Platelet aggregation by platelet-clumping substance

vol .

THRONEBOSTS RESEARCH Printed in the United States

4, pp . 625-637,

1974

Pergamon Press, Inc .

PLATELET AGGREGATION BY PLATELET-CLUMPING SUBSTANCE HIROH YAMAZAKI, TADAHIRO SANO, TATSUO SHIMAMOTO and TAKIO SHIMAMOTO The Insititute for Cardiovascular Diseases, Tokyo Medical and Dental University, School of Medicine Tokyo, Japan (Received 12 .2 .1974 ; in revised form 4 .3 .1974 . Accepted by Editor S . Okamoto)

ABSTRACT The platelet-clumping substance isolated from rabbit plasma by the authors aggregated washed rabbit platelets, while ADP did not . The presence of AMP or adenosine did riot inhibit the appearance of platelet' aggregation induced by the clumping substance . Pretreatment of phosphoenol-pyruvate and pyruvate kinase on unwashed platelets did not affect the platelet aggregation by the clumping substance . It clumped human platelets and also shrimp thrombocytes . Electron microscopic findings showed that platelets to which the clumping substance was added aggregated in their original form as falt disc shapes with few pseudopods . While fusion of membrane was present, microtubules were observed in its regular form . These results showed the existence of a platelet-clumping substance in blood that is different from ADP, collagen, thrombin, adrenaline etc . and may play an important role in hemostasis and thrombogenesis .

INTRODUCTION Previously we have separated a platelet-clumping substance from rabbit plasma and analysed its chemical property

(1) .

It showed a close relationship

to acid mucopolysaccharide bound to protein . It clumped washed or unwashed platelets equally, even in a concentration of 20 }ig/ml . In the present paper, we have observed a pattern of platelet aggregation induced by the plateletclumping substance and compared it to platelet aggregation induced by adenosine diphosphate(ADP) .

625



626

MODE OF PLATELET-CLUMPING

Vol .4,IVo .S

METHODS Methods to detect aggregation of washed and unwashed platelets were described previously (1) .

Citrated platelet-rich plasma (CPRP, final concen-

tration of sodium citrate : 0 .38 %) and washed platelet suspension in physiological saline were obtained from rabbits and healthy male, whose blood was type 0 . Blood was collected from shrimp also to observe aggregation of the thrombocytes . Platelet-saline suspension and CPRP from rabbits were added with 20, 10, 5, 2,5, 1 .25 and 0 .625 mM of AMP (Sigma Chemical Co ., St . Louis, Missouri, U .S .A .) or adenosine (Sigma Chemical Co ., St . Louis, Missouri, U .S .A .) . Immediately and 30 minutes afterwards, 50 µg/ml of the platelet-clumping substance was added and platelet aggregations were observed . Platelet-saline suspension and CPRP were added with pyruvate kinase (Sigma Chemical Co ., St . Louis, Missouri, U .S .A .) and phosphoenolpyruvate (Sigma Chemical Co ., St . Louis, Missouri, U .S .A .) according to Haslam (2) .

After incubation for 30

0

minutes at 3! C, platelet aggregability was examined with an addition of the preparation of the platelet-clumping substance, ADP (Sigma Chemical Co ., St . Louis, Missouri, U .S .A .) and adrenaline (Sankyo Co ., Ltd ., Tokyo, Japan) . A platelet aggregation meter (Model 300, Chrono-Log Corp . Broomall, Pennsylvania, U .S .A .), connected with a pen-writing electronic recorder (Model U-125U, Shimadzu Co ., Ltd ., Kyoto, Japan) was also used, following Born (3) and O'Brien (4) . To observe morphological changes in platelet aggregation, CPRP was added with each 50 }tg/ml of ADP or the platelet-clumping substance at room temperature . When platelet aggregation occurred, 0 .4 ml of CPRP was added to 15 ml of 0 .5 % glutaraldehyde solution . After 90 minutes, the mixture was centrifuged (500 x g, 5 minutes) and then the precipitate was washed with distilled water three times . The washed samples were dried to take a thin film on a slide glass . The specimens were covered with a layer of gold and examined by a scanning electron microscope (JSM-U3, Japan Electron Optics Laboratory, Tokyo, Japan) under a scanning electron beam of 2 .5 KV . The residual 10 ml of CPRP, added to 40 ml of 4 .0 % chilled glutaraldehyde solution, was used to prepare specimens for the electron microscope . Thirty minutes after the addition of the glutaraldehyde solution, the mixture was centrifuged and washed with phosphate buffer solution (pH 7 .4) three times and finally with veronal buffer (pH 7 .4) once . Then the precipitates were added to 2 ml of 20 % osmic acid solution . Sixty minutes later, the



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MODE OF PTITELET-CLT21PTSG

£27

mixture was centrifuged and then dehydrated in a graded series of ethanols . The specimens were embedded in Epon mixture (Taab Lab . Reading, England) in the usual wav . Sections were stained with uranyl acetate and observed with an electron microscope (HU-11A, Hitachi Indust . Co ., Ltd, Tokyo, Japan) .

RESULTS Fig . 1 shows platelet aggregation curves in platelet saline suspension

Platelet Aggregation in Washed Rabbit Platelets Suspended in Saline

Platelet-Clumping Substance 50 }19/ml FIG . 1 The platelet aggregation curves of washed rabbit platelets suspended in saline were demonstrated using an aggregation meter . Sixty jig/ml of the platelet-clumping substance aggregated washed platelets, while the same platelets were not aggregated by 50 Pg/ml of ADP .

induced by the platelet-clumping substance and ADP . Fifty pg/ml of ADP did not induce aggregation of washed platelets, while 50 49/ml of the clumping substance did aggregate washed platelets . The washed platelets were aggregated immediately after addition of the substance . The platelets clumping did not disappear during the observation time of up to four hours . The clumping substance isolated from rabbit plasma clumped even human platelets . It also clumped invertebrate platelets such as shrimp thrombocytes . On the one hand, ADP did not clump shrimp thrombocytes .



MODE OF PLATELET-CLUMPING

628

Vol .k,No,5

The presence of AMP and adenosine did not inhibit the appearance of platelet clumping after addition of 50 1+9/ml of the platelet-clumping substance . Following Haslam's method (2), pretreatment of phosphoenolpyruvate •and pyruvate kinase on unwashed

platelets

did not affect the

platelet aggregation induced by the clumping substance, while it inhibited the aggregation induced by adrenaline (Table 1) . Moreover, washed platelets

TABLE 1

Adrenaline

ADP

retreatment

Platelet-clumping Substance

500 )19/ml 50 ug/ml 500 )
,o

None

+

+

+

PEP +PK

+

+

..

+

50

p9/ml

+

+

+

+

+

+

+

+

S a~ m d ro++a ~a

c

m

c

None

m

V m n +' VI .d ., a'

c

PEP + PK

-

-

-

-

After pretreatment in the form of phosphoenolpyruvate (PEP) and pyruvate kinase (PK) on the platelets, unwashed platelets were aggregated by ADP and the platelet-clumping substance and not by adrenaline . On the one hand, washed platelets were not aggregated by ADP or adrenaline, while they were aggregated by the clumping substance regardless of the pretreatment .

were not aggregated by ADP or adrenaline, while they were aggregated by the clumping substance regardless of the pretreatment . Fig . 2 shows scanning electronmicroscopic findings of aggregated platelets added with 50 pg/ml of ADP (left) and the platelet-clumping substance (right) . The same degree of platelet aggregation was observed in both the photographs . Even under the same magnification, the size of each platelet in the aggregated mass induced by ADP appears to be smaller than that induced by the clumping substance . The platelets in ADP-induced aggregation

Vol .-t,No .5

629

MODE OF PLATELET-CLUMPING

A 0 P

Platelet-Clumping Substance

(

SEM x 3,000

)

FIG 2 Scanning electronmicroscopic findings of aggregated platelet to which 50 yg/ml of ADP (left) or the platelet-clumping substance (right) was added . Even under the same magnification, the size of each platelet in the aggregated mass induced by ADP appears to be smaller than that induced by the clumping substance . The platelets in ADP-induced aggregation were shrunk to a round form, while the platelets in the clumping-substance-induced aggregation showed little change from their original flat form .



63 0

MODE OF PLATELET-CLLMPIN G

.4 val

were shrunk to a round form, while the platelets in the aggregation induced by the platelet-clumping substance showed little change from their original flat form . There were many pseudopods in the platelets in ADP-induced aggregation . The platelets to which the clumping substance was added had a fewer number of pseudopods . The above differences were observed in transmission electronmicroscopic findings also . Platelets in ADP-induced aggregation showed irregular forms with many pseudopods . It appears that platelets were connected with each other by their pseudopods or protrusions as shown in Fig . 3 . Platelets in

FIG . 3 Platelet aggregation induced by ADP . Platelets showed irregular forms with many pseudopods .

the clumping substance-induced aggregation showed a flat form with few pseudopods (Fig . 4) . It appears that platelets were connected with each



o

MODE OF PL?TFLET-CT MPT.~O

FIG . 4 Platelet aggregation induced by the platelet-clumping substance . Platelets showed flat form with few pseudopods . Platelets were connected with each other by their surface and not by pseudopods . Abundant vacuolization was observed .

other by thier surface and not by pseudopods . Abundant vacuolization was observed . Microtubules were regular and fusion of membrane was observed locally . Amorphous or fine fibrillary material was present between the adjacent platelets . Under higher magnification, these findings are clearly seen (Fig . 5) . While fusion of membrane was present, microtubules was observed in its regular form . In ADP aggregation, fusion of membrane was less frequent when the membranes of adjacent platelets showed a tight adhesion (Fig .

6) .

The circumferential band of microtubules has been reduced

in the circumference of the platelets . Vacuolization was observed less frequently .

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MODE OF PLATELET-CLUMPING

Vol . 4, No -

FIG . 5 Platelet aggregation induced by the platelet-clumping substance under higher magnification . While fusion of membrane (F) was present, microtubules (MT) were observed in its regular form .

5

MODE OF PLATELET-CLUMPING

633

FIG . 6 Platelet aggregation induced by ADP under higher magnification . When the membranes of adjacent platelets showed a tight adhesion, fusion of membrane was less frequently observed . The circumferential band of microtubules has been reduced in the circumference of the platelets . Vacuolization appeared less .



634

MODE OF PLATELET-CLTTMPINC

vol .4,N0 . 5

DISCUSSION The platelet-clumping substance behaved differently from thrombin, adrenaline and ADP-cofactors .

ADP,

The clumping substance aggregated

washed platelets even without calcium ions or any kind of cofactors . aggregated shrimp thrombocytes . cytes (5) .

It also

ADP did not aggregate invertebrate thrombo-

The appearance of platelet aggregation by the clumping substance

was not prevented by the presence of AMP or adenosine, which is known to inhibit platelet aggregation by ADP (6) .

The preparation of the clumping

substance did not show any distinctive absorption peak at around 260 mµ due to nucleotide base (1) .

Following Haslam's method (2), pretreatment of

phosphoenolpyruvate and pyruvate kinase on platelets did not affect platelet aggregation induced by the clumping substance, while it inhibited aggregation induced by adrenaline, thrombin or collagen . Moreover, washed platelets were not aggregated by ADP or adrenaline, while they were aggregated by the clumping substance regardless of the pretreatment . The result suggests that the platelet aggregation induced by the platelet-clumping substance was not due to ADP released from the platelets . Moreover, the morphological differences in aggregated platelets induced by ADP and the clumping substance are also clearly seen in their shape and ultrastructures . Platelets in ADP-induced aggregation showed irregular form with many pseudopods . It appears that platelets were connected with each other by their pseudopods or protrusions .

Platelets in the clumping

substance-induced aggregation showed a flat form with few pseudopods . It appears that platelets were connected with each other by their surfaces and not by pseudopods . Abundant vacuolization was observed . While fusion of membrane is present, microtubules is observed in its regular form . In ADP aggregation, fusion of membrane was less frequently observed . The circumferential band of microtubules has been reduced in the circumference of platelets . These findings suggest that the platelet-clumping substance aggregated platelets in their original flat form

(7) .

After platelets are

exposed to collagen, thrombin, ADP, serotonin, adrenaline or noradrenaline, the cells lose their discoid shape and become irregular with multiple pseudopods . Changes in surface contour are accompanied by the movement of granules toward the cell center where they are closely encircled by the centrally displaced circumferential band of microtubules and microfilaments (8) . Following Behnke

(9),

the microtubules may play a role in maintaining

the shape of platelets ; he also observed whether chemicals which affect

Vol .

b,No,5

635

MODE OF PLATELET-CLUMPING

platelet function also affect platelet shape and platelet microtubules .

He

concluded that an intact marginal bundle of microtubules was essential for the maintenance of platelet shape, but that intact microtubules were not essential for ADP-induced platelet aggregation . In our previous experiment (10), platelets treated with KCN, NaF or ouabain had regular microtubules and they were aggregated by ADP . Platelets treated with N-ethylmaleimide, lidocaine or colchicine did not show regular microtubules and they were not aggregated by ADP . However, platelets treated with monoiodoacetate or HgCI

2

had regular microtubules, while they were not aggregated by ADP . On the contrary, the platelet-clumping substance can aggregate only platelets with regular microtubules . Such finding suggests the difference in biological behaviour of the clumping substance from ADP and its physiological role on platelets . In the present paper, we have reported the existence of the plateletclumping substance in blood, which differs from ADP and so on . Our previous (1, 11, 12,

14, 15) and present studies have led us to the conclusion

13,

that there exists a platelet-clumping substance precursor in plasma which is activated by the invasion of high-molecular-weight substances, such as endotoxin or agar, or adrenaline in vivo,

and also by some kind of proteoly-

tic enzymes or acidification of plasma and serum in vitro (Fig .

I

PLATELET-CLUMPING SUBSTANCE PRECURSOR

7) .

When

I

Invasion of Bacterial Endotoxln (SHIMAMOTO & YAMAZAKI . 1958) Invasion of High Molecular Weight Substances (YAMAZAKI et al ICI) Chondro .rm Sulfate a or Heparin

Adrena line (YAMAZAKI at al 1967)

Activation (Colamor

+Calcium Ions

Proteolytic Enzymes (YAMAZAKI t al . 1970)

(MURASE et al . 1970)

Acidification of Plasma or Serum (YAMAZAKI et el 1966)

urvmecesary la. La")

Clumping of Platelets

FIG .

7

Schema of the appearance of platelet-clumping substance in blood .



636

MODE OF PLATELET-CLUMPING

Vol . 4 ,No .3

the serum or plasma was activated, washed and unwashed platelets were equally aggregated by the activated clumping substance even in the absence of calcium ions . The aggregation was not related to released ADP from platelets . To clarify the hemostasis and thrombogenesis, the existence of the plateletclumping substance in blood implies the importance of further research, suggesting the existence of a phylogenetically old hemostatic mechanism which counteracts the bleeding from acidotic tissues damaged by inflammatory or ischemic processes, regardless of the fibrinogen-fibrin system . ACKNOWLEDGEMENT The authors wish to thank Dr . T . SAKAI, Dr . S . FUJINO and Dr . Y . SHIOYA for experimental assistance for the electronmicrographs . REFERENCES 1 . YAMAZAKI,H ., IJIRI, H ., SANO, T ., ANAN,K . and SHIMAMOTO,T . Isolation and chemical analyses of platelet-clumping substance in blood . Thrombosis Research : to be contributed . 2, HASLAM,R .J, Role of adenosine diphosphate in the aggregation of human blood-platelets by thrombin and by fatty acids . Nature : 202,765 . 1964 . 3 . BORN,G .V .R . Quantitative investigations into aggregation of blood platelets . J .Physiol . : 162,67, 1962 . 4 . O'BRIEN,J .R . Platelet aggregation . Part 2 . Some results from a new method of study, J . Clin . Path,: 15,452 . 1962 . 5 . MAUPIN,B . Blood platelets in man and animals . Oxford, Pergamon Press . 1969 . P . 516 . 6, BORN,G .V,R . and CROSS,M .J . The aggregation of blood platelets . J . Physiol,: 168,178 . 1963 . 7 . SHIMAMOTO,T ., YAMAZAKI,H . and SHIMAMOTO,T . Scanning electron microscopic observation of platelets in hemostasis . Thrombos . Diath . haemorrh .: in press . 8, WHITE,J .G . Platelet morphology . In Johnson S .A .(Ed .) The circulating platelet . New York and London . Academic Press . 1971 . P . 45 . 9 . BEHNKE,O . Effects of some chemicals on blood platelet microtubules, platelet shape and some platelet functions in vitro . Scand . J . Haemat, 7,123 . 1970 . 10 . YAMAZAKI,H . Vessels and platelets . Platelet-clumping substance in blood . In Plenary session in the 14th International Society of Hematology . Sao Paulo . Brasil . 1972 . P . 18 .



Vol . 4,Vo .5

11 .

MODE OF PLATELET CLLNPING

YAMAZAKI,H ., SHIMAMOTO,T ., MURASE,H .

and SHIMAMOTO,T .

637

Appearance of

platelet-clumping substance in plasma of rabbit after the intravenous injection of agar-solution, bacterial endotoxin or adrenaline . Blood :

30,792 . 1967 . 12 . YAMAZAKI,H ., MURASE,H ., SHIMAMOTO,T . and SHIMAMOTO,T . Appearance of platelet-clumping substance after acidification of plasma . Blood :

31,348 .

1968 . 13 . Y .AMAZAKI,H ., MURASE,H ., SHIMAMOTO,T . and SHIMAMOTO,T . Appearance of platelet-clumping substance in plasma from human with thrombosis, infection or tissue destruction, Preceed . Jap . Acad . :

44,1066 . 1968 .

14 . Y.A)UZAKI,H ., MURASE,H ., IJIRI,H . and SHIMAMOTO,T . The appearance and destruction of platelet-clumping substance in serum added with trypsin . Thrombos . Diathes . haemorrh . :

23,469 . 1970 .

15 . MURASE,H ., SHIMAMOTO,T ., KOBAYASHI,I ., IJIRI,H ., SHIMAMOTO,T . and Y .AMAZAKI,H . Acid mucopolysaccharides as a cofactor in formation of platelet-clumping substance . Blood : 37,684 . 1971 .