Gray Platelet Syndrome Demonstration of Alpha Granule Membranes That Can Fuse with the Cell Surface Jean-Philippe Rosa,* James N. George,* Dorothy F. Bainton,t Alan T. Nurden,I Jacques P. Caen,I and Rodger P. McEver* *Departments ofMedicine and Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas 78284-7880; $Department ofPathology, University of California School ofMedicine, San Francisco, California 94143; §Unite 150 INSERM, H6pital Lariboisiere, Paris, France Abstract have shown that some of the vesicles contain small quantities of the alpha-granule proteins fibrinogen and von Willebrand Platelets from patients with the gray platelet syndrome have factor (9). Immunocytochemical analysis has also documented decreased recognizable alpha granules and are markedly defi- the presence of fibrinogen in the surface-connected canalicular cient in some alpha-granule secretory proteins. Using immuno- system of gray platelets and von Willebrand factor in the Golgi cytochemical techniques with antibodies to an alpha-granule region and some small vesicles of megakaryocytes from pa- membrane protein, GMP-140, we identified the membranes of tients with the gray platelet syndrome (9). Although these ob- intracellular vesicles in gray platelets as alpha-granule mem- servations did not provide direct evidence for alpha-granule branes. Gray platelets contained normal amounts of GMP-140 membranes in gray platelets, they led to the hypothesis that the as measured by electroimmunoassay. The activation of gray alpha-granule abnormality arises from a defect in the mecha- platelets with thrombin caused GMP-140 to be redistributed nisms for transferring proteins into developing alpha granules, to the plasma membrane surface, as in normal platelets. In rather than defective synthesis of alpha-granule membranes (2, agreement with previous studies, an endogenously synthesized 8, 9). It was proposed that instead of being transferred to alpha secretory protein, platelet factor 4, was undetectable in gray granules, these proteins were constitutively secreted from platelets. However, the alpha-granule proteins albumin and megakaryocytes. IgG, which are thought to be derived from endocytosis of It has been thought that most, if not all, alpha-granule plasma proteins into megakaryocytes, were present in sub- secretory proteins are synthesized by megakaryocytes, and stantial quantities and were secreted efficiently from gray there is direct evidence that some of the proteins are synthe- platelets. Therefore, the fundamental defect in the gray plate- sized endogenously (10-14). However, we have recently let syndrome may be in the targeting of endogenously synthe- shown that a plasma protein can be endocytosed by megakar- sized secretory proteins to developing alpha granules in mega- yocytes and incorporated into alpha granules (15). Major karyocytes. plasma proteins such as albumin and IgG may be routed into platelet alpha granules by this endocytotic pathway (16, 17), Introduction since the concentrations of these proteins in the platelet are directly related to their concentrations in plasma (18). The The gray platelet syndrome is a rare congenital bleeding dis- concentration and disposition of presumably exogenously ac- order first described by Raccuglia (1). Gray platelets are mark- quired proteins in gray platelets are less well understood than edly deficient in morphologically recognizable alpha granules the concentration and disposition of the endogenously synthe- (2) and in the alpha-granule secretory proteins platelet factor 4, sized alpha-granule proteins. The platelet albumin concentra- beta-thromboglobulin, fibrinogen, von Willebrand factor, tion in one patient with the gray platelet syndrome was re- platelet-derived growth factor, fibronectin, and thrombospon- ported to be approximately half the normal value (4), but din (3-7). However, ultrastructural and biochemical studies albumin secretion was not studied in this patient. have indicated that lysosomes, peroxisomes, dense bodies, and We have previously described a monoclonal antibody, des- their contents are normal in gray platelets (2, 3, 5, 8). ignated S12, which reacts with a platelet membrane protein of The cause of the defect accounting for the alpha-granule 140,000 relative molecular mass (Mr) that is localized to the abnormality in gray platelets is unknown. Many gray platelets membranes of alpha granules in unstimulated platelets, but is are highly vacuolated (2, 5), and immunocytochemical studies redistributed to the plasma membrane of activated platelets during secretion (19-21). The protein has been named GMP- 140,' to indicate that it is a granule membrane protein. Address reprint requests to Dr. McEver, Cardiovascular Biology Pro- Similar findings have been described by Furie and co-workers gram, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, in Oklahoma City, OK 73104. (22, 23). GMP- 140 appears to be localized to alpha granules A preliminary report of this study was presented at the meeting of megakaryocytes and is presumed to be synthesized by these the Council on Thrombosis of the American Heart Association, 19 cells (24, 25). In this study, using antibodies to GMP-140 as a November 1986, Dallas, TX, and published in abstract form. (1986. marker, we show that many vesicles in gray platelets contain Circulation. 74:11-423.) alpha-granule membrane proteins and that these vesicles fuse Received for publication 7 February 1987 and in revised form 27 with the plasma membrane after the cells have been activated April 1987. with thrombin, resulting in the normal distribution of GMP- 140 over the platelet surface. We also show by enzyme- J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/87/10/1138/09 $2.00 1. Abbreviation used in this paper: GMP-140, alpha-granule mem- Volume 80, October 1987, 1138-1146 brane protein. 1138 Rosa, George, Bainton, Nurden, Caen, and McEver linked immunoassay (ELISA) and electroimmunoassay that antibodies to human platelet proteins (28, 33). Plates were then gray platelets contain substantial quantities of IgG and albu- washed, stained with Coomassie Blue, dried, and, when indicated, min and that these proteins are secreted appropriately from subjected to autoradiography. Crossed immunoelectrophoresis was activated cells. These findings suggest that the alpha-granule performed as described previously ( 19). abnormalities in the gray platelet syndrome may be due to Immunocytochemistry. Immediately fixed, washed, or thrombin- stimulated platelets were fixed in 8% paraformaldehyde in 0.1 M abnormal targeting of endogenously synthesized secretory PIPES buffer (pH 7.2) and were washed in the same buffer containing proteins to developing alpha granules in megakaryocytes. 10% (wt/vol) sucrose (20, 34). They were infiltrated for 30 min with 2.3 M sucrose, embedded in the sucrose solution, frozen, and stored in Methods liquid nitrogen. The frozen thin-section techniques described by To- kuyasu (35) were used, with the modifications described by Griffiths et Patients. Platelets from two unrelated patients with gray platelet syn- al. (36). When sections were incubated with polyclonal antibodies, the drome were studied. H.B. has been the subject of several reports (4, 5, probe was 5 nm colloidal gold conjugated with protein A-5; for sec- 8, 9). A preliminary report on S.S. has been published (26). Blood tions incubated with monoclonal antibody, the probe was 5 nm colloi- samples were taken from the patients and from normal volunteers with dal gold conjugated with goat anti-mouse IgG (Janssen Pharmaceuti- informed consent. Platelets from H.B. were used in immunocyto- cals, Beerse, Belgium). Control measures for all procedures included chemical studies and for analysis by crossed immunoelectrophoresis. the substitution of buffer, preimmune rabbit serum, or purified mouse All other studies were performed on platelets from S.S. IgG for specific primary antibody. Antibodies. Two monoclonal antibodies were used: S12, which ELISA. Platelets were prepared and assayed for IgG and albumin recognizes the platelet alpha-granule membrane protein GMP- 140 (19, by an antibody-capture ELISA system as described previously (16). 20); and Tab, which recognizes platelet membrane glycoprotein IIb For the albumin assay, affinity-purified goat anti-human albumin was (27, 28). Rabbit polyclonal antibodies to platelet factor 4 (29) were a used to coat the wells, and the same antibody was biotinylated for use gift from Dr. Shirley P. Levine (University of Texas Health Science as the detection reagent. Center at San Antonio). Rabbit polyclonal antibodies to GMP-140, Protein determination. Proteins were determined by the method of glycoprotein lIb, and glycoprotein Illa were prepared as described Markwell (37). previously (20, 30). A monoclonal antibody to human albumin was obtained from Accurate Chemical and Scientific Corp., Westbury, NY. Goat affinity-purified polyclonal antibodies to human albumin Results and IgG were obtained from Cappel Laboratories, Malvern, PA. Bio- tinylated antibodies and avidin D-biotinylated horseradish peroxidase were obtained from Vector Laboratories, Burlingame, CA. To determine the content and subcellular location of Platelet isolation. In one instance, blood from patient H.B. was GMP- 140 in gray platelets, we performed direct platelet-bind- drawn immediately into fixative and the platelets were isolated as ing studies with 125I-S12 IgG. As shown in Fig. 1 A, very few