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From www.bloodjournal.org by guest on August 1, 2017. For personal use only. Platelet Dense Granule Membranes Contain Both Granulophysin and P-Selectin (GM P 140) - ByS.J. Israels, J.M. Gerrard, Y.V. Jacques, A. McNicol, B. Cham, M. Nishibori, and D.F. Bainton We recently reported the characterizationof a platelet granule membrane protein of molecular weight (mol wt) 40.000 called granulophysin (Gerrard et al: Blood 77:101, 1991). identified by a monoclonal antibody (MoAb D545) raised to purified dense granule membranes. Using immunoelectronmicroscopic techniques on frozen thin sections, this protein was localizedin restingand thrombin-stimulatedplatelets. In resting platelets, labeled with antigranulophysin antibodies and immunogold probes, label was localized to the membranes of one or two clear granules per platelet thin section. D545 also labeled dense granules in permeabilized whole platelets and isolated dense granule preparations examined by whole-mount techniques. Expression of granulophysin on the platelet surface paralleled dense granule secretion as measured by ‘%-serotonin release under conditions in which lysosomal granule release, as measured by B-glucuronidase secretion, was less than 5%. After thrombin stimulation, both the surface-connected canalicular system and the plasma membrane were labeled, demonstrating redistribution of granulophysin associated with degranulation. Double labeling experiments with D545 and antibodies to the *granule membrane protein, P-selectin, demonstrated labelingof both P-selectin and granulophysin on dense granule membranes. Distribution of both proteins on the plasma membrane after platelet stimulation was similar. The results demonstrate that granulophysin is localized to the dense granules of platelets and is redistributed to the plasma membrane after platelet activation. 8 1992 by The American Society of Hematology. P granulophysin in resting and thrombin-activated human platelets. We also compared the distribution of granulophysin with that of P-selectin (previously known as GMP140 or PADGEM), a well-characterized a-granule membrane protein10-12that mediates adhesion of activated platelets with n e ~ t r o p h i l s . ~ ~ J ~ LATELETS CONTAIN a varied assortment of biologically active molecules stored in several types of granules. Release of these molecules after platelet stimulation results in their interaction with other platelets, blood cells, and the vessel wall. These secreted components contribute to promotion of hemostasis, wound healing, and formation of atherosclerotic plaques. In particular, secretion of dense granule contents plays a pivotal role in formation of platelet thrombi. These storage organelles (also called dense bodies or &granules) contain serotonin, adenine and guanine nucleotides, calcium, magnesium, and inorganic phosphate.’ The inherent density of the core in these granules in human platelets when viewed in thin section or by whole-mount technique results from their calcium content? A decrease in the number of dense granules or their contents has clinical implications because many patients with this condition have a bleeding diathe~is.3,~ This condition, called storage pool deficiency (SPD), may be acquired or congenital, and several different congenital types have been described, including those associated with albinism, such as the Hermansky-Pudlak those associated with a-granule deficiency: and those that are associated with an autosomal dominant inheritance pattern but have no associated abnormalities.*We recently described a platelet dense granule membrane protein, granulophysin, identified using monoclonal antibodies (MoAbs) D545 and D503, raised against purified human platelet dense granule membranes? As analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), the protein has a molecular weight (mol wt) of 40 Kd and was shown to be deficient in a patient with the HermanslyPudlak syndrome. Our earlier studies using immunofluorescent techniques demonstrated that the antigranulophysin antibodies reacted minimally with unstimulated human platelets unless the platelets were made permeable with saponin. After thrombin activation, however, nonpermeable platelets showed staining with the fluorescent-tagged MoAb. In the present study, we used immunoelectron-microscopytechniques to investigate the ultrastructural localization of Blood, Vol80, No 1 (July I), 1992:pp 143-152 MATERIALS AND METHODS Antibodies. MoAbs D545 and D503, both directed against granulophysin,were prepared after immunization of BAL.B/c mice with platelet dense granule protein as described in detail previously? D541, an MoAb produced using the same dense granule protein preparation, was shown to be directed against P-selectin as analyzed by Western blot using purified P-selectin provided by Dr R. P. McEver (Oklahoma City, OK), who also provided the polyclonal rabbit antiserum to P-seIectin.lo Colloidal gold conjugates were obtained from Janssen Pharmaceutica (Beerse, Belgium). Preparation of platelets. Blood was obtained from aspirin-free normal adult volunteers by venipuncture and fixed immediately15 or drawn into acid-citrate-dextrose(ACD) anticoagulant. Plateletrich plasma (PRP) was obtained by centrifugation of blood at 8OOg for 5 minutes. Platelets were washed and resuspended in 0.1 mol/L phosphate buffer, pH 7.4. Resting platelets were allowed to incubate at 37°C for 1 hour before fixation. Some platelet samples were stimulated with 1 U/mL bovine thrombin (Sigma, St Louis, MO) for 1 or 5 minutes before fixation. Platelets used for From the Department of Paediatrics and the Manitoba Institute of Cell Bwlogy, University of Manitoba, Winnipeg, Manitoba, Canada; and the Department of Pathology, University of Califontia School of Medicine, San Francisco, CA. Submitted June 24,1991; accepted March 11, 1992. Suppored by Grants No. MA7396 from the Medical Research Council of Canada and No. HLB316lOfrom the National Institutes of Health. Address reprint requests to Sara J. Israels MD, Manitoba Institute of Cell Biology, 100 Olivia St, Winnipeg, Manitoba, Canada, R3E OW. The publication costs of this article were &frayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section I734 solely to indicate this fact. 0 1992 by The American Sociey of Hematology. 0006-4971/92/8001-0025$3.00/0 143 From www.bloodjournal.org by guest on August 1, 2017. For personal use only. 144 ISRAELS ET AL Fig 1. (a) Tnwmiuion electron micrograph (TEM) of a human p1.t.M to lllu"ta the morphology of typkl organelleawhen emboddod In Epon (drrctibed Inthe Materialsand Methodsd o n ) . Thh cell contains many trgnnuies (a), as well aa one seratonln-containing d e w gnnuk (arrow) and the SCCS. (Originalmagnification x40,OOO.) (b) TEM showing localizationof MoAb 0545 to Mw(y identified dense granule membrane protein of 40 Kd. Platelets were dripped into 8% paraformaldehyde and prepared for frozen thin-section lmmunocytochemimy. The gold label (GAR-10) is contained in the dense granule (arrow) and is not evident in the ogranulea (a), SCCS, or on the plasma membrane (pm). The MoAb was used at a concentretion of 10 FglmL, and a bridge antibody rabbit-anti mouse IgG was used at a dilution of 1:lOO; GAR-10 was then applied. (Original magnification x50,OOO.) From www.bloodjournal.org by guest on August 1, 2017. For personal use only. PLATELET DENSE GRANULE MEMBRANE PROTEINS immunocytochemistry were fixed in 4% or 8% paraformaldehyde in phosphate buffer, infiltrated with 2.1 mollL sucrose, and embedded in sucrose solution before freezing and storage in liquid NZ.'~ Platelets prepared for morphology alone were fixed in 1.4% glutaraldehyde and 1.9% paraformaldehyde in 0.2 mollL sodiumcacodylate buffer with 0.025% CaC12, post fixed in OSOSstained with uranyl acetate, and embedded in Epon.I6 Immunocytochemical techniques on f i z e n thin sections. Frozen thin sectionswere cut as previously described.I5Immunocytochemical procedures were performed on frozen thin sections" with the following antibodies. The primary antibody, D545, was used in a concentration of 10 &mL. lmmunogold probes, goat anti-mouse IgG-gold (10 nm, GAMIO) or protein A conjugated to 10 nm gold (Janssen Pharmaceutica) were used at a dilution of 1:50. For some experiments, a bridging second antibody, rabbit antimouse, was used, followed by gold conjugated to goat antirabbit IgG (10 nm, GARIO). For double-label experiments, D545 was used as above, with polyclonal antiserum to P-selectin. After washing, the cells were labeled with goat antirabbit IgG-gold (5 nm, GARS) and goat anti-mouse IgG-gold (10 mm, GAMIO). Controls for each experiment included substitution of buffer or nonimmune serum for the specific primary antibody. 145 Reparation of densegmnules. Dense granules were prepared as described previouslypusing a modification of the method of Rendu et allRby centrifugation of a whole platelet homogenate through 40% metrizamide at 1lO.ooqS for 30 minutes. The sedimented pellet was markedly enriched in dense granules. The material above the metrizamide layer contained the remaining platelet membranes and was enriched in a-granules. It was used as a dense granule-depleted membrane fraction for some studies. Western blors. Westem blots were performed as previously described! Electrophoresis was performed on a 7.5% polyacrylamide gel with a 4% stacking gel according to the method of Laemmli,I9 after initial incubation of protein for 30 minutes in a Weber-Osbome bufferm (three parts sample to one part 6% SDS. 40 mmollL NaP04, pH 7.0,20% glycerol, and 0.01% bromophenol blue). Proteins were transferred to nitrocellulose at 100 V for 1 hour at 15°C. The nitrocellulose was blocked for 1 hour using 10% nonfat powdered milk, washed with tris-buffered saline (TBS),and incubated with monoclonal anti-P-selectin (D541). After washing with TBS, the nitrocellulose was incubated with biotinylated goat antimouse IgG from Vectastain ABC Kit (Dimension Laboratories, Mississauga. Ontario, Canada) and then with Vector ABC peroxidase-conjugate. Blotting was completed by incubating with 6 Flg 2. TEM of thrombin-rtimulated(1 mi-) platelets 1llUrh.h.mdhMbuHon of gmnuloph@n. Aftof the frozenthin Maion waa Incubated wkh MoAb D545, GAM-10 gold was added. Label is evident along the plasma membranes (pm) of activated platelets 88 well 88 in intracellular vacuoles, preaunublyportions of the SCCS. (Original magnification x60,OOO.) From www.bloodjournal.org by guest on August 1, 2017. For personal use only. ISRAELS ET AL 146 mg 4-chloro-I-napthol in 2 mL methanol and 50 FL 30% H202 in 10 mL TBS until color development was achieved. hpamrion of whole mounts for elecmn micmcopy. PRP or isolated dense granules were fixed in I% paraformaldehydelO.l% glutaraldehyde. Platelets or isolated dense granules were made permeable by incubation in Triton X-100 (Mallinkrodt, Mississauga, Ontario, Canada) (0.1%) for 3 minutes before beingwashed with TBS. Platelets or dense granules were then allowed to settle on formvar-coated (J.B. EM Services Inc, St Laurent, Quebec, Canada) grids before incubation with D545 (or nonimmune ascites control) and GAMIO. Differential secretion of semronin and pglucumnidase. PRP was aliquoted for parallel analysis of serotonin (dense granule) release, B-glucuronidase (lysosomal granule) release, and flow cytometric analysis of granulophysin expression on the platelet surface. Each of these samples was washed and resuspended in a HEPESTyrode’s buffer?’ and aliquots from each were then treated as follows: ( I ) 0.5 mmollL colchicine for 45 minutes, then 5 U/mL thrombin for 5 seconds; (2) 0.5 mmollL colchicine for 45 minutes, then 5 UlmL thrombin for 30 seconds; (3) 0.4 mmol/L RGDSP peptide and 1 mmol/L CaClz for 1 minute, then 5 U/mL thrombin for 5 minutes; or (4) no treatment. Colchicine was used to inhibit lysosomal granule secretion,u and the RGDSP peptide was used to minimize aggregation under conditions in which secretion was maximum.l’ Serotonin release was measured from platelets prelabeled with 14C-serotoninas previously and B-glucuronidase secretion was measured by the method of Hoehn and Kanfer.2s In each case, values were expressed as the amount released into the supernatant as a percentage of the total platelet content. Flow cyromerry analysis of gmnulophysin dbtriburion. Samples for analysis were treated as described above, and activation was terminated by addition of an equal volume of cold ACD. The platelets were pelleted, resuspended in HEPES-Tyrodes buffer containing 10% ACD, and incubated for 1 hour on ice in the dark with D545 (80 pglmL) conjugated directly to FlTC or indirectly labeled with D545, biotinylated goat anti-mouse IgG (1:IW). and FITC-avidin (1:3.000). To some samples, unlabeled D545 was added before the fluorescent labeling was performed. Samples were fixed with 1% paraformaldehyde and analyzed using an EPICS model 753 flow cytometer (Coulter Electronics, Hialeah, FL) equipped with an argon ion laser (500 mW, 488 nm). Fluorescencewas detected at 525 nm. Forward and 90” light scatter measurements were used to establish gates for intact viable platelets. Single-parameter,255channel. log-integralgreen fluorescent histogramswere obtained, each based on 1 x I@ gated events. RESULTS Distribution of P-selectin on unstimulated platelets was previously studied by frozen thin section and shown to be on the membranes of a-granules.10Although in previous studies the dense granule population could not be identified because the granules do not retain their dense core during this type of tissue preparation, some unidentified clear vesicles were labeled and the possibility that they were dense granules was considered.In These “dense” granules are easily identified in platelets prepared by conventional fine-structural techniques (Fig la). In this study, we used Fig 3. Preparationsimilar to that d d b o d In legend to Fig 2 but fixed 5 minutus after exporun to thrombin. Again the label h on the plasma membraneand in SCCS. Bridges (arrow)betweencoils do not contain the label. (Originalmagni&.tion x50,OOO.) From www.bloodjournal.org by guest on August 1, 2017. For personal use only. PLATELET DENSE GRANULE MEMBRANE PROTEINS the recently developed MoAb D545, raised to purified dense (serotonincontaining) granule membranes which identifies a 40-Kd protein, granulophysin, to determine the ultrastructural localization of granulophysin. To accomplish this, we incubated frozen thin sections of platelets with D545 followed by a second bridging antibody and a colloidal goldconjugated third antibody. Resting platelets examined by electron microscopy showed that immunoreactive granulophysin was localized in one or two granules per platelet (Fig lb). Other identifiable granules, the surfaceconnected canalicular system (SCCS) and plasma membrane were not labeled. When platelets were stimulated with thrombin, the immunogold label was redistributed to the SCCS and plasma membrane (Figs 2 and 3). After 1-minutestimulation, some label could still be observed in granules, although it had also appeared in the SCCS and on the plasma membranes. Five minutes after exposure to thrombin, labeling of granules was rare. Distribution along the membrancs of the SCCS and plasma membrane appeared to be homogeneous when evaluated with protein A-gold as the immunoprobe for the primary 147 antibody because protein A-gold does not clump as IgGgold probes may.%There was no apparent concentration of label on pseudopods. At some sites of cellcell contact no label was observed (Fig 3). Double labeling of resting platelets with D545 and polyclonal antiserum to P-selectin, a protein previously identified in the membrane of a-granules, showed that P-selectin was present not only along the a-granule membranes but also along the membranes of granules labeled by D545 (Fig 4). However, D545 did not label a-granules. The double-labeled granules often appeared as empty vesicles on the frozen scctions, with no evidence of a dense core. Occasionally,small vesicularstructureswere observed within the granules (Fig 4). These structures have been observed previously in densecore chromaffin granules, particularly after freezing.27Five minutes after thrombin stimulation, double labeling with the two antibodies demonstrated distribution of both granulophysin and P-selectin to the SCCS and plasma membrane (Fig 5). Labeling of P-selectin was more abundant than that of granulophysin, but it is not possible to draw conclusions from this because it may Flg4. Frozenthin d o n of a restingplatelet incubatedwith two antibodies: a polydonai antibody against P - w M n iabded wwh OAR4 and MoAb OS45 against granulophysin labeled with GAM-10. The 5-nm gold (P-seldn) is evident in a-granules and the dense granule, whereas the 10-nm gold (arrows), denoting the presence of granulophysin, is evident In dense granules only. (Original magnification x80,OOO; inset x77,OOO.) From www.bloodjournal.org by guest on August 1, 2017. For personal use only. Fig 5. Double Iabellng of thrombln-stimulatd (5 minute) platelets &om dhhlbution of both anti-P-wldn (5 nm gold) and a d granulophyain(10 nm gold) adbodies tothe plasma membrane (pm), aa wail as in some SCCS. (Odginalmagnf&.tion x65,OOO.) represent only the difference in binding between polyclonal antibodies and MoAbs. In addition, P-selectin is distributed all over the plasma membrane, even at cellcell contact points. Western blot analysis showed that P-selectin was present in the whole platelet homogenate, the dense granuledepleted fraction and the dense granule-enriched preparation (Fig 6), in contrast to granulophysin which has previously been shown to be specific to the dense granuleenriched fraction, with very low amounts in the dense granule-depleted fractions? Because the serotonincontaining granules do not retain their electron-dense core after preparation for cryosectioning, we also examined, by electron microscopy, whole mounts of permeabilized platelets and isolated dense granules labeled with D545 and colloidal goldconjugated second antibody (Fig 7). Labeling with D545 of identifiable electron-dense granules could be observed in quiescent platelets permeabilized with Triton X-100 for 3 minutes before incubation with the antibody (Fig 7a). Isolated dense granules prepared from dense granule-enriched fractions also were labeled with D545 when granules were made permeable with Triton X-100before incubation with D545 (Fig 7b-e). To investigate the possibility that some of the clear vesicles labeled on cryosections might represent lysosomal membranes, we evaluated redistribution of granulophysin to the platelet plasma membrane after stimuli that produced either dense granule secretion only or dense granule plus lysosomal secretion. Differential secretion was evaluated by quantifying I4C-serotoninrelease from dense granules and p-glucuronidase release from lysosomes. Redistribution of granulophysin to surface membranes of platelets was evaluated by flow cytometry using FITC-tagged D545 (Fig 8,Table 1): 5.5% to 12.5% of quiescent platelets were positive after labeling with D545-FITC depending on the labeling method. After stimulation with thrombin for 5 and 30 seconds (with colchicine and without external calcium), conditions that produced significant dense granule secretion (71%, 80%) but minimal lysosomal granule secretion (2.5%. 4.4%), the percentage of fluorescent-positivecells increased to 66% and 69%. Maximal dense granule secretion (91.1%) as well as release of 31.8% of p-glucuronidase from lysosomes caused an additional 7.8% of platelets to express granulophysin on their surface (Fig 8, C through E). Experiments using indirect labeling of D545 with FlTC yielded substantial expression of D545 expression under conditions of selectivc serotonin granule secretion, but a From www.bloodjournal.org by guest on August 1, 2017. For personal use only. PLATELET DENSE GRANULE MEMBRANE PROTEINS kDa -205 -116 -77 -46 A B C Fig 6. Western blot with antCP-..l.cWn MoAb 0541. I . "A contrlns a whole platelot homogenate, lam B contains a dense granuledepleted fraction (rich in egranu1.r). and lane C contains the dense gmnule-enrichedfraction. Equivalentamounts of protein (10 pg per lane) were applied to each lane. Samples were solubilized in Weber-hbome buffer and separated, nonredud, on a 7.5% hemmli gel as described in the Materials and Methods section. relatively larger increment in association with lysosomal granule secretion. DISCUSSION Identification of granulophysin by MoAbs as a protein specific to dense granules in platelets has provided a tool to further evaluate dense granules at an ultrastructural level. Although frozen thin sections have been very useful in examining the a-granule and its fusion with the SCCS and 149 plasma membrane through observation of P-selectin, an integral granule membrane protein, identifying dense granules has not been possible because they lose their distinctive dense core during the preparati~n'~,''and cannot be distinguished from other vesicles. Using D545 and immunoelectron microscopy techniques, we were able to identify granulophysin-containing membrane vesicles specifically. These vesicles did not contain electron-dense material (indeed, they usually appeared less electron dense than a-granules) and lacked other morphologically distinguishing features. Usually one or two were evident per platelet, which coincides with the mean dense granule count in thin sections of resting platelets.zB There was no evidence of granulophysin present in a-granules, SCCS or on the plasma membrane of resting platelets. Using whole mount techniques that allow dense granules to be visualized on transmission electron microscopy by their inherent electron density, we were able to demonstrate labelingof dense granules both in whole platelets and isolated dense granule preparations made permeable with Triton X-100. Neither quiescent platelets nor isolated dense granules show labeling unless made permeable. The evidence provided both by the cryosections showing membrane staining of specific vesicles and by the whole mounts showing specific labeling of dense granules support the localization of granulophysin to the dense granule membrane. Double labeling with antibodies to both P-selectin and granulophysin demonstrated the presence of P-selectin in the granulophysin-positive granules, as well as the more numerous granulophysin-negative a-granules. This is supported by the results of Western blot analysis, which demonstrates the presence of P-selectin in both dense granule-enriched and dense granule-depleted fractions. Therefore, P-selectin appears to be common to both types of granule membranes. This finding is also supported by the study of Lages et alZPdemonstratingboth decreased expression of P-selectin in a patient with severe a8 SPD and labeling by anti-P-selectin of both a-granules and unidentified clear vesicles in control platelets.10*29 With the antigranulophysin antibody, the clear vesicles that label with both anti-P-selectin and antigranulophysin as dense granules can now be identified. Failure of the a-granules to stain with D545 also supports our previous finding that granulophysin is not present in the membranes of all granules. Because the vesicles labeled with D545 on cryosections could not be specifically identified and because lysosomes also lack identifying morphologiccharacteristics on cryosections, we investigated the possibility that some of these vesicles might represent lysosomes, using conditions that produce differential secretion of dense granules (as measured by 14C-serotoninrelease) and lysosomes (as measured by secretion of p-glucuronidase). With addition of colchicine, which inhibits lysosomal secretion, brief stimulation with thrombin (5 and 30 seconds) produced significant dense granule secretion ( > 70% serotonin release) accompanied by redistribution of granulophysin to the platelet plasma membrane in 69% of cells quantified by flow From www.bloodjournal.org by guest on August 1, 2017. For personal use only. ISRAELS nAL 150 -7. WMomountsdpbtaktta)orbd.t.d donla grm(.r (b tlNOl#t 0 ) f l r d wkh 1% pM(* nukkhyd.IO.1K gMu.ldrhld. and lre8t.d wkh triton X-100 tor 3 minut- fa. c through 01 Mom a l n g pl.t.d on gcldr. L.b.Hngwkh Ds45 m n d GAM 10 (0. b, d. 0 ) 01 " u n o "sand GAM 10 n control IC). ~OdgIrulNgnlfkdons: lo) x27.OOO. fb) xS6.OOO. IC) x 16O.OOO. Id) x66.OOO. lo) x68.OOO.1 1&1. UmtimuW Thrombin 5 m+ cokhkine Thrombin 30 seconds + colchicine Thrombin 5 minutes Values am meen L. SE. - & e n t l o n d s o " & O ~ m d ~ d O r m ( o O h * J n o n P I . h k ( ~ Saaonln s.o*m 1%) In * 81 7.71 :1.73 71.29 t 3.22 80.50 L. 1.37 91.14 L. 1.03 - SmHOn 1%) In S, 0.80 t 0.32 2.48 L. 0.81 4.42 t 0.89 31.82 t 4.87 C.m L.b.cd Wflh DYSflTC1%) kb*.ccLMml In * 6) Di..ccL.b.l In * I) 6.54 :2.18 23.17 t 7.18 30.69 t 5.95 51.67 t 4.0s 12.5 66.1 69.0 76.8 From www.bloodjournal.org by guest on August 1, 2017. For personal use only. PLATELET DENSE GRANULE MEMBRANE PROTEINS 1 I A\ I cl 151 B/ membranes and its expression on the platelet surface paralleling dense granule secretion. However, some granulophysin may also be present on lysosomal membranes and some of the clear vesicles labeled on the cryosections may represent lysosomes. In this regard, Nieuwenhuis et a130,3i demonstrated that lysosomal membranes are redistributed to the platelet plasma membrane after activation. After stimulation with thrombin, granulophysin was redistributed to the SCCS and plasma membrane as was previously described for P-selectin.lo Like a-granules, dense granule membranes apparently fuse with the SCCS on platelet stimulation and release their contents into the extracellular space through the SCCS. After degranulation, granulophysin, like P-selectin, becomes expressed on the plasma membrane. Distribution of granulophysin on the plasma membrane did not demonstrate any clustering or concentration in specific sites that might suggest a specific function. This homogeneous distribution is similar to that of P-selectin. P-Selectin, which on the platelet plasma membrane mediates adhesion of activated platelets to neutrophils, has been identified as a member of the selectin family of vascular cell surface re~ept0rs.l~ The function of granulophysin is unknown, and whether its presence on the plasma membrane of stimulated platelets plays a functional role or is simply a marker of dense granule membrane fusion with external membranes is not clear. The role of granulophysin, which may be related to granule integrity, is probably of broader significance than to the platelet alone because the presence of granulophysin has been identified in endothelial cells, n e ~ t r o p h i l s ,and ~ ~ lymphokine-activated killer cells.9 LL ’ I I Fig 8. Flow cytometric analysis of D545-FITC binding to quiescent and activated platelets as described in the Materials and Methods section. Representative histograms of fluorescence recorded after binding of directly labeled 0545-FITC. Vertical line separates negative and positive fluorescent populations. Fluorescence intensity is displayed on a three-decade logarithmic scale. (A) Resting platelets and (B) resting platelets incubated with unlabeled D545 before addition of D545-FITC. (C) Plateletspretreated with colchicine before exposure to thrombin for 5 seconds. (D) Plateletspretreated with colchicine before exposure to thrombin for 30 seconds. (E) Platelets exposed to thrombin for 5 minutes without colchicine and with external calcium. (F) Platelets treated as in (E) but incubated with D545-FITC in the presence of excess unlabeled D545. cytometry. These conditions produced minimal lysosomal secretion ( < 5%). When conditions also allowed lysosomal secretion to occur (thrombin stimulation for 5 minutes), fluorescence increased by 7.8% more. These results support the presence of granulophysin on the dense granule ACKNOWLEDGMENT We thank C. Robertson and E.M. McMillan for technical assistance, A. Lundy for manuscript preparation, and Dr E. Rector for expert assistance with flow cytometry analysis. REFEREiNCES 1. White JG, Gerrard JM: Ultrastructural features of abnormal blood platelets. Am J Pathol83:590, 1976 2. Gerrard JM, Rao GH, White JG: The influence of reserpine and ethylenediaminetetraacetic acid (EDTA) on serotonin storage organelles of blood platelets. Am J Pathol87:633,1977 3. Holmsen H, Weiss HJ: Further evidence for a deficient storage pool of adenine nucleotides in platelets from some patients with thrombocytopathia: ‘Storage pool disease.’ Blood 39:197,1972 4. Israels SJ, McNicol A, Robertson C, Gerrard JM: Platelet storage pool deficiency: Diagnosis in patients with prolonged bleeding times and normal platelet aggregation. Br J Haematol 75:118, 1990 5. Hermansky F, Pudlak P: Albinism with hemorrhagic diathesis and unusual pigmented reticular cells in the bone marrow: Report of two cases with histochemical studies. Blood 14:162, 1959 6. Witkop CJ, Krumwiede M, Sedano H, White JG: Reliability of absent platelet dense bodies as a diagnostic criterion for Hermansky-Pudlak syndrome. Am J Haematol26:305,1987 7. Weiss HJ, Witte LD, Kaplan KL, Lages BA, Chernoff A, Nossel HL, Goodman DL, Baumgartner HR: Heterogeneity of storage pool deficiency studies on granule bound substances in 18 patients including variants deficient in a-granules, platelet factor 4, p-thromboglobulin, and platelet-derived growth factor. Blood 54:1296,1979 8. Weiss HJ, Chervenick PA, Zalusky R, Factor A A familial platelet defect in platelet function associated with impaired release of adenosine diphosphate. N Engl J Med 281:1264,1969 9. Gerrard JM, Lint D, Sims PJ, Weidmer T, Fugate RD, McMillan E, Robertson C, Israels SJ: Identification of a platelet dense granule membrane protein that is deficient in a patient with the Hermansky-Pudlak syndrome. Blood 77:101,1991 10. Stenberg PE, McEver RP, Shuman MA, Jacques YV, Bainton D F A platelet a-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J Cell Biol 101:880,1985 11. McEver RP, Beckstead JH, Moore KL, Marshall-Carlson L, Bainton DF: GMP-140, a platelet a-granule membrane protein is also synthesized by vascular endothelial cells and localized in the Weibel-Palade bodies. J Clin Invest 84:92,1989 12. 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For personal use only. 1992 80: 143-152 Platelet dense granule membranes contain both granulophysin and Pselectin (GMP-140) SJ Israels, JM Gerrard, YV Jacques, A McNicol, B Cham, M Nishibori and DF Bainton Updated information and services can be found at: http://www.bloodjournal.org/content/80/1/143.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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