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1.
Biochem Soc Trans ; 35(Pt 3): 555-8, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17511650

ABSTRACT

Coagulation proteases are involved in a highly orchestrated proteolytic cascade which is essential for haemostasis and blood clotting. In particular, the initiator of the coagulation cascade, Factor VIIa, binds to its cofactor, tissue factor, and its substrate, Factor X, via exosite interactions to form a ternary catalytic complex named extrinsic Xase. These exosite interactions have also been shown to allosterically induce the active conformation of the catalytic site of Factor VIIa. We have developed a direct continuous fluorescence polarization-based extrinsic Xase assay, which has been used to screen in excess of 1 million structurally diverse low-molecular-mass compounds as a potential starting point for the development of anticoagulants. The primary screen hits were categorized with deconvolution assays into either active-site or exosite inhibitors. The latter category of hits displayed both competitive and uncompetitive modalities of inhibition with respect to Factor X activation. An uncompetitive mechanism of action is of particular interest as it offers a hypothetical inhibitory advantage in the context of inhibiting a proteolytic cascade such as the blood coagulation pathway.


Subject(s)
Factor VIIa/antagonists & inhibitors , Allosteric Regulation , Binding Sites , Drug Evaluation, Preclinical , Factor VIIa/chemistry , Factor VIIa/metabolism , Factor X/chemistry , Factor X/metabolism , Fluorescence Polarization/methods , Humans , In Vitro Techniques , Kinetics , Models, Molecular , Multiprotein Complexes , Thromboplastin/chemistry , Thromboplastin/metabolism
2.
J Thromb Haemost ; 4(9): 1989-96, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16961606

ABSTRACT

BACKGROUND: A significant need exists for new chronic oral anticoagulation therapies to replace warfarin. Previous studies have shown that beta-D-xylosides, which prime glycosaminoglycan (GAG) synthesis, have antithrombin and antithrombotic activity. In the following report, a new orally active beta-D-xyloside (odiparcil) has been characterized in a rat model of venous thrombosis and its efficacy and bleeding liability compared to warfarin. Additionally, studies were conducted to investigate odiparcil's ex vivo antithrombin and antiplatelet activity, and also to explore the potential utility of protamine sulfate as a neutralizing agent. METHODS AND RESULTS: In vivo thrombosis studies were conducted in a rat inferior vena cava model, and bleeding studies in a rat tail transection model. Following oral dosing, warfarin and odiparcil produced dose-related suppression of thrombus formation. A therapeutically relevant dose of warfarin in this model (international normalized ratio; INR 3.0) achieved approximately 65% inhibition of thrombus formation. Warfarin caused dose-related significant increases in bleeding indices. Odiparcil antithrombotic activity was limited by its mechanism to a maximum suppression of thrombus formation of 65-70%, and did not prolong bleeding indices. Additionally, odiparcil-induced heparin cofactor II (HCII)-dependent antithrombin activity was shown to be a function of dermatan sulfate-like GAG production. Other than thrombin-related effects, no odiparcil effects on platelet function were observed. In antidote studies, it was demonstrated that odiparcil-induced antithrombotic activity could be partially neutralized by protamine sulfate. CONCLUSIONS: These experiments suggest that an antithrombotic approach based upon xyloside induction of circulating GAGs may have the potential to approximate the efficacy of warfarin and yet with a reduced risk to hemostasis.


Subject(s)
Glycosides/therapeutic use , Venous Thrombosis/drug therapy , Warfarin/therapeutic use , Animals , Anticoagulants/adverse effects , Anticoagulants/therapeutic use , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Glycosaminoglycans/blood , Glycosides/adverse effects , Hemorrhage/chemically induced , Heparin Cofactor II , Protamines/therapeutic use , Rats , Vena Cava, Inferior , Warfarin/adverse effects
3.
J Thromb Haemost ; 4(4): 882-90, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16634759

ABSTRACT

P2X1 receptors are ATP-gated channel demonstrated to be involved in multiple platelet responses, although in vitro analysis has been complicated by the effects of rapid desensitization. To further investigate potential roles of P2X1 receptors in platelet activation, the current study employed methods which maximally preserved P2X1 functionality. In preliminary in vivo studies, P2X1-deficiency reduced thrombus formation following the laser-induced, but not FeCl3-induced injury. Given the multiple potential mechanisms involved in thrombus formation in vivo, including tissue-factor/thrombin generation pathways, subsequent studies were designed to investigate the effects of P2X1 inhibition or stimulation on platelet activation in vitro; specifically, the interaction of P2X1 with thrombin receptor stimulation. Aggregation initiated by low/threshold levels of a protease-activated receptor (PAR)4 agonist was reduced in P2X1-deficient murine platelets, and inhibition of P2X1 in wild-type platelets similarly reduced PAR4-mediated aggregation. In human platelets, aggregation to low/threshold stimulation of PAR1 was inhibited with the P2X1 antagonist MRS2159. In addition, P2X1 stimulation primed human platelet responses, such that subsequent sub-threshold PAR1 responses were converted into significant aggregation. Selective ADP receptor inhibitors attenuated P2X1-mediated priming, suggesting that the synergy between P2X1 and sub-threshold PAR1 stimulation was in part because of enhanced granular release of ADP. Overall, the present study defines a novel interaction between platelet P2X1 and thrombin receptors, with P2X1 functioning to amplify aggregation responses at low levels of thrombin receptor stimulation.


Subject(s)
Platelet Aggregation , Receptors, Purinergic P2/metabolism , Receptors, Thrombin/metabolism , Animals , Blood Platelets/metabolism , Chlorides , Ferric Compounds/pharmacology , Humans , Lasers , Mice , Purinergic P2 Receptor Antagonists , Receptors, Purinergic P2X , Species Specificity , Thrombosis/metabolism
4.
J Thromb Haemost ; 2(10): 1782-9, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15456490

ABSTRACT

During normal hemostasis, the coagulation protease factor (F)XIa activates FIX. Hereditary deficiency of the FXIa precursor, FXI, is usually associated with reduced FXI protein in plasma, and circulating dysfunctional FXI variants are rare. We identified a patient with < 1% normal plasma FXI activity and normal levels of FXI antigen, who is homozygous for a FXI Gly555 to Glu substitution. Gly555 is two amino acids N-terminal to the protease active site serine residue, and is highly conserved among serine proteases. Recombinant FXI-Glu555 is activated normally by FXIIa and thrombin, and FXIa-Glu555 binds activated factor IX similarly to wild type FXIa (FXIa(WT)). When compared with FXIa(WT), FXIa-Glu555 activates factor IX at a greatly reduced rate ( approximately 400-fold), and is resistant to inhibition by antithrombin. Interestingly, FXIa(WT) and FXIa-Glu555 cleave the small tripeptide substrate S-2366 with comparable k(cat)s. Modeling indicates that the side chain of Glu555 significantly alters the electrostatic charge around the active site, and would sterically interfere with the interaction between the FXIa S2' site and the P2' residues on factor IX and antithrombin. FXI-Glu555 is the first reported example of a naturally occurring FXI variant with a significant defect in FIX activation.


Subject(s)
Factor IX/metabolism , Factor XI Deficiency/genetics , Mutation, Missense , Antithrombin III/pharmacology , Binding Sites , Factor XI/analysis , Factor XI/genetics , Factor XI/metabolism , Homozygote , Humans , Kinetics , Models, Molecular , Protein Binding/genetics , Static Electricity
5.
J Thromb Haemost ; 1(12): 2626-35, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14675100

ABSTRACT

The platelet P2X1 purinergic receptor is a ligand-gated ion channel that responds to ATP. The precise role of P2X1 in platelet function is unknown, though stimulation with the P2X1 agonist alpha,beta-Me-ATP is known to result in platelet shape change through elevation of calcium levels. The aim of the present study was to examine further the effects of P2X1 stimulation on platelet activation. Stimulation of P2X1 with alpha,beta-Me-ATP resulted in shape change and small aggregate formation in heparin-anticoagulated platelet preparations. Given the ability of heparin to potentiate platelet activation, subsequent experiments were performed in hirudin. In these platelet preparations, aggregate formation in response to alpha,beta-Me-ATP alone was less than that observed in heparin; however, alpha,beta-Me-ATP significantly potentiated platelet aggregate formation when added in conjunction with other weak platelet agonists [epinephrine or thrombopoietin (TPO)]. Platelet aggregate formation was confirmed by single platelet loss (microaggregate formation), microscopy, and light transmittance studies. Further, the P2X1 antagonist MRS-2159 inhibited platelet shape change and aggregation responses induced by alpha,beta-Me-ATP. Overall, this study demonstrates that P2X1 stimulation can induce/potentiate platelet activation in combination with other platelet agonists. These results are the first demonstration of platelet aggregation mediated through direct P2X1 stimulation, supporting a role for this receptor in regulating platelet activation.


Subject(s)
Adenosine Triphosphate/analogs & derivatives , Platelet Activation , Receptors, Purinergic P2/physiology , Adenosine Triphosphate/pharmacology , Calcium Signaling , Cell Size/drug effects , Epinephrine/pharmacology , Heparin/pharmacology , Humans , Platelet Activation/drug effects , Platelet Adhesiveness/drug effects , Platelet Function Tests , Receptors, Purinergic P2/drug effects , Receptors, Purinergic P2X , Thrombopoietin/pharmacology
7.
Thromb Res ; 100(1): 73-9, 2000 Oct 01.
Article in English | MEDLINE | ID: mdl-11053619

ABSTRACT

A humanized inhibitory anti-factor IX(a) antibody (SB 249417) has been compared to enoxaparin (Lovenox) in a rat model of arterial thrombosis. Pretreatment of rats with either SB 249417 (3.0 mg/kg, i. v.) or enoxaparin (30.0 mg/kg, i.v. or s.c.) resulted in comparable and significant reductions in thrombus formation. However, the efficacious dose of enoxaparin resulted in >30-fold increase in the aPTT over baseline, while the efficacious dose of SB 249417 prolonged the aPTT by only approximately 3-fold. Additionally, pretreatment with SB 249417 resulted in sustained blood flow and arterial patency throughout the experiment in >80% of rats treated. In contrast, <30% of rats pretreated with enoxaparin remained patent throughout the experiment. The data in this report indicate that the selective inhibition of factor IX(a) with the monoclonal antibody SB 249417 produces a superior antithrombotic profile to that of the low molecular weight heparin enoxaparin.


Subject(s)
Antibodies, Monoclonal/pharmacology , Carotid Artery Thrombosis/drug therapy , Coronary Disease/drug therapy , Enoxaparin/pharmacology , Factor IXa/immunology , Fibrinolytic Agents/pharmacology , Animals , Antibodies, Monoclonal/therapeutic use , Blood Coagulation Tests , Blood Flow Velocity/drug effects , Carotid Artery Thrombosis/chemically induced , Disease Models, Animal , Dose-Response Relationship, Drug , Enoxaparin/standards , Enoxaparin/therapeutic use , Fibrinolytic Agents/standards , Fibrinolytic Agents/therapeutic use , Heparin, Low-Molecular-Weight/immunology , Heparin, Low-Molecular-Weight/pharmacology , Heparin, Low-Molecular-Weight/therapeutic use , Humans , Male , Rats , Rats, Sprague-Dawley , Time Factors
8.
Thromb Haemost ; 82(5): 1443-5, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10595635

ABSTRACT

An inhibitory anti-factor IX/IXa antibody (BC2) has been investigated as an anti-thrombotic agent in a rat venous thrombosis model. The treatment of rats post-injury with a single bolus dose of BC2 (3 mg/kg, i.v.) resulted in an approximately 4 fold reduction in venous thrombus mass (P = 0.043). This efficacy was matched by a minimal (<2.5 fold) prolongation of the aPTT and had no effect on the prothrombin time (PT). Heparin by comparison, given as a bolus followed by continuous infusion, at doses comparable in efficacy at reducing thrombus formation, prolonged the aPTT >50 fold. These results demonstrate that the anti-factor IX/IXa antibody (BC2), when compared to heparin, can effectively reduce venous thrombosis with less disruptive consequences on blood clotting.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Factor IX/antagonists & inhibitors , Vena Cava, Inferior , Venous Thrombosis/prevention & control , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antibody Specificity , Drug Evaluation, Preclinical , Epitopes/immunology , Factor IX/immunology , Factor IX/physiology , Factor IXa/immunology , Heparin/administration & dosage , Heparin/therapeutic use , Infusions, Intravenous , Injections, Intravenous , Male , Mice , Partial Thromboplastin Time , Protein Structure, Tertiary , Prothrombin Time , Rats , Rats, Sprague-Dawley
9.
Arterioscler Thromb Vasc Biol ; 19(10): 2554-62, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10521387

ABSTRACT

A murine antihuman factor IX monoclonal antibody (BC2) has been generated and evaluated for its capacity to prolong the activated partial thromboplastin time (aPTT) in vitro and ex vivo and to prevent arterial thrombosis in a rat model in vivo. BC2 extended aPTT to a maximum of 60 to 80 seconds at 100 to 1000 nmol/L in vitro (rat and human plasma, respectively) and ex vivo (rat) after dosing of rats up to 6 mg/kg in vivo. BC2, administered as bolus (1 to 6 mg/kg) followed by infusion (0.3 to 2 mg x kg(-1) x h(-1)), dose-dependently prevented thrombosis of an injured rat carotid artery (FeCl(3)-patch model), increased time to artery occlusion, and reduced incidence of vessel occlusion. BC2 efficacy in preventing arterial thrombosis exceeded that of heparin (bolus 15 to 120 U/kg followed by infusion 0.5 to 4.0 U x kg(-1) x min(-1)), whereas the latter rendered the blood incoagulable (aPTT>1000 seconds). BC2 demonstrated complete antithrombotic efficacy also as a single bolus given either as prevessel or postvessel injury as evidenced by reduction of thrombus mass (from 4.18+/-0.49 to 1.80 +/-0.3 mg, P<0.001), increasing vessel patency time (from 14.9+/-0.9 minutes to 58.3+/-1.7 minutes, P<0.001) and decreasing incidence of vessel occlusion from 100% to 0% in vehicle- versus BC2-treated rats, respectively. BC2 (3 mg/kg, IV) administered in a single bolus resulted in 50% reduction in thrombus mass (P<0.01), extended vessel patency time (P<0.001), extended aPTT only 4-fold, and had no effect on blood loss via a tail surgical wound; heparin, at doses that reduced thrombus mass to a similar extent, extended aPTT beyond 1000 seconds (over 500-fold) and increased blood loss from 1.8+/-0.7 to 3.3 +/-0.6 mL (P<0.001). These data suggest that BC2 may provide enhanced therapeutic efficacy in humans at lesser interference with blood hemostasis than heparin.


Subject(s)
Antibodies, Monoclonal/pharmacology , Factor IX/immunology , Thrombosis/immunology , Anesthesia , Animals , Anticoagulants/pharmacology , Arterial Occlusive Diseases/chemically induced , Arterial Occlusive Diseases/drug therapy , Arterial Occlusive Diseases/immunology , Aspirin/pharmacology , Blood Loss, Surgical , Carotid Arteries , Disease Models, Animal , Dose-Response Relationship, Immunologic , Factor IX/metabolism , Fibrinolytic Agents/pharmacology , Heparin/pharmacology , Humans , Immunoglobulin G/pharmacology , Iron , Male , Microscopy, Electron, Scanning , Partial Thromboplastin Time , Prothrombin Time , Rats , Rats, Sprague-Dawley , Thrombosis/chemically induced , Thrombosis/drug therapy
10.
Proc Natl Acad Sci U S A ; 94(13): 6922-6, 1997 Jun 24.
Article in English | MEDLINE | ID: mdl-9192667

ABSTRACT

Previous reports suggest that tissue factor (TF) may play an essential role in embryonic vascular development and tumor angiogenesis. To further examine this relationship, the morphology of fully developed TF-deficient embryos and the growth of TF-deficient teratomas and teratocarcinomas were analyzed. In a 129/Sv genetic background, TF null embryos do not survive beyond mid-gestation. In contrast, 14% of 129/Sv x C57BL/6 TF-deficient embryos escape this early mortality and survive to birth. On gross and microscopic inspection, these late gestation, TF-deficient embryos appear normal. The growth and vascularity of TF(+/+), TF(+/-), and TF(-/-) teratomas and teratocarcinomas are indistinguishable. Thus, tumor-derived TF is not required for tumor growth and angiogenesis and the combined data do not support an essential role for TF in embryonic vascular development.


Subject(s)
Embryonic and Fetal Development/genetics , Teratocarcinoma/pathology , Teratoma/pathology , Testicular Neoplasms/pathology , Thromboplastin/genetics , Animals , Female , Gene Deletion , Gene Expression Regulation, Developmental , Male , Mice , Mice, Inbred C57BL , Pregnancy , Teratocarcinoma/genetics , Teratoma/genetics , Testicular Neoplasms/genetics , Thromboplastin/deficiency
11.
Blood ; 88(5): 1583-7, 1996 Sep 01.
Article in English | MEDLINE | ID: mdl-8781413

ABSTRACT

Tissue factor (TF) is an integral membrane glycoprotein that is believed to be the physiologic initiator of the blood coagulation cascade. Disruption of the mouse tissue factor gene leads to embryonic lethality between days E9.5-E11.5 of gestation. On E9.5, TF(-/-) embryos appear indistinguishable from their TF(+/+) and TF(+/-) littermates. By E10.5, TF(-/-) embryos are severely growth retarded, appear nearly bloodless, and are in most cases dead. Initial observations suggest that TF(-/-) embryos are dying of circulatory failure. Approximately 15% of the TF(-/-) embryos survive beyond E10.5, but none complete gestation. Heterozygotes appear normal and free of bleeding complications.


Subject(s)
Fetal Death/genetics , Gene Targeting , Thromboplastin/genetics , Animals , Blood Coagulation/genetics , Chimera , Embryo, Mammalian/blood supply , Female , Fetal Growth Retardation/genetics , Genetic Vectors/genetics , Gestational Age , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Thromboplastin/deficiency
12.
J Biol Chem ; 271(16): 9497-502, 1996 Apr 19.
Article in English | MEDLINE | ID: mdl-8621621

ABSTRACT

Coagulation factor Xa is a plasma serine protease that catalyzes prothrombin to thrombin conversion, which, in turn, leads to the generation of the fibrin clot. Of the several parameters that govern the plasma level of factor Xa, control of its catabolism is of crucial importance. However, little is known regarding the mechanisms by which factor Xa is catabolized. In the present study we examine the cellular basis for the uptake and degradation of factor Xa. 125I-Factor Xa was degraded by hepatoma cells and embryonic fibroblasts via a process which required cell surface-bound tissue factor pathway inhibitor (TFPI), a potent inhibitor of factor Xa. Uptake and degradation of cell surface-bound 125I-TFPI was also markedly stimulated in response to factor Xa binding. The intracellular kinetics of 125I-factor Xa and cell surface-bound 125I-TFPI display a strikingly similar pattern, suggesting that factor Xa and cell surface-bound TFPI are taken up as a bimolecular complex. Using cell lines either deficient in low density lipoprotein receptor-related protein, an endocytic receptor that mediates the degradation of uncomplexed TFPI (Warshawsky, I., Broze, G.J., Jr., and Schwartz, A.L. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6664-6668), or deficient in tissue factor (TF), an integral membrane protein capable of forming quarternary complexes with factor Xa, TFPI, and factor VIIa, we demonstrated that the receptor that mediates the uptake and degradation of factor Xa-TFPI complex was neither low density lipoprotein receptor-related protein nor TF. As the vascular endothelial cell surface retains a substantial pool of TFPI (Sandset, P.M., Alildgaard, U., and Larsen, M.L. (1988) Thromb. Res. 50, 803-813; Novotny, W.F., Brown, S.G., Miletich, J.P., Rader, D.J., and Broze, G.J., Jr. (1991) Blood 78, 387-393), our data suggest that endothelial cell surface TFPI may be actively involved in the clearance of factor Xa from the circulation via mediated uptake and degradation.


Subject(s)
Endocytosis , Factor Xa/metabolism , Lipoproteins/metabolism , Receptors, Immunologic/metabolism , Cell Line , Cell Membrane/metabolism , Fibrinolytic Agents/metabolism , Humans , Iodine Radioisotopes , Kinetics , Low Density Lipoprotein Receptor-Related Protein-1 , Receptors, LDL/metabolism , Recombinant Proteins/metabolism , Tumor Cells, Cultured
13.
Biochemistry ; 31(6): 1806-8, 1992 Feb 18.
Article in English | MEDLINE | ID: mdl-1310619

ABSTRACT

The blood coagulation factor IX(a) binds specifically to a site on endothelial cells with a Kd of 2.0-3.0 nM. A number of previous studies have attempted to define the region(s) of factor IX(a) that mediate this interaction. These studies suggested that there are two regions of factor IX(a), the gamma-carboxyglutamic acid (Gla) domain and the epidermal growth factor like (EGF-like) domains, that mediate high-affinity binding to endothelial cells. Recently, however, the participation of the EGF1 domain has been excluded from the interaction. This indicated that if there was an EGF component of factor IX contributing to the binding affinity, then it must be in the second EGF-like domain. In order to further evaluate this relationship, we performed competitive binding experiments between 125I plasma factor IX and a set of six chimeric proteins composed of portions of factor VII and factor IX. Our data suggest that the high-affinity interaction between factor IX and the endothelial cell binding site is mediated by the factor IX Gla domain and that the factor IX EGF domains are not involved in binding specificity.


Subject(s)
1-Carboxyglutamic Acid/metabolism , Endothelium, Vascular/metabolism , Factor IX/chemistry , Binding Sites , Binding, Competitive , Factor IX/metabolism , Factor VII/metabolism , Humans , Receptors, Cell Surface/metabolism , Recombinant Proteins/metabolism
14.
J Biol Chem ; 266(29): 19198-202, 1991 Oct 15.
Article in English | MEDLINE | ID: mdl-1918037

ABSTRACT

Tissue factor is an integral membrane glycoprotein that serves as an essential cofactor for the blood coagulation factor VIIa. Recent studies have attempted to localize the tissue factor recognition determinant of human factor VIIa. While several regions of factor VIIa have been implicated as important for tissue factor binding, the high affinity tissue factor recognition determinant of human factor VIIa is unknown. In order to define the determinant, we constructed a set of six chimeric proteins composed of portions of factor VII and factor IX. We then utilized the chimeras in competition experiments with 125I-labeled factor VIIa for recombinant tissue factor bound to an Immobilon-P membrane. The data indicate that the high affinity tissue factor recognition determinant of human factor VIIa is within the epidermal growth factor domains.


Subject(s)
Factor VIIa/metabolism , Thromboplastin/metabolism , Base Sequence , Chimera , Cloning, Molecular , DNA/genetics , Electrophoresis, Polyacrylamide Gel , Epidermal Growth Factor/genetics , Factor VIIa/genetics , Humans , Molecular Sequence Data , Mutagenesis , Polymerase Chain Reaction
15.
Thromb Haemost ; 61(2): 225-9, 1989 Apr 25.
Article in English | MEDLINE | ID: mdl-2473538

ABSTRACT

In order to define the epitopes recognized by anti-coagulation factor VIII (FVIII) monoclonal antibodies, we have constructed a recombinant DNA epitope library from random fragments of the FVIII cDNA. The characterization of 33 different clones producing recombinant antigens in the expression vector, lambda gt11, has identified the epitopes for six different anti-FVIII monoclonal antibodies. The antigenic determinant for each antibody was defined by the overlapping or shared DNA sequence of multiple-immunoreactive clones. One weak inhibitor of FVIII coagulant activity binds within the gly701-ser750 sequence of the FVIII sequence. An antibody which recognizes the amino-terminus of FVIII heavy-chain (within trp14-tyr46) does not inhibit FVIII activity. Two non-neutralizing antibodies both map within residues asp807-ser817, while two other non-neutralizing antibodies bind within lys1673-pro1688. The general usefulness of this strategy for mapping FVIII antigenic determinants is discussed.


Subject(s)
Antibodies, Monoclonal , Epitopes/analysis , Factor VII/immunology , Recombinant Proteins , Base Sequence , Cloning, Molecular , Genetic Vectors
16.
Proc Natl Acad Sci U S A ; 85(9): 3165-9, 1988 May.
Article in English | MEDLINE | ID: mdl-2452445

ABSTRACT

We have used a recombinant DNA epitope library to localize the binding region of a factor VIII (FVIII) monoclonal antibody that neutralizes coagulant activity. The antibody, C5, has previously been described and has been shown to have a FVIII neutralizing potency of 1488 Bethesda units per mg of purified immunoglobulin. A recombinant DNA epitope library was constructed from short, random FVIII cDNA fragments and immunologically screened with C5 to identify bacteriophage expressing the antigenic determinant. The isolation and characterization of immunoreactive bacteriophage restricted the C5 epitope to the overlapping or shared DNA sequence of nine different clones and corresponded to amino acid residues 338-362 of the mature FVIII peptide. The defined epitope is between the proposed activated protein C cleavage site (Arg-336) and thrombin cleavage site (Arg-372) on the amino-terminal 90-kDa FVIII heavy-chain subunit. The identification of the epitope of an inhibiting anti-FVIII antibody between two critical cleavage sites suggests that this amino acid sequence plays a role in regulating FVIII coagulant activity.


Subject(s)
Epitopes/analysis , Factor VIII/immunology , Amino Acid Sequence , Antibodies, Monoclonal , Bacteriophages/genetics , Base Sequence , Binding Sites , DNA, Recombinant/analysis , DNA, Viral/analysis , Enzyme-Linked Immunosorbent Assay , Humans
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