THROMBOSIS RESEARCH Volume 10, Pages325-335.

PLATELET

FUNCTION

IN

Pergamon Press,

SICKLE

CELL

R. A. Gruppo, H. I. Glueck, S. M. Granger, Departments of Pediatrics and University of Cincinnati College Cincinnati General Hospital, and Sickle Children's Hospital Research Foundation,

1977. Printed in Gt. Britain.

ANEMIA

and M. A. Miller

Medicine, of Medicine Cell Disease Center Cincinnati, Ohio, USA

(Received 1.12.1976; in revised form 3.1.1977 Accepted by Editor M.I. Barnhart)

ABSTRACT Platelet function was serially followed in a group of 13 children with SCD. Defective platelet aggregation was seen in all 4 patients studied during painful crisis and in 45 percent of patients during "asymptomatic" periods. Stypven times were shorter (perhaps reflecting increased PF-3) in the PRP of patients; however, Stypven times were slightly prolonged following freeze-thawing. Recombination experiments in 8 patients revealed no inhibition of SCD plasma on the function of platelets derived normal controls. These findings are suggestive of partial activation of platelets in SCD and may suggest a role of platelets in the genesis of the vaso-occlusive crisis common in this disorder. INTRODUCTION

Thrombotic phenomena appear to be an important factor in many of the vaso-occlusive problems associated with sickle cell disease (SCD). Although sickling of red cells is primarily responsible for this phenomenon, the individual roles, although secondary, of the platelets, coagulation factors, and components of the fibrinolytic system have been suggested (1). In particular, the role of platelets has been recently reappraised. Green and associates (2) have reported thrombocytosis in SCD patients whether in crisis or asymptomatic, while Van der Sar (3) observed a post-crisis thrombocytosis in 8 patients with SCD. Barnhart and associates (4, 5, 6) found a marked increase in the percentage of platelets circulating in the "activated" dendritic or aggregated state regardless of whether the patient was in crisis or asymptomatic. Haut and associates (7) reported normal autologous platelet survival during non-crisis periods 325

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compared to a marked decreased survival during crisis. There are, however, conflicting reports regarding platelet function in SCD. Haut and associates (7) noted platelet aggregation in both crisis and non-crisis periods to be normal, while aggregation in three subjects during post-crisis thrombocytosis was greater than normal. Stuart and co-authors (8) found an impairment in the rate and percent of first phase platelet aggregation using adenosine diphosphate (ADP) as the aggregating agent in ten patients during vaso-occlusive crises, compared to normal first phase aggregation in asymptomatic patients. Kisker and Glueck (9) from our laboratories studied 16 patients with SCD with reference to several aspects of their hemostatic mechanisms including platelet aggregation. Their preliminary studies revealed a frequent abnormality in platelet function consisting of decreased aggregation to epinephrine, ADP and collagen. These preliminary studies led to the more detailed investigations described in this report. In addition, the relationship between defective platelet aggregation, serotonin release, and the effect of SCD plasma on normal platelets will be discussed. MATERIALS AND METHODS Informed consent was obtained from the patient and parent or guardian prior to enrollment in the study as approved by the Committee on Human Experimentation, Children's Hospital Research Foundation, Cincinnati, Ohio. Patient and control groups were divided as follows: Group I (controls): 10 normal volunteers (5 male and 5 female who had ingested no medication for one week prior to and during the duration of the study followed weekly for five consecutive weeks. Group II: 13 patients (11 SS, 1 S-thal, 1 SD by hemoglobin electrophoresis) ages 5 to 21 who were asymptomatic at the time of the study and had ingested no medication other than acetaminophen for at least one week. Group III: 4 patients from Group II who were restudied during the first 48 hours after the onset of a painful crisis. They had ingested no medication other than acetaminophen for at least one week and were on no parenteral analgesics except merperidine. In addition, a separate control subject was also studied simultaneously with the patient on the day of testing. Hence, control and patient platelet rich plasma (PRP) were studied under identical conditions of time, centrifugation, storage, and testing. All results were analyzed statistically by the nested analysis of covariants (10). The following studies were performed on all patients: complete blood counts, reticulocyte and platelet counts, bilirubin (total, direct and indirect), platelet aggregation with epinephrine, ADP and collagen, "C-serotonin uptake and release and PF-3 availability. In addition, recombination studies were done in 8 subjects following platelet separation and washing on albumin gradients. Venous blood for platelet aggregation and "C-serotonin uptake and release was collected in 0.1 M buffered citrate, pH 5.0 (1 part citrate and 9 parts blood). The titrated blood was spun at 40 x g for 15 minutes to obtain PRP. Platelet Poor Plasma (PPP) was prepared by respinning at 600 g for 25

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PPP contained less than 1.0 x lo3 platelets per mm3 minutes. All platelet counts were performed using a Thrombowith PPP. AggregaInc., Hialeah, Florida. Counter, Coulter Electronics, tion was performed with epinephrine (Parke Davis and Co., Detroit, Mich.), 1:150,000 dilution (30 micromolar) final concentration, ADP (Sigma Chemical Co., St. Louis, MO.), 2.0, 4.0 and 20 micromolar final concentration, and collagen. A collagen suspension was prepared by suspending 2 grams commercial collagen from bovine achilles tendon (Sigma Chemical Co., St. Louis, MO.) in 100 ml saline and blended in a Waring blender (Waring Products Div., New Hartford, Connecticut) for 5 minutes. The mixture was centrifuged for 15 minutes and the supernatant diluted with saline to a concentration which produced maximal All aggregation was measured aggregation with control PRP. with 2 Chrono-Log aggregometers with a dual pen Chrono-Log Platelet recorder (Chrono-Log Corporation, Broomall, Pa.) separation and washing was performed on albumin density gradients as described by Walsh (11). Following washing in calcium-free Tyrode's solution, the platelets were resuspended in patient or control PPP and platelet counts adjusted to 3 to 4 x 10' per "C-serotonin uptake and release was performed according mm3. to a modification of Tschopp and Zucker (12). Twenty lambda 14C-serotonin (50 mCi/mmole, Radiochemical Center, Amersham) was added per 10 ml PRP and incubated for 10 minutes at room temperature. After completion of labeling PRP was stored during Following aggregation (5 the procedures at 20 degrees C. minutes) PRP was kept on ice and then spun at 4 degrees C at 600 g for 10 minutes. A 50 lambda sample of PPP was placed in 10 ml of liquid scintillation fluid (21 gm PPO, 900 ml 95% ethanol, 2100 ml toluene) and radioactivity was measured by liquid scintillation and spectrophotometry with a Beckman LS 230 liquid scintillation counter. All samples were done in duplicate and results averaged. PF-3 was measured according to the method of Sixma and Nijessen (13) using Russell's viper venom (Stypven). Stypven times were initially determined after diluting one part PPP (platelet count less than 100) or PRP adjusted to 3 - 4 x lo5 per mm3 with 3 parts normal PPP. In addition, Stypven times were similarly measured on PRP following freezing and thawing 5 times, presumably releasing maximal quantities of PF-3. RESULTS The chief platelet abnormality found in SCD patients was an impairment of aggregation with epinephrine, ADP and less frequently to collagen. Figure 1 demonstrates a patient with abnormal aggregation to all three agents. Abnormalities occurred in all 4 patients during crisis (Fig. 2), but in addition, abnormalities were often seen in the patients during symptom free periods. Table I summarizes the aggregation data in 29 separate studies in 13 patients during non-crisis periods. In the Group I controls, following the addition of the aggregating agent, maximal aggregation was obtained by the 4 minute interval. Hence, the percent aggregation at this interval was used to compare the patient to the control group. Results were considered abnormal when the level of aggregation at the 4

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minute interval fell 2 S.D. below the normal mean. The mean percent aggregation at 4 minutes was statistically significantly depressed when compared to controls for each aggregating agent. The percentage of patients with abnormal aggregation (below 2 to one S-D.) is also shown. In the 13 patients, abnormalities or more of the aggregating agents occurred in a total of 45 percent of observations. An abnormality to a single aggregating agent was noted in only 15% observations whereas multiple abnormalities were seen in the remainder. There was no statistical correlation between aggregation abnormalities and the level of hematocrit, reticulocyte or platelet count, or bilirubin.

IO 0

90 &a ra 60 !a * Y) 20 10 0

FIG. 1 Platelet aggregationdenon&rating abnormalitiesto epinephrine,ADP and collagen inanasyqkcsntic patient with SCD. 2 represents +2 S.D. innormal (Group I) controls.

Aggregation response of PRP to epinephrine and collagen has been shown to be dependent on the time interval between collection of the sample and testing (14). Therefore, in all aggregation experiments, the patient's PRP and a separate control were run simultaneously on each testing day. However, there was no statistically significant difference in percent aggregation (P>O.5) with any aggregating agent between these random "day-to-day" controls versus the Group I controls which were used for the statistical determinations (cf. Methods).

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100 -

0'

m

Epinephrine

ADP

ADP

30x10-‘M

2XKT.M

4X10-‘M

ADP 20 x IO-’ M

Collagen

FIG. 2 Maxknalpercentaggregation (4min.J in 4 SCDpatients + 2 S.D. in nonral studiedduringcrisis. 3 represents (GroupI) controls.

TABLE I Mean percent aggregation in 13 asymptomatic patients (33 observations) compared to controls. Aggregation was considered abnormal if the percent aggregation at 4 minutes fell 2 S.D. below the normal mean. lean Percent Aggregation + 2 S.D. t 4 min. j.9qresntih.

Normal

l-

Patients

P Value

Percent Abnormal

Epi., 30 x 10-6M

63.1 + 18.8

41.9 f 26.0

< 0.001

30.3

ADP, 2 x 10-6M

63.2 f 15.5

40.5 k 24.2

< 0.02

39.4

ADP, 4 x 10-6M

70.8 + 10.7

53.6 f 22.7

< 0.001

33.3

ADP, 20 x 10-6M

75.1 +

8.7

63.3 + 17.2

< 0.001

33.3

Collagen

68.9 f 11.5

57.4 + 23.1

< 0.001

24.2

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Table II demonstrates the results of "C-serotonin uptake and release which were simultaneously measured during all aggregation experiments. Except for epinephrine, there were no statistically significant differences between the SCD patients versus controls. TABLE II "C-serotonin release following platelet aggregation in SCD. Data represents a summary of 13 asymtomatic patients (20 observations) versus normal (Group I) controls.

Percent "C-serotonin Release f 2 S.D. Aggregating Agent Normal 1 Patients

P Value

I

Epinephrine

43.8211.3

30.7219.4

0.015

ADP, 2 x 10-6M

33.9f11.2

19.5f12.5

0.170

ADP, 4 x 10-6M

26.7f 8.7

26.4k13.0

0.130

ADP, 20 x 10-6M

37:7f 8.9 I

Collagen

33.4flO.l I

0.255

32.3k14.0 I

I

39.9f15.3 I

0.136 I

To determine if the aggregation abnormalities were secon_ . . dary to plasma factors, recombination studies were performed on eight patients with abnormal aggregation. Platelets were separated on albumin gradients and washed once in Tyrode's buffer solution. These washed platelets were then recombined with plasma as shown in Figure 3. Platelet counts were adjusted to 3 to 4 x 10' and following a 30 minute incubation period without stirring at 37', aggregation studies were performed. In all eight recombination studies, SCD.platelets resuspended in normal plasma showed no functional improvement, whereas normal platelets resuspended in SCD plasma for the same interval consistently revealed no inhibition. Platelets possess several distinct coagulant activities (15). One of them, PF-3, is thought to catalyze the reaction of Factor X and V to activate prothrombin in the presence of calcium. T!%s activity is normally present in platelets in latent form but made available on the membrane during platelet activation.

x_x 100

Patient 0.P Epinrphrins I : 150,000

z? 00 t

g 9

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I

Normal platelets Normal plasma ._. Normal atelsts Sickle $ asma 8-A Sickk plotelrts Normal plasma 0---o Sickle ~atelats Sickle plasma

FIG.

Aggregatian response to epine&ri.ne (A) and ADF’ (B) follcwbq platelet separationonalbunin density gradients, washing, and reambination with various plasrtxis. The results are representative of typical aggregation responses cbserwd in eight similar mixing

experilwnts

TIME

80

(min.)

Patient D.P. t 4 x IO-% ADP

(B)

A_A

Sickle platelets Normal plasma

0_-o

Sickle plotelets Sickle plasma

TIME

3

( min.)

.

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PF-3 activity was determined using Stypven on samples of PRP and PPP prior to freeze-thawing. Such samples presumably contain only minimal amounts of available pro-coagulant activity. Similarly PF-3 activity was determined on PRP following freeze-thawing five times, thus releasing maximal amounts of residual activity from platelets. Because of the day-to-day variance in the Stypven tests, all statistical analyses were performed on simultaneously matched (patient vs. control) samples. The differences were calculated and analyzed by the paired t test. As shown in Figure 4, the mean Stypven time of patient PPP prior to freezethawing was 30.1 f 4.1 sec. vs. 38.02 + 4.2 sec. in controls. The mean of the differences on paired samples was 7.5 sec., p < 0.001. Similarly the mean Stypven time of patient PRP prior to freeze-thawing was 29.5 ?;4.9 sec., vs. 32.6 ?:4.1 sec. in controls. The mean difference was 3.0 sec., p = 0.013. Following release of residual PF-3 activity in PRP with freeze-thawing, the values of the patients' Stypven times were now slightly but consistently longer than the controls (16.7 + 2.2 sec. vs. 16.3 + 2.2 sec. The mean of the differences on paired samples was 0.47 sec., p = 0.02.

50 T

40 1

0I

0

Control

m

SCD Potients

3

PPP

f:

I S.D.

PRP

Re-Freeze-Thaw Pre-Freeze-Thaw Post-Freeze-Thaw

FIG. 4 StypvenClotting times UT-3 activity)on PPP and PRP prior tofreeze-thawingvs.pRpafterfreeze-thawing(maximally releasingPF-3)inSCDversus controls.

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DISCUSSION One of the chief platelet abnormalities found in our patients with SCD were impairments of platelet aggregation to epinephrine, ADP and less frequently to collagen. These abnormalities were often seen during asymptomatic periods, and were observed in all 4 patients studied during crisis. Furthermore, we have shown that platelet refractoriness could not be conferred on normal platelets incubated in SCD plasma. Stuart and co-authors (8) noted impaired rate and percent of first phase platelet aggregation with ADP in SCD patients during vaso-occlusive crises. It is difficult to compare our data with theirs, since they used amounts of ADP (0.8 micromolar) suboptimal for secondary aggregation in their patients, all of whom were on aspirin. Aggregation changes in their patient group was dissimilar to that seen in the control group while on aspirin, and was interpreted as representing true platelet refractoriness. Stypven times in non-frozen PRP and PPP of our patients were shortened whereas the Stypven times following freezethawing were slightly longer than the controls. Although the mean differences were minute, the differences between paired samples of patient vs. control (paired t test) were statistically consistent. The shorter Stypven times in non-frozen PRP and PPP in our patients suggest prior partial activation with increased available PF-3, although the presence of other activated clotting factors, particularly X, cannot be excluded. Following freeze-thawing, decreased residual activity of PF-3 in SCD platelets is suggested by slightly but significantly prolonged Stypven times. This partial activation may relate to the earlier findings of Barnhart and associates (4, 5, 6) who reported an increased percentage of morphologically "activated" dendritic and aggregated platelets in the circulating blood of SCD patients, either asymptomatic or in crisis. The pathophysiology of the vaso-occlusive crisis in SCD has been recently reviewed by Klug and associates (16). Decreased red cell deformability promotes stasis, which in turn augments local hypoxia and acidosis,. leading to increased sickling. With continued stagnation, propogation of the vasoocclusive mass occurs by the addition of red cells,'platelets and leukocytes until the situation becomes irreversible. The role of platelets in the clinical vaso-occlusive crisis of SCD is still uncertain. We have found suggestive evidence for increased available procoagulant (PF-3) suggesting prior activation and decreased aggregation to epinephrine, ADP and collagen. The finding of decreased platelet function in a disease characterized by a thrombotic tendency is not necessarily contradictory. A "refractory state" has been reported in patients prone to postoperative thrombosis, particularly those with malignancy (17). Possible causes of this refractory state in SCD have been reviewed by Stuart and associates (8) and may be related to ADP induced refractoriness described by O'Brien (18). It is possible that ADP is released in vivo from hemolyzed erythrocytes

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or released at the site of focal vaso-occlusion from activated platelets. Whether platelets are only passively affected by the vascular events accompanying SCD or play a contributory role in the pathophysiology and clinical manifestations of this disease needs to be further defined. ACKNOWLEDGEMENTS This study was supported in part by Research Grants HL15996-01 from the National Heart and Lung Institute and USPHS Grant HL-2904-19; and in part by Training Grant 5 TO1 CA 0519607 from the National Institutes of Health. REFERENCES 1.

RICKLES, F. R., and O'LEARY, D. S.: Role of coagulation system in pathophysiology of sickle cell disease. Arch. Intern. Med., 133, 635, 1974.

2.

GREEN, D., KWAAN, H. D., and RURIZ, G.: Impaired fibrinolysis in sickle cell disease. Relation to crisis and infection. Thromb. Diath. Haemorrh., 24, 10, 1970.

3.

VAN DER SAR, A.: The sudden rise of platelets and reticulocytes in different sickle cell crises. Blood, 34, 733, 1969.

4.

WALSH, R. T., LUSHER, J. M., and BARNHART, M. I.: Coagulation and fibrinolysis studies in sickle cell anemia. Thromb, Diath. Haemorrh., Supplement 53, 271, 1973.

5.

LUSHER, J. M., and BARNHART, M. I.: Evaluation of oral urea in the management of sickle cell anemia. Adv. Exp. Med. Biol., 28, 303, 1972.

6.

BARNHART, M. I., VETTRAINO, A., and LUSHER, J. M.: Microscopy as an aid in the evaluation and management of sickle cell anemia. Thromb. Diath. Haemorrh., Supplement 53, 193, 1973.

7.

HAUT, M. J., COWAN, D. H., and HARRIS, J. W.: Platelet function and survival in sickle cell disease. J. Lab. Clin. Med., 82, 44, lP73.

8.

STUART, M. J., STOCKMAN, J. A., and OSKI, F. A.: Abnormalities of platelet aggregation in the vaso-occlusive crisis of sickle cell anemia. J. Pediat., 85, 629, 1974.

9.

KISKER, C. T., and GLUECK, H. I.:

Unpublished observations.

10. OSTLE, B.: Statistics in research, Iowa, 1963. University Press, page 288.

Iowa State

11. WALSH, P. N.: Albumin density gradient separation and washing of platelets and the study of platelet coagulant activities. Brit. J. Haemat., 22, 205, 1972.

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12. TSCHOPP, T. B., and ZUCKER, M. B.: platelet function in rats. Blood,

Hereditary defect 40, 217, 1972.

in

13. SIKMA, J. J., and NIJESSEN, J. G.: Characteristics of platelet factor 3 release during ADP-induced aggregation. Comparison with 5-hydroxytryptamine release. Thromb. Diath. Haemorrh., 24, 206, 1970. 14. ROSSI, E. C., and LOUIS, G.: A time-dependent increase in the responsiveness of platelet-rich plasma to epinephrine. J. Lab. Clin. Med., 85, 300, 1975. 15. WALSH, P. N.: a hypothesis.

Platelet coagulant activities Blood, 43, 597, 1974.

and hemostasis:

16. KLUG, P. P., LESSIN, L. S., RADICE, P.: Rheologic aspects of sickle cell disease. Arch. Intern. Med., 133, 577, 1974. 17. O'BRIEN, J. R., TULEVSKI, V. G., ETHERINGTON, M., MADGWICK, T ., ALKJAERSIG, N., and FLETCHER, A.: Platelet function studies before and after operation and the effect of postoperative thrombosis. J. Lab. Clin. Med., 83, 342, 1974. 18. O'BRIEN, J. R.: Refractory state of platelet aggregation with major operations. Lancet, 2, 741, 1971.