Activation of the coagulation cascade after infusion of a factor... concentrate in congenitally deficient patients

Activation of the coagulation cascade after infusion of a factor... concentrate in congenitally deficient patients

From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1994 84: 1314-1319
Activation of the coagulation cascade after infusion of a factor XI
concentrate in congenitally deficient patients
PM Mannucci, KA Bauer, E Santagostino, E Faioni, S Barzegar, R Coppola and RD Rosenberg
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Copyright 2011 by The American Society of Hematology; all rights reserved.
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
Activation of the Coagulation Cascade After Infusion of a Factor
Concentrate in Congenitally Deficient Patients
XI
By Pier Mannuccio Mannucci, Kenneth A. Bauer, Elena Santagostino, Elena Faioni, Samad Barzegar,
Raffaella Coppola, and Robert D. Rosenberg
Virally inactivated, high-purity factor XI concentrates are
available for treatment of patients with factor XI deficiency.
However, preliminary experience indicates that some preparations may be thrombogenic. We evaluated whether a
highly purified concentrate produced signs of activation of
the coagulation cascade in two patients with severe factor
XI deficiency infused before and after surgery.Signs of
heightened enzymatic activity of the common pathway of
coagulation (elevated plasma levels of prothrombin fragment 1+2 and fibrinopeptide A) developed in theearly postinfusion period, accompanied by more delayed signs offibrin
formation with secondary hyperfibrinolysis (elevated D-dimer and plasmin-antiplasmin complex). These changes occurred in both patients, but were more severe in the older
patient with breast cancer when she underwent surgery,
being accompanied by fibrinogen and platelet consumption.
There were no concomitant signs of heightened activity of
the factor VII-tissue factor mechanism on the factor Xase
complex (plasma levels of activated factor VI1 and of factor
IX and X activation peptides did not increase).The observed
changes in biochemical markers of coagulation activation
indicate that concentrate infusions increased thrombin generation and activity and that such changes were magnified
by malignancy and surgery. Because somefactor XI concentrates may bethrombogenic, they should be used with caution, especially in patients with other risk factors for thrombosis.
0 1994 by The American Society of Hematology.
P
heightened factor Xa and thrombin enzymatic activity accompanied by secondary fibrinolysis. In one patient with
breast cancer, the laboratory abnormalities were more severe,
particularly during and after surgery when signs of fibrinogen and platelet consumption developed.
ATIENTS WITH congenital factor XIdeficiency are
usually treated with fresh-frozen plasma, which contains the protein lacking in these patients and necessary for
hemostasis. Large amounts of plasma, suchas those that
may be repeatedly needed after major surgical procedures,
may lead to volume overload. Most importantly, plasma carries a risk of transmitting bloodborne viral infections. To
circumvent these problems, virally inactivated plasma concentrates, purified with chromatographic techniques to contain almost exclusively factor XI, have been developed and
used clinically, particularly during and after surgical procedures.’.* However, a preliminary report has shown that in
two patients concentrate infusions were accompanied by the
occurrence of laboratory signs of intravascular coagulation
with secondary fibrinolysis, expressed as a postinfusion increase in fibrinogen-fibrin degradation products and D-dimer.3 In one patient, who died after coronary artery bypass
surgery, all the grafts were found during emergency thoracotomy to be occluded by thrombi.3
Prompted by this report indicating that some factor XI
concentrates may be thrombogenic, we have monitored the
hemostatic systems of two patients with congenital factor
XI deficiency during treatment with large doses of a factor
XI concentrate.’ Both patients developed laboratory signs of
pathologic activation of coagulation after the infusion, with
From the Angelo Bianchi Bonomi Hemophilia and Thrombosis
Center, Institute of Internal Medicine, IRCCS Maggiore Hospital
and University of Milan, Italy; the Charles A. Dana Research Institute and the Harward-Thorndike Laboratory, Department of Medicine, Beth Israel Hospital and Harvard Medical School, Boston,
MA; and the Department of Biology and Whitaker College, Massachussetts Institute of Technology, Cambridge, MA.
Submitted October 27, 1993; accepted April 22,1994.
Address reprint requests to Pier M. Mannucci, MD, Via Pace 9,
20122 Milano, Italy.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-497//94/8404-0037$3.00/0
1314
PATIENTS AND METHODS
Patients. Patient no. I was a 19-year-old womanwith severe
factor XI deficiency ( < l U/dL), expressed clinically as prolonged
bleeding after dental extractions and superficial cuts. She was referred for the surgical removal of a large pilonidal cyst of the anus.
Patient no. 2 was a 69-year-old woman with severe factor XI deficiency (< 1 U/dL), expressed as prolonged bleeding after appendectomy and tooth extractions, which on one occasion required transfusions of whole blood. She was referred with a diagnosis of breast
adenocarcinoma for total mastectomyand axillary lymphadenectomy. The patient was in good clinical condition and there was no
evidence of metastatic disease by computed tomography scans or
bone scintigraphy. Neither of thetwo patients was anti-human immunodeficiency virus seropositive, nor was there clinical or serologic
evidence of previous infection with the hepatitis B and C viruses.
Measurements and methods. Factor XI functional activity was
measured in plasma using the microplate assay described by Scott
and C ~ l m a n In
. ~ brief, plasma factor XI was activated by Kaolin,
in the presence of soybean trypsin inhibitor to inhibit formation of
Kallikrein and in the presence of corn trypsin inhibitor to inhibit
activated factor XII. Cleavage of the chromogenic substrate S-2366
(Chromogenix, Molndal, Sweden) was then evaluated by measuring
absorbance at 405 nm. The between-assay coefficient of variation
of this method was 7%, which is lower than that of factor XI assays
based on the activated partial thromboplastin time (15% to 20%).
Activated factor XIwas measured in the concentrate withan
amidolytic method, using as a reference standard a purified preparation of activated human factor XI (Enzyme Research Laboratories.
South Bend, IN). Aliquots of different dilutions of activated factor
XI were incubated at 37°C with S-2366. Absorbances were read at
405 nm over 10 minutes and plotted against concentrations of activated factor XI on a logarithmic scale. The factor XI concentrate
was treated in the same way, and concentrations of activated factor
XI were read on the standard curve and expressed as a percentage
of factor XI by weight. Measurements were performed in triplicate
and the results were averaged.
Samples of reconstituted concentrate were electrophoresed on SOdium dodecyl sulfate ( l % ) polyacrylamide gel (10%) (SDS-PAGE)
under reducing and nonreducing conditions and protein bands were
Blood, Vol 84, NO 4 (August 15), 1994: pp 1314-1319
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
ACTIVATION
COAGULATION
FACTOR
AFTER
XI CONCENTRATE
shown by silver staining. Purified preparations of antithrombin 111,
factor XI, and activated factor XI (Enzyme Research Laboratories)
were run on the same gel.
The factor IX activation peptide (FMP; the product of the enzymatic action on factor M of factor VIWIIa-tissue factor or of factor
XIa) and the factor X activation peptide (FXP; the product of the
enzymatic action on factor X of factor VIWIIa-tissue factor or of
the factor Ma-VIIIa-activated surface complex) were measured by
double-antibody radioimmunoassay (RIA) in plasma stored at -80°C
until assay and extracted to eliminate nonspecific effects from other
plasma proteins.5,6The plasma half-lives of these moieties are 15
and 30 minutes, respectively?.6 Prothrombin fragment 1+2 (F1+2),
a measure of the enzymatic action of factor Xa on prothrombin with
a plasma half-life of 90 minutes, was measured on stored plasma
by double-antibody RIA.' Fibrinopeptide A (FPA), a measure of the
enzymatic action of thrombin on fibrinogen with a plasma half-life
of 3 to 5 minutes, was measured by enzyme immunoassay (Stago,
Asnieres, France) in plasma extracted twice with bentonite to remove
fibrinogen. The plasma concentrations of FXP and FIXP are given
in picomoles and those of FPA and F1 + 2 are given in nanomoles.
For these methods, venous blood was collected into an anticoagulant
mixture containing aprotinin, a synthetic thrombin inhibitor, and
EDTA (purchased from Byk-Sangtec, Dietsenbach, Germany). Factor VIIa, the two-chain enzymatic form of factor VI1 with a plasma
half-life of 180 minutes, was measured in citrated plasma by a onestage, prothrombin time-based assay using a truncated and soluble
form of recombinant tissue factor that, on relipidation, reacts with
factor VIIa but not with one-chain factor VII.* The normal laboratory
ranges for these measurements (see Table 1) are based on the calculation of log means -C SD.
We also measured fibrinogen, antithrombin 111, and platelets by
previously published standard methods'; D-dimer and plasmin-antiplasmin complex were measured by enzyme immunoassays (Dimertest; Ortho Diagnostic System, Milan, Italy; Behring Institute,
Scoppito, Italy).
Concentrate. The factor XI concentrate, not licensed in Italy,
was provided by the Centre Regional de Transfusion Sanguine (Lille,
France; courtesy of Prof M. Goudemand) as a single lot (30010226)
in vials containing approximately 1 0 0 W/mL factor XI after reconstitution in 10 mL of distilled water. The concentrate was produced
from cryoprecipitate-poor pooled plasma obtained from whole blood
donations or by plasmapheresis of unpaid donors. A detailed description of the manufacturing process has been published by BurnoufRadosevich and Burnouf.' In brief, the process involves filtration on
a negatively charged cellulose filter followed by chromatography on
a cation exchange resin. A solvent-detergent mixture [0.3% tri(-nbutyl) phosphate and 1% Tween 801 was added before chromatography to inactivate lipid-enveloped viruses." The concentrate has a
high specific activity (120 to 160 IU of factor XI per milligram of
protein) and before lyophilization pasteurized antithrombin 111 and
heparin are added, both at final concentrations of 2 U/mL of reconstituted concentrate.' According to the manufacturer, the concentrate
lot used in this study contained no measurable levels of activated
factors XI, M, X, thrombin, and plasmin.' Activated factor XI was
also measured by us and was found to be present in small amounts
(0.003% wt/wt of factor XI). On SDS-PAGE performed under reducing and nonreducing conditions, the only bands shown by silver
staining corresponded in electrophoretic mobility to purified factor
XI and antithrombin I11 run in the same gel, as previously demonstrated by manufacturers.*We detected a small amount of prothrombin F1 +2 in the concentrate (0.87 pmol/L). No thrombogenic activity
was found in a stasis model after infusing rabbits with concentrate
doses of 900 to 1,100 U of factor XI per killogram of body weight.
The only lot used in this study was not thrombogenic after infusion
in rats at a dose of 300 Ukg, in contrast with a prothrombin complex
1315
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From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1316
MANNUCCI ET AL
concentrate, which at the same dosage generated more than 600 ng/
mL of FPA (unpublished data from the manufacturers).
CASE REPORTS
Patientno. 1. The first concentrate infusion (50 U k g )
was administered on an out-patient
basis to evaluaterecovery
and half-life. The total amount of heparin infused with this
dose (50 IU) is not large enough to
affect coagulation per
se.
Table 1 shows that factor XI levels increased to greater
than 100 U/dL in the first postinfusion day, with increments
of factor XI of 1.9 U/dL per U k g administered. Factor XI
declined slowly in the next 10 days (Table l), with a halflife of 61 hours, but8 U/dL couldstill be measuredin plasma
14 days after the infusion (Table 1). A second concentrate
infusion was then administered (50 U k g ) and surgical removal of the cystwas performed under localanesthesia.
At 6 hours postinfusion, factor XI was 138 U/dL, with an
increment of 2.4 U/dL per U k g administered. Although the
patient did not bleed abnormally during or after surgery, a
supplementary dose of factor XI was administered on postoperativeday 4(day 19fromthe first infusion)tokeep
factor XI levels above 50 U/dL (preinfusion levels, 62 U/
dL; postinfusion levels, 120 U/dL). The wound healed uneventfully and thepatient was discharged on the
postoperative
day 7.
Table 1 also shows that, after the first infusion of factor
XI, there was no important change in routine measurements
of hemostasis (platelets,fibrinogen,andantithrombin
111).
However,whenmoresensitivemarkers
were measured,
there were highpostinfusion values of D-dimer from normal
preinfusion levels (41 to 319 ng/mL), plasmin-antiplasmin
complex (258 to 385 ng/mL), and F1+ 2 (0.61 to 2.07 nmol/
L). There were also high postinfusion values of FPA (up to
3.6 nmoVL), althoughno preinfusionvalue had been obtained. The increase of FPA and F1+2 occurredalready
1 hour after the infusion, whereas those of the remaining
measurements were more delayed (at 2 to 6 hours). There
were no changes in FXP, FIXP, and factor VIIa, which remained normal. After the second and
third infusion, we monitored only fibrinogen, antithrombin 111, and platelets, all of
which remained normal (data not shown).
Patient no. 2. This patient received the first concentrate
infusion 12 days before surgery. As in patient no. 1, factor
XI increased to more than 1 0 0 U/dL in the earlypostinfusion
period (with an increase of 2.1 U/dL per U k g infused) and
declined slowly over thenext 12 days, with a half-life of 62
hours. There were slight and delayed decreases in platelet
154 X 109/L) and plasmafibrinogen
count(from209to
(from 355 to 289 mg/dL)4 to
at 6 hours, but not inantithrombin 111 (Table 2). On the postinfusion day 13, when factor
XI levels were still 10 U/dL, the patient received a second
infusion (50 U k g ) and underwent mastectomy and lymphadenectomy. Histologic examination confirmed the presence
of breast adenocarcinoma, with no metastatic involvement
of axillary lymphnodes. With a peak factor XI level of 159
U/dL attained in the early postinfusion period, the patient
did not bleed abnormally at the time of surgery. However,
there were clear signs of consumption coagulopathy during
the day of operation, seen as a progressive decline of fibrinogen from 371 to 143 mg/dL and of platelets from 190 to
130 X 10y/L over the first X postoperative hours, with little
change in antithrombin 111. Although there was some oozing
from the drainage during the postoperative
period,it was
only on postoperative day 6 (day 19 after the first infusion)
that the hematocrit decreasedto 22% and alarge wound
hematoma developed. Because plasma factor XI was 16 U/
dL, with a normal platelet count and fibrinogen levels ( I 88
X IO’/L and 402 mg/dL),bleeding was thought to bemainly
caused by low factor XI levels rather than by consumption
of
coagulopathy, so thatpatientreceivedathirdinfusion
factor XI concentrate and 2 U of packed red blood cells. In
the first postinfusion hours there were marked decreases in
fibrinogen (to a nadir of 187 mg/dL at 8 hours) and platelets
( 1 12 X 109/Lat 12 hours), with little change in antithrombin
111. In the next few days, there was no further decrease in
hematocrit, the wound hematoma absorbed slowly, and the
patient was discharged 20 days after surgery. At all times,
renal and liver functions (as assessed by measuring plasma
creatinine and liver function tests) remained normal.
The results for the more sensitive molecular markers of
coagulation activation are shown in Table 2. After the first
test infusion, there were clear signsof heightened factor Xa
and thrombin activity (increasing levels of FPA and F1 +2)
and of secondary hyperfibrinolysis (increasing levels of Ddimer and plasmin-antiplasmin complex). FPA and F1 +2
peaked earlier (at 1 hour) than the remaining measurements
(at 4 to 6 hours). The same pattern of changes was seenafter
the second infusion,administered atthetime of surgery,
and after the third infusion, administered at the time of the
hematocrit decrease. At the time of the latter infusion, however,some values were alreadymarkedlyelevatedbefore
infusion (FPA, D-dimer, plasmin-antiplasmin complex, and,
to alesser extent, F1+2). At no time were there any important changesintheplasmalevels
of FIXP and factor
VIIa, which remained normal. There were slightly elevated
values of FXP in some samples, with no clearpattern of
change in relation to infusions and surgery (Table 2).
DISCUSSION
The infusion into two patients with congenital factor X1
deficiency of large doses of factor XI concentrate was followed by the development of signs of heightened activity of
the enzymes of the common pathway of blood coagulation,
accompanied by signs of fibrin formationand secondary
fibrinolysis.
In patient no. 1, heightened enzymatic activity was discrete, developedprogressively throughout theearly postinfusion period, and could only be detected
by sensitive markers
such as F1 +2, FPA, and D-dimer,
with no signs of consumption coagulopathy (normal fibrinogen, antithrombin 111, and
platelets). The patient was young andhealthy, with no underlying condition predisposing to hypercoagulability, and developed the abnormalities after a test concentrate infusion
administered on anout-patientbasis,when
shewas fully
mobile and considerably before the planned surgical procedure. Exogenous F1 +2 in the concentrate cannot explainthe
postinfusion increase of this peptide in plasma, because a
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
COAGULATIONACTIVATION AFTERFACTOR
1317
XICONCENTRATE
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From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1318
small amount of F1+2 wasmeasuredin
theconcentrate
(0.87 pmoVL) and the F1+ 2 peak occurred relatively late in
the postinfusion period (at 2 hours), suggesting endogenous
generation triggered by the concentrate itself.
Patient no. 2 was much older than patient no. I and had
localized breast cancer, two conditions predisposing to hypercoagulability. The elevations of FPA, F1+2, D-dimer,
and plasmin-antiplasmin complex that occurred afterfirst
the
concentrate infusion, administered on an out-patient basis,
were much more marked than those elicited in patient no. 1
by thesameconcentratedoseand
were accompanied by
signs of platelet and fibrinogen consumption comparable to
those seen in patientswith disseminated intravascular coagulation. It is unlikely thatincreases of hypercoagulability
markers were caused by impaired clearance from plasma,
because renal and hepatic functions remained normal. It appears, therefore, that in the patient pre-existing hypercoagulability caused by malignancyinteracted with the concentrate
infusion to increase theseverity of the hemostatic alterations.
This is further illustrated by the occurrence of even more
marked and persistent alterations after the two subsequent
infusions, administeredin connection with major surgery,
ie, another thrombogenic stimulus.
The alterations of the coagulation cascade seen were characterized by the delayed appearance,at 1 to 2 hours postinfusion, of signs of heightened thrombin activity (expressed as
elevated FPA) and heightened factor Xa activity (elevated
F1 +2), with resolution beginning by 4 to 6 hours. There was
not heightened factor X activation, as demonstrated by small
and inconsistent changes of factor X activation peptide. A
similar pattern of ITA and F1+2 increases has previously
been observed in patients with hemophilia B infused with
potentially thrombogenic prothrombin complexconcentrates,butin
themtherewasalso
a delayedincrease of
the factor X activation peptide." There are several possible
mechanisms to explain the hypercoagulable state observed
in ourpatientswithfactorXI
deficiency. Giventhatthe
product infused was a factor XI concentrate,activated factor
XI might be expected to bea trigger for hypercoagulability.
We have directly measured factor XIa
in the concentrate and
found this enzyme to be present in small amounts (0.003%
of the zymogen). These may not be
sufficient to activate
massively factor IX, because the factor
IX activation peptide
(ie,theproduct of the enzymatic action of factor XIa on
factor IX) was normal in
both patients postinfusion. Massive
contamination of the concentrate with activated factor IX is
also unlikely, because this enzymatic activity was unmeasurable according to the concentrate
manufacturer and the postinfusion plasma levels of factor Xactivation peptide (the
product of the enzymatic action of factor IXa on factor X)
measured by us werenormal or only modestly and inconsistently elevated. Because factor VIIa
was also normal, the
mechanisms by which the concentrate caused the activation
of the last phaseof coagulation remainunclear, even though
the assaysused may be too insensitive to exclude that
activation proceeded through the generation of trace quantities of
activated factors IX or X.
Signs of secondary fibrinolysis and thrombotic complications have been observed by Gitel et al' after the infusion of
MANNUCCI ET AL
a factor XI concentrate
produced by a different manufacturer.
Because the method of preparation of that concentrate has
not been reported and its production has been discontinued,
we cannot fully compare the characteristics of the two concentrates. However, weknow that thatconcentrate was much
less pure than the one we used (specific activity, 7.5 v 120
to 160 U of factor XI per milligram of protein) and that it
exhibited proteolytic activity against S-2238, a chromogenic
substrate sensitive to the enzymatic activity of thrombin.'
No such activity could be detected in our concentrate.' The
biochemical characteristics of another factor XIconcentrate,
produced by a British manufacturer, are knownin detail,' but
there is no study of the postinfusion behavior of coagulation
activationmarkers.' The main biochemicaldifference between the British product and our product is that the former
contains a more than 50-fold molar excess of antithrombin
111over factor XI, meant to inactivate
any coagulation serine
protease that may be generated during concentrate manufacture.' Even though the clinical record of the British product
indicates safety in humans,' some batcheselicited significant
thrombogenic responses, similar in magnitude to those seen
after prothrombin complex concentrates, in rabbits infused
with doses 2 to 4 times the highest dose recommended for
humans (50 Ukg).'' Following these observations, the manufacturer has recently adopted the precautionary measure of
adding approximately 10 IU/mL of heparin to the concentrate to accelerate inhibition of coagulation proteases by antithrombin" (theconcentratewe
used contains a smaller
amount of heparin, 2 IU/mL).
On thebasis of theseresults, we recommend
that manufacturersperform furtherbiochemicaland
animal studiesto
identify thrombogenic component(s) that may be present in
some factor XI concentrates, and try
to to eliminateit. Meanwhile, concentrates should be used only when repeated infusions of fresh-frozenplasma might cause significant fluid
overload. Plasma virally inactivated with solvent/detergent''
or paste~rization'~
is now availableand should be considered
as an alternative treatment. When concentrates are needed,
it is advisable to monitorpatients for clinical and laboratory
signs of coagulation activation, particularly patients undergoing surgery or carrying other risk factors for thrombosis.
ACKNOWLEDGMENT
We thank Dr Myriana Burnouf-Radosevich (Lille, France) for
providing information on the concentrate and for stimulating discussions.
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