Eur J Haematol 1988;40:65-68

Key words: hematologic disorders - intraplatelet ADP and ATP - platelet volume

Platelet volume and intraplatelet adenine nucleotides in various hematologic disorders

Hiroyuki Nozaki, Tadarni Nagao i3 Shigeru Arimori

Department of Internal Medicine, School of Medicine, Tokai University, Japan

By recent advanced techniques, blood platelets have proved to be varied in size and metabolism in various hematologic disorders. We examined platelet volume and intraplatelet adenine nucleotides in 36 patients with various hematologic disorders in order to clarify the quantitative platelet abnormalities. Platelet volumes were smaller in patients with acute leukemia and aplastic anemia, and larger in patients with immune thrombocytopenic purpura (ITP). The amount of intraplatelet ADP was decreased and ATP/ADP ratio was increased in acute leukemia, aplastic anemia and myeloproliferative disoders (MPD), which strongly suggested the presence of storage pool deficiency in these patients. Intraplatelet ADP per volume was decreased in acute leukemia, aplastic anemia and MPD, and ATP per volume was decreased in aplastic anemia. ATP content was increased in ITP in proportion to the increased platelet volume. These parameters were examined in 36 patients with the following hema- tologic disorders: 7 acute leukemia treated with cytotoxic chemotherapy, 5 aplastic anemia, 3 paroxysmal nocturnal hemoglobinuria (PNH), 9 immune thrombocytopenic purpura (ITP), 5 hypersplenism and 7 myeloproliferative disorders (MPD).

Accepted for publication September 3. 1987

Using recent advanced techniques, blood plate- lets have been shown to be varied in size, func- tion and metabolism in various hematologic dis- orders. Investigators in several laboratories have demonstrated large platelets in patients with immune thrombocytopenic purpura (ITP), and

nucleotides remains to be established. In the present study qualitative platelet abnormalities were evaluated with platelet volume and adenine nucleotides in various hematologic disorders.

Patients and methods small platelets in patients with acute leukemia (1- 6) . The lower concentration of intraplatelet ade- nine nucleotides has been described in patients

This study involved 36 patients with the following hematologic disorders: 7 acute leukemia treated with

mal nocturnal hemoglobinuria (PNH), 9 ITP, 5 hyper-

with acute leukemia and myeloproliferative dis- cytotoxic chemotherapy, 5 aplastic anemia, 3 paroxys- Orders (MPD) 7-1 ')* However$ there were few - reports O f intraplatelet adenine nucleotides in other hematologic disorders; moreover, the rele- vance of platelet volume to intraplatelet adenine

splenism, and 7 MPD. 48 healthy adult volunteers served as controls. None of these patients had under- gone platelet transfusion during the 2 wk before plate-

Eur J Huemutol 1988;40 5

66 NOZAKI ET AL

let measurements were made. Details are shown in Table 1.

Platelet samples Blood for platelet sizing and intraplatelet adenine nu- cleotides was collected by venipuncture into siliconized glass tubes containing 3.2% anticoagulant. The blood was centrifuged at 120 x g for 10 min at room tem- perature to provide platelet rich plasma (PRP).

Platelet counts and volumes Platelet counts and volumes from PRP were performed electronically with a Coulter Counter ZBI, Channelyzer C-1000 and a 70 aperture (Coulter Electronics, Inc., Hialeah, Fla) within 2 h of venipuncture. PRP was diluted to 1:3000 with Isoton (Coulter Electronics, Inc.). A portion of the 1:3000 dilution was subse- quently aspirated by the ZBI and channelyzed until a volume of approximately 15 000-30 000 platelets was determined. The median platelet volumes were evalu- ated in this study because mean platelet volumes could not be obtained easily using the channelyzer. Details of the procedures and calibration routines have been re- ported by Mundschenk et al (12).

lntraplatelet adenine nucleotides lntraplatelet adenosine 5’-diphosphate (ADP) and ade- nosine 5’-triphosphate (ATP) contents were measured by the method of Holmsen et al (13). In brief, ADP was converted into ATP by the pyruvate kinase system, and initial light flash provoked by the reaction of ATP and firefly lantern extract (Sigma Chemical Co., St. Louis, Mo) was measured with a liquid scintillation spectrometer (Packard Instrument, Inc., Downers Grove, ILL).

Statistical analysis The significance of difference between mean values was tested by Student’s t test.

TABLE 1 Details of subjects studied

Results The median platelet volume was significantly smaller in patients with acute leukemia and aplastic anemia, and larger in patients with ITP, than that of healthy subjects.

The amount of intraplatelet ADP was signifi- cantly decreased, and ATP/ADP ratio was increased, in patients with acute leukemia, aplas- tic anemia and MPD. ATP content was de- creased in patients with acute leukemia or aplas- tic anemia, and increased in patients with ITP.

The value of intraplatelet ADP per platelet volume was significantly decreased in patients with acute leukemia, aplastic anemia or MPD, and ATP per volume was decreased in aplastic anemia.

There were no signifcant differences in platelet volume and adenine nucleotides between patients with P N H or hypersplenism and normal sub- jects. Details are given in Table 2.

Discussion Platelet volume has been gradually introduced as a variable in clinical hematology following recent advances in automated particle sizing technol- ogy. In the present study, the platelet volume was significantly smaller in patients with aplastic anemia and acute leukemia treated with cytotoxic chemotherapy, and larger in patients with ITP, when compared with healthy subjects, which confirmed previous investigations (1-5). The het- erogeneity of platelet size in these patients may be affected by the distinct marrow megakaryo- cytopoiesis. Bessman has demonstrated that the

Normal Acute Aplastic PNH ITP Hypersplenism MPD subjects leukemia anemia

Number studied 48 7 ANLL 5 5 3 9 5 7 CML 3 ALL 2 PV 4

51 ? 12 35 ? 21 56 f 21 29 + 9 50f I 8 40+ 14 55 f 14

Platelet count 34 8 f 4 6+3 16 + 3 8 + 4 1 1 + 6 149 + 48

Results are given as mean? SD. PNH = Paroxysmal nocturnal hemoglobinuria. ITP = immune thrombocytopenic purpura. MPD = Myeloproliferative disorders. ANLL = Acute nonlymphocytic leukemia.

Sex male/female 24/24 6/ 1 2/3 310 4/5 2/3 3/4 Age (YO

(10 x 1WA)

PLATELET VOLUME AND ADENINE NUCLEOTIDES 67

mean platelet volume correlates directly and lin- early with the mean megakaryocyte ploidy in normal subjects and in patients with aplastic anemia, acute leukemia, megaloblastic anemia or ITP (6) . A number of studies have shown that larger platelets are more active than smaller ones

The level of intraplatelet adenine nucleotides, especially ADP, was significantly decreased, and ATP/ADP ratio was increased in patients with acute leukemia, aplastic anemia and MPD. These findings strongly suggested the presence of stor- age pool deficiency in these patients (7). Orig- inally, congenital storage pool deficiency was described in the Hermansky-Pudlak syndrome, the Chediak-Higashi syndrome and others (7). Thereafter, acquired storage pool deficiency syn- dromes have been demonstrated in patients with acute leukemia, MPD or disseminated intra- vascular coagulation, as well as in patients who have a circulating antiplatelet antibody (7-1 1). However, there have been few reports of low amounts of intraplatelet adenine nucleotices in patients with aplastic anemia. At present, the storage pool deficiency has been accounted for by structural or functional abnormalities of gran- ules in defective marrow megakaryocytopoiesis

( 14- 18).

TABLE 2 Platelet volume and intraplatelet adenine nucleotides

or by platelet activation in vivo and consequent partial depletion of dense granule contents (7, 11). As patients with aplastic anemia apparently have defective marrow megakaryocytopoiesis, the reduction in intraplatelet adenine nucleotides appears to be attributed to the marrow defect.

There have been few reports concerning the relationship between platelet volume and dense body contents in hematologic disorders. In healthy subjects, Thompson et a1 have shown that small-sized platelets have small numbers of dense bodies and large-sized platelets have large numbers of dense bodies per platelet, and that there is significant correlation between platelet volume and ATP and B-TG content (16, 17). If this is also true of the patients with hematologic disorders, the decreased platelet adenine nucleo- tides in patients with aplastic anemia or acute leukemia may be partially owing to the smaller platelet volume in addition to the storage pool deficiency syndromes. Carty & Gear have re- ported that large platelets are enriched in ATP even when normalized per unit volume in healthy subjects (1 8). Decreased ATP/volume in patients with aplastic anemia might be explained by these findings, while normal ATP/volume in patients with acute leukemia can not. This discrepancy

MPD Hyper-

ITP splenism PNH

Normal Acute Aplastic subjects leukemia anemia

Platelet volume (Median, p3) ADP content (nmol/ I09 platelets)

5.79 f 0.50 3.93 f 0.77' 4.40 f 1.504 5.37 2 0.43 6.39 f 0.804 6.09 f 0.39 5.49 f 0.37

32.5 f 6.8 12.8 f 5.81 9.8 f 8.21 21.7 f 12.2 35.5 f 13.0 43.3 2 14.8 17.6 f 5.4'

65 .7 f10 .7 43.5f20.32 26.2f19.01 51 .8 f17 .3 74.8f10.13 73 .6 f13 .2 6 0 . l f l 2 . 0 ATP content (nmol/lO9 platelets)

(nmoVlO9 platelets) ADP/volume (nmol/109 p,atelets/~3) 5 . 6 2 1 . 1 3 . 3 5 1.31 2.0+ 1 . 1 ' 4 . 0 f 2 . 2 5 .6 f2 .1 7.1 f 2 . 5 3.2f0.91

ATPholume (nmol~lOsplatelets~p,) 11.421.7 10.724.0 5.422.31 9 . 5 2 2 . 6 11.922.2 12.1k2.2 10.6f2.4

ADP t ATP/volume (nmol,109 platelets/p3) 17.0 f 2.4 14.3 f 3.9 7.4 f 3.31 12.8 f 5.9 17.5 f 3.9 19.2 f 4.7 14.1 k 2.32

ATP/ADP 2 .08 f0 .40 3.98f2.034 2.86f0.452 2 .80 f1 .10 2 .31 f0 .72 1.78f0.33 3.5720.951

Results are given as mean f SD. PNH = Paroxysmal nocturnal hemoglobinuria. ITP = Immune thrombocytopenic purpura. MPD = Myeloproliferative disorders. Comparison of hematologic disorders and normal subjects, Significant to p < 0.001, 2Significant to p < 0.01. 3Significant to p < 0.02, 4Significant to p < 0.05.

+ 'Ontent 98.2 2 15.3 56.3 f 23.61 36.1 f 27.21 73.6 f 29.1 110.3 f 21.6 116.9 f 27.4 77.1 f 15.62

68 NOZAKI ET AL

remains to be cl'arilfied. Decreased ADPholume and normal ATPholume in patients with MPD may be attributed simply to the storage pool deficiency.

Platelet volume and adenine nucleotides have also been examined in order to clarify the qualitative platelet abnormalities in patients with PNH, ITP or hypersplenism, in whom a platelet aggregation test can not be measured because of the thrombocytopenia. Normal results were obtained in patients with PNH or hypersplenism, while the level of intraplatelet ATP content in patients with ITP was significantly increased in proportion to the increased platelet volume. The normal results obtained in PNH or hypersple- nism are presumably due to the relatively high platelet counts or to the not so seriously affected marrow megakaryocytopoiesis. The change in ITP is probably attributable to the accelerated thrombocytopoiesis.

Evaluation of platelet volume and intraplatelet adenine nucleotides appears to be useful in assessing qualitative platelet abnormalities in various hematologic disorders, especially with thrombocytopenia.

Acknowledgment This work was supported in part by Tokai University General Research Grant no. 55.

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Correspondense to: Hiroyuki Nozaki Department of Internal Medicine School of Medicine Tokai University Bohseidai, lsehara Kanagawa 259-1 1 Japan