THROMBOSIS RESEARCH 14; 283-297 @Pergamon Press Ltd.1979. printed
in Great Britain 0049-3848/79/ 0301-0283
902.0,0/o
ABNORMALITIES OF PLATELET AGGREGATION IN SICKLECELLANEMIA PRESENCE OF A PLASMAFACTORINHIBITINGAGGREGATION BY RISTOCETIN K. E. Sarji,
K. Eurenius,
C. 0. Fullwood, H. B. Schraibman, and J. A. Colwell
Veterans Administration Hospital and of Endocrinology-Metabolism-Nutrition and Hematology Department of Medicine Medical University of South Carolina, Charleston, South Carolina USA Divisions
(Received 8.5.1978; in revised Accepted by Editor K.M.
form 18.10.1978. Brinkhous)
ABSTRACT Platelet aggregation in plasma from subjects with sickle cell anemia, not in crisis, was tested using the aggregometer. Aggregation induced by adenosine diphosphate (ADP) , epinephrine, or collagen was decreased in platelet-rich plasma (PRP) from these subjects. Aggregation induced by ristocetin (1.09 mg/ml final cont.) was absent or was preceded by a lag phase in sickle cell PRP but not in normal PRP. Washed platelets from subjects with sickle cell anemia mixed with normal platelet-poor plasma (PPP) showed normal ristocetin-induced Washed normal platelets mixed with sickle cell PPP aggregation. showed progressive inhibition of ristocetin aggregation with increasing aaxnmts of plasma. Dilution of the sickle cell PPP or increasing the final concentration of ristocetin to 2.4 mg/ml resulted in normal aggregation. Factor VIII antigen levels (196+16% S.E.M.) and factor VIII procoagulant activity (286+82%) were elevated in sickle cell plasma. Fibrinogen levels (239+42 mg%) were normal or slightly decreased; no relationship to aggregation was seen. These results suggest that an abnormally high factor VIII antigen/van Willebrand factor activity ratio is present in sickle cell anemia. It is postulated that inhibition of ristocetin-induced platelet aggregation by sickle cell plasma is related to competition for available ristocetin. I NTRODUCI ION The most Cowon clinical anemia is the painful thrombosis tiated
is of early
by sickled
to alterations
complication
thrombotic significance
erythrocytes.
in the hemostatic
occlusive
seen in patients crisis.
or a result
with sickle
It is uncertain of vascular
occlusion
There are conflicting
reports
system in sickle
anemia.
283
cell
cell
whether ini-
with regard Studies on
PLATELETS IN SICKLE CELL ANEMIA
284
patients counts,
in crisis factor
VIII,
(1) and factor including
have indicated
that there is an increase
and fibrinogen
in extent of aggregation
and collagen
not in crisis in crisis
and normal aggregation
gen in precrisis post crisis
or crisis
period,
ADP, epinephrine,
(4) reported
drome has also been described
investigated
but an increased
(3)
(ADP),
a decrease
in patients
aggregation
thrombin,
during the
In contrast, aggregation
with
A von Willebrand
in crisis.
with sickle
cell
data reported,
and factor
not
and colla-
sensitivity
in platelet
in four patients
in patients
in patients
by Haut -et al (10).
In view of the conflicting
platelet
and turnover
with ADP, epinephrine,
periods,
and collagen
hematuria (11).
phase aggregation
with adenosine diphosphate
survival
have been reported
Gruppo and coworkers
aggregation
system have also been reported
Normal platelet
(S-9).
activity
(4).
Changes in the coagulation
patients
in platelet
an impairment of rate and extent of first
epinephrine,
in platelet
and a decrease of fibrinolytic
XIII (2) as well. as abnormalities
and a decrease
Vol.l&,No.2/3
VIII related
trait
syn-
and
we have further activities
in
not in crisis. MATERIALS ANDMETHODS
Plasma samples Asymptomatic patients
with sickle
patients
with well-documented
studies,
normal hemoglobin
patients
have chronic
ly elevated
indicated
evidence
for
routine
counts, of
without
medical
for
and off
cell
anemia were studied. anemia as indicated
and absence
of
of homolysis
increased All
signs
to study. medications
These are
by kindred such
splenomegaly.
as indicated
by consistent-
indirect
bilirubin
patients
were studied
and in no instance
symptoms,
one week prior
disease
levels
GI bleeding.
checkups,
at a time when additional no medication
A2 levels,
constant
reticulocyte
and negative
sickle
cell
were these
or problems Controls were also
concentrations at times
patients
were evident. of
similar
tested.
studied They had
age and sex A two syringe
vo1.14,~0.2/3
with plastic
technique, citrate ture
all
were kept
PPP was stored
platelet-rich
Platelet
assay
within
at -2O’C
later
assay.
for
ADP (1.0
factor
4 hours
of
Platelet
3.8%
at room tempera-
10 minutes
for
into
at 4°C for
VIII
procoaguAll
collection. counts
were done on
(12).
was performed
For studies
nM) (Sigma),
acid
(Applied
using
using
epinephrine Science
gation
(1.0
ml plasma
glycerate
(2,3-DPG)
(SO-400
or plasma (Sigma)
collagen
.4S ml PRP.
ml platelet
suspension
dilutions
and 0.1
was prepared
(Sigma),
ml ristocetin
to
washed platelets
by centrifugation with
for
.05 ml arachi-
(Abbott,
For platelet in buffer
aggrewas used
2,3-diphospho-
ml ristocetin.
in saline
final
with
resuspension
chromatography
sickle
alone,
were prepared
and resuspension
These platelets
platelets/mm3. some studies
.l
by adding
use in some experi-
preparation
For most studies,
EDTA (If),
0.1
was induced
PM/ml).
Washed platelet
followed
or
pH 7.3)
at 37"C, stirring
aggregometer
uM) (Sigma),
Laboratories),
with washed platelets, 0.3
a Payton
PRP, aggregation
6 mg/ml in Tris-saline-dextrose,
ments
directly
aggregation
at 1000 rpm.
with
plasmas
at 4’C until
285
10 minutes
(PRP) and 1000 X g for Platelet-poor
(PPP) .
plasmas
Aggregation
donic
was used to draw blood was at 100 X g for
plasma
plasma
assay
other
syringes,
platelet-rich
platelet-poor lant
IN SICKLE CELL ANEMIA
Centrifugation
(1:9).
for
PLATELETS
cell
by column chromatography
in Tris-saline-dextrose
in Tris-saline-dextrose do not aggregate
platelets,
due to the smaller
with
platelets
with
at l,OOO,OOO ristocetin
alone.
For
were washed by column
volume of PRP available
from these
patients. Plasma analyses A pooled used to define antigen,
titrated
plasma
100 percent
and von Willebrand
from 10 normal
activity factor
for
factor
donors VIII
(vWF) activity.
(5 males,
5 females)
procoagulant, Factor
VIII
factor
a was VIII
procoagulant
286
PLATELETS
(VIII:C)
IN SICKLE
was assayed using an activated
CELL ANEMIA
PTT (14).
vol.lh,No.2/3
Factor VIII antigen
(VIIIR:AG) was determined using electroimmunodiffusion
(1.5)) with rabbit
factor
VIII antibody
NJ).
factor
activity
macroscopic
(Behring Diagnostics,
Somerville,
von Willebrand
(VIIIR:WF) was determined using the previously
aggregation
Fibrinogen
levels
method of Ellis
technique
with ristocetin
were done on a fibrometer
described
(13). (Sherwood) according
to the
et al (16). -RESULTS
All subjects
with sickle
with ADP, epinephrine,
5
4
I
ADP
cell
or collagen.
anemia showed some decrease This was seen primarily
I.OpM
in aggregation
as defective
I.5 #bY
5 ADP
E ip
2.0pM
1
FIG. 1 ADP-induced platelet anemia.
aggregation
in PRP from a subject
with sickle
cell
Vo1.14,No.2/3
PLATELETS
IN SICKLE CELL ANEMIA
287
second phase aggregation. Aggregometerrecordingswith ADP from one such patient are shown in Figure 1. Aggregationincreaseswith increasinglevels of ADP, but a clearly biphasic recording is not obtained at any concentration. Figure 2 shows results obtained in PRP from another patient with increasing concentrationsof epinephrine. Normal aggregationis not obtained even with 10 uM epinephrine, Collagen-inducedplatelet aggregationin sickle cell PRP is shown in Figure 3. Aggregationin PRP from sickle cell anemia with a l/10 dilution of the stock collagen preparationwas comparableto that obtained in control PRP with a l/100 dilution. In control PRP, a l/SO dilution produced produced a somewhat greater degree of aggregationwith a shorter lag period than the l/100 dilution. Aggregationwith .5 mM arachidonicacid was normal in sickle cell PRP.
“I
Eplnephrme
1.0 ,A
2.0pM
10.0 pM
I
2
i
4
0
I
2
3
4
Minuhs
FIG. 2 Epinephrine-inducedplatelet aggregationin PRP from a subject with sickle cell anemia.
288
PLATELETS
COLLAGEN
IN SICKLE
CELL ANEMIA
l/IO
vol.&No.2/3
l/50
d
i
i
i
4
Minutes
FIG. 3 Collagen-inducedplatelet aggregationin PRP from a subject with sickle cell anemia.
In some sickle cell PRP little or no aggregationwas seen with ristocetin (final concentration1.09 mg/ml) (Figure 4), while in others, a lag phase of two to three minutes was noted, followed by aggregation. Four minutes after ristocetinaddition, mean height of aggregometercurves was 26*20 S.E.M. with sickle cell PRP (n = 17) and 73*6 S.E.M. with normal PRP (n = 14) (p < -001). In order to determine whether this abnormal aggregationof sickle cell PRP with ristocetinwas intrinsicor extrinsic to the platelet, further studies were performed. One such experiment is shown in Figure 5. No ristocetin-inducedaggregationwas seen in PRP from the donor with sickle cell anemia. Next, normal PPP was added to the sickle cell PRP in the aggregometer, and ristocetin added. Again, no aggregationwas seen. Finally,
Vo1.14,No.2/3
6 1
PLATELETS
IN SICKLE
289
CELL ANEMIA
RISTOCETIN ~armal
Sickle Cell Anemia 5-
.s .-t E E e lE .P J
4-
3-
2II
9
I-
O
7 0
I
2
3
4
0
I
2
3
4
Minutes
FIG. 4 Collagen-inducedplatelet aggregationin PW anemia.
6 ss-
PRP
SS-PRP+N.PfQ
from a subject with sickle cell
jS- Plls
FIG. 5 Ristocetin-inducedplatelet aggregationin (a) PRP from a subject with sickle cell anemia (on left), (b) PRP from the same subject with sickle cell anemia with normal PPP (.35 ml PRP + .l ml PPP) and (c) column washed platelets from the subject with sickle cell anemia and normal.plasma(.35 ml platelet suspension + .l ml PPP).
PLATELETS
290
IN
SICKLE
CELL ANEMIA
N+N
I
N+SS
r
01
Vol.l&No.2/3
I
0
2
I
4
3
FIG. 6 Ristocetin-induced aggregation PPP (on left) or “sickle cell”
normal
PPP was added to column washed sickle
was then added. To confirm induced plasma
Aggregation
or sickle
cell
inhibition
normal
cell
aggregation Aggregation
in this
the inhibitory
aggregation,
inhibit
sickle
of normal washed platelets PPP (on right).
PPP. (Figure
of
plasma occurred
case
effect
of
In this
platelets
pooled
and ristocetin
was normal. sickle
washed platelets system,
cell
PRP on ristocetin-
were exposed sickle
cell
with
different
to normal
PPP
WES
pooled
found
to
6).
normal washed platelets in Tris-saline-dextrose at 70% sickle
cell
permitted
with
less
than 20% sickle
increased
with
less
sickle
seen with
20-40% sickle
cell
cell
with normal
cell
cell
is
plasma.
shown in Figure
plasma, plasma.
plasma present,
dilutions
while
normal
Aggregation until
7.
of Complete
aggregation
was
progressively
normal aggregation
was
Vol.lb,No.2/3
PLATELETS
IN S.ICKLE CELL ANEMIA
291
470%
”
3s -PPP
,
0
60%
3
2
4
4
3
2
I
0
0
I
2
3
4
MmJtes
FIG. 7 Ristocetin-induced dilutions of sickle
aggregation cell PPP.
of normal washed platelets
VIIIR:WF measured by macroscopic determined of
in 12 sickle
20%, lo%,
times
cell
plasmas.
and 5% are routinely
1 pool
cell
plasmas
(100% activity).
cell
of ristocetin
of ristocetin
assay,
ristocetin final
determinations
was
plasma of
dilutions
aggregation
and compared to the same dilutions assay,
VIIIR:WF was normal
aggregation
in sickle
PPP upon normal washed platelets
ing concentrations Since
In this
with
different
of
a
in sickle
(83+11% S.E.M.).
Inhibition by s ckle
In this
used for
with normal washed platelets,
nors
aggregation
with
of the
fibrinogen (17)
could
PRP (Figure
be reversed
8) or
by increas-
drug.
has been shown to precipitate and might
and found to be normal
cell
in all
affect cases
our assay, (x = 239t42
these
with high levels
mg% S.E.M.).
concentrations
were measured Other activities
PLATELETS TN SICKLE
292
CELL ANEMIA
2.4 mg/mt
1.2mg/ml
0
vol.lb,No.2,'3
2
I
3
4 0 Minutes
I
2
3
4
FIG. 8 Platelet aggregation induced in sickle cell.PRP left) or 2.4 mg/ml ristocetin (on right). related
to VIIIR:WF were determined in sickle
was 187&14% (n = 22) and factor Aggregation sickle
cell
hemoglobin was inhibitory Aggregation
in this
saline
threshold
Factor VIII antigen
was 286S82% (n = 6).
was determined in PRP from one subject
to determine whether the presence of sickle to ristocetin-induced
that 2,3-DPG inhibits
aggregation
platelet
or 2,3=DPG was added to normal PRP prior
DPGat 100-400 pM/ml generally epinephrine,
PPP.
(on
with
cell
(Figure 9).
case was normal,
In view of reports either
cell
VIII procoagulant
with ristocetin
thallesemia
by 1.2 mg/ml ristocetin
and collagen
concentrations
had little
unless
threshold
effect
aggregation to ristocetin.
on aggregation
concentrations
of these aggregating
agents,
(la), 2,3-
with ADP,
were used.
With
2,3-DPG was usually
Vo1.14,No.2/3
PLATELETS IN SICKLE CELL ANEMIA
293
RISTOCETIN
0
I
2 Minutes
3
4
FIG. 9 Ristocetin (1.09 mg/ml) -induced aggregation sickle cell thallesemia.
in PRP from a subject
inhibitory.
with ristocetin
In one normal PRP, aggregation
with
was unaffected
50 uM/ml 2,3-DPG, was decreased with 100 or 200 uM/ml and prevented by 400 Wml.
In PRP from two other normal subjects,
on ristocetin-induced
aggregation.
2,3-DPG had little
effect
by
PLATELETS IN SICKLE CELL ANEMIA
294
Vo1.14,No.2/3
DISCUSSION Platelet
aggregation
in PBP from subjects
depressed with ADP, epinephrine, Aggregation
with ristocetin
from subjects intrinsic correct
the defect
cell
ability
either
two possibilities; on the platelet
at higher levels
plasma (19),
The inability cell
to ristocetin-induced aggregation,
cell
with ristocetin.
PPP, eliminating
of normal plasma to
plasma indicates
aggregation.
by increasing
ristdcetin
to produce aggregation,
to produce aggregation
the
The correction concentration,
that at high concentrations,
or that an abnormal factor
anemia was
by suspending washed platelets
in the presence of sickle
of abnormal ris tocetin
acts directly
and especially
anemia in normal control
changes as causal.
presence of an inhibitor
suggests
was corrected
with sickle
platelet
collagen,
with sickle
ristocetin
as suggested by its
in von Willebrand’s
VIII molecule
disease
is present which might
compete for ristocetin. Minimal inhibition that this
of ristocetin-induced
aggregation
is not the mechanism by which ristocetin-induced Another possibility
inhibited.
might be ADP, liberated
since ADP has been shown to inhibit
ristocetin-induced
by 2,3-DPG suggests aggregation by chronic
aggregation
hemolysis, (20).
however, in view of the rapid degradation
This also appears unlikely,
is
of ADP
in plasma. The abnormal aggregation rather than to a platelet platelet
defect
epinephrine, this,
since
aggregation
with ristocetin
and collagen. aggregatien
Gruppo -et al (4) reported
defect.
was responsible
for the decrease
Cur results
by these other agents,
Cur results
(ZZ),
that an intrinsic
in aggregation
with ristocetin
induced by ristocetin
aggregate with ristocetin
‘appears to be due to the plasma,
do not conflict
is by a different
since metabolically
seen with ADP,
inactive
with
mechanism than platelets
(21). as well as independent findings
Brewer (23, 24) and Miotti
et al (25), --
from Leichtman and
show that abnormal ristocetin-induced
will
vo1.14,No.2/3
platelet
aggregationis
PLATELETS
IN SICKLE CELL ANEMIA
295
seen in plasma from subjects with sickle cell anemia.
This abnormal aggregationis due to an inhibitoryeffect of the plasma. This inhibitionis seen although there are high levels of VIIIR:AG in sickle cell plasma and normal levels of VIIIR:WF in dilute samples of sickle cell plasma. The clarificationof these findingsmay prove to be helpful in understanding the pathogenesisof thromboticphenomena in sickle cell anemia. ACKNOWLEDGEMENTS This work was supportedby the Medical Research Service of the Veterans Administration. REFERENCES 1.
ALKJAERSIG,N., FLETCHER,A., JOIST, H. and CHAPLIN, H. Hemostatic alterationsaccompanyingsickle cell pain crises. J. Lab. Clin. Med. 88, 440, 1976.
2.
ITTYERAH, R., ALKJAERSIG,N., FLETCHER,A. and CHAPLIN, H. Coagulation factor XIII_concentrationin sickle-celldisease. 3. gab. Clin. Med. 88, 546, 1976.
3.
STUART, M.J., STOCKMAN,J.A. and OSKI, F.A. Abnormalitiesof platelet aggregationin the vaso-occlusivecrisis of sickle-cellanemia. J. Pediatr. 85, 629, 1974.
4.
GRUPPO, R.A., GLUECK, H.I., GRANGER, S.M. and MILLER, M.A. Platelet function in sickle cell anemia. Tbromb. &es. 10, 325, 1977.
5.
ABILDGAARD,C.F., SIMONE, J.V. and SCHULMAN, I. Factor VIII (antihaemophilic factor) activity in sickle-cellanaemia. or. J. Saemetol. 13, 19, 1967.
6.
GREEN, D., KUMN, H.C. and RUIZ, G. Impaired fibrinolysisin sickle cell disease. Thromb. Diath. Haemorrh. 24, 10, 1970.
7.
LESLIE, J., LANGLER, D. and SERTEANT, G.R. Coagulationchanges during the steady state in homozygous sickle-celldisease in Jamaica. Br. J. Saematol. 30, 159. 1975.
8.
RICKLES, F.R. and D'LEARY, D.S. Role of coagulationsystem in pathophysiology of sickle cell disease. Arch. Intern. Med. 133, 635, 1974.
9.
WALSH, R.T., LUSHER, J.M. and BARNHART,MI. Coagulationand fibrinolysis studies in sickle cell anemia. Thromb. Diath. Raemorrh. (Suppl. 53) 271, 1973.
10. HAUT, M.J., COWAN, D.H. and HARRIS, J.W. Platelet function and survival in sickle cell disease. J. Lab. Clin. Med. 82, 44, 1973.
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LEVISON,S.P. and JUNG, C.J. Sickle cell trait and hema11. BRODY,J.I., turia associated with von Willebrand syndromes. Ann. Intern. Med. 86, 529, 1977. 12. BRECHER,G. and CRONKITE,E.P. Morphology and enumeration of human blood platelets. J. Appl. Physiol. 3, 365, 1950. 13.
K.M. Nature of SARJI, K.E., STRATTON, R.D.. WAGNER,R.H. and BRINKHOUS, von Willebrand factor: A new assay and a specific inhibitor. Proc. Nat. Acad. Sci. 71, 2937, 1974.
14.
DACIE, J.V. and LEWIS, S.M. PracticalHaematology. Grune and Stratton, New York, 1968.
15.
ZIMMERMAN,T.S., HOYER, L.W., DICKSON, L. Determinationof the von Willebrand'sdisease antigen (factor VIII-relatedantigen) in plasma by quantitativeimmunoelectrophoresis.J. gab. Clin. Med. 86, 152, 1975.
16.
ELLIS, B.C. and STRANKSKY,A. A quick and accurate method for the determinationof fibrinogenin plasma. J. Lab. Clin. Med. 58, 477, 1961.
17.
STIBBE, J. and KIRBY, E.P. The influence of haemaccel, fibrinogen,and albumin on ristocetin-inducedplatelet aggregation. Relevance to the quantitativemeasurementof the ristocetin cofactor. Thromb. Res. 8, 151, 1976.
18.
IATRIDIS, P.G. The in vivo increase of factor VIII activity by 2,3diphosphoglycerate. Thromb. Res. 9, 533, 1976.
19.
MEYER, D., JENKINS, C.S.P., DREYFUS, M.D. et al. Willebrand factor and ristocetin. II. Relationship between Willebrand factor, Willebrand antigen and factor VIII activity. Br. J. Haematol. 28, 579, 1974.
20.
GRANT, R-A., ZUCKER, M.B. and MCPHERSON,J. ADP-induced inhibitionof von Willebrand factor-mediatedplatelet agglutination. Am. J. Physiol. 230, 1406, 1976.
21.
ALLAIN, J.P., COOPER. H.A., WAGNER, R.H. and BRINKHOUS,K.M. Platelets fixed with paraformaldehyde: A new reagent for assay of von Willebrand factor and platelet aggregatingfactor. J. Lab. Clin. Med. 85, 318, 1975.
22.
SARJI, K-E., SCHRAIBMAN,H.B., FULLWOOD, C.O., COLWELL, J.A., O'DELL, R. and EURENIUS, K. Sickle cell anemia: Presence of a plasma factor which inhibits platelet aggregation. Nineteenth Annual Meeting, American Society of Hematology, Boston, Massachusetts,December 4-7, 1976,p. 207.
23.
LEICHTMAN,D.A. and BREWER, G.J. A plasma inhibitorof ristocetininduced platelet aggregationin patients with sickle hemoglobinopathies. Am. J. Hematol. 2, 251, 1977.
24.
LEICHTMAN, D.A. and BREWER, G.J. Abnormal ristocetin-inducedplatelet aggregationin patients with sickle hemoglobinopathies. Nineteenth Annual Meeting, American Society of Hematology, Boston, Massachusetts, December 4-7, 1977. p. 201.
Va1.14,No.2/3
PLATELETS
IN SICKLE CELL ANEMIA
Factor VIII and sickle cell disease. Nineteenth Annual Meeting, American Society Hematology, Boston, Massachusetts, December 4-7, 1977, p. 177.
297
25. MIO'ITI,A., WALKER, M., KRISHNAMMURTHY,M. and DOSIK, H.
of
in Great Britain 0049-3848/79/ 0301-0283
902.0,0/o
ABNORMALITIES OF PLATELET AGGREGATION IN SICKLECELLANEMIA PRESENCE OF A PLASMAFACTORINHIBITINGAGGREGATION BY RISTOCETIN K. E. Sarji,
K. Eurenius,
C. 0. Fullwood, H. B. Schraibman, and J. A. Colwell
Veterans Administration Hospital and of Endocrinology-Metabolism-Nutrition and Hematology Department of Medicine Medical University of South Carolina, Charleston, South Carolina USA Divisions
(Received 8.5.1978; in revised Accepted by Editor K.M.
form 18.10.1978. Brinkhous)
ABSTRACT Platelet aggregation in plasma from subjects with sickle cell anemia, not in crisis, was tested using the aggregometer. Aggregation induced by adenosine diphosphate (ADP) , epinephrine, or collagen was decreased in platelet-rich plasma (PRP) from these subjects. Aggregation induced by ristocetin (1.09 mg/ml final cont.) was absent or was preceded by a lag phase in sickle cell PRP but not in normal PRP. Washed platelets from subjects with sickle cell anemia mixed with normal platelet-poor plasma (PPP) showed normal ristocetin-induced Washed normal platelets mixed with sickle cell PPP aggregation. showed progressive inhibition of ristocetin aggregation with increasing aaxnmts of plasma. Dilution of the sickle cell PPP or increasing the final concentration of ristocetin to 2.4 mg/ml resulted in normal aggregation. Factor VIII antigen levels (196+16% S.E.M.) and factor VIII procoagulant activity (286+82%) were elevated in sickle cell plasma. Fibrinogen levels (239+42 mg%) were normal or slightly decreased; no relationship to aggregation was seen. These results suggest that an abnormally high factor VIII antigen/van Willebrand factor activity ratio is present in sickle cell anemia. It is postulated that inhibition of ristocetin-induced platelet aggregation by sickle cell plasma is related to competition for available ristocetin. I NTRODUCI ION The most Cowon clinical anemia is the painful thrombosis tiated
is of early
by sickled
to alterations
complication
thrombotic significance
erythrocytes.
in the hemostatic
occlusive
seen in patients crisis.
or a result
with sickle
It is uncertain of vascular
occlusion
There are conflicting
reports
system in sickle
anemia.
283
cell
cell
whether ini-
with regard Studies on
PLATELETS IN SICKLE CELL ANEMIA
284
patients counts,
in crisis factor
VIII,
(1) and factor including
have indicated
that there is an increase
and fibrinogen
in extent of aggregation
and collagen
not in crisis in crisis
and normal aggregation
gen in precrisis post crisis
or crisis
period,
ADP, epinephrine,
(4) reported
drome has also been described
investigated
but an increased
(3)
(ADP),
a decrease
in patients
aggregation
thrombin,
during the
In contrast, aggregation
with
A von Willebrand
in crisis.
with sickle
cell
data reported,
and factor
not
and colla-
sensitivity
in platelet
in four patients
in patients
in patients
by Haut -et al (10).
In view of the conflicting
platelet
and turnover
with ADP, epinephrine,
periods,
and collagen
hematuria (11).
phase aggregation
with adenosine diphosphate
survival
have been reported
Gruppo and coworkers
aggregation
system have also been reported
Normal platelet
(S-9).
activity
(4).
Changes in the coagulation
patients
in platelet
an impairment of rate and extent of first
epinephrine,
in platelet
and a decrease of fibrinolytic
XIII (2) as well. as abnormalities
and a decrease
Vol.l&,No.2/3
VIII related
trait
syn-
and
we have further activities
in
not in crisis. MATERIALS ANDMETHODS
Plasma samples Asymptomatic patients
with sickle
patients
with well-documented
studies,
normal hemoglobin
patients
have chronic
ly elevated
indicated
evidence
for
routine
counts, of
without
medical
for
and off
cell
anemia were studied. anemia as indicated
and absence
of
of homolysis
increased All
signs
to study. medications
These are
by kindred such
splenomegaly.
as indicated
by consistent-
indirect
bilirubin
patients
were studied
and in no instance
symptoms,
one week prior
disease
levels
GI bleeding.
checkups,
at a time when additional no medication
A2 levels,
constant
reticulocyte
and negative
sickle
cell
were these
or problems Controls were also
concentrations at times
patients
were evident. of
similar
tested.
studied They had
age and sex A two syringe
vo1.14,~0.2/3
with plastic
technique, citrate ture
all
were kept
PPP was stored
platelet-rich
Platelet
assay
within
at -2O’C
later
assay.
for
ADP (1.0
factor
4 hours
of
Platelet
3.8%
at room tempera-
10 minutes
for
into
at 4°C for
VIII
procoaguAll
collection. counts
were done on
(12).
was performed
For studies
nM) (Sigma),
acid
(Applied
using
using
epinephrine Science
gation
(1.0
ml plasma
glycerate
(2,3-DPG)
(SO-400
or plasma (Sigma)
collagen
.4S ml PRP.
ml platelet
suspension
dilutions
and 0.1
was prepared
(Sigma),
ml ristocetin
to
washed platelets
by centrifugation with
for
.05 ml arachi-
(Abbott,
For platelet in buffer
aggrewas used
2,3-diphospho-
ml ristocetin.
in saline
final
with
resuspension
chromatography
sickle
alone,
were prepared
and resuspension
These platelets
platelets/mm3. some studies
.l
by adding
use in some experi-
preparation
For most studies,
EDTA (If),
0.1
was induced
PM/ml).
Washed platelet
followed
or
pH 7.3)
at 37"C, stirring
aggregometer
uM) (Sigma),
Laboratories),
with washed platelets, 0.3
a Payton
PRP, aggregation
6 mg/ml in Tris-saline-dextrose,
ments
directly
aggregation
at 1000 rpm.
with
plasmas
at 4’C until
285
10 minutes
(PRP) and 1000 X g for Platelet-poor
(PPP) .
plasmas
Aggregation
donic
was used to draw blood was at 100 X g for
plasma
plasma
assay
other
syringes,
platelet-rich
platelet-poor lant
IN SICKLE CELL ANEMIA
Centrifugation
(1:9).
for
PLATELETS
cell
by column chromatography
in Tris-saline-dextrose
in Tris-saline-dextrose do not aggregate
platelets,
due to the smaller
with
platelets
with
at l,OOO,OOO ristocetin
alone.
For
were washed by column
volume of PRP available
from these
patients. Plasma analyses A pooled used to define antigen,
titrated
plasma
100 percent
and von Willebrand
from 10 normal
activity factor
for
factor
donors VIII
(vWF) activity.
(5 males,
5 females)
procoagulant, Factor
VIII
factor
a was VIII
procoagulant
286
PLATELETS
(VIII:C)
IN SICKLE
was assayed using an activated
CELL ANEMIA
PTT (14).
vol.lh,No.2/3
Factor VIII antigen
(VIIIR:AG) was determined using electroimmunodiffusion
(1.5)) with rabbit
factor
VIII antibody
NJ).
factor
activity
macroscopic
(Behring Diagnostics,
Somerville,
von Willebrand
(VIIIR:WF) was determined using the previously
aggregation
Fibrinogen
levels
method of Ellis
technique
with ristocetin
were done on a fibrometer
described
(13). (Sherwood) according
to the
et al (16). -RESULTS
All subjects
with sickle
with ADP, epinephrine,
5
4
I
ADP
cell
or collagen.
anemia showed some decrease This was seen primarily
I.OpM
in aggregation
as defective
I.5 #bY
5 ADP
E ip
2.0pM
1
FIG. 1 ADP-induced platelet anemia.
aggregation
in PRP from a subject
with sickle
cell
Vo1.14,No.2/3
PLATELETS
IN SICKLE CELL ANEMIA
287
second phase aggregation. Aggregometerrecordingswith ADP from one such patient are shown in Figure 1. Aggregationincreaseswith increasinglevels of ADP, but a clearly biphasic recording is not obtained at any concentration. Figure 2 shows results obtained in PRP from another patient with increasing concentrationsof epinephrine. Normal aggregationis not obtained even with 10 uM epinephrine, Collagen-inducedplatelet aggregationin sickle cell PRP is shown in Figure 3. Aggregationin PRP from sickle cell anemia with a l/10 dilution of the stock collagen preparationwas comparableto that obtained in control PRP with a l/100 dilution. In control PRP, a l/SO dilution produced produced a somewhat greater degree of aggregationwith a shorter lag period than the l/100 dilution. Aggregationwith .5 mM arachidonicacid was normal in sickle cell PRP.
“I
Eplnephrme
1.0 ,A
2.0pM
10.0 pM
I
2
i
4
0
I
2
3
4
Minuhs
FIG. 2 Epinephrine-inducedplatelet aggregationin PRP from a subject with sickle cell anemia.
288
PLATELETS
COLLAGEN
IN SICKLE
CELL ANEMIA
l/IO
vol.&No.2/3
l/50
d
i
i
i
4
Minutes
FIG. 3 Collagen-inducedplatelet aggregationin PRP from a subject with sickle cell anemia.
In some sickle cell PRP little or no aggregationwas seen with ristocetin (final concentration1.09 mg/ml) (Figure 4), while in others, a lag phase of two to three minutes was noted, followed by aggregation. Four minutes after ristocetinaddition, mean height of aggregometercurves was 26*20 S.E.M. with sickle cell PRP (n = 17) and 73*6 S.E.M. with normal PRP (n = 14) (p < -001). In order to determine whether this abnormal aggregationof sickle cell PRP with ristocetinwas intrinsicor extrinsic to the platelet, further studies were performed. One such experiment is shown in Figure 5. No ristocetin-inducedaggregationwas seen in PRP from the donor with sickle cell anemia. Next, normal PPP was added to the sickle cell PRP in the aggregometer, and ristocetin added. Again, no aggregationwas seen. Finally,
Vo1.14,No.2/3
6 1
PLATELETS
IN SICKLE
289
CELL ANEMIA
RISTOCETIN ~armal
Sickle Cell Anemia 5-
.s .-t E E e lE .P J
4-
3-
2II
9
I-
O
7 0
I
2
3
4
0
I
2
3
4
Minutes
FIG. 4 Collagen-inducedplatelet aggregationin PW anemia.
6 ss-
PRP
SS-PRP+N.PfQ
from a subject with sickle cell
jS- Plls
FIG. 5 Ristocetin-inducedplatelet aggregationin (a) PRP from a subject with sickle cell anemia (on left), (b) PRP from the same subject with sickle cell anemia with normal PPP (.35 ml PRP + .l ml PPP) and (c) column washed platelets from the subject with sickle cell anemia and normal.plasma(.35 ml platelet suspension + .l ml PPP).
PLATELETS
290
IN
SICKLE
CELL ANEMIA
N+N
I
N+SS
r
01
Vol.l&No.2/3
I
0
2
I
4
3
FIG. 6 Ristocetin-induced aggregation PPP (on left) or “sickle cell”
normal
PPP was added to column washed sickle
was then added. To confirm induced plasma
Aggregation
or sickle
cell
inhibition
normal
cell
aggregation Aggregation
in this
the inhibitory
aggregation,
inhibit
sickle
of normal washed platelets PPP (on right).
PPP. (Figure
of
plasma occurred
case
effect
of
In this
platelets
pooled
and ristocetin
was normal. sickle
washed platelets system,
cell
PRP on ristocetin-
were exposed sickle
cell
with
different
to normal
PPP
WES
pooled
found
to
6).
normal washed platelets in Tris-saline-dextrose at 70% sickle
cell
permitted
with
less
than 20% sickle
increased
with
less
sickle
seen with
20-40% sickle
cell
cell
with normal
cell
cell
is
plasma.
shown in Figure
plasma, plasma.
plasma present,
dilutions
while
normal
Aggregation until
7.
of Complete
aggregation
was
progressively
normal aggregation
was
Vol.lb,No.2/3
PLATELETS
IN S.ICKLE CELL ANEMIA
291
470%
”
3s -PPP
,
0
60%
3
2
4
4
3
2
I
0
0
I
2
3
4
MmJtes
FIG. 7 Ristocetin-induced dilutions of sickle
aggregation cell PPP.
of normal washed platelets
VIIIR:WF measured by macroscopic determined of
in 12 sickle
20%, lo%,
times
cell
plasmas.
and 5% are routinely
1 pool
cell
plasmas
(100% activity).
cell
of ristocetin
of ristocetin
assay,
ristocetin final
determinations
was
plasma of
dilutions
aggregation
and compared to the same dilutions assay,
VIIIR:WF was normal
aggregation
in sickle
PPP upon normal washed platelets
ing concentrations Since
In this
with
different
of
a
in sickle
(83+11% S.E.M.).
Inhibition by s ckle
In this
used for
with normal washed platelets,
nors
aggregation
with
of the
fibrinogen (17)
could
PRP (Figure
be reversed
8) or
by increas-
drug.
has been shown to precipitate and might
and found to be normal
cell
in all
affect cases
our assay, (x = 239t42
these
with high levels
mg% S.E.M.).
concentrations
were measured Other activities
PLATELETS TN SICKLE
292
CELL ANEMIA
2.4 mg/mt
1.2mg/ml
0
vol.lb,No.2,'3
2
I
3
4 0 Minutes
I
2
3
4
FIG. 8 Platelet aggregation induced in sickle cell.PRP left) or 2.4 mg/ml ristocetin (on right). related
to VIIIR:WF were determined in sickle
was 187&14% (n = 22) and factor Aggregation sickle
cell
hemoglobin was inhibitory Aggregation
in this
saline
threshold
Factor VIII antigen
was 286S82% (n = 6).
was determined in PRP from one subject
to determine whether the presence of sickle to ristocetin-induced
that 2,3-DPG inhibits
aggregation
platelet
or 2,3=DPG was added to normal PRP prior
DPGat 100-400 pM/ml generally epinephrine,
PPP.
(on
with
cell
(Figure 9).
case was normal,
In view of reports either
cell
VIII procoagulant
with ristocetin
thallesemia
by 1.2 mg/ml ristocetin
and collagen
concentrations
had little
unless
threshold
effect
aggregation to ristocetin.
on aggregation
concentrations
of these aggregating
agents,
(la), 2,3-
with ADP,
were used.
With
2,3-DPG was usually
Vo1.14,No.2/3
PLATELETS IN SICKLE CELL ANEMIA
293
RISTOCETIN
0
I
2 Minutes
3
4
FIG. 9 Ristocetin (1.09 mg/ml) -induced aggregation sickle cell thallesemia.
in PRP from a subject
inhibitory.
with ristocetin
In one normal PRP, aggregation
with
was unaffected
50 uM/ml 2,3-DPG, was decreased with 100 or 200 uM/ml and prevented by 400 Wml.
In PRP from two other normal subjects,
on ristocetin-induced
aggregation.
2,3-DPG had little
effect
by
PLATELETS IN SICKLE CELL ANEMIA
294
Vo1.14,No.2/3
DISCUSSION Platelet
aggregation
in PBP from subjects
depressed with ADP, epinephrine, Aggregation
with ristocetin
from subjects intrinsic correct
the defect
cell
ability
either
two possibilities; on the platelet
at higher levels
plasma (19),
The inability cell
to ristocetin-induced aggregation,
cell
with ristocetin.
PPP, eliminating
of normal plasma to
plasma indicates
aggregation.
by increasing
ristdcetin
to produce aggregation,
to produce aggregation
the
The correction concentration,
that at high concentrations,
or that an abnormal factor
anemia was
by suspending washed platelets
in the presence of sickle
of abnormal ris tocetin
acts directly
and especially
anemia in normal control
changes as causal.
presence of an inhibitor
suggests
was corrected
with sickle
platelet
collagen,
with sickle
ristocetin
as suggested by its
in von Willebrand’s
VIII molecule
disease
is present which might
compete for ristocetin. Minimal inhibition that this
of ristocetin-induced
aggregation
is not the mechanism by which ristocetin-induced Another possibility
inhibited.
might be ADP, liberated
since ADP has been shown to inhibit
ristocetin-induced
by 2,3-DPG suggests aggregation by chronic
aggregation
hemolysis, (20).
however, in view of the rapid degradation
This also appears unlikely,
is
of ADP
in plasma. The abnormal aggregation rather than to a platelet platelet
defect
epinephrine, this,
since
aggregation
with ristocetin
and collagen. aggregatien
Gruppo -et al (4) reported
defect.
was responsible
for the decrease
Cur results
by these other agents,
Cur results
(ZZ),
that an intrinsic
in aggregation
with ristocetin
induced by ristocetin
aggregate with ristocetin
‘appears to be due to the plasma,
do not conflict
is by a different
since metabolically
seen with ADP,
inactive
with
mechanism than platelets
(21). as well as independent findings
Brewer (23, 24) and Miotti
et al (25), --
from Leichtman and
show that abnormal ristocetin-induced
will
vo1.14,No.2/3
platelet
aggregationis
PLATELETS
IN SICKLE CELL ANEMIA
295
seen in plasma from subjects with sickle cell anemia.
This abnormal aggregationis due to an inhibitoryeffect of the plasma. This inhibitionis seen although there are high levels of VIIIR:AG in sickle cell plasma and normal levels of VIIIR:WF in dilute samples of sickle cell plasma. The clarificationof these findingsmay prove to be helpful in understanding the pathogenesisof thromboticphenomena in sickle cell anemia. ACKNOWLEDGEMENTS This work was supportedby the Medical Research Service of the Veterans Administration. REFERENCES 1.
ALKJAERSIG,N., FLETCHER,A., JOIST, H. and CHAPLIN, H. Hemostatic alterationsaccompanyingsickle cell pain crises. J. Lab. Clin. Med. 88, 440, 1976.
2.
ITTYERAH, R., ALKJAERSIG,N., FLETCHER,A. and CHAPLIN, H. Coagulation factor XIII_concentrationin sickle-celldisease. 3. gab. Clin. Med. 88, 546, 1976.
3.
STUART, M.J., STOCKMAN,J.A. and OSKI, F.A. Abnormalitiesof platelet aggregationin the vaso-occlusivecrisis of sickle-cellanemia. J. Pediatr. 85, 629, 1974.
4.
GRUPPO, R.A., GLUECK, H.I., GRANGER, S.M. and MILLER, M.A. Platelet function in sickle cell anemia. Tbromb. &es. 10, 325, 1977.
5.
ABILDGAARD,C.F., SIMONE, J.V. and SCHULMAN, I. Factor VIII (antihaemophilic factor) activity in sickle-cellanaemia. or. J. Saemetol. 13, 19, 1967.
6.
GREEN, D., KUMN, H.C. and RUIZ, G. Impaired fibrinolysisin sickle cell disease. Thromb. Diath. Haemorrh. 24, 10, 1970.
7.
LESLIE, J., LANGLER, D. and SERTEANT, G.R. Coagulationchanges during the steady state in homozygous sickle-celldisease in Jamaica. Br. J. Saematol. 30, 159. 1975.
8.
RICKLES, F.R. and D'LEARY, D.S. Role of coagulationsystem in pathophysiology of sickle cell disease. Arch. Intern. Med. 133, 635, 1974.
9.
WALSH, R.T., LUSHER, J.M. and BARNHART,MI. Coagulationand fibrinolysis studies in sickle cell anemia. Thromb. Diath. Raemorrh. (Suppl. 53) 271, 1973.
10. HAUT, M.J., COWAN, D.H. and HARRIS, J.W. Platelet function and survival in sickle cell disease. J. Lab. Clin. Med. 82, 44, 1973.
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LEVISON,S.P. and JUNG, C.J. Sickle cell trait and hema11. BRODY,J.I., turia associated with von Willebrand syndromes. Ann. Intern. Med. 86, 529, 1977. 12. BRECHER,G. and CRONKITE,E.P. Morphology and enumeration of human blood platelets. J. Appl. Physiol. 3, 365, 1950. 13.
K.M. Nature of SARJI, K.E., STRATTON, R.D.. WAGNER,R.H. and BRINKHOUS, von Willebrand factor: A new assay and a specific inhibitor. Proc. Nat. Acad. Sci. 71, 2937, 1974.
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DACIE, J.V. and LEWIS, S.M. PracticalHaematology. Grune and Stratton, New York, 1968.
15.
ZIMMERMAN,T.S., HOYER, L.W., DICKSON, L. Determinationof the von Willebrand'sdisease antigen (factor VIII-relatedantigen) in plasma by quantitativeimmunoelectrophoresis.J. gab. Clin. Med. 86, 152, 1975.
16.
ELLIS, B.C. and STRANKSKY,A. A quick and accurate method for the determinationof fibrinogenin plasma. J. Lab. Clin. Med. 58, 477, 1961.
17.
STIBBE, J. and KIRBY, E.P. The influence of haemaccel, fibrinogen,and albumin on ristocetin-inducedplatelet aggregation. Relevance to the quantitativemeasurementof the ristocetin cofactor. Thromb. Res. 8, 151, 1976.
18.
IATRIDIS, P.G. The in vivo increase of factor VIII activity by 2,3diphosphoglycerate. Thromb. Res. 9, 533, 1976.
19.
MEYER, D., JENKINS, C.S.P., DREYFUS, M.D. et al. Willebrand factor and ristocetin. II. Relationship between Willebrand factor, Willebrand antigen and factor VIII activity. Br. J. Haematol. 28, 579, 1974.
20.
GRANT, R-A., ZUCKER, M.B. and MCPHERSON,J. ADP-induced inhibitionof von Willebrand factor-mediatedplatelet agglutination. Am. J. Physiol. 230, 1406, 1976.
21.
ALLAIN, J.P., COOPER. H.A., WAGNER, R.H. and BRINKHOUS,K.M. Platelets fixed with paraformaldehyde: A new reagent for assay of von Willebrand factor and platelet aggregatingfactor. J. Lab. Clin. Med. 85, 318, 1975.
22.
SARJI, K-E., SCHRAIBMAN,H.B., FULLWOOD, C.O., COLWELL, J.A., O'DELL, R. and EURENIUS, K. Sickle cell anemia: Presence of a plasma factor which inhibits platelet aggregation. Nineteenth Annual Meeting, American Society of Hematology, Boston, Massachusetts,December 4-7, 1976,p. 207.
23.
LEICHTMAN,D.A. and BREWER, G.J. A plasma inhibitorof ristocetininduced platelet aggregationin patients with sickle hemoglobinopathies. Am. J. Hematol. 2, 251, 1977.
24.
LEICHTMAN, D.A. and BREWER, G.J. Abnormal ristocetin-inducedplatelet aggregationin patients with sickle hemoglobinopathies. Nineteenth Annual Meeting, American Society of Hematology, Boston, Massachusetts, December 4-7, 1977. p. 201.
Va1.14,No.2/3
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IN SICKLE CELL ANEMIA
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297
25. MIO'ITI,A., WALKER, M., KRISHNAMMURTHY,M. and DOSIK, H.
of
