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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Division of Cell and Molecular Medicine,
Center for Molecular Medicine, Jichi Medical School,
Minamikawachi-machi; the Third Department of Internal Medicine, Dokkyo
University School of Medicine, Mibu-machi, Tochigi; and the First
Department of Internal Medicine, Faculty of Medicine, Tokyo Medical and
Dental University, Bunkyo-ku, Tokyo, Japan.
Acquired coagulation factor inhibitors include pathologic
immunoglobulins that specifically bind to coagulation factors and either neutralize their procoagulant activity, accelerate their clearance from the circulation, or have proteolytic activity to degrade
them into inactive polypeptides. Here, an autoantibody against
prothrombin is described in a patient with serious hemorrhagic diatheses. The autoantibody exerts its influence by a previously unknown mechanism in which it inhibits coagulation through aberrant activation of the proenzyme in a catalytic manner. The antibody-bound prothrombin formed a stable stoichiometric complex with antithrombin III, consisting of intact prothrombin and an antithrombin III molecule
cleaved at the 393Arg-394Ser bond. The antibody
dissociated from prothrombin after the complex formation with
antithrombin III. Although the bound antibody elicited protease
activity from prothrombin, the complex was not able to convert
fibrinogen to fibrin or to activate protein C. Thus, this is the first
description of an autoantibody that induces protease-like activity from
a human proenzyme, permitting subsequent neutralization by its
physiological inhibitor.
(Blood. 2001;97:3783-3789) Circulating anticoagulants are usually defined as
abnormal substances that directly interfere with mechanisms of blood
coagulation.1 They include abnormal immunoglobulins that
specifically bind to coagulation factors and either neutralize their
procoagulant activity,2 accelerate their clearance from
the circulation,3 or have proteolytic activity to degrade
them into inactive polypeptides.4
Enzymatic conversion of prothrombin to an active enzyme, thrombin, is
essential in the final step of blood coagulation. Patients with
hereditary abnormalities of prothrombin generate decreased amounts of
thrombin, which typically results in a lifelong bleeding disorder.5 By contrast, acquired inhibitors of thrombin or prothrombin may have variable effects on the blood coagulation system.
Nonneutralizing, antiprothrombin antibodies are the most common
inhibitors that lead to a prothrombotic state that is associated with
the lupus anticoagulant in patients with systemic lupus erythematosus and various other diseases.6 In contrast, neutralizing
thrombin antibodies rarely occur. In addition to the antibodies induced by exposure to bovine topical thrombin such as fibrin glue during surgical procedures,7 isolated cases of antithrombin
autoantibodies have been reported in patients with collagen diseases,
liver cirrhosis, and paraproteinemia, or even without any apparent
disease.8-10 In some instances, these inhibitors have been
associated with a serious bleeding disorder.3,11-13
However, the specificity of these antibodies has never been extensively investigated.
We report a patient in whom a potent human prothrombin inhibitor
developed that was associated with recurrent bleedings including retroperitoneal and pulmonary hematoma in the apparent absence of
underlying disease. The present observation is the first description of
an autoantibody directed toward prothrombin, which induces protease-like activity to facilitate a stable stoichiometric complex formation with its physiological inhibitor antithrombin III without Case history
Prothrombin inhibitor
Characterization of the aberrant thrombin-antithrombin complex Human prothrombin and IgG were pretreated with 1 mM di-isopropyl fluorophosphate (DFP; Wako Pure Chemical Industries, Tokyo, Japan) at 25°C for 1 hour and dialyzed extensively before additional experiments were conducted. The prothrombin was radiolabeled with sodium iodide I 125 using Iodobeads (Pierce, Rockford, IL) according to the manufacturer's directions. To observe the aberrant thrombin-antithrombin (TAT') complex formation in vitro with patient IgG, a mixture of 1.0 µM nonlabeled and 10 nM 125I-labeled prothrombin was incubated with 750 µg/mL IgG from patient or normal plasma at 4°C for 2 hours, and each mixture was then incubated with 5 µM antithrombin III at 37°C for various intervals (0-24 hours). Samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and the gel was dried and exposed for autoradiography at 25°C. We purified the TAT' complex from patient plasma using immobilized antihuman antithrombin III antibody Sepharose followed by chromatography on affinity resins containing antihuman prothrombin antibody (Nordic Immunology, Tilburg, CA). Patient plasma was also fractionated by BaSO4 absorption, and the barium eluate was applied to an affinity column of immobilized anti-TAT monoclonal antibody (JITAT-17).16 Each sample was analyzed by 10% SDS-PAGE under reducing and nonreducing conditions; this was followed by immunoblotting with antihuman antithrombin III and antihuman prothrombin polyclonal antibodies.3H-DFP incorporation into IgG antibody-prothrombin Before use, 310.8 kBq/µmol 3H-DFP (NEN Life Science Products, Boston, MA) was diluted 4 times with nonlabeled DFP to a concentration of 1 mM DFP. One micromolar prothrombin was incubated with 750 µg/mL patient IgG at 4°C for 2 hours; this was followed by the addition of 100 µM 3H-DFP and further incubation at 25°C for 2 hours. The sample was fractionated on a Sephacryl S-200HR gel filtration column, and the radioactivity of each fraction was determined with a liquid scintillation counter (LSC-3500; Aloka, Tokyo, Japan).Prothrombin derivatives and recombinant deletion mutants Prothrombin was digested with human factor Xa (Calbiochem, La Jolla, CA) into fragment 1, fragment 2, fragment 1 + 2, and -thrombin. Then the fragments were separated by high-performance liquid chromatography (Waters 996 Alliance system; Milford, MA) using a
reverse-phase column (phenyl-5PW RP; Toso, Tokyo, Japan). Meizothrombin
was prepared by incubating 3.3 µM Echinus carinatus venom
(ECV) with prothrombin (22 µM) and 0.36 mM Dansyl-arginine N,
N-(3-ethyl-1,5-pentanediyl)amide (DAPA) (Hematologic Technologies, Essex, VT) in 550 µL Tris-HCl, pH 7.4, for 15 minutes at room temperature. The reaction was quenched by the addition of 1 mL wheat
germ agglutinin gel slurry (Vector Laboratories, Burlingame, CA), which
binds ECV. Bound ECV was removed by filtration. Immediately before use,
the excess DAPA was removed by gel filtration through Sephadex
G-50.17 Human cDNA clones for full-length prothrombin were
kindly provided by Dr J. E. Sadler (Washington University School
of Medicine, St Louis, MO). The recombinant human prothrombin deletion
mutants used in this study were constructed by site-directed mutagenesis (Quikchange Site-Directed Mutagenesis Kit; Stratagene, La
Jolla, CA) with mutagenic primers 5'-GATGACTCCACGCTCCTAAGGCTCCAGTGTG-3' (nucleotides 563-593), 5'-CAGACAGGGCCATCTAAGGGCGTACCGCC-3' (nucleotides 901-929), and 5'-GACGGGCGCATTGTGTAGGGCTCGGATGC-3' (nucleotides 1062-1090) to produce fragment 1, fragment 1 + 2, and fragment 1 + 2 with the light chain of -thrombin. Chinese hamster ovary cells, which lacked the enzyme dihydrofolate reductase, were
transfected with the expression constructs in pCDNA3 (Invitrogen,
Carlsbad, CA). For production of recombinant prothrombin and its
deletion mutants, each cell line was grown in serum-free medium
containing 10 µg/mL vitamin K2 for 24 hours, as described
previously.18
Amino terminal sequence analysis One micromolar human prothrombin was incubated with 750 µg/mL patient IgG in the presence of 5 µM antithrombin III at 37°C for 12 hours. The mixture was applied to JITAT-17 monoclonal antibody immobilized on Sepharose, and the eluate was separated by 10% SDS-PAGE under reducing and nonreducing conditions. The gel was electroblotted onto a polyvinylidene difluoride membrane at 50 mA for 16 hours. The membrane was stained with 0.25% Coomassie in 50% methanol without acetic acid, and visible bands were cut and processed for NH2-terminal sequencing in an ABI model 496A sequencer (Foster City, CA). The amount of protein sequenced ranged from 4 to 8 pmol, depending on the fragments.
Mixing patient plasma with normal plasma generates TAT complex We initially suspected that the markedly prolonged prothrombin and activated partial thromboplastin times in a patient with hemorrhagic diatheses were caused by a coagulation factor(s) deficiency because the prolonged clotting times were corrected after 1-hour incubation at 37°C with normal plasma (Figure 1). However, in further incubation, 25% of the patient plasma led to prolongation of PT and APTT in a time-dependent manner (data not shown). In the mixing study, TAT complex formation markedly increased in a similar manner (Figure 2A). Therefore, we measured residual prothrombin activity in the mixture. Five percent of patient plasma added to normal plasma caused a slow loss of total prothrombin coagulant activity (Figure 2B). Surprisingly, the TAT complex immunopurified from patient plasma showed a higher molecular weight (130 kd) than the standard TAT complex. Thus, we designate this aberrant high-molecular-weight complex as TAT' complex.
Antibody against prothrombin makes a high-molecular-weight complex of prothrombin in the presence of antithrombin III Gel filtration analysis of the patient's plasma demonstrated that the TAT' complex generating factor was present in a high-molecular-weight fraction (150 kd). This factor did not bind to a protein A-Sepharose resin but specifically bound to protein G-Sepharose resin (data not shown). Patient and normal IgG were purified using antihuman immunoglobulin polyclonal antibody-Sepharose. Pass-through fractions of patient plasma from the column neither decreased the coagulant activity of prothrombin nor increased the formation of TAT' in the mixing studies described above. Purified IgGs were incubated with 125I-labeled human prothrombin in the absence or in the presence of a molar excess of antithrombin III (Figure 3, panels A and B, respectively). Incubation of 125I-prothrombin and patient IgG with antithrombin III resulted in a shift of the 72 kd prothrombin band to 130 kd in a time-dependent manner, as judged by SDS-PAGE. The amount of 130-kd complex reached 17% of total prothrombin after 24 hours (Figure 3B). In contrast, normal IgG had no effect on the migration of prothrombin or on the complex formation as judged by an enzyme-linked immunosorbent assay (ELISA) in the presence of antithrombin III (Figure 3C). The generation of the high-molecular-weight complex was not accelerated by heparin (data not shown). We separated the 130-kd complex from the patient plasma by barium absorption followed by affinity chromatography on antiprothrombin polyclonal IgG-Sepharose resin or immobilized monoclonal antibody against TAT complex (JITAT-17). It has been shown that JITAT-17 recognizes the heavy chain of modified antithrombin III in the TAT complex but that it does not react with free thrombin or intact antithrombin III.15 The 130-kd molecule was present solely in the barium-absorbed fractions and specifically bound to both antibody-affinity columns, indicating the presence of a prothrombin molecule with an intact NH2-terminal -carboxyglutamic acid (Gla) domain in a
complex with antithrombin III. This prothrombin-antithrombin complex
was also stable to treatment with glycine HCl, pH 3.0, 5.0 M urea, or
2.0% SDS.
Amino terminal sequencing of the high-molecular-weight complex Amino acid sequencing of the first 10 residues of the 130-kd protein revealed that it consisted of an equimolar complex of prothrombin and antithrombin III cleaved between 393arginine and 394serine (Table 1; Figure 4). Autoradiographic analysis of 125I-labeled prothrombin, a 72-kd single-chain molecule, incubated with patient IgG in the absence of antithrombin III revealed prothrombin-derived bands at 32.5 and 40 kd (Figure 3A). The result was similar, even if the patient IgG and prothrombin had been pretreated separately with 1 mM DFP and dialyzed extensively (data not shown). The amount of native prothrombin at 24 hours was decreased to less than 80% of initial level. Amino acid sequencing of the first 5 residues of the 32.5- and 40-kd peptides showed that prothrombin was cleaved between 284arginine and 285threonine (Table 2). Antithrombin III did not form a complex with either the 32.5-kd or the 40-kd prothrombin derivatives, which were generated by incubation of prothrombin with purified patient IgG in the absence of antithrombin III. Furthermore, 125I-antithrombin III was not cleaved, nor did it form a high-molecular-weight complex with patient IgG even after a 24-hour incubation. These results suggest that patient IgG induces a conformational change in the prothrombin molecule sufficient to permit the formation of a complex with antithrombin III.
Antibody-bound prothrombin is unable to convert fibrinogen to fibrin or to activate protein C After incubation of 125I-fibrinogen with the patient IgG-prothrombin mixture for various time periods, each sample was analyzed by SDS-PAGE subjected to autoradiography (Figure 5). The IgG-prothrombin mixture could not convert fibrinogen to fibrin even after a 6-hour incubation. This result is consistent with the clinical data ("Materials and methods"). Moreover, protein C was not activated by either the patient IgG or an IgG-prothrombin mixture even if soluble thrombomodulin (a kind gift of Mochida Pharma, Tokyo, Japan) was present, as judged by an amidolytic assay using a substrate specific for activated protein C (data not shown).
Serine protease activity appears in antibody-bound prothrombin without its prior conversion to a 2-chain molecule Pretreatment of patient IgG with a variety of protease inhibitors, such as antipain-dihydrochloride (100 µM), bestatin (150 µM), chymostatin (100 µM), E-64 (25 µM), leupeptin (10 µM), pepstatin (1 µM), phosphoramidon (400 µM), Pefabloc SC (4 mM), EDTANa2 (1 mM), and aprotinin (0.3 µM) for 1 hour did not decrease the TAT' complex-forming activity of the IgG (data not shown). Patient IgG pretreated with the serine protease inhibitor DFP (final concentration, 1 mM) also generated TAT' complex on the addition of prothrombin and antithrombin III. In contrast, when the mixture of prothrombin and patient IgG was treated with the same concentration of DFP or 10 mM p-amidino phenylmethylsulfonyl fluoride (pAPMSF; Wako Pure Chemical Industries) before it was added to antithrombin III, the TAT' complex generation was completely inhibited (data not shown). These results indicate that IgG itself does not have enzymatic activity analogous to the alloantibodies against factor VIII described previously4 and that it appears to elicit protease activity in the prothrombin zymogen without peptide bond cleavage.To address this question of aberrant IgG-induced prothrombin
activation, we examined whether 3H-DFP could be
incorporated into the antibody-bound prothrombin. Prothrombin was mixed
at 1 µM with 750 µg/mL normal IgG or patient IgG at 4°C for 2 hours, and the mixture was incubated with either DFP or
3H-DFP (37 kBq/µL) at 25°C for 2 hours followed by gel
filtration analysis. There was prothrombin antigen in the fractions
corresponding not only to 70-kd but also to 220-kd molecules that were
supposed to be the complex of prothrombin and the patient IgG (Figure
6A). However, prothrombin antigen existed
only in the fractions corresponding to 70-kd molecules when we used
normal IgG (data not shown). When we mixed prothrombin with patient
IgG, 3H-DFP coeluted with fractions corresponding to 220-kd
molecules (Figure 6B), and there was no 3H-DFP signal in
the 150-kd fractions (IgG) or in the 70-kd fractions (prothrombin).
Considering the amount of prothrombin antigen in the fractions
corresponding to 220-kd molecules, we estimated less than 1% of total
IgG made complex with prothrombin (Figure 6A). In the absence of
DFP, we further confirmed that these 220-kd fractions had TAT' complex
generation activity when antithrombin III was added (Figure 6C).
Therefore, these results are consistent with the observation that the
serine protease activity appears in the IgG-bound prothrombin, without
its prior conversion to a 2-chain molecule, and that this active
molecule subsequently cleaves antithrombin III at its reactive center
to make a stable complex.
The patient IgG showed specific and saturable binding to immobilized
prothrombin, dissociating from prothrombin after its complex formation
with antithrombin III (Figure 7A). This
result and the observations shown in Figures 2 and 3 suggest that the effect of the prothrombin autoantibody is catalytic. Prothrombin fragment 2 was recognized by the antibody (Figure 7B), but this recognition was far poorer than that of prothrombin. Interestingly, meizothrombin bound to the patient IgG more efficiently than
prothrombin did (Figure 7A). This may imply that another
conformation-dependent epitope exists near the catalytic domain of
prothrombin because the antibody did not bind to the prothrombin
complexed with antithrombin III.
Kinetic analysis of antibody-bound prothrombin Because antibody-bound prothrombin acquires serine protease activity and hydrolyzes an arginine-serine peptide bond in antithrombin III, we evaluated its hydrolytic activity toward various synthetic substrates. Antibody-bound prothrombin was unable to hydrolyze the fluorescent peptide Bz-Arg-methylcoumarylamide (trypsin substrate), Z-Pyr-Gly-Arg-MCA (factor Xa), or Pyr-Gly-Arg-MCA (urokinase and tissue-type plasminogen activator), but it could cleave Boc-Val-Pro-Arg-MCA (substrate for-thrombin). This hydrolytic
activity was completely inhibited in the presence of a molar excess of
antithrombin III. -Thrombin hydrolyzed the substrate with
Km = 13.5 ± 2.7 µM and kcat = 26.5 ± 1.5 s 1, which is
consistent with previous results.19 Incubation of IgG-prothrombin with increasing concentrations of the
Boc-Val-Pro-Arg-MCA peptide led to saturation of the hydrolytic
activity toward the substrate. The curves of the reciprocal of the
velocity plotted as a function of the reciprocal of the substrate
concentration were linear (r = 0.99), indicating that the
reaction conformed to simple Michaelis-Menten kinetics (data not
shown). The calculated average apparent Km and
kcat for the reaction were 42.5 ± 10.1 µM and
0.132 ± 0.065 s1, respectively. This apparent
kcat value was obtained based on the total prothrombin
concentration. Because less than 1% of total IgG seemed to be
concerned with prothrombin activation as shown (Figure 6), the apparent
kcat obtained was likely to be a significant underestimate
of the true kcat. However, the IgG-bound prothrombin exhibited quite a different macromolecular specificity toward physiological substrates such as fibrinogen and protein C.
Acquired coagulation factor inhibitors include pathologic immunoglobulins that specifically bind to coagulation factors and either neutralize their procoagulant activity or accelerate their clearance from the circulation.2,3,12 These inhibitors may develop as alloantibodies in patients who have congenital deficiencies and undergo replacement therapy, or as autoantibodies in patients with no known previous factor deficiency.20,21 The prothrombin autoantibody described here is distinct from staphylocoagulase because it causes fibrinogen-fibrin conversion22 and is unable to be neutralized by any in vivo inhibitors.23 Thus, to our knowledge, this is the first description of an acquired inhibitor that induces protease activity from the human zymogen protein, which is then neutralized by its own physiological inhibitor. We found a large amount of TAT' complex and a decreased level of prothrombin and antithrombin III without any consumption of fibrinogen in patient plasma. It is, however, almost impossible to estimate how much this antibody contributes to the low level of prothrombin antigen in the patient plasma because the IgG-bound prothrombin should be rapidly catabolized from reticuloendothelial systems in vivo. From a structural standpoint, characterization of this antibody-zymogen complex could provide insight into the molecular transition of the active site pocket from the inactive zymogen form to the active enzyme for which there is little data even from crystallographic studies.
We thank Dr Toshiyuki Miyata (National Cardiovascular Center, Osaka, Japan) for his useful comments on prothrombin mutants.
Submitted September 28, 2000; accepted January 26, 2001.
Supported in part by a Grant-in-Aid from the Ministry of Health and Welfare of Japan.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Yoichi Sakata, Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical School, Minamikawachi-machi, Tochigi 329-0498, Japan; e-mail: yoisaka{at}jichi.ac.jp.
1. Feinstein DI, Rapaport SI. Acquired inhibitors of blood coagulation. Prog Haemost. 1972;1:75-95. 2. Margolius A, Jackosn DP, Rantoff OD. Circulating anticoagulants: a study of 40 cases and a review of the literature. Medicine. 1960;40:145-202.
3.
Bajaj SP, Rapaport SI, Barclay S, Herbst KD.
Acquired hypoprothrombinemia due to nonneutralizing antibodies to prothrombin: mechanism and management.
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1985;65:1538-1543 4. Lacroix-Desmazes S, Moreau A, Sooryanarayana, et al. Catalytic activity of antibodies against factor VIII in patients with hemophilia A. Nat Med. 1999;5:1044-1047[CrossRef][Medline] [Order article via Infotrieve].
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Rabiet MJ, Furie BC, Furie B.
Molecular defect of prothrombin Barcelona: substitution of cysteine for arginine at residue 273.
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6.
Fleck RA, Rapaport SI, Rao VM.
Anti-prothrombin antibodies and the lupus anticoagulant.
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Lawson JH, Pennell BJ, Olson JD, et al.
Isolation and characterization of an acquired antithrombin antibody.
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1990;76:2249-2257 8. Struzik TZ, Hanicki J, Hawiger J, et al. Cryo-coagulopathy with presence of immunoantithrombin in the course of lupus erythematosus disseminatus. Acta Medica Polona. 1964;V:61-80. 9. Barthels M, Heimburger N. Acquired thrombin inhibitor in a patient with liver cirrhosis. Haemostasis. 1985;15:395-401[Medline] [Order article via Infotrieve]. 10. Gabriel DA, Carr ME, Cook L, et al. Spontaneous antithrombin in a patient with benign paraprotein. Am J Hematol. 1987;25:85-93[Medline] [Order article via Infotrieve]. 11. Scully MF, Ellis V, Kakkar VV, et al. An acquired coagulation inhibitor to factor II. Br J Haematol. 1982;50:655-664[Medline] [Order article via Infotrieve]. 12. Sie P, Bezeaud A, Dupouy D, et al. An acquired antithrombin autoantibody directed toward the catalytic center of enzyme. J Clin Invest. 1991;88:290-296.
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La Spada AR, Skalhegg BS, Henderson R, et al.
Fatal hemorrhage in a patient with an acquired inhibitor of human thrombin.
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1995;333:494-497 14. Madoiwa S, Mimuro J, Nakamura Y, et al. Abberant activation of prothrombin without fibrinogen-fibrin conversion. Thromb Haemost. 1999;82(suppl 2):704. 15. Pelzer H, Schwarz A, Heimburger N. Determination of human thrombin-antithrombin III complex in a patient with an enzyme-linked immunosorbent assay. Thromb Haemostat. 1988;59:101-106[Medline] [Order article via Infotrieve]. 16. Asakura S, Yoshida N, Matsuda M, Murayama H, Soe G. Preparation and characterization of monoclonal antibodies against the human thrombin-antithrombin III complex. Biochem Biophys Acta. 1988;952:37-47[CrossRef][Medline] [Order article via Infotrieve].
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1991;88:6775-6779 19. Kawabata S, Miura T, Morita T, et al. Highly sensitive peptide-4-methlycoumaryl-7-amide substrates for blood-clotting proteases and trypsin. Eur J Biochem. 1988;172:17-25[Medline] [Order article via Infotrieve]. 20. Exner T. Diagnostic methodologies for circulating anticoagulants. Thromb Haemostat. 1995;74:338-344[Medline] [Order article via Infotrieve]. 21. Lee MT, Nardi MA, Hu G, Hadzi-Nesic J, Karpatkin M. Transient hemorrhagic diathesis associated with an inhibitor of prothrombin with lupus anticoagulant in a 11/2-year-old girl. Am J Hematol. 1996;51:307-314[CrossRef][Medline] [Order article via Infotrieve].
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© 2001 by The American Society of Hematology.
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  R. Toso, H. Zhu, and R. M. Camire The Conformational Switch from the Factor X Zymogen to Protease State Mediates Exosite Expression and Prothrombinase Assembly J. Biol. Chem., July 4, 2008; 283(27): 18627 - 18635. [Abstract] [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
; 5%, 
; 10%, 
; 50%
final, 
) and normal plasma were incubated at 37°C for 0 to 24 hours. The amount of TAT' complex generated in each sample was measured
by ELISA for normal TAT. (inset) TAT complex and TAT' complex isolated
from patient plasma were subjected to SDS-PAGE (10% separating gels
under reducing conditions). (B) Patient plasma (0%, 
