercial heparins, the mode of antithrombotic actions, pharmacokinetics and physiologic disposition of these agents is distinct from the parent materials. The antithrombotic potency of these fractions is generally assigned in terms of their ability to inhibit serine proteases such as factor Xa and thrombin in the presence of antithrombin III (AT III). However, these agents are also reported to exert their antithrombotic actions via non AT III mediated inhibition of serine proteases (XIIa, IXa), activation of fibrinolysis via the release of plasminogen activators and charge transition on vascular surfaces. In order to evaluate the relative pharmacologic actions of heparin and its derivatives, we utilized defined animal models to obtain data in terms of pharmacologic parameters. Significant differences between heparin and its derivatives were evident. Human studies on the pharmacokinetics and the endogenous actions of these agents confirmed our preclinical findings. These studies suggest that well-defined preclinical studies are an essential element in the development of newer antithrombotic agents.

Animals↗
PubMed1983

Direct radioimmune detection in human plasma of the association between factor VIII procoagulant protein and von Willebrand factor, and the interaction of von Willebrand factor-bound procoagulant VIII with platelets.

The predominant procoagulant factor VIII (VIII:C) form in normal human plasma containing various combinations of anticoagulants and serine/cysteine protease inhibitors is a protein with mol wt 2.6 +/- 0.2 X 10(5). This protein can be detected by 125I-anti-VIII:C Fab binding and gel electrophoresis in the presence and absence of sodium dodecylsulfate (SDS) and is distinct from the subunit of factor VIII/von Willebrand factor (VIII:vWF) multimers. No larger VIII:C form is present in plasma from patients with severe congenital deficiencies of each of the coagulation factors, other than VIII:C. The mol wt approximately 2.6 X 10(5) VIII:C form is, therefore, likely to be the in vivo procoagulant form of VIII:C, rather than a partially proteolyzed, partially activated derivative of a larger precursor. About 60% of this procoagulant mol wt approximately 2.6 X 10(5) VIII:C form in plasma is present in noncovalent complexes with larger VIII:vWF multimers, which attach reversibly to platelet surfaces in the presence of ristocetin. This VIII:vWF-bound protein of mol wt approximately 2.6 X 10(5) may be the plasma procoagulant form of VIII:C which, after proteolytic activation, accelerates the IXa-mediated cleavage and activation of X postulated to occur on platelet surfaces.

Antigens↗