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Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 2026-2031
Prothrombin Greenville, Arg517 Gln, Identified
in an Individual Heterozygous for Dysprothrombinemia
By
R.A. Henriksen,
C.K. Dunham,
L.D. Miller,
J.T. Casey II,
J.B. Menke,
C.L. Knupp, and
S.J. Usala
From the Section of Allergy, Asthma and Immunology, the Section of
Hematology, and the Section of Endocrinology, Department of Medicine,
East Carolina University, Greenville, NC.
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ABSTRACT |
A 64-year-old white male was referred for evaluation of prolonged
prothrombin time (PT) and activated partial thromboplastin time (aPTT)
obtained before elective surgery with initial PT and PTT results of
14.9 and 38.4 seconds, respectively, which corrected to normal in 1:1
mixes with normal plasma. Functional prothrombin assay indicated a
level of 51% with thromboplastin as an activator. The prothrombin
antigen was 102%. This discordance in the functional and immunologic
prothrombin levels was evidence for dysprothrombinemia. Western
blotting showed that thrombin was formed at a normal rate in diluted
plasma consistent with a mutation within the thrombin portion of
prothrombin. DNA was isolated from leukocytes and the thrombin exons
were amplified by polymerase chain reaction, cloned, and sequenced. For
exon 13, eight clones were sequenced with four clones showing a point
mutation in the codon for Arg517, which would result in
substitution by Gln. Arg517 is part of the
Arg-Gly-Asp(RGD) sequence in thrombin and contributes to
an ion cluster with aspartic acid residues 552 and 554. Mutation at
this residue most probably distorts the structure of the
Na+ binding site in thrombin. This is the first report
indicating the critical role of Arg517 in the normal
physiological interaction of thrombin with fibrinogen. This
dysprothrombin is designated Prothrombin Greenville.
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INTRODUCTION |
PROTHROMBIN, M.W. 72,000, is the plasma
glycoprotein zymogen form of the enzyme thrombin, a critical enzyme in
the regulation of hemostasis with both procoagulant and anticoagulant
activities. In addition, thrombin also stimulates cellular activation
through a cell surface thrombin receptor. The gene for prothrombin is located on an autosomal chromosome at 11p11-q121 and severe
(homozygous) deficiencies with phenotypic manifestations are
encountered only rarely. In cases of heterozygosity, functional deficiencies in prothrombin of 50% or less do not result in
physiological manifestations and they are not usually detected by the
routine screening tests for coagulation factor deficiency, the
prothrombin time (PT), or the activated partial thromboplastin time
(aPTT). Thus, although mutations resulting in dysprothrombinemia and
hypoprothrombinemia may occur with a frequency approximating that of
hemophilia,2 these mutations are not readily identified.
Because of its functional importance, mutations in prothrombin that
result in alterations of thrombin function are of particular interest.
Congenitally mutant thrombins have been identified previously in eight
pedigrees with the primary structure defect identified for six distinct mutations.3-11 All of these mutants have been identified
because of decreased fibrinogen clotting activity, the thrombin
function that is monitored in clinical coagulation tests.
Site-specific mutants of proteins are of great value in increasing our
understanding of structure-function relationships. Because of the
several interactions of thrombin with other proteins, there are
numerous surface residues that may be implicated in these interactions,
the mutation of which may result in decreased function. Both the
congenital and other site-specific mutants of thrombin together with
knowledge of the thrombin crystal structure12 have
contributed to our understanding of critical features required for
normal thrombin function. Some features of thrombin that are known to
regulate its activity include the catalytic triad residues, primary
substrate binding site, anion binding exosite I (which interacts with
fibrinogen and the thrombin receptor/substrate), anion binding exosite
II, the heparin binding site, the thrombomodulin binding site, and the
more recently identified Na+ binding site.13,14
The studies described here were undertaken to determine an explanation
for the observed low functional prothrombin level in a patient who was
evaluated for abnormal prothrombin and partial thromboplastin times
before elective surgery. DNA sequencing showed that the patient was
heterozygous for dysprothrombinemia and normal prothrombin with
identification of a G A transition in exon 13 that predicts a
substitution of Gln for Arg517(187) (Residues of thrombin
are numbered according to the prothrombin sequence15 with the chymotrypsinogen
numbering12 in parentheses). As reported
previously,16 this residue forms part of an ionic cluster
in thrombin that is adjacent to the Na+ ion
ligands.13
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MATERIALS AND METHODS |
Reagents.
Fibrinogen from KabiVitrum was purchased from Helena Laboratories
(Beaumont, TX). Phospholipid was obtained as rabbit brain cephalin from
Sigma Chemical Co (St Louis, MO) and a stock solution was prepared
according to the manufacturer's directions. Taipan venom from
Oxyuranus scutellatus was obtained from Miami Serpentarium Laboratories (Punta Gorda, FL), and Echis carinatus venom was obtained from Sigma Chemical Co. Dade factor assay reference plasma for
preparation of prothrombin standard curves was obtained from Scientific
Products (McGaw Park, IL). The chromogenic substrate, tos-Gly-Pro-Arg-pNA (Chromozym TH) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN). Oligonucleotides were prepared by the DNA core laboratory, East Carolina University (Greenville, NC). The restriction enzyme Mnl I was obtained
from Amersham Life Science (Arlington Heights, IL). Other chemicals were obtained from domestic suppliers.
Blood specimens for plasma and DNA.
Phlebotomy was performed after obtaining informed consent. For
coagulation assays, plasma was obtained from citrated whole blood,
subjected to a second centrifugation at 10,000g and stored in
aliquots at  80°C. For isolation of DNA, whole blood was
anticoagulated with acid citrate dextrose solution (A).
Coagulation assays.
Prothrombin assays were performed by one stage assay with either taipan
venom2,17 or Echis carinatus venom18 as
activators and fibrinogen as substrate. The chromogenic substrate
tos-Gly-Pro-Arg-pNA was used in a two-stage assay and prothrombin
activation was accomplished with a taipan venom activator reagent
consisting of 0.2 mg/mL taipan venom in 0.1 mol/L NaCl, 0.04 mol/L
Tris HCl, pH 7.5, 2 mmol/L Ca2+, and rabbit brain cephalin
at 1/5 dilution of the stock solution. For activation, 0.1 mL of the
activator reagent was incubated with 0.1 mL of plasma diluted in 0.1 mol/L NaCl, 0.05 mol/L TrisHCl, and pH 7.5. After 1.0 minute
incubation at 37°C, 100 µL of the incubation mixture was
transferred to 1.1 mL 0.1 mol/L NaCl, 0.05 mol/L TrisHCl, and pH 8.3 containing 0.14 mmol/L tos-Gly-Pro-Arg-pNA. The rate of substrate
hydrolysis was monitored at 405 nm. Percent activity was determined
from a standard curve prepared with factor assay reference plasma.
Other coagulation assays were performed by established clinical
laboratories.
Western blotting for prothrombin.
Plasma samples from the proband and a normal individual were diluted
1/30 with 0.1 mol/L NaCl, 0.05 mol/L TrisHCl, and pH 7.5 and treated
with one half volume of the taipan venom activator reagent containing 3 mmol/L Ca2+. Samples were incubated at 37°C and at 30, 60, and 600 seconds 100-µL aliquots were added to a 25-µL
electrophoresis sample solution, 0.24 mol/L TrisHCl, pH 6.8, 5 mmol/L
EDTA, and 8% sodium dodecyl sulfate. Electrophoresis on 12%
polyacrylamide gels was performed as described19 without
reduction of disulfide bonds. After electrophoresis, proteins were
transferred to nitrocellulose. The membrane was blocked with 5% nonfat
dry milk in phosphate buffered saline, pH 7.4. The primary antibody was
polyclonal goat antihuman prothrombin antiserum obtained from ICN
(Costa Mesa, CA). The secondary antibody was peroxidase conjugated
rabbit antigoat IgG (Sigma Chemical Co) with detection by enhanced
chemiluminescence (Amersham Life Science).
DNA sequencing.
DNA was isolated from leukocytes after proteinase K
digestion.20 Thrombin exons were amplified by the
polymerase chain reaction (PCR) with primers derived from the adjacent
noncoding regions of the prothrombin gene. The primers were modified
from those used previously8 and are listed in
Table 1. Temperature conditions for PCR of
all exons were denaturation, 1 minute at 94°C; annealing, 1 minute
at 55°C; and extension, 2 minutes at 72°C for 30 cycles. PCR amplified DNA fragments for the proband were purified by
electrophoresis and cloned by using the Original TA Cloning Kit Ver 2.0 from Invitrogen (San Diego, CA). DNA sequencing was performed by the
dideoxy method.21 Sequences were read manually and compared
with the published prothrombin gene sequence.22 Both coding
and noncoding strands were sequenced.
Restriction fragment length polymorphism analysis.
Restriction enzyme digestion of PCR amplified prothrombin exon 13 was
performed with Mnl I. Hydrolysis was monitored by
electrophoresis on 2% agarose gel with ethidium bromide staining. The
reaction mixtures were incubated at 37°C for a total of 7 hours to
ensure complete hydrolysis.
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RESULTS |
Case report.
The proband was a 64-year-old white male referred to the East Carolina
University School of Medicine Hematology/Oncology Clinic for evaluation
of an increased PT and aPTT obtained before elective surgery for a
right rotator cuff repair. He had no prior history of significant
bleeding or bruising and had undergone previous herniorrhaphy, right
heel surgery, traumatic amputation of the distal portions of two
fingers, and multiple tooth extractions without significant bleeding or
the need for blood transfusions. There was no history of liver disease.
Diet was normal with only remote use of small amounts of alcohol. No
family history of abnormal bleeding was noted. Physical examination was
not remarkable for jaundice, hepatosplenomegaly, or signs of chronic
liver disease. No ecchymoses or evidence of recent bleeding was
present. No joint deformities or evidence of hemarthrosis was noted.
Limitation of motion of the right shoulder was present because of the
rotator cuff injury. Laboratory studies performed by a reference
laboratory before referral indicated a low prothrombin activity with
normal factor V and X activity levels.
The laboratory results of the hemostasis evaluation were as follows
with normal ranges in parenthesis: platelets 220 (150 - 440) × 103/µL, bleeding time 3.2 (2.5 - 9.5) minutes, PTT 14.9 (11.6 - 13.2) seconds, and for a 1:1 mix with control plasma 12.3 seconds, aPTT 38.8 (22.6 - 33.0) seconds and for a 1:1 mix with control
plasma 32.5 seconds, fibrinogen 2.73 (2.00 - 4.00) mg/mL. Values
obtained for factor assays with >50% considered normal were as
follows: factor V 90%, factor VII 73%, factor IX 64% and factor X
101%. The prothrombin activity assay was 51% (normal >50%) in a
one-stage assay with thromboplastin as the activator. In contrast the
prothrombin antigen was 102% with a normal range of 75% to 130%.
Fibrinogen was the substrate for all factor assays. Dilute Russell's
viper venom time, antinuclear antibody, and anticardiolipin antibody panel testing were normal indicating that a lupus anticoagulant was not
present.
The normal values for the vitamin K-dependent coagulation factors other
than prothrombin indicate that the low prothrombin level was not caused
by vitamin K deficiency. Thus, the discordance between the prothrombin
antigen and activity assays indicates the presence of
dysprothrombinemia in this otherwise healthy individual. The
dysprothrombin has been designated Prothrombin Greenville. Because the
surgeon did not wish to risk bleeding complications, and because the
prothrombin activity was at or near the hemostatic threshold, the
patient was given fresh frozen plasma in the perioperative period. The
surgery was successfully completed with no unusual bleeding noted.
Additional prothrombin assays.
To further characterize the dysprothrombin identified in the proband,
additional prothrombin assays were performed with venom activators and
both fibrinogen and tos-Gly-Pro-Arg-pNA as thrombin substrates. The
results of these assays are presented as mean ± standard deviation
for three separate assays: one-stage assay with Echis carinatus
venom as activator and fibrinogen as substrate 36% ± 1%,
one-stage assay with taipan venom/PL/Ca2+ as activator and
fibrinogen as substrate 50% ± 2%, and two-stage assay with the
latter activator and tos-Gly-Pro-Arg-pNA as substrate 76% ± 4%.
The taipan venom assay with fibrinogen as substrate, 50%, was similar
to the 51% obtained initially with thromboplastin as activator. If the
dysprothrombinemia were the consequence of a mutation in the activation
peptide or a defect preventing hydrolysis of the
Arg271( )-Thr bond of prothrombin, it was predicted
that activation by Echis carinatus venom would yield a normal
thrombin level.23,24 This was not observed, which suggests
that the molecular defect is within the thrombin portion of the
molecule. When a chromogenic substrate was used in a two-stage assay,
the prothrombin activity for the proband was greater than observed with
fibrinogen as substrate. This result suggested that the defect in the
predicted dysthrombin probably affected thrombin-fibrinogen interaction
to a greater extent than the interaction with a low molecular weight
substrate. However, there is apparently also some defect in catalysis
by the dysthrombin because the activity toward tos-Gly-Pro-Arg-pNA is
not equivalent to that expected for the observed antigen level.
Prothrombin activation and Western blotting.
To further examine the nature of the defect in the dysprothrombin, the
time course of prothrombin cleavage was determined by Western blotting.
The results following activation of diluted whole plasma by taipan
venom and electrophoresis on a nonreducing gel shown in
Fig 1, indicate that prothrombin is cleaved
at a similar rate for both the proband and the control and that the products for both have similar molecular weights. A mutation at Arg271 would result in an activation defect and
approximately half of the total prothrombin in this apparently
heterozygous individual should have remained as the higher molecular
weight proteins, meizothrombin (with the same molecular weight as
prothrombin), or prethrombin 1 at the end of the incubation period. A
mutation at Arg320(16) would result in the accumulation of
prethrombin 2 (uncleaved thrombin A and B chains). Under reducing
conditions, prethrombin 2 should appear at a higher molecular weight
than the thrombin heavy chain. Electrophoresis performed under reducing
conditions indicated conversion of all prothrombin to thrombin (results
not shown). Thus, the results are consistent with the presence of a
dysthrombin. This dysthrombin was predicted to be the consequence of a
point mutation because of the similar molecular weights for prothrombin
and its hydrolysis products obtained from plasma of the control and the
proband.
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| Fig 1.
Prothrombin activation determined by Western blotting.
The activation of prothrombin in diluted whole plasma and Western
blotting were performed as described in the Materials and Methods. The location and size of molecular weight standards is indicated on the
left. The activation reaction was stopped at the time in seconds indicated at the top of the figure. Prothrombin, a glycoprotein is seen
corresponding to the molecular weight marker 97.2 kD. The lane marked
thr is purified thrombin and serves as a marker for this product. The
band just above the 50 kD marker that appears after activation is
prethrombin-1 and the decreased amount seen in the proband (P) is
consistent with the decreased thrombin activity in this sample. C
indicates control plasma. The lower molecular weight bands seen
primarily at 600 seconds correspond to the activation peptides,
fragment 1, and fragment 2. The highest molecular weight band in this
unreduced sample corresponds to cross-reacting human IgG present in the
plasma. The other bands present both before and after activation are
not identified.
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DNA sequencing.
Attempted screening of PCR products for the thrombin exons was not
successful in identifying a point mutation. Because the proband was
expected to be heterozygous for a dysprothrombin, at least six clones
were sequenced for each exon to verify the presence of a unique point
mutation. The following list gives the number of the prothrombin exon
with the number of clones sequenced indicated in parenthesis: 8 (7), 9 (6), 10 (14), 11 (12), 12 (11), 13 (8), and 14 (8). The nucleotide at
position 8908 in exon 1022 was C in all the clones that
were sequenced as originally reported for the human prothrombin cDNA
sequence.15 This does not result in an amino acid
substitution. For exon 13, 4 of 8 clones sequenced yielded a
transitional mutation at nucleotide 19777 corresponding to a codon
change from CGA to CAA at amino acid residue 517(187), which results in
an ArgGln mutation. The finding of 50% normal sequences
confirms that the proband is heterozygous for the mutation. The results
for sequencing two clones for exon 13 amplified from DNA of the proband
are shown in Fig 2. A mutation at
Arg517(187), a surface residue that forms part of an ionic
cluster16 in thrombin, has not been previously reported.
This region of thrombin, which is near the base of the primary
substrate binding pocket, has not been specifically associated with the
interaction of thrombin with fibrinogen.
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| Fig 2.
Results of DNA sequencing. Nucleotide sequence for a
portion of two clones obtained for prothrombin exon 13, which was
amplified by PCR from DNA of the proband. Sequencing was by the dideoxy method of Sanger.21 The asterisk (*) indicates the mutated
amino acid residue.
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Structural consequences of the Arg517(187)Gln
mutation.
The mutated arginine residue in this dysprothrombin is a part of an RGD
sequence in thrombin. However, the aspartic acid residue of this
sequence is located at the base of the primary substrate binding pocket
and neither this residue nor the adjacent glycine are accessible to the
molecular surface, which makes it unlikely that this sequence
contributes to binding interactions in native thrombin.16
The ionic cluster formed by Arg517(187) with
Asp552(221) and Asp554(222) is shown
schematically in Fig 3. A Na+
binding site that contributes to optimal coagulant activity and involves an adjacent region of thrombin has been
identified13,25 in which one of the Na+ ligands
is provided by the carbonyl oxygen of Arg553(221A).
Examination of the thrombin structure indicates that the correct positioning of this Na+ ligand must depend on formation of
the ionic cluster between the two adjacent aspartic acid residues and
Arg517(187). Thus, in Thrombin Greenville, where this ionic
interaction is lost because of the substitution of the positively
charged arginine residue by the neutral residue glutamine, there is
most probably a significant perturbation of the structure of the
Na+ binding site, which appears to result in an enzyme
lacking significant fibrinogen clotting activity as indicated by the
functional prothrombin assays. Previous results from Di Cera's
laboratory26,27 indicate that in the absence of
Na+ the rate of release of fibrinopeptide A by thrombin is
decreased to a greater extent than is the hydrolysis of the low
molecular weight substrate
H-D-Phe-pipecolyl-Arg-p-nitroanilide. Similarly, the results
obtained here for the prothrombin assays suggest that structural
features associated with the ionic cluster around
Arg517(187) are apparently less critical for the normal
hydrolysis by thrombin of the low molecular weight substrate
tos-Gly-Pro-Arg-pNA than for the release of fibrinopeptide A from
fibrinogen.
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| Fig 3.
Structure of thrombin showing location of mutated ionic
cluster. Ribbon structure for thrombin is shown as produced by the RasMol modeling program with coordinates obtained from the Brookhaven database. Side chains are shown at the bottom left for residues Arg517(187) marked by the arrow head,
Asp552(221), and Asp554(222). In this view the
active site serine residue is located at the center, anion binding
exosite I extends across the center right, and anion binding exosite II
is along the upper left edge.
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Restriction enzyme digestion.
Sequence analysis of the normal and Prothrombin Greenville nucleotide
sequences identified an Mnl I restriction site in the normal
sequence that was lost from Prothrombin Greenville. The recognition
sequence for Mnl I is CCTC. The recognition sequence on the
complementary strand, GAGG corresponds to the prothrombin gene coding
sequence for nucleotides 19777 to 19780 in exon 13. The Prothrombin
Greenville mutation converts this sequence to AAGG resulting in the
loss of the Mnl I restriction cleavage site. The PCR amplified
exon 13 for the proband and several family members were subjected to
Mnl I digestion. The results shown in
Fig 4 indicate that in the proband and one
son, digestion of this fragment is incomplete, which is consistent with
the loss of the Mnl I restriction site as predicted by the
observed mutation and the expected heterozygosity for this mutation.
Figure 5 shows the pedigree for the proband
with prothrombin assays for several family members. These results are
consistent with the findings obtained by Mnl I digestion.
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| Fig 4.
Restriction digest with Mnl I. The mutation
identified in Prothrombin Greenville predicts the loss of an
Mnl I restriction site where the sequence GAGG in the normal is
the enzyme recognition site. This site is converted to AAGG in the
mutant. Exon 13 for the proband and five family members was amplified
by PCR and subjected to digestion with Mnl I. Hydrolysis was
continued for 7 hours at 37°C. Additional enzyme was added to
samples for lanes 3, 4, and 6 after 6 hours of hydrolysis to ensure
complete hydrolysis. Shown are the final hydrolysis products after
agarose gel electrophoresis and ethidium bromide staining. Lane numbers
correspond to samples obtained from individuals shown in the pedigree
(Fig 5). Shown in the lane at left is a 100-base pair ladder.
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| Fig 5.
Pedigree for Prothrombin Greenville. The upper numbers
beside the numbered symbols are prothrombin antigen levels as percent and the lower numbers are percent prothrombin activity obtained after
activation by Echis carinatus venom with fibrinogen as the substrate. Shading indicates heterozygotes. ND, not determined;  ,
proband; /, deceased. Individuals without assay values were not
studied.
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DISCUSSION |
The dysprothrombin identified in these studies has been designated
Prothrombin Greenville for the North Carolina location where this
mutant protein was characterized. It was anticipated that a dysthrombin
with decreased fibrinogen clotting activity would have an altered
residue associated with either the catalytic triad residues, anion
binding exosite I, or the primary substrate binding pocket as has been
observed with other dysthrombins.2 This did not appear to
be the case for the mutation that we identified, so other structural
features were examined in an attempt to relate the identified mutation
to defective fibrinogen clotting activity. Examination of the results
of structural studies of thrombin13,25 suggest that this
mutation most probably affects the Na+ binding site.
Binding of Na+ at this site is proposed to participate in
the regulation of the procoagulant and anticoagulant forms of thrombin
in which fibrinogen or protein C are the respective thrombin
substrates.14 This is the first report of a dysthrombin in
which the Na+ binding site appears to be the locus of the
mutation. These findings are also consistent with a critical structural
role for the ionic cluster involving residues 517 (187), 552 (221), and
554 (222) located on the surface of thrombin. From comparison of the
prethrombin 2 and thrombin structures, it was proposed25
that this ionic cluster is a crucial driving force for the structural
rearrangement that occurs in this region of thrombin on cleavage of the
thrombin A and B chains to yield the active enzyme thrombin. This
structural rearrangement results not only in formation of the
Na+ binding site by positioning one of the Na+
ligands, R553 (221A), but also results in a shift of the position of
D519 (189) at the base of the primary substrate (arginine) binding
pocket. It is probable that improper positioning of D519 (189) also
contributes to the functional defect observed in Thrombin Greenville.
This ionic cluster is not conserved in the primary structure of other
serine proteases,13 although it is conserved in bovine
thrombin as is the Arg that provides a Na+ ligand
corresponding to Arg 553(221A) of human thrombin.28 A
related mutant thrombin with the sequence Ala552(221)ArgLys
has been prepared by recombinant DNA methods. In this mutant, the
allosteric effect of Na+ is lost with the result that the
kinetics for release of fibrinopeptide A are similar in the presence
and absence of Na+.13 It appears that this
latter mutant has a relatively greater activity in the release of
fibrinopeptide A than does Thrombin Greenville. As with other
dysprothrombins that have been reported, this dysprothrombin was
identified because of its decreased fibrinogen clotting activity. This
heterozygous defect does not appear to have resulted in any phenotypic
manifestation in the proband. There is no history of either excessive
bleeding or thrombosis in the proband.
The GA transitional mutation has most probably arisen on the
noncoding DNA strand where the sequence CpG occurs, a sequence that has
been identified as a mutational "hot spot." These mutations are
thought to arise when a methylcytosine is deaminated and then recognized as a T with the result that A is incorporated on the complementary strand during DNA replication. Other dysthrombins that
have arisen by a similar mechanism are Tokushima3 and Quick
I.4
The identification of individuals heterozygous for dysprothrombinemia
is somewhat fortuitous because they are not generally manifested by
prolonged coagulation screening tests. For Prothrombin Greenville, the
dysthrombin may have been inhibitory contributing to the prolonged PT
and aPTT tests or the mildly decreased factor VII and factor IX levels
in combination with the low prothrombin level may have resulted in the
prolonged times.29
Further characterization of the enzymatic activity of this mutant
thrombin will require isolation of Prothrombin Greenville from plasma
or the expression of the mutant protein by recombinant DNA techniques.
These studies are currently planned and will permit a more definitive
analysis of the role of the ionic cluster formed around
Arg517(187) in normal thrombin function.
| |
FOOTNOTES |
Submitted July 18, 1997;
accepted November 5, 1997.
Supported by National Institutes of Health Grant No. HL 45194 (to
R.A.H.). R.A.H. is an Established Investigator of the American Heart
Association.
Presented at the American Society for Hematology Annual Meeting,
Seattle, WA, December 5, 1995 (Blood 86:71a, 1995).
Address reprint requests to R.A. Henriksen, PhD, Department of
Medicine, East Carolina University, Greenville, NC 27858-4354.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank the proband and his family for their generous
cooperation in making this study possible, Dr Bruce D. Wilhelmsen for
referring the proband to us for further study, and Scott Eaves for
performing the Western blotting studies after prothrombin activation.
| |
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Abstract
Introduction
Abstract