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Thrombocytosis and platelet aggregates in the circulation of adjuvant arthritic rats.

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256
THROMBOCYTOSIS AND PLATELET
AGGREGATES IN THE
CIRCULATION OF ADJUVANT
ARTHRITIC RATS
PETER GORC)G and IREN B. KOVACS
A marked increase in platelet count was observed
in adjuvant-induced polyarthritic rats. Elevation of platelet count was still significant 3 months after onset of
disease. Increased number and larger size of platelets
reflect an increase in the rate of platelet production. By
means of the platelet-count ratio technique, circulating
platelet aggregates were demonstrated in rats with severe
arthritis. Aspirin treatment prevented thrombocytosis and
formation of platelet aggregates.
Elevated platelet count is encountered in a number of clinical conditions. A platelet count in excess of
normal may occur in association with myeloproliferative disorders or secondary to certain underlying diseases. Among the latter a recent study has shown that
thrombocytosis occurs in rheumatoid arthritis ( I ) . In
our previous studies (2,3) platelet function abnormalities (decreased bleeding time, increased aggregation
caused by various stimuli, and increased susceptibility to
formation of thrombus) were estimated in adjuvant arthritic rats. The polyarthritic syndrome induced in rats
From the Department of Pharmacology. EGYT Pharmacochemical Works, H-1475 Budapest, and Korvin Otto Hospital.
H-1071 Budapest, Hungary.
Peter GGrGg, Ph.D.: Researcher, Department of Pharmacology, EGYT Pharmacochemical Works, Budapest; Iren B. Kovics,
M.D.: Head internist, Korvin Otto Hospital, Budapest.
Address reprint requests to Dr. Peter GorGg, Department of
Pharmacology, EGYT Pharmcochemical Works, H-1475. Budapest,
Hungary .
Submitted for publication February 28. 1977: accepted April
22. 1977.
Arthritis and Rheumatism, Vol. 21, No. 2 (March 1978)
by the administration of Freund’s adjuvant is the most
common model for testing therapeutic substances. In
contrast to the absence of rheumatoid factors, adjuvant
arthritis as a cell-mediated immune reaction is at present
the nearest experimental approximation to human rheumatoid arthritis ( 4 3 ) .
The possibility that the elevated platelet count
may be associated with a predisposition to the thrombotic state must be considered. I t therefore seemed desirable to investigate the platelet count in established
adjuvant arthritis. The observed thrombocytosis and the
detection of circulating platelet aggregates in the blood
of arthritic rats are considered significant factors in the
time-course and vascular complications of this animal
disease.
METHODS
Adjuvant-Induced Polyarthritis in the Rat. Male,
Sprague-Dawley (CFY) rats weighing 150-160 grams at the
start of the experiment were used. Adjuvant (suspension of 0.5
mg heat-killed Mycobacterium tuberculosis in 0.1 ml paraffinoil) was injected intradermally into the base of the tail. The
severity of the arthritis in each paw was subjectively assessed in
units of 0.5 on a scale from 0 to 4. Maximal score for an
individual rat was 16. Severely arthritic rats were defined as the
score/rat > 12.
Platelet Count. Platelet count was determined from the
arterial blood by the sedimentation method of Bull et al. (6).
Four milliliters of blood were drawn into a syringe containing
50 U of heparin, transferred to a tube containing 20 mg of
solid E D T A and 0.05 ml of 40% polyvinyl-pyrrolidone, and
shaken thoroughly. After standing for 1 hour a 10 &aliquot
of platelet-rich plasma ( P R P ) was diluted with 5 ml of 10%
THROMBOCYTOSIS
257
photograph on to a millimeter grid and measuring the longest
dimension of cells. One hundred cells were counted and those
having a size larger than 5 p m were considered to be megathrombocytes.
RESULTS AND DISCUSSION
601
I
0
I
5
I
13
DAYS
2;
’++
Figure I . Throtnhocyto.sis in adjuvant-induced polyarthritic rats. Rats
Itwe inoc.ulutrd \t.itli M tuberculosis adjuvant at daj. 0. Mean values are
plotrrcl 1tY1h runge k SE shoitw bj, vertical bars. Number of rats are
= controls: 0 = arthritic rats. V = rats
indicated heside the ralues.
with mild or slightlj* obserirable arthritic reactions; = daily doses of
aspirin. 200 tnggihg. ( A S A I for 5 days.
EDTA and a I ml-aliquot of this primary dilution further
diluted with 23 ml saline. This gave a final platelet dilution of 1
in 12.000. The erythrocyte sedimentation rate of severely arthritic rats was very high. A I h o u r sedimentation time was
therefore chosen to eliminate the possible error arising from
the difference in the sedimentation rates among the samples.
Platelets were counted electronically. using a Celloscope 401
automatic particle counter.
Detection of Platelet Aggregates. The simple plateletcount ratio technique of Wu and Hoak ( 7 ) was used to demonstrate circulating platelet aggregates. Arterial blood was drawn
into two polypropylene syringes, one containing 2 ml buffered
EDTA/formalin solution and the other buffered EDTA solution only. Circulating platelet aggregates were fixed when exposed to formalin and sedimented at a faster rate than the
individual cells. After centrifugation the platelet counts on
both PRP samples were determined using !he Celloscope 401
counter. The results were expressed as follows:
Platelet aggregate ratio =
Platelet count in EDTA/formalin PRP
Platelet count in EDTA PRP
Platelet Size. The Celloscope 401 counter was used to
determine platelet size, based on the relationship between the
cell volume and discriminator setting (8). I n addition size and
distribution of platelets were determined from the Giemsastained film of EDTA anticoagulated PRP; EDTA was cmployed as anticoagulant to avoid the problem of clumping and
spreading of platelets with glass contact (9). Four different
microscopic fields were photographed (phase-microscope,
X
600). The size of the cells was determined by projecting the
Platelet counts were determined a t different intervals after the injection of adjuvant. T h e platelet count
was significantly elevated 13 days after the inoculation
of adjuvant. the time when the first signs of arthritic
swellings were observable (Figure I ). The highest platelet count, 1,375,620 per mm3 was found at 21 days, when
arthritic changes were fully developed. The highest
single counts were obtained in those animals showing
the greatest generalized arthritic reaction. Rats that had
a mild articular reaction to adjuvant had only a moderately elevated platelet count. The difference i n the platelet count between arthritic and control rats of similar
weight and ages declined after the first month but was
still significant 3 months after onset of the disease. Subacute treatment of severely arthritic rats with acetylsalicylic acid (aspirin) for 1 week reduced the platelet
count to normal.
I t has been suggested that the degree of thrombocytosis could be estimated as a reflection of an increase in the rate of platelet production and release
rather than a prolongation of the platelet life span. This
suggestion was confirmed by examination of the blood
films. Young platelets, known a s megathrombocytes.
account for roughly 10% of the number normally present on the stained smear of a healthy man. An increase
in the number is caused by accelerated platelet production. T h e number of large platelets was signilicaritly
elevated in the blood films from severely arthritic rats
(mean f SE for controls: 6.7 f 0.4%: for arthritic rats:
36.5 f 2.1%). Platelets. identified as niegathronibocytes.
were generally not larger than 8-10 p m .
An increased number o f large platelets in the
P R P of severely arthritic rats was also confirmed by the
electronic platelet volume determination. When the cell
volume (discriminator setting) was plotted against the
relative number of cells (readout), the slope of the two
curves-representing platelet volume distribution o f arthritic and normal rats-differed
significantly (Figure
2 ).
I t may be assumed that a premature discharge of
young cells from the bone marrow underlies the observed phenomenon. To clarify this assumption, the collection and counting of megakaryocytes in bone marrow
a n d morphologic studies of “platelet-like” cells in the
G o R o G AND KOVACS
258
blood smears of diseased rats are the subject of further
examinations. Nevertheless, increased numbers of circulating megathrombocytes in the clinical situation are
thought to be associated with clotting abnormalities,
which indicate a predisposition to the thrombotic state.
The same can be supposed to exist in the investigated
chronic animal disease.
Circulating platelet aggregates were demonstrated in the blood of severely arthritic rats (Figure 3).
Whether or not these aggregates are irreversible is an
open question at this stage. The fact that detection of
circulating platelet aggregates is associated with elevated
platelet count can be considered in two ways: I ) irreversible platelet aggregates are formed and sequestered in
the capillary bed causing a secondary stimulus for increased production or release of platelets, or 2 ) reversible aggregates are formed which are not retained in the
capillaries and cause no drop in the platelet count.
Short-term aspirin treatment was ineffective, whereas
prolonged treatment returned the decreased plateletcount ratio in severely arthritic rats to normal.
Adjuvant-induced polyarthritis in the rats, which
is an immunologic disease involving delayed hyper-
’*’
1
P<
0
00
C
P<
P>
0.001 0.01 0.05
SA
i
I
M A SA+ASA
Figure 3. Circularing plaieler aggregaies in rhe blood o j a r t h r i i i c rurs.
D r c n w s i d plureler wunr ratio ineans increased nunrber o/ circulating
pkuri~leruggregurrs. C’ = conrrols; SA = seaere!,. arthritic rats; MA =
rars t t i r l r mild o r .vlighr!,~obsercahle arthritic reacrion; SA + ASA =
~ ~ ~ c e r earrhriric
!).
rars rreared n,irh dai/y doses of 200 m g / kg aspirin
lAS,4 /or 5 duI.s. The lusr doses were udtninistered 4 hours heJorc. hlood
cdlec~rion.E.vpi~rinrrtrrstt’ere done .from 22 to 26 davs a j k r /he irrocularion with uc/;itautir.
0
10
20
30
la
i
50
60
70
DISCRIMINATOR SETTINGS
Figure 2. Platelei volume distribution curves measured by electronic cell
counrer. Discriminator sertings can be expressed in terms ofplatelet size
in p 3 ajier appropriate calibration. but in this report discriminator settings have been used lo designate relative platelet size. Shaded area
represents the niean f SE of five conrrol curves. Each line represents
[he plurelet aolutiie distribution curve o j a severel.)? arthritic rar.
sensitivity, is clinically and morphologically much like
rheumatoid arthritis in human beings (43). I n a recent
study Hutchinson et al. ( I ) found thrombocytosis in a
high percentage of rheumatoid arthritic patients and an
association appeared to exist between thrombocytosis
and extraarticular manifestation (arteritis) of rheumatoid disease. Thrombocytosis seen in the most severe
cases of rheumatoid disease was suggested to be the
result of a compensatory increase in platelet production
associated with active intravascular coagulation. The
recent results of Conn et al. (10) suggest that overcompensated intravascular coagulation and fibrinolysis
occur in rheumatoid arthritis. Thirty-seven percent of
their patients had elevated platelet count (>400.000 per
mm3). Under experimental conditions Selroos and Wegelius (II ) observed thrombocytosis in fibrinogen-induced arthritis in rabbits.
Our present data confirm these results in the
most suitable and frequently used animal model of rheu-
THROMBOCYTOSIS
matoid arthritis. Elevated platelet count a n d platelet
aggregates in the circulation of arthritic rats, together
with increased aggregability in vitro (2), increased tendency t o form thrombi in vivo (3), and increased size of
platelets, appear t o be of theoretical interest a n d practical importance. Alterations in the number and behavior of platelets indicate a distinct tendency t o shift the
hemostatic equilibrium in favor of thrombosis in severe
arthritic disease. Whether these changes mean that
platelets are closely involved in the pathogenesis o r there
exists a real risk for thrombosis in adjuvant arthritis,
cannot be answered at present and further studies are
required.
259
4.
5.
6.
7.
8.
REFERENCES
I . Hutchinson RM, Davis P, Jayson MIV: Thrombocytosis
in rheumatoid arthritis. Ann Rheum Dis 35: 138-142, 1976
2. Giiriig P, Kovlcs IB: Alteration of platelet behaviour
during various conditions, Inflammation Biochemistry
and Drug Interaction. Edited by A Bertelli, JC Houck.
Amsterdam, Excerpta Med, Congress Series 188, 1969,
pp 197-203
3. G6rog P, Kovlcs IB: Thrombus formation, hemostasis,
9.
10.
11.
adhesiveness of leukocytes and morphological abnormalities in the microcirculation of adjuvant arthritic rats.
Agents Actions 6:607-613, 1976
Schmidt K, Ott VR: Vergleichbarkeit der Adjuvanskrankheit der Ratte mit Krankheiten des Menschen. Schweiz
Med Wschr 103:1099-1103, 1973
Mohr W, Wild A: Adjuvant arthritis. Arzneim Forsch
(Drug Res) 26: 1860-1 866, 1976
Bull BS, Schneidermann MA, Brecher G: Platelet counts
with the Coulter counter. Am J Clin Path 44:678-688,
I965
Wu KK, Hoak JC: A new method for the quantitative
detection of platelet aggregates in patients with arterial
insufficiency. Lancet 2:924-925, 1974
McDonald TP, Odell TT, Gosslee DG: Platelet size in
relation to platelet age. Proc SOC Exp Biol Med
115:680-689, 1964
Garg SK, Amorosi EL, Karpatkin S: Use of megathrombocyte as an index of megakaryocyte number. N Engl J
Med 284:11-17, 1971
Conn DL, McDuffie FC, Kazmier FJ, Schroeter AL, Sun
NCJ: Coagulation abnormalities in rheumatoid disease.
Arthritis Rheum 19:1237-1243, 1976
Selroos 0, Wegelius 0: Thrombocytosis in fibrinogen
induced arthritis in rabbits. Scand J Rheumatol
2:167-173, 1974
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