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Heterogeneity of rabbit platelets
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
164, 752-755 (1974)
Heterogeneity Effect of Chronic
Blood
of Rabbit
Loss on Glycolytic
Platelets
and Related
Enzyme
Activity’-
’
S. KARPATKIN Department
of Medicine,
New York University
Medical
Center, New York, New York 10016
Received May 17, 1974 Rabbits were chronically bled, with Fe replacement, every 3-4 days for 21 days. Their platelet count and megathrombocyte number (large-heavy, young platelets) increased 1.7and 2.3-fold, respectively. Eight of the 11 enzymes of the Embden-Meyerhof pathway increased 2-5-fold in activity per g wet weight during the period of blood letting. The five related enzymes (phosphoglucomutase, glucose 6-P dehydrogenase, 6-P gluconic dehydrogenase, glutathione reductase, and o-glycerol-P dehydrogenase) as well as the three Embden-Meyerhof pathway enzymes (aldolase, enolase, and pyruvate kinase) did not increase in activity over basal values. It is concluded that chronic blood loss with Fe replacement leads to a specific increase in enzyme activity of 8 of the 11 Embden-Meyerhof pathway enzymes.
Iron is required for maximum platelet production during the “platelet-production stimulus” of acute or chronic blood loss via the production of megathrombocytes (large-heavy, young platelets (1, 2)). Whereas blood loss without Fe replacement led to a rise in platelet count and megathrombocyte number of 1.4- and 1.7fold, respectivley, blood loss with iron replacement led to a rise in platelet count and megathrombocyte number of 2.5 and 3.8-fold, respectively, in guinea pigs (3-5). It was postulated that iron was necessary for the synthesis or production of an integral part of the platelet (4, 5). Recently published studies have revealed that iron is required for platelet protein synthesis (6). The chelation of iron with 2,2’-bipyridine reduced platelet protein synthesis by 80%, whereas transferrin or hemin could both reverse this inhibition as well as independently stimulate protein synthesis by 48-170%, respectively (6). ‘Supported by Grant HL‘13336 of the National Heart and Lung Institute and Contract DADA C-8163 of the U. S. Army Research and Development Command. 2This is paper III in a series. Paper II appeared in J. Lab. Clin. Med. 83, 896-901 (1974).
Since the platelet is an aerobic glycolytic tissue, which derives approximately half of its energy from glycolysis (7-lo), it was postulated that key glycolytic enzymes might be increased in activity during the accelerated platelet and megathrombocyte production induced by blood loss plus iron replacement. All 11 enzymes of the Embden-Meyerhof (EM) pathway, as well as five related enzymes, were measured at several time points during blood letting. The data reveal a 2%&fold increase in enzyme activity per g wet weight of 8 of the 11 enzymes of the EM pathway. This rise was fairly specific in that no change in enzyme activity was noted in eight other enzymes measured. MATERIALS
AND
METHODS
White New Zealand rabbits, weighing approximately 4 kg, were bled via ear artery for basal values and then chronically bled every 3-4 days by removal of approximately 15% of their whole blood volume (30-40 ml). Fe loss was calculated and immediately replaced by the intramuscular injection of Imferon. Rabbit platelets were obtained and washed in a modified Ringer solution as described for human platelets (11). Red blood cell and white blood cell contamination was negligible, as described previously (8), and could not have interfered with platelet cell
BLOOD LOSS: PLATELET sap enzyme activity. Platelet volume was determined by centrifugation at 2OOOgin 12.5 cm x 1 mm glass capillary tubes. One milliliter packed platelets was equivalent to 1 g wet weight. All operations were performed at 0-4°C and enzyme extraction performed by ultrasonic disruption as described previously (12). Briefly, the platelet pellet was suspended in 3 vol per platelet volume of Ringer solution containing 5 mM EDTA, 5 mM mercaptoethanol, pH adjusted to 7.5. This suspension was sonicated at maximal intensity at 4°C with a Bronwill Biosonik microprobe for two loset intervals. The disrupted cell suspension was then centrifuged at 105,OOOgfor 60 min in a Beckmrn L2 ultracentrifuge to obtain cell sap. Enzymatic assays employed classical techniques for coupling of enzymatic reactions to pyridine nucleotide oxidation-reduction changes at 340 nm and 30°C as described in detail previously (12). These were minor modifications of the methods of Lowry and Passonneau (13). A Gilford power supply and photo-cell, Beckman spectral system, and Honeywell recorder were employed to measure initial enzyme rates. These were linear with time and “extract” concentration (105,OOOg supematant) and were zero in the absence of substrate(s). Optimal dilutions for concentration-dependent linearity were made with 50 mM Tris buffer, 5 mM EDTA, 5 mM mercaptoethanol, pH 7.5, as described previously (except for phosphoglyceromutase, pyruvate kinase, and phosphoglucomutase which were diluted 1:20, 1:5, l:lOO, and 1:5, respectively). Enzymatic rates were assayed in microcuvettes of 0.5-ml volume in 50 mM Tris buffer, 5 mM mercaptoethanol, pH 7.5, at 30°C. All rates were assayed in triplicate, employing lo-, 20-, and 30-~1portions of tissue extract. Data are expressed as pmoles substrate converted/g wet weight/hr at 30°C. Distilled deionized water was used at all times. All chemicals were reagent grade. Enzyme reagents were
P=cOS
COOI COO1
NS
~001
GLYCOLYTIC
ENZYMES
753
obtained from either Sigma Chemical Co., Boehringer Mannheim Corp., or Worthington Biochemical Corp. Platelet counts were performed manually under phase contrast optics as described previously (14). Megathrombocyte number was measured with a Coulter Counter Model B and 70-pm aperture tube (15), at window threshold settings representing approximately 10% of the platelet volume distribution (14-25 lrm3). RESULTS
Basal values and comparison with human platelets. A plot of rabbit platelet cell sap
EM pathway enzyme activity at pH 7.5 is depicted in Fig. 1. This is similar to that reported for human platelets (12) with peaks of enzyme activity for phosphohexoisomerase and triose phosphate isomerase. The apparent rate-limiting enzymes (those with lowest cell sap activity) were also similar to those reported for human platelets in that they included hexokinase, phosphofructokinase, glyceraldehyde-3-P dehydrogenase, and phosphoglyceromutase. Of interest are further comparisons of total cell sap activity, The first five enzymes of the EM pathway are approximately the same for both human (12) and rabbit platelets (hexokinase to triosephosphate isomerase). The last six enzymes were considerably less for rabbit platelets. The non-EM pathway enzymes (phosphoglucomutase, glucose 6-P dehydrogenase, 6-P gluconic dehydrogenase, and glutathione reductase) were also considerably less
(001
COOI tOOI
NS
NS
(05
FIG. 1. Platelet cell sap enzyme activity fof the Embden-Meyerhof pathway at pH 7.5. Data for basal rabbit platelet enzyme activity, nine experiments (O----O), are compared to maximal increase in enzyme activity, four experiments (vertical arrows) after chronic blood loss. The interrupted line represents a ratio of 1 for the increase in enzyme activity over basal activity. The SD range for each determination was +15-25%. P values are given for the significance of the increase in enzyme activity over basal values (vertical arrows) after chronic blood loss, and were determined by Student’s t test. Abbreviations, reading from left to right, refer to the consecutive enzymes of the Embden-Meyerhof pathway.
754
S. KARPATKIN
for rabbit platelets and averaged (eight experiments) 129, 124, 110, and 85 ~moleslglhr, respectively. Alpha glycerophosphate dehydrogenase activity was approximately the same for both platelet systems. Effect of chronic blood loss. The basal platelet count and megathrombocyte number for 10 rabbits averaged 479,000/mms and 53,000/mms, respectively. With chronic blood loss plus iron replacement, the platelet count rose to 1.3-, 1.6-, 1.7-, and 1.&fold basal levels on days 4, 7, 11, and 14 respectively. The megathrombocyte number rose at a greater rate to 1.7-, 1.5, 2.1-, and 2.3-fold basal levels, respectively. The effect of chronic blood loss on glycolytic and related enzyme activity of a typical experiment from four different experiments is shown in Fig. 2. All the enzymes of the EM pathway, except aldolase, enolase, and pyruvate kinase, increased significantly over basal values. The five non-EM pathway enzymes (phosphoglucomutase, glucose 6-P dehydrogenase, 6-P gluconic dehydrogenase, glutathione reductase, and cw-glycerol-P dehydrogenase) did not increase significantly over basal values. Increase in enzyme activity generally began between the second and third blood letting, peaked on days 7 through 20 (third through fifth bloodletting), and then declined during the third week, after the fifth bloodletting. The peak average of four such experiments is shown in Fig. 1, in which the vertical arrows reflect the increase over basal values. The enzymes with the greatest increase in activity over basal values were phosphoglyceromutase, 4% fold; phosphoglycerokinase, 3.4-fold; phosphofructokinase, 3.0-fold; glyceraldehyde3-P dehydrogenase, 2.9-fold; phosphohexoisomerase, 2.8-fold; and triose-P-isomerase, 2.0-fold. DISCUSSION
The data clearly indicate that chronic blood loss with Fe replacement leads to a platelet population with enhanced glycolytic enzyme activity for 8 of the 11 enzymes of the EM pathway. This response was fairly specific in that none of the five
OSI.,,,.,,,.,.,.,.,.,.,., 0 2 4
‘am 6
8
IO
12
14
16
IS
20
22
Days
FIG. 2. Effect of chronic blood loss on EmbdenMeyerhof pathway enzyme activity and related enzyme activity, over a 21-day period of blood letting with Fe replacement (see Methods for details). Abbreviations, top panel: hexokinase (HK), phosphohexoisomerase (PHI), phosphofructokinase (PFK), aldolase (ALD), triose-P isomerase (TPI). Middle panel: glyceraldehyde-3-P dehydrogenase (GABP), phosphoglycerokinase (PGK), phosphoglyceromutase (PGLYM), enolase (ENOL), pyruvate kinase (PK), lactic dehydrogenase (LDH). Bottom panel: phosphoglucomutase (PGM), glucose 6-P dehydrogenase (G6P), 6-P-gluconic dehydrogenase (6PG), glutathione reductase (Glut. Red.), cy-glycerolP dehydrogenase ((YGP).
other related enzymes measured, increased in activity. The increase in glycolytic enzyme activity might conceivably be related to the increase in protein synthesis required for the production of 1.7-fold more platelets and 2.3-fold more megathrombocytes after the stimulus to platelet production of blood loss plus Fe replacement. Platelets contain 52% protein per gram dry weight (9). It is conceivable that the specific increase in enzyme activity of key enzymes (as well as others) of the EM pathway is required for the enhanced platelet production and concomitant protein synthesis. Alternatively,
BLOOD
LOSS: PLATELET
enhanced glycolytic enzyme activity might reflect increased synthesis of enzymes as part of a generalized increase in protein synthesis required for increased platelet production. The relative specificity of the increase in EM enzyme activity makes this suggestion less likely. Other considerations would include a prolongation of enzyme half-life and/or activity by the removal of specific inhibitors. The increase in glycolytic enzyme activity might also be explained, at least in part, by the production of a platelet population enriched with megathrombocytes. These large-heavy platelets have been isolated under basal conditions by use of a density gradient with human platelets, and have been shown to have a greater rate of glycolysis (1) and a 2-fold greater content of key enzymes (as well as others) of the EM pathway (12). It is of interest that the three EM pathway enzymes of the megathrombocyte-enriched rabbit platelet population which did not increase in activity after blood loss plus Fe replacement, similarly were not increased in megathrombocyte-enriched human platelets obtained under basal conditions after isolation by density gradient (12). Similarly, 6 of the 11 EM pathway enzymes which were increased in basal human megathrombocytes, were also increased in the rabbit “stress-induced” platelet population. It is, therefore, conceivable that the increase in rabbit glycolytic enzyme activity reflects the early release from the bone marrow of a young megathrombocyte-enriched platelet population know to contain greater EM pathway enzyme activity. This does not rule out the possibility that other as yet unidentifiable platelets with enhanced glycolytic enzyme activity might also be re-
GLYCOLYTIC
ENZYMES
755
leased from the bone marrow megakaryocytes at greater than basal rates after the stimulus of blood loss plus Fe replacement. Regardless of the mechanism(s) involved, it is clear that blood loss plus Fe replacement leads to an increase in specific glycolytic enzyme activity. This is accompanied by an increase in platelet and megathrombocyte production with its concomitant increase in protein synthesis. REFERENCES 1. KARPATKIN, S. (1969) J. Clin. Invest. 48, 1073-1082. 2. AMOROSI, E. L., GARG, S. K., AND KARPATKIN, S. (1971) Brit. J. Haematol. 21, 227-232. 3. GARG, S. K., WEINER, M., AND KARPATKIN, S. (1973) J. Ckn. Inuest. 52, 31a. 4. GARG, S. K., WEINER, M., AND KARPATKIN, S. (1972/73) Haemostasis 1, 121-135. 5. KARPATKIN, S., AND GARG, S. K. (1974) Brit. J. i?aematol. 26.307-311. 6. FREEDMAN, M. L., AND KARPATKIN, S. (1973) Biochem. Biophys. Res. Common. 54,475-481. 7. KARPATKIN, S. (1967) J. Clin. Inuest. 46,409-417. 8. KARPATKIN, S., AND LANCER, R. M. (1968) J. Ctin. Invest. 47, 2158-2168. 9. KARPATKIN, S. (1972) in Hematology (Williams, W. J., Beutler, E., Erslev, A. J., and Rundles, New R. W., eds.), pp. 999-1013, McGraw-Hill, York. 10. DETWILER, T. C., AND ZIVKOVIC, R. V. (1970) Biochim. Biophys. Acta 197, 117-126. 11. LYMAN, B., ROSENBERG, L., AND KARPATKIN, S. (1971) J. Ckn. Inoest. 50, 185441863. 12. KARPATKIN, S., AND STRICK, N. (1972) J. Clin. Inuest. 51, 1235-1243. 13. LOWRY, D. H., AND PASSONNEAU,J. V. (1964) J. Biol. Chem. 239, 31-42. 14. KARPATKIN, S., GARG, S. K., AND SISKIND, G. W. (1971) Amer. J. Med. 51, l-4. 15. GARG, S. K., AMOROSI, E. L., AND KARPATKIN, S. (1971) N. Engl. J. Med. 284, 11-17.
OF BIOCHEMISTRY
AND
BIOPHYSICS
164, 752-755 (1974)
Heterogeneity Effect of Chronic
Blood
of Rabbit
Loss on Glycolytic
Platelets
and Related
Enzyme
Activity’-
’
S. KARPATKIN Department
of Medicine,
New York University
Medical
Center, New York, New York 10016
Received May 17, 1974 Rabbits were chronically bled, with Fe replacement, every 3-4 days for 21 days. Their platelet count and megathrombocyte number (large-heavy, young platelets) increased 1.7and 2.3-fold, respectively. Eight of the 11 enzymes of the Embden-Meyerhof pathway increased 2-5-fold in activity per g wet weight during the period of blood letting. The five related enzymes (phosphoglucomutase, glucose 6-P dehydrogenase, 6-P gluconic dehydrogenase, glutathione reductase, and o-glycerol-P dehydrogenase) as well as the three Embden-Meyerhof pathway enzymes (aldolase, enolase, and pyruvate kinase) did not increase in activity over basal values. It is concluded that chronic blood loss with Fe replacement leads to a specific increase in enzyme activity of 8 of the 11 Embden-Meyerhof pathway enzymes.
Iron is required for maximum platelet production during the “platelet-production stimulus” of acute or chronic blood loss via the production of megathrombocytes (large-heavy, young platelets (1, 2)). Whereas blood loss without Fe replacement led to a rise in platelet count and megathrombocyte number of 1.4- and 1.7fold, respectivley, blood loss with iron replacement led to a rise in platelet count and megathrombocyte number of 2.5 and 3.8-fold, respectively, in guinea pigs (3-5). It was postulated that iron was necessary for the synthesis or production of an integral part of the platelet (4, 5). Recently published studies have revealed that iron is required for platelet protein synthesis (6). The chelation of iron with 2,2’-bipyridine reduced platelet protein synthesis by 80%, whereas transferrin or hemin could both reverse this inhibition as well as independently stimulate protein synthesis by 48-170%, respectively (6). ‘Supported by Grant HL‘13336 of the National Heart and Lung Institute and Contract DADA C-8163 of the U. S. Army Research and Development Command. 2This is paper III in a series. Paper II appeared in J. Lab. Clin. Med. 83, 896-901 (1974).
Since the platelet is an aerobic glycolytic tissue, which derives approximately half of its energy from glycolysis (7-lo), it was postulated that key glycolytic enzymes might be increased in activity during the accelerated platelet and megathrombocyte production induced by blood loss plus iron replacement. All 11 enzymes of the Embden-Meyerhof (EM) pathway, as well as five related enzymes, were measured at several time points during blood letting. The data reveal a 2%&fold increase in enzyme activity per g wet weight of 8 of the 11 enzymes of the EM pathway. This rise was fairly specific in that no change in enzyme activity was noted in eight other enzymes measured. MATERIALS
AND
METHODS
White New Zealand rabbits, weighing approximately 4 kg, were bled via ear artery for basal values and then chronically bled every 3-4 days by removal of approximately 15% of their whole blood volume (30-40 ml). Fe loss was calculated and immediately replaced by the intramuscular injection of Imferon. Rabbit platelets were obtained and washed in a modified Ringer solution as described for human platelets (11). Red blood cell and white blood cell contamination was negligible, as described previously (8), and could not have interfered with platelet cell
BLOOD LOSS: PLATELET sap enzyme activity. Platelet volume was determined by centrifugation at 2OOOgin 12.5 cm x 1 mm glass capillary tubes. One milliliter packed platelets was equivalent to 1 g wet weight. All operations were performed at 0-4°C and enzyme extraction performed by ultrasonic disruption as described previously (12). Briefly, the platelet pellet was suspended in 3 vol per platelet volume of Ringer solution containing 5 mM EDTA, 5 mM mercaptoethanol, pH adjusted to 7.5. This suspension was sonicated at maximal intensity at 4°C with a Bronwill Biosonik microprobe for two loset intervals. The disrupted cell suspension was then centrifuged at 105,OOOgfor 60 min in a Beckmrn L2 ultracentrifuge to obtain cell sap. Enzymatic assays employed classical techniques for coupling of enzymatic reactions to pyridine nucleotide oxidation-reduction changes at 340 nm and 30°C as described in detail previously (12). These were minor modifications of the methods of Lowry and Passonneau (13). A Gilford power supply and photo-cell, Beckman spectral system, and Honeywell recorder were employed to measure initial enzyme rates. These were linear with time and “extract” concentration (105,OOOg supematant) and were zero in the absence of substrate(s). Optimal dilutions for concentration-dependent linearity were made with 50 mM Tris buffer, 5 mM EDTA, 5 mM mercaptoethanol, pH 7.5, as described previously (except for phosphoglyceromutase, pyruvate kinase, and phosphoglucomutase which were diluted 1:20, 1:5, l:lOO, and 1:5, respectively). Enzymatic rates were assayed in microcuvettes of 0.5-ml volume in 50 mM Tris buffer, 5 mM mercaptoethanol, pH 7.5, at 30°C. All rates were assayed in triplicate, employing lo-, 20-, and 30-~1portions of tissue extract. Data are expressed as pmoles substrate converted/g wet weight/hr at 30°C. Distilled deionized water was used at all times. All chemicals were reagent grade. Enzyme reagents were
P=cOS
COOI COO1
NS
~001
GLYCOLYTIC
ENZYMES
753
obtained from either Sigma Chemical Co., Boehringer Mannheim Corp., or Worthington Biochemical Corp. Platelet counts were performed manually under phase contrast optics as described previously (14). Megathrombocyte number was measured with a Coulter Counter Model B and 70-pm aperture tube (15), at window threshold settings representing approximately 10% of the platelet volume distribution (14-25 lrm3). RESULTS
Basal values and comparison with human platelets. A plot of rabbit platelet cell sap
EM pathway enzyme activity at pH 7.5 is depicted in Fig. 1. This is similar to that reported for human platelets (12) with peaks of enzyme activity for phosphohexoisomerase and triose phosphate isomerase. The apparent rate-limiting enzymes (those with lowest cell sap activity) were also similar to those reported for human platelets in that they included hexokinase, phosphofructokinase, glyceraldehyde-3-P dehydrogenase, and phosphoglyceromutase. Of interest are further comparisons of total cell sap activity, The first five enzymes of the EM pathway are approximately the same for both human (12) and rabbit platelets (hexokinase to triosephosphate isomerase). The last six enzymes were considerably less for rabbit platelets. The non-EM pathway enzymes (phosphoglucomutase, glucose 6-P dehydrogenase, 6-P gluconic dehydrogenase, and glutathione reductase) were also considerably less
(001
COOI tOOI
NS
NS
(05
FIG. 1. Platelet cell sap enzyme activity fof the Embden-Meyerhof pathway at pH 7.5. Data for basal rabbit platelet enzyme activity, nine experiments (O----O), are compared to maximal increase in enzyme activity, four experiments (vertical arrows) after chronic blood loss. The interrupted line represents a ratio of 1 for the increase in enzyme activity over basal activity. The SD range for each determination was +15-25%. P values are given for the significance of the increase in enzyme activity over basal values (vertical arrows) after chronic blood loss, and were determined by Student’s t test. Abbreviations, reading from left to right, refer to the consecutive enzymes of the Embden-Meyerhof pathway.
754
S. KARPATKIN
for rabbit platelets and averaged (eight experiments) 129, 124, 110, and 85 ~moleslglhr, respectively. Alpha glycerophosphate dehydrogenase activity was approximately the same for both platelet systems. Effect of chronic blood loss. The basal platelet count and megathrombocyte number for 10 rabbits averaged 479,000/mms and 53,000/mms, respectively. With chronic blood loss plus iron replacement, the platelet count rose to 1.3-, 1.6-, 1.7-, and 1.&fold basal levels on days 4, 7, 11, and 14 respectively. The megathrombocyte number rose at a greater rate to 1.7-, 1.5, 2.1-, and 2.3-fold basal levels, respectively. The effect of chronic blood loss on glycolytic and related enzyme activity of a typical experiment from four different experiments is shown in Fig. 2. All the enzymes of the EM pathway, except aldolase, enolase, and pyruvate kinase, increased significantly over basal values. The five non-EM pathway enzymes (phosphoglucomutase, glucose 6-P dehydrogenase, 6-P gluconic dehydrogenase, glutathione reductase, and cw-glycerol-P dehydrogenase) did not increase significantly over basal values. Increase in enzyme activity generally began between the second and third blood letting, peaked on days 7 through 20 (third through fifth bloodletting), and then declined during the third week, after the fifth bloodletting. The peak average of four such experiments is shown in Fig. 1, in which the vertical arrows reflect the increase over basal values. The enzymes with the greatest increase in activity over basal values were phosphoglyceromutase, 4% fold; phosphoglycerokinase, 3.4-fold; phosphofructokinase, 3.0-fold; glyceraldehyde3-P dehydrogenase, 2.9-fold; phosphohexoisomerase, 2.8-fold; and triose-P-isomerase, 2.0-fold. DISCUSSION
The data clearly indicate that chronic blood loss with Fe replacement leads to a platelet population with enhanced glycolytic enzyme activity for 8 of the 11 enzymes of the EM pathway. This response was fairly specific in that none of the five
OSI.,,,.,,,.,.,.,.,.,.,., 0 2 4
‘am 6
8
IO
12
14
16
IS
20
22
Days
FIG. 2. Effect of chronic blood loss on EmbdenMeyerhof pathway enzyme activity and related enzyme activity, over a 21-day period of blood letting with Fe replacement (see Methods for details). Abbreviations, top panel: hexokinase (HK), phosphohexoisomerase (PHI), phosphofructokinase (PFK), aldolase (ALD), triose-P isomerase (TPI). Middle panel: glyceraldehyde-3-P dehydrogenase (GABP), phosphoglycerokinase (PGK), phosphoglyceromutase (PGLYM), enolase (ENOL), pyruvate kinase (PK), lactic dehydrogenase (LDH). Bottom panel: phosphoglucomutase (PGM), glucose 6-P dehydrogenase (G6P), 6-P-gluconic dehydrogenase (6PG), glutathione reductase (Glut. Red.), cy-glycerolP dehydrogenase ((YGP).
other related enzymes measured, increased in activity. The increase in glycolytic enzyme activity might conceivably be related to the increase in protein synthesis required for the production of 1.7-fold more platelets and 2.3-fold more megathrombocytes after the stimulus to platelet production of blood loss plus Fe replacement. Platelets contain 52% protein per gram dry weight (9). It is conceivable that the specific increase in enzyme activity of key enzymes (as well as others) of the EM pathway is required for the enhanced platelet production and concomitant protein synthesis. Alternatively,
BLOOD
LOSS: PLATELET
enhanced glycolytic enzyme activity might reflect increased synthesis of enzymes as part of a generalized increase in protein synthesis required for increased platelet production. The relative specificity of the increase in EM enzyme activity makes this suggestion less likely. Other considerations would include a prolongation of enzyme half-life and/or activity by the removal of specific inhibitors. The increase in glycolytic enzyme activity might also be explained, at least in part, by the production of a platelet population enriched with megathrombocytes. These large-heavy platelets have been isolated under basal conditions by use of a density gradient with human platelets, and have been shown to have a greater rate of glycolysis (1) and a 2-fold greater content of key enzymes (as well as others) of the EM pathway (12). It is of interest that the three EM pathway enzymes of the megathrombocyte-enriched rabbit platelet population which did not increase in activity after blood loss plus Fe replacement, similarly were not increased in megathrombocyte-enriched human platelets obtained under basal conditions after isolation by density gradient (12). Similarly, 6 of the 11 EM pathway enzymes which were increased in basal human megathrombocytes, were also increased in the rabbit “stress-induced” platelet population. It is, therefore, conceivable that the increase in rabbit glycolytic enzyme activity reflects the early release from the bone marrow of a young megathrombocyte-enriched platelet population know to contain greater EM pathway enzyme activity. This does not rule out the possibility that other as yet unidentifiable platelets with enhanced glycolytic enzyme activity might also be re-
GLYCOLYTIC
ENZYMES
755
leased from the bone marrow megakaryocytes at greater than basal rates after the stimulus of blood loss plus Fe replacement. Regardless of the mechanism(s) involved, it is clear that blood loss plus Fe replacement leads to an increase in specific glycolytic enzyme activity. This is accompanied by an increase in platelet and megathrombocyte production with its concomitant increase in protein synthesis. REFERENCES 1. KARPATKIN, S. (1969) J. Clin. Invest. 48, 1073-1082. 2. AMOROSI, E. L., GARG, S. K., AND KARPATKIN, S. (1971) Brit. J. Haematol. 21, 227-232. 3. GARG, S. K., WEINER, M., AND KARPATKIN, S. (1973) J. Ckn. Inuest. 52, 31a. 4. GARG, S. K., WEINER, M., AND KARPATKIN, S. (1972/73) Haemostasis 1, 121-135. 5. KARPATKIN, S., AND GARG, S. K. (1974) Brit. J. i?aematol. 26.307-311. 6. FREEDMAN, M. L., AND KARPATKIN, S. (1973) Biochem. Biophys. Res. Common. 54,475-481. 7. KARPATKIN, S. (1967) J. Clin. Inuest. 46,409-417. 8. KARPATKIN, S., AND LANCER, R. M. (1968) J. Ctin. Invest. 47, 2158-2168. 9. KARPATKIN, S. (1972) in Hematology (Williams, W. J., Beutler, E., Erslev, A. J., and Rundles, New R. W., eds.), pp. 999-1013, McGraw-Hill, York. 10. DETWILER, T. C., AND ZIVKOVIC, R. V. (1970) Biochim. Biophys. Acta 197, 117-126. 11. LYMAN, B., ROSENBERG, L., AND KARPATKIN, S. (1971) J. Ckn. Inoest. 50, 185441863. 12. KARPATKIN, S., AND STRICK, N. (1972) J. Clin. Inuest. 51, 1235-1243. 13. LOWRY, D. H., AND PASSONNEAU,J. V. (1964) J. Biol. Chem. 239, 31-42. 14. KARPATKIN, S., GARG, S. K., AND SISKIND, G. W. (1971) Amer. J. Med. 51, l-4. 15. GARG, S. K., AMOROSI, E. L., AND KARPATKIN, S. (1971) N. Engl. J. Med. 284, 11-17.