THROMBOSIS RESEARCH Printed in the United States
vol. 8, pp. 719-724, 1976 Pergamon Press, Inc.
PLATELET MEMBRANE GLYCOPROTEINS: ALTERATION DURING STORAGE OF HUMAN PLATELET CONCENTRATES James N. George Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78284
(Received 18.2.1976; in reVised form 30.3.1976. Accepted by Editor M.J. Larrieu)
Human blood platelets are routinely stored in hospital blood banks for transfusion to prevent thrombocytopenic hemorrhage. We have investigated the effect of storage of platelet concentrates at 22' on the glycoproteins of the plasma membrane. These proteins are exposed on the surface192 and are thought to be involved in platelet contact interactions.3-5 METHODS Platelet concentrates which had been stored in citratephosphate-dextrose at 22' with continual agitation for '72hours were provided by Wilford Hall Air Force Hospital, San Antonio. Fresh platelets were obtained from normal donors and used immediately. Platelets were washed, disrupted by sonication and plasma membranes were isolated from the top of 30$ sucrose after centrifugation at 130,000 x g for two hours. The isolated membranes were washed once, resuspended in buffer, solubilized by adding an equal volume of 66 SDS and an aliquot was assayed for protein.' Samples were reduced with 40 II% dithiothreitol at 100' for 10 minutes just before electrophoresis and 100 ug of protein were applied to SDS-polyacrylamide gels. Gels were stained for carbohydrate using periodic acidSchiff (PAS) reagent' and scanned at 550 nm. Detailed methods have been described.2
PLATELET MEMBRANE GLYCOPROTEIN
The quantity of membrane glycoprotein present was determined using fetuin (Sigma Chemical Co.) as an internal standard.8 After electrophoresis, fetuin in the range 2-20 ug gave a linear relationship between the amount of protein and the PAS band density as determined by the area under the peak (weight of paper). Since the molecular weight of fetuin (50,500 daltons) is less than that of the platelet membrane glycoproteins no interference occurs, thus 10 ug of fetuin were applied to each gel with the membrane sample. The addition of the internal control was critical since the area under the fetuin peaks varied between 1460-3160 ug (weight of paper) on different days. The variation among samples on the same day was small and it was assumed that the degree of PAS staining of the fetuin accurately reflected the staining of the platelet glycoproteins on the same gel. RESULTS AND DISCUSSION The results are presented in Table 1. ReproducibEe values were obtained for membrane glycoproteins of normal platelets. Since only carbohydrate was stained and the fraction of carbohydrate is probably different in each membrane glycoprotein, no comparison of the relative,amounts of the various glycoproteins in the membrane can be made. However this method is valid for comparison of the same glycoprotein among different samples. Glycoprotein I (150,000 daltons) was markedly decreased in the stored platelets with almost no overlap with&he values for fresh platelets. Glycoprotein III (92,000 daltons) was also significantly decreased but with many overlapping values between stored and fresh platelets. In contrast the values for glycoprotein II (118,000 daltons) were the same in both fresh and stored platelets. When platelet membrane samples are fully reduced, as in these experiments, only the lower molecular weight form of glycoprotein II (termed glycoprotein IIb, 118,000 daltons2) is prominent. Analysis of unreduced samples will be required to quantitatively evaluate glycoprotein IIa (125,000 daltons), which may be a separate protein or only a part of glYCOprOtein II which migrates faster after reduction.2 Although glycoproteins could be lost from stored platelets during membrane preparation as well as during blood bank storage, this would
PLATELET MEMBRANE GLYCOPROTEIN
TABLE I Quantitative Estimates of Membrane Glycoproteins in Fresh and Stored Platelets Fresh
6.9 2 0.5 (4.5-8.8)
3.4 2 0.3 (2.1~5.J)
3.4 2 0.4 (2.0-6.1)
3.2 + 0.3 (1.7-5.3)
5.2 f 0.6 (1.9-8.0)
3.3 _t 0.b. (1.7-6.3)
Values are given in arbitrary units, equivalent to fetuin weight, per 100 ug of membrane protein. 100 ug of membrane protein, plus 10 ug of fetuin were applied to each gel (4% acrylamide, 5 x 130 mm), electrophoresed, stained for carbohydrate and scanned. The areas under the peaks were cut out and weighed. The platelet glycoproteins were then assigned values relative to the weight of paper of the fetuin peak. The data are the mean i standard error (with the range of values in parentheses) for 10 samples of fresh platelets from 10 different donors and samples from 12 different bags of stored platelets. p-values were calculated by the Student t test.
also indicate an abnormal instability of the membrane proteins. Quantitative analysis of membrane proteins either by staining or by immunological techniques' will be necessary to determine if intact glycoprotein molecules, or only the carbohydrate moieties as seen with the PAS reaction, are being lost from the membrane. In these initial studies we could not determine if the protein stained by Coomassie blue was altered in stored platelets. The scanning baseline of the Coomassie-stained gels was more irregular making analysis of the peak area difficult, the protein band associated with glycoprotein I is very small192 , and the protein band of approximately 95,000 daltons did not always coincide with the PAS reaction of glycoprotein III and may
PLATELET MEMBRANE GLYCOPROTEIN
represent a different protein. No change in glycoprotein molecular weight or new PAS bands were observed in the membrane samples from stored platelets. In view of two earlier observations, the unequal loss of membrane glycoproteins was unexpected. In red cells, incubation is accompanied by fragmentation of unaltered plasma membrane." Also we have postulated that platelets lose pieces of membrane by fragmentation during in vivo aging, based on the equal loss of diazotized (125I)-diiodosulfanilic acid from the three labeled glycoproteins during circulation in the rabbit.1' The lack of any quantitative change in glycoprotein II is strong evidence against loss of unaltered pieces of membrane during storage, although if the glycoproteins are not distributed uniformly within the membrane then possibly pieces of membrane could fragment from the platelet without loss of glycoprotein II. The preferential loss of glycoprotein I is consistant with its exposed position on the membrane, which has been suggested by its greater susceptibility to trypsin hydrolysis than either glycoproteins II or 1II.3 However the loss of glycoprotein III is in contrast to its greater resistance to trypsin hydrolysis than either glycoproteins I or II.293 The functional effect of a decreased concentration of intact glycoprotein I in the platelet membrane may be significant since this protein has been found to be decreased in platelets from patients with Bernard-Soulier syndrome and it therefore may be involved in platelet adhesion to subendothelium.395 Three-day-old stored platelets are a commonly available source of large quantities of platelets for biochemical studies, such as isolation of membrane glycoproteins.12 Our demonstration of changes of the membrane glycoproteins indicates that studies of stored platelets may not accurately reflect the membrane condition of fresh platelets, similar to the problems encountered in the studies of platelet actomyosin due to proteolysis of myosin during blood bank storage.13 Optimal conditions for preservation of platelet function are still being actively investigated. Storage of platelet concentrates at 22' for up to 72 hours is a common clinical practice, and platelets stored in this way survive normally when reinfused into the normal donor.14 However others have demonstrated a functional abnormality in platelets stored at
22' while platelets stored at 4' retained better hemostatic function in spite of shortened circulation time.15916 The structural changes reported here may help to define the alterations in platelet function and survival which are associated with different methods of preservation. AKNOWLEDGEMENTS I thank Mrs. P. C. Lewis for technical assistance and Drs. D. A. Sears, C. S. P. Jenkins, K. J. Clemetson and E. F. Liischer for review of this manuscript. This work was supported by the American Heart Association and by an NIH Grant 1 ROl AM17137OlAl. Dr. George is currently on leave to the Theodor Kocher Institut, Universitat Bern, 3000 Bern, Switzerland. REFERENCES 1.
PHILLIPS, D.R. Effect of trypsin on the exposed polypeptides and glycoproteins in the human platelet membrane. Biochem. 11, 4582, 1972. GEORGE, J.N., POTTERF, R.D., LEWIS, P.C. and SEARS, D.A. Studies on platelet plasma membranes. I. Characterization of surface roteins of human platelets labeled with diazotiztd P125-I)-diiodosulfanilic acid. J. Lab, Clin. Med. In press. NURDEN, A.T. and CAEN, J.P. Specific roles for platelet surface glycoproteins in platelet function. Nature 255, 720, 1975. PHILLIPS, D.R., JENKINS, C.S.P., LUSCHER, E.F. and LARRXEU, M-J. Molecular differences of exposed surface proteins on thrombasthenic platelet plasma membranes. Nature 257, 599, 1975. JENKINS, C.S.P., PHILLIPS, D.R., CLl%lETSON,X.3., MEYER, D LARRIEU, M-J. and LUSCHER, E.F. Platelet membrane giicoproteins implicated in ristocetin-induced aggregation: studies of the proteins on platelets from patients with Bernard-Soulier syndrome and von Willebrand's disease. J. Clin. Invest. 57, 112, 1976. LOWRY, O.H., ROSEBROUGH, N.J. PARR, A.L. and RANDALL, R.J. Protein measurement with the #'Olin phenol reagent. J. Biol. Chem. 193, 265, 1951. FAIRBANKS, G., STECK, T.L. and WALLACH, D.F.H. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochem. 10, 2606, 1971. MATHIEU, J-M. and QUARLES, R.H. Quantitative scanning of glycoproteins on polyacrylamide gels stained with periodic acid-Schiff reagent. Anal. Biochem. 55, 313, 1973. BHAKDI S., BflG-HANSEN,T.C., KNUFFERMANN, g??%"#A%&!l D F H' Quantitative immunoelge;;;;~;oresis of proteins in'h& &ythrocyte membranes. . Biophys. Acta 406, 489, 1975.
PLATELET MEMBRANE GLYCOPROTEIN
WEED, R.I. and BOWDLER, A.J. Metabolic dependence of the critical hemolytic volume of human erythrocytes: relationship to osmotic fragility and autohemolysis in hereditary spherocytosis and normal red cells. J. Clin. Invest. 45,
1137, 1966. Studies on platelet plasma membranes. II. Characterization of surface proteins of rabbit platelets in vitro and during circulation in vivo using diazotized (125-I)-diiodosulfanilic acid as a label. J. Lab. Clin. Med. In press. 12. NACHMAN, R.L., HUBBARD, A. and FERRIS, B. Iodination of the human platelet membrane: studies of the major surface glycoprotein. J. Biol. Chem. 248, 2928, 1973. 13. ABRAMOWITZ, J.W., STRACHER, A. and DETWILER, T.C. Proteolysis of myosin during platelet storage. J. Clin. Invest.
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53, 1493, 1974. 14. MURPHY, S. and GARDNER, F.H. Platelet preservation effect of storage temperature on maintenance of platelet viability - deleterious effect of refrigerated storage. New Enp. J. Med. 280, 1094, 1969. 15. BECKER, G.A., TUCCELLI, M., KUNICKI, T., CHALOS, M. and ASTER, R.H. Studies of platelet concentrates stored at 22 C and 4 C. Transfusion 13, 61, 1973. 16. VALERI, C.R. Hemostatic effectiveness of liquid-preserved and previously frozen human platelets. New Eng. J. Med. 290, 353, 1974.