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Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4581-4592
Blood Coagulation in Hemophilia A and Hemophilia C
By
Kevin M. Cawthern,
Cornelis van `t Veer,
Jennifer B. Lock,
Maria E. DiLorenzo,
Richard F. Branda, and
Kenneth G. Mann
From the Department of Biochemistry, College of Medicine, and the
Department of Medicine, University of Vermont, Burlington, VT.
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ABSTRACT |
Tissue factor (TF)-induced coagulation was compared in contact
pathway suppressed human blood from normal, factor VIII-deficient, and
factor XI-deficient donors. The progress of the reaction was analyzed
in quenched samples by immunoassay and immunoblotting for
fibrinopeptide A (FPA), thrombin-antithrombin (TAT), factor V
activation, and osteonectin. In hemophilia A blood (factor VIII:C <1%) treated with 25 pmol/L TF, clotting was significantly delayed versus normal, whereas replacement with recombinant factor VIII (1 U/mL) restored the clot time near normal values. Fibrinopeptide A
release was slower over the course of the experiment than in normal
blood or hemophilic blood with factor VIII replaced, but significant
release was observed by the end of the experiment. Factor V activation
was significantly impaired, with both the heavy and light chains
presenting more slowly than in the normal or replacement cases.
Differences in platelet activation (osteonectin release) between normal
and factor VIII-deficient blood were small, with the midpoint of the
profiles observed within 1 minute of each other. Thrombin generation
during the propagation phase (subsequent to clotting) was greatly
impaired in factor VIII deficiency, being depressed to less than 1/29
(<1.9 nmol TAT/L/min) the rate in normal blood (55 nmol TAT/L/min).
Replacement with recombinant factor VIII normalized the rate of TAT
generation. Thus, coagulation in hemophilia A blood at 25 pmol/L TF is
impaired, with significantly slower thrombin generation than normal
during the propagation phase; this reduced thrombin appears to affect
FPA production and factor V activation more profoundly than platelet
activation. At the same level of TF in factor XI-deficient blood (XI:C
<2%), only minor differences in clotting or product formation (FPA, osteonectin, and factor Va) were observed. Using reduced levels of
initiator (5 pmol/L TF), the reaction was more strongly influenced by
factor XI deficiency. Clot formation was delayed from 11.1 to 15.7 minutes, which shortened to 9.7 minutes with factor XI replacement. The
maximum thrombin generation rate observed (~37 nmol TAT/L/min) was
approximately one third that for normal (110 nmol/L TAT/min) or with
factor XI replacement (119 nmol TAT/L/min). FPA release, factor V
activation, and release of platelet osteonectin were slower in factor
XI-deficient blood than in normal blood. The data demonstrate that
factor XI deficiency results in significantly delayed clot formation
only at sufficiently low TF concentrations. However, even at these low
TF concentrations, significant thrombin is generated in the propagation
phase after formation of the initial clot in hemophilia C blood.
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INTRODUCTION |
IN VITRO, DISTINCT PLASMA coagulation
pathways lead to the generation of thrombin: the intrinsic (or contact)
pathway and the extrinsic (or tissue factor [TF])
pathway.1 In recent years, the TF pathway has been
considered to be central to thrombin generation in normal hemorrhage
control in vivo.2 The TF pathway is thought to proceed by
assembly of three distinct surface-dependent complexes.3,4 The initiating complex is the extrinsic tenase (factor VIIa and cofactor TF on a membrane), which assembles when circulating plasma factor VIIa encounters TF at the site of injury.5 During an initiation phase, this complex activates a fraction of the circulating zymogens factors X and IX to their respective active forms, factors Xa
and IXa.6-9 Factor Xa, in complex with factor
V/Va,10 potentiates factor V and factor VIII activation
indirectly via the generation of limited amounts of
thrombin.11,12 Additional factor Xa is generated by the
intrinsic tenase complex, composed of factor IXa assembled with factor
VIIIa on the platelet surface. Factor Xa assembles with factor Va into
the prothrombinase complex, the major activator of prothrombin to
 -thrombin during a propagation phase. The importance of the
extrinsic and intrinsic tenase complexes and prothrombinase in thrombin
generation and coagulation is underscored by the observation that
deficiencies in factor VII, factor VIII, factor IX, factor X, and
factor V are associated with hemorrhagic tendencies.
Current theories of coagulation and hemostasis propose that, in the
quiescent state, low-level thrombin generation is suppressed by
endogenous inhibitory mechanisms, including antithrombin-III (AT-III),13,14 TF pathway inhibitor
(TFPI),15,16 and the protein C pathway.17
Ongoing generation of factor IXa and factor Xa by the factor VIIa/TF
complex is limited due to inhibition by TFPI and
AT-III.2,18-20 At high concentrations of TF, such as are
used in a prothrombin time (PT) assay, levels of extrinsic tenase
formed are sufficient to lead to thrombin in excess of the threshold
levels necessary for hemostasis.21 However, at lower TF
concentrations, the activity of the intrinsic tenase is essential for
above-threshold levels of thrombin and hemostasis.8,9,22
Whereas the extrinsic and intrinsic tenases have distinct roles in
factor Xa and thrombin generation in coagulation, evidence for the
involvement of the initiating members of the contact pathway is
limited. In factor XI deficiency (hemophilia C), severe spontaneous bleeding is rarely observed; affected patients typically exhibit significant hemorrhage only upon surgical challenge or extreme trauma.23-25 No associations are found between factor XII
or prekallikrein deficiency and abnormal bleeding, a peculiar
circumstance given that factor XIIa is a potent activator of factor
XI.26-28 Recent work has sought to clarify the role of
factor XI during coagulation in vivo. Slow activation of factor XI by
thrombin has been demonstrated in vitro, and the reaction is greatly
accelerated on negatively charged surfaces such as dextran
sulfate.29,30 After its activation by limited amounts of
thrombin, factor XIa may support TF-initiated thrombin generation by
elevating levels of factor IXa and consequently the intrinsic tenase.
Thus, substantial contributions to thrombin generation may be provided
by both the intrinsic tenase and factor XIa. Whereas these observations
explain the bleeding that may accompany factor XI deficiency, they have
been challenged on the basis that the rate of factor XI activation by
thrombin in vitro is extremely slow in the absence of exogenously added
surfaces.31 Other than recent studies in plasma systems,
little physiological evidence exists to support or refute this
mechanism.32,33
The current study was undertaken to evaluate the established role of
factor VIII and the potential contribution of factor XI during whole
blood coagulation in vitro, using a model of the TF pathway in which
factor XIIa is blocked.34 The advantage of this system is
that the native state of all the blood components is preserved,
providing a physiologically relevant platform to test theories of blood
coagulation.
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MATERIALS AND METHODS |
Materials.
Recombinant human TF and recombinant factor VIII were provided as gifts
by Drs Roger Lundblad and Shu-Len Liu (Hyland Division, Baxter
Healthcare Corp, Duarte, CA) and human factor XI was a gift from Dr Richard Jenny (Hematologic Technologies, Inc, Essex Junction, VT). Trypsin inhibitor from corn was either obtained from
Fluka (Ronkonkoma, NY) or prepared as described below.
1-Palmitoyl-2-oleoyl phosphatidylserine (PS) and 1-palmitoyl-2-oleoyl
phosphatidylcholine (PC) were purchased from either Sigma Chemical Co
(St Louis, MO) or Avanti Polar Lipids, Inc (Birmingham, AL).
D-Phenylalanyl-L-prolyl-L-arginine chloromethyl ketone
(FPRck35) was obtained as a gift from Hematologic Technologies, Inc (Essex Junction, VT) or purchased from Calbiochem (La
Jolla, CA). Diisopropyl fluorophosphate (DFP) was obtained from Sigma Chemical Co, diluted to working concentration (1 mol/L) in
anhydrous isopropanol, and stored at  20°C. Pooled
standardized normal (FACT, lot no. D12S1) and factor XI-deficient (lot
no. GK1122-N17P1) plasmas were obtained from George King Biomedical (Overland Park, KS). Thromboplastin (Simplastin Excel) and activated partial thromboplastin time (aPTT; Automated APTT)
reagents were purchased from Organon Teknika (Durham, NC). The
following analytes were estimated using enzyme-linked immunosorbent
assay (ELISA) kits obtained from the manufacturers
according to the instructions provided: thrombin-AT-III (Enzygnost TAT;
Behring, Westwood, MA); fibrinopeptide A (Asserachrom FPA;
Diagnostica Stago/American Bioproducts, Parsippany, NJ);
and platelet osteonectin (a gift from Dr Richard Jenny, Hematologic
Technologies, Inc).
A murine monoclonal antibody (FVaHC#17; 5 to 10 µg/mL)
that recognizes an epitope between residues 307 and 506 in the heavy chain (HC) of factor V/Va36 was prepared according to
previously published procedures.37 The reactivity and
specificity of this antibody in Western analyses are similar to another
antibody described previously (FVaHC#6).38 A
second murine monoclonal antibody, directed against the light chain of
the cofactor (FVaLC#9; 5 to 10 µg/mL), was prepared as
described elsewhere.39
Human donors.
All donors, normal and deficient, were recruited and advised according
to a protocol approved by the University of Vermont Human Studies
Committee. Normal individuals (age range, 22 to 36 years) were selected
so as to exclude donors with a personal or familial history of
thrombosis/hemorrhage or regular aspirin or drug use. All individuals
exhibited values in the normal range for the PT (11.6 to 13.8 seconds),
aPTT (27 to 36 seconds), and fibrinogen and platelet counts (172,000 to
376,000/µL). Subsequent to each control experiment, factor XI levels
were assayed for the normal donors (range, 95 to 119 U/dL), falling
within the accepted normal adult range (75 to 130 U/dL).23
Two hemophilia A donors have been examined to date. The first
hemophilia A donor detailed in this report (patient A1) was a
46-year-old man with severe factor VIII deficiency (VIII:C <0.5%) who exhibited a life-long tendency toward bleeding. The propositus suffered recurrent hemarthroses in the elbows, knees, and ankles and
has a limited range of motion with pain in the shoulders, elbows, and
ankles; he had received no replacement therapy for 2.5 weeks before the
experiment. As is common among hemophilia A patients transfused with
human products, this individual had developed a CDC class A-III HIV
infection. Treatment with indinevir resulted in thrombocytopenia, which
partially resolved upon discontinuation of the drug. The platelet count
on the day of the experiment was 97,000 platelets/µL, but has
decreased since then (December 1995); there is no evidence of
antiplatelet antibodies. Current medications are
trimethoprim-sulfamethoxazole, zidovudine, lamivudine, and zalcitabine.
A second hemophilia A donor has been examined (patient A2) and is
discussed briefly. This 18-year-old man has tested positive for
hepatitis C. However, he exhibits normal fibrinogen levels, platelet
count, and tissue thromboplastin time (prothrombin time), although he
is severely factor VIII-deficient (prolonged aPTT; VIII:C <1%). This
donor has a history of bleeding and joint pain and routinely
self-administers recombinant factor VIII products when symptomatic.
Before our study, he had not received recombinant factor VIII products
for at least 4 days (50% replacement dose, >7 half-lives). There was
no evidence of inhibitors (eg, anti-factor VIII antibodies).
Three hemophilia C (factor XI-deficient) donors have been studied;
results with two of these patients are described here in detail.
Patient C1 is a 53-year-old woman with no family history of bleeding.
She had exhibited easy bruising as well as frequent nosebleeds, but no
menorrhagia. At age 33, she had surgical correction of a deviated nasal
septum because of the nosebleeds, but afterward continued to experience
mild nose and gum bleeding. At age 48, she was found to have a
prolonged aPTT during a preoperative evaluation for osteoarthritis of
the left hip. The patient had a PT of 12.4 seconds and prolonged aPTT
(factor XI:C = 2%). Factor XII, fibrinogen, and platelet
counts are in the normal range; there was no evidence of an inhibitor.
The patient underwent successful total hip replacement after
replacement with fresh frozen plasma to normalize the aPTT and
experienced no complications.
Patient C2 is a 49-year-old man with a personal history of episodic
bleeding but no family history of hemorrhage. At age 8, he had a
tonsillectomy that required an extra day of hospitalization due to
excessive bleeding. At age 10, he experienced 2 days of oozing after a
dental extraction. A fracture of the clavicle at age 25 was not
accompanied by significant bleeding. After his 38th birthday, he was
evaluated for a possible bleeding disorder, which yielded a PT of 11.1 seconds, a prolonged aPTT (factor XI:C = 10%), a bleeding time of 4.5 minutes, and levels of factors VIII, IX, and XII in the normal range
(90% to 103%, with no evidence of an inhibitor). At the time of the
experiment, patient C2 also presented with a platelet count
(145,000/µL) slightly below the normal range (172,000 to
376,000/µL); this platelet count was observed for this individual on
two separate occasions.
Preparation of corn trypsin inhibitor.
For repeat experiments with the second hemophilia A patient, corn
trypsin inhibitor was prepared according to the procedure of Hojima et
al,40 with a few minor modifications. Dry popcorn seed was
obtained from a local grocery and was repeatedly extracted until no
further prolongation of the aPTT in normal plasma was observed (see
below). Acetone precipitation was performed as described,40 and the resuspended material was applied to a column (2.5 × 60 cm) of DEAE-Sepharose. The major peak of inhibitory
activity was pooled and applied to a second column (2.5 × 94 cm)
of Sephadex G-50. This final pooled fraction was dialyzed (3,000 molecular weight cutoff) versus 10 mmol/L ammonium bicarbonate, pH 7.8, and then lyophilized to dryness. The isolated protein exhibited a
single band at 14,000 molecular weight and was reconstituted in
HBS (HEPES-buffered saline; 20 mmol/L HEPES, 150 mmol/L
NaCl, pH 7.4). No attempt was made to further purify the isoforms of this inhibitor, all of which exhibit similar inhibitory potential with
factor XIIa.40
Preparation of TF/lipid reagent.
TF (5 nmol/L) was relipidated into small unilammelar
vesicles41 of 25 mol% PS/75 mol% PC (10 µmol/L total
lipid) in HBS plus calcium (2 mmol/L) for 30 minutes at
37°C.8 Concentrated sucrose (60% wt/vol) was
subsequently added to the relipidation mixture to 10% final to
stabilize the vesicles for long-term freezer storage (up to 12 months).
Aliquots of the reagent (200 µL) were stored at 20°C,
which could be rehydrated 60 minutes before each experiment and used
with reproducible results.
Factor XI preparations.
Using a factor Xa amplification assay, other
investigators32,33 have shown that traces of contaminating
factor XIa in factor XI preparations (picomoles down to femtomoles) can
significantly affect levels of the intrinsic tenase, leading to
artificially high levels of factor Xa generation. Factor XI
preparations were routinely treated to remove traces of factor XIa.
Concentrated factor XI stocks (~300 to 350 U/mg, ~3 to 5 mg/mL)
were loaded into a Slide-A-Lyzer dialysis cassette (molecular weight
cutoff 10,000; Pierce Chemical, Rockford, IL) and were treated
initially with FPRck (10 µmol/L for 30 minutes at 25°C) in HBS.
After dialysis versus 4 L HBS (2 changes for 2 hours each at 4°C),
an additional treatment was performed with DFP (2 mmol/L) for a
15-minute interval. Final dialysis was performed versus 4 L HBS (2 changes for 2 hours each at 4°C), and the factor XI stock was
aliquotted into capped nonstick microcentrifuge tubes (VWR Scientific,
West Chester, PA), quick-frozen inside the capped tubes by immersion in
a dry ice/methanol slurry, and stored at 70°C. aPTT clotting
assays indicated that the specific activity of factor XI was unaffected by these treatments. After FPRck and DFP treatments, analysis of
residual factor XIa activity was performed using an
aminonaphthalenesulfonamide derivative12,42 of the
tripeptide D-Leu-L-Pro-L-Arg, with kcat/Km = 7.10 × 105 mol/L 1
s1. In factor XI preparations (typically 200 nmol/L), factor XIa concentrations were below the limits of the assay
(<100 fmol/L factor XIa activity). Therefore, at plasma
concentrations of factor XI (25 to 30 nmol/L), potential contamination
by factor XIa was less than 12.5 fmol/L. No clotting (>20 minutes)
was observed in factor XI-deficient blood when factor XI and corn
trypsin inhibitor were added in the absence of TF. However, as a final
precaution for the replacement experiments using a very low TF
concentration (5 pmol/L), human 1-protease inhibitor
(0.5 mg/mL, 9.6 µmol/L) was added to the factor XI preparations (1.5 mg/mL, 9.4 µmol/L).43 The 1-protease
inhibitor added to the blood with these factor XI preparations was
insignificant (22 nmol/L) relative to the level plasma inhibitor
already present (~47 µmol/L).
Assay for TF dependence in factor XI-deficient plasma.
Relipidated TF reagent (see above) was rehydrated (200 µL) and
diluted with HBS/calcium (2 mmol/L) to obtain a stock reagent containing 552 pmol/L TF and 1.10 µmol/L PCPS. This initial stock was
further diluted with PCPS (1.10 µmol/L) in HBS/calcium (2 mmol/L) to
obtain a set of working TF stock concentrations ranging from 17.25 to
552 pmol/L TF at constant lipid concentration. The assay was performed
according to the following protocol. Into a polystyrene tube (12 × 75 mm) was added 200 µL human plasma (XI-deficient, <1%
XI:C; or XI-deficient with factor XI replaced at 3.5 µg/mL, 100 U/dL)
and 10 µL corn trypsin inhibitor (1.15 mg/mL in HBS). After 30 seconds of equilibration at 37°C, 10 µL of working TF stock was
added, followed immediately by the addition of 10 µL of 390 mmol/L
CaCl2 in water. Upon addition of the calcium, a timer was
started and the tube was rocked in the 37°C water bath until
strands of fibrin or a solid clot could be identified, at which point
the time was noted.
PT and aPTT assays.
Assays were performed in plastic tubes (VWR Scientific, no. 60818-270)
on 100-µL samples of fresh frozen, citrated human plasma using the
manual tilt-tube approach described by the manufacturer of the PT or
aPTT reagent. When testing the effect of buffer or inhibitor, no more
than 10 µL of the additive was mixed with 90 µL of plasma to
minimize dilution errors.
Coagulation in whole blood.
The protocol used is a modification of the protocol of Rand et
al34 performed under the supervision of one of the authors (R.F.B.) at the Clinical Research Center, Fletcher Allen Health Care
(Burlington, VT). Clotting in freshly drawn, nonanticoagulated whole
blood was performed in 32 capped polystyrene culture tubes as
described, except that two series were performed per experiment (16 tubes/series). Reagents were loaded in the following amounts: corn
trypsin inhibitor (all tubes, to give 50 µg/mL blood); relipidated TF
(lipid:protein = 2,000) in HBS with 5 mmol/L calcium (all tubes in each
series except phlebotomy control tube, to give 25 or 5 pmol/L TF/mL
blood); factor VIII or factor XI (all tubes, replacement series only,
to give 1 U/mL final); and equivalent volume factor VIII/XI dilution
buffer (HBS, pH 7.4, all tubes, deficiency series only). No more than
45 µL of reagent was loaded in each tube. The zero tube of each
series was pretreated using 1 mL of inhibitor cocktail (containing 50 mmol/L EDTA and 20 mmol/L benzamidine-HCl in HBS, pH 7.4) and 10 µL
of 10 mmol/L FPRck (diluted in 0.01 mol/L HCl).
Patient or normal donor blood was drawn by venipuncture under a
protocol approved by the Human Studies Committee at the University of
Vermont, as described.34 Clotting was initiated by delivery into the reagent-loaded tubes and with periodic quenching of the tubes
with inhibitor cocktail and FPRck as described above. Two series of
quenched samples were obtained after reaction progress up to 20 minutes
after initiation; both reducing (1%  -mercaptoethanol) and
nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) samples were prepared from each tube (60 µL supernatant, 190 µL 2% SDS-PAGE sample solution,34 heated exactly 5 minutes at 98°C ± 2°C). An aliquot from each tube was
filtered to remove cellular contaminants for osteonectin assays (200 µL; 0.2-µm AcroDisc; Gelman Sciences, Ann Arbor, MI). The remaining
serum and cell pellets/clots were aliquotted to screw cap tubes,
frozen, and stored at 20°C for immunoblot or immunoassay
analysis.
Immunoassays and Western analysis.
Commercial ELISAs for fibrinopeptide A (FPA), thrombin-AT-III (TAT),
and platelet -granule release (osteonectin) were performed according
to manufacturers protocols, with corrections for sample dilution by
added quench solution (1.00 mL) and hematocrit (typically 40% of the
total blood volume). Results were analyzed on a Vmax microtiter plate
reader (Molecular Devices, Menlo Park, CA) equipped with SOFTMax ver.
2.0 software and an IBM Personal System 2 Model 30/286 PC (IBM Corp,
Armonk, NY). In each assay, a minimum of 5 standard
concentrations were run in duplicate, with duplicate sample
determinations. The relationship between concentration of standard and
optical density was established by fitting the data to either a
four-parameter or log-logit fit, as described by the manufacturer
(Molecular Devices). For analysis of factor Va, samples were separated
on SDS-PAGE according to Laemmli44 as modified by our
laboratory.34 Separate gels were run for heavy-chain and
light-chain analysis. Each gel containing samples was loaded along with
a prestained molecular weight standard mixture (14 to 200 kD) and
dilute standards (3 to 4 samples) allowing comparison and quantitation
of analyte amounts horizontally on the immunoblots. Transfer from the
gel to nitrocellulose (BioRad, Hercules, CA) was performed for 1.5 to 3 hours via an SDS-free tank transfer procedure as
described,45,46 with subsequent immunoblot analysis
according to Rand et al.34
Transmittance scans of immunoblot images on Reflection film (product # NEF-496; NEN Life Science Products, Boston, MA) were performed on a
Hewlett-Packard ScanJet 4C/T equipped with a transparency adapter for
backlighting the x-ray film (Hewlett-Packard, Palo Alto,
CA). Analysis of the .TIFF files was performed on a Power Macintosh 9500/200 computer (Apple Computers, Cupertino,
CA) using the public domain NIH Image program (v. 1.60, Spring 1994, developed at the US National Institutes of Health and
available from the internet by anonymous FTP from zippy.nimh.nih.gov or
on floppy disk from the National Technical Information Service,
Springfield, VA, part no. PB95-500195GEI). The recommendations of the
software developers were followed to minimize nonlinearity of scanned
data with respect to sample concentration (see addendum to the NIH Image manual, "Using Image for Densitometric Analysis of 1-D
gels"). Concentrations were estimated by comparison of sample band
density with a minimum of four serial dilutions of purified standard
proteins loaded on the same gel (internal standard method). Standard
curves were obtained from plots of the logarithm of the standard
concentration versus scanned density, which are typically linear. From
fits of these plots, a relationship between scanned density and
concentration is obtained, allowing conversion of sample density to
concentration. From these values, relative concentrations were
determined by normalizing the data relative to the maximum. Limits of
detectability are also estimated from the standard curve, typically 0.9 nmol/L or lower for factor VaHC and 0.3 nmol/L or lower for
factor VaLC.
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RESULTS |
Blood coagulation in hemophilia A.
Coagulation in blood from a patient with severe hemophilia A (patient
A1, <0.5% VIII:C) was compared with coagulation in blood from normal
donors after initiation with 25 pmol/L TF
(Fig 1). Depicted in Fig 1A are the time
courses for TAT generation in normal and factor VIII-deficient blood,
with and without factor VIII replacement. The normal profile ( ) is
constructed from averaged data (±SEM) from a series of 14 experiments conducted over a period of 18 months with 4 normal
subjects. Little TAT is detected throughout the initiation phase;
subsequently, the bulk of the TAT produced is generated explosively (55 nmol/L/min) during the propagation phase after clot time (4.0 ± 0.2 minutes, arrow a). In factor VIII-deficient blood ( ), clot time
occurred at 6.5 minutes (arrow c), representing an increase in
the length of the initiation phase of 60% over the normal case. The
explosive thrombin generation ordinarily observed in normal blood after
clot time is greatly depressed (maximum rate, 1.9 nmol/L/min; 4% of
normal). Replacement with recombinant factor VIII ( ; 1 U/mL)
shortened the clot time (4.1 minutes, arrow b) and increased TAT
formation to 46 nmol/L/min between 5 and 16 minutes.
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| Fig 1.
Coagulation in normal and hemophilia A blood, with and
without replacement. Using 25 pmol/L TF, coagulation was initiated in
normal and hemophilia A blood (see the Materials and Methods and Rand
et al34) with and without recombinantfactor VIII
replacement (1 U/mL blood). Smooth curves have been drawn by hand
through the points to approximate the data. (A) Time courses for TAT in normal blood (), hemophilia A blood (patient A1; ), and
hemophilia A blood with recombinant factor VIII (). Each point on
the normal curve represents an average TAT from 14 experiments on four
normal subjects (with error bars, SEM). Average clot time for the
composite normal curve is 4.0 ± 0.2 minutes (arrow a). Clotting in
factor VIII-deficient blood occurred at 6.5 minutes (arrow c), which was shortened to 4.1 minutes with factor VIII replacement (arrow b); a
control tube without TF in the factor VIII-deficient experiment did not
clot (>20.6 minutes), and the replacement control clotted at 17.8 minutes. (B) FPA generation in normal blood, factor VIII-deficient blood, and factor VIII-deficient blood with replacement (symbols as in
[A]). The normal curve represents the averaged results from nine
experiments performed on two individuals, with error bars (SEM). The
average clot time for the normal profile was 4.1 ± 0.2 (SEM) minutes
(arrow a), with the other clot times as in (A). (C) Osteonectin release
was measured to examine platelet activation (symbols as in [A]). The
normal curve is constructed from blood taken from a single normal donor
(clot time = 4.1 minutes, arrow a) drawn contemporaneously with the
factor VIII-deficient patient. Other clot times and symbols are as in
(A). (D) After analysis of factor V activation by immunoblotting,
profiles were constructed by densitometric analysis as in the Materials
and Methods. Time courses are given for formation of the heavy ( ,
) and light chains (, ) of factor Va (factor VaHC
and VaLC), with factor VIII replacement (solid symbols) and
without (open symbols). For clarity, the normal profile is omitted, but
is similar to the profile with factor VIII replacement. Clot times are
4.1 minutes (hemophilia A with factor VIII replacement, arrow a) and 6.5 minutes (hemophilia A, arrow b).
|
|
Progress curves for FPA release in these experiments are given in Fig
1B. The normal profile is the average of a series of nine experiments
on two individuals. FPA is liberated between 4 and 6 minutes at a
maximum rate of 5.2 µmol/L/min, with about 4.8 µmol/L (30%
maximum) observed at clot time (arrow a). In factor VIII deficiency,
the maximum FPA release rate is 1.6 µmol/L/min (30% of normal),
reflecting fibrin formation at a slower rate than normal over the
course of the experiment (20 minutes). The extent of FPA release at
clot time in the hemophilic case (arrow c) is the same as in the normal
profile (~30% maximum in both cases). Replacement of factor VIII
increases the maximum FPA rate to 6.4 µmol/L/min, exceeding the
normal rate by 23%. At clot time the extent of FPA release is 35%
(7.4 µmol/L), similar to the estimates from normal and factor
VIII-deficient blood.
Profiles for osteonectin release as a measure of platelet
activation47,48 are given in Fig 1C. In the blood of a
normal individual (contemporaneous control), osteonectin release is
approximately 50% by clot time (arrow a) and is complete by 5 minutes.
In factor VIII-deficient blood, the progress curve is slightly delayed
versus normal. Complete osteonectin release is observed by 6 minutes, reaching maximum levels before clot time (6.5 minutes, arrow c). A
curve similar to the control was obtained when factor VIII was replaced
in the deficient blood. Taken together, the similarity of these
profiles indicates that platelet activation at 25 pmol/L TF is only
slightly affected by the absence of factor VIII.
Time courses for factor VaHC generation in hemophilia A
blood are shown in Fig 1D with and without factor VIII replacement. When factor VIII is present, significant generation of factor VaHC begins at 3 minutes and is complete by 6 minutes
(). In the absence of factor VIII (), generation of factor
VaHC is slowed and does not reach a maximum until 10 to 12 minutes. At clot time in each experiment, approximately 50% to 55% of
the heavy chain is generated. When factor VIII is present, formation of
the light chain (LC; , Fig 1D) is first detected at clot time (4.1 minutes, arrow a) and is complete at 6 minutes. In contrast, factor
VaLC generation is dramatically delayed in factor
VIII-deficient blood (), with traces observed at 8 minutes and rapid
generation after 9 minutes. Thus, LC production, the limiting step in
expression of factor Va cofactor activity,34,49 is
significantly delayed in the absence of factor VIII.
Impaired factor V activation may contribute to the limited prothrombin
conversion observed in hemophiliacs. Earlier studies34 in
normal blood have shown that, during the propagation phase of thrombin
generation, prothrombinase concentration is estimated at 7 pmol/L at
clot time, increasing rapidly to a maximum of 150 pmol/L 3 minutes
later. As a result, factor Xa was proposed to be the limiting component
of prothrombinase in normal blood, because factor Va heavy chain levels
and platelet activation could be demonstrated at concentrations well in
excess of 150 pmol/L.34 Calculations based on TAT data in
the present study for the normal case indicate approximately 35 pmol/L
prothrombinase at clot time from TAT measurements, which increases to
106 pmol/L before the reaction levels off (12 minutes into the
reaction). In hemophilic blood with factor VIII replacement,
prothrombinase estimates similar to the normal case were calculated
from the TAT data (19 pmol/L at clot time, 136 pmol/L at maximum).
Similar to the case in normal blood,34 the heavy chain of
factor Va is present in excess of these concentrations (50% to 55% of
maximum at clot time, ~12 to 13 nmol/L). Light chain is not
significantly converted until after clot time, but is rapidly converted
once thrombin generation reaches explosive levels (quantitative by 3 minutes after clot time).
However, the present studies in factor VIII-deficient blood suggest
approximately 1 pmol/L prothrombinase at clot time, which did not
exceed 6 pmol/L during the experiment. Previous model studies have
shown that factor Xa generation is impaired when either factor VIII or
factor IX is present at an abnormally low levels8,9,50;
therefore, the limited prothrombinase activity inferred for this
patient probably results in part from limited factor Xa generation. Whereas factor Va heavy chain was observed at nearly 75% by clot time,
light chain was virtually undetectable until after clot time,
increasing only slowly thereafter. Therefore, the low levels of
prothrombinase observed in hemophilia A blood throughout the course of
the experiment are likely the result of reduced factor Xa generation in
the absence of factor VIII, with a possible contribution from the
limited levels of fully activated factor Va that are only slowly
generated within the clot. The slowed formation of fully activated
factor Va suggests an additional contribution to the impaired thrombin
generation, because free factor Xa alone is virtually ineffective in
converting prothrombin.3,4 Furthermore, free factor Xa
lacks the relative protection against inactivation by TFPI and AT-III
that fully formed factor Va provides in the prothrombinase
complex.51,52
We have confirmed these results in whole blood from a second severe
hemophilia A patient (patient A2, data not shown). Under conditions
that induced normal blood clotting at 5.2 minutes (~12.5 pmol/L TF),
clotting in hemophilia A blood was observed at 9.8 minutes. Explosive
thrombin generation was absent, as evidenced by low levels of TAT
detected throughout the course of the experiment. Replacement with
natural human factor VIII (Hemofil M, 1 U/mL blood) restored clotting
to 5.8 minutes, as well as explosive TAT generation. As a result of the
reduced thrombin levels, final levels of FPA generated were lower in
the factor VIII-deficient case (~15 µmol/L) than with replacement
(nearly 20 µmol/L). Platelet activation was delayed approximately 3 minutes without factor VIII replacement, but nevertheless reached
maximal levels by the end of each experiment, with identical
fluid-phase osteonectin within 2 to 3 minutes after clot time.
Therefore, reduced thrombin generation in hemophilia A blood was
reflected in a lower final level of fibrin formation, whereas platelet
activation was delayed but not significantly reduced.
Clotting in factor XI-deficient plasma as a function of TF.
von dem Borne et al33 showed an effect of factor XI on
fibrin formation in plasma at very low thromboplastin concentrations. Using decreasing concentrations of relipidated TF, a similar dependence on factor XI was demonstrated for clotting in factor XI-deficient plasma, measured with suppression of contact activation in the presence
of corn trypsin inhibitor (Fig 2). Clot
times with () and without () factor XI lie along curves that
intersect near 24 pmol/L TF, but diverge at concentrations of TF below
24 pmol/L. Clotting at 24 pmol/L is 28 seconds slower without factor
XI, whereas by 6 pmol/L TF, the difference reaches 5.7 minutes. Based on these observations, blood coagulation in hemophilia C was
investigated at 25 and 5 pmol/L.
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| Fig 2.
Clotting in factor XI-deficient plasma as a function of
TF and factor XI. Clot time in factor XI-deficient plasma (<1%) was measured as a function of TF and factor XI (as described in the Materials and Methods). The ordinate is shown in seconds (left-hand axis) and minutes (right-hand axis). Two curves are presented: one for
factor XI-deficient plasma without factor XI replacement (<1%, )
and a second curve for the same plasma with 1 U/mL factor XI (25 nmol/L, ). Smooth curves following each set of data were drawn,
meeting near 24 pmol/L TF. The curves become increasingly divergent as
TF concentration is reduced to 6 pmol/L TF, where the difference is
most pronounced.
|
|
Coagulation in factor XI-deficient and normal whole blood at 25 pmol/L TF.
Table 1 reports the clot times for
coagulation in factor XI-deficient blood, deficient blood with
replacement, and normal whole blood initiated at 25 and 5 pmol/L TF
with suppression of contact activation by corn trypsin inhibitor. At 25 pmol/L TF, at which concentration deficiency of factor VIII results in
impaired clot formation and suppression of the propagation phase of
thrombin generation, factor XI deficiency has a negligible effect. Clot time is 3.5 minutes (contemporaneous normal control = 3.3 minutes) and
is not shortened by factor XI replacement (3.7 minutes). In the control
tubes (corn trypsin inhibitor present, no TF added), clotting is
significantly prolonged or nonexistent, indicating that other sources
of initiation contribute negligibly in these experiments. At 25 pmol/L
TF, thrombin generation profiles for the normal and factor XI-deficient
(patient C1) donors are almost identical, whereas the profile for
factor XI replacement exhibits somewhat faster thrombin generation
(Fig 3A). The maximum rates of thrombin
generation are nearly identical in the normal (61 nmol/L/min) and
factor XI-deficient (63 nmol/L/min) experiments, whereas factor XI
replacement increased the rate of thrombin generation to 85 nmol/L/min.
This increase leads to a 65% higher concentration of final TAT in the
replacement case (950 nmol/L) than in the deficient case (575 nmol/L).
These results indicate that factor XI is not required for explosive
thrombin generation in blood initiated with 25 pmol/L, but modestly
influenced the rate of thrombin generation after clot formation in this
individual.
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| Fig 3.
Coagulation in normal and hemophilia C blood at 25 pmol/L
initiator, with and without replacement. Using 25 pmol/L TF,
coagulation was initiated in normal and hemophilia C blood (see the
Materials and Methods; Rand et al34), with and without
factor XI replacement (1 U/mL blood). Time courses for TAT are provided
(A) after immunoassay analysis of quenched samples from the normal
blood (), hemophilia C blood (patient C1, ), and hemophilia C
blood with factor XI replacement (). Clot times for the experiments
and control tubes are as given in Table 1 and are denoted by arrows for
blood from normal (arrow a), hemophilia C (arrow c), and hemophilia C
donors with factor XI replacement (arrow b, 1 U/mL). In addition to
TAT, FPA (B) and platelet osteonectin (C) profiles are provided.
(Symbols and clot times as in [A].)
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|
Figure 3B shows the FPA profiles obtained at 25 pmol/L TF. For each
case, the reaction progress occurs over similar time scales and to
similar extents. In factor XI-deficient blood, FPA is released at a
maximum rate of 6.1 µmol/L/min (5.6 µmol/L/min in normal blood),
whereas replacement of factor XI increased this rate to 7.3 µmol/L/min. Fibrinogen conversion at clot time was between 30% and
41% in all cases. In addition, osteonectin release profiles (Fig 3C)
show that platelet activation is not strongly influenced by the
presence or absence of factor XI when the reaction is initiated with 25 pmol/L TF. In all cases, the profiles were similar and exhibited
maximal release by 5 minutes. Evaluation of factor Va generation in
normal and factor XI-deficient blood showed identical activation
profiles. Densitometric profiles of the heavy chain (Fig 4A) show that factor VaHC
is detectable within 1 minute of initiation in all reactions, and by
clot time approximately 33% to 50% is observed in each profile.
Likewise, the profiles for light chain formation in the factor
XI-deficient, replacement, and normal experiments are similar to each
other (Fig 4B). Levels of factor VaLC are below the limits
of detection throughout the initiation phase and only a small fraction
is generated at clot time. After clot time, light chain is generated
quantitatively within 1 to 2 minutes (4 and 5 minutes postinitiation).
Together, the factor VaHC and VaLC profiles
demonstrate that cofactor activation is unaffected by factor XI.
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| Fig 4.
Factor Va generation during coagulation in normal and
hemophilia C blood at 25 pmol/L initiator, with and without
replacement. For the experiments described in Fig 3, analysis of factor
V activation was performed by immunoblotting. Profiles following factor
Va heavy chain (A) and light chain (B) were constructed by
densitometric analysis as in the Materials and Methods. Time courses
are given for formation of the heavy and light chains in normal blood
() and hemophilia C blood (patient C1), with () and without ( ) factor XI replacement. Clot times are as in Table 1 and Fig 3, and
curves have been drawn through the points by hand.
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|
Coagulation in factor XI-deficient and normal blood at 5 pmol/L TF.
Coagulation in normal and factor XI-deficient blood (patient C2) after
initiation at 5 pmol/L TF are summarized in Table 1 and
Fig 5. Confirmation of the data has been
obtained in a separate experiment with a third factor XI-deficient
individual (data not shown). Clotting in factor XI-deficient blood at 5 pmol/L initiator (15.7 minutes, Table 1) was delayed 4.6 minutes
relative to normal (11.1 minutes, contemporaneous donor). Factor XI
replacement shortened the clot time in hemophilia C blood by 6 minutes
(9.7 minutes), in agreement with the predictions of the plasma assay
(Fig 2). In the absence of TF (corn trypsin inhibitor only), the
controls for hemophilia C did not clot with or without factor XI
replacement (over 20.5 minutes), confirming that factor XIa
contamination was not significant in the factor XI preparations.
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| Fig 5.
Coagulation in normal and hemophilia C blood at 5 pmol/L
initiator, with and without replacement. Using 5 pmol/L TF, coagulation was initiated in normal and hemophilia C blood (see the Materials and
Methods; Rand et al34), with and without factor XI
replacement (1 U/mL blood). Time courses for TAT are provided (A) after
immunoassay analysis of quenched samples from the normal blood (),
hemophilia C blood (patient C2, ), and hemophilia C blood with
factor XI replacement (). Clot times for the experiments and control
tubes are as given in Table 1 and are denoted by arrows for blood from normal (arrow a), hemophilia C (arrow c), and hemophilia C donors with
factor XI replacement (arrow b, 1 U/mL). In addition to TAT, FPA (B)
and platelet osteonectin (C) profiles are provided. (Symbols and clot
times as in [A].) Clot times are as in Table 1 and (A), and curves
have been drawn through the points by hand.
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|
In the TAT profiles of Fig 5A, the bulk of the thrombin is formed after
clot time, and factor XI replacement increases the rate of thrombin
generation during the propagation phase in hemophilia C blood. In
normal blood (Fig 5A), TAT is generated at 110 nmol/L/min after clot
time (arrow b), compared with approximately 37 nmol/L/min after clot
time (arrow c) in factor XI-deficient blood. Factor XI replacement
increases the TAT rate to 119 nmol/L/min at clot time (arrow a). The
result is that final levels of TAT are higher in the normal and
replacement experiments (750 and 600 nmol/L), but only reached
approximately 150 nmol/L when the factor XI-deficient experiment was
terminated. But even at 5 pmol/L TF, thrombin production in hemophilia
C blood is in excess of levels observed in hemophilia A blood at 25 pmol/L, consistent with the relative clinical severity of the two
congenital diseases.
Figure 5B displays profiles for the release
of fibrinopeptide A, which also becomes dependent on factor XI at 5 pmol/L TF. FPA release occurs more slowly in hemophilia C blood at 5 pmol/L initiator (maximum rate, 2.7 µmol/L/min) than in normal blood (15.5 µmol/L/min) and is incomplete by the end of the experimental period. Replacement of factor XI increases FPA formation to 7.4 µmol/L/min and provides complete fibrinogen conversion within 2 to
2.5 minutes of clot time.
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| Fig 6.
Factor Va generation during coagulation in normal and
hemophilia C blood at 5 pmol/L initiator, with and without replacement. For the experiments described in Fig 5, analysis of factor V activation was performed by immunoblotting. Profiles following factor Va heavy
chain (A) and light chain (B) were constructed by densitometric analysis as in the Materials and Methods. Time courses are given for
formation of the heavy and light chains in normal blood () and
hemophilia C blood (patient C2), with () and without () factor XI
replacement. Clot times are as in Table 1 and Fig 5, and curves have
been drawn through the points by hand.
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|
Platelet activation in blood initiated at 5 pmol/L TF was estimated by
osteonectin release (Fig 5C) and contrasted starkly with the data at
higher levels of initiator (Fig 3C). In factor XI-deficient blood,
osteonectin release is very slow during the initiation phase, reaching
approximately 65% by clot time (arrow c). During this period,
aggregates were noted as grainy accumulations at the walls of the
reaction tubes. With factor XI replacement, osteonectin again increases
slowly over the initiation phase, reaching 67% by clot time (arrow a);
the remaining osteonectin is released within 2 minutes. This was
similar to the normal profile in which 42% fluid phase osteonectin is
detected at clot time (arrow b) followed by rapid and immediate release
of the remaining protein. In general, at 5 pmol/L TF, platelet
activation is slower and less complete at clot time (42% to 67%),
associated with limited thrombin generation during the initiation
phase. After clot formation, the observed thrombin burst activates the
platelets rapidly, ensuring maximal activation. Thus, unlike
hemophilias A and C at 25 pmol/L TF, platelet activation at 5 pmol/L TF
is significantly influenced by the absence of factor XI.
At 5 pmol/L TF, the generation of factor VaHC in hemophilia
C blood, like FPA release and platelet activation, is slower than normal (Fig 6A). The bulk of the heavy chain does not appear until 20 minutes (), whereas in the replacement () and normal () profiles the maximum occurs after clot time in each case
near 12 and 15 minutes, respectively. Only small amounts
of factor VaHC appear before clot formation in these
experiments. In contrast, factor VaLC (Fig 6B) is
undetectable until after clot time in each of the three experiments and
again appears to be the limiting step in cofactor activation. Thus,
factor V activation is affected by the presence or absence of factor
XI, with the bulk of factor V activation occurring after clot time in
each case.
| |
DISCUSSION |
Previously, our laboratory had described coagulation in normal blood
after initiation by TF under conditions in which contact activation was
suppressed by corn trypsin inhibitor.34 In those studies,
clotting was observed at the end of an initiation phase in which
limited amounts of thrombin (~15 nmol/L) had been produced. Immediately after clot formation, a period of explosive thrombin generation was observed (ie, the propagation phase) in which
substantial thrombin (up to 360 nmol/L total) was produced within 8 minutes. Using this approach, we investigated coagulation in hemophilia A and hemophilia C blood. In severe hemophilia A at 25 pmol/L TF,
clotting was delayed versus normal (~2.4 minutes), evidence of
modestly reduced levels of thrombin during the initiation phase leading
to clot formation. A more striking observation was the severely
depressed thrombin generation during the propagation phase (ie, after
the clot had been detected), measuring less than 4% of the normal
rate. Consequently, this impaired thrombin generation was reflected in
a reduced rate of fibrinogen cleavage and drastically delayed factor Va
generation. Replacement of factor VIII restored normal clotting and
thrombin generation in the propagation phase after clotting. These
results support earlier observations of reduced prothrombinase activity
in the absence of functional intrinsic tenase.6,8,21,22,51
In addition, our study in whole blood shows that reduced thrombin
generation in the absence of factor VIII also leads to severely reduced
factor Va generation, which further reduces the effectiveness of the
limited factor Xa produced during coagulation in hemophilia A.
Although thrombin generation, fibrinogen cleavage, and platelet
activation were retarded during coagulation in hemophilia A blood, we
found only a relatively slight delay in platelet activation versus
normal (~1 minute). In a model system containing zymogens, cofactors,
and platelets, Hoffman et al50 observed that factor IXa is
not an effective initiator of platelet activation. Our results at 25 pmol/L TF parallel theirs, showing that the progress of platelet
release is virtually unaffected in factor VIII deficiency, with 100%
activation observed before clot time. Thus, platelet activation is
largely independent of the intrinsic tenase. Together, the observations
of complete platelet activation and slow fibrinogen formation correlate
with the description of clotting in the bleeding time wounds of
hemophiliacs,53,54 in which the primary platelet plug is
devoid of normal fibrin stabilization, leading to a friable clot that
is subject to rupture.
The effect of factor XI deficiency on fibrin formation in plasma
coagulation has recently been shown to depend on the level of TF
used.33 We have now extended these observations to include observations on thrombin, factor Va, and platelets in hemophilia C
blood. In contrast with the results for hemophilia A at 25 pmol/L TF,
coagulation was hardly affected in hemophilia C blood. Clotting was
identical in hemophilia C and normal experiments, as was explosive thrombin generation in the propagation phase. Replacement of factor XI
modestly increased thrombin generation after clot time, but all other
products of the reaction (factor V activation, osteonectin release from
activated platelets, and FPA) were unaffected by the presence or
absence of factor XI. Reducing the initiator concentration to 5 pmol/L
prolongs the initiation phase of hemophilia C blood coagulation by 4.6 minutes versus normal, and factor XI replacement shortens this clot
time by nearly 6 minutes. The results indicate that impaired
coagulation in hemophilia C will occur at lower initiator
concentrations than those observed for impaired coagulation in
hemophilia A. Furthermore, we observed that, in hemophilia C, maximum
thrombin generation rates decrease as TF is reduced from 25 to 5 pmol/L. This agrees with results in a reconstituted model of the TF
pathway without factor XI,21 in which the combination of
TFPI and AT-III decrease the rate of thrombin generation as the
initiating TF concentration was reduced. However, in normal blood as
well as in hemophilia C blood with factor XI replacement, such a
decrease in thrombin generation was not observed. In fact, an increase
was detected: from 61 and 85 nmol/L TAT/min in the normal and
replacement experiments using 25 pmol/L TF, respectively, to 110 and
119 nmol/L TAT/min with 5 pmol/L TF. These observations indicate that
factor XI plays an increasingly significant role in supplementing
prothrombinase levels as the initiator concentration is reduced.
|
Abstract
Introduction
Abstract