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Biochemical Studies of Two Patients with the Gray Platelet Syndrome

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Biochemical Studies of Two Patients with the Gray Platelet Syndrome Biochemical studies of two patients with the gray platelet syndrome. Selective deficiency of platelet alpha granules. J M Gerrard, … , L A Harker, J G White J Clin Invest. 1980;66(1):102-109. https://doi.org/10.1172/JCI109823. Research Article The biochemistry of platelets from two unrelated patients with the gray platelet syndrome, a deficiency of platelet alpha- granules, has been evaluated. Ultrastructural studies of their platelets revealed the number of alpha-granules to be less than 15% of normal, whereas the number of dense bodies was within normal limits. Platelets from both patients had severe deficiencies of platelet factor 4 and beta-thromboglobulin (less than 10% of normal). Sodium dodecyl sulfate- polyacrylamide gel electrophoresis showed a marked deficiency of thrombin-sensitive protein in both patients. Analysis of the platelet-derived growth factor in one patient showed it was also markedly reduced. Levels of lysosomal enzymes, adenine nucleotides, serotonin, and catalase, and conversion of arachidonic acid by the lipoxygenase and cyclo- oxygenase enzymes, were within normal limits. The results provide important evidence to define the contents of alpha- granules and to differentiate these contents from the contents of lysosomal granules, dense bodies, and peroxisomes. Functional studies of these platelets showed deficiencies in ADP, thrombin, and collagen aggregation. The results suggest that alpha-granules or their contents make a contribution to normal platelet aggregation. Find the latest version: https://jci.me/109823/pdf Biochemical Studies of Two Patients with the Gray Platelet Syndrome SELECTIVE DEFICIENCY OF PLATELET ALPHA GRANULES JONATHAN M. GERRARD, DAVID R. PHILLIPS, GUNDU H. R. RAO, EDWARD F. PLOW, DANIEL A. WALZ, RUSSELL ROSS, LAURENCE A. HARKER, and JAMES G. WHITE, Departments of Pediatrics, Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455; Department of Biochemistry, St. Jude Children's Hospital, Memphis, Tennessee 38101; Department of Molecular Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92137; Department of Physiology, Wayne State University, Detroit, Michigan 48201; Department of Pathology and Medicine, University of Washington, Seattle, Washington 98195 A B S T R A C T The biochemistry of platelets from two INTRODUCTION unrelated patients with the gray platelet syndrome, a deficiency of platelet a-granules, has been evaluated. Platelets are circulating blood cells primarily involved Ultrastructural studies of their platelets revealed the in hemostatic and thrombotic functions (1, 2). Dis- number of a-granules to be <15% of normal, whereas orders of platelet function may predispose to bleeding the number of dense bodies was within normal limits. or to thrombotic and atherosclerotic disorders (3, 4). Platelets from both patients had severe deficiencies of Understanding of the biology of this cell has been platelet factor 4 and f-thromboglobulin (<10% of facilitated by careful study of congenital abnormalities normal). Sodium dodecyl sulfate-polyacrylamide gel of platelets as in thrombasthenia, a deficiency of plate- electrophoresis showed a marked deficiency of throm- let surface glycoproteins (5-7), and the Hermansky- bin-sensitive protein in both patients. Analysis of the Pudlak syndrome or storage pool deficiency in which a platelet-derived growth factor in one patient showed it selective absence of platelet-dense granules occurs was also markedly reduced. Levels of lysosomal en- (8-11). These natural experiments provide important zymes, adenine nucleotides, serotonin, and catalase, clues to the role of the missing structures in cellular and conversion of arachidonic acid by the lipoxy- physiology. The gray platelet syndrome, first described genase and cyclo-oxygenase enzymes, were within by Raccuglia in 1971 (12), is a disorder characterized normal limits. The results provide important evidence by large platelets that contain few granules and, there- to define the contents of a-granules, and to differen- fore, appear gray when viewed under the light micro- tiate these contents from the contents of lysosomal scope, the characteristic that gives the disorder its name. Electron microscopic studies of this disorder granules, dense bodies, and peroxisomes. Functional and studies of these platelets showed deficiencies in ADP, have revealed a relative deficiency of a-granules, thrombin, and collagen aggregation. The results sug- normal numbers of dense bodies and peroxisomes in gest that a-granules or their contents make a contribu- these platelets (3, 12-14). We now report an evalua- to normal platelet aggregation. tion of the biochemistry of these platelets, including tion analysis of adenine nucleotides and serotonin con- Preliminary reports of part of this work have appeared in stituents of platelet-dense bodies, hydrolytic enzymes abstract form in Clin. Res. 26: 503A, 1978, and in Blood. present in platelet lysosomes, catalase believed to be 54(Suppl. 1): 241a, 1979. in the platelet peroxisomes, and various polypeptides Dr. Gerrard and Dr. Plow are established Investigators of present in a-granules. The selective absence of a- the American Heart Association. Reprint requests should be granules and their contents in this disorder provides a addressed to Dr. Gerrard, Manitoba Institute of Cell Biology, Manitoba R3E OV9, Canada. new approach to subcellular localization of certain Received for publication 15 November 1979 and in revised platelet proteins, and provides a natural experiment to form 17 March 1980. probe the role of a-granules in platelet function. 102 J. Clin. Invest. C The American Society for Clinical Investigation, Inc. - 0021-9738/80/07/0102108 $1.00 Volume 66 July 1980 102-109 METHODS were frozen-thawed three times, the homogenate centrifuged at 16,000 rpm in a Sorvall SS-34 rotor (DuPont Instruments- Patients. Two patients with the gray platelet syndrome Sorvall Biomedical Div., DuPont Co., Newtown, Conn.) for were studied. The first was a 22 yr-old male originally 30 min, the pellet resuspended in 1.09 M NaCl, 0.01 M Tris, described by Raccuglia (12). This patient had petechiae and pH 7.4, and spun again for 30 min using the same conditions. ecchymoses as a newborn and a bruising tendency with re- The combined supernates were dialyzed against phosphate- current knee pain, presumably intraarticular bleeding, buffered saline for 24 h. The frozen-thawed supernate was throughout childhood. His platelet count during this period equivalent to 0.34 x 109 platelets/ml, and the salt extract ranged from 25,000- 150,000/mm3. It increased temporarily on equivalent to 0.70 x 109 platelets/ml. two occasions after prednisone and permanently after splenec- Platelet factor 4 (PF4)1 and f-thromboglobulin were meas- tomy at age nine, though it has been slightly below normal ured by radioimmunoassay. The procedure used in Lab B in the last few years (range 100,000- 150,000/mm3). He for both PF4 and P-thromboglobulin has been described else- has had only one severe bleeding episode (a large hematoma) where (29). The assay for PF4 used in Lab A involved its puri- since then, after a football injury, although his bleeding times fication according to the procedure of Hermondson et al. (30), have remained slightly longer than normal. The second pa- then preparation of antisera by injecting 3-kg New Zealand tient studied was a girl with Goldenhar's syndrome (oculo- white rabbits with 1 mg purified protein mixed with complete auriculovertebral dysplasia). She developed a recurrent Freund's adjuvant. Serum, collected after antibody was petechial rash associated with frequent bruises at about demonstrated by a precipitin line on agar gel electrophoresis, 8 mo of age, a time when she was frequently falling in her was stored in 0.3-ml aliquots in a -70° freezer. For the radio- efforts to learn to walk. This bruising tendency continued. immunoassay, the PF4 was labeled using lactoperoxidase, About 2 yr of age she developed a large black eye after her by combining 10 ,g of purified PF4 with 1 mCi of '25I in 40 face had been hit lightly. She was then referred for evalua- ,ul of 0.4 M Na acetate. 10 ,lA of H202 (-3 x 10-6g) was added tion and found to have large gray platelets on a Wright to the mixture for 5 min at room temperature, after which stained blood smear. This patient is now age four. She has an additional 5 ,ul of H202 was added. At 8 min total time, continued to have a bruising tendency, and on one occasion, the reaction mixture was run over a heparin agarose column after a small laceration to one toe, she bled for a "long time" and washed with phosphate-buffered saline, pH 6.4, to re- before it stopped. Her platelet count has ranged between move the unbound '25I and to stop the lactoperoxidase reac- 100,000 and 150,000/mm3. tion. When the eluate had <1,000 cpm/10 1,u, the '2I-PF4 Experimental. For most experiments, blood drawn from was eluted with 2 M NaCl and collected in 0.5-ml fractions the antecubital vein from patients or normal donors was in plastic tubes coated with 0.2% bovine serum albumin. The anticoagulated with citrate-citric acid dextrose, pH 6.5, to '25I-PF4 had about 6 x 107 counts/4g. The labeled fraction achieve final concentrations of 9.3 mM sodium citrate, 0.7 with the greatest activity was used in the assay after dilution mM citric acid, and 14 mM dextrose. For study of platelet with standard buffer to give 20,000 cpm/20 ,ul and stored at polypeptides and surface glycoproteins, 17.2 ml of blood was -70°. '25I-PF4 was stable for 3-4 mo. drawn from each normal donor and each patient and mixed For the radioimmunoassay itself, standard unlabeled PF4 with 2.8 ml of acid citrate dextrose (15). The blood was then was diluted in buffer (0.1 M Tris, 0.15 M NaCl, 0.2% Na azide, centrifuged immediately at 100 g for 20 min at room tempera- 0.5 U heparin/ml, and 2% dog plasma) to the PF4 concentra- ture to obtain platelet-rich plasma.
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