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Prepublished online as a Blood First Edition Paper on April 30, 2002; DOI 10.1182/blood-2002-01-0243.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Angelo Bianchi Bonomi Hemophilia and
Thrombosis Center, and Fondazione Luigi Villa, IRCCS Maggiore Hospital
University of Milan, Milan, Italy, and the Hemostasis Research Centre,
Catholic University School of Medicine, Rome, Italy.
In a patient who presented with a severe coagulation deficiency in
plasma contrasting with a very mild hemorrhagic diathesis a homozygous
Arg67His mutation was identified in the prothrombin gene.
Wild-type (factor IIa [FIIa]-WT) and mutant Arg67His thrombin (FIIa-MT67) had similar amidolytic activity. By contrast, the kcat/Km value of fibrinopeptide A hydrolysis by
FIIa-WT and FIIa-MT67 was equal to 2.1 × 107
M Prothrombin deficiency is an autosomal recessive
bleeding disorder characterized by 2 phenotypes: hypoprothrombinemia,
with concomitantly low levels of coagulant activity and antigen (type I), and dysprothrombinemia, with low activity but borderline or normal
antigen levels (type II). These disorders are rare and there is always
residual prothrombin procoagulant activity measurable in patients, in
agreement with the fact that the phenotype found in
prothrombin-deficient mice indicates that complete prothrombin deficiency may be lethal in humans.1,2 Genetic and
biochemical analyses show that these disorders are the result of
substitution, deletion, or insertion of single nucleotides in the
prothrombin gene, resulting in the substitution of an amino acid in the
protein or in a premature stop codon. To date, 32 such defects in
prothrombin have been identified.3-5 Homozygous
hypoprothrombinemia (type I) is always characterized by severe bleeding
manifestations, whereas the bleeding tendency in dysprothrombinemia is
more variable, even though there is usually a good correlation between
the levels of prothrombin activity and clinical
severity.3,6
Recently, we identified a homozygous Arg67His missense mutation in the
prothrombin gene of an Iranian girl with
dysprothrombinemia.3 The only symptoms in this patient
were sporadic ecchymosis and one episode of buttock hematoma following
a major trauma. Arginine 67 (chymotrypsin numbering) is centrally
located on the surface of the thrombin domain referred to as fibrinogen
recognition site (FRS). Its guanidyl side chain is engaged in an
extended internal salt bridge cluster and interacts with many thrombin
macromolecular substrates, modulators, and inhibitors, such as
fibrinogen, thrombomodulin, and heparin cofactor II.7
Previously, a substitution of Arg67 with cysteine was identified in the
compound heterozygous dysprothrombins referred to as Quick I and
Corpus Christi.8,9 Moreover, alanine and glutamine
scanning mutagenesis studies, in which the highly exposed charged or
polar residues on thrombin were individually mutated to neutral
residues, demonstrated the relevance of Arg67 for thrombin activity
toward various substrates.10-15 The peculiar substitution
Arg67His observed in the homozygous state in our patient was never
identified and studied before. This prompted us to investigate both in
vivo and in vitro the functional properties of this natural thrombin
variant, which might be regarded as an FRS-knockout model of
thrombin in humans. To this end we investigated the functional
properties of the variant, by means of in vitro expression analysis and
evaluation of the function of purified recombinant wild-type (WT) and
mutant thrombin, tested on different substrates and modulators to
verify the change in specificity of the Arg67His mutant.
Patient
Coagulation assays
DNA analysis Following DNA extraction from leukocytes, the coding region, intron/exon boundaries, and the 5' and 3' untranslated regions (UTRs) of the prothrombin gene were amplified by polymerase chain reaction (PCR) and screened for mutations by single-strand conformation polymorphism (SSCP) analysis.3Site-directed mutagenesis and construction of expression vectors Full-length complementary DNA (cDNA) of human prothrombin (including 38 base pairs [bp] of 5'-UTR and 97 bp of 3'-UTR) was obtained by PCR amplification of M13mp18 (kindly provided by Dr Barbara C. Furie, Harvard Medical School, Boston, MA). The entire cDNA generated by PCR amplification (2021 bp) was sequenced before transfection. To clone the product of PCR amplification into the SalIPT7EcoRI vector, we designed the forward and reverse primers, with SalI and EcoRI restriction sites, respectively (forward: 5'-ACGCGTCGACGACAGACAATTCCTCAGTGACCCAGGAGCTGACACACTATGGCGCACGTCCGAG-3' and reverse: 5'-GGAATTCCGCTGAGAGTCACTTTTATTGGGAACCATAGTT TTAGAAACACAAAAATAA-3'). The product of PCR amplification was purified, digested with SalI and EcoRI and ligated into the SalIPT7EcoRI vector that had been digested with the same enzymes, to make the clone pT7-SalIFII-WTEcoRI.To investigate the influence of the Arg67His substitution on prothrombin activity, mutant factor II (FII) His67 was obtained by site-directed mutagenesis of PT7-SalIFII-WTEcoRI using a commercially available kit (Clontech, Palo Alto, CA). Oligonucleotide (5'-CCGAGAATGACCTTCT GGTACACATTGGCAAGCACT-3') spanning nucleotides 1301-1336 of the human FII cDNA were used to introduce a G to A at position 1322 (bold letters) coding for His67. This primer also introduced a KpnI restriction site (underlined), arising from a silent GTG-to-GTA mutation at nucleotide 1319, to facilitate screening for clones carrying the mutation. The cloned insert was sequenced and sequencing confirmed that the mutation had been introduced. SalIFII-WTEcoRI and SalIFII-MT67EcoRI fragments were prepared from the corresponding pT7 vectors (pT7-SalIFII-WTEcoRI and pT7-SalIFII-MT67EcoRI), and ligated separately into the expression plasmid pED-mtxr. The resulting plasmids, pED-FII-WT and pED-FII-MT67, are dicistronic messenger RNA mammalian expression vector carrying the WT or MT67 FII cDNAs at the 5' open reading frame and the dihydrofolate reductase (DHFR) gene at the 3' open reading frame. Cell culture and transfection assays For transient transfection experiments, African green monkey COS-7 cells (CRL1650; American Type Culture Collection, Manassas, VA) were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES, pH 7.2, 100 U/mL penicillin G, 100 µg/mL streptomycin, and 8 µg/mL vitamin K1 (phytonadione; Abbott Laboratories, North Chicago, IL) in a 5% CO2 atmosphere at 37°C. The DNA of pED-FII-WT or pED-FII-MT67 (30 ng) was transfected by electroporation into 5 × 106 COS-7 cells according to the manufacturer's instructions. After 72 hours, supernatants and cell lysates were assayed for FII antigen level.Purification of WT and Arg67His prothrombin Recombinant human WT and Arg67His mutant prothrombin were isolated from about 100 mL cell supernatant concentrates using an affinity chromatography column containing activated agarose coupled to monoclonal antibody human factor II (HFII) lot 103.55 from Enzyme Research (Indianapolis, IN), followed by activation of prothrombin by ecarin and ion-exchange purification by high-performance liquid chromatography (HPLC), as previously described.15 WT and Arg67His were purified and characterized as previously described.17 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 4% to 20% gradient gels showed that both WT and Arg67His thrombins were pure with an apparent molecular weight of 35 kd. The purified enzyme was immediately aliquoted and frozen at 80°C until use. The concentration of recombinant
thrombins was measured spectrophotometrically at 280 nm, using an
extinction coefficient (0.1%) equal to 1.83.
Activation of purified WT and Arg67His prothrombins by the prothrombinase complex Initial rates of prothrombin activation by human factor Xa (FXa) both in the presence and absence of factor Va (FVa) were measured by discontinuous method of the formed thrombin, assessed from the increase in the steady-state hydrolysis rate of 100 µM D-Phe-Pip-Pro-Arg-pNA at 405 nm. On the basis of a recent report suggesting the involvement of prothrombin pro-FRS in FVa interaction,18 no phospholipid reagent was used in this assay to investigate the direct prothrombin interaction with either FXa or FVa and unequivocally attribute the observed effects to each of these interactions only. Reaction mixtures contained 50 nM of either purified WT or Arg67His prothrombin, 5 nM FVa (American Diagnostica, Instrumentation Laboratory, Milan, Italy), 10 nM FXa (Calbiochem, Inalco, Milan, Italy) in 50 mM Tris-HCl, 0.15 M NaCl, 5 mM CaCl2, 0.1% PEG 6000, pH 7.50. Reactions were started by the addition of FXa. At various times (5-30 minutes when FXa alone was used; 1-5 minutes when both FXa and FVa were used), 100-µL aliquots of the reaction mixtures were quenched by addition to 0.9 mL 50 mM Tris-HCl, 0.15 M NaCl, 5 mM EDTA, 0.1% PEG 6000, pH 7.50, containing 100 µM D-Phe-Pip-Pro-Arg-pNA. The rate of active thrombin production was measured at 405 nm.Michaelis constants pertaining to WT and Arg67His thrombins for synthetic substrate hydrolysis The kcat and Km values for substrate hydrolysis by WT and Arg67His thrombins were measured as previously detailed, in 10 mM Tris-HCl, 0.15 M NaCl, 0.1% PEG 6000, pH 7.50 at 25°C (buffer A).19 The synthetic substrate used was D-Phe-Pip-Arg-pNA. Thrombin was used at a concentration of 1 nM.Binding affinity of thrombin for thrombomodulin and glycoprotein
Ib 
(GpIb) 1-282 fragment was purified from outdated platelet
concentrates, as previously detailed.15 Binding of
thrombin to human TM and purified GpIb fragment was performed by
solid-phase assays, as described.17,20 Detection of
thrombin was accomplished by incubating each well with 150 µL 100 µM D-Phe-Pip-Arg-pNA in 50 mM Tris, 0.2 M NaCl,
0.1% PEG 6000, pH 8.00, at 25°C. The absorbance was measured at 405 nm using a microplate reader Auto-Reader III system (Ortho Clinical
Diagnostics, Milan, Italy). Each determination (sample and blank) was
performed in duplicate.
Measurement of kcat/Km for PC activation by WT and Arg67His thrombin forms Hydrolysis of PC by free WT and Arg67His thrombin forms was monitored using 50 nM-thrombin, 0.5 µM PC in buffer A at 25°C. At appropriate time intervals the reaction was stopped with 0.3 M
HClO4. The solution was then centrifuged at 14 000 rpm and
the supernatant aspirated and filtered through 0.22-µm membranes for reversed-phase high-performance liquid chromatography (RP-HPLC) analysis, using a Bio-Rad C18 column (Bio-Rad Laboratories,
Hercules, CA). The eluants were 0.1% trifluoroacetic acid
(TFA) and 40% acetonitrile in 0.1% TFA. The gradient was from
0% to 100% of 40% acetonitrile in 30 minutes. The
activation peptide was monitored at 205 nm and quantified by a
reference curve using the synthetic activation peptide, DTEDQEDQVDPR,
purchased from Primm (Milan, Italy). Concentration of the activation
peptide at a given time, [aP]t, was fitted to the
following equation: [aP]t = [aP]max [1 exp(kobs)], where the pseudo first-order rate
constant kobs is equal to
e0kcat/Km
(e0 = thrombin concentration), as the data were obtained
at PC concentration less than Km of the hydrolytic reaction. When the kcat/Km value was calculated
in the presence of TM, the experimental conditions were the same as
indicated above, except that the solution contained 100 nM TM, 2 mM
CaCl2 and 10 nM thrombin forms.
Fibrinopeptide release by thrombin The rate of fibrinogen hydrolysis was studied by monitoring the rate of fibrinopeptide A release at different fibrinogen concentrations ranging from 0.5 to 64 µM, using a RP-HPLC method, previously detailed.21 WT and Arg67His thrombin forms were used in buffer A at 0.2 and 2 nM concentrations, respectively. The Michaelis parameters for steady-state hydrolysis were calculated by the GRAFIT software (Erithacus Software, Staines, United Kingdom).Inhibition of WT and Arg67His thrombin forms by AT and heparin cofactor II Pseudo-first-order kinetics formalism was used to investigate the interaction of WT and Arg67His thrombin forms with 2 serpins, that is, AT and heparin cofactor II (HCII) in the absence of GAG. HCII (American Diagnostica) was incubated at 1 µM concentration with 0.1 µg/mL polybrene (Sigma-Aldrich) in 10 mM Tris-HCl, 0.15 M NaCl, 0.1% PEG 6000, pH 7.50 (buffer A) at 37°C. The reaction was started by adding 50 nM wild-type or Arg67His. At various time points, a 25-µL aliquot was removed and added to a solution containing 100 µM D-Phe-Pip-Arg-pNA in buffer A at 37°C to measure the velocity of amidase activity of the residual thrombin in a thermostatted Varian Cary 2200 spectrophotometer. The amount of residual thrombin present in solution was proportional to the initial velocity of the amidase reaction, following the equation12: Vt = V0 exp(-k't), where Vt and V0 are the velocity at time t and zero, respectively, while k' is the apparent first-order rate constant of thrombin-HCII interaction. The value of the second-order rate constant, kon, was calculated by dividing k' by the serpin concentration.For experiments using AT, progress curve kinetics was used to analyze the experimental data. AT was used between 10 and 15 µM in buffer A at 37°C. The reaction with thrombin was started by adding 0.1 nM WT or Arg67His and the release of p-nitroaniline was measured as a function of time at 405 nm. Progress curves for the thrombin-AT complex formation were analyzed by the following relation12: pNAt = vst + [(v0 - vs)(1 - exp-k't)/k', where pNA is the concentration of p-nitroaniline at time t, k' has the same meaning as the immediately preceding equation, and v0 and vs are the initial and steady-state velocities of substrate hydrolysis, respectively. Progress curves fitted to this equation provided estimates for k', v0, and vs. The association second-order rate constant, kon, for thrombin-AT interaction was calculated by using the relation12: kon = k'(1-vs/ v0)(1+[S]/Km)/[AT], where S is the Phe-Pip-Arg-pNA concentration and Km is the Michaelis constant of its hydrolysis by thrombin, calculated in separate experiments. The measurements were also performed in duplicate. Progress curve kinetics was also used to derive the kon of
both AT and HCII interaction with WT and mutant thrombin in the presence of high-molecular-weight heparin from porcine intestinal mucosa (Sigma-Aldrich; sodium salt grade I-A, 170 USP/mg, average molecular weight, 16 500 d) and porcine dermatan sulfate
(Sigma-Aldrich; alternating copoly( Measurement of the PAR1 peptide hydrolysis by WT and mutant Arg67His thrombins Hydrolysis of the protease-activated thrombin receptor-1 (PAR1) peptide (PAR1P, NH2-LDPRSFLLRNPNDKYEPFWEDEE-COOH; single-letter amino acid codes), purchased from Primm (Milan, Italy) by the different thrombin forms was followed by measuring the release of the peptide LDPR, resulting from the cleavage of the NH2-terminus of PAR-1, according to a previously described method.17 Briefly, 0.5 µM PAR1P peptide was incubated with 50 to 100 pM WT or mutant thrombins (1 nM for the Arg67Ala form) in 10 mM HEPES, 0.15 M NaCl, 0.1% PEG 6000, pH 7.5, at 25°C. At time intervals (1, 2, 3, 4, 8, 12, and 15 minutes) the reaction was stopped with 0.3 M HClO4 and the cleaved peptide was measured by RP-HPLC, using a 250 × 4.6 mm RP-304 column (Bio-Rad), as previously detailed.17Interaction of WT and Arg67His thrombin forms with platelets We investigated the effect of Arg67His thrombin on the enzyme capacity to activate gel-filtered platelets. The latter were prepared in 10 mM HEPES, 0.15 M NaCl, 5.5 mM glucose, 0.2% BSA, pH 7.50, at 25°C as previously detailed.15 In these experiments, thrombin concentration ranged from 0.25 to 128 nM. Aggregation of gel-filtered platelets (3 × 105/µL) was studied in a PACKS 4 aggregometer (Helena Laboratories, Milan, Italy), using a final volume of 500 µL. Aggregation response was evaluated by taking into account the maximum velocity of transmittance increase per minute (expressed as percent of transmittance, set by using plain buffer).
Coagulation and genomic studies Coagulation assays repeated in Italy in the patient's fresh-frozen plasma confirmed a marked prolongation of both the prothrombin time and the activated partial thromboplastin time and severe prothrombin deficiency as measured by a classical coagulant assay (< 1%) contrasting with almost normal levels of prothrombin as antigen (61% ± 7%) and active enzyme (66% ± 8%) as measured by a 2-stage ecarin assay. On the other hand, the prothrombin concentration measured by the Taipan venom amidolytic assay was lower than the control, being 44% ± 5%. The dissociation between these results suggested the presence of a dysfunctional molecule and prompted us to study the activation of purified prothrombin by the prothrombinase complex, as reported below. A thrombophilic condition was ruled out in the patient, who had AT (115%), PC (71%), and free protein S (70%) levels within the normal range. The patient had neither G20210A mutation in the prothrombin gene nor the G1691A mutation in the factor V gene.To determine the molecular abnormality associated with the deficiency, the entire coding region and 5'-3'UTR of the prothrombin gene were amplified and screened by SSCP analysis. An abnormally migrating fragment was detected in exon 10. Sequence analysis revealed the patient to be homozygous for a nucleotide substitution that resulted in the Arg67His substitution. Both the parents were heterozygous for the same substitution. Expression studies To investigate the function of the mutant proteins, the pED-FII-WT and pED-FII-MT67 vectors were expressed in COS-7 cells by transient transfection. Enzyme immunoassay of the cell lysates and conditioned media demonstrated that the prothrombin antigen level of mutant Arg67His was similar to WT prothrombin (data not shown).Enzymatic activity of the mutant Arg67His thrombin toward synthetic and natural substrates The Michaelis parameters pertaining to the hydrolysis of the synthetic substrate Phe-Pip-Arg-pNA are given in Table 1. The Arg67His mutation did not significantly alter the amidase activity of thrombin, suggesting that this substitution does not affect the interaction of the substrate with S1-S4 of the enzyme. By contrast, the interaction of thrombin with natural substrates, such as fibrinogen and thrombin receptor (TR), showed profound changes compared to WT. In fact, fibrinopeptide A hydrolysis was characterized by an approximate13-fold increase of the Km value (38 µM vs 3 µM) and by a 2-fold decrease of the kcat value (32.5 s 1 vs 64 s1), as shown in Figure 1A.
Thus, the kcat/Km value decreased from 2.1 × 107 M1 s1 to
9 × 105 M1 s1, that is,
22-fold, as shown in Table 1. This finding may explain the loss of the
clotting capacity of the mutant thrombin in patient plasma.
Another macromolecular substrate, the TR-derived 38-60 peptide, which
interacts with both the catalytic pocket and FRS of thrombin, showed a
severe defect in interacting with the enzyme, as shown in Figure 1B. TR
38-60 hydrolysis was characterized by an approximate 23-fold increase
of the Km value (62 µM versus 2.7 µM) and by a 2-fold
decrease of the kcat value (50 s Hydrolysis of PC in the absence of TM was not affected by the mutation, because the kcat/Km value was very close to that observed with the WT enzyme, as shown in Table 1. This finding is in substantial agreement with previous data, showing that thrombin does not involve its FRS in its interaction with free PC, except when the enzyme forms a ternary complex with TM and PC, as shown in experiments using TM as cofactor (see below). Interaction of the mutant Arg67His thrombin with TM Solid-phase binding experiments showed that Arg67 plays a central role in the interaction with TM, because the Kd decreased 33-fold in the Arg67His mutant, as shown in Figure 2A. The defective binding of the mutant thrombin severely affected the catalytic competence of the enzyme in PC hydrolysis. Under experimental conditions using 100 nM TM and either 10 nM WT or mutant thrombin, the apparent kcat/Km value pertaining to PC hydrolysis decreased from 2.3 × 105 M1 s1 to
3.7 × 104 M1 s1 as shown in
Figure 2B. This inhibitory effect was attributed to a defective TM
interaction, because PC hydrolysis by the Arg67His mutant enzyme, as
reported above, was unchanged compared to the wild-type form.
Interaction of WT and Arg67His thrombin with AT and HCII The best-fit second-order rate constants for both WT and Arg67His interaction with AT and HCII in the presence and absence of heparin and dermatan sulfate, respectively, are listed in Table 3. HCII interaction with Arg67His thrombin decreased roughly 3-fold compared to WT, in the absence of dermatan sulfate. This result was in agreement with previous findings that the N terminus of HCII interacts with the thrombin FRS.21 This defect was even more evident in the presence of dermatan sulfate, where Arg67His showed a 500-fold decrease of its kon value compared to WT thrombin. On the contrary, the Arg67His interaction with AT did not show such drastic decrease both in the absence and presence of heparin. This finding is in agreement with previous findings that FRS is not engaged in thrombin interaction with this serpin.12,22
Interaction of WT and mutant Arg67His thrombin with gel-filtered platelets The Arg67His mutant thrombin showed a defect in its capacity to activate gel-filtered platelets, the EC50 value being increased 10-fold compared to WT (Figure 3). This biologic effect was attributed to a defective PAR-1 interaction only, because solid-phase binding experiments showed a normal interaction with GpIb, the other major
thrombin receptor involved in the activation of human
platelets.23 The Kd value of this binding was
equal to 109 ± 12 nM in WT and 123 ± 18 nM in Arg7His. This
finding is in agreement with recent data showing that a similar but not
isosteric and isoelectric mutation at Arg67, that is Arg67Ala, does not
affect thrombin interaction with GpIb.15
Activation of purified WT and Arg67His prothrombin with the FXa-FVa complex The Arg67His mutation caused a net decrease of the initial rates of prothrombin activation by FXa only when FVa was present in the activation mixture. When FXa alone was used at concentrations of 10 nM in the activation mixture, the initial rates of prothrombin activation were 6.5 ± 0.9 pM/min and 5.7 ± 0.1 pM/min for WT and Arg67His prothrombin, respectively (Table 4). By contrast, when 10 nM FXa plus 5 nM FVa was used, the initial rates of prothrombin activation were 500 ± 90 pM/min and 100 ± 15 pM/min for WT and Arg67His, respectively (Table 4). These experiments showed that Arg67His mutation reduced the cofactor effect of FVa in the FXa-catalyzed prothrombin activation.
In a recent study aimed at screening natural variants of prothrombin in a large cohort of families,3 we identified and investigated a woman with undetectable plasma levels of prothrombin activity, borderline levels of prothrombin antigen, and a very mild bleeding diathesis. The latter finding is in contrast with what we found in other patients with hyperprothrombinemia, who usually suffer from more severe spontaneous and posttraumatic bleeding symptoms, despite the fact that, at variance with our patient, they have measurable levels of prothrombin activity.3 A homozygous Arg67His mutation in the prothrombin gene, which results in the loss of a positive charge within the FRS of thrombin, was found. The amino acid substitution involves an arginine and can be hypothesized to alter the enzyme interaction with natural substrates and modulators that bind to FRS, such as fibrinogen, PAR-1, or thrombomodulin. The positively charged FRS residues contribute significantly to enhance the rate of complex formation with thrombin ligands through steering with complementary electrostatic fields and a coordinated combination of molecular contacts within the complex that is peculiar for each macromolecular ligand. A severe reduction of the catalytic competence of thrombin toward the
fibrinogen A The Arg67His mutant showed also a severely defective interaction with
PAR-1, associated with a net reduction of platelet-activating capacity,
that was not due to a defective interaction with GpIb Activation of Arg67His prothrombin by the FXa-FVa prothrombinase complex was severely impaired. This finding is in agreement with recent results suggesting a role of pro-FRS in the molecular recognition of prothrombin by the complex, possibly by direct involvement of pro-FRS in FVa binding.18 The 5-fold reduction of the initial rate of the Arg67His prothrombin activation by FXa only in the presence of FVa corroborates the recently proposed role, direct or allosteric, of the pro-FRS domain in prothrombin interaction with FVa.18 The present findings would suggest that Arg67 participates indeed in this interaction. It is also of interest that activation of the mutant Arg67His prothrombin by the Taipan snake venom gave a value of plasma prothrombin concentration lower than that obtained with the ecarin assay. These snake venoms belong to different classes of prothrombin activators. Ecarin converts prothrombin into meizothrombin, which autocatalytically produces active thrombin, in absence of FVa or phospholipids.28 By contrast, Taipan snake venom needs Ca++ and phospholipids to activate prothrombin, being associated with a FVa-like cofactor activity.29 The Arg67His mutation could thus inhibit the FVa-like activity of this venom. These findings suggest also that the initial characterization of prothrombin variants should include the use of different classes of venom activators to disclose dysfunctional features of the variants. Despite the major functional defects discussed above, the Arg67His mutant was associated with very mild bleeding symptoms. This prompted us to investigate other thrombin interactions with modulators and natural inhibitors involved in the regulation of the anticoagulant functions as well as in the inhibition of the enzyme. TM is an integral glycoprotein of the endothelial cell membrane and binds to thrombin, switching the function of this enzyme from procoagulant to anticoagulant through the activation of the PC pathway. Previous nuclear magnetic resonance (NMR) and x-ray diffraction studies showed that the endothelial growth factor (EGF) 5-6 domains of TM make many hydrogen and salt bridges with FRS thrombin residues. Arg67 is involved in this bond network because the hydroxyl group of TM Tyr413 makes a hydrogen bond with the guanidyl side chain of thrombin Arg67.30-32 In our experiments the Arg67His variant showed a reduced interaction with TM, expressed by a Kd value 30-fold higher than in the WT form. This defect caused a defective TM-catalyzed PC hydrolysis. The activation of PC alone, in the absence of TM, did not undergo a reduction of the apparent kcat/Km value, as reported in Table 1. On the other hand, in the presence of a high TM concentration, the apparent kcat/Km value was 6-fold lower for the Arg67His mutant than for the WT. It may be thus hypothesized that in vivo the TM-linked physiologic formation of activated PC undergoes a drastic impairment. Thus, the most relevant TM-linked anticoagulant function of thrombin was as defective as that pertaining to the clotting capacity of the enzyme. This anticoagulant dysfunction might in part counterbalance the procoagulant defect of the mutant, leading to surmise that the formation of active thrombin in the patient is not down-regulated by activated PC. This may help explain the very mild bleeding tendency of the patient carrying this natural variant. The study of HCII and AT interaction with the Arg67His mutant might further unravel the apparent enigma of the absence of a severe hemorrhagic syndrome in the patient. Kinetic experiments showed for the Arg67His mutant a strong decrease of the second-order rate constant of HCII association both in the absence (3-fold) and especially in the presence of dermatan sulfate (500-fold). In addition, a moderate decrease of the AT association rate constant in the presence of heparin (1.5-fold) was observed. The severely defective interaction of the mutant thrombin with HCII is in agreement with the involvement of the N-terminus of HCII in the ligation of thrombin FRS.12,33 The HCII inhibitory activity toward thrombin is strongly enhanced by the presence of glycosaminoglycans (GAGs), such as dermatan sulfate, which allosterically "activates" HCII. A specific dermatan sulfate hexasaccharide sequence composed of repeats of iduronic acid 2-sulfate and N-acetylgalactosamine 4-sulfate has been shown to bind to a cationic domain of HCII.34 This binding disrupts the intramolecular docking, allowing the free N-terminal acidic domain of the inhibitor to bind to the FRS of thrombin.33 Given the selectivity with which dermatan sulfate activates HCII among all serpins interacting with GAGs, and the presence of dermatan sulfate in the extracellular matrix of a wide variety of tissues, HCII has been proposed to inhibit "extravascular" thrombin activity. This inhibitory mechanism has been proposed to oppose the proatherogenic activity of thrombin.35,36 In our patient, the impaired thrombin-HCII interaction might oppose the defective procoagulant capacity of the thrombin mutant, increasing the half-life of free thrombin. The interaction of Arg67Ala thrombin with AT showed only a modest reduction, consistent with the fact that the thrombin-AT interaction does not involve directly the FRS.22 The modest reduction of the association rate of the interaction in the presence of heparin might further contribute to a reduced inhibition in vivo of the thrombin variant. In conclusion, the experimental data showed that both procoagulant and anticoagulant functions of the Arg67His mutant are impaired. Moreover, the mutation induced a strongly defective inhibition of thrombin by HCII. Altogether, these functional abnormalities might somewhat counterbalance each other so that ultimately the hemostatic equilibrium does not undergo drastic changes, explaining the very mild clinical phenotype associated with this congenital type II prothrombin deficiency.
Submitted January 28, 2002; accepted April 9, 2002.
Prepublished online as Blood First Edition Paper, April 30, 2002; DOI 10.1182/blood-2002-01-0243.
Supported by a grant (COFIN 2000) of the Ministry of the University and Scientific and Technological Research of Italy (R.D.C.), IRCCS Maggiore Hospital, Milan, Italy; and Fondazione Italo Monzino (P.M.M.).
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: Sepideh Akhavan, A. Bianchi Bonomi Hemophilia and Thrombosis Center, Via Pace 9, 20122 Milan, Italy; e-mail: sepidehakhavan{at}yahoo.it or sakhavan{at}bichat.inserm.fr.
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Abstract
Introduction
-iduronic
acid-[1
3]-N-acetyl-
) and Arg67His mutant (
)
thrombin. Continuous lines were drawn according to the best-fit
kcat and Km values listed in Table 
