IHROMBOSIS RESEARCH Printed in the United
Suppl. II, Vol. 8, 19'76 Pergamon Press, Inc.
States
SECTION
ROLE
OF BLOOD
PLATELET
IN THE
V
FORMATION
OF THROMBUS
T. Maekawa,
N. Kobayashi, H. Arai and T. Takeuchi Third Department of Internal Medicine Gunma University School of Medicine Maebashi, Japan 371
ABSTRACT In the busulfan-induced thrombocytopenic rabbits, the femoral artery was completely occluded after injection with ellagic acid at the site of stenosing ligature in 5 out of 15 rabbits, while in the control rabbits with normal platelet count, a large thrombus was present around the stricture in 12 out of 17 rabbits. Occluding thrombus was produced in only 2 out of 15 rabbits which ingested more than 0.05 g of aspirin 6 hours before surgery. Rate of occluding thrombus formation on the chemically intimectomized femoral artery with diluted sulfuric acid was evidently high in the rabbits with rebound thrombocytosis after transient thrombocytopenia induced by intravenous injection with anti-platelet serum. In the case of stasis thrombus formation, both in thrombocytopenit rabbits and in aspirin-ingested rabbits, percentage of rabbits producing the thrombus was not significantly different from that of the control. From the results described above, it is suggested that both quantitatively and qualitatively normal platelets are required for the production of arterial thrombosis. On the other hand, blood platelets may not contribute to the formation of stasis thrombosis.
INTRODUCTION Among the numerous factors which are relevant in the genesis of arterial thrombosis, it is suggested that platelet occupies a central portion. The importance of the role of platelets in the thrombus formation has been well accepted. This suggestion comes from the pathomorphological investigations on both clinical and experimental thrombosis. Indirect evidence supports this concept, but direct proof is lacking. Therefore, the following experiments have been performed in order to make 227
228
Suppl.
the relationship between platelet and thrombus The results obtained in these experiments will
i-i
formation clearer. be reported.
METHODS Production of arterial thrombosis: Under pentobarbital anesthesia the femoral artery of normal rabbits weighing 2 to 3 kg was exposed. After all branches from the artery were ligated, a Steel wire of 0.90 mm in diameter was placed alongside the femoral artery. The artery was loosely ligated near the distal end together with steel wire by thick silk thread. A stricture was made in the femoral artery by pulling out the steel wire. Ten mg of water soluble ellagic acid (Yamanouchi Pharmaceutical Co.) dissolved in 5 ml of distilled water was injected intravenously. Twenty-four hours after these procedures the rabbits were again anesthetized and the femoral arteries with stenosing ligature were exposed and examined macroscopitally for the presence of thrombosis. Stasis thrombus formation: A modified method of Wessler was employed. Normal adult rabbits were used. A segment of the jugular vein was freed from its surrounding structures and its tributaries were ligated under pentobarbital anesthesia. Ten mg of water soluble ellagic acid dissolved in 5 ml of distilled water was injected into the contralateral ear vein. Within 10 to 15 seconds after completion of the injection, the previously exposed jugular vein was gently isolated with two small vascular clamps. After isolation in situ for 30 minutes, the segment was then removed and its contents emptied into a Petri dish containing 30 ml of 5 % sodium citrate solution. The amount of clot formed (stasis thrombus) was scored on a scale from 0 to 4; a score 0 represented no clot, and a score 4 represented a single thrombus forming a cast of the isolated segment. Chemical intimectomy of the femoral artery: About 1.5 cm of the arterial segment was gently isolated in situ by two small vasSulfuric acid solution of 0.2 % was injected incular clamps. to the segment after the blood in the segment was withdrawn. the sulfuric acid solution was withExactly 2 minutes later, drawn and blood flow of the femoral artery was recovered by reThe formation of the occluding thrombus was moving the clamps. examined 24 hours after the chemical intimectomy. Induction of thrombocytopenia: Thrombocytopenia was induced by two intraperitoneal injections with busulfan in normal adult TwentyThe method of Evensen et al (1) was employed. rabbits. five mg per kg of body weight of busulfan dissolved in polyethylene glycol in a concentration of 10 mg per ml were injected Platelet counts decreased to less than 124,000 every other day. Leukopenia /cmm in 10 to 14 days after two shots of busulfan. while change in hematocrit values was minimal. accompanied, Aspirin ingestion: A capsule containing 0.01 g to 1.0 g of acetylsalicylic acid (Shionogi Pharmaceutical Co.) was given Various experimental procedures orally in the early morning. were started 6 hours after the aspirin ingestion. is esPreparation of anti-platelet serum: The method employed EDTA-PRP sentially similar to that described by Ohkuma (2). was separated from the femoral artery of normal adult rabbits. Washed platelet suspension was prepared from the EDTA-PRP by
suppI_.
II
PLATELETS
229
IN THROMBUS FORMATION
TABLE 1 Effecstof Aspirin Ingestion on Experimental Thrombosis of the Femoral Artery with Stenosing Ligature by 10 mg of Ellagic Acid Injection :abbit No Injection with Normal Saline
Body Weight (kg)
Sex
Occluding Thrombus
ET ET ET ET ET
9 10 11 13 14
2.1 2.4 2.2 2.3 2.1
M F F M F
;:; (+ 1 (- 1 (- 1
ET ET
1 2
3.1 2.9
F M
(+ (-
1 1
ET ET ET ET ET ET ET ET ET ET ET
3 4 5 6 7 8 21 22 23 24 25 26
3.3 2.7 2.0 2.2 1.9 2.0 2.2 2.2 2.0 2.0 2.1 2.0
F M F F M F F F F F F
i;; ((+ (+ (+ (+ (+
> > 1 > > >
ET 27 28 ET 29
2.3 2.4 2.1
F F
ir; (+ >
ET ET ET ET ET ET ET
31 32 34 35 36 37 38
2.4 2.2 2.2 2.0 2.2 2.2 2.2
F M M M F M F
;:; (((+ ((-
ET ET ET ET
41 42 43 44
2.5 2.2 2.1 2.3
M M M M
[r,' (- > (- >
ET ET ET ET
51 52 53 54
2.2
(-
;:! 2.3
F F F F
ET ET ET ET
61 62 63 64
2.4 2.1 2.3 2.4
F M F M
(+ (+ (+ (+
1;:' (+
>
1 > 1 1 >
1
;:; C - 1 1 1 1 >
MAEKAWAET AL
230
Suppl.
II
low speed centrifugation with EDTA-normal saline. One ml of platelet suspension containing 500,000 to l,OOO,OOO platelets per cmm was injected twice a week in lst, Znd, 5th, 6th, 9th and 10th week. At the end of 11th week, blood was collected from the immunized guinea pigs. The anti-platelet serum separated from thus collected blood was absorbed with rabbit erythrocytes. Antibody titer of the anti-platelet serum was 1 : a. Rabbits with rebound thrombocytosis: Platelet counts of the normal adult rabbits were rapidly decreased to 2 to 4 % of the prior values after single intravenous injection with 2 ml of anti-platelet serum. Following marked transient drop, platelet counts rose rapidly to more than prior values 4 days after the injection with anti-platelet serum. RESULTS (1) the
Effect of aspirin ingestion on experimental thrombosis of femoral artery with stricture. Results obtained in the control rabbits injected with normal saline instead of ellagic acid are presented at the top of the Table 1. A small thrombus was found at the site of the stricture in 1 out of 5 rabbits. On the other hand, a large thrombus was present around the stricture in 12 out of 17 The femoral artery was rabbits after injection of ellagic acid. completely occluded in only 2 out of ii rabbits which ingested Ingest ion more than 0.05 g of aspirin 6 hours before surgery. of 0.01 g of aspirin which is a dose having no effect on coliagen-induced aggregation of platelets, did not reduce the formation rate of the arterial thrombus by eliagic acid injection. (2) Effect of busulfan-induced thrombocytopenia on experimental thrombosis of the femoral artery with stricture. In the thrombocytopenic rabbits induced by the two shots of busulfan, production rate of the occluding thrombus was As shown in the lower part of the Table 2, a markedly reduced. large thrombus was produced in only 1 out of 5 rabbits with platelet count of less than 50,000 per cmm. (3) Effect of thrombocytosis on the formation of thrombosis at the chemically intimectomized femoral artery. As listed in the upper part of the Table 3, occluding thrombus yielded only in 33 % of the experimental rabbits with Yield of occluding thrombosis at the normal platelet count. site of the femoral artery intimectomized with 0.2 % solution of sulfuric acid significantly rose to 60 %, among the rabbits in rebound thrombocytosis induced by the injection with antiplatelet serum. (4) Effect of aspirin ingestion or busulfan-induced thrombocytopenia on the formation of stasis thrombus. ingestion had no effect As shown in the Table 4, aspirin Reduction of the on stasis thrombus by eliagic acid injection. circulating platelet counts also had no effect on the formation of stasis thrombus (Table 5). DISCUSSION It
is
presumed
that
the
initial
process
in the
formation
II
Suppl.
PLATELETS
231
IN 'TI-IROMBUSFORMATION
TABLE 2 Effect of Thrombocytopenia on Experimental Thrombosis of the Femoral Artery with Stenosing Ligature by 10 mg of Ellagic Acid Injection Rabbit No. B B B B B B B B B B B B B B B
30 31 32 33 34 35 36 37 38 35 40 41 42 43 44
Body Weight Sex Platelet Count Occluding Thrombus per cmm (kg) 2.0 2.3 2.3 2.4 2.3 2.2 2.3 2.3 2.3 2.3 2.0 2.3 2.4 2.3 2.3
F F M F F F M M F F F F F F F
82,000 88,000 66,000 124,000 98,000 68,000 84,000 96,000 64,000 116,000 24,000 42,000 7,000 30,000 40,000
TABLE 3 Effect of Thrombocytosis on Experimental Thrombosis of the Femoral Artery Chemically Intimectomized by 0.2 % of Sulfuric Acid Solution (Rabbits with Normal Platelet Count) Rabbit Body Weight Sex Platelet Count Occluding Thrombus No. (kg) CI 1 CI 2 CI 3 CI 4 CI 5 CI 16 CI 17 CI 18
3.0 2.8 2.9 2.7 2.6 3.2 2.5 2.9
Normal Normal Normal Normal Normal Normal Normal Normal
[;I ( - 1 ( + >
[I; ( + 1 ( - 1
(Rabbits with Rebound Thrombocytosis after anti-platelet Serum Injection) Rabbit Body Weight Sex ;,;:~;~;s;,;;~ Occluding Thrombus No. (kg) CI CI CI CI CI
25 26 27 28 29
3.0 2.4 2.4 2.5 3.0
F F F M M
214 104 72 129 128
% % % % %
(+) (+)
of occluding thrombus is adhesion and aggregation of platelets to the site of stenosing ligature. In this case, it is suggested that slight endothelial injury and disturbance of blood
232
WKAWA
ET AL
Suppl.
Ji
TABLE 4 Effect
of
Aspirin
Amount of Aspirin Ingested (g) 0
1.0 0.3 0.05 0.01
Ingestion No.
of
on Stasis
Thrombus
Formation
Rabbits
with
Thrombi
Scored
0
1
2
3
4
1 0 0 1 0
0 0 1 0 0
0 1 0 0 0
0 1 1 0 0
4 3 2 3 4
Total
5 5 4 4 4
TABLE 5 Effect Rakiit
ST ST ST ST ST
of
Thrombocytopenia
Body Weight
.
(kg)
1 2 3 4 5
2.0 2.3 2.4 2.3 2.3 Number of
on Stasis
Sex Platelet per M F F M F Rabbits
Count cmm
82,000 88,000 119,000 66,000 99,000
Thrombus
Formation
Score
Thrombus
0
of 1
2
3
4 X X X
X
0
0
0
1
X
4
flow due to placing of the stenosing ligature may accelerate the In addition to transient adhesion and aggregation of platelets. hypercoagulability induced by ellagic acid injection (3), disturbance of blood flow plays a definite role in the production In this experimental procedure, of the occluding thrombosis. which was devised by us, the most important things are the constancy of the production rate of occluding thrombosis. It is noted that thrombocytopenia of experimental animals evidently reduced the production rate of the occluding thrombosis. No occluding thrombosis was observed in the rabbits In these animals, with platelet count of less than 40,00O/cmm. it can be excluded that leukopenia may possibly contribute to the reduction in the production rate of the occluding thrombosis, as leukocyte counts in these animals were around 50 % of prior value and no significant relationship was observed between leuThe thrombotic kocyte count and production rate of thrombus. process in the femoral artery under the condition of the experimental procedure is clearly prevented by ingestion of more than As reported elswhere (4), collagen-induced 0.05 g of aspirin. aggregation of platelets was completely inhibited in the platelet rich plasma obtained from rabbits which ingested more than As above mentioned, the 0.05 g of aspirin 6 hours previously. adhesion and aggregation of platelets to the endothelium of the artery injured at the time of placing the stenosing ligature is Thus, the preone of the most important contributing factors. vention of thrombus formation by aspirin, which is one of the Simmost powerful anti-aggregating agents, was not surprising. ilar experimental results have already been reported (5, 6, 7).
Suppl.
II
PLATELETS
IN THROMBUS
FORMATION
233
it is suggested that both quantitatively From these results, and qualitatively normal platelets are required for the production of occluding arterial thrombosis. On the other hand, Moschos et al (8) reported that aspirin did not affect the incidence of coronary or femoral artery thrombosis in dogs inThe difference might duced by means of a catheter electrode. It is recogbe due to their technique of producing thrombosis. nized that the occurrence of thrombi can be determined by other variables which may not necessarily relate to the mode of thrombus production in the present study. It was impossible to use the animals with marked thrombocytopenia induced by injection with anti-platelet serum for as severe antigen-antibody reaction might the experiment, trigger intravascular coagulation. intimectomy Thus, chemical was performed 4 days after anti-platelet serum injection, when Histological examithe animals were in rebound thrombocytosis. nation revealed that the injury was restricted in the intima, when 0.2 % solution of suifuric acid was applied. Occluding rabbits with thrombus yielded only in 33 % of the experimental Either additional procedure of stenosing normal platelet count. ligature at the site of chemically intimectomized artery or increase in the concentration of sulfuric acid for chemical intimectomy to 1.0 % resulted in a significant increase in the production rate of occluding thrombosis (9). Similar increase in yield of occluding thrombosis at the site of chemically intimectomized artery with 0.2 % solution of sulfuric acid was observed in the rabbits with rebound thrombocytosis induced by injection with anti-platelet serum. This also supports directly the concept that platelets occupy a central position in the development of thrombosis. In contrast to arterial thrombosis, either aspirin ingestion or busulfan-induced thrombocytopenia had no effect on stasis thrombus formation. It is believed that the production of venous thrombosis depends mainly upon stasis and hypercoagulability. The results obtained in the present study suggest that the adhesion and aggregation of platelets are not involved in the evolution of stasis thrombosis.
REFERENCES 1.
Evensen, S.A., Jeremic, M. and Hjort, F.F. Experimental thrombocytopenia induced by busulfan (myleran) in rabbits: extremely low platelet levels and intact clotting system. Throm. Diath. Haemorrh. 19, 570, 1968.
2.
Ohkuma, M. Studies on experimental Acta haematol. iap. a, 800, 1967.
3.
Kobayashi, N., Itoh, T., Suzuki, Y. and Maekawa, T. Experimental studies on hypercoagulable state induced by ellagic acid administration. J. Jap. Coil. Angiol. ll, 3, 1971.
4.
Maekawa, T. Arai, H. and Kobayashi, N. The effect on platelets and experimental thrombosis. Throm. 363, 1974. orrh. Suppl. 60,
immune thrombocytopenia.
of aspirin Diath. Haem-
234
MAEXAWA
ET AL
Suppl.
iI
5. Evans, G.E., Packham, M.A., Nishizawa, E.E., Mustard, J.F. and Murphy, E.A. The effect of acetylsalicylic acid on platelet function. J. Exp. Med. 127, 877, 1968. 6. Danese, C.A., Voleti, Ch.D. and Weiss, M.D. Protection by aspirin against experimentally induced arterial thrombosis in dogs. Throm. Diath. Haemorrh. 25, 288, 1971 7. Bousser, H.G. et Lecrubier, C. AgrCgation plaquettaire experimentale et thrombose arterielle. Nouv. Presse Med. 2, 1687, 1973. 8. Moschos, C.B., Lahiri, K.? Peter, A., Jesrani, M.V. and Regan, T.J. Effect of aspirin upon experimental coronary and noncoronary thrombosis and arrythmia. Amer. Heart J. 84, 525, 1972. The role of platelets in the 9. Kobayashi, N. and Maekawa, 'I'. thrombus formation. J. Jap. Coll. Angiol. 2, 575, 1974.
Suppl. II, Vol. 8, 19'76 Pergamon Press, Inc.
States
SECTION
ROLE
OF BLOOD
PLATELET
IN THE
V
FORMATION
OF THROMBUS
T. Maekawa,
N. Kobayashi, H. Arai and T. Takeuchi Third Department of Internal Medicine Gunma University School of Medicine Maebashi, Japan 371
ABSTRACT In the busulfan-induced thrombocytopenic rabbits, the femoral artery was completely occluded after injection with ellagic acid at the site of stenosing ligature in 5 out of 15 rabbits, while in the control rabbits with normal platelet count, a large thrombus was present around the stricture in 12 out of 17 rabbits. Occluding thrombus was produced in only 2 out of 15 rabbits which ingested more than 0.05 g of aspirin 6 hours before surgery. Rate of occluding thrombus formation on the chemically intimectomized femoral artery with diluted sulfuric acid was evidently high in the rabbits with rebound thrombocytosis after transient thrombocytopenia induced by intravenous injection with anti-platelet serum. In the case of stasis thrombus formation, both in thrombocytopenit rabbits and in aspirin-ingested rabbits, percentage of rabbits producing the thrombus was not significantly different from that of the control. From the results described above, it is suggested that both quantitatively and qualitatively normal platelets are required for the production of arterial thrombosis. On the other hand, blood platelets may not contribute to the formation of stasis thrombosis.
INTRODUCTION Among the numerous factors which are relevant in the genesis of arterial thrombosis, it is suggested that platelet occupies a central portion. The importance of the role of platelets in the thrombus formation has been well accepted. This suggestion comes from the pathomorphological investigations on both clinical and experimental thrombosis. Indirect evidence supports this concept, but direct proof is lacking. Therefore, the following experiments have been performed in order to make 227
228
Suppl.
the relationship between platelet and thrombus The results obtained in these experiments will
i-i
formation clearer. be reported.
METHODS Production of arterial thrombosis: Under pentobarbital anesthesia the femoral artery of normal rabbits weighing 2 to 3 kg was exposed. After all branches from the artery were ligated, a Steel wire of 0.90 mm in diameter was placed alongside the femoral artery. The artery was loosely ligated near the distal end together with steel wire by thick silk thread. A stricture was made in the femoral artery by pulling out the steel wire. Ten mg of water soluble ellagic acid (Yamanouchi Pharmaceutical Co.) dissolved in 5 ml of distilled water was injected intravenously. Twenty-four hours after these procedures the rabbits were again anesthetized and the femoral arteries with stenosing ligature were exposed and examined macroscopitally for the presence of thrombosis. Stasis thrombus formation: A modified method of Wessler was employed. Normal adult rabbits were used. A segment of the jugular vein was freed from its surrounding structures and its tributaries were ligated under pentobarbital anesthesia. Ten mg of water soluble ellagic acid dissolved in 5 ml of distilled water was injected into the contralateral ear vein. Within 10 to 15 seconds after completion of the injection, the previously exposed jugular vein was gently isolated with two small vascular clamps. After isolation in situ for 30 minutes, the segment was then removed and its contents emptied into a Petri dish containing 30 ml of 5 % sodium citrate solution. The amount of clot formed (stasis thrombus) was scored on a scale from 0 to 4; a score 0 represented no clot, and a score 4 represented a single thrombus forming a cast of the isolated segment. Chemical intimectomy of the femoral artery: About 1.5 cm of the arterial segment was gently isolated in situ by two small vasSulfuric acid solution of 0.2 % was injected incular clamps. to the segment after the blood in the segment was withdrawn. the sulfuric acid solution was withExactly 2 minutes later, drawn and blood flow of the femoral artery was recovered by reThe formation of the occluding thrombus was moving the clamps. examined 24 hours after the chemical intimectomy. Induction of thrombocytopenia: Thrombocytopenia was induced by two intraperitoneal injections with busulfan in normal adult TwentyThe method of Evensen et al (1) was employed. rabbits. five mg per kg of body weight of busulfan dissolved in polyethylene glycol in a concentration of 10 mg per ml were injected Platelet counts decreased to less than 124,000 every other day. Leukopenia /cmm in 10 to 14 days after two shots of busulfan. while change in hematocrit values was minimal. accompanied, Aspirin ingestion: A capsule containing 0.01 g to 1.0 g of acetylsalicylic acid (Shionogi Pharmaceutical Co.) was given Various experimental procedures orally in the early morning. were started 6 hours after the aspirin ingestion. is esPreparation of anti-platelet serum: The method employed EDTA-PRP sentially similar to that described by Ohkuma (2). was separated from the femoral artery of normal adult rabbits. Washed platelet suspension was prepared from the EDTA-PRP by
suppI_.
II
PLATELETS
229
IN THROMBUS FORMATION
TABLE 1 Effecstof Aspirin Ingestion on Experimental Thrombosis of the Femoral Artery with Stenosing Ligature by 10 mg of Ellagic Acid Injection :abbit No Injection with Normal Saline
Body Weight (kg)
Sex
Occluding Thrombus
ET ET ET ET ET
9 10 11 13 14
2.1 2.4 2.2 2.3 2.1
M F F M F
;:; (+ 1 (- 1 (- 1
ET ET
1 2
3.1 2.9
F M
(+ (-
1 1
ET ET ET ET ET ET ET ET ET ET ET
3 4 5 6 7 8 21 22 23 24 25 26
3.3 2.7 2.0 2.2 1.9 2.0 2.2 2.2 2.0 2.0 2.1 2.0
F M F F M F F F F F F
i;; ((+ (+ (+ (+ (+
> > 1 > > >
ET 27 28 ET 29
2.3 2.4 2.1
F F
ir; (+ >
ET ET ET ET ET ET ET
31 32 34 35 36 37 38
2.4 2.2 2.2 2.0 2.2 2.2 2.2
F M M M F M F
;:; (((+ ((-
ET ET ET ET
41 42 43 44
2.5 2.2 2.1 2.3
M M M M
[r,' (- > (- >
ET ET ET ET
51 52 53 54
2.2
(-
;:! 2.3
F F F F
ET ET ET ET
61 62 63 64
2.4 2.1 2.3 2.4
F M F M
(+ (+ (+ (+
1;:' (+
>
1 > 1 1 >
1
;:; C - 1 1 1 1 >
MAEKAWAET AL
230
Suppl.
II
low speed centrifugation with EDTA-normal saline. One ml of platelet suspension containing 500,000 to l,OOO,OOO platelets per cmm was injected twice a week in lst, Znd, 5th, 6th, 9th and 10th week. At the end of 11th week, blood was collected from the immunized guinea pigs. The anti-platelet serum separated from thus collected blood was absorbed with rabbit erythrocytes. Antibody titer of the anti-platelet serum was 1 : a. Rabbits with rebound thrombocytosis: Platelet counts of the normal adult rabbits were rapidly decreased to 2 to 4 % of the prior values after single intravenous injection with 2 ml of anti-platelet serum. Following marked transient drop, platelet counts rose rapidly to more than prior values 4 days after the injection with anti-platelet serum. RESULTS (1) the
Effect of aspirin ingestion on experimental thrombosis of femoral artery with stricture. Results obtained in the control rabbits injected with normal saline instead of ellagic acid are presented at the top of the Table 1. A small thrombus was found at the site of the stricture in 1 out of 5 rabbits. On the other hand, a large thrombus was present around the stricture in 12 out of 17 The femoral artery was rabbits after injection of ellagic acid. completely occluded in only 2 out of ii rabbits which ingested Ingest ion more than 0.05 g of aspirin 6 hours before surgery. of 0.01 g of aspirin which is a dose having no effect on coliagen-induced aggregation of platelets, did not reduce the formation rate of the arterial thrombus by eliagic acid injection. (2) Effect of busulfan-induced thrombocytopenia on experimental thrombosis of the femoral artery with stricture. In the thrombocytopenic rabbits induced by the two shots of busulfan, production rate of the occluding thrombus was As shown in the lower part of the Table 2, a markedly reduced. large thrombus was produced in only 1 out of 5 rabbits with platelet count of less than 50,000 per cmm. (3) Effect of thrombocytosis on the formation of thrombosis at the chemically intimectomized femoral artery. As listed in the upper part of the Table 3, occluding thrombus yielded only in 33 % of the experimental rabbits with Yield of occluding thrombosis at the normal platelet count. site of the femoral artery intimectomized with 0.2 % solution of sulfuric acid significantly rose to 60 %, among the rabbits in rebound thrombocytosis induced by the injection with antiplatelet serum. (4) Effect of aspirin ingestion or busulfan-induced thrombocytopenia on the formation of stasis thrombus. ingestion had no effect As shown in the Table 4, aspirin Reduction of the on stasis thrombus by eliagic acid injection. circulating platelet counts also had no effect on the formation of stasis thrombus (Table 5). DISCUSSION It
is
presumed
that
the
initial
process
in the
formation
II
Suppl.
PLATELETS
231
IN 'TI-IROMBUSFORMATION
TABLE 2 Effect of Thrombocytopenia on Experimental Thrombosis of the Femoral Artery with Stenosing Ligature by 10 mg of Ellagic Acid Injection Rabbit No. B B B B B B B B B B B B B B B
30 31 32 33 34 35 36 37 38 35 40 41 42 43 44
Body Weight Sex Platelet Count Occluding Thrombus per cmm (kg) 2.0 2.3 2.3 2.4 2.3 2.2 2.3 2.3 2.3 2.3 2.0 2.3 2.4 2.3 2.3
F F M F F F M M F F F F F F F
82,000 88,000 66,000 124,000 98,000 68,000 84,000 96,000 64,000 116,000 24,000 42,000 7,000 30,000 40,000
TABLE 3 Effect of Thrombocytosis on Experimental Thrombosis of the Femoral Artery Chemically Intimectomized by 0.2 % of Sulfuric Acid Solution (Rabbits with Normal Platelet Count) Rabbit Body Weight Sex Platelet Count Occluding Thrombus No. (kg) CI 1 CI 2 CI 3 CI 4 CI 5 CI 16 CI 17 CI 18
3.0 2.8 2.9 2.7 2.6 3.2 2.5 2.9
Normal Normal Normal Normal Normal Normal Normal Normal
[;I ( - 1 ( + >
[I; ( + 1 ( - 1
(Rabbits with Rebound Thrombocytosis after anti-platelet Serum Injection) Rabbit Body Weight Sex ;,;:~;~;s;,;;~ Occluding Thrombus No. (kg) CI CI CI CI CI
25 26 27 28 29
3.0 2.4 2.4 2.5 3.0
F F F M M
214 104 72 129 128
% % % % %
(+) (+)
of occluding thrombus is adhesion and aggregation of platelets to the site of stenosing ligature. In this case, it is suggested that slight endothelial injury and disturbance of blood
232
WKAWA
ET AL
Suppl.
Ji
TABLE 4 Effect
of
Aspirin
Amount of Aspirin Ingested (g) 0
1.0 0.3 0.05 0.01
Ingestion No.
of
on Stasis
Thrombus
Formation
Rabbits
with
Thrombi
Scored
0
1
2
3
4
1 0 0 1 0
0 0 1 0 0
0 1 0 0 0
0 1 1 0 0
4 3 2 3 4
Total
5 5 4 4 4
TABLE 5 Effect Rakiit
ST ST ST ST ST
of
Thrombocytopenia
Body Weight
.
(kg)
1 2 3 4 5
2.0 2.3 2.4 2.3 2.3 Number of
on Stasis
Sex Platelet per M F F M F Rabbits
Count cmm
82,000 88,000 119,000 66,000 99,000
Thrombus
Formation
Score
Thrombus
0
of 1
2
3
4 X X X
X
0
0
0
1
X
4
flow due to placing of the stenosing ligature may accelerate the In addition to transient adhesion and aggregation of platelets. hypercoagulability induced by ellagic acid injection (3), disturbance of blood flow plays a definite role in the production In this experimental procedure, of the occluding thrombosis. which was devised by us, the most important things are the constancy of the production rate of occluding thrombosis. It is noted that thrombocytopenia of experimental animals evidently reduced the production rate of the occluding thrombosis. No occluding thrombosis was observed in the rabbits In these animals, with platelet count of less than 40,00O/cmm. it can be excluded that leukopenia may possibly contribute to the reduction in the production rate of the occluding thrombosis, as leukocyte counts in these animals were around 50 % of prior value and no significant relationship was observed between leuThe thrombotic kocyte count and production rate of thrombus. process in the femoral artery under the condition of the experimental procedure is clearly prevented by ingestion of more than As reported elswhere (4), collagen-induced 0.05 g of aspirin. aggregation of platelets was completely inhibited in the platelet rich plasma obtained from rabbits which ingested more than As above mentioned, the 0.05 g of aspirin 6 hours previously. adhesion and aggregation of platelets to the endothelium of the artery injured at the time of placing the stenosing ligature is Thus, the preone of the most important contributing factors. vention of thrombus formation by aspirin, which is one of the Simmost powerful anti-aggregating agents, was not surprising. ilar experimental results have already been reported (5, 6, 7).
Suppl.
II
PLATELETS
IN THROMBUS
FORMATION
233
it is suggested that both quantitatively From these results, and qualitatively normal platelets are required for the production of occluding arterial thrombosis. On the other hand, Moschos et al (8) reported that aspirin did not affect the incidence of coronary or femoral artery thrombosis in dogs inThe difference might duced by means of a catheter electrode. It is recogbe due to their technique of producing thrombosis. nized that the occurrence of thrombi can be determined by other variables which may not necessarily relate to the mode of thrombus production in the present study. It was impossible to use the animals with marked thrombocytopenia induced by injection with anti-platelet serum for as severe antigen-antibody reaction might the experiment, trigger intravascular coagulation. intimectomy Thus, chemical was performed 4 days after anti-platelet serum injection, when Histological examithe animals were in rebound thrombocytosis. nation revealed that the injury was restricted in the intima, when 0.2 % solution of suifuric acid was applied. Occluding rabbits with thrombus yielded only in 33 % of the experimental Either additional procedure of stenosing normal platelet count. ligature at the site of chemically intimectomized artery or increase in the concentration of sulfuric acid for chemical intimectomy to 1.0 % resulted in a significant increase in the production rate of occluding thrombosis (9). Similar increase in yield of occluding thrombosis at the site of chemically intimectomized artery with 0.2 % solution of sulfuric acid was observed in the rabbits with rebound thrombocytosis induced by injection with anti-platelet serum. This also supports directly the concept that platelets occupy a central position in the development of thrombosis. In contrast to arterial thrombosis, either aspirin ingestion or busulfan-induced thrombocytopenia had no effect on stasis thrombus formation. It is believed that the production of venous thrombosis depends mainly upon stasis and hypercoagulability. The results obtained in the present study suggest that the adhesion and aggregation of platelets are not involved in the evolution of stasis thrombosis.
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5. Evans, G.E., Packham, M.A., Nishizawa, E.E., Mustard, J.F. and Murphy, E.A. The effect of acetylsalicylic acid on platelet function. J. Exp. Med. 127, 877, 1968. 6. Danese, C.A., Voleti, Ch.D. and Weiss, M.D. Protection by aspirin against experimentally induced arterial thrombosis in dogs. Throm. Diath. Haemorrh. 25, 288, 1971 7. Bousser, H.G. et Lecrubier, C. AgrCgation plaquettaire experimentale et thrombose arterielle. Nouv. Presse Med. 2, 1687, 1973. 8. Moschos, C.B., Lahiri, K.? Peter, A., Jesrani, M.V. and Regan, T.J. Effect of aspirin upon experimental coronary and noncoronary thrombosis and arrythmia. Amer. Heart J. 84, 525, 1972. The role of platelets in the 9. Kobayashi, N. and Maekawa, 'I'. thrombus formation. J. Jap. Coll. Angiol. 2, 575, 1974.
