Life Sciences Vol . 20, pp . 1571-1580, 1977 . Printed in the U.S .A .
Pergamon Press
RELATIONSHIPS BETWEEN BLOOD PLATSLET AND ERYTHROCYTE FORMATION* George W. Cooper and Barbara Cooper The City College of the City University of New York, New York, N.Y . 10031 (Received in final form March 31, 1977) Summary The rate of thrombopoiesis was measured by platelet incorporation of 35S-sulfate in mice having markedly stimulated or suppressed erythropoiesis, and compared to controls . Twenty-four hr exposure to 0.4 atm in a low pressure chamber caused increased platelet incorporation of 35 S, while exposure for 2 to 5 days caused reductions in both 35S uptake and platelet counts at time of sacrifice. These values were progressively reduced with the longer periods of hypoxia. Two units of human urinary erythropoietin (ESF) wer injected twice daily for 1 to 5 days and also caused reduced platelet 5S uptake in mice given the ESP for 2,`4, or 5 days . Mice rendered polycythemic by hypertransfusion or previous exposure to low barometric pressure had increased platelet 35E uptake and increased platelet counts . One possible explanation is that the severe hypoxia or the administration of ESP strongly stimulated erythropoiesis, but might have inhibited thrombopoiesis by depleting megakaryocyte precursors in the bone marrow. The formation of erythrocytes and of platelets is believed to be regulated by different humoral mechanisms (1) . However, a number of instances have been reported of interrelations between the rates of thrombopoiesis and erythropoi esis . A relationship was reported (2) between hemoglobin and platelet levels in iron deficiency anemia, where the lowest platelet counts were found in severely anemic patients, while thrombocytosis was reported in the moderately anemic patients . In a second study of hypoxic patients with cyanotic congenital heart disease, platelet counts were reported to be directly related to the degree of oxygen saturation and inversely related to mean hemoglobin levels (3) . Platelet levels tended to be elevated in younger patients having hemoglobins lower than 16 g/dl and mildly reduced oxygen saturations, while platelet counts were quite low in children having hemoglobin levels higher than 16 g/dl and long standing hypoxia. Rats given an experimental iron deficiency anemia, where erythropoletis was blocked, had increased platelet counts and increased platelet and megakaryocyte production, which was reversed after iron administration (4,5) . Rats rendered severely anemic by exchange transfusion with platelet rich, erythrocyte deficient plasma (o .22 PCV) were reported to have significantly increased platelet counts (6) . Reduced numbers of bone marrow megakaryocytes were reported in the rats sacrificed three days after the induction of the anemia . Platelet levels were
in
*Presented part at the meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April 15-20, 1973, Fed . Proc . 32 413 (1973) .
1571
1572
Platelet and Erythrocyte Relationships
Vol . 20, No . 9, 1977
not significantly changed in less severely anemic rats, unless erythropoissis was simultaneously blocked by the injection of actinomycin D. Materials and Methods Young female mice of the CF 1 strain, obtained from Carworth Farms, New City, N.Y . were used in these studies. Platelet counts were done on freely flowing §1il vein blood by the phase contrast method (7) . Platelet incorporation of S-sulfate wan determined3 as previously described (8) . Mice were 5 given a iv injection of 25 pCi Nat 804, and bled 48 hr later by cardiac punc ture . The washed platelet suspension was dissolved in 1 ml of NCS reagent (Amersham-Searle), 15 ml of a toluene-PPO-dimethyl POFOF scintillator was Backman L8 250 liquid scintillation added, and the sample was counted in counter. Platelet incorporation of 3 %-sulfate was calculated by the following equation :
% uptake x 103. .
sample dpm x blood vol/g x body vt standard dos vol of sample (3/4 ml x 10/11)
x 100,000
The 0.75 ml sample volume was multiplied by the factor 10/11, which represented the fraction of the platelets recovered in the sample (8) . Blood volume was measured using red calls labeled in vivo with 2 pCi 59Fe-ferrous citrate . The tagged cells (0 .2 ml) were iâjectoa into the tail of 5 CF 1 female mice 2 days after a 5 day exposure to continuous hypoxia of 0.4 atm in the low pressure chamber (equivalent to an altitude of 6,400 m) and in 7 control mice . The cardiac blood (0 .5 ml) was counted in a Packard Auto-Gamma automatic gamma scintillation counter . The blood volume was 58 ± . 4 .2 pl/g in the hypoxic mice (PCV : 0.52 + .028), and was 52 ± 3 .8 pl/g in the controls (PCV: 0.46 + .008) . The former value (58 pl/g) was used for calculating the radiosulfate uptake in the hypomic, exhypoxic, red cell transfused, and 987 injected mice, while the latter value (52 pl/g) was used for the controls . Control mice were always sacrificed simultaneously with the experimental animals .
win
In one experiment, the mice were subjected to 0 .4 atm in the low pressure chamber for either 24 or 96 hr, and were injected iv with 25 rCi li~3580 within 10 min after removal from the chamber. The mice were sacrificed h hr later. In a second, repeat experiment, the mice were subjected to continuous S when hypoxia (0 .4 atm) for 1 to 5 days, almost immediately injected with removed from the chamber, and axsanguinated 48 hr later . The experimental groups were placed in the low pressure chamber on different days, since all of the mice were removed from the chamber at the same time . In another experiment, 2 units of human urinary erythropoistin (SSF) dissolved in isotonic saline were administered ip twice daily for 1 to 5 days . The specific activity of the =SF was 58 .8 units/mg . Control mice received the same volume (0 .5 ml) of saline twice daily for 5 days . The radioisotope was injected iv into the mice 24 hr after the final B8F or saline injection, and the mica were sacrificed 48 hr later. All animals ware sacrificed on the same day. The dose of ESF that was injected into the mice (2 units, twice daily) was a dose calculated to achieve plasma levels of =SF that were of the sass order of magnitude as in animals exposed to hypoxia of 0.4 atm . Bats subjected to 0.4 atm for 8 hr had 0.77 units 981/mî of their plasma (9) . The 25 g mice had blood volumes of 1.3 ml and plasma volumes of 0.72 ml, implying that
Vol . 20, No . 9, 1977
Platelet and Erythrocyte Relationships
1573
a plasma level of 0 .5 units/souse should be maintained . Since exogenous ISF administered to dogs was found to have a rapid biphasic disappearance from the plasma, with an initial phase having a half-time from 20-45 min and a slower phase with a half-time of 9-10 .5 hr (10), 4 times this estimated level of ESF (2 units) were injected twice daily into the mice . In a fourth experiment, mice were given a single ip injection of a red blood cell suspension that had been vambed 4 times with isotonic saline, and had been shown to be free of platelets . Radiosulfate was administered 4 days after the hypertransfusion . Mice were also rendered polycythemic by subjection to 0 .4 atm for 19 br/day in the low pressure camber for 12 days in the fifth experiment . The mice were injected iv with 5S four days after removal from the chamber, and sacrificed 2 days later . The significance of differences between mean values of the experimental and control groups was determined by the Student t test (11) .
Reaulits Two-day platelet 35 S-sulfate incorporation values and platelet counts at sacrifice of control mice and mice exposed to 24 or 96 hr of hypoxia are shown in Table 1 . Acute exposure to hypoxia (24 hr) seems to stimulate platelet production., as judged by increased incorporation of 5S that was 183% of the control value (p .< .02) . Ninety-six hr of continuous exposure to hypoxia resulted in reduced sulfate uptake to 54% of the controls (p
Pergamon Press
RELATIONSHIPS BETWEEN BLOOD PLATSLET AND ERYTHROCYTE FORMATION* George W. Cooper and Barbara Cooper The City College of the City University of New York, New York, N.Y . 10031 (Received in final form March 31, 1977) Summary The rate of thrombopoiesis was measured by platelet incorporation of 35S-sulfate in mice having markedly stimulated or suppressed erythropoiesis, and compared to controls . Twenty-four hr exposure to 0.4 atm in a low pressure chamber caused increased platelet incorporation of 35 S, while exposure for 2 to 5 days caused reductions in both 35S uptake and platelet counts at time of sacrifice. These values were progressively reduced with the longer periods of hypoxia. Two units of human urinary erythropoietin (ESF) wer injected twice daily for 1 to 5 days and also caused reduced platelet 5S uptake in mice given the ESP for 2,`4, or 5 days . Mice rendered polycythemic by hypertransfusion or previous exposure to low barometric pressure had increased platelet 35E uptake and increased platelet counts . One possible explanation is that the severe hypoxia or the administration of ESP strongly stimulated erythropoiesis, but might have inhibited thrombopoiesis by depleting megakaryocyte precursors in the bone marrow. The formation of erythrocytes and of platelets is believed to be regulated by different humoral mechanisms (1) . However, a number of instances have been reported of interrelations between the rates of thrombopoiesis and erythropoi esis . A relationship was reported (2) between hemoglobin and platelet levels in iron deficiency anemia, where the lowest platelet counts were found in severely anemic patients, while thrombocytosis was reported in the moderately anemic patients . In a second study of hypoxic patients with cyanotic congenital heart disease, platelet counts were reported to be directly related to the degree of oxygen saturation and inversely related to mean hemoglobin levels (3) . Platelet levels tended to be elevated in younger patients having hemoglobins lower than 16 g/dl and mildly reduced oxygen saturations, while platelet counts were quite low in children having hemoglobin levels higher than 16 g/dl and long standing hypoxia. Rats given an experimental iron deficiency anemia, where erythropoletis was blocked, had increased platelet counts and increased platelet and megakaryocyte production, which was reversed after iron administration (4,5) . Rats rendered severely anemic by exchange transfusion with platelet rich, erythrocyte deficient plasma (o .22 PCV) were reported to have significantly increased platelet counts (6) . Reduced numbers of bone marrow megakaryocytes were reported in the rats sacrificed three days after the induction of the anemia . Platelet levels were
in
*Presented part at the meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April 15-20, 1973, Fed . Proc . 32 413 (1973) .
1571
1572
Platelet and Erythrocyte Relationships
Vol . 20, No . 9, 1977
not significantly changed in less severely anemic rats, unless erythropoissis was simultaneously blocked by the injection of actinomycin D. Materials and Methods Young female mice of the CF 1 strain, obtained from Carworth Farms, New City, N.Y . were used in these studies. Platelet counts were done on freely flowing §1il vein blood by the phase contrast method (7) . Platelet incorporation of S-sulfate wan determined3 as previously described (8) . Mice were 5 given a iv injection of 25 pCi Nat 804, and bled 48 hr later by cardiac punc ture . The washed platelet suspension was dissolved in 1 ml of NCS reagent (Amersham-Searle), 15 ml of a toluene-PPO-dimethyl POFOF scintillator was Backman L8 250 liquid scintillation added, and the sample was counted in counter. Platelet incorporation of 3 %-sulfate was calculated by the following equation :
% uptake x 103. .
sample dpm x blood vol/g x body vt standard dos vol of sample (3/4 ml x 10/11)
x 100,000
The 0.75 ml sample volume was multiplied by the factor 10/11, which represented the fraction of the platelets recovered in the sample (8) . Blood volume was measured using red calls labeled in vivo with 2 pCi 59Fe-ferrous citrate . The tagged cells (0 .2 ml) were iâjectoa into the tail of 5 CF 1 female mice 2 days after a 5 day exposure to continuous hypoxia of 0.4 atm in the low pressure chamber (equivalent to an altitude of 6,400 m) and in 7 control mice . The cardiac blood (0 .5 ml) was counted in a Packard Auto-Gamma automatic gamma scintillation counter . The blood volume was 58 ± . 4 .2 pl/g in the hypoxic mice (PCV : 0.52 + .028), and was 52 ± 3 .8 pl/g in the controls (PCV: 0.46 + .008) . The former value (58 pl/g) was used for calculating the radiosulfate uptake in the hypomic, exhypoxic, red cell transfused, and 987 injected mice, while the latter value (52 pl/g) was used for the controls . Control mice were always sacrificed simultaneously with the experimental animals .
win
In one experiment, the mice were subjected to 0 .4 atm in the low pressure chamber for either 24 or 96 hr, and were injected iv with 25 rCi li~3580 within 10 min after removal from the chamber. The mice were sacrificed h hr later. In a second, repeat experiment, the mice were subjected to continuous S when hypoxia (0 .4 atm) for 1 to 5 days, almost immediately injected with removed from the chamber, and axsanguinated 48 hr later . The experimental groups were placed in the low pressure chamber on different days, since all of the mice were removed from the chamber at the same time . In another experiment, 2 units of human urinary erythropoistin (SSF) dissolved in isotonic saline were administered ip twice daily for 1 to 5 days . The specific activity of the =SF was 58 .8 units/mg . Control mice received the same volume (0 .5 ml) of saline twice daily for 5 days . The radioisotope was injected iv into the mice 24 hr after the final B8F or saline injection, and the mica were sacrificed 48 hr later. All animals ware sacrificed on the same day. The dose of ESF that was injected into the mice (2 units, twice daily) was a dose calculated to achieve plasma levels of =SF that were of the sass order of magnitude as in animals exposed to hypoxia of 0.4 atm . Bats subjected to 0.4 atm for 8 hr had 0.77 units 981/mî of their plasma (9) . The 25 g mice had blood volumes of 1.3 ml and plasma volumes of 0.72 ml, implying that
Vol . 20, No . 9, 1977
Platelet and Erythrocyte Relationships
1573
a plasma level of 0 .5 units/souse should be maintained . Since exogenous ISF administered to dogs was found to have a rapid biphasic disappearance from the plasma, with an initial phase having a half-time from 20-45 min and a slower phase with a half-time of 9-10 .5 hr (10), 4 times this estimated level of ESF (2 units) were injected twice daily into the mice . In a fourth experiment, mice were given a single ip injection of a red blood cell suspension that had been vambed 4 times with isotonic saline, and had been shown to be free of platelets . Radiosulfate was administered 4 days after the hypertransfusion . Mice were also rendered polycythemic by subjection to 0 .4 atm for 19 br/day in the low pressure camber for 12 days in the fifth experiment . The mice were injected iv with 5S four days after removal from the chamber, and sacrificed 2 days later . The significance of differences between mean values of the experimental and control groups was determined by the Student t test (11) .
Reaulits Two-day platelet 35 S-sulfate incorporation values and platelet counts at sacrifice of control mice and mice exposed to 24 or 96 hr of hypoxia are shown in Table 1 . Acute exposure to hypoxia (24 hr) seems to stimulate platelet production., as judged by increased incorporation of 5S that was 183% of the control value (p .< .02) . Ninety-six hr of continuous exposure to hypoxia resulted in reduced sulfate uptake to 54% of the controls (p
