the effect of adenine nucleotides on thrombus formation, platelet count, and blood coagulation

EXPERIMENTAL AND MOLECULAR PATHOLOGY 5. 43-60 (1966)

The Effect of Adenine Nucleotides on Thrombus Formation,

Platelet Count, and Blood CoagulationlS2

J. F. MUSTARD, H. C. ROWSELL, F. LOTZ, AND B. HEGARDT

Department of Physiological Sciences, Ontario Veterinary College, Guelph; Blood and Vascular

Disease Research Unit, Departments of Medicine and Pathology, University of Toronto; Sunnybrook Hospital, Department of Veterans Aflairs, Toronto

AND

E. A. MURPHY

Department of Biostatistics, Johns Hopkins University, :i School of Plrblic Health and Hygiene, Baltimore, Maryland

Received May 3, 1965

There is evidence that platelets are a necessary and perhaps primary factor in the formation of thrombi from flowing blood (Mustard et al, 1962a). Knowledge of the mechanisms involved in the adherence of platelet to each other and to surfaces is important in understanding thrombogenesis in arterial systems. Hellem (1960) and Ollgaard (1961) demonstrated that a factor which can be isolated from platelets, red cells and other cells, causes platelets to adhere to each other. Subsequent work by Gaarder et al. (1961) showed this factor to be adenosine diphosphate (ADP). Kaser-Glanzmann and Luscher (1962) have provided evidence that thrombin, by converting platelet adenosine triphosphate (ATP) to ADP some of which escapes into the ambient fluid causes platelet aggregation. Haslam (1964) has recently published confirmatory evidence on this point. It therefore appears from this and other work (Born, 1962; O’Brien, 1962; Mitchell and Sharp, 1964), that ADP is an important factor in causing platelet aggregation.

Evidence that this is also true in viva is provided by the experiments of Honour and Mitchell (1963). Using the microcirculation technique in rabbits, they demonstrated that addition of small amounts of ADP at points of vessel injury enhanced “white body” formation, and that poisons which inhibit metabolic reactions involved in the production of ADP, had the converse effect; topical extravascular application of ADP solutions did not lead to platelet aggregation if the vessel wall was intact. Recently in collaboration with Born (1964), they have shown that pretreatment of animals with adenosine or 2-chloradenosine which inhibit *ADP-induced platelet aggregation in vitro, suppresses white body formation in injured vessels in the microcirculation (Born et al., 1964). Zucker and Borrelli (1962), Spaet et al. (1962) and

1 This work was supported by grants from the US. Public Health Service, H-4964 and H-6912; the Medical Research Council, M.4-1309; The Ontario Heart Foundation and the Department of Veterans Affairs.

2 Presented in part at the .4nnual Meeting of the Canadian Society for Clinical Investigation, Quebec City, 1964.

a This is paper number 369 from the Department of Biostatistics.

43

44 J. F. MUSTARD ET AL.

Hovig (1963) and co-workers have shown that platelets after they come in contact with collagen, adhere to it and to each other. Hovig’s work (1963) suggests that this is probably mediated through release of platelet ADP.

Born and Cross (1963) found that the injection of ADP into the circulation of cats caused a rapid decrease in the number of circulating platelets and that neither adenosine monophosphate (AMP) or adenosine had this effect. They suggested that the disappearance of platelets from the circulation following the infusion of ADP could be due to the adherence of platelets to the endothelium and the trapping of platelet clumps in small vessels (Born and Cross, 1963).

It seems then that ADP may be important in the initiation and growth of thrombi from flowing blood. We have, therefore, studied the effect of ATP, ADP, and AMP on thrombus formation using the extracorporeal shunt technique which has been described (Downie et d., 1963). In brief, this method provides a means of testing whether or not changes in the characteristics of the circulating blood influences thrombus formation. The results of these investigations form the subject of the present communication.

MATERIALS AND METHODS

Animals. The animals used in these studies were either Landrace, Yorkshire or crossed Landrace-Yorkshire pigs of 15-40 kg body weight, reared at the Ontario Agricultural College. They were maintained on the standard basic low-fat hog ration, details of which have been given elsewhere (Mustard et al., 1963). The animal was anesthetized with intravenous pentobarbital sodium (approximately 150 mg/kg body weight) and a carotid artery and jugular vein were cannulated with size PE 320 silicone-coated polyethylene catheters. An hour later when any transient circulatory disturbance had subsided, the flow chamber was connected to the catheters and flow through them established. Immediately a specimen of blood was drawn from the sampler at the venous end of the shunt through a la-gage stainless steel needle into a paraffin-coated, glass syringe. This blood sample was used for the base line studies. One minute later, the infusion was given over a la-minute period through the proximal sampler. In some studies, the material was infused through the distal sampler and when this is so, reference will be made in the text. One minute after completion of the injection, a second sample of blood was taken. Subsequent samples were always taken 20 minutes after completion of the infusion and usually at 3 and at 10 minutes as well. Twenty minutes after completion of the infusion, the shunt was removed, dismantled and the thrombus collected and weighed in accordance with the usual procedures which have been described in detail elsewhere (Downie et al., 1963 Mustard et al., 1963). Each animal was used for one study.

Coagulation studies. A portion of each blood sample was placed in glass and silicone-coated glass tubes for determination of the clotting times, and the rest was placed in 3.87, trisodium citrate (one part citrate to nine parts blood) in silicone- coated glass centrifuge tubes. The following coagulation tests were carried out by methods which have been described: the whole blood clotting time in glass and silicone-coated glass tubes (Murphy and Mustard, 1960), the prothrombin time (Murphy and Mustard, 1960), the Russell’s viper venom time (Mustard, 1957). the platelet count (Mustard, 1957), and the activity of Al(OH),-treated plasma and serum in the thromboplastin generation test (Mustard and Hoeksema, 1962).

THE EFFECT OF ADENINE NUCLEOTIDES 45

The partial thromboplastin time was carried out on titrated plasma prepared from whole blood which had been centrifuged at 3,600 rpm (RCF = 1,500) at 4°C for 12 minutes. One part of a kaolin suspension (20 mg/ml) was added to two parts of titrated plasma in a glass tube at 37°C and the mixture incubated for 9.5 minutes. Then one part of a thromboplastic phospholipid prepared from brain (50 11,g//ml) was added followed 10 seconds later by l/‘lOth part &t/4 CaCl,. A stop-watch was started immediately and the interval until the appearance of fibrin recorded.

When the partial thromboplastin time was used for factor VIII and 1,X assays, the following plasma mixtures were used. Factor VIII- or factor IS-deficient plasmas obtained from our hemophilic dog colony (Mustard et al., 1960: Mustard rt al.. 1962b) were used to dilute the test plasma in the proportion of 1: 1, 1:2, 1:4, 1:8, and 1: 16. The clotting time of the test plasma was converted with the aid of a standard curve into a percentage of the reference plasma. The reference plasma was pooled from several normal pigs and stored in bulk in silicone-coated glass tubes at -196°C in a liquid nitrogen refrigerator. The standard curve was prepared from the reference plasma on each test day.

Glass contact activity was estimated by preparing on the day of each experiment, platelet-poor titrated plasma which had not been allowed to come in contact with glass surfaces (all surfaces being silicone-coated). One part of M,‘4 CaCl- was added to ten parts of plasma in uncoated and in siliconed glass tubes at 37°C and the clotting time determined. One part of the test plasma was added to nine parts of the noncontacted plasma used as the standard. The test plasma was collected and prepared in the same fashion as the noncontacted plasma standard.

Statistical considerations. In the calculation of the data, the logarithms of the weights of the thrombi and the clotting times have been used. The reciprocals of the values for the thromboplastin generation test were used. Nean values have been converted back to the original units, prior to their insertion in tables and charts. The reasons for these transformations have been given in detail elsewhere (Downie et al., 1963; Mustard et al., 1963; Murphy and Mustard, 1960).

Flow. Blood flow was estimated by the method described previously (Downie et al.. 1963) which involves measurement of the speed of movement of a 2-ml air bubble through a measured segment of the tubing.

.-ldenine nucleotides. The preparation of ATP, .4DP, and AMP used in these experiments were obtained from the California Corporation for Biochemical Research, or from Sigma Chemicals. Prior to their infusion. they were dissolved in sterile, imidazole-buffered saline (pH 7.3). The solutions used in each experiment were freshly prepared on the day of the study. Each infusion was made up in the same volume of buffered saline (25 ml).

Heparin. In some experiments, heparin infusions were given before administration of the .4DP. The heparin was obtained from Connaught Medical Research Laboratories or Riker Laboratories. In these studies, the heparin was given through the venous cannula 20 minutes before the extracorporeal shunt was connected up and the nucleotide infusion. Samples of blood were taken before, 1 minute and 20 minutes after the heparin infusion. -4 similar set of specimens were taken after the nucleotide infusion.

Blood pressure. In come of the experiments, cannulas were placed in the femoral


artery and coupled to a strain-gage manometer and blood pressure continuously recorded with a Grass Polygraph. At the same time records were made of respiration and the electrocardiograph.

Histology. Some animals not used in the flow chamber experiments were destroyed one minute after ADP infusion, and sections taken immediately from various organs. In other studies, the animals were destroyed after longer intervals. The tissues were fixed in Zenker’s solution.

TABLE I EFFECT OF ADENINE NUCLEOTIDES ON THE WEIGHT OF THROMBUS FORMED IN THE

EXTRACORPOREAL CIRCULATIONS

Treatment

Weight of Marginal 95 %

deposit confidence limits Comparison

No. of geometric on mean (pg) with controlb

Studies mean (wLg) Lower Upper t P

Control (Saline) 12 351 135 911 -

AMP 15 394 192 807 0.214 < 0.9

ADP 30 1,750 1,071 2,859 3.438 < 0.002

ATP 11 704 277 1.788 1.151 < 0.3

Q The means and confidence limits are computed on the logarithms of the values, but converted

back into the original units for this table. b The three t tests are not orthogonal. However, only one is of prime interest, ADP versus the

control, and neither of the other two comparisons approaches significance.

Radioisotope studies. Donor swine of the same blood group as the recipient animals were injected with diisopropylfluorophosphate as Ps3 (DFP32)” (4 nc/kg body weight). Four to 12 hours after the injection, 500 to 1,000 ml of blood were with- drawn into sterile silicone-coated glass bottles containing buffered 1.5 per cent EDTA (nine parts blood to one part EDTA) . Platelet-rich plasma was prepared by centri- fuging the blood at 800 rpm (RCF = 77) for 12 minutes at room temperature. The platelet-rich plasma was removed, concentrated and transfused into the recipient animal 2-4 hours before the shunt experiment. Blood samples were taken from the recipient animals 15 minutes after the transfusion, just before the shunt experiment and 20 minutes after the ADP infusion. The radioactivities of the platelets in these samples were determined by methods which have been described (Mustard and Murphy, 1963). A sample of platelet-poor plasma was prepared from the donor blood. To this sample of platelet-poor plasma, an equal volume of unlabeled platelet- rich plasma was added. This mixture was incubated at 37°C for 15 minutes, following which the platelets were isolated and their radioactivity determined. These platelets did not show any significant labeling with DFP3”. It was therefore con- cluded that the transfused donor plasma was unlikely to produce any significant labeling of the recipient’s platelets. The platelets labeled with the amount of DFP3? used in these studies were found to clump in the same manner as normal platelets when tested with .4DP in vitro.

RESULTS

Thrombus. Following the intravenous infusion of ADP, the average amount of deposit formed in the extracorporeal shunt was approximately five times as great as that formed following saline or AMP (Table I). The mean value for the AMP ex-

4 Obtained from the Radiochemical Centre, Amersham, Buckinghamsbire, England


periments was not significantly different from the mean for the animals given buffered saline. The amount of deposit formed after ATP was intermediate in amount (Table I). In these experiments, the deposit formed mainly at the classic site at the bifurcation not in the regions where flow tended to be larninar.

Platelet count. Immediately following the infusion of ADP, there was a fall in the circulating platelet count which usually returned to near the pre-infusion level within 10 minutes (Fig. 1). In samples prepared for platelet counts 1 minute after

PLATELET COUNT

NO./CU. MM. x 103

f - PER00 OF INFUSION 0 I

0 2 4 6 6 10 12 14 16 18 20

TIME - MINUTES

FIG. 1. The change in the platelet count of two pigs during and after an infusion of adenosine

diphosphate (ADP) into the proximal end of the extracorporeal shunt. The period of infusion is

indicated by the black bar.

the infusion, it was not unusual to find platelet clumps in the counting chambers. In the ADP experiments, the mean platelet count at 1 minute was significantly less than the pre-infusion value (Table II). There was also a considerable decrease in the platelet count in the ATP experiments, but the difference was not significant at 1 minute, although it was at 3 minutes (Table II). Adenosine monophosphate infusions did not significantly affect the platelet count.

In order to determine whether the platelets which returned to the circulation were from body stores or were those which disappeared from the circulation after the infusion of ADP, six pigs were given donor platelets labeled with DFP3”. The mean radioactivity per milligram platelet material was found 20 minutes after the .4DP infusion to be similar to what it was beforehand (Table III).

Clotting time. Immediately after the infusion of ADP, a shortening of the clotting time in glass and silicone-coated glass tubes was observed (Table IV). The degree of acceleration in glass was not so great after the ,4DP infusion. The clotting time in


silicone approached that in glass (Table V). This acceleration of clotting diminished about as rapidly as the platelet count returned towards the pre-infusion levels. .4 similar but less prolonged acceleration of clotting occurred after the infusion of ATP but the infusion of AMP did not lead to any significant acceleration of clotting (Table IV).

TABLE II EFFECT OF ADENINE NUCLEOTIDES ON THE PLATELET COUNT (No./mm3 X lo”)

Nucleotide infused

Time after infusion AMP ADP ATP

1 Min

No. of paired reading+ 18 21

Mean change - 49.0 - 255.0

t 1.74 7.19

P << 0.001

3 Min

No. of paired readings” 14 8

Mean change -31.0 - 167.0

t 0.75 1.90

P < 0.2

20 Min

No. of paired readings” 14 15

Mean change -61.0 - 59.0

t 1.74 2.65

P < 0.02

n Includes va!ues from experiments in which flow chambers were not used.

TABLE III PLATELET RADIOACTIVITY (DFPas) pm- AND POST-.4DP

12

- 147.0

2.19

< 0.1

10

- 133.0

2.91

< 0.02

11

- 12.0

0.30

< 0.8

Radioactivity

counts/min/mg platelet material

Post-ADP

Platelet count

No./mm3 X lOa

Post-ADP

Experiment Pre-ADP 20 Min Pre-ADP 1 Min 20 Min

1

2

3

4

5

6

Mean difference

Pre-20 Min

t P

40.2 40.9 230 40 225

38.6 36.8 600 190 605

54.3 55.2 270 65 210

72.1 71.0 275 110 355

29.2 28.1 565 55 540

33.4 35.6 405 195 390

0.03 3.33

0.048 0.206

< 1.0 < 0.9

Prothrombin time. Following the infusion of ATP, ADP, and AMP, there was on the average shortening of the one-stage prothrombin time (Table VI). However, these changes were not significant.

Partial thromboplastin time. The values for the partial thromboplastin tended to be shortened 1 and 3 minutes after the infusion of ADP (Table VII).


T.4BLE I\ CHANCE IN GLASS AND SILICONE-COATED GLASS CLOTTIN TIMES FOLLOWAX SUCLEOTIDE INFUSIONS

Clotting time mean percentage change

Nucleotide 1 Min 3 Min 20 Min

Adenoeine

monophosphate Observations 18

Mean change + 0.6

t 0.11

P < 1.0

Glass

1 5

- S.5

0.62

< 0.6

14

- 15.6

2.27

< 0.05

.4denosine Observations

diphosphate Meanchange

.4denosine

triphosphate

Adenosine monophosphate

Adenosine

diphosphate

Adenosine triphosphate

P

Observations

Mean change

P

Observations Mean change

t P

Observations

Mean change

t P

Observations

Mean change t P

Time after nucleotide infusion

19 x 12

- 25.5 - 28.7 - 8.9

7.06 5.13 2.48

< 0.001 < 0.002 < 0.05

12

-28.2

3.82

< 0.005

9

- 24.2

2.19

< 0.1

11

- 6.6

1.01

< 0,s

18

- 16.0

1.78

< 0.1

20

- 53.7

5 230

< 0.001

13

-44.1

3.59

< 0.005

Silicone

12

- 6.7

0.73

< 0.j

1 5

- 8.1

0.56

< 0.6

9

-- 55.2

4.51

< 0.002

1.3

- 21.1

2.99

< 0.02

9 10

- 29.9 - 21.6

1.82 1.53

< 0.2 < 0.2

T.4BLE V EXAMPLES OF CHANCE LV GLASS AND SILICONE CLOTTING TIMKS

.~FTER NucLE~TIDE INFUSION

Clotting time

Glass Silicone

Experiment Pre-

nucleotide

Post-nucleotide

1 Min .< Min

Pre-

nucleotide

Post-nucleotide

1 Min 3 Min

1 6.5 S .O 4.4 23.5 5.2 10.0

2 6.0 3 5 4.0 34.0 7.8 3.5

3 5.5 4.2 4.1 18.0 4.8 4.5

4 6.0 4.3 3.; 15.0 6 .3 5.8

5 6.2 S .O 4.8 26.5 7.5 8.5

6 5.7 4.0 4.0 20.5 9.5 10.8

7 S.0 5.9 6.5 13 0 7.0 14.3


Activity of Al(OH)3-trcated plasma in the thromboplastin generation test. When the maximum activities of the samples prepared before and after the ADP infusion were compared, there was evidence of a decrease (Table VII). This, however, was not quite significant.

Factor F’ZZZ activity. The ADP infusion produced a significant diminution of factor VIII activity at one minute as determined in the partial thromboplastin time assay (Table VII).

Nucleotide infused

TABLE VI PROTHROMBIN TIE

1 Min

Mean change (set) time post infusion

3 Min 20 Min

AMP

ADP

ATP

Observations

Mean change

t P

Observations Mean change

t P

Observations

Meanchange

P

3 3

- 1.18 + 4.40

2.70 0.92

< 0.1 < 0.5

11

- 0.78

2.15

< 0.1

7

- 0.49

2.04

< 0.1

- - -

3

- 0.25

0.93

< 0.5

3

- 0.80

1.19

< 0.5

6

- 1.15

3.31

< 0.05

6

0.29

2.01

< 0.1

TABLE VII

THE EFFECT OF ADP ON THE PARTIAL THROMBOPLASTIN TIME, ALL-TREATED

PLASMA Acrrvrrv AND FACYTOR VIII ACTIVITY

Partial thromboplastin time

(set)

Control

1 Min 3 Min 10 Min 20 Min

Observations 16 15 15 15

Mean change 0.20 0.45 0.33 -0.11

t 0.28 0.81 0.34 0.15

P < 0.8 < 0.5 < 0.8 < 0.9

Activity of AI(Otreated plasma

(set) in thromboplastin generation test

Observations 8 - 6

Meanchange 0.93 - - 0.66

t 1.91 - - 2.15

P < 0.1 - - < 0.1

Observations 14

Meanchange - 12.9

t 2.78

P < 0.02

Factor VIII activity (%)

14 14

- 3.4 - 10.6

0.44 1.44

< 0.7 < 0.2

14

-4.6

0.56

< 0.6


Glass contact activity. The ADP infusions produced an appreciable increase in glass contact activity in nine out of ten cases studied. A significant increase in activity was demonstrable at 1 minute. Neither AMP nor .4TP infusions produced any consistent response (Table VIII).

Blood pressure and electrocardiogram changes. Examples of the changes in electrocardiogram pattern and blood pressure following infusions of ADP are shown in Fig. 2. There was a fall in blood pressure which lasted for the duration of the in-

TABLE VIII GLASS CONTACT ACTIVITY

Nucleotide infused

Time post-infusion

1 Min 3 Min

AMP Observations

Control Ratios

Treated

t

P

ADP Observations

Control Ratio __-

Treated t P

,4TP Observations

Ratio

t tJ

10 6

0.961 0.942

O.Sl < 0.5

1.25 < 0.3

10

1.182

3.40

< 0.01

6 6

1.036

0.531 < 0.7

7

1.128

1.24

< 0.3

1.084

0.984 < 0.4

Control (pre-infusion value set) N Ratio =

Treated (post-infusion value set)

(This is the same as taking the difference between the logarithm of the pre and post infusion values.)

fusion of each of the nucleotides. It seemed to be in part related to changes in function of the pulmonary vasculature and heart. This effect on blood pressure was considerably greater with ADP than with ,4MP infusions. The pattern of the electrocardiogram was altered during the infusion of ADP and the effect lasted for 1 to 2 minutes which is also the period in which the platelet count was depressed. There were changes compatible with ischemia, bradycardia, extrasystoles or coupled beats with changes in amplitude. Adenosine monophosphate infusion induced a slight fall in arterial pressure and some slowing of cardiac rate.

Some of the animals developed apnea and required artificial respiration for up to a minute following the .4DP infusion. Some, in addition, showed a transient, blotchy erythema of the skin at the same time. In general, if the dosage was small or the infusion of .4DP was given slowly, the overall reaction was not severe.

Histology. Some animals not used in the thrombus study were killed immediately after the infusion of ADP. Examination of the small vessels in the lungs and heart showed extensive and numerous platelet aggregates (Fig. 3). No platelet clumps


were found in animals examined immediately after the AMP infusion, but some were found immediately after ATP. Animals killed 20 minutes after the ADP infusion did not show any evidence of such aggregates.

The deposits which were found in the shunts after the ADP infusion were predominantly white thrombi rich in platelets. The proportion of platelet material appeared to be greater than that in shunts from animals not given ADP, but this impression is difficult to assess critically.

EKG

LEA01

EKG

LEAD n

FEMORAL ARTERY

PRESSURE MUG.

FIG. 2. The changes in electrocardiogram and blood pressure immediately and 10 minutes after the infusion of ADP into the proximal end of the shunt. The depression of the S-T segment in lead T, seen immediately after the infusion was not present at 10 minutes.

The effect 01 diferent concentrations of ADP. The effects of smaller doses of ADP were studied. The results are shown in Table IX. There was increased thrombus formation with a fall in the platelet count and acceleration of the silicone clotting time. The animals showed much less systemic reaction than that seen with the larger doses.

In order to observe whether ADP would exert an effect if allowed to circulate through the body first, it was injected into the distal end of the shunt. Given in this way in doses ranging from 17 to 108 us/kg body weight, the usual systemic circulation reaction, fall in the circulating platelet count and acceleration of the clotting time in silicone-coated glass tubes occurred (Table X). The weight of thrombus formed, however, was no greater and if anything less than the mean in normal con- trols.

T he efiect of heparin. The intravenous infusion of heparin in doses ranging I be- twec :n 200 and 40,000 units/kg body weight did not effect the fall in platelet cou nt, but did suppress the formation of thrombi in the shunt following the infusion of A\Dl P (Table XI). There were no detectable changes in the clotting time in gl; ass


FI LG. 3. A section through a small myocardial vessel immediately after an intravenous A infu! sion. An aggregate of platelets surrounded by white cells and red cells can be seen in

lumc :n of the vessel (H & E; X 480).

,DP the


or silicone. Although there was a tendency for the platelet count to decrease immediately after the infusion of heparin, the platelet count was allowed to return towards the preheparin level before the ADP was infused. It may be noted that with heavy intravenous dosage of heparin, even though the bleeding time was

TABLE IX THE EFFECT OF VARIOUS SMALL DOSES OF ADP

Treatment

Weight of thrombus (pg)

95% Confidence Comparison

No. of Mean limits on mean (fig) with

studies (geometric) Lower UDDer control

Control (saline) 12 351 135 911 t= 3.414

ADPa 13 2,520 1,090 5,827 p <o.oos

Mean change of platelet count 1 min after ADP

Mean = - 145,OOO/mms n= 13

t= 7.68

p = < 0.001

Mean change of silicone clotting time 1 min after ADP

Mean percentage change = - 61 .O

n= 13 t= 8.80

p = < 0.001

a Dosage: 6 animals were given 500 pg of ADP and 7 animals 2 mg of ADP.

TABLE X

THE EFFECT OF ADP INFUSIONS INTO DISTAL END OF SHUNT

ADP dose (pg/kg body weight)

17.5 16.9

105.7 107.8

Weight of

deposit

(Pd

140 1,030

1 800

Platelet count Clotting time (No./mms x 103) minutes - silicone

1 Min 1 Min Pre- post- Pre- Post- ADP ADP ADP ADP

250 390 21.0 17.2 230 185 13.0 17.5

415 233 27.0 6.0

51.5 205 29.5 9.5

Appropriate mean 104

95% confidence limits 1.96547.3

Significancea of

difference between means

a Paired differences.

352.5 253.2 21.59 11.45

t 1.030 t 1.530

P < 0.4 P < 0.3

markedly prolonged and thrombus formation in the shunt totally suppressed, platelets exposed to ADP still undergo aggregation as manifested by the fall in the platelet count in all but one experiment (Table XII).

DISCUSSION

These experiments show that when increased ADP is present in circulating blood, there is an enhanced effect locally on thrombus formation in areas of disturbed flow. However, there was no evidence of deposit formation in regions of the shunt where


TABLE XI

HEPARIN AND ADP

Saline

Thrombus weight (kg)

Heparin

ADP and ADP

Observations

Mean (geometric)

95% Confidence limits Upper

Lower

12 30 19

351 1,750 < 05

911 2,859 -

135 1,071 -

Change in platelet count No./mm3 X lo3 (Prel min)

Heparin

ADP and ADP

No. of paired differences 21 17 Mean change - 255 - 155

t 7.19 5.266

P << 0.001 < 0.001

Note. Exact confidence limits on the mean weight of thrombus after heparin + ADP cannot be

calculated, since no thrombus was formed in 10 of the cases. The upper value on the sample mean is computed by setting the weight at 1 ug in all such cases.

TABLE XII

EFFECTS OF HEPARIN AND ADP ON PLATELET COUNT, WEIGHT OF THROMBUS,

AND BLEEDING TIME

Dose of heparin Dose of ADP (units/kg) (w’k.4

Platelet count

No./mm3 X 103

20 Min

after heparin

23% Min after

heparin,

1 Min after ADP

Weight of

thrombusa

w

Bleeding

timeb (min)

4,400c

28,600c

42,600e

7,3ow

44od t.i.d. for 3 days

4-W

t.i.d. for 3 days

2,200c

1,800”

2.75 235 60 <l 2.75 125 50 <l 1.78 120 25 <l 1.84 120 20 <l

1.38 250 160 <1

1.12 210 225 <l 6.0

1.38 330 90 <l 25.0

1.12 475 400 <l 25.0

25.0

25.0

19.0

19.0

3.5

(1 No detectable thrombus was found in the shunts in these experiments. b The bleeding time in 20 normal pigs gave the following values: mean 3.2 min, range 1.5-4.8 min.

(This is determined by making a l-inch cut to the subcutaneous tissue on the inside of the thigh. The bleeding from the edges is blotted with filter paper every 30 set until it stops.)

* Intravenous heparin.

d Subcutaneous heparin.


flow is predominantly laminar. Thus it appears that the effect of .4DP in enhancing thrombus formation in these studies required an additional factor, that of disturbed blood flow,

If none of the dose were destroyed until after completion of the injection, the smallest amount of ADP infused in these experiments could have been expected to produce a plasma level of the order of 50 rig/ml. About one tenth of this concentration is sufficient to produce platelet clumping in native platelet-rich plasma in vitro (Mustard et al., 1964). With complete mixing and no destruction when blood flow

through the shunt is 400 ml/minute, the average concentration at the bifurcation achieved would be of the order of 1,600 rig/ml of plasma. In view of the rapid rate at which ADP is changed in blood, it is unlikely this maximum concentration was ever attained. The possibility exists that levels of the order discussed above, may appear at times in parts of the circulation when there is cell injury such as with ischemia and infarction or hemolysis. Honour and Mitchell (1963) and Born et al. (1964), in studies of rabbit’s cerebral microcirculation have adduced evidence that traces of ADP are important and perhaps essential in the production of “white bodies.”

The failure of AMP to enhance thrombus formation is compatible with the fact that it also does not cause platelet aggregation (Born, 1962: O’Brien, 1962: Mitchell and Sharp, 1964). The intermediate effect seen with .4TP is probably at least in part due to the tendency it has, particularly in ZGVO, to be converted into ADP. Our commercial preparations of ATP have usually been contaminated with trace amounts of ADP when examined by paper chromatography. It must not; therefore, be inferred that the effect seen in these experiments was due to ATP as such.

The sharp fall in platelet count following ADP administration probably means as suggested by Born and Cross (1963) that clumps of platelets are momentarily arrested in parts of the circulation, presumably the small vessels. The histological studies carried out in the present investigations are compatible with this conclusion. Nordoy and Chandler (1964) in their recently reported study came to a similar conclusion. That the same platelets which disappear from the circulation return at the end of the transient thrombocytopenia has been demonstrated by studying the be- havior of donor platelets labeled with DFP”‘. Since platelets so prepared can still be clumped by ADP in vitro, there is no reason to suppose that they are treated any differently from untreated platelets in viva. In experiments in which we have given thrombin to pigs, a more sustained thrombocytopenia occurs but the platelets which return to the circulation after 1-2 hours appears to be the same as those which were present before the thrombin infusion (Robinson et aZ., 1962). These platelets have been found to have a life span much the same as normal after their return to the circulation. The slower reversal of the thrombocytopenia and platelet aggregates in the thrombin experiment may perhaps be due to the formation of fibrin in the aggregates which must by lyzed before the platelets can return to the circulation.

The nature of the factors involved in the breaking up of platelet aggregates in viva is not at present known. However, it is of some interest in the present experiment that unlike the aggregates in the vessels, the deposits which formed in the shunts after administration of .4DP did not break up spontaneously. Since the blood bath- ing the clumped platelets in the vessels and in the shunt is the same, this dis-


crepancy may mean that some of the factors involved in the breaking up of aggregates lie in the vessel wall. There is good evidence that enzymes with phosphatase

activity occur in the subendothelial tissues (Kirk, 1963). We have found that the addition of acid or alkaline phosphatase to plasma in vitro accelerates the disintegra- tion of ADP-induced platelet a ggregates (Blakely ct al., unpublished observations). However, it should be pointed out that even in the absence of added .4DP, deposits invariably form and persist at the bifurcation in the silicone-coated shunt. It is possible that the persistence of deposits in the shunts may be attributable to continual release of small amounts of .4DP as a result of injury to the cellular elements of the blood by disturbances in flow in the region of the bifurcation. R-e have elsewhere reported that while stagnant blood in the flow chamber may not coagulate flowing blood from the same animal, in the same flow chamber will form deposits, in an equal period of time (Murphy et al., 1963).

The marked acceleration following intravenous ADP of the clotting time in silicone-coated, in contrast with uncoated glass tubes was striking. This effect diminished as the platelets returned to circulation. The addition of ADP to native platelet plasma in vitro accelerates clotting in the presence of platelets but not otherwise (Mustard et al., 1964). Experiments in progress have so far indicated that ADP infusions do not accelerate blood coagulation in thrombocytopenic animals (Rowsell et al., 1965).

In the present study, 4DP infusions did not produce any marked acceleration of the one-stage prothrombin time, partial thromboplastin time, or thromboplastin generation test. We have evidence that L4DP releases clotting activity from platelets in z&o (Mustard et at., 1964). In the present experiment, moreover, the clumping of the platelets was associated with an increase in glass contact activity and nearly abolished in some instances the difference between the glass- and silicone-clotting times. It remains to be determined how this effect of ADP is mediated. It may act by releasing or otherwise making available platelet-clotting factors, or it may be that in some way the physical changes produced by clumping of platelets activate the contact factor in blood coagulation.

Furthermore, the available morphological evidence does not indicate that ADP causes major structural alterations in the platelet (Hovig, 1962). The marked acceleration of clotting seen with the ADP infusions could be taken as evidence of a hyper- coagulable state. Despite this, there was no tendency for the platelet aggregates in the intact body to persist and grow in size. This observation further emphasizes the difficulty of trying to relate thrombus formation to in z@t~ tests of clotting.

There was a small but consistent fall in factor VIII activity concomitant with the fall in platelet count. This in some respects parallels the much greater fall in factor VIII activity seen with thrombin infusions (Robinson et al., 1962). There are no indications of whether the fall in factor VIII activity with .4DP is a primary or secondary phenomenon.

The results from the present experiment do not support the claim that adenine nucleotides, in particular ATP and &4DP, are inhibitors of blood coagulation (Pil- geram, 1963: Quick. 1963: Quick, 1964). Since nucleotides are very rapidly cleared from the blood (J+rgensen, 1956), it would be difficult to achieve plasma levels in z~iz~ equal to those in Pilgeram’s (1963) and Quick’s ( 1963; 1964) in z&vu esperi- ments.


Although the infusion of large amounts of heparin before administration of ADP prevented thrombus formation in the shunts, the ADP infusion still caused a fall in the platelet count. Since the aggregation of platelets by ADP was unaffected by pretreatment with heparin, it appears that heparin can more easily block the adherence of platelets to a silicone-coated surface than to each other. That this dis- tinction is not merely an artifact is suggested by the fact the bleeding time which reflects in part the formation of platelet plugs at sites of injury and is therefore in part a surface effect, may be grossly prolonged while the fall in platelet count induced by ADP is little affected. It seemed likely therefore that platelet adherence to other surfaces is more readily suppressed by heparin than is ADP-induced aggregation. We have found that high concentrations of heparin of the order of 2000 units/ ml of plasma are necessary before there is any inhibition of ADP-induced platelet aggregation in vitro. This has been the experience of others (Owren, 1964).

The effect of ADP infusions on thrombosis in the extracorporeal shunt seems to be mediated through aggregation of platelets which then come in contact with surfaces and accumulate at points of disturbed flow. However, the occurrence of the circulatory upset during the first minute after ADP infusion raises the possibility of reflex epinephrine secretion. We have found that epinephrine accelerates clotting and en- hances thrombus formation in the shunt experiments (Rowsell et al., to be published). However, the experiments in which small doses of ADP were administered produced little hemodynamic reaction in the animals but still promoted increased thrombus formation, marked acceleration of clotting and a transient thrombocytopenia. Con- versely, in the experiments in which ADP was injected distal to the flow chamber, the usual circulatory disturbances occurred but thrombus formation was not enhanced. In the epinephrine experiments the acceleration of clotting was much less, and there was no evidence of platelet aggregation and fall in the platelet count (Rowsell et al., to be published). These observations support the conclusion that the effect of ADP infusions on clotting and thrombosis is primarily mediated through platelet aggregation and its consequences.

The persistence of the shortened clottin, u time in silicone-coated glass after return of the platelet count could be related to activation of clotting or alteration in the platelet which persist for a time after the aggregates have broken up. Here again, epinephrine release could be a factor. It seems important that the effect of ADP on platelets and the relationship of this to acceleration of clotting should be studied in some detail.

SUMMARY

Adenosine diphosphate (ADP) produces a transient fall in the circulating platelet count, and locally promotes thrombus formation in the extracorporeal shunt. If present in the pulmonary or

coronary circulation in high concentrations, it may produce a marked fall in blood pressure and disturbances in cardiac rhythm. It produces comparatively little effect on in vitvo tests of coagulation, except that there is activation of contact factor with acceleration of the clotting time in

silicone so that it is almost as short as that in glass. Adenosine triphosphate (ATP) has a similar but less marked effect that is possibly due to the spontaneous conversion of ATP to ADP.

Adenosine monophosphate (AMP) has no demonstrable effect. Infusion of heparin in sufficient doses suppresses the promotion of thrombus formation by ADP, produces a prolonged bleeding time but fails to prevent platelet aggregation and the occurrence of transient thrombocytopenia.

The thrombocytopenia and the plugging of small vessels by platelet aggregates demonstrable at

post mortem performed immediately after ADP infusion are rapidly reversed, and it can be shown


that platelets returning to the circulation are the same as those present beforehand. The thrombi

produced in the extracorporeal circulation, however, are not reversible. The vital properties of the

vessel wall may account for this difference.

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the effect of adenine nucleotides on thrombus formation, platelet count, and blood coagulation

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