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The Effects of Activators and Inhibitors of Platelet Aggregation on Platelet Protein Kinase
98
THE ROYAL COLLEGE O F PATHOLOGISTS OF AUSTRALIA
SOME BIOPHYSICAL CHARACTERISTICS OF THE AGAR COLONY FORMING CELL IN MAN
RICKARD, K. A., BROWN,R. D., DUNLEAVY, L. D. & KRONENBERG, H.
Department of Haematology, Royal Prince AIfred Hospital, Sydney, New South Wales When unfractionated human bone marrow is cultured in nutrient agar on peripheral leucocyte feeder layers granulocytic colonies are obtained. A linear relationship exists between the number of cells plated and the number of colonies obtained. A range for the number of colony-forming cells (CFC) in normal marrow has been established. A large series of non-neoplastic samples suggest a higher concentration of marrow CFC in males than females. Cell cycle characteristics based on in vitro thymidine suicide experiments indicate the human CFC arises from a proliferating population of cells. This kinetic similarity with murine marrow and the granulocytic nature of the colonies suggests that the CFC in man arises from a granulocyte progenitor or committed myeloid stem cell compartment. The radiation sensitivity in air, expressed as a Do value, for the human CFC was 127r. The cell survival curve exhibits an initial shoulder and yields an extrapolation number between 1 and 2 consistent with the diploid nature of the CFC. There tended to be a negative correlation between the Do value and the proliferative status of the CFC. After fractionation on discontinuous albumin gradients the CFC population from normal marrow consistently appeared in fraction 3. Cells in this fraction had the light microscopic characteristics of small lymphocytes. Gradient fractions rich in monocytes and macrophages when placed in feeder layers tended to give high colony stimulatory activity. THE MANAGEMENT OF HAEMOPHILIAC PATIENTS WITH ANTIBODIES AGAINST FACTOR Vlll
DAVEY, M. G., JACKSON, J. M. & THOMPSON, D. S.
Department of Haematology, Royal Perth Hospital, and Red Cross Blood Transfusion Service, Perth, Western Australia One of the most serious problems that can arise in a patient with haemophilia A (factor VIII deficiency) is formation of antibodies capable of neutralizing factor VIII activity. Such immunization makes it difficult to control bleeding, since infused factor VIII activity disappears rapidly so that frequent large doses are required to maintain haemostatic levels. Two patients with such antibodies have recently been treated. The first was a boy with severe haemophilia who was found to have a circulating factor VIII inhibitor at the age of 15. He has since required J treatment on 4 occasions; the second was a man with less severe haemophilia (AHF assays 8 - 1 2 ~ ~who formed antibody following prostatectomy at the age of 58. His bleeding could not be stopped. Measures used in treating these patients included large doses of human, bovine and porcine factor VIII, cyclophosphamide, corticosteroids, epsilonaminocaproic acid and plasma exchange. Such treatment has many hazards for the patients, such as allergic reactions to animal and human proteins, bone marrow depression, haemolysis, thrombocytopenia, and serum hepatitis. Investigations of other means of treatment must be undertaken, with particular attention to more efficient methods of plasma exchange, and specific suppression of the immune response to factor VIII. THE EFFECTS OF ACTIVATORS AND INHIBITORS OF PLATELET AGGREGATION ON PLATELET PROTEIN KINASE
BISHOP,G. A, & ROZENBERG, M. C. Division of Haematology, Prince Henry Hospital, New South Wales One possible mode of action of cyclic AMP in platelet aggregation is through cyclic AMP-dependent protein phosphokinase. A protein kinase has been partially purified from platelets and the effect of ADP, AMP, adenosine, the pyrimido-pyrimidine RA233 and calcium on enzyme activity has been investigated. The platelet protein kinase had a basal activity of approximately 320 pmol of 3EPincorporated/mg protein/min and 5 )c lo-' M cyclic AMP stimulated this activity by approximately 120°,,. The Km for cyclic AMP ranged from 3.0 V. lo--@ M to 1.1 Y lo-' M and the Km for ATP ranged from 6.6 \' 10-B M to 9.0 Y M.
ABSTRACTS OF ANNUAL MEETING
1973
99
ADP (5 \ M) inhibited protein kinase activity by 24y0 (SD4.60/0)in the absence of cyclic AMP and by 187, (SD 4.1",) with 5 x M cyclic AMP. AMP (5 v M) inhibited protein kinase activity M cyclic AMP. 5 Y by 16:; (SD 1.60n)without cyclic AMP and by 26% (SD 1.70,;))with 5 i M adenosine inhibited enzyme activity by approximately So, (SD0.9",) and this inhibition was independent of cyclic AMP concentration. Cyclic AMP-stimulated protein kinase activity had an absolute requirement for magnesium compared to calcium. Activity in the presence of Mg++and absence of cyclic AMP was inhibited approximately 80", by an equimolar (10 mM) concentration of Ca++. 5 mM Ca++ inhibited activity approximately 4"" while 5.5 mM Ca++ inhibited activity approximately 60n,. Protein kinase activity in the presence of Mg++ and cyclic AMP was similarly inhibited by Ca++. Calcium and cyclic AMP have antagonistic effects on platelet protein phosphokinase activity, an observation which may be important in the context of current knowledge of platelet aggregation. SCREENING FOR VARIANT HAEMOGLOBINS IN THE AUSTRALIAN PO PU LATlO N
FLEMING, PATRICIA, MORGAN, LEONIE & ARNOLD, BARBARA. The Institute of Clinical Pathology & Medical Research, Lidcornbe, New Scuth Wales Using the Beckman Microzone apparatus, large numbers of bloods have been screened for electrophoretic haemoglobin variants. It has successfully separated known examples of haemoglobins A, S , C , H, Kempsey and Norfolk. F separates only when in relatively high proportions. Without an elaborate automatic scanner the technique cannot be satisfactorily adapted to quantitation of H b A, and is not suitable for diagnosis of thalassaemia minor. Over a period of 18 mth, 2,500 bloods have been tested from patients in whom haemoglobin electrophoresis was neither clinically nor haematologically indicated and, apart from a small number of cord blood specimens, 4 electrophoretically abnormal haemoglobins were detected. One patient was a haematologically normal Italian woman of 55 whose cells were found to sickle and who had haemoglobin A and haemoglobin S . One 'fast' haemoglobin is still being investigated. Identification of the abnormalities in the other two bloods is proceeding. Both families are of pure British stock, both have a 'fast' haemoglobin with an x chain substitution and all carriers are clinically and haematologically normal and have approximately 7On0 of the abnormal haemoglobins. One family has the substitution z 16 Lys * glu (Haemoglobin I). The variant in the other family has mobility between Hb A and Hb I suggesting a single charge variation. CHROMOSOME CHANGES I N THE ELASTIC TRANSFORMATION STAGES OF CHRONIC GRANULOCYTIC LEUKAEMIA
Department of Medicine, St Vincent's Hospital, GARSON, 0. MARGARET. Fitzroy, Victoria Approximately 70", of patients with Ph' 've chronic granulocytic leukaemia (CGL) develop a terminal phase of blastic transformation or metamorphosis of the disease, in which additional chromosome changes usually occur. Investigation of bone marrow chromosomes in 30 patients entering this phase was undertaken to determine whether chromosome changes provided a reliable indication of transformation and to follow these changes if remission was obtained; satisfactory mitoses were obtained from 28. All showed chromosome abnormalities additional to the 46 Ph' f v e line of the chronic leukaemic state. Nineteen of the patients had hyperdiploid cell lines, 7 hypodiploid and 2 pseudodiploid. Most of the hyperdiploid lines contained 47 chromosomes/cell and all the hypodiploid lines were 45. The chromosome group most frequently involved in change was the C group and additional Ph' chromosomes were found in 13 patients. Remission of blastic transformation was achieved in 11 patients-the average time in remission being 6.9 mth. In all these patients, remission marrows reverted to the 46 Ph' line, but in 3 patients, Ph'-vr cells appeared initially and persisted together with the Ph'+ve lines. These 3 patients had the longest periods of remission, one being for 22 mth. With relapse, chromosome abnormalities recurred. It is concluded that chromosome studies are useful in the diagnosis of the blastic transformation stage of CGL and may have a prognostic significance in remission.
THE ROYAL COLLEGE O F PATHOLOGISTS OF AUSTRALIA
SOME BIOPHYSICAL CHARACTERISTICS OF THE AGAR COLONY FORMING CELL IN MAN
RICKARD, K. A., BROWN,R. D., DUNLEAVY, L. D. & KRONENBERG, H.
Department of Haematology, Royal Prince AIfred Hospital, Sydney, New South Wales When unfractionated human bone marrow is cultured in nutrient agar on peripheral leucocyte feeder layers granulocytic colonies are obtained. A linear relationship exists between the number of cells plated and the number of colonies obtained. A range for the number of colony-forming cells (CFC) in normal marrow has been established. A large series of non-neoplastic samples suggest a higher concentration of marrow CFC in males than females. Cell cycle characteristics based on in vitro thymidine suicide experiments indicate the human CFC arises from a proliferating population of cells. This kinetic similarity with murine marrow and the granulocytic nature of the colonies suggests that the CFC in man arises from a granulocyte progenitor or committed myeloid stem cell compartment. The radiation sensitivity in air, expressed as a Do value, for the human CFC was 127r. The cell survival curve exhibits an initial shoulder and yields an extrapolation number between 1 and 2 consistent with the diploid nature of the CFC. There tended to be a negative correlation between the Do value and the proliferative status of the CFC. After fractionation on discontinuous albumin gradients the CFC population from normal marrow consistently appeared in fraction 3. Cells in this fraction had the light microscopic characteristics of small lymphocytes. Gradient fractions rich in monocytes and macrophages when placed in feeder layers tended to give high colony stimulatory activity. THE MANAGEMENT OF HAEMOPHILIAC PATIENTS WITH ANTIBODIES AGAINST FACTOR Vlll
DAVEY, M. G., JACKSON, J. M. & THOMPSON, D. S.
Department of Haematology, Royal Perth Hospital, and Red Cross Blood Transfusion Service, Perth, Western Australia One of the most serious problems that can arise in a patient with haemophilia A (factor VIII deficiency) is formation of antibodies capable of neutralizing factor VIII activity. Such immunization makes it difficult to control bleeding, since infused factor VIII activity disappears rapidly so that frequent large doses are required to maintain haemostatic levels. Two patients with such antibodies have recently been treated. The first was a boy with severe haemophilia who was found to have a circulating factor VIII inhibitor at the age of 15. He has since required J treatment on 4 occasions; the second was a man with less severe haemophilia (AHF assays 8 - 1 2 ~ ~who formed antibody following prostatectomy at the age of 58. His bleeding could not be stopped. Measures used in treating these patients included large doses of human, bovine and porcine factor VIII, cyclophosphamide, corticosteroids, epsilonaminocaproic acid and plasma exchange. Such treatment has many hazards for the patients, such as allergic reactions to animal and human proteins, bone marrow depression, haemolysis, thrombocytopenia, and serum hepatitis. Investigations of other means of treatment must be undertaken, with particular attention to more efficient methods of plasma exchange, and specific suppression of the immune response to factor VIII. THE EFFECTS OF ACTIVATORS AND INHIBITORS OF PLATELET AGGREGATION ON PLATELET PROTEIN KINASE
BISHOP,G. A, & ROZENBERG, M. C. Division of Haematology, Prince Henry Hospital, New South Wales One possible mode of action of cyclic AMP in platelet aggregation is through cyclic AMP-dependent protein phosphokinase. A protein kinase has been partially purified from platelets and the effect of ADP, AMP, adenosine, the pyrimido-pyrimidine RA233 and calcium on enzyme activity has been investigated. The platelet protein kinase had a basal activity of approximately 320 pmol of 3EPincorporated/mg protein/min and 5 )c lo-' M cyclic AMP stimulated this activity by approximately 120°,,. The Km for cyclic AMP ranged from 3.0 V. lo--@ M to 1.1 Y lo-' M and the Km for ATP ranged from 6.6 \' 10-B M to 9.0 Y M.
ABSTRACTS OF ANNUAL MEETING
1973
99
ADP (5 \ M) inhibited protein kinase activity by 24y0 (SD4.60/0)in the absence of cyclic AMP and by 187, (SD 4.1",) with 5 x M cyclic AMP. AMP (5 v M) inhibited protein kinase activity M cyclic AMP. 5 Y by 16:; (SD 1.60n)without cyclic AMP and by 26% (SD 1.70,;))with 5 i M adenosine inhibited enzyme activity by approximately So, (SD0.9",) and this inhibition was independent of cyclic AMP concentration. Cyclic AMP-stimulated protein kinase activity had an absolute requirement for magnesium compared to calcium. Activity in the presence of Mg++and absence of cyclic AMP was inhibited approximately 80", by an equimolar (10 mM) concentration of Ca++. 5 mM Ca++ inhibited activity approximately 4"" while 5.5 mM Ca++ inhibited activity approximately 60n,. Protein kinase activity in the presence of Mg++ and cyclic AMP was similarly inhibited by Ca++. Calcium and cyclic AMP have antagonistic effects on platelet protein phosphokinase activity, an observation which may be important in the context of current knowledge of platelet aggregation. SCREENING FOR VARIANT HAEMOGLOBINS IN THE AUSTRALIAN PO PU LATlO N
FLEMING, PATRICIA, MORGAN, LEONIE & ARNOLD, BARBARA. The Institute of Clinical Pathology & Medical Research, Lidcornbe, New Scuth Wales Using the Beckman Microzone apparatus, large numbers of bloods have been screened for electrophoretic haemoglobin variants. It has successfully separated known examples of haemoglobins A, S , C , H, Kempsey and Norfolk. F separates only when in relatively high proportions. Without an elaborate automatic scanner the technique cannot be satisfactorily adapted to quantitation of H b A, and is not suitable for diagnosis of thalassaemia minor. Over a period of 18 mth, 2,500 bloods have been tested from patients in whom haemoglobin electrophoresis was neither clinically nor haematologically indicated and, apart from a small number of cord blood specimens, 4 electrophoretically abnormal haemoglobins were detected. One patient was a haematologically normal Italian woman of 55 whose cells were found to sickle and who had haemoglobin A and haemoglobin S . One 'fast' haemoglobin is still being investigated. Identification of the abnormalities in the other two bloods is proceeding. Both families are of pure British stock, both have a 'fast' haemoglobin with an x chain substitution and all carriers are clinically and haematologically normal and have approximately 7On0 of the abnormal haemoglobins. One family has the substitution z 16 Lys * glu (Haemoglobin I). The variant in the other family has mobility between Hb A and Hb I suggesting a single charge variation. CHROMOSOME CHANGES I N THE ELASTIC TRANSFORMATION STAGES OF CHRONIC GRANULOCYTIC LEUKAEMIA
Department of Medicine, St Vincent's Hospital, GARSON, 0. MARGARET. Fitzroy, Victoria Approximately 70", of patients with Ph' 've chronic granulocytic leukaemia (CGL) develop a terminal phase of blastic transformation or metamorphosis of the disease, in which additional chromosome changes usually occur. Investigation of bone marrow chromosomes in 30 patients entering this phase was undertaken to determine whether chromosome changes provided a reliable indication of transformation and to follow these changes if remission was obtained; satisfactory mitoses were obtained from 28. All showed chromosome abnormalities additional to the 46 Ph' f v e line of the chronic leukaemic state. Nineteen of the patients had hyperdiploid cell lines, 7 hypodiploid and 2 pseudodiploid. Most of the hyperdiploid lines contained 47 chromosomes/cell and all the hypodiploid lines were 45. The chromosome group most frequently involved in change was the C group and additional Ph' chromosomes were found in 13 patients. Remission of blastic transformation was achieved in 11 patients-the average time in remission being 6.9 mth. In all these patients, remission marrows reverted to the 46 Ph' line, but in 3 patients, Ph'-vr cells appeared initially and persisted together with the Ph'+ve lines. These 3 patients had the longest periods of remission, one being for 22 mth. With relapse, chromosome abnormalities recurred. It is concluded that chromosome studies are useful in the diagnosis of the blastic transformation stage of CGL and may have a prognostic significance in remission.