THE PLATELET D E F E C T ASSOCIATED WITH ALBINISM

Roger M. Hardisty Deportment of Hematology, Institute of Child Health, Hospital for Sick Children

London, W.C. 1. England

and

David C . B. Mills* Department of Pharmacology, Royal College of Surgeons

London, England

In 1967, Hardisty and Hutton’ described a series of patients with mild bleed- ing tendencies whose platelets failed to aggregate on exposure to collagen and gave no second phase of aggregation on exposure to adenosine diphosphate ( A D P ) , epinephrine, or thrombin. They suggested that the bleeding tendency of these patients, among whom were two unrelated women with total albinism, might reflect a defect of the release of A D P from their platelets. We present here the results of further work that confirms this suggestion and offers some explana- tion of the underlying mechanisms involved.

Clinical and Routine Laboratory Findings The patients PH and AB were aged 37 and 20, respectively, when first ex-

amined. Both had suffered since early childhood from excessive bruising follow- ing minor trauma, and both had required transfusion because of postoperative hemorrhage. The younger patient had had recurrent severe epistaxes, which were eventually controlled by estrogen therapy, and both had had menorrhagia, on account of which they were taking oral contraceptives. Neither had any family history of albinism or of a hemorrhagic tendency; the elder patient’s parents were first cousins.

All tests of the blood coagulation mechanism, including factor VIII assays, gave normal results in both patients. Their bleeding times were prolonged each time they were measured, while their platelet counts were always above 240,000/ pl. Both patients’ platelet “adhesiveness” (Salzman’s method) and platelet factor 3 availability were reduced.

Platelet Aggregation The platelets of both patients gave good first-phase aggregation with A D P and

epinephrine, but no second phase with either of these reagents, and practically no aggregation with collagen at ten times the concentration that produced good aggregation in normal platelet-rich plasma ( P R P ) (FIGURE 1) . There was also no second-phase aggregation with thrombin, but the small reversible aggregation caused by serotonin (5-hydroxytryptamine, 5 H T ) did not differ from the normal pattern.

Platelet Adenine Nucleotides The adenine nucleotide content of the two patients’ platelets is compared in

TABLE 1 with that of a control subject, and the proportion of total nucleotides released after five minutes’ incubation with thrombin is also shown. The total amount of adenosine triphosphate ( ATP) in the patients’ platelets was slightly

Present address: Temple University School of Medicine, Philadelphia, Pa. 19140.

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430 Annals New York Academy of Sciences

ADP ADRENALINE COLLAGEN

ATP (pmoles/lO1I platelets) ADP (gmoles/lOll platelets)

Percentage of nucleotides released ATP/ADP ratio

by thrombin

s

Albino patients - Normal AB PH

7.04 4.02 6.70 4.01 0.55 0.85 1.76 1.3 7.9

65 16 4

C

-0.05

4.4 -0.2 L'l

, L ....... ~ .... .... ......

3.3 83 --

t t

115

t

t I I

I M I ' ' ' ' ' ' 0 2 4 6 8 1 0 0 2 4 6 8 1 0 0 2 1 6 8 1 0

llm lmln.1 llm In1n.l llm h l n . 1

FIGURE 1. Aggregation curves obtained after the addition of ADP, epinephrine and collagen to stirred platelet-rich plasma from a normal subject (A, C, and E) and from an albino patient (B, D, and F) . The final concentrations of ADP and epinephrine are shown as p M and the concentrations of collagen added as dilutions of a stock suspension.

Hardisty & Mills: Albinism 43 1

reduced compared with the control, but that of ADP was very greatly reduced, resulting in a much increased ATPIADP ratio. Moreover, a much smaller propor- tion of the total nucleotides was released from the patients’ platelets than from normal control platelets after incubation with thrombin.

In FIGURE 2 is shown the effect of incubation with thrombin on the release of individual adenine nucleotides from the platelets. It can be seen that no more nucleotides were released from the patients’ platelets by thrombin than by saline, and, moreover, that the amounts of the various nucleotides originally present in the patients’ platelets were closely similar to those remaining in normal platelets after incubation with thrombin.

Serotonin Uptake and Release The results of an experiment in which PRP from the patients and a normal

subject was incubated with serotonin in vitro are shown in FIGURE 3. Although the initial rate of serotonin uptake by the patients’ platelets was not significantly dif- ferent from that of the control, they were capable of taking up a much smaller total amount, and rapidly reached saturation.

FIGURE 4 shows the percentage release of I4C-labeled serotonin from normal and patients’ platelets after incubation for five minutes with either thrombin (1 unit/ml) or buffered saline; taking the difference between saline and thrombin release as an indication of the release specifically attributable to thrombin, that of the patients’ platelets was little more than half the normal.

In order to determine the extent to which the radioactive material that the abnormal platelets took up was present within the cells as serotonin itself, or to what extent it was metabolized, platelets that had been incubated with I4C- serotonin were spun down and extracted with N-HC104 and the extracts were

*r n NORMAL

T D M PRP .

Y n6r il T D M -l --1 T D M 9 T D M T D M m T D M T D M T D M

FIGURE 2. Adenine nucleotides in whole PRP (left-hand row of columns) and washed platelets, before incubation (second row) and after incubation with saline (third row) or thrombin (fourth row). The solid columns show the amount of each nucleotide released from the platelets after incubation, and the open columns the amount remaining within the platelets. T, ATP; D, ADP; M, AMP.

432 Annals New York Academy of Sciences

Patients

0 20 40 60 Minutes

FIGURE 3. Uptake of W-labeled serotonin by platelets at

'"I Jf 0.2

31°C.

FIGURE 4. Release of '%-labeled serotonin from platelets after five minutes' incubation with saline (solid columns) or thrombin (open columns).

Hardisty & Mills: Albinism 433

neutralized and chromatographed on cellulose phosphate paper together with serotonin and 5-hydroxyindole acetic acid as markers. The results of this experiment are given in FIGURE 5, which shows that in the patients’ platelets about 12% less activity than in the control was found as serotonin itself, but that a corresponding increase in activity was found at a spot corresponding to a fluorescent material running between serotonin and 5-hydroxyindole acetic acid. This probably represents 5-hydroxytryptophol, which is the principal product of metabolism of serotonin by platelets.

Uptake and Release of Epinephrine When normal platelets are incubated with 3H-epinephrine, they take up the

label over a few hours; it can be recovered from the platelets partly as epinephrine itself and partly as an acidic metabolite of unknown structure.2 Such an experi- ment was carried out on the platelets of one of our albino patients by Dr. Brian Smith: the results are shown in FIGURE 6. It can be seen that the overall rate of uptake was similar in normal and patients’ platelets, but whereas rather more than half of the activity taken up into normal platelets was in the form of epinephrine, in the case of the patient’s platelets it was almost entirely as the metabolite. FIGURE 6 also shows the release of radioactivity from platelets that have been incubated with labeled epinephrine and then exposed to either buf- fered saline or thrombin; 63% of the total radioactivity was released by thrombin from control platelets, compared with only 23% by saline, but in the case of the patient’s platelets there was no difference between the effect of saline and thrombin, each of which released about 26%.

Release of Lysosomal Enzymes Another effect of thrombin on normal platelets is to release certain hydrolytic

enzymes that are thought to be contained within the platelet lysosomes or alpha-

NORMAL ALBINO -(AB)----SOLVENT FRONT

5HT

1.6

ORIGIN FIGURE 5. Cellulose-phosphate chromatogram of platelet extracts following incubation with

14C-labeled serotonin. The percentage of recovery of radioactivity is shown for normal platelets on the left, and patient’s platelets on the right.

434

10-

30 W Lo

U W

CL d 20-

s 10-

Annals New York Academy of Sciences

-

. . U r n RELEASE

NORMAL

n

16

12 1. FWIENT AB.

n

bin

FIGURE 6. Uptake and release of [3H]-epinephrine by platelets. The three left-hand columns show the platelet content of epinephrine (A) and metabolite ( M ) before incubation and after one and two hours' incubation with 3H-epinephrine. The two right-hand columns show the activity released into the supernatant (S) and remaining in the platelets (P) after incubation of labeled platelets with saline or thrombin.

0 i

PGLUCURONIOASE

6r

PN-ACETYLGLUCOSAMINIOASE

n

20 ]li NO

J[! rLBlNO

FIGURE 7. Platelet content (lower columns) of beta-glucuronidase and beta-N-acetyl- glucosaminidase, and release of these enzymes (upper columns) after incubation for five minutes with saline (solid columns) or thrombin (open columns).

Hardisty & Mills: Albinism 43 5

granules. The total content in normal and albino platelets of two of these enzymes-beta-glucuronidase and beta-N acetylglucosaminidase-and their re- lease by thrombin are shown in FIGURE 7; there was no striking difference be- tween the total amounts of these two enzymes in the patients’ and normal platelets, but the proportion of each enzyme released by thrombin was rather less in the albino platelets.

Discussion These findings show that the aggregation defect of these albino patients’

platelets is associated with a deficiency of adenine nucleotides in the storage pool, so that the total nucleotide content resembles that of normal platelets from which nucleotides have already been released. This, in turn, is associated with a defect of the storage mechanism for serotonin, so that these platelets are not only very deficient in serotonin, but also limited in the amount which they are capable of taking up in vitro, though not in the rate of uptake. Taken to- gether, these findings suggest a deficiency of the organelles in which the non- metabolic pool of nucleotides and serotonin are normally stored. These are thought to be the densely osmiophilic granules, and a deficiency of these has already been described in other albino patients with a similar d e f e ~ t . ~ . ~ We have seen no dense bodies on electron microscopy of our patients’ platelets, but the technique used also revealed very few in normal human platelets, so that we hesitate to draw firm conclusions from this observation.

Platelets also contain mono-amine oxidase activity, and Pletscher5 has suggested that it is the localization of serotonin in the storage granules that protects it from the action of this enzyme. If this is so, and if our patients’ platelets lack these storage organelles, then one would expect a greater proportion of the relatively small amount of serotonin that they are capable of taking up to be rapidly metabolized within the platelets. This is borne out by our findings in one of the patients reported here.

The abnormalities of uptake and release of epinephrine by albino platelets are in several respects strikingly similar to those of serotonin; both are taken up at a normal initial rate by albino platelets, but are metabolized to a greater extent within them, and much less of each is released by thrombin from albino platelets than from normal platelets. Together with other evidence,2 this suggests that epinephrine, like serotonin, is normally stored in the intact form in platelet organelles, and that these are deficient in the platelets of our albino patients. It seems likely that the storage site for epinephrine is the same as that for serotonin and adenine nucleotides.

The hemostatic defect associated with albinism thus appears to be due to a failure of release of ADP and serotonin, which results from a defect in the mechanism by which these substances, and probably also epinephrine, are stored within the platelets, presumably in the densely osmiophilic granules. We have found no evidence in these patients’ platelets of an abnormality in the changes that occur in the metabolically active fraction of the adenine nucleotides during the release reaction, and that have been held to provide the energy necessary for When our patients’ platelets were incubated with 14C-adenine and then exposed to thrombin, the breakdown of rapidly labeled nucleotides to IMP and hy?oxanthine was both qualitatively and quantitatively similar to that in normal platelets. There was therefore no evidence for a block in this energy-producing mechanism, such as may underlie the action of aspirin in preventing the release reaction.8

It remains only to consider the relation of this platelet abnormality to albinism.

436 Annals New York Academy of Sciences

Sufficient cases have now been r e p ~ r t e d ~ > * . ~ l ~ to suggest that there is a real association between the two defects, but we have no evidence to suggest what the biochemical or genetic basis might be. We have studied platelet aggregation and nucleotide content and release in four other albinos who had normal bleeding times and no history of a hemorrhagic tendency, and in none of them did we find any abnormality. We also found no abnormality of the platelets of either of the parents of one of our patients, who were first cousins, but neither of whom suffered from albinism or from a bleeding tendency. It is clear, therefore, that this platelet defect is associated with only occasional cases of albinism, and also that it is responsible for the bleeding tendency from which a few of these patients suffer. As has recently been pointed out,14 the most likely explanation of the association, on the basis of reported cases, is that it results from a mutation involving closely linked recessive genes. A very similar, if not identical, platelet defect has been described in a number of nonalbino patients, and in some of these cases15-17 it appears to occur as a hereditary defect of as yet uncertain genetic pattern.

References 1. HARDISTY, R. M. & R. A. HUTTON. 1967. Bleeding tendency associated with “new”

abnormality of platelet behaviour. Lancet I: 983. 2. BORN, G. V. R. & J. B. SMITH. 1970. Uptake, metabolism and release of [3Hl-epinephrine

by human platelets. Brit. J. Pharmacol. 39: 765. 3. MAURER, H. M., S. BUCKINGHAM, E. MCGILVRAY, A. SPIELVOGEL & J. A. WOLFF. 1968.

Prolonged bleeding time, abnormal binding of platelet serotonin (5HT), absent platelet “dark body,” defective platelet factor 3 activation, bone marrow inclusions and chromosome breaks in albinism. XI1 Congr. Internat. SOC. Haematol. (abstr.) : 198. New York, N. Y.

1971. Studies of platelets in a variant of the Hermansky-Pudlak syndrome. Amer. J. Path. 63: 319.

1968. Metabolism, transfer and storage of 5-hydroxytryptamine in blood platelets. Brit. J. Pharmacol. 32: 1.

1967. Effects of thrombin on the radioactive nucleotides of human washed platelets. Biochem. J. 105: 857.

1969. Adenine nucleotide metabolism of blood platelets. VI. Subcellular localization of nucleotide pools with different functions in the platelet release reaction. Biochim. Biophys. Acta 186: 254.

1969. Effect of some inhibitors of platelet aggregation on platelet nucleotides. Biochem. J. 114: 669.

Albinism associated with hemorrhagic diathesis and unusual pigmented reticular cells in the bone marrow: report of two cases with histo- chemical studies. Blood 14: 162.

10. LARSEN, M. C., A. B. LEY, M. B. ZUCKER & L. E. LOSEKE. 1962. The association of albinism with pseudohemophilia. Ann. Intern. Med. 56: 504.

11. VERLOOP, M. C., A. VON WIERINGEN, J. VWLSTEKE, H. C. HART & J. HUIZINGA. 1964. Albinismus, hamorrhagische Diathese und anomale Pigmentzellen im Knochenmark. Med. Klin. 59: 408.

12. SOBOTKOWSKA, K. & S. KOSSMAN. 1966. Przypadek albinizmu ze wspotistinejaca angio- hemofilia. Pol. Arch. Med. Wewn. 36: 703.

13. Mufirz, F. J., J. FRADERA, N. MALDONADO & E. PEREZ-SANTIAGO. 1970. Albinism, bleeding tendency and abnormal pigmented cells in the bone marrow: a case report. Texas Rep. Biol. Med. 28: 167.

14. LOGAN, L. J., S. I. RAPAPORT & I. MAHER. 1971. Albinism and abnormal platelet func- tion. New Eng. J. Med. 284: 1340.

15. HARDISTY, R. M. 1969. Haemorrhagic disorders due to functional abnormalities of platelets. J. Roy. Coll. Phycns. London 3: 182.

16. WEISS, H. J., P. A. CHERVENICK, R. ZALUSKY & A. FACTOR. 1969. A familial defect in platelet function associated with impaired release of adenosine diphosphate. New Eng. J. Med. 281: 1264.

17. HOLMSEN, H. & H. J. WEISS. 1970. Hereditary defect in the platelet release reaction caused by a deficiency in the storage pool of platelet adenine nucleotides. Brit. J. Haemat. 19: 643.

4. WHITE, J. G., J. R. EDSON, S. J. DESNICK & C. J. WITKOP.

5. PLETSCHER, A.

6. IRELAND, D. M.

7. HOLMSEN, H., H. J. DAY & E. STORM.

8. BALL, G. M., M. FULWOOD, D. M. IRELAND & P. YATES.

9. HERMANSKY, F. & P. PUDLAK. 1959.