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Thrombocyte aggregation in hereditary spherocytosis
Clrnrca Chrmwza Acta,
Elsevier
Biomedical
119 (1982) Press
257-262
251
CCA 2039
Thrombocyte aggregation in hereditary spherocytosis Riitta y Department
Johnsson
of Clinical ChemtstT,
b
Unrversrty of Helsrnkr and h Fmnrsh Red Cross Blood Transfusion Servrce.
(Received
a,* and J.-J. Himberg Helsmkr (Fmland)
May 27th; revision October
24th. 1981)
Summary Thrombocyte function was studied in seven splenectomized patients and one unsplenectomized patient with hereditary or congenital spherocytosis (HS). Adenosine diphosphate (ADP) concentrations, which trigger the aggregation of control thrombocytes, induced only a release reaction in thrombocytes from six of the HS patients. Collagen-induced thrombocyte aggregation was also diminished in two patients, and epinephrine-induced thrombocyte aggregation in one patient. Two patients whose thrombocytes responded normally to ADP, collagen and epinephrine, were identical twins with spherocytosis of equivocal heredity. Ristocetin-induced aggregation was normal in all the patients studied. Xanthines (pentoxifylline, theophylline) had no effect on the aggregation of thrombocytes from HS patients or controls. Half-life of thrombocytes was normal in the two splenectomized patients studied. The results demonstrate that not only red cells but also thrombocytes are defective in HS.
Introduction Red cells from patients with hereditary or congenital spherocytosis (HS) display several physico-chemical abnormalities [ 11, but the primary defect is still not known. In a previous study we observed that the response of spherocytic red cells to the xanthine derivative pentoxifylline was different from that of normal red cells: this drug, which improved the flexibility of normal red cells, made the rigid HS erythrocytes even more inflexible [2]. The effect of pentoxifylline, like that of other xanthines, is considered to be mediated by the inhibition of phosphodiesterase, resulting in an increase in the concentration of CAMP in red cells [3]. Since thrombocytes are also rich in phosphodiesterase, we undertook to study the effect of * Correspondence: Dr. Riitta Johnsson, Department Meilahti Hospital, SF-00290 Helsinki 29, Finland.
0009-898
I /82/oooO-0000/$02
75 Q 1982 Elsewer
of Clinical
Biomedxal
Press
Chemistry,
Umversity
of Helsinki,
25X
pentoxifylline tients.
and theophylline
on the aggregation of thrombocytes
from HS pa-
Material and methods Patients Seven splenectomized subjects (5 females and 2 males, aged 23-48 yr) and one 2 1-year-old unsplenectomized female with well-compensated haemolytic anaemia (Hb 132 g/l, reticulocytes 16%) were studied. The splenectomized patients included a set of identical twins. Two healthy subjects, splenectomized because of traumatic rupture, were also studied. The control subjects were healthy laboratory personnel (6 females and 4 males, aged 24-38 yr) with a normal blood picture. None of the subjects studied received any medication two weeks prior to the study. Thrombocyte aggregation tests Fasting blood samples (10 ml) were collected in the morning in plastic tubes containing 1 ml of 3.13% sodium citrate. Platelet rich plasma (PRP) was prepared by centrifugation at 400 X g for 8 min. Platelet poor plasma (PPP) was obtained by recentrifugation of the pellet at 1400 X g for 18 min, after which the supernatant was separated and recentrifuged at 15000 X g for 15 min. Platelet aggregation tests were carried out in a Payton aggregometer (Payton Associates, Buffalo, NY, USA). The final platelet concentration in the PRP was corrected to 200 X 109/1 with the patients’ own PPP. All aggregations were performed at 37°C using siliconized l-ml cuvettes and siliconized bars rotating at 1000 r-pm. The aggregometer recorder was set at 0% and 100% using PRP and PPP, respectively, before each aggregation experiment. PRP, 0.9 ml, was incubated in the cuvette and the baseline readings were followed for 3 min, after which lo-25 ~1 of trigger solution was added. The concentrations of the trigger agents were selected on the basis of dose response experiments with platelets from control subjects. All measurements were made in duplicate within 3 h from the preparation of PRP. The aggregation curve was followed up to 10 min after the addition of the aggregating agent. The release reaction is seen as a primary wave in the curve. Secondary phase aggregation is calculated from the stable or slowly increasing level of optical transmissionafter the primary wave. The percentage of maximum aggregation was defined as the degree of secondary phase aggregation 6 mm after the addition of the trigger agent. Thrombocyte half-life was measured using “0-labelled autologous thrombocytes [41. Material Radioactive chromium (Na”Cr0,) was purchased from the Radiochemical Centre (Amersham). Bovine tendon collagen and adenosine diphosphate (ADP) were from Sigma Chemical Co. (St. Louis, MO, USA), epinephrine from Medica Ltd. (Helsinki) and ristocetin from Lundbeck Ltd. (Copenhagen).
259
Results The thrombocyte aggregation results, percentage of maximum aggregation, are presented in Table I. The ADP-induced aggregation was decreased in all cases except for the identical twins. Two patients also showed reduced aggregation with collagen and one with epinephrine. Ristocetin-induced aggregation was not altered in any of the HS patients studied. In order to assess the reproducibility of the findings, two patients (IL and ArH) were restudied after three months; the results from the two studies were similar. Pentoxifylline (20 mg/l) and theophylline (20 mg/l), when added to PRP before the trigger substance, did not change the aggregation pattern either in HS patients or in controls (Fig. la patient ArH, Fig. lb patient IL). In patients IL and JJ the thrombocyte half-life was 4.7 and 5.4 days, respectively. In two splenectomized healthy subjects the aggregation of thrombocytes was normal (Table I).
Fig. I. (A) Patient cyte aggregation.
(ArH) with normal
thrombocyte
aggregation.
(B) Patient
(IL) with decreased
thrombo-
260
TABLE
I
THE PERCENT MAXIMUM SECONDARY PHASE FROM HEREDITARY SPHEROCYTOSIS PATIENTS Patient
Sex
AnH ’ ArH a JPh JR JJ KE IL LL Control n=lO RE’ SH’ a b ’ d e
AGGREGATION
ADP
Collagen
1 Wml
10Wm*
Epinephrme 0.9 pg/ml
70 70 OC 0’ OC OC 0’ 0’
71 73 78 70 75 40 0 72
65 72 62 0’ 22 21 16 60 d
20-50 68 77
60-80 77 74
15-75 27 70
OF
THROMBOCYTES
Ristocetin 1.25 mg/ml 70 70 _ _ 100 72 80 6X
values
m/f f f
Identical twins. Unsplenectomized patient. Only primary wave and return to baseline: Delayed aggregation after 6 min. Splenectomized healthy subjects.
15-80 x7 23
release reaction.
Discussion The aggregation of thrombocytes from HS patients was decreased in six of the eight patients studied. This finding demonstrates that not only red cells but also thrombocytes are defective in HS. The two patients, identical twins, with normal thrombocyte aggregation tests were in fact those who in our earlier study [5] demonstrated low red cell membrane Ca-Mg-ATPase activity, whereas other HS patients had normal or elevated enzyme activity. It should also be noted that the heredity of the disease could not be established with certainty in these twins since their father had died and their mother and six siblings were healthy. HS is inherited as a mendelian dominant. However, occasionally the red cell abnormality cannot be demonstrated in either parent, possibly due to variation of penetrance or new mutation [6]. In spite of considerable variation in the severity of haemolysis in HS, the majority of patients is considered to have the same disease. However, in rare cases unresponsive to splenectomy [7] and with an atypical glucose autohaemolysis test [8,9], a different etiology is possible. It is therefore possible that the two cases with normal aggregation of thrombocytes differ in etiology from cases of HS with decreased aggregation of thrombocytes. Since two splenectomized healthy subjects had normal aggregation of thrombocytes, the observed decrease in aggregation of thrombocytes in the majority of the HS patients studied cannot be due to the splenectomy of these patients. The
261
decreased aggregation of thrombocytes in an unsplenectomized case of HS is further evidence that the abnormal platelet function in HS patients is the result of a basic abnormality and not splenectomy. It is also interesting to note that improvement rather than deterioration of platelet function has been observed in patients splenectomized because of hairy cell leukaemia [ 10,l l] and in autosplenectomized sickle-cell anaemia [ 121. Previous studies of HS have been confined only to red cells. However, McCann and Jacob [ 131observed two patients with spherocytosis and spinal cord disease and suggested that in HS there might be a stroma protein defect of plasma membranes also affecting cells other than erythrocytes. One of these patients also had decreased thrombocyte aggregation prior to splenectomy. However, no defect in the stroma proteins of HS red cells has been observed in spite of intensive research (see review by Zail [l]). According to some recent studies, the defects in the HS red cell membrane actually involve the lipids: a decrease in phospholipids [ 14,151, long-chain fatty acids [ 16) and lipid fluidity [17,18] has been observed. Increased lipase activity in HS red cells was recently reported by Etienne et al [ 191. In normal thrombocytes diglyceride lipase is involved in arachidonate metabolism [20] and, therefore, in the metabolism of prostaglandines, which are known to modulate thrombocyte aggregation [21]. If in HS the lipase activity of thrombocytes is also altered this might result in abnormal thrombocyte aggregation, Wenzel et al [22] have reported that pentoxifylline in vitro decreased the aggregation of normal human thrombocytes. However, the concentrations used by them were higher than those recommended for clinical practice. Pentoxifylline in uiuo has been reported to decrease the ADP-induced aggregation of thrombocytes from patients with obstructive cerebrovascular disorders [23]. These authors also observed a pentoxifylline-induced alteration in serum neutral fats and suggested that this drug effect on thrombocytes might be due to lipid changes and not to the pentoxifyllineinduced increase in CAMP. The fact that theophylline did not affect thrombocyte aggregation in any of the tests made by us also supports our view that mechanisms other than those mediated by the in~bition of phosphodiesterase are involved. Our observation that in HS the plasma lipid composition f24] is also disturbed is further evidence that in this disease the basic defect is in the lipid metabolism. Acknowledgements
We thank Mrs. Arja Siekkinen for skillful technical assistance. The work was supported by grants from the Stipend Fund Oy Hoechst Fennica Ab-Medical Factory. References I Zail SS. Annotation. The erythrocyte membrane abnormality of hereditary spherocyto&. Br J Haematol 1977; 37: 305-310. 2 Johnsson R Effect of pentoxifylline on red cell flexlbllity and canon tramport in healthy subjects and patients with hereditary spherocytosis. Stand J Haematol 1979: 23: Xi -87.
262 3 Popendiker K, Boksay I, Bollman V. Zur Pharmakologie des neuen peripheren Gefbsdilatators 3,7-Dimethyl- I -(5-oxohexyl)xanthin. Arzneim-Forsch (Drug Res) 197 I ; 2 I : 1160- I 17 I. 4 Kotilainen M. Platelet kinetics in normal subjects and haematological disorders with special reference to thrombocytopenia and to the role of spleen. Stand J Haematol 1969; Suppl No. 5. 5 Johnsson R, Santaholma S, Saris N-E. Calcium transport and adenosine triphosphatase activities of erythrocyte membranes in congenital spherocytosis. Stand J Clin Lab Invest 1978; 38: 121- 125. 6 Dacie JV. The haemolytic anaemias: congenital and acquired, Part 2. London: Churchill, 1954. 7 Carwicz S. Atypical spherocytosis, a disease of spleen as well as of red blood cells. Lancet 1975; I: 956-957. 8 Young LE. Observations on inheritance and heterogeneity of chronic spherocytosis. Trans Assoc Am Phscns 1955; 68: 141-148. 9 Langley GR, Felderhof CH. Atypical autohemolysis in hereditary spherocytosis as a reflection of two cell populations: relationship of cell lipids to conditioning by the spleen. Blood 1968; 32: 569-585. IO Sweet DL, Golome HM. Correction of platelet defect after splenectomy in hairy cell leukemia. J Am Med Assoc 1979; 241: 1684. 11 Rosove MH, Naeim F, Harwig S, Zighelboim J. Severe platelet dysfunction in hairy cell leukemia with improvement after splenectomy. Blood 1980; 55: 903-906. 12 Kenny MW, George AJ, Stuart J. Platelet hyperactivity in sickle-cell disease: a consequence of hyposplenism. J Clin Path01 1980; 33: 622-625. 13 McCann SR, Jacob HS. Spinal cord disease in hereditary spherocytosis: report of two cases with a hypothesized common mechanism for neurologic and red cell abnormalities. Blood 1976; 48: 259-263. 14 Cooper RA, Jandl JH. The role of membrane lipids in the survival of red cells in hereditary spherocytosis. J Clin Invest 1969; 48: 736-744. 15 Johnsson R. Red cell membrane proteins and lipids in spherocytosis. Stand J Haematol 1978; 20: 341-350. 16 Kuiper PJC, Livne A. Differences in fatty acid composition between normal human erythrocytes and hereditary spherocytosis affected cells. Biochim Biophys Acta 1972; 260: 755-758. 17 Aloni B, Shinitzky M, Moses S, Livne A. Elevated microviscosity in membranes of erythrocytes affected by hereditary spherocytosis. Br J Haematol 1975; 31: 117-123. 18 Jansson S-E, Johnsson R, Gripenberg J, Vuopio P. The fluidity gradient in erythrocyte membranes in hereditary spherocytosis: a spin label study. Br J Haematol 1980; 46: 73-78. 19 Etienne J, Noe L, Debray J, Polonovski J. Increased lipase activity in erythrocytes of patients with hereditary spherocytosis. Clin Chem 1979; 25: 1520. 20 Bell RL, Kennerly DA, Stanford N, Majerus PW. Diglyceride lipase: A pathway for arachidonate release from human platelets. Proc Nat1 Acad Sci USA 1979; 76: 3238-3241. 21 Emmons PR, Hampton JR, Harrison MJG, Honour AJ, Mitchell JRA. Effect of prostaglandin E, on platelet behaviour in vitro and in viva. Br Med J 1967; 2: 468-472. 22 Wenzel E, Rietkotter U, Otte B, Holzhtiter H, Bahre G, Volkmer R, Kaminsky R. Einfluss von Pentoxifyllin auf charakteristische Thrombozytenaggregationsund Fragmentierungsph%nomene. Med Welt 1975; 26: 2100-2102. 23 Itoh T, Satoh T. Influence of pentoxifylline (“Trental”) on platelet aggregation and serum lipids m patients with obstructive cerebrovascular disorders. Pharmatherapeutica 1979; 2: 159-258. 24 Johnsson R, Saris N-E. Plasma and erythrocyte lipids in hereditary spherocytosis. Chn Chim Acta 1981; 114: 263-268.
Elsevier
Biomedical
119 (1982) Press
257-262
251
CCA 2039
Thrombocyte aggregation in hereditary spherocytosis Riitta y Department
Johnsson
of Clinical ChemtstT,
b
Unrversrty of Helsrnkr and h Fmnrsh Red Cross Blood Transfusion Servrce.
(Received
a,* and J.-J. Himberg Helsmkr (Fmland)
May 27th; revision October
24th. 1981)
Summary Thrombocyte function was studied in seven splenectomized patients and one unsplenectomized patient with hereditary or congenital spherocytosis (HS). Adenosine diphosphate (ADP) concentrations, which trigger the aggregation of control thrombocytes, induced only a release reaction in thrombocytes from six of the HS patients. Collagen-induced thrombocyte aggregation was also diminished in two patients, and epinephrine-induced thrombocyte aggregation in one patient. Two patients whose thrombocytes responded normally to ADP, collagen and epinephrine, were identical twins with spherocytosis of equivocal heredity. Ristocetin-induced aggregation was normal in all the patients studied. Xanthines (pentoxifylline, theophylline) had no effect on the aggregation of thrombocytes from HS patients or controls. Half-life of thrombocytes was normal in the two splenectomized patients studied. The results demonstrate that not only red cells but also thrombocytes are defective in HS.
Introduction Red cells from patients with hereditary or congenital spherocytosis (HS) display several physico-chemical abnormalities [ 11, but the primary defect is still not known. In a previous study we observed that the response of spherocytic red cells to the xanthine derivative pentoxifylline was different from that of normal red cells: this drug, which improved the flexibility of normal red cells, made the rigid HS erythrocytes even more inflexible [2]. The effect of pentoxifylline, like that of other xanthines, is considered to be mediated by the inhibition of phosphodiesterase, resulting in an increase in the concentration of CAMP in red cells [3]. Since thrombocytes are also rich in phosphodiesterase, we undertook to study the effect of * Correspondence: Dr. Riitta Johnsson, Department Meilahti Hospital, SF-00290 Helsinki 29, Finland.
0009-898
I /82/oooO-0000/$02
75 Q 1982 Elsewer
of Clinical
Biomedxal
Press
Chemistry,
Umversity
of Helsinki,
25X
pentoxifylline tients.
and theophylline
on the aggregation of thrombocytes
from HS pa-
Material and methods Patients Seven splenectomized subjects (5 females and 2 males, aged 23-48 yr) and one 2 1-year-old unsplenectomized female with well-compensated haemolytic anaemia (Hb 132 g/l, reticulocytes 16%) were studied. The splenectomized patients included a set of identical twins. Two healthy subjects, splenectomized because of traumatic rupture, were also studied. The control subjects were healthy laboratory personnel (6 females and 4 males, aged 24-38 yr) with a normal blood picture. None of the subjects studied received any medication two weeks prior to the study. Thrombocyte aggregation tests Fasting blood samples (10 ml) were collected in the morning in plastic tubes containing 1 ml of 3.13% sodium citrate. Platelet rich plasma (PRP) was prepared by centrifugation at 400 X g for 8 min. Platelet poor plasma (PPP) was obtained by recentrifugation of the pellet at 1400 X g for 18 min, after which the supernatant was separated and recentrifuged at 15000 X g for 15 min. Platelet aggregation tests were carried out in a Payton aggregometer (Payton Associates, Buffalo, NY, USA). The final platelet concentration in the PRP was corrected to 200 X 109/1 with the patients’ own PPP. All aggregations were performed at 37°C using siliconized l-ml cuvettes and siliconized bars rotating at 1000 r-pm. The aggregometer recorder was set at 0% and 100% using PRP and PPP, respectively, before each aggregation experiment. PRP, 0.9 ml, was incubated in the cuvette and the baseline readings were followed for 3 min, after which lo-25 ~1 of trigger solution was added. The concentrations of the trigger agents were selected on the basis of dose response experiments with platelets from control subjects. All measurements were made in duplicate within 3 h from the preparation of PRP. The aggregation curve was followed up to 10 min after the addition of the aggregating agent. The release reaction is seen as a primary wave in the curve. Secondary phase aggregation is calculated from the stable or slowly increasing level of optical transmissionafter the primary wave. The percentage of maximum aggregation was defined as the degree of secondary phase aggregation 6 mm after the addition of the trigger agent. Thrombocyte half-life was measured using “0-labelled autologous thrombocytes [41. Material Radioactive chromium (Na”Cr0,) was purchased from the Radiochemical Centre (Amersham). Bovine tendon collagen and adenosine diphosphate (ADP) were from Sigma Chemical Co. (St. Louis, MO, USA), epinephrine from Medica Ltd. (Helsinki) and ristocetin from Lundbeck Ltd. (Copenhagen).
259
Results The thrombocyte aggregation results, percentage of maximum aggregation, are presented in Table I. The ADP-induced aggregation was decreased in all cases except for the identical twins. Two patients also showed reduced aggregation with collagen and one with epinephrine. Ristocetin-induced aggregation was not altered in any of the HS patients studied. In order to assess the reproducibility of the findings, two patients (IL and ArH) were restudied after three months; the results from the two studies were similar. Pentoxifylline (20 mg/l) and theophylline (20 mg/l), when added to PRP before the trigger substance, did not change the aggregation pattern either in HS patients or in controls (Fig. la patient ArH, Fig. lb patient IL). In patients IL and JJ the thrombocyte half-life was 4.7 and 5.4 days, respectively. In two splenectomized healthy subjects the aggregation of thrombocytes was normal (Table I).
Fig. I. (A) Patient cyte aggregation.
(ArH) with normal
thrombocyte
aggregation.
(B) Patient
(IL) with decreased
thrombo-
260
TABLE
I
THE PERCENT MAXIMUM SECONDARY PHASE FROM HEREDITARY SPHEROCYTOSIS PATIENTS Patient
Sex
AnH ’ ArH a JPh JR JJ KE IL LL Control n=lO RE’ SH’ a b ’ d e
AGGREGATION
ADP
Collagen
1 Wml
10Wm*
Epinephrme 0.9 pg/ml
70 70 OC 0’ OC OC 0’ 0’
71 73 78 70 75 40 0 72
65 72 62 0’ 22 21 16 60 d
20-50 68 77
60-80 77 74
15-75 27 70
OF
THROMBOCYTES
Ristocetin 1.25 mg/ml 70 70 _ _ 100 72 80 6X
values
m/f f f
Identical twins. Unsplenectomized patient. Only primary wave and return to baseline: Delayed aggregation after 6 min. Splenectomized healthy subjects.
15-80 x7 23
release reaction.
Discussion The aggregation of thrombocytes from HS patients was decreased in six of the eight patients studied. This finding demonstrates that not only red cells but also thrombocytes are defective in HS. The two patients, identical twins, with normal thrombocyte aggregation tests were in fact those who in our earlier study [5] demonstrated low red cell membrane Ca-Mg-ATPase activity, whereas other HS patients had normal or elevated enzyme activity. It should also be noted that the heredity of the disease could not be established with certainty in these twins since their father had died and their mother and six siblings were healthy. HS is inherited as a mendelian dominant. However, occasionally the red cell abnormality cannot be demonstrated in either parent, possibly due to variation of penetrance or new mutation [6]. In spite of considerable variation in the severity of haemolysis in HS, the majority of patients is considered to have the same disease. However, in rare cases unresponsive to splenectomy [7] and with an atypical glucose autohaemolysis test [8,9], a different etiology is possible. It is therefore possible that the two cases with normal aggregation of thrombocytes differ in etiology from cases of HS with decreased aggregation of thrombocytes. Since two splenectomized healthy subjects had normal aggregation of thrombocytes, the observed decrease in aggregation of thrombocytes in the majority of the HS patients studied cannot be due to the splenectomy of these patients. The
261
decreased aggregation of thrombocytes in an unsplenectomized case of HS is further evidence that the abnormal platelet function in HS patients is the result of a basic abnormality and not splenectomy. It is also interesting to note that improvement rather than deterioration of platelet function has been observed in patients splenectomized because of hairy cell leukaemia [ 10,l l] and in autosplenectomized sickle-cell anaemia [ 121. Previous studies of HS have been confined only to red cells. However, McCann and Jacob [ 131observed two patients with spherocytosis and spinal cord disease and suggested that in HS there might be a stroma protein defect of plasma membranes also affecting cells other than erythrocytes. One of these patients also had decreased thrombocyte aggregation prior to splenectomy. However, no defect in the stroma proteins of HS red cells has been observed in spite of intensive research (see review by Zail [l]). According to some recent studies, the defects in the HS red cell membrane actually involve the lipids: a decrease in phospholipids [ 14,151, long-chain fatty acids [ 16) and lipid fluidity [17,18] has been observed. Increased lipase activity in HS red cells was recently reported by Etienne et al [ 191. In normal thrombocytes diglyceride lipase is involved in arachidonate metabolism [20] and, therefore, in the metabolism of prostaglandines, which are known to modulate thrombocyte aggregation [21]. If in HS the lipase activity of thrombocytes is also altered this might result in abnormal thrombocyte aggregation, Wenzel et al [22] have reported that pentoxifylline in vitro decreased the aggregation of normal human thrombocytes. However, the concentrations used by them were higher than those recommended for clinical practice. Pentoxifylline in uiuo has been reported to decrease the ADP-induced aggregation of thrombocytes from patients with obstructive cerebrovascular disorders [23]. These authors also observed a pentoxifylline-induced alteration in serum neutral fats and suggested that this drug effect on thrombocytes might be due to lipid changes and not to the pentoxifyllineinduced increase in CAMP. The fact that theophylline did not affect thrombocyte aggregation in any of the tests made by us also supports our view that mechanisms other than those mediated by the in~bition of phosphodiesterase are involved. Our observation that in HS the plasma lipid composition f24] is also disturbed is further evidence that in this disease the basic defect is in the lipid metabolism. Acknowledgements
We thank Mrs. Arja Siekkinen for skillful technical assistance. The work was supported by grants from the Stipend Fund Oy Hoechst Fennica Ab-Medical Factory. References I Zail SS. Annotation. The erythrocyte membrane abnormality of hereditary spherocyto&. Br J Haematol 1977; 37: 305-310. 2 Johnsson R Effect of pentoxifylline on red cell flexlbllity and canon tramport in healthy subjects and patients with hereditary spherocytosis. Stand J Haematol 1979: 23: Xi -87.
262 3 Popendiker K, Boksay I, Bollman V. Zur Pharmakologie des neuen peripheren Gefbsdilatators 3,7-Dimethyl- I -(5-oxohexyl)xanthin. Arzneim-Forsch (Drug Res) 197 I ; 2 I : 1160- I 17 I. 4 Kotilainen M. Platelet kinetics in normal subjects and haematological disorders with special reference to thrombocytopenia and to the role of spleen. Stand J Haematol 1969; Suppl No. 5. 5 Johnsson R, Santaholma S, Saris N-E. Calcium transport and adenosine triphosphatase activities of erythrocyte membranes in congenital spherocytosis. Stand J Clin Lab Invest 1978; 38: 121- 125. 6 Dacie JV. The haemolytic anaemias: congenital and acquired, Part 2. London: Churchill, 1954. 7 Carwicz S. Atypical spherocytosis, a disease of spleen as well as of red blood cells. Lancet 1975; I: 956-957. 8 Young LE. Observations on inheritance and heterogeneity of chronic spherocytosis. Trans Assoc Am Phscns 1955; 68: 141-148. 9 Langley GR, Felderhof CH. Atypical autohemolysis in hereditary spherocytosis as a reflection of two cell populations: relationship of cell lipids to conditioning by the spleen. Blood 1968; 32: 569-585. IO Sweet DL, Golome HM. Correction of platelet defect after splenectomy in hairy cell leukemia. J Am Med Assoc 1979; 241: 1684. 11 Rosove MH, Naeim F, Harwig S, Zighelboim J. Severe platelet dysfunction in hairy cell leukemia with improvement after splenectomy. Blood 1980; 55: 903-906. 12 Kenny MW, George AJ, Stuart J. Platelet hyperactivity in sickle-cell disease: a consequence of hyposplenism. J Clin Path01 1980; 33: 622-625. 13 McCann SR, Jacob HS. Spinal cord disease in hereditary spherocytosis: report of two cases with a hypothesized common mechanism for neurologic and red cell abnormalities. Blood 1976; 48: 259-263. 14 Cooper RA, Jandl JH. The role of membrane lipids in the survival of red cells in hereditary spherocytosis. J Clin Invest 1969; 48: 736-744. 15 Johnsson R. Red cell membrane proteins and lipids in spherocytosis. Stand J Haematol 1978; 20: 341-350. 16 Kuiper PJC, Livne A. Differences in fatty acid composition between normal human erythrocytes and hereditary spherocytosis affected cells. Biochim Biophys Acta 1972; 260: 755-758. 17 Aloni B, Shinitzky M, Moses S, Livne A. Elevated microviscosity in membranes of erythrocytes affected by hereditary spherocytosis. Br J Haematol 1975; 31: 117-123. 18 Jansson S-E, Johnsson R, Gripenberg J, Vuopio P. The fluidity gradient in erythrocyte membranes in hereditary spherocytosis: a spin label study. Br J Haematol 1980; 46: 73-78. 19 Etienne J, Noe L, Debray J, Polonovski J. Increased lipase activity in erythrocytes of patients with hereditary spherocytosis. Clin Chem 1979; 25: 1520. 20 Bell RL, Kennerly DA, Stanford N, Majerus PW. Diglyceride lipase: A pathway for arachidonate release from human platelets. Proc Nat1 Acad Sci USA 1979; 76: 3238-3241. 21 Emmons PR, Hampton JR, Harrison MJG, Honour AJ, Mitchell JRA. Effect of prostaglandin E, on platelet behaviour in vitro and in viva. Br Med J 1967; 2: 468-472. 22 Wenzel E, Rietkotter U, Otte B, Holzhtiter H, Bahre G, Volkmer R, Kaminsky R. Einfluss von Pentoxifyllin auf charakteristische Thrombozytenaggregationsund Fragmentierungsph%nomene. Med Welt 1975; 26: 2100-2102. 23 Itoh T, Satoh T. Influence of pentoxifylline (“Trental”) on platelet aggregation and serum lipids m patients with obstructive cerebrovascular disorders. Pharmatherapeutica 1979; 2: 159-258. 24 Johnsson R, Saris N-E. Plasma and erythrocyte lipids in hereditary spherocytosis. Chn Chim Acta 1981; 114: 263-268.