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From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Identification of Primary Lysosomes and By Mary The presence well documented; of lysosomal has been the determine tion the subject the of two time platelets, and blood we examined was degree reaction product was Golgi-associated were sometimes reaction product was LOOD bodies to reactive. to vesicles of a-granules storage are in with one or other a two enzymes, phosphate arylsulfatase bone undergoing From chest the Berkeley, nia School in part mammals cavity directly Dense drawn from 10 ml into to be lysocytochemical Address ment San surgery. Cells ofMedicine, in part April reprint of Pathology, Francisco, /4, under 1981; requests University Francisco, Calif 94143. by Grune & Stratton, 0006-9971/5903-0003$O1.OO/O the Berkeley fixed with M fixation, detail the buffer, incubation according preparations were (while suspended water bath postfixed was pipetted at Cells to either room washed 7.4, method in 0504; 6.0; 10 of wash stained with dehydrated in before uranyl buffer, pH ethanols; Sections citrate, were cut with a diamond and examined with a Siemens cells then 30 mm. After in 0.1 AS are described in for trimetaphosphatase Oliver.’8 without mm When to cytochemical 4#{176}Cfor and incubated incubated CaCI2.16 into fixative; at 22#{176}C,and 7% sucrose. AcPase were Karnovsky’s paraformalde- ( 1 hr to overnight) containing for of 0.025% The Na-cacodylate M Prior at was centrifuge; 1 .9% dripped to cool out in the postfixation 90#{176}Cfor 0.1 temperature. carried directly blood 5- 1 0 vol of fixative. and with was either U heparin/ the or a modification buffer Blood into clinical in glutaraldehyde the bones. used, into glutaraldehyde pH platelet of anticoagulants g on a table-top procedures elsewhere.’2”7 (TMPase) use ACD the cells were thoroughly M Na-cacodylate The was in primary during (50-100 then at that are or a heparinized 1% sucrose, fixation participated they volunteers Na-cacodylate for 3 hr more incubations, had on the were omitted, blood was 30 mm at 37#{176}C,allowed for lysosomes organelles. normal 250 I .4% the enzyme- Cells substrate buffer) were in or heated incubation. acetate and knife, stained 101 electron control the in a Cells in veronal embedded cells were acetate in briefly with microscope. Epon. lead HL-24577 Contract accepted to Dorothy RESULTS marrow Megakaryocytes nucleated cells tures of MK Laboratory. University of Califor- Research W-7405-ENG-48. October 21, 1981. F. Bainton, of California School M.D., Depart- of Medicine, constitute 0.l%-0.5% of the of the bone marrow. The general feamaturation in human bone marrow resemble those described for tion in this cell line involves and AM-10486 and Environmental Inc. in 0.2 of or of patients from Calif Grants of Health ofEnergy rib fragments Lawrence by USPHS © 1982 472 hyde disease’4 this conenzymes 7.4, containing anticoagulants were fixed markers. In the evidence that two lysosomal expressed of Pathology, San by the Office from were Division, pH fixative lysosomes analyses was were the platelet secreted by pressure heparin 1.5% profiles. reaction. or without either that that are in about examined they in Golgi containing constitute that vein at found of platelet other believe fixative plasma was cells release 10 mm 1 5% from antecubital for buffer, conclude contents When fixative. fixative We we syringe, platelet-rich platelets (ACD) blood) centrifuged and the acid-citrate-dextrose distinct, also about MK in the vesicles in only no evidence into the were all event, vesicles METHODS collected the Department the Department Submitted AND was Biomedical and Supported and marrow was digestive and rat, and that these vesicles are distinct from the time of their formation. MATERIALS Human there AS distinct enzyme, small be demonstrated in these are whose acid phosphatase (AcPase) (with B-glyceroand trimetaphosphate as substrates) and (AS), in small vesicles similar to those we reported in the from a-granules vesicles they lysosomal in similar whereas enzymes. lysosomes, of platelets from patients with storage pool and the “gray platelet syndrome”5 support clusion since these platelets have lysosomal absent a-granule we present cytochemical and platelets contain containing both aggregation indicate that Furthermore, or of MK, study’2 that rat megakaryocytes (MK) and platelets store lysosomal enzymes in vesicles distinct from both dense bodies and a-granules. Recent cell fractionation but reduced present article, human MK found size, organelles.’ not containing for Since it could profiles, were any localized Vesicles a-granules that Another was platelet variable morphologically studies of human platelets also and a-granules are separable.’3 of negative and Golgi a-granules. (AS), In mature of and many of their components have been Although a-granules are often considered somes, we concluded from a previous than of all stages; The Heavy vesicles; MK AcPase Acid in those F. Bainton smaller complex. region of man and other a variety and confined lightly PLATELETS contain as but 25% marrow maturation. coated limited to (MK) Golgi Dorothy arylsulfatase localiza- bone in the as well and order cytochemistry. cytoplasmic usually cisternae B human present cisternae In subcellular and MK, of is megakaryocytes normal microscopy recognizable considerable and in and however, disagreement. enzymes (AcPase) youngest some platelets “lysosome,” Megakaryocytes Platelets E. Bentfeld-Barker in human of the of appearance by electron phosphatase the of lysosomal and enzymes identity in Human of ploidy and the development mic organelles; electron cytoplasmic maturation other species.’9 Maturaincreasing nuclear poly- of a unique microscopy begins during set of cytoplashas shown that the ploidization phase, but continues after nuclear development is complete. Since the two processes are neither synchronous nor sequential, classification of the maturational stages of MK is difficult. The nomenclature used in the Blood, Vol. 59. No. 3 (March), 1982 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. MEGAKARYOCYTES AND PLATELET LYSOSOMES 473 Fig. 1 . Golgi region of an immature human MK tested for AcPase. Several stacks of Golgi cisternae (Gc) are evident. In most atacks, reaction product is confined to one cisterns (arrows) and nearby coated vesicles (cv), while the remaining cisternae show little or no reaction. No typical a-granules can be identified in this field; many of the mitochondria (m) have been disrupted by cold fixation or the acid incubation medium. Tissue fixed in 1 .5% glutaraldehyde for 30 mm at 4’C and incubated in the Barks-Anderson medium for 3 hr at 30”C (n, nucleus; nu, nucleolus; x22,000). Lower Inset: Higher magnification of part of same cell. Reactive and unreactive coated vesicles can be identified ( x 36,000). Upper Inset: Light micrograph of 1 m Epon section of an immature MK similar to the cell shown in the electron micrograph. It has scant cytoplasm and a bibbed nucleus with several large nucleoli ( x2000). heavy From www.bloodjournal.org by guest on June 16, 2017. For personal use only. BENTFELD-BARKER 474 present study is based on the system proposed Paulus2#{176}and used in our previous paper on rat Briefly: (1) “immature MK” are small (-15-18 diameter) cells that have a nucleus:cytoplasm greater than I and few cytoplasmic organelles than MK” large are nucleus, rough numbers of polyribosomes; heterogeneous in size, a well endoplasmic lobes are fully granulated Immature a “maturing lobulated developed Golgi complex, abundant reticulum (RER), and variable numbers of a-granules (3) “mature MK” are nuclear (2) have by MK.’2 sm in ratio other and also closely and cells demarcation heterogeneous apposed, divided constitute into platelet a minority is fields. of the total MK population,2’ and are very difficult to identify morphological criteria, especially in thin sections. youngest cell we identified by electron microscopy by The (not often present is shown same localized cytoplasm. can in Fig. stage 4A. in vesicles At higher be seen that the MK; tion therefore, for AS the (Fig. Golgi can region, be is depicted identified (-80-120 as nm) inset). that Reaction containing in Fig. small are I Forming . perinuclear is also (Figs. region cation a small cisternae. cisterna, 2 and 3) have with accumulating membranes, and is lysosomes vesicles (Fig. evident in each amount The mature variable vesicles and a-granules may RER, unreactive. a large well a-granules, well developed were never 1, lower probably reticulum, Golgi equivalyso- be present including Maturing in the MK developed Golgi vesicles, demarRER. As can be seen in Fig. 3A and B, they showed a similar tion of AcPase. In favorable sections, reaction was found in vesicles and a few Golgi cisternae, RER to the coated stack, chiefly in one or two cisternae, lent to GERL (Golgi, endoplasmic some22), although some of the other nuclear limited AcPase-positive usually product more activity reactive. distribuproduct but the In more cells, the AcPase-positive vesicles were more in size ( 1 75-250 nm), suggesting that smaller may fuse to form larger vesicles. All appeared homogeneous boundaries. was difficult showing no cellular As we had found to demonstrate debris with rat in mature within MK, cells their AcPase by the lead-salt methods used for electron microscopy. Nevertheless, a few small reactive vesicles could usually be seen in thin sections of such cells. At this stage of maturation, Golgi complexes considerably in size and are trol preparations incubated heated showed have generally decreased difficult to sample. Conwithout substrate or no reaction. In MK ofall maturational stages, from the youngest recognizable cells to the most mature, AS was always present, confined to small vesicles. Although reactive illustrates a cell Reaction size we of the reflect AS in rats, Part of mature at product throughout (inset, Fig. structures is the it 4B), are distinct densities seen in MK after 4B) are very probably unidentified cell, AcPase 4B AS. reactive some larger showing region, from the a-granules, which are unreactive and usually larger. Dense bodies (illustrated in Fig. 5, inset) are normally not seen in routine electron microscopy of region. also for of variable magnification for lysosomal not be completely without substrate, A slightly Figure reacted product could bated but Golgi product in any component We presume that this may shown) had a bibbed nucleus similar to that of the cell shown in the upper inset of Fig. 1 . It measured 1 8 tm in diameter and showed AcPase reaction in the Golgi lobes, in the BAINTON limited sampling, since we previously observed the Golgi region of MK from thombocytopenic which have a higher proportion of young MK. the highly demarcated cytoplasm of a typical MK the cytoplasm were never saw reaction secretory apparatus. the membranes; in size, the and vesicles AND enzyme. The abolished suggesting endogeneous incubareaction reaction product in controls incuthe presence of substrate. However, cells heated in a water bath for 10 mm at 90#{176}C prior to incubation in complete medium were totally devoid of reaction product, indicating enzyme inactivation; morphological preservation under these conditions excellent. Vesicles reactive for both enzymes were also strable in circulating platelets. Figure 5 shows let from blood dripped directly into fixative anticoagulant. preserved, body.* The disc shape of the as are numerous a-granules Figures 6, 7, and AcPase, enzymes were AS, than 8 show the a-granules. We did bodies are characteristic similar light dense (Fig. body platelets not test (see halo incubated MK All for three smaller for TMPase. of lysosomes in platelets density of reaction prodand the platelet dense Fig. 5). is helpful 5, inset). demona platewithout platelet is well and one dense and TMPase, respectively. present in vesicles somewhat Cytochemical identification poses problems because the ucts for lysosomal enzymes was When present, in identifying In preparations the the incubated for AS, we noted densities that we consider due to reaction product in about 25% of platelet profiles. In preparations processed with our standard fixation and embedding techniques, but not incubated for enzymatic activity, we saw the represent serotonin-containing about 7% incubated 5In granules; density, contents,7 our of platelet profiles. for AS, even if all earlier paper’2 although other and densities dense this components therefore, Thus, in the dense we identified these amine contribute appear we have may to form dropped that we bodies preparations bodies were organdIes this as serotonin to a major assume in only their portion designation. electron of their From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Fig. 2. Golgi region of a maturing MK. Surrounding a centriole (cc) in the perinuclear area are numerous stacks of Golgi cisternae (Gc) and abundant vesicles similar to, but more extensive than those seen early in maturation. The cytoplasm is replete with a-granules (a), demarcation membranes (dma) and rough endoplasmic reticulum (rer) ( x 1 6,000). Inset ( x 38,000): Higher magnification of a few Golgi cisternae with numerous adjacent vesicles (ye), some of which are coated (cv). Occasionally, a Golgi cisterna is swollen and contains flocculent material (arrow). Fixed in glutaraldehyde at 4’C, postflxed in 0s0, and stained en bloc with uranyl acetate (n, nucleus). From www.bloodjournal.org by guest on June 16, 2017. For personal use only. BENTFELD-BARKER 476 Fig. 3. AND (A) maturing Golgi MK. which complex AcPase product is confined nae (arrows) and (cv). BAINTON of a reaction to two cistercoated vesicles appear to bud from a reactive cisterna. Alpha granules (a) are unreactive. Tissue prepared as in Fig. 1 ( x 35,000). Inset: Light micrograph similar of maturation has increased. toplasm ratio compared a cell stage. at Cell a size but the nucleus:cyhas decreased as with the immature cell in Fig. 1 (x1500). (B) Golgi complex of another maturing MK. but at a more advanced stage of maturation than Fig. 3A. Distribution of reaction product lar to that negative (arrows) shown is simi- in Fig. 3A. a-granules (a). Note Tissue prepared as in Fig. 1 (x33,000). Inset: Light micrograph of a similar cell. which exhibits a Iobult.ted nucleus and a greatly decreased nucleus:cytoplasm ratio ( x 1500). misclassified would as account When lysosomes (which for less than platelets were 30% incubated deposits similar to those were found in only about is unlikely), of the total for they cisterna, lysosomes. AcPase, size dense seen after incubation I 5% of platelet profiles. for AS Since it was in both always MK and present platelets. in vesicles of variable Unfortunately, we could not determine whether the same organelles for both enzymes or whether there are two are reactive populations more limited experiments were performed with TMPase, we have not attempted to quantitate our results. With our methods, it is impossible to determine whether the sites of these different enzyme activities of reactive organelles. Nevertheless, it is clear that a-granules were not reactive for either enzyme. Previous cytochemical studies232t have reported AcPase to be present in what appeared to be platelet agranules; however, this demonstration usually required are the same. The smaller percentage of AcPasereactive platelets may reflect a narrower distribution of this enzyme as compared with AS or it may be due enzyme distribution. Furthermore, micrographs of platelets25 clearly to technical latency. of the enzyme outside of these investigations problems, such as partial cytochemical platelet activation identification DISCUSSION These platelets studies provide and MK contain that can be designated shown to be present immature and maturing in platelets further evidence a distinct class primary in the MK, Recent that human of organelles also lysosomes. AcPase was secretory apparatus of as well as in vesicles of mature MK and some platelets. As we had found in rat MK, heaviest reaction product was confined to one or two cisternae and coated vesicles in the Golgi complex.t Although AS was never found in any Golgi tin GERL. also always reported our previous However, appeared possible. in other paper’2 since reactive other Golgi in some Inasmuch cell we designated types,22 cases, as similar and the cisternae reactive of MK a clear-cut uncertainties we have done cisternae in both distinction have no further as or injury Fig. that studies platelet platelet a-granules. fractionation studies presence product have as well, and none to MK, where is less equivocal of fractionated lysosomes are than Although some of platelets2728 of lysosomal enzymes platelets distinct from of the early demonstrated in the “granule tion,” new separation procedures’3 have made ble to obtain a-granule fractions of much purity. Such fractions show little lysosomal but rather are rich in fl-thromboglobulin, factor platelet altered some published show a distribution 4). biochemical indicate may the a-granules was extended of reaction (see that cell the frac- it possigreater activity, platelet 4, platelet growth factor, and fibrinogen.’#{176}29 The lysosomal enzymes that have been studied species was now not been cytochem- ical tests to define reactive sites GERL in MK more precisely, as Golgi-associated cisternae. we here refer to the From www.bloodjournal.org by guest on June 16, 2017. For personal use only. MEGAKARYOCYTES AND PLATELET LYSOSOMES 477 Fig. 4. (A) Mature human MK. Note the numerous a-granules (a) and demarcation system (dms) ( x 1 7,000). Inset: Light micrograph of a mature MK showing morphology typical of a thrombogenic or mature MK. The nuclear lobes are close together at one pole of the cell, and the voluminous cytoplasm appears to be fragmenting ( x 1000). (B) Mature human MK processed for AS. The cytoplasm is replete with channels of the demarcation system (dms). Numerous a-granules (a) are also present. AS is found in vesicles of variable size (ly), but usually somewhat smaller than the a-granules, which are unreactive. Tissue fixed in paraformaldehyde-glutaraldehyde for 30 mm at 4’C and incubated for 2 hr at 250’C in Goldfischer’s medium for AS. Inset: Higher magnification of small portion of another MK showing numerous unreactive a-granules and two lysosomal vesicles (ly) containing AS reaction product ( x 17,000). From www.bloodjournal.org by guest on June 16, 2017. For personal use only. BENTFELD-BARKER 478 AND BAINTON Fig. 5. a-granules Human platelet. The (a) vary in size and density, and some contain nucleoids (small arrows). The core of the dense body (d) nearly fills the membrane-bound vesicle. A much more prominent halo is frequently seen (inset). Fixation at 37’C in 1 .5% glutaraldehyde without anticoagulant (m. mitochondrion; mt, microtubules) ( x24,000; inset, x 52.000). Fig. 6. Human platelet incubated for AS. In this specimen the a-granules (a) are clearly seen with their characteristic nucleoids (small arrows). Two densities (ly) probably represent AS reaction product; the third (d) a dense body. Tissue fixed hyde Fig. and 48 in I .5% further glutaralde- processed as in (x24,000). Fig. 7. Human platelet incubated for AcPase. Two densities are seen: one (Iy) is considered to be due to lead phosphate and hence represents a lysosome. while the other (d) is probably a dense body with its characteristic clear halo (a,a-granule). Tissue prepared as in Fig. 1 ( x24,000). Fig. 8. Human platelet incubated for demonstration of TMPase. This enzyme, which prosumably also.” (ly) demonstrates is localized identical lysosomes in small to those vesicles showing AcPase and AS activity. Fixation as in Fig. 48; incubation for 90 mm at 30CC sediment in fractions of lower shown by electron microscopy of mitochondria and membrane that these vesicles probably enzymes found in that fraction. try has tions. not In clinical yet been performed investigations pool deficiencies, described whose dense bodies, but density, which have been to be composed mostly vesicles.’3 contain However, on the of patients We the believe lysosomal cytochemis- various with frac- storage at least one patient has been platelets lack both a-granules and contain normal amounts of lysosmal enzymes.’4 described other both The “gray platelet in I 97 1 and since then patients,’5’3’ platelets all, while (x24,000). is another syndrome,” first identified in several disorder in which the contain either very few a-granules or none the platelet lysosomal enzymes are normal biochemical32 and cytochemical “experiments of nature” also point somal organdIes in platelets. criteria.’5 to distinct at by These lyso- Studies of secretion kinetics indicate that thrombin, but not ADP, causes platelets to secrete a certain amount of each lysosomal enzyme, generally about From www.bloodjournal.org by guest on June 16, 2017. For personal use only. MEGAKARYOCYTES AND PLATELET 50%. acid 3-glycerophosphatase Although be distributed other enzymes Accordingly, two in the tested, same only Fukami classes and of platelet glycerophosphatase, This could of $-glycerophosphatase Unlike form a granules,34 similar vesicles to those of variable subcellular about 10% Salganicoff’ the other is certainly also reflect to the the primary population MK and of 479 appears lysosomes: and somal enzymes. secretion kinetics LYSOSOMES one the marker subsequent studies immunocytochemical have in our postulated containing remaining filyso- but the binding membrane. lysosomes of leukocytes, which of morphologically identifiable platelet lysosomes appear to be most other cell types. They are size, which apparently are pinched off from certain Golgi cisternae, perhaps GERL membranes.35 Compared with ct-granules, they constitute a minor proportion of the MK or platelet organelles, and they probably are not recognizable without specific cytochemical stains. Their contents (without the cytochemical stains) may have an electron density similar to that of a-granule contents; however, especially as seen in MK, they are in no way identical to a- On is known about the basis of osmium-fixed in the Golgi tissue, complex. the formation morphological Jones36 proposed However, since ofa-granules observations clear that maturation. immature been found, (Halverson results of We hope that being developed et al.37) formation relationship the will soon make ofa-granules and to lysosomes. lysosomes are present very early A few a-granules may also be MK, but they become more prominent it thus It is in MK seen in later in maturation. The lysosomes platelets digestive of both rat show none of the debris activity that is normally lysosomes. We therefore believe lysosomes in both cells. Although have attempted to demonstrate lets,38#{176} most appear to be canalicular platelets tion of has have taken system. never platelet be extracellular) current diagrammed Cytochemically, Dense body concluded up are human enzymes is still unknown.4’ of the they lysosomes are small and of that they are primary several investigators phagocytosis in platesubstances retained and (which in that the material by the funcappears organelles by ultrastructural the diagram in labeled MK or other evidence seen in secondary that actually lysosomal concept and and Digestion ofexogenous been demonstrated, platelets, as revealed is summarized in in in that they arise no cytochemical yet have been equivocal. procedures now to follow the clarify their Our Little has laboratory possible further granules. MK. for them fractions as the of it is secreted. possible, differential lysosome to to of human cytochemistry, Fig. 9. We have as distinct entities. acid-hydrolase-con- I Surface connected Canalicular System tubular IX membrane Fig. 9. Diagram of a human platelet showing components visible by electron microscopy and cytochemistry. In addition to membranous components. mitochondria, microtubules. and glycogen. we have illustrated four types of storage organdIes: a-granules, dense bodies, lysosomes, and peroxisomes. While the first two can be Identified morphologically, their nomenclature has been inconsistent and confusing. Of the original terminology adopted for platelet organeIIes, only “a-granulomere,” modified to “a-granule” has survived and is now the almost universally accepted designation for the most prominent platelet granule. Likewise, dense bodies have been called various names, none of which, including the recently proposed “-granule,”4 is indicative of their chemical nature. In view of the widespread use of the term “dense body,” we suggest it be retained until a more definitive name is devised. Peroxisom.s and Iysosomes are recognizable only by cytochemical stains. We have included peroxisomes. although thus far only one typical peroxisome component. catalase, has been demonstrated in MK and platelets of normal individuals.TM Platelct lysosomes are small vesicles without a recognizable “granule” morphology. Although the term “X-granule”4 emphasizes their individuality, we consider it superfluous; platelet lysosomes are similar to primary lysosomes of other cells, for which no other name has proven necessary. From www.bloodjournal.org by guest on June 16, 2017. For personal use only. BENTFELD-BARKER 480 taming somes vesicles of most AND BAINTON ACKNOWLEDGMENT that appear similar to primary lysoother cell types.3435 On the basis of the of evidence discussed above, we believe We several lines that they should be recognized as separate organelles and not be considered a subpopulation of a-granules. wish technical for to thank Yvonne assistance, preparation of prepared by Bob 19. Breton-Gorius Jacques as well the as and Barbara Ivy Hsieh for and Susan Turner (Fig. 9) Poetter manuscript. The diagram excellent was Stevens. REFERENCES I . Fukami dIes. MH, A review. Thromb 2. Fukami improved Bauer, for platelets. White electron Human platelet 38:963, 1977 Stewart isolation Biol The dense JG: iS, the J Cell opacity L: Haemostas MH, method human 3. Salganicoff GJ, of dense 77:389, storage organ- Salganicoff storage marrow cell L: An 20. Paulus from in guinea granules bodies of of the serotonin human platelets. storage particles. 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Broekman ogen Emmines platelets. localization in nucleotide localization platelet Dangelmaier subcellular amine-storing tus Cytol Rev megakaryocyte Hand lysosomal PD, in human pyrophosphate platelets. 1969 Peters 7. Fukami in blood 22. New 23. Hi, I 86:254, phosphorus JP: 1971 H, Day blood platelets. VI. Subcellular with different functions in the Biophys Tranzer 5-hydroxytryptamine of lar of F: DNA Feinendegen . Reyes Int megakaryocytes: and Kinetics 1969 4. J-M: pig diographic 1978 i, maturation. 4 in endoplasmic Acad of reticulum: Sci 73:2781, megakaryocyte 1960 MA, Levine localization normal human A cytochemist’s 1976 granules SD, from Beckstead JH, offl-thromboglobulin megakaryocytes. Golgi Bainand i Cell Biol 1981 Mustard 1:168, iF, 1968 Packham MA: Platelet phagocytosis. 5cr Hae- From www.bloodjournal.org by guest on June 16, 2017. For personal use only. MEGAKARYOCYTES 39. Lewis platelets: JC, latex JG: Phagocytosis 41 Annu 42. . JE, KG: Med Blood Uptake Nachmias H, Weiss 30:1 Mann acid Phagocytosis in human phagosomes 50:39, i Pathol Secretable by 69:439, storage blood platelets. 1972 pools Asch A: Shape and Rodman mechanisms destruction in platelets. 44. in i, in control cytoplasmic dine and lidocaine-treated human platelets. BlOOd 1977 43. particles 19, 1979 V, Sullender filaments 1976 latex Am Hi: 481 phosphatase-positive 47:833, of or sequestration? Holmsen Rev LYSOSOMES of uptake. White PLATELET Maldonado Localization following 40. AND NF, of the Breton-Gorius megakaryocytes method. Mason of white RG, thrombus platelet. Fed Brinkhous Proc i, Guichard and platelets i Microscop Biol KM: formation: 22:1 356, J: Two as Cell pathogenic and self- 1963 different revealed 23:197, Some Agglutination by 1975 types the of granules diaminobenzi- From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 1982 59: 472-481 Identification of primary lysosomes in human megakaryocytes and platelets ME Bentfeld-Barker and DF Bainton Updated information and services can be found at: http://www.bloodjournal.org/content/59/3/472.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. Copyright 2011 by The American Society of Hematology; all rights reserved.