Platelet aggregability in chronic arterial occlusive diseases of the extremities

0049-3848/80/220363-11$02.00/O THROMBOSIS RESEARCH 20; 363-373, 1980 Printed in the USA. All rights reserved. Copyright (c) 1980 Pergamon Press Ltd

IN CHRONIC ARTERIAL OCCLUSIVE DISEASES OF THE EXTREMITIES

PLATELET AGGREKABILITY

Hiroshi Kobayashi and Yoshio Mishima Department of Surgery, University of Tokyo, 7-3 Hongo, Bunkyo-ku, Tokyo, Japan (Received 20.8.1980; in revised form 25.8.1980. Accepted by Editor N. Aoki. Received by Executive Editorial Office 10.11.1980)

ABSTRACT Platelet aggregability in 120 male patients with either Buer er's disease (TAO) or arteriosclerosis obliterans (AS0B was studied by optical density method. The results were compared with the clinical courses of the diseases. Hypoaggregability was observed with an addition of small amounts of ADP or collagen in TAO patients at the chronically aggravating and stationary stages. This hypoaggregability suggested to indicate some pathologic changes in vivo which may precede and promote thrombosis. Remarkably enhanced aggregability was noted in AS0 patients at acute occlusive stage and in TAO patients at acute stage of thrombophlebitis migrains. Some differences in platelet aggregability between TAO and AS0 were also discussed.

INTRODUCTION The cases experienced in Japan as the chronic arterial occlusive diseases of the extremities are mostly regarded as Buerger's disease(l-3). Progressive aggravation has been pointed out as one of the clinical characteristics of the disease, but it has not yet been fully understood whether this aggravation is due to the relapsing arteritis itself or to the development of simple secondary thrombosis. In any event, platelets are considered to be involved in the process of the aggravation. It has been widely accepted that platelets play an

Key words : platelet aggregability, Buerger's disease, arteriosclerosis obliterans, ADP, collagen 363

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important role in arterial thrombosis, especially in the initial process (4-7). Although extensive studies have been carried out on platelet functions in various thromboembolic diseases (8-13), no systematic study in Buerger's disease has been reported yet. So this report concerns platelet function in the patients with arterial occlusive diseases of the extremities, especially with Buerger's disease.

MATERIALS

and METHODS

One hundred and twenty male cases with either Buerger's disease(TA0) or arteriosclerosis obliterans(ASO) were studied repeatedly. The patients were classified into four stages according to the clinical course of the disease at the time of the study, as follows. (1) Stationary stage : no clinical change could be detected more than 2 months both before and after the study. (2) Improving stage : apparent improvement was noted both in the clinical and laboratory findings at the time of the study. (3) Aggravating stage : chronic aggravation was proved both in the clinical and laboratory findings at the time of the study. (4) Acute occlusive or TPm stage : acute main arterial occlusion occurred within 2 weeks before the study. Among the patients with TAO, the cases at the acute stage of thrombophlebitis migrans(TPm) were included in this group and compared with the cases of AS0 at the acute occlusive stage. The normal control group was composed of 50 healthy males in whom no abnormality was found by general screening examinaThese are shown in Table 1. tions. All drugs which were claimed to affect platelet function were withheld more than 10 days before the study and cigarettesmoking was discontinued more than 24 hours before the study. Blood was drawn from a vein into a plastic syringe containing 1 part of 3.8 percent sodium citrate to 9 parts of Platelet-rich plasma(PRP) was obtained by centrifugation blood. at 100G. for 10 minutes, and the blood residue was further centrifuged at 1000G. for 20 minutes to obtain platelet-poor plasma(PPP). Platelet aggregation was measured by optical density method (14) by using an aggregometer (EEL Model-169, Evans, Essex, England). Platelet aggregation was induced by two kinds of aggregating agents, adenosine 5' -diphosphate(ADP) and collagen suspension. ADP(Sigma, St. Louis, U.S.A.) was dissolved in physiological saline and 4 different amounts of ADP were used: 0.5, 1.0, 2.0 and 5.9 pg in 0.6 ml of PRP. Collagen suspension was prepared by adding 200 mg of bovine Achilles-tendon collagen '(Sigma, St. Louis, U.S.A.) to 30 ml of physiological saline followed by homogenization and centrifugation at 100G. for 5 minutes, and the resultant supernatant was used as the original The original suspension was diluted 2 and 4 suspension(C3). times(C2 and Cl, respectively), and 10 ~1 of each suspension was added to 0.6 ml of PBP. To evaluate platelet aggregability, two parameters were employed, maximum aggregation rate (MAR, the maximum change in

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TABLE 1 Number of the Cases at Each Stage

Age

Case

Control Group

48.5*12.0

50

TAO

43.2k9.7

60

(Mean*S.D.)

Stationary

47

Stage

Aggravating

24

Stage

Improving Stage

11

TPm Stage

15 62.5k8.1

AS0 Stationary

35

Stage

6

With Graft Aggravating

60

10

Stage

8

Improving Stage Acute Occlusive

4

Stage

transmittance in 5 minutes after an addition of the aggregating agents, expressed as percentage of the transmittance of PPP) and the appearance rate of the secondary aggregation at each amount of ADP. Other examinations routinely carried out were bleeding time, coagulation time, hematocrit, white and red blood cell count, platelet count, prothrombin time, capillary fragility test (Rumpel-Leede), serum fibrinogen and fibrin/fibrinogen degradation products(FDP) determinations.

RESULTS Normal control group The distribution of maximum aggregation rates (MAR) of the control group is shown in Fig. 1. The mean values were 20.1 f 5.3 (Mean * S.D.)% at 0.5 pg, 47.9 * 12.5% at 1.0 yg, 63.7 * 9.4% at 2.0 pg and 77.4 * 9.7% at 5.9 p$ ADP, and 39.7 4 28.2% with Cl, 71.7 * 10.3% with C2 and 81.5 - 8.0% with C3 collagen suspension. The appearance rates of the secondary aggregation were 2.0% at 0.5 pg, 62.0% at 1.0 ug, 94.0% at 2.Opg and 100% al~,5.C)~{gADP (Fig. 2). P R The results are shown in Table 2. The mean MAR values in the patients at the aggravating and stationary stages were significantly lower than those in the control group at 0.5, 1.0 and 2.0 pg ADP, and with Cl and C2 collagen suspension, whereas no significant difference was noted between the results in TAO patients at the improving stage and those in the control group.

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PLATELET AGGREGATION IN TAO

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U

i!

Ii3 . bIi’ : . .: i F fzI . ”

:

.

7

: . .

:. i:

:.

.

f-l

*

ip-’

”

()

.

9.0

1

w

, Mean k S.D.

.

.

.

7 T

0’

-

; .

095

LO

2.0

5,9

ADP

rg

'Yfi-

c2

c3

Collagen

FIG. 1 Distribution of the maximum aggregation rate in the normal control group

FIG. 2 Appearance rate of the secondary aggregation in the normal control group

ADP

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The mean MAR values in the patients at the TPm stage were significantly higher than those in the control group at any amount of ADP or collagen suspension. The appearance rates of the secondary aggregation obtained by 1.0 pg ADP in the patients at the stationary and aggravating stages were 19.1 and 12.5%, respectively, and both 'E;;r;hzignificantly lower than those in the control group. contrary, the rates in the patients at the improving and TPm stages obtained by 0.5 pg ADP were 27.3 and 53.3%, respectively, and both were significantly higher than those in the control group. -results are shown in Table 3. No significant difference from the control group could be detected in the patients at the aggravating stage. The mean MAR values in the patients at the improving and stationary stages were significantly higher than those in the control group at 0.5, 1.0 and 2.0 pg ADP. The mean MAR values with C3 in the patients at the stationary stage and with Cl at the improving stage were also significantly higher. The mean MAR values in 6 patients with preceding dacron bypass grafting, who were at the stationary stage at the time of the study, were significantly lower at 1.0 and 2.0 pg Remarkably high ADP, and with C2 and C3 collagen suspension. mean hiAR values were noted in 4 patients at the acute occlusive stage at all amounts of ADP or collagen suspension. The appearance rates of the secondary aggregation at 0.5 pg of ADP were significantly higher than those in the control group, irrespective of the stage.

DISCUSSION The chronically aggravating stage of TAO, which is a gradual impairment of the peripheral circulation, may be explainer3 best by arterial thrombus formation, which is considered to be promoted either by relapsing inflammatory process of the artery or by development of secondary thrombosis. The thrombus formed may be partially resolved and, at the same time, partially organized and thus produce gradual narrowing of the arterial lumen. And it may be also possible that even if thrombus formation does not take place, platelet aggregation and adhesion to the impaired endothelial cells produce thickening of the intima followed by narrowing of the arterial lumen(l5). At the improving stage of TAO, these pathologic processes may not take place and the development of collateral circulation or recanalization in some cases will produce the improvement of the peripheral circulation. This notion will be partially su ported by the finding that significantly elevated (>lO pg/ ml P FDP was detected in 20.8% of TAO cases at the aggravating stage but in no case at the improving stage. This was also true in AS0 patients. Elevated FDP was detected in 4% of the cases at the aggravating stage but in no case at the improving stage. As for platelet aggregability, hypoaggregability was discovered at relatively small amounts of ADP or collagen (0.5 2.0 pg ADP, Or Cl and C2 collagen suspension) in TAO atients at the aggravating stage. When sufficient amounts of AD p or

50

47

24

11

15

Control Group

Stationary Stage

A=;;;ting

Improving Stage

TPm Stage

n=

2.0

5.9 (I-lg)

24.7+9.f

8.M.;

86.1+6.2

c2

c3

54.4+26.2*82.0212.4

+ O.O5>P

53.f

27.y

0

2.1

2.0

0.5

80.0

45.5

12.;

19.1"

62.0

1.0

72.8 100.0

62.5 100.0

63.8 100.0

94.0 100.0

2.0

* O.O05>P

100.0 100.0

ADP

Appearance Rate of Secondary Aggregation (.%I

itO.O25>P>O.O1

61.8+26.,+54.5*7.2* 93.6k4.i

32.6k33.9 67.1224.5 82.3k10.2

8.3212.;

14.7222.; 55.6+26.9*79.8212.3

39.7228.2 71.7k10.3 e1.5+5.0

Cl

Collagen

Difference from control group

29.7217.0k59.9f18.Zt76.4k8.7

17.8k15.1 45.3221.9 64.2k14.6 78.0+7.8

51.8~16.& 79.327.5

29.7+13.$54.4+16.3* 77.527.9

47.9212.5 63.7t9.4' 77.429.7

1.0

9.5t5.z

2O.lk5.3

0.5

ADP

MAR (%>

TABLE 2 Results in the Patients with TAO

84.8r6.r

91.3k3.r

Difference from control group

78.3k6.r

4

Acute Occlusive Stage

72.5k6.y

47.1~21.~68.0f10.~73.3+12.~80.5~12.5

8

c2

Collagen

81.528.0

c3

73.2U5.T

itO.O25>P>O.O1

60.0

50.0

71.4

62.0

1.0

1oF

100.0

62.?100.0

30.8

33.y

37.y

2.0

0.5

94.0 100.0

2.0

** O.O05>P

100.0 100.0

100.0 100.0

100.0 100.0

83.3 100.0

100.0 100.0

ADP

Appearance Rate of Secondary Aggregation (X>

* 0.01>P>0.005

73.5+11.r83.0+9.1+ 92.026.7

61.6?16.?74.8?12.1 83.3k13.6

45.8232.3 69.4219.7 82.9k14.0

32.2k32.6 57.5+2&y

50.9228.1 73.8k14.5 86.0?10.?

39.7k28.2 71.7UO.3

Cl

+ O.O5>P

45.8k20.1 68.6k12.1 78.0+10.1

17.5k13.2 33.3+15.-!-52.2kll.t73.7ilO.l

32.6?20.~58.0+19.tiJ(70.7t12.4k 80.129.2

77.4k9.7

5.9 (1.18)

Improving Stage

6

With Graft

2.0

47.9212.5 63.7k9.4

1.0

17.327.1

35

Stationary Stage

2O.lk5.3

0.5

Aggravating Stage 10

50

Control Group

n=

ADP

MAR (%>

TABLE 3 Results in the Patients with AS0

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PLATELET AGGREGATION TAO

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collagen (5.9 ng ADP or C3 collagen suspension) were used, the results were identical to those obtained in the control group. In contrast with this, in TAO patients at the improving stage, the mean MAR values were not significantly different from those in the control group and, moreover, the appearance rate of the secondary aggregation at 0.5 ug ADP was even significantly higher than that in the control group. Thus, hypoaggregability at small amounts of the aggregating agents was considered to be characteristic of TAO patients at the aggravating stage. This relative difference of platelet aggregability between these two stages was confirmed also in AS0 patients. These findings are considered to show that the hypoaggregability measured in vitro has close relationship to the clinical chronic aggravation of the disease and, at the same time, to indicate some pathologic changes in vivo which promote thrombosis. The exact mechanisms causing this hypoaggregability are not yet clear, but something like "partial release reaction" (16) may be one of the possibilities. And it may be possible that when platelets are repeatedly exposed to some kinds of minimal stimulation, minimal enough not to induce irreversible changes in platelets such as metamorphosis, they may be functionally exhausted and their aggregability by small amounts of aggregating agents can be reduced. And if the lifespan of these platelets is not significantly shortened (17), these affected platelets can constantly occupy a certain portion of the platelet population in the peripheral blood. O'Brien et a1.(12) reported decreased aggregation response to thrombin in patients after recovery from myocardial infarction. They proposed "refractory" state as the mechanism of this phenomenon. In TAO patients at the stationary stage, platelet aggregability was noted to be comparable to that at the aggravating stage. This result must be underlined, because, from the standpoint of platelet aggregability, it may be reasonably accepted that at both stages there are almost the same pathologic changes preceding actual thrombus formation. In fact, chronic relapsing and aggravating episodes have been observed later in many of TAO patients who were clinically stationary at the time of the study. Enhanced aggregability was noted in AS0 patients at each stage except the aggravating one. This finding cannot be attributed to aging itself, because we had already confirmed no significant correlation between the age and MAR or the appearance rate of the secondary aggregation in the subjects of the control group (18). One of the causative factors for this hyperaggregability in AS0 patients may be in the plasma such as lipids (8) (19-24). Both in TAO patients at the TPm stage or AS0 patients at the acute occlusive stage, remarkably enhanced aggregability was observed. It is probable that greatly increased platelet consumption at the site of thrombosis promotes accelerated turnover of platelets followed by increased number of young platelets in the circulating blood. Young platelets are functionally more active than old ones (25, 26), and thus enhanced aggregability can be explained. But considering that the patients at such acute stages are in a severely stressed condition, we presume many other unknown factors may contribute

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to the enhanced platelet aggregability.

CONCLUSION (1) Platelet aggregability in 120 male patients with arterial occlusive diseases of the extremities (60 cases with TAO and 60 cases with ASO) was studied by the optical density method. The results were discussed mainly with respect to TAO by comparing with close observation of the clinical courses of the diseases. (2) Hypoaggregability at small amounts of ADP or collagen was noted in TAO patients at the chronically aggravating stage. This finding was considered to indicate some pathologic changes in vivo which may precede and promote thrombosis. (3) Platelet aggregability in TAO patients at the stationary stage was identical to that at the aggravating stage. This suggested to indicate that some pathologic changes similar to those at the aggravating stage exist also in the patients at the stationary stage, even though their clinical conditions were seemingly stationary. (4) Enhanced platelet aggregability was found in AS0 patients except those at the aggravating stage. Some factors such as li ids in plasma may be attributable. (57 R emarkably enhanced aggregability was noted both in AS0 patients at the acute occlusive stage and in TAO patients at the TPm stage. (6) Based on the findings stated above, it was concluded from the standpoint of platelet aggregability that TAO has a pathophysiologically different aspect from ASO.

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7. DIDISHEIM. P., KAZMIER. F.J., and FUSTER, V. Platelet inhibition in the management of thrombosis. Thromb. Diath. Haemorrh., 32, 21-33, 1974. 8. MUSTARD, J.F. and PACKHAM, M.A. Factors influencing function: Adhesion, release, and aggregation. Pharmacol. Rev., 22: 97-187, 1970.

platelet

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