Prevention by Aspirin of the Classic Generalized Shwartzman Reaction Jean-Gilles Latour, PhD, and Fran9ois Bernard, MD

The classic generalized Shwartzman reaction induced in the rabbit was prevented with large doses of aspirin (250 mg/kg) when administered at the time of the first and preparing injection of endotoxin. Such a result was not observed when the drug was given at the time of the second and provoking injection of endotoxin. Our investigations on platelet and coagulation indicate that aspirin prevents disseminated intravascular coagulation through an interference with the blood coagulation and Hageman factor activation rather than by the inhibition of platelet aggregation and availability of platelet procoagulant activity. (Am J Pathol 91:595-606, 1978)

THE GENERALIZED SHWARTZMAN REACTION (GSR) iS characterized by bilateral renal cortical necrosis produced by the thrombotic occlusion of the glomerular capillaries.' The phenomenon is the result of a complex interplay among coagulation, fibrinolysis, platelets, and vasomotor activity of the microcirculation subsequent to the entrance of bacterial endotoxin in the circulation.2'3 The process by which endotoxin triggers coagulation in vivo involves primarily the Hageman factor 6 and leukocytes,6'7 but the role played by the platelets is not clear. Thrombocytopenia induced by specific platelet antibody 8 but not by busulphan 9 prevents the Shwartzman reaction, and the injection of platelet factor 3 extracts or substitutes in animals has failed in most instances to induce significant intravascular coagulation.'0 However, in addition to making factor 3 available,1" endotoxin is known to induce platelet aggregation 12 and the release of several other platelet constituents which are active on the microcirculation and blood coagulation "I and, therefore, capable of taking part in the pathogenesis of the Shwartzman reaction. The outcome of several drugs interfering with platelet function has led to the hypothesis that these could be beneficial in the prevention and treatment of intravascular coagulation. Aspirin, the most investigated platelet inhibitor, has been already reported to antagonize some hemodynamic changes in experimental endotoxin shock 14,1" and to prevent the From the Laboratory of Experimental Pathology, Institut de Cardiologie de Montreal and the Department of Pathology, University of Montreal Faculty of Medicine, Montreal, Quebec. Supported by Grant MA-4478 from the Medical Research Council of Canada and the Jean-Louis

Levesque Foundation. Dr. Latour is Chercheur Boursier, Conseil de la recherche en sante du Quebec. Accepted for publication February 10, 1978. Address reprint requests to Dr. Jean-Gilles Latour, Institut de Cardiologie de Montr6al, 5000 Belanger Street East, Montreal, Quebec HIT 1C8, Canada. 0002-9440/78/0608-0595$01.00 595

596

LATOUR AND BERNARD

American Journal of Pathology

Shwartzman reaction.16'17 However, this last observation has been challenged by Slabber et al.'8 To clarify the role of the platelets and to assess the therapeutic value of aspirin in endotoxin-induced intravascular coagulation, the drug was tested on the classic generalized Shwartzman reaction in rabbits. Our results show that aspirin, given in large doses at the time of the first and preparing injection of endotoxin, prevents the Shwartzman reaction through an action on the coagulation system and Hageman factor rather than by inhibitions of platelet aggregation and availability of platelet procoagulant activity. Materials and Methods Animals

Male New Zealand albino rabbits, 1.9 to 2.1 kg, from the Canadian Breeding Farm were used. They were housed in a constant-temperature environment and given standard chow (Ralston Purina Canada Ltd.) and water ad libitum. With the rabbits under pentobarbital anesthesia (30 mg/kg), blood samples were collected from the jugular vein after a small skin incision was made. Endotoxin

Escherichia coli endotoxin (0.55:B5, Boivin type, Difco Laboratories, Detroit) was dissolved in physiologic sterile saline and injected through the marginal ear vein at the dosage of 100 ,ug in a 2-ml volume. Aspirin

Acetylsalicylic acid (Brickman & Companies, Montreal) was suspended in distilled water. The drug was fed to the rabbits at the dosage of 250 mg/kg via a stomach tube. Clotting Tests and Factor Assays Plasma recalcification times, cephalin times (in plastic tubes, in unsiliconized glass tubes, or in the presence of celite), Hageman factor, platelet counts, platelet procoagulant activity, and hematocrits were determined as previously described." Bell and Alton " rabbit brain cephalin extract was used as the platelet phospholipid substitute. The platelet coagulant activity was expressed in percentage of the pre-experimental or control animal values, which were set arbitrarily as 100%. For this purpose, dilution curves (100 X 108 to 600 X 101 platelets/cu mm) from control and treated animals were run simultaneously. Prothrombin times were measured on a fibrometer (BBL, Becton Dickinson Co., Cockeysville, Md.) with the use of simplastin (Warner Chilcott, Scarborough, Ontario). Fibrinogen was assayed according to the method described by Ratnoff and Menzie.20 Platelet Aggregation

Aggregation of platelets in plasma (PRP) was tested with collagen as previously described.' The assays were performed with platelet counts adjusted to 700 X 10'/cu mm. The surface area under the curve after 3 minutes was estimated and taken as the measurement for the extent of platelet aggregation.

Vol. 91, No. 3 June 1978

ASPIRIN AND THE SHWARTZMAN REACTION

597

Histology

At the end of the experiments, the rabbits were killed with an overdose of pentobarbital. The kidneys were fixed in 10% neutral formalin, and histologic sections stained with phosphotungstic-acid-hematoxylin were examined for fibrin thrombi. Experiments Series 1: Effects of Aspirin on Platelet Aggregation to Collagen and on Blood Coagulation

Four hundred sixty rabbits were divided into two groups (Table 1, Text-figure 1). The first group was given aspirin and the second received water. Blood was taken after 1, 2, 4, 12, and 21 hours in, respectively, 50, 42, 39, 45, and 53 rabbits from each group. All the investigations listed in Table 1 were conducted at each time in 8 to 14 rabbits from Groups I and II. To avoid effects of successive blood removals and manipulations, each animal was used only once and at a specific time. However, groups of assays and tests were performed simultaneously on the same animal as follows: Experiment 1: platelet aggregation; Experiment 2: platelet procoagulant activity; Experiment 3: clotting times, fibrinogen, Hageman factor, and hematocrit. Series 2: Prevention of the Generalized Shwartzman Reaction by Aspirin: Effects on Hageman Factor, Fibrinogen, and Platelet Consumption

Three groups of 35 rabbits were treated as illustrated in Text-figure 2. All the rabbits were given two intravenous injections of endotoxin 21 hours apart. One hour before and again 2 hours after the first injection of endotoxin, Groups I and III were given water and Group II received aspirin. One hour prior to the second injection of endotoxin and again after 2 hours, Group III was given aspirin and Groups I and II received water (Table 2). Two hours before the experiment, at 4, 12, and 20 hours after the first injection of endotoxin and at 2, 4, and 8 hours after the second injection of endotoxin, blood was collected from 18 animals from each group for the measurement of Hageman factor (Textfigure 3), fibrinogen (Text-figure 4), and the platelet counts (Text-figure 5). Eight hours after the last injection of endotoxin, the animals were killed and the kidneys were removed for histologic examination.

Results Series 1. As indicated in Table 1 and illustrated in Text-figure 1, aggregation to collagen was decreased by 73% (P < 0.001), 88% (P < 0.001), 81% (P < 0.001), 48% (P < 0.001), and 30% (P < 0.05) at 1, 2, 4,

12, and 21 hours after administration of aspirin, respectively. The platelet procoagulant activity was reduced by 50% (P < 0.001) at 1, 2, and 4 hours after aspirin. The activity gradually returned to normal and exceeded the pre-experimental range after 21 hours by 50% (P < 0.001). These changes were paralleled by prolongations of the plasma recalcification time at 1 (32%, P < 0.001), 2 (47%, P < 0.001), and 4 hours (43%, P < 0.001) and by a shortening of 20% (P < 0.001) 21 hours after administration of aspirin. Evaluations of cephalin times carried out in plastic tubes were prolonged by 20% (P < 0.001) at 1 hour, by 23% at 2 hours, and by 10% at 4 hours after aspirin. They returned to normal thereafter. When determinations of cephalin times were performed in unsiliconized

598

American Journal of Pathology

LATOUR AND BERNARD

(V). 0 V C)00 -H -H -H -H -H -H -H -H 0 0 0 10 0o0-V.o 0060 It cV0 10 0

N'0 -H -H cV)_

CO)

-H

I-

.

0 o o o o o oCV) ° C#V) y-0 CV04 CNO V

0

08

o 0)

.

_

N-

CD

--H -H

0

0

I N 2

1%-'0'N CI8 ts v o V) 00) 00 CM t-t

N

_

-

N

eu

4+ -H )

N

0 0

_OR 00

-H -H -H

-H -H

.

D

.

'-CV -

0 C

0

Ul

0

'_ N CV0)

b-

N

C

e

r 00 -H -H

-H -H -H

-H

-H

c

C-)Ir) -C

-H

-H H-H

10

Cj

_

O

OO

aM0N

_

-

-H-H -H-H -f -H -H -H H -H 10V) 00 CV) CCV) 0 0 Go CO)( v 0 o0o ') ' 00 ,- CVP) 10) '- N ' N -CV) O

O

O.~ N -

N CM0 3')

t _

o)

CO) 00 0

cm'N

4t

0

-H-

H-H

-H

V) 0

0)

cV)'0CO 0

11-

0

O

'-0 OO t-H -H

CO It

o t

CV-_) -H I

0 Ot NY

Le v'-

.t _No'

**

*

V)

100

40 o

CV)

to

0o

-H -H -H GoCV)LO 00 ^ 4.

-H

-H H -H

-H 0

_-i-

8Co 0

0

-H -H .z

010t

0T~N cn' NO r 1 ' . : CV)'-0

1%

-H -H C00 O 00

CO O c0'4 O L OM 0

Cj C,NM

OM N

t-.

00

H

-

0

N

C

'°V)

r.-' Nm 0 '4- 0 04 0 +HH-H -H -H 10 0- 0D'4' 0o 001c CV)O 0)0 VW ON '-) _10 o'-'4' N0CM 0No '4ClV) _ _ OO CO 0 '-0

0

-H -H

CO) N-

0

0

Ua

-H- -H 00 O o '-,

'EL co 0 0:

0

_

-H H

-

0

% 0 0 N t)

H1

c

'C

4H

C)0

0o ) '4 N '-4 C) 1Vtoi-

0

0

cm

_-I_

._

0

T-

-0 -H-H 0G

-

-H-H

CM

C

co

CM

1

U)

0:

NO 00

N0

H -H+1 -H-H rr-to t.. '- 0) 00I C O C'N 0 0 an)

H-

-

c 0

o

CO0

c c i

*

0

*

NO

0

q

,

-H

H -H

4-

Nm

10

CN 0Q v

H

4

0 CM

Ov N

1

Co-LcE

0=o UJN

1- t H -H-H oNM V-'

-H

'NO0I c LO CV)h -

C0

>cE as>'

0 E C_

w

o

'F.

o :1,-mt

s-

0

o )0

-I-

C.

.0 .0

0E 0

a. 1

V V000 V,

1"

(L L L O) V V++C

t.l

ASPIRIN AND THE SHWARTZMAN REACTION

Vol. 91, No. 3 June 1978

TEXT-FIGURE 1-Effect of aspirin on platelet aggregation induced by collagen in vitro in the rabbit. ASA (250 mg/kg) was fed to 60 rabbits; the aggregation to collagen was tested on groups of 12 animals after 1 (D), 2 (F), 4 (E), 12 (C), and 21 hours (B). Simultaneously, five groups of 12 rabbits were given distilled water, respectively, at 1, 2, 4, 12, and 12 hours before the test (A). Results: mean and typical curve responses.

I

0

1

599

A

B

C D E F

2

3

MINUTES

glass tubes, prolongations of the clotting time of 37% (P < 0.001), 47% (P < 0.001), and 16% (P < 0.001) were also observed at 1, 2, and 4 hours after administration of aspirin. However, the celite-activated cephalin times and prothrombin times remained unchanged after the administration of aspirin. No significant changes in Hageman factor, fibrinogen, platelet counts, and hematocrit were encountered after administration of aspirin. Series 2. As shown in Table 2, the incidence of the GSR was only 8.5% (P < 0.001) in Group II given aspirin at the time of the first injection of endotoxin, compared with 65.8% and 68.5%, respectively, in Groups I and GROUP -1 _

0 2

::S

GROUP Water Endotoxin Water

:

GROUP

N

_.- ASA -

Endotoxin

---ASA

0

Water

Endotoxin Water

::

::::

-:

20-.

21 23

....

..

Water Endotoxin Water

...

X

:::.

-Water -Endotoxin - Water

l: ..4

*: -l

.:-:@l

_ ASA -

Endotoxin

_ ASA

>::.4

,-.

*.-:-4 *--:: ::::

29

-Sacrifice

ASA: 250 mg/kg orally

i.,

-Sacrifice

:.-:. -::-:

;;|;

_ Sacrifice

Endotoxin: E coli 055: B5, 0.1 mg IV

TEXT-FIGURE 2-Experimental design for the study of the effects of aspirin on the Shwartzman reaction.

600

LATOUR AND BERNARD

American Journal of Pathology

Table 2-Prevention of the Generalized Shwartzman Reaction by Aspirin Group

Treatment

I 11 IlIl

Water ASA (-1, 2)* ASA (20, 23)*

Glomerular No. rabbits thrombosis (%) 35 35 35

* Hours before or after the first NS = not significant.

injection

65.8 8.5 68.5

P -

Prevention by aspirin of the classic generalized Shwartzman reaction.

Prevention by Aspirin of the Classic Generalized Shwartzman Reaction Jean-Gilles Latour, PhD, and Fran9ois Bernard, MD The classic generalized Shwart...
1MB Sizes 0 Downloads 0 Views