Scand J H a e m a t o l ( l 9 7 6 ) 17, 285-292
Platelet Production Capacity at Intervals after Acute Thrombocytopenia CARLW. JACKSON& CAROL C. EDWARDS Laboratory of Hematology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
Platelet producing capacity was examined at intervals after acute platelet depletion by reinduction of acute thrombocytopenia and assessment of the subsequent platelet recovery rates or Y h u l f a t e incorporation into platelets. Platelet producing capacity was increased on days 1 and 2, somewhat decreased on days 3 and 4 and had returned to baseline by day 5. These studies suggest that there is an initial increase followed by a decrease on days 3 and 4 in cells which can respond to thrombopoietic stimulating factor after induction of acute thrombocytopenia. Key words: platelets - thrombocytopenia
- thrombopoiesis
Accepted for publication July 6 , 1976 Correspondence to: Dr. Carl W. Jackson, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, Tennessee 38101, USA
Acute thrombocytopenia stimulates megakaryocytopoiesis (Ebbe et a1 1968a/b, Odell et al 1969, Penington & Olson 1970, Rolovic et a1 1970) and platelet production (Harker 1968, Peningtcm 1969; McDonald 1973). There is a maximum platelet production rate which is attained after a single acute thrombocytopenic episode (Odell et a1 1975). This maximum rate of platelet output is referred to as the platelet producing capacity. The platelet producing capacity after a single episode of acute thrombocytopenia should represent the normal platelet producing capacity. This study was designed
to determine whether acute thrombocytopenia alters the platelet producing capacity. The relative platelet producing capacity has been estimated at intervals after an initial platelet depletion in rats by reinducing acute thrombocytopenia and studying the subsequent platelet recovery rate or 35S-sulfate incorporation into platelets. The assumption has been made that platelet recovery rate for the first 4 days after acute platelet depletion is a direct reflection of the platelet production rate based on the following observations: 1) platelet recovery after acute thrombocytopenia represents new platelet
Supported in part by NIH Childhood Cancer Research Center Grant CA 08480 and by ALSAC.
286
CARL W. JACKSON & CAROL C. EDWARDS
production (Penington 1969, Harker 1970, McDonald 1973) and 2) 51Cr-labelled platelets from rats rendered thrombwytopenic with antiplatelet antiserum have a survival time of 4% days (Ginsburg & Aster 1969). MATERIALS AND METHODS Male Long-Evans hooded rats weighing 198479 g were obtained from Blue Spruce Farms, Inc., Altamont, New York. The largest range of body weight in a single experiment was 97 g. Platelets were counted by the phase-contrast method of Brecher & Cronkite (1950). Animals were anaesthetized with ether for injections and platelet counts. Blood was obtained by puncturing a vasolinecoated tail vein with a 25-gauge needle. Blood was collected using the Unopette system. Average initial platelet count in the groups that received two APS injections was 1.016 f 0.130 x 106 per ,d for 67 rats and 1.042 f 0.125 x 1CF per pl for 56 rats receiving a single APS injection. Antiserum against intact rat platelets was prepared in rabbits according to Ode11 et a1 (1961). Amount of antiplatelet antiserum injected
For all experiments the amount of antiserum administered was adjusted so that the platelet count would be reduced to less than 5 % (usually less than 1 %) of baseline levels after 4 to 6 h and would recover to approximately 1OO,OOO/p1 by 24 h. Since the platelet count prior t o the second APS injection varied depending on the number of days that had elapsed after the first injection of APS, the amount of APS given the second time was adjusted appropriately to reduce the platelet count t o the same level as with the initial APS. For example, in the experiment depicted in Figure lb, rats were given an initial APS injection of 0.1 ml with a second APS injection of 0.06 ml on day 2 or 0.11 ml on day 3. In the experiment depicted in Figure lc, rats were given an initial APS injection of 0.11 ml with a second APS injection of 0.06 ml on day 1 or 0.15 ml on days 4 or 5. APS was administered i.v. except in 1 experiment in which the second injection was given intraperitoneally.
35S-sulfateincorporation in to platelets
Rats were given 1 /cCi of 35S-sulfate (New England Nuclear) per g of body weight i.v. and sacrificed 36 h later. Blood was obtained from the dorsal aorta in acid citrate. The platelets were separated by centrifugation, washed 3 times with saline containing 0.1 % Naz EDTA, precipitated with 10 % trichloroacetic acid, solubilized in 2 ml of NCS tissue solubilizer (AmershadSearle) and placed in scintillation fluid (4 g PPO, 0.1 g POPOP, 340 ml absolute ethanol, 660 ml toluene). The radioactivity was determined in a Packard Tri-Carb Scintillation Spectrometer. 35S-sulfate incorporation into platelets is expressed as per cent of the total 35S-sulfate injected. RESULTS
Representative platelet recovery curves for the various APS injection schedules are shown in Figure 1. Only the recovery after the second APS injection is presented except for the 7-day interval for which the recovery after both the first and second APS is given. Platelet recovery after 2 APS injections spaced 1 day apart was more rapid and showed a higher peak platelet count than any other schedule. With this injection schedule, the platelet count usually peaked on day 5 while the peak usually occurred on day 6 after a single APS injection. The' peak platelet count after APS injections on days 0 and 2 occurred on day 4. The peak platelet count wcurred on days 5 or 6 with all other APS injection schedules. The daily increases in platelet count during the 1-4 day interval after a second platelet reduction are shown in Table 1. Table 1 is arranged so that the platelet recovery rates for the various groups can be compared using the initial APS as well as the second APS as reference points. These daily recovery rates were compared by the student t-test using the second APS as the reference point with those during the 1-4
f
f
.f
4 (4)
5 (19)
7 (5)
t
.f 286f70
292 f 70
278k654)
459k100 224k1342) P
Platelet Production Capacity at Intervals after Acute Thrombocytopenia CARLW. JACKSON& CAROL C. EDWARDS Laboratory of Hematology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
Platelet producing capacity was examined at intervals after acute platelet depletion by reinduction of acute thrombocytopenia and assessment of the subsequent platelet recovery rates or Y h u l f a t e incorporation into platelets. Platelet producing capacity was increased on days 1 and 2, somewhat decreased on days 3 and 4 and had returned to baseline by day 5. These studies suggest that there is an initial increase followed by a decrease on days 3 and 4 in cells which can respond to thrombopoietic stimulating factor after induction of acute thrombocytopenia. Key words: platelets - thrombocytopenia
- thrombopoiesis
Accepted for publication July 6 , 1976 Correspondence to: Dr. Carl W. Jackson, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, Tennessee 38101, USA
Acute thrombocytopenia stimulates megakaryocytopoiesis (Ebbe et a1 1968a/b, Odell et al 1969, Penington & Olson 1970, Rolovic et a1 1970) and platelet production (Harker 1968, Peningtcm 1969; McDonald 1973). There is a maximum platelet production rate which is attained after a single acute thrombocytopenic episode (Odell et a1 1975). This maximum rate of platelet output is referred to as the platelet producing capacity. The platelet producing capacity after a single episode of acute thrombocytopenia should represent the normal platelet producing capacity. This study was designed
to determine whether acute thrombocytopenia alters the platelet producing capacity. The relative platelet producing capacity has been estimated at intervals after an initial platelet depletion in rats by reinducing acute thrombocytopenia and studying the subsequent platelet recovery rate or 35S-sulfate incorporation into platelets. The assumption has been made that platelet recovery rate for the first 4 days after acute platelet depletion is a direct reflection of the platelet production rate based on the following observations: 1) platelet recovery after acute thrombocytopenia represents new platelet
Supported in part by NIH Childhood Cancer Research Center Grant CA 08480 and by ALSAC.
286
CARL W. JACKSON & CAROL C. EDWARDS
production (Penington 1969, Harker 1970, McDonald 1973) and 2) 51Cr-labelled platelets from rats rendered thrombwytopenic with antiplatelet antiserum have a survival time of 4% days (Ginsburg & Aster 1969). MATERIALS AND METHODS Male Long-Evans hooded rats weighing 198479 g were obtained from Blue Spruce Farms, Inc., Altamont, New York. The largest range of body weight in a single experiment was 97 g. Platelets were counted by the phase-contrast method of Brecher & Cronkite (1950). Animals were anaesthetized with ether for injections and platelet counts. Blood was obtained by puncturing a vasolinecoated tail vein with a 25-gauge needle. Blood was collected using the Unopette system. Average initial platelet count in the groups that received two APS injections was 1.016 f 0.130 x 106 per ,d for 67 rats and 1.042 f 0.125 x 1CF per pl for 56 rats receiving a single APS injection. Antiserum against intact rat platelets was prepared in rabbits according to Ode11 et a1 (1961). Amount of antiplatelet antiserum injected
For all experiments the amount of antiserum administered was adjusted so that the platelet count would be reduced to less than 5 % (usually less than 1 %) of baseline levels after 4 to 6 h and would recover to approximately 1OO,OOO/p1 by 24 h. Since the platelet count prior t o the second APS injection varied depending on the number of days that had elapsed after the first injection of APS, the amount of APS given the second time was adjusted appropriately to reduce the platelet count t o the same level as with the initial APS. For example, in the experiment depicted in Figure lb, rats were given an initial APS injection of 0.1 ml with a second APS injection of 0.06 ml on day 2 or 0.11 ml on day 3. In the experiment depicted in Figure lc, rats were given an initial APS injection of 0.11 ml with a second APS injection of 0.06 ml on day 1 or 0.15 ml on days 4 or 5. APS was administered i.v. except in 1 experiment in which the second injection was given intraperitoneally.
35S-sulfateincorporation in to platelets
Rats were given 1 /cCi of 35S-sulfate (New England Nuclear) per g of body weight i.v. and sacrificed 36 h later. Blood was obtained from the dorsal aorta in acid citrate. The platelets were separated by centrifugation, washed 3 times with saline containing 0.1 % Naz EDTA, precipitated with 10 % trichloroacetic acid, solubilized in 2 ml of NCS tissue solubilizer (AmershadSearle) and placed in scintillation fluid (4 g PPO, 0.1 g POPOP, 340 ml absolute ethanol, 660 ml toluene). The radioactivity was determined in a Packard Tri-Carb Scintillation Spectrometer. 35S-sulfate incorporation into platelets is expressed as per cent of the total 35S-sulfate injected. RESULTS
Representative platelet recovery curves for the various APS injection schedules are shown in Figure 1. Only the recovery after the second APS injection is presented except for the 7-day interval for which the recovery after both the first and second APS is given. Platelet recovery after 2 APS injections spaced 1 day apart was more rapid and showed a higher peak platelet count than any other schedule. With this injection schedule, the platelet count usually peaked on day 5 while the peak usually occurred on day 6 after a single APS injection. The' peak platelet count after APS injections on days 0 and 2 occurred on day 4. The peak platelet count wcurred on days 5 or 6 with all other APS injection schedules. The daily increases in platelet count during the 1-4 day interval after a second platelet reduction are shown in Table 1. Table 1 is arranged so that the platelet recovery rates for the various groups can be compared using the initial APS as well as the second APS as reference points. These daily recovery rates were compared by the student t-test using the second APS as the reference point with those during the 1-4
f
f
.f
4 (4)
5 (19)
7 (5)
t
.f 286f70
292 f 70
278k654)
459k100 224k1342) P
