Leukemia & Lymphoma
ISSN: 1042-8194 (Print) 1029-2403 (Online) Journal homepage: http://www.tandfonline.com/loi/ilal20
Platelet Storage Pool Deficiency, Leukemia, and Myelodysplastic Syndromes J. M. Gerrard & A. McNicol To cite this article: J. M. Gerrard & A. McNicol (1992) Platelet Storage Pool Deficiency, Leukemia, and Myelodysplastic Syndromes, Leukemia & Lymphoma, 8:4-5, 277-281, DOI: 10.3109/10428199209051007 To link to this article: http://dx.doi.org/10.3109/10428199209051007
Published online: 01 Jul 2009.
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Citing articles: 1 View citing articles
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Date: 06 November 2015, At: 09:44
0 1992 Harwood
Leukemia and Lymphoma, Vol. 8 , pp. 277-281 Reprints available directly from the publisher Photocopying permitted by license only
Academic Publishers G m b H Printed in the Unjted Kingdom
Platelet Storage Pool Deficiency, Leukemia, and Myelodysplastic Syndromes J. M. GERRARD and A. McNICOL Downloaded by [University of California, San Diego] at 09:44 06 November 2015
Manitoba Institute of Cell Biology, Winnipeg, Manitoba, Canada (Received 15 April 1992)
Abnormalities in platelet dense granules, small intracellular organelles containing ATP, ADP, calcium, serotonin, and pyrophosphate, have frequently been reported in patients with leukemia and myeloproliferative disorders, particularly acute and chronic myelogenous leukemia. Recent studies of a family which includes several members with an autosomal dominant dense granule deficiency condition show an association between the presence of this form of dense granule deficiency and the development of acute myelogenous leukemia. Studies in two additional patients, one with the Monosomy 7 syndrome and the second with a myelodysplastic syndrome, revealed a defect in platelet dense granules. This defect appears to be due to an abnormality in the formation of these granules rather than the presence of empty vesicular structures or decreased contents due to activation associated secretion. The results suggest that the defect in platelet dense granules associated with leukemia or myelodysplastic syndromes may result from a chromosome alteration in the megakaryocyte cell line leading to decreased formation of dense granules. Studies in the family with an inherited bleeding disorder suggest that a gene coding for a protein important for the formation of dense granules is located adjacent to a gene which, when abnormal, may predispose to the development of leukemia. K E Y WORDS:
Platelet storage pool deficiency MDS
Human platelets contain four types of storage organelles: alpha granules, dense granules, lysosomes, and peroxisomes. Dense granules contain ATP, ADP, calcium, serotonin, and pyrophosphate, which are secreted when platelets are stimulated'. Inherited or acquired abnormalities in the platelet dense granule occur fairly c o m m ~ n l y ~Recent - ~ . studies in mice have suggested that there are at least 12 distinct genetic defects which can lead to abnormal dense granule^^-^; alterations in lysosomes, defects in pigment formation (e.g. hair colour) and inner ear function may also occur. Dense granule defects have also been found in patients with leukemia and myeloproliferative disAddress for correspondence: Jon M. Gerrard, M.D., Ph.D., Manitoba Institute of Cell Biology, 100 Olivia Street, Winnipeg, Manitoba, R3E OV9, Canada
leukemia
myelodysplastic syndromes
orders, particularly acute and chronic myelogenous leukemia7-' '. This relationship may suggest that the leukemia is associated with either the presence of a factor which activates platelets and causes discharge of their granule contents or with a chromosomal defect resulting in an abnormality of a protein critical to dense granule formation. For many years, prevailing opinion held that the dense and alpha granule deficiency seen in leukemia and myelodysplastic syndromes was secondary to in uiuo platelet a~tivation''-'~, a view bolstered by the finding of elevated levels of platelet alpha granule proteins in plasma' 3 , 1 4 . Also supporting this concept was a study which showed that, in 4 of 6 patients, autologous platelets labelled with ''C-serotonin (serotonin is taken up by dense granules) had a shorter apparent half-life than those labelled with 51Cr'2, suggesting that the platelets were activated in vivo and
277
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278
J. M. GERRARD AND A. McNlCOL
preferentially lost the contents of their dense granules”. However, in two patients the calculated platelet half-life was similar or longer using 14Cserotonin. Furthermore, an apparent decrease in the half-life of serotonin-labelled platelets compared to 51Crlabelled platelets could be due to the presence of deficient or defective dense granules in which uptake and/or retention of serotonin is abnormal. Other studies of patients with chronic myeloproliferative disorders rarely show elevated fibrinopeptide A level^'^-'^, arguing against significant ii? vivo thrombin generation. Wehmeier et al. have used this data to argue against in vivo platelet activation and in favour of primary megakaryocyte abnormalities leading to an inability to store platelet granule contents properly”. Consistent with this,concept is the frequent presence of hypogranular megakaryocytes in myelodysplastic s y n d r ~ m s ’ ~ * ’ ~ . We recently studied a family with an inherited dense granule defect in which seven members have developed leukemia (either acute myeloblastic or acute myelomonocytic leukemia)”. Cytogenetic studies revealed that one of the patients who developed leukemia had the Monosomy 7 syndrome’O*’l. The only other patient to have cytogenetics performed showed a population of bone marrow cells with an extra G chromosome or G chromosome-like fragment. Several members of the family also have an inherited platelet storage pool deficiency. In one patient (female) with a lifelong history of easy bruising and bleeding (severe hemorrhage with tonsillectomy at age 5, with appendectomy at age 21, and after childbirth at age 23) the platelet storage pool deficiency was clearly identified three years before the onset of the leukemia at age 41 and, based on the history, was presumably present since birth. Two other family members (both female) with a lifelong history of easy bruising and bleeding developed leukemia at ages 64 and 75. Three other family members (one male, two female), who developed leukemia at ages 5, 6, and 7 respectively, had a previous history of easy bruising and/or bleeding. Studies in one of these children a year before the onset of leukemia found defective platelet aggregation, consistent with a storage pool deficiency. One patient (male), who developed leukemia at age 23, had no history of significant bleeding or bruising before the onset of leukemia. The platelets of three family members who had lifelong histories of bleeding and bruising but had not developed leukemia were found to have low levels of dense granules. Interpretation of these findings in the various
patients is facilitated by understanding how dense granules and their contents are evaluated. Low levels of platelet serotonin and ADP indicate a deficiency in the contents of the platelet dense granules. Since both ATP and ADP are present in dense granules, but ATP is predominant in the metabolic pool, a deficiency of platelet dense granule contents is associated with an increase in the ATP:ADP ratio. A decrease in the amount of ATP secreted in response to a strong stimulus like thrombin is also indicative of a dense granule deficiency4. Counts of dense granules as viewed under the electron microscope using whole mounts of platelets have been used to enumerate these organelles”. The opaque appearance of the dense granule in whole mount preparations results from their content of calciumz3. The whole mount technique has proved to be very useful, but does not distinguish between the absence of dense granules and the presence of empty vesicles. In this respect, two other techniques provide additional information. Quinacrine, a fluorescent dye selectively taken up by dense granulesz4, is incorporated into both full granules and empty vesicles, provided that they have the ability to transport and retain the dye. With this method of analysis, findings in affected family members (Table 1) show that their dense granule vesicles are low in number. The low number of dense granules in affected family members is, therefore, not due to the presence of empty vesicles. A second technique employing D545, an antibody to the dense granule protein, granulophysin, supports the results using quinacrine. D545 has the advantage in that unlike quinacrine it can also detect activated platelets in which the granule contents have been secreted. After secretion, granulophysin, the protein recognized by D545 and present in dense granule membranes, is translocated to the platelet surface25. The number of dense granules labelled by D545 was similar to that seen using quinacrine and whole mount preparations. Furthermore, there was no evidence of granulophysin on the platelet surface. The results strongly suggest a primary decrease in dense granule numbers in the affected members of this family. That the numbers of dense granules were consistently decreased by 50% suggests the defective condition may result from a heterozygote state, leading to production of half the normal amount of a protein structurally important for dense granule formation. We have recently had the opportunity to study two additional patients with dense granule deficiencies associated with leukemia or myelodysplasia; one of them was a child with the Monosomy 7 syndrome.
Serotonin (ng/109 platelets)
Monosomy 7 syndrome Myleodysplastic syndrome
9 mos 74 yrs
Diagnosis
Patient I1 Normal Range
Age
patient I
Table 2
0.42 p M 1.9-5.4 PM
0 Pm
ATP secretion
1.1-1.6
0.23 0.30 _+ 0.30 2.5-5
1.95
Whole mounts
2.5-5
4.2-8.1
2.71
1.51 1.53 ND 1.15
2.6-6.7
256720
1.36 1.87 ND
3.00 4.00 6.15 2.42
2.3 _+ 0.32 4 6
ND
D545
0.23 1.35 _+ 0.48 34.5
1.45
immunofiuorescence
0545
ND 34.5
5.45 k 0.30
0.85 0.20 1.90 0.23 ND 1.40 k 0.27
immunojluorence
4 6
1.76 0.22 1.85 k 0.39 ND 1.63 k 0.24
Quinucrine technique
Quinacrine immunoJuorescence
0.52
0.39
0.29 0.46
Whole amounts
Normal Range
6.54 7.58 ND
5.78 4.56 3.61 4.23
Ratio A T P / AD P
4.80 4.01 ND
1.93 1.14 0.58 1.75
ADP ATP (nmol/lOOplatelets)
Those with normal aggregation 111-4 ND IV-6 ND IV-8 789 1v-1
Those with abnormal platelet aggregation 111-1 110 111-8 180 111-9 228 IV-3 ND
Family members
Table 1
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5 Pdml 9-30
ND
Granulophysin (pg dense granule equivalents
9-30
ND
12 16 ND 10
Granulophysin levels ( p g dense granule equivalents)
Downloaded by [University of California, San Diego] at 09:44 06 November 2015
280
J. M. GERRARD AND A. McNICOL
Both patients had low levels of dense granules on whole mount examination and employing D545.In addition, no significant distribution of granulophysin to the platelet surface was observed in either case. In one of the patients, the total platelet content of granulophysin was very low. The patient with the Monosomy 7 syndrome showed an improvement in dense granules after chemotherapy. The results are most consistent with the platelet defect being the result of abnormal genetic material in the megakaryocytes. Two other families with an inherited platelet defect and a high incidence of acute myeloid leukemias have been d e ~ c r i b e d ~ ~However, .~’. the platelet defect was not precisely characterized in either of these families. Two members of another family with an inherited dense granule defect not associated with albinism28 have developed leukemia, while an affected member of a family with an autosomal dominant inherited storage pool defect originally described by Weiss et developed acute myelogenous leukemia and a second affected family member was also said to have died of leukemia2’ or a myelodysplastic condition. A possible explanation for the association of a platelet dense granule defect and leukemia could be a concomitant defect in natural killer cells, as occurs in the Chediak-Higashi syndrome. However, a natural killer cell defect was not detected in the affected members of the family that we studied. We speculate that the platelet dense granule defect assoicated with leukemia results from a chromosomal alteration in the megakaryocyte cell line. One possibility might be the deletion of a gene coding for a protein necessary for dense granule formation and deletion of an adjacent tumor suppressor gene. Acknowledgements We thank Dr. Lorne Brandes for his help in providing platelets from one of the patients and for his helpful comments on the manuscript. We are also grateful to Barbara Doan for editorial assistance. This work was supported by grants from the Medical Research Council and the National Cancer Institute. Jon M. Gerrard is a Children’s Hospital of Winnipeg Research Foundation Professor.
REFERENCES 1. Da Prada, M., Richards, J. G. and Kettler, R. Amino
storage organelles in platelets. In Gordon, J. L., ed. Platelets in Biology and Pathology 2. Amsterdam, The Netherlands: Elsevier/North Holland, 1981, pp. 107-146. 2. Weiss, H. J., Chervenick, P. A., Zalusky, R. and Factor, A. (1969). A familial defect in platelet function associated with impaired release of adenosine diphosphate. Ntw, E~rq/..1. Met/.. 281. 1264-1270. 3. Nieuwenhuis, H. K., Akkerman, J. W. N. and Sixma, J. J. (1987).
Patients with a prolonged bleeding time and normal aggregation tests may have storage pool deficiency: Studies on one hundred and six patients. Blood, 70, 62G 623. 4. Israels, S. J., McNicol, A,, Robertson, C. and Gerrard, J. M. (1990). Platelet storage deficiency: Diagnosis in patients with prolonged bleeding times and abnormal aggregation. Br. J . Haemutol., 75, 1 18 121 5. Novak, E. K., Hui, S. W. and Swank, R. T. (1984). Platelet storage deficiency in mouse pigment mutations associated with seven distinct genetic loci. Blood, 63, 536544. 6. Swank, R. T., Reddington, M., Howlett, 0. and Novak, E. K. (1991). Platelet storage pool deficiency associated with inherited abnormalities of the inner ear in the mouse pigment mutants mated a mocha. Blood, 78, 2036-2044. 7. Cowan, D. H., Graham, R. C., Jr. and Baunach, D. (1975).Thc platelet defect in leukemia. Platelet ultrastructure, adenine nucleotide metabolism and the release reaction. J . Clin. Invest., 56, 188-200. 8. Gerrard, J. M., Stoddard, S. F., Shapiro, R. S., Coccia, P. F., Ramsay, N. K. C., Nesbit, M. E., Rao, G . H. R., Drivit, W. and White, J. G . (1978). Platelet storage pool deficiency and prostaglandin synthesis in chronic granulocytic leukemia. Br J . Haematol., 40, 597-607. 9. Rendu, F., Lebret, M., Nurden, A. and Caen, J. P. (1979). Detection of an acquired platelet storage pool disease in three patients with an myeloproliferative disorder. Throtnh. Hoemost., 42, 794-795. 10. Russell, N. H., Salmon, J., Keenan, J. P. and Bellingham, A. J. (1981). Platelet adenine nucleotides and arachidonic acid metabolism in myeloproliferative disorders. Thromh. Rex, 22, 389-397. 11. Pareti, F. I., Gugliotta, L., Mannucci, L., Guarini, A,, and Mannucci, P. M. (1982). Biomedical and metabolic aspects of platelet dysfunction in chronic myeloproliferative disorders. Thromb. Haemost., 47, 8 4 8 9 . 12. Malpass, T. W., Savage, B., Hanson, S. R., Slichter, S. J. and Harker, L. A. (1984). Correlation between prolonged bleeding time and depletion of platelet dense granule ADP in patients with myelodysplastic myeloproliferative disorders. J. Lab. Clbt. Med., 103, 894904. 13. Boughton, B. J., Allington, M. J. and King, A. (1978). Platelet and plasma /?-thromboglobulin in myeloproliferative syndromes and secondary thrombocytosis. Br. J. Hnemntol. 40. t 25- I 32. 14. Najean, Y., Poirier, 0. and Lokiec, F. (1983). The clinical significance of beta-thromboglobulin and platelet factor- 4 in polycythaemic patients. Scund. J. Hacvnutol., 31, 298- 304. 15. Wehmeier, A., Scharf, R. E., Fricke, S. and Schneider, W. (1980). Bleeding and thrombosis in chronic myeloproliferative disorders: Relation of platelet disorders to clinical aspects of the disease. Harmostasis, 19, 25 1-259. 16. Ireland, H., Land, D. A. A,, Wolff, S. and Foadi, M . (1982). I n vivo platelet release in myeloproliferative disorders. Thromh. Haemost., 48, 41-45. 17. Maldonado, J. E. (1974). Dysplastic platelets and circulating megakaryocytes in chronic myeloproliferative diseases. 11. Ultrastructure of circulating megakaryocytcs. Blood, 43, 8 1 1-820. 18. Wong, K. F. and Cham, J. K. C. (1991). Are dysplastic and hypogranular megakaryocytes specific markers for myelodysplastic syndrome? Br. J. Hcrernatol., 77, 509 514. 19. Gerrard, J. M., Israels, E. D., Bishop. A. J., Schroeder, M. L., Beattie, L. L., McNicol, A., Israels, S. J., Walz, D., Greenberg. A. H., Ray, M. and Israels, L. G. (1991). Inherited platelet-storage pool deficiency associated with a high incidence of acute myeloid leukemia. Br. J . Haematol., 79, 246 -255. 20. Sieff, C. A., Chessells, J. M., Harvey, B. A. M., Pickthall, V. J. and Lawler, S. D. (1981). Monosomy 7 in childhood: A myeloproliferative disorder. Br. J . H a e t h o l . , 49, 235- 249.
PLATELET STORAGE P O O L DEFICIENCY, LEUKEMIA AND MDS
Downloaded by [University of California, San Diego] at 09:44 06 November 2015
21. Hutter, J. J., Hecht, F., Kaiser-McCaw, B., Hays, T., Baranko, P., Cohen, J. and Durie, B. (1984). Bone marrow monosomy 7: Hematologic and clinical manifestations in childhood and adolescence. Hemurot. Oncot., 2, 5- 12. 22. Witkop, C. K., Krumwiede, M., Sedano, H. and White, J . G. (1987). Reliability of absent dense bodies as a diagnostic criterion for Hermansky-Pudlak Syndrome. Am. J. Hemurot., 26, 305-31 1. 23. Gerrard, J. M., Rao, G. H. R. and White, J. G. (1977). The influence of reserpine and ethylenediaminetetra acetic acid (EDTA) on serotonin storage organelles of blood platelets. Am. J. Puihol., 87, 633. 24. Skaer, R. J., Flemans, R. J. and McQuilkan, S. (1981). Mepacrine stains the dense bodies of human platelets and not platelet lysosomes. Br. J. Huernurol., 49, 435-438. 25. Gerrard, J. M., Lint, D., Sims, P. J., Wiedmer, T., Fugate, R. D., McMillan, E., Robertson, C. and Israels, S. J. (1991).
26. 27. 28.
29.
28 1
Identification o f a platelet dense granule membrane protein that is deficient in a patient with the Hermansky-Pudlak syndrome. Blood, 77, 101-1 12. Luddy, R. E., Champion, L. A. and Schwartz, A. D. (1978). A fatal myeloproliferative syndrome in a family with thrombocytopenia and platelet dysfunction. Cancer, 41, 1959-1963. Dowton, S. B., Beardsley, D., Jamison, D., Blattner, S. and Li, F. P. (1985). Studies of a familial platelet disorder. Blood, 65, 557-563. White, J. G., Gerrard, J. M., Rao, G. H. R., Edson, J. R. and Witkop, C. J. (1975). Differences in platelet storage pool deficiency (SPD) of Hermansky-Pudlak syndrome (HPS) and non-albjnos. Thromb. Diath. Huemorrh., 34, 36G361. Lages, F., Shattil, S. J., Bainton, D. F. and Weiss, H. J. (1991). Decreased content and surface expression of cc-granule membrane protein GMP-140 in one of two types of platelet a6 storage pool deficiency. J. Clin. Invest., 87, 919-929.
ISSN: 1042-8194 (Print) 1029-2403 (Online) Journal homepage: http://www.tandfonline.com/loi/ilal20
Platelet Storage Pool Deficiency, Leukemia, and Myelodysplastic Syndromes J. M. Gerrard & A. McNicol To cite this article: J. M. Gerrard & A. McNicol (1992) Platelet Storage Pool Deficiency, Leukemia, and Myelodysplastic Syndromes, Leukemia & Lymphoma, 8:4-5, 277-281, DOI: 10.3109/10428199209051007 To link to this article: http://dx.doi.org/10.3109/10428199209051007
Published online: 01 Jul 2009.
Submit your article to this journal
Article views: 37
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ilal20 Download by: [University of California, San Diego]
Date: 06 November 2015, At: 09:44
0 1992 Harwood
Leukemia and Lymphoma, Vol. 8 , pp. 277-281 Reprints available directly from the publisher Photocopying permitted by license only
Academic Publishers G m b H Printed in the Unjted Kingdom
Platelet Storage Pool Deficiency, Leukemia, and Myelodysplastic Syndromes J. M. GERRARD and A. McNICOL Downloaded by [University of California, San Diego] at 09:44 06 November 2015
Manitoba Institute of Cell Biology, Winnipeg, Manitoba, Canada (Received 15 April 1992)
Abnormalities in platelet dense granules, small intracellular organelles containing ATP, ADP, calcium, serotonin, and pyrophosphate, have frequently been reported in patients with leukemia and myeloproliferative disorders, particularly acute and chronic myelogenous leukemia. Recent studies of a family which includes several members with an autosomal dominant dense granule deficiency condition show an association between the presence of this form of dense granule deficiency and the development of acute myelogenous leukemia. Studies in two additional patients, one with the Monosomy 7 syndrome and the second with a myelodysplastic syndrome, revealed a defect in platelet dense granules. This defect appears to be due to an abnormality in the formation of these granules rather than the presence of empty vesicular structures or decreased contents due to activation associated secretion. The results suggest that the defect in platelet dense granules associated with leukemia or myelodysplastic syndromes may result from a chromosome alteration in the megakaryocyte cell line leading to decreased formation of dense granules. Studies in the family with an inherited bleeding disorder suggest that a gene coding for a protein important for the formation of dense granules is located adjacent to a gene which, when abnormal, may predispose to the development of leukemia. K E Y WORDS:
Platelet storage pool deficiency MDS
Human platelets contain four types of storage organelles: alpha granules, dense granules, lysosomes, and peroxisomes. Dense granules contain ATP, ADP, calcium, serotonin, and pyrophosphate, which are secreted when platelets are stimulated'. Inherited or acquired abnormalities in the platelet dense granule occur fairly c o m m ~ n l y ~Recent - ~ . studies in mice have suggested that there are at least 12 distinct genetic defects which can lead to abnormal dense granule^^-^; alterations in lysosomes, defects in pigment formation (e.g. hair colour) and inner ear function may also occur. Dense granule defects have also been found in patients with leukemia and myeloproliferative disAddress for correspondence: Jon M. Gerrard, M.D., Ph.D., Manitoba Institute of Cell Biology, 100 Olivia Street, Winnipeg, Manitoba, R3E OV9, Canada
leukemia
myelodysplastic syndromes
orders, particularly acute and chronic myelogenous leukemia7-' '. This relationship may suggest that the leukemia is associated with either the presence of a factor which activates platelets and causes discharge of their granule contents or with a chromosomal defect resulting in an abnormality of a protein critical to dense granule formation. For many years, prevailing opinion held that the dense and alpha granule deficiency seen in leukemia and myelodysplastic syndromes was secondary to in uiuo platelet a~tivation''-'~, a view bolstered by the finding of elevated levels of platelet alpha granule proteins in plasma' 3 , 1 4 . Also supporting this concept was a study which showed that, in 4 of 6 patients, autologous platelets labelled with ''C-serotonin (serotonin is taken up by dense granules) had a shorter apparent half-life than those labelled with 51Cr'2, suggesting that the platelets were activated in vivo and
277
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278
J. M. GERRARD AND A. McNlCOL
preferentially lost the contents of their dense granules”. However, in two patients the calculated platelet half-life was similar or longer using 14Cserotonin. Furthermore, an apparent decrease in the half-life of serotonin-labelled platelets compared to 51Crlabelled platelets could be due to the presence of deficient or defective dense granules in which uptake and/or retention of serotonin is abnormal. Other studies of patients with chronic myeloproliferative disorders rarely show elevated fibrinopeptide A level^'^-'^, arguing against significant ii? vivo thrombin generation. Wehmeier et al. have used this data to argue against in vivo platelet activation and in favour of primary megakaryocyte abnormalities leading to an inability to store platelet granule contents properly”. Consistent with this,concept is the frequent presence of hypogranular megakaryocytes in myelodysplastic s y n d r ~ m s ’ ~ * ’ ~ . We recently studied a family with an inherited dense granule defect in which seven members have developed leukemia (either acute myeloblastic or acute myelomonocytic leukemia)”. Cytogenetic studies revealed that one of the patients who developed leukemia had the Monosomy 7 syndrome’O*’l. The only other patient to have cytogenetics performed showed a population of bone marrow cells with an extra G chromosome or G chromosome-like fragment. Several members of the family also have an inherited platelet storage pool deficiency. In one patient (female) with a lifelong history of easy bruising and bleeding (severe hemorrhage with tonsillectomy at age 5, with appendectomy at age 21, and after childbirth at age 23) the platelet storage pool deficiency was clearly identified three years before the onset of the leukemia at age 41 and, based on the history, was presumably present since birth. Two other family members (both female) with a lifelong history of easy bruising and bleeding developed leukemia at ages 64 and 75. Three other family members (one male, two female), who developed leukemia at ages 5, 6, and 7 respectively, had a previous history of easy bruising and/or bleeding. Studies in one of these children a year before the onset of leukemia found defective platelet aggregation, consistent with a storage pool deficiency. One patient (male), who developed leukemia at age 23, had no history of significant bleeding or bruising before the onset of leukemia. The platelets of three family members who had lifelong histories of bleeding and bruising but had not developed leukemia were found to have low levels of dense granules. Interpretation of these findings in the various
patients is facilitated by understanding how dense granules and their contents are evaluated. Low levels of platelet serotonin and ADP indicate a deficiency in the contents of the platelet dense granules. Since both ATP and ADP are present in dense granules, but ATP is predominant in the metabolic pool, a deficiency of platelet dense granule contents is associated with an increase in the ATP:ADP ratio. A decrease in the amount of ATP secreted in response to a strong stimulus like thrombin is also indicative of a dense granule deficiency4. Counts of dense granules as viewed under the electron microscope using whole mounts of platelets have been used to enumerate these organelles”. The opaque appearance of the dense granule in whole mount preparations results from their content of calciumz3. The whole mount technique has proved to be very useful, but does not distinguish between the absence of dense granules and the presence of empty vesicles. In this respect, two other techniques provide additional information. Quinacrine, a fluorescent dye selectively taken up by dense granulesz4, is incorporated into both full granules and empty vesicles, provided that they have the ability to transport and retain the dye. With this method of analysis, findings in affected family members (Table 1) show that their dense granule vesicles are low in number. The low number of dense granules in affected family members is, therefore, not due to the presence of empty vesicles. A second technique employing D545, an antibody to the dense granule protein, granulophysin, supports the results using quinacrine. D545 has the advantage in that unlike quinacrine it can also detect activated platelets in which the granule contents have been secreted. After secretion, granulophysin, the protein recognized by D545 and present in dense granule membranes, is translocated to the platelet surface25. The number of dense granules labelled by D545 was similar to that seen using quinacrine and whole mount preparations. Furthermore, there was no evidence of granulophysin on the platelet surface. The results strongly suggest a primary decrease in dense granule numbers in the affected members of this family. That the numbers of dense granules were consistently decreased by 50% suggests the defective condition may result from a heterozygote state, leading to production of half the normal amount of a protein structurally important for dense granule formation. We have recently had the opportunity to study two additional patients with dense granule deficiencies associated with leukemia or myelodysplasia; one of them was a child with the Monosomy 7 syndrome.
Serotonin (ng/109 platelets)
Monosomy 7 syndrome Myleodysplastic syndrome
9 mos 74 yrs
Diagnosis
Patient I1 Normal Range
Age
patient I
Table 2
0.42 p M 1.9-5.4 PM
0 Pm
ATP secretion
1.1-1.6
0.23 0.30 _+ 0.30 2.5-5
1.95
Whole mounts
2.5-5
4.2-8.1
2.71
1.51 1.53 ND 1.15
2.6-6.7
256720
1.36 1.87 ND
3.00 4.00 6.15 2.42
2.3 _+ 0.32 4 6
ND
D545
0.23 1.35 _+ 0.48 34.5
1.45
immunofiuorescence
0545
ND 34.5
5.45 k 0.30
0.85 0.20 1.90 0.23 ND 1.40 k 0.27
immunojluorence
4 6
1.76 0.22 1.85 k 0.39 ND 1.63 k 0.24
Quinucrine technique
Quinacrine immunoJuorescence
0.52
0.39
0.29 0.46
Whole amounts
Normal Range
6.54 7.58 ND
5.78 4.56 3.61 4.23
Ratio A T P / AD P
4.80 4.01 ND
1.93 1.14 0.58 1.75
ADP ATP (nmol/lOOplatelets)
Those with normal aggregation 111-4 ND IV-6 ND IV-8 789 1v-1
Those with abnormal platelet aggregation 111-1 110 111-8 180 111-9 228 IV-3 ND
Family members
Table 1
Downloaded by [University of California, San Diego] at 09:44 06 November 2015
5 Pdml 9-30
ND
Granulophysin (pg dense granule equivalents
9-30
ND
12 16 ND 10
Granulophysin levels ( p g dense granule equivalents)
Downloaded by [University of California, San Diego] at 09:44 06 November 2015
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J. M. GERRARD AND A. McNICOL
Both patients had low levels of dense granules on whole mount examination and employing D545.In addition, no significant distribution of granulophysin to the platelet surface was observed in either case. In one of the patients, the total platelet content of granulophysin was very low. The patient with the Monosomy 7 syndrome showed an improvement in dense granules after chemotherapy. The results are most consistent with the platelet defect being the result of abnormal genetic material in the megakaryocytes. Two other families with an inherited platelet defect and a high incidence of acute myeloid leukemias have been d e ~ c r i b e d ~ ~However, .~’. the platelet defect was not precisely characterized in either of these families. Two members of another family with an inherited dense granule defect not associated with albinism28 have developed leukemia, while an affected member of a family with an autosomal dominant inherited storage pool defect originally described by Weiss et developed acute myelogenous leukemia and a second affected family member was also said to have died of leukemia2’ or a myelodysplastic condition. A possible explanation for the association of a platelet dense granule defect and leukemia could be a concomitant defect in natural killer cells, as occurs in the Chediak-Higashi syndrome. However, a natural killer cell defect was not detected in the affected members of the family that we studied. We speculate that the platelet dense granule defect assoicated with leukemia results from a chromosomal alteration in the megakaryocyte cell line. One possibility might be the deletion of a gene coding for a protein necessary for dense granule formation and deletion of an adjacent tumor suppressor gene. Acknowledgements We thank Dr. Lorne Brandes for his help in providing platelets from one of the patients and for his helpful comments on the manuscript. We are also grateful to Barbara Doan for editorial assistance. This work was supported by grants from the Medical Research Council and the National Cancer Institute. Jon M. Gerrard is a Children’s Hospital of Winnipeg Research Foundation Professor.
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