Immunoglobulin G Is a Platelet Alpha Granule-Secreted Protein
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Immunoglobulin G is a platelet alpha granule-secreted protein. J N George, … , L K Knieriem, D F Bainton J Clin Invest. 1985;76(5):2020-2025. https://doi.org/10.1172/JCI112203. Research Article It has been known for 27 yr that blood platelets contain IgG, yet its subcellular location and significance have never been clearly determined. In these studies, the location of IgG within human platelets was investigated by immunocytochemical techniques and by the response of platelet IgG to agents that cause platelet secretion. Using frozen thin-sections of platelets and an immunogold probe, IgG was located within the alpha-granules. Thrombin stimulation caused parallel secretion of platelet IgG and two known alpha-granule proteins, platelet factor 4 and beta-thromboglobulin, beginning at 0.02 U/ml and reaching 100% at 0.5 U/ml. Thrombin-induced secretion of all three proteins was inhibited by prostaglandin E1 and dibutyryl-cyclic AMP. Calcium ionophore A23187 also caused parallel secretion of all three proteins, whereas ADP caused virtually no secretion of any of the three. From these data and a review of the literature, we hypothesize that plasma IgG is taken up by megakaryocytes and delivered to the alpha-granules, where it is stored for later secretion by mature platelets. Find the latest version: https://jci.me/112203/pdf Rapid Publication Immunoglobulin G Is a Platelet Alpha Granule-secreted Protein James N. George, Sherry Saucerman, Shirley P. Levine, and Linda K. Knieriem Division ofHematology, Department ofMedicine, University of Texas Health Science Center, and Audie L. Murphy Veterans Hospital, San Antonio, Texas 78284 Dorothy F. Bainton Department ofPathology, University of California School ofMedicine, San Francisco, California 94143 Abstract The significance and subcellular location of platelet IgG re- main unclear (1 1). Various assays have shown the IgG concen- It has been known for 27 yr that blood platelets contain IgG, tration of normal platelets to be 1-10 fg/platelet (4,000-40,000 yet its subcellular location and significance have never been molecules/platelet) (9, 12). Comparisons of assays on intact and clearly determined. In these studies, the location of IgG within lysed platelets have suggested that one-third to two-thirds of human platelets was investigated by immunocytochemical tech- platelet IgG is on the cell surface (13, 14). However some assays niques and by the response of platelet IgG to agents that cause have demonstrated markedly less surface IgG, 146-235 mole- platelet secretion. Using frozen thin-sections of platelets and an cules/platelet (15-17). If platelet IgG is contained within secre- immunogold probe, IgG was located within the a-granules. tory granules, then the techniques of platelet washing and in- Thrombin stimulation caused parallel secretion of platelet IgG cubation, which can easily cause platelet secretion (18), may be and two known a-granule proteins, platelet factor 4 and ft- critical and uncontrolled variables affecting these data. thromboglobulin, beginning at 0.02 U/ml and reaching 100% at The objective of our experiments was to further characterize 0.5 U/ml. Thrombin-induced secretion of all three proteins was the IgG associated with normal platelets. We have demonstrated inhibited by prostglandin El and dibutyryl-cyclic AMP. Calcium that IgG is located within the platelet a-granules and that its ionophore A23187 also caused parallel secretion of all three secretion from platelets is parallel to that oftwo known a-granule proteins, whereas ADP caused virtually no secretion of any of proteins (19-21): platelet factor 4 (PF4)' and P-thromboglob- the three. From these data and a review of the literature, we ulin (BTG). hypothesize that plasma IgG is taken up by megakaryocytes and delivered to the a-granules, where it is stored for later secretion Methods by mature platelets. Immunocytochemical procedures. Platelets were fixed in 8% paraform- Introduction aldehyde in 0.1 M Pipes buffer, pH 7.2, by dripping blood directly from the needle into the fixative maintained at 37°C, as described by Stenberg IgG was first identified in blood platelets by Salmon (1) in 1958. et al. (20, 21). Platelets were washed in the same buffer containing 10% Davey and LUscher (2, 3) confirmed this observation in 1966 (wt/vol) sucrose. They were infiltrated for 30 min with 2.1 M sucrose, and demonstrated that IgG was secreted from platelets in re- embedded in the sucrose solution, frozen, and stored in liquid nitrogen. Sections were cut on an ultracut E (A. 0. Reichert, Los Angeles, CA). sponse to thrombin. Later, it was suggested that IgG was a surface The frozen thin-section techniques described by Tokuyasu (22) were component of platelets because it was removed from platelets used, with the modifications for the use of colloidal gold described by by trypsin treatment (4); however, this removal may have been Griffiths et al. (23). The primary antibody, which was prepared against a result of trypsin-induced platelet secretion (5). The clinical human IgG (heavy and light chains) in the rabbit, was obtained from relevance of quantitative assays for platelet-associated IgG was Miles Scientific, Inc., Naperville, IL. It was used at dilutions of 1:10-1: promoted by the demonstration of increased concentrations of 1,000. The immunogold probe, which was used in a dilution of 1:50, platelet IgG in patients with immune thrombocytopenic purpura was goat anti-rabbit IgG linked to 5-nm colloidal gold particles (GAR- (6-8). These studies assumed that IgG was an antiplatelet an- 5) (Jansson Pharmaceuticals, Beerse, Belgium). The immunological tibody and that the assays were analogous to the Coombs' an- techniques were performed as previously described (20, 21). tiglobulin test used with erythrocytes in autoimmune hemolytic Platelet preparationfor secretion studies. Blood was drawn from nor- mal volunteers who had not ingested aspirin or other agents that inhibit disease. Since that time, numerous techniques for measuring platelet function for the previous 10 d. The blood was anticoagulated platelet IgG have been developed to identify patients with dis- with 0.1 volume of 3.8% sodium citrate and 0.5 gg/ml prostaglandin orders of immunologic platelet destruction (reviewed by (PGE), (Sigma Chemical Co., St. Louis, MO). Platelet-rich plasma was McMillan [9] and by Kelton [10]). obtained by centrifugation, and the platelets were washed twice in Ty- rode's buffer (138 mM NaCl, 29 mM KC1, 12 mM NaHCO3, 0.4 mM 1% 0.35% bovine serum albumin to Dr. NaHPO4, glucose, [BSA]) (24), pH Address correspondence and reprint requests George. 50 to as "wash Platelets Receivedfor publication 1I June 1985. 6.5, containing ng/ml PGE, (referred buffer"). J. Clin. Invest. 1. Abbreviations used in this paper: BTG, f3-thromboglobulin; db-cAMP, © The American Society for Clinical Investigation, Inc. N6OZ-dibutyryl adenosine 3',5'monophosphate cyclic AMP; ELISA, en- 0021-9738/85/11/2020/06 $1.00 zyme-linked immunosorbant assay; PF4, platelet factor 4; PG, prosta- Volume 76, November 1985, 2020-2025 glandin. 2020 George, Saucerman, Levine, Knieriem, and Bainton were resuspended in Tyrode's buffer with added CaC12 (2 mM) and MgC12 except that goat serum was omitted from the wash buffers and 1% gelatin (1 mM) and without PGE1 at pH 7.4 (referred to as "resuspension buffer"). was substituted for BSA. Wells were coated with rabbit anti-human al- Platelet secretion. Human a-thrombin, 2,600 U/mg, was a gift from bumin (Cappel Laboratories). A human serum albumin standard was Dr. John W. Fenton II, Albany, NY. Calcium ionophore A23187 and obtained from Sigma Chemical Co. The assay was performed with goat ADP were obtained from Sigma Chemical Co. Purified human fibrinogen anti-human albumin (Cappel) and biotinylated rabbit anti-goat IgG was a gift from Dr. Rodger McEver, San Antonio, TX. The following (Vector Laboratories, Inc.). reaction mixtures were used: thrombin, 0.01-0.5 U/ml; ionophore Immunoblotting. Whole platelets and platelet fractions were solu- A23 187, 0.1-5.0 MM, with leupeptin (0.4 mM), an inhibitor of the en- bilized in 2% sodium dodecyl sulfate (the Lubrol-insoluble pellet was dogenous platelet calcium-dependent protease (25); and ADP, 2-50 MM, suspended by sonication before solubilization) and electrophoresed on with fibrinogen (0.5 mg/ml). To inhibit thrombin-induced platelet se- a 7-12% exponential gradient acrylamide gel, either unreduced or after cretion in some experiments, PGE1 and N6O-dibutyryl adenosine reduction with fl-mercaptoethanol (29). Following electrophoresis, pro- 3',5'monophosphate cyclic AMP (db-cAMP) (Sigma Chemical Co.) were teins were transferred onto nitrocellulose (29, 30) and IgG was detected added to the platelet suspension in final concentrations of 10 Mug/ml and with biotinylated goat anti-human IgG and the "Vectastain" immuno- I mM, respectively (26). The final suspensions, I ml containing 108 peroxidase system (Vector). platelets, were incubated with occasional shaking for 10 min at 370C. Then, 1.5 ml of Tyrode's resuspension buffer was added, the tubes were Results centrifuged at 1,500 g for 10 min at room temperature, and the super- natant fluids were frozen in and aliquots for assay of PF4, BTG, IgG. localization The use of The platelet pellet was washed once, resuspended in 1 ml of Tyrode's Morphological ofplatelet IgG. colloidal resuspension buffer by sonication, solubilized in 0.5% Lubrol PX (Sigma gold immunoconjugates on frozen thin-sections of resting plate- Chemical Co.) at 37°C for 30 min, centrifuged in an Eppendorf microfuge lets allowed the study of the intracellular distribution of IgG (12,000 g) for 5 min, and the supernatant fluid was frozen for assay of antigen. Immunogold particles were found mainly within the platelet-associated IgG. matrix of most, but not all, a-granules (Fig.