Development of platelet secretory granules

seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 13, 2002: pp. 293–302 doi:10.1016/S1084–9521(02)00059-9, available online at http://www.idealibrary.com on

Development of platelet secretory granules Sarah M. King and Guy L. Reed ∗

Platelet granule exocytosis plays a critical role in thrombosis and wound healing. Platelets have three major types of secretory granules that are defined by their unique molecular contents, kinetics of exocytosis and morphologies. Although the ontogeny of platelet granules is poorly understood, a convergence of new insights into megakaryocyte development, the molecular mechanisms of vesicle trafficking and the genetic basis of platelet granule defects, is beginning to define the cellular and molecular pathways responsible for platelet granule ontogeny.

system.8 As megakaryocytes enlarge multivesicular bodies become fewer, alpha and dense granules become numerous, a demarcating membrane system develops and granules are transported into the developing pro-platelet.8–10 The mature platelet has no nucleus, few remnant Golgi stacks and minimal protein translation.

Key words: alpha granule / dense granule / lysosome / platelet secretion / secretory pool disorders

Alpha granules are the largest (200–500 nm) and most abundant secretory granules (∼80 per cell) in platelets.11, 12 They contain growth factors, coagulation proteins, adhesion molecules, cytokines, cell activating agents, angiogenic factors, etc.13 These molecules include platelet-specific secretory proteins that are synthesized only in megakaryocytes, plateletselective molecules that are synthesized by megakaryocytes and relatively few other cells and molecules not synthesized by platelets that appear to be taken up into platelets through vesicle trafficking processes. Platelet-specific proteins include platelet factor IV and beta-thromboglobulin which arise from platelet basic protein by proteolytic processing.14 Platelet-selective molecules, which are found in much higher concentrations in platelets than the blood, include coagulation factor V, thrombospondin, P-selectin, and von Willebrand factor (vWF).15 Finally, alpha granules also contain molecules (e.g. fibrinogen) that are synthesized by other cells and taken up into granules through endocytosis.16–18 Alpha granules appear to develop from vesicles budding from the megakaryocyte trans-Golgi apparatus.19, 20 Alpha granules also acquire proteins from the plasma through fluid-phase endocytosis for IgG and albumin and receptor-mediated uptake for molecules such as fibrinogen (mediated by integrin α IIb β 3 or glycoprotein IIb–IIIa).16–18, 21, 22 Platelets contain coated pits and vesicles; molecules taken up by endocytosis maybe targeted to the alpha granule by a clathrin-coated vesicle pathway which is nondegradative or by a clathrin-independent, degrading,

Alpha granules

© 2002 Elsevier Science Ltd. All rights reserved.

Development of granules in megakaryocytes and platelets Platelets contain three types of secretory organelles— lysosomes, alpha and dense granules. These secretory organelles have distinctive: (1) molecular composition; (2) genetic diseases; (3) ultrastructural morphology; (4) kinetics of exocytosis and secretory responses to different stimuli.1–3 Granules develop in megakaryocytes, the progenitor cells of platelets. Megakaryocytes are polyploid bone marrow cells (averaging 16 sets of chromosomes) that undergo a process of endomitosis. Megakaryocyte development requires a cell-selective pattern of gene expression and is regulated by cytokines, particularly thrombopoietin.4–7 Young megakaryocytes have multivesicular bodies but relatively few alpha and dense granules with an immature demarcating membrane From the Cardiovascular Biology Laboratory, Harvard School of Public Health, Bldg. II-127, 677 Huntington Ave., Boston, MA 02115, USA. * Corresponding author. E-mail: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1084–9521 / 02 / $– see front matter

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endosomal–lysosomal route.16, 23–27 Further evidence for vesicle trafficking between the alpha granule and plasma membrane is provided by the observation that platelet alpha granule membranes contain proteins synthesized in the megakaryocyte and found on the plasma membrane such as α IIb β 3 28 , CD3629 , Rap130 , CD9, PECAM 131 and the glycoprotein Ib–IX–V complex.32 Alpha granules have four distinguishable morphologic zones.33–36 Moving from the outside of the granule inward there are (1) the peripheral membrane of the granule, (2) tubular and vesicular structures, (3) an electron-lucent area and, (4) an electron-dense nucleoid. The external granule membrane contains P-selectin and α IIb β 3 .8 The peripherally distributed tubules (up to 20 nm) contain multimeric forms of vWF34, 37 and resemble structures seen in Weibel–Palade bodies (see Reference 38) in endothelial cells.39 Some alpha granules also contain small vesicles (40–100 nm) or exosomes (containing the lysosomal/dense granule protein CD63) that are released in response to thrombin.40 Exosomes are secreted by a multitude of cell types as a consequence of fusion of multivesicular bodies or late endosomes/lysosomes with the plasma membrane (41, Murk et al. this issue). Exosomes are distinguishable from secreted microvesicles (100 nm–1 µm) that contain platelet membrane proteins (e.g. α IIb β 3 , P-selectin) and appear to be derived (or shed) from the plasma membrane after platelet activation.40 The adjacent, less electron dense region contains fibrinogen, thrombospondin and albumin; the electron dense nucleoid contains β-thromboglobulin, PF-4 and proteoglycans.42

molecules because when cultured with BSA-gold particles, there is trafficking first into the endosome, then into MVB1, then into MVB2 and then into the alpha granule.8 Indeed trafficking appears to occur through all membrane systems in the platelet. For example, the plasma membrane glycoproteins Ib and IIb–IIIa (integrin α IIb β 3 ) can be detected in both alpha granules and dense granules.44 The alpha granule membrane protein P-selectin can also be found on dense granule membranes.44, 45 Small amounts of CD63 and serotonin staining have been detected in atypical alpha granules located near MVBs (from which they may originate) although mature alpha granules typically do not contain CD63.8, 43 Thus, MVBs may provide a sorting compartment for dense granule as well as alpha granules during granule maturation.43 Curiously, platelet MVB1 and MVB2 do not contain significant amounts of lysosomal enzymes despite the fact that lysosomes are typically the stage following MVBs in the endocytic pathway in other cells.8

Dense granule development Dense granules contain high concentrations of small molecules such as ADP/ATP, calcium, magnesium and serotonin that give these vesicles an electron-opaque appearance in ultrastructural studies. There are typically 3–9 dense granules per platelet46 in humans and 5–6 dense granules per platelet in mice.47 Dense granules are slightly acidic (pH of 6.1) and contain membrane proteins typically found in lysosomes such as CD63 (LAMP 3) and LAMP 2, but not LAMP 1.48–51 Less is known about dense than alpha granule development. In cultured human megakaryocytes molecular markers of dense granules (CD63 and serotonin) appeared early concomitant with alpha granule formation but these vesicles appeared relatively empty.9, 52 MVBs in early and intermediate megakaryocytes also contain CD63 and serotonin.43 As the megakaryocyte matures and the platelet develops, dense granule staining becomes more intense suggesting the specific transport and storage mechanisms required for content loading improve as the megakaryocyte matures.53, 54

Development of multivesicular bodies Multivesicular bodies (MVBs) are more abundant in early megakaryocytes and decline with cellular maturation. The MVB appears to be a key megakaryocyteplatelet storage and sorting compartment. It contains molecules typically sorted to alpha granules (e.g. β-thromboglobulin, vWF), dense granules (CD63) and lysosomes.43 Type 1 MVBs have many internal vesicles (30–70 nm) that only contain the alpha granule proteins β-thromboglobulin and vWF. Type 2 MVB have peripherally distributed internal vesicles (containing the dense granule—lysosomal marker CD63) and also have electron-dense material containing β-thromboglobulin and vWF.8 Megakaryocyte MVBs appear to be key sorting organelles for endocytosed

Platelet storage pool deficiencies (SPD) Congenital deficiency of alpha and/or dense granules (or storage pools) in humans is a rare condition 294

Platelet granules development

often associated with a tendency for abnormal bleeding. SPD is due to abnormal, absent or empty granules and is distinguished from conditions associated with platelet secretion defects due to impaired cellular activation or signaling.48, 55, 56 Identification of the genes responsible for SPDs will permit elucidation of the molecular pathways responsible for the development of alpha and dense granules (see Reference 57).

lack of pigment in the eyes and skin (oculocutaneous albinism), deficiency in platelet dense granules and a cellular accumulation of ceroid lipofuscin.59 Lysosomes and related vesicles are affected such as platelet dense granules, and melanosomes. In humans HPS has variable features and geographical distributions.50 In mice also there is heterogeneity in the severity of the dense granule and lysosomal defects.60 The genetic basis for many of these disorders has been identified (Table 1). HPS-1 is found in northwestern Puerto Rican patients who have a mutation in the HPS-1 gene, that encodes an 80 kDa protein of unknown function.61–63 The mouse model of this disease is the pale ear mouse.64 Humans with HPS-2 have mutations in the β3A subunit of adaptor complex 3 (AP-3).65, 66 Pearl is the mouse homologue67 but mocha, another dense SPD mouse, also has a defect in the delta subunit of

Dense SPD Platelet dense SPDs are rare and heterogeneous.58 The dense SPD may be an isolated defect or it may be associated with disorders of pigmentation, immune function, etc. such as the Chediak–Higashi (CHS) and Hermansky–Pudlak syndromes (HPS). HPS is an autosomal recessive disorder in humans characterized by

Table 1. Granule defects associated with human and mouse platelet dense SPD Dense SPD syndromea

HPS-1114 Pale ear47, 110, 115, 116 HPS-275 Pearl47, 60, 110, 116 HPS-373 Cocoa60, 112 HPS-476 Light ear47, 60, 110 CHS80, 117 Beige47, 110, 118 Subtle gray60, 113 Cappuccino60, 107, 119 Ashen77 Gunmetal94, 95 Ruby eye47, 60, 110 Ruby eye-247, 60, 110 Sandy60, 108 Mocha106 Muted106 Pallid47, 60, 110, 116

Affected protein

HPS-1p HPS-1p AP-3 β3A subunit AP-3 β3A subunit HPS-3p HPS-3p HPS-4p HPS-4p Lyst Lyst Unknown Unknown Rab27a RabGGTase α Unknown Unknown Unknown AP-3 δ subunit Unknown Palladin

Lysosomesb

Dense granules

Platelet

Kidney

Liver

Granule numberd

C

S

C

S

C

Whole mountf

N

↑

↑

↓

N

N

↓

↑

↓

N

N

N

N

N

N

↑

↑

↓

↓ N

↑ N ↑

↓ N ↓

N N ↑

N N N ↓ ↓ ↓ ↓

N ↑ ↑ ↑ ↑ ↑ ↑

N ↓ ↓ ↓ ↓ ↓ ↓

N

N N N N N N N N N

a

N N N N

S

↓ ↓ ↓ ↓↓ ↓ ↓↓

N

↓↓ ↓ ↓ ↓ ↓↓ ↓↓ ↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓

Mepacrine stainingg

Serotonin contente

N

↓

N

↓↓

N

↓↓

N

↓ ↓ ↓↓ ↓ ↓↓ ↓↓ ↓

N

N N N N N N

Alpha granulesc

↓↓ ↓↓ ↓ ↓↓

Human diseases are in bold, mouse models in plain text. C, lysosomal enzyme content; S, lysosomal enzyme secretion; N, normal; ↑, increased; ↓, reduced. c Alpha granule abnormalities have not been noted in mouse models of dense SPD with the exception of gunmetal.60 d Results vary according to method used. e Serotonin concentration: ↓↓ highly reduced (<1 ␮g per platelet). f Whole mount: ↓↓ highly reduced (<3 granules per platelet on average). g All dense granules stained with mepacrine exhibited reduced fluorescence intensity and reduced flashing. b

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the AP-3 complex.68 The AP-3 complex is a heterotetramer with homology to two adaptor protein complexes (AP-1 and AP-2) that associate with clathrin and are involved in vesicle trafficking (see Reference 57). AP-1 is involved in protein sorting in the trans-Golgi network and AP-2 is involved in endocytosis at the plasma membrane. AP-3 may be involved in sorting cargo proteins that contain dileucine-based signals to lysosomes and lysosome-related organelles.69 AP-3 is involved in targeting P-selectin to secretory granules in endothelial cells but does not appear to be required for targeting to alpha granules in platelets; this suggests that sorting mechanisms may be mechanistically different in these cells despite their related granule contents70–72 (see Reference 38). HPS-3 has recently been described in central Puerto Rico.73 The HPS-3 gene encodes a 114 kDa protein with no homology to any functional proteins; it is predicted to function in vesicle trafficking due to several consensus protein sorting signals.73 The cocoa mouse is the model for this disease.74 HPS-3 disease is milder than HPS-1, while HPS-2 is intermediate in severity.73, 75 Human HPS-4 is another variant that shares the same defective gene (HPS-4), of unknown function, found in light ear mice.76 Light ear mice also show reduced levels of HPS-1 and there is evidence for partial co-localization of HPS-1 and HPS-4 in transfected melanoma cells suggesting that they may function in the same pathway.76 Other genetic causes of dense SPD have been identified in mice. The ashen mouse has a mutation that leads to an abnormal transcript for Rab27a, a member of a family of vesicle trafficking proteins.77 The pallid mouse has a dense granule and lysosomal defect associated with abnormal pallidin gene which codes for a protein that associates with syntaxin 13, a SNARE protein involved in vesicle trafficking.13, 78 CHS is also associated with dense granule abnormalities, hypopigmentation, immune deficiency, giant lysosomes and melanosomes, and reduced or absent platelet granules.79 Human CHS and mouse CHS (beige) are due to a mutation in Lyst, a 430 kDa cytoplasmic protein which has no known function.80, 81

Golgi-associated alpha granule precursors are found in α-SPD megakaryocytes.83, 85 α-SPD platelets have severely decreased levels of megakaryocyte synthesized soluble proteins (such as β-thromboglobulin) but these molecules can be found in the plasma suggesting impaired alpha granule storage mechanisms or premature release.85–88 Normal levels of pinocytosed proteins (IgG and albumin)71, 84, 86 are found in small granules in the megakaryocytes and platelets. In some α-SPD patients the alpha granule membrane protein P-selectin redistributes to the platelet membrane following cell activation71, 72 which also argues that the defect in alpha granule storage pool deficiency is not in exocytosis per se, but in the packing or formation of the alpha granule.71, 85 Dense granules appear to take up serotonin normally even though dense granule secretion may be reduced.87, 89 The Weibel–Palade body (see Reference 38), a secretory granule of endothelial cells which is closely related to the alpha granule, appears to form and develop normally in α-SPD.90 Relatively few families with α-SPD have been described and the molecular defects responsible have not been elucidated.5, 91 Mixed alpha and dense SPD is rare in humans and the molecular causes are unknown. Gunmetal mice have features of alpha and dense SPD with quantitative reductions in alpha granule proteins and dense granule content.92 Gunmetal mouse platelets are larger, reduced in number and contain bizarre alpha granules with striated inclusions; there is also reduced secretion from cytotoxic T cells.92–94 Gunmetal mice have a defect in the gene for Rab geranylgeranyl transferase (RABGGTA)95 leading to reduced geranylgeryanylation of a number of Rabs, including Rab27a.13

Lysosomes Platelets contain only a few primary and secondary lysosomes.96 Lysosomes are formed early in the maturation of megakaryocytes even before alpha granule development.52 Platelet lysosomes are typically 175–250 nm in diameter, and primary lysosomes are small, electron dense granules.97 Platelet lysosomes are more heterogeneous in nature and composition than alpha or dense granules.98 Platelet lysosomes contain the ubiquitous lysosomal membrane proteins LAMP-1, LAMP-2, and CD63 (LAMP-3).50 In contrast to trafficking to the alpha granule, endocytic trafficking to platelet lysosomes via endosomes appears to be clathrin-independent.25 Lysosomal secretion can be elicited in vitro from platelets with strong,

Human α-SPD Human α-SPD (or gray platelet syndrome82 ) is a heterogeneous disorder with markedly reduced alpha granules and a variable tendency for bleeding.71, 83 Platelet dense granules and lysosomes appear normal in α-SPD.84 Small abnormal vesicles that resemble 296

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somewhat non-physiologic stimuli but, in vivo there is less evidence for lysosomal exocytosis than for release of alpha and dense granules.99 Platelet lysosomes, like lysosomes in other cells, contain a number of acid hydrolases (at least 13), cathepsins D and E100 , and other proteins.101, 102 MVBs found in platelets and a number of other cells may represent endosomal/lysosomal compartments41 that contain small vesicles (exosomes) derived from endocytosis.103–105 These exosomes may be eventually sorted to dense or alpha granules.8, 43 The mouse models of HPS or dense SPD provide additional information on lysosome biogenesis. AP-3 plays a role in targeting the lysosomal membrane protein LAMP-1 to lysosomes, yet the exact mechanism is still unknown (see Reference 57). AP-3 deficient mice (mocha and pearl) both show decreased platelet lysosome secretion (Table 1), but have normal concentrations of platelet lysosomal enzymes despite absent dense granule content.106 Lysosomal sorting/biogenesis pathways may differ in platelets and other cells because many dense SPD mice show tissue-restricted lysosomal abnormalities (Table 1). Dense SPD mice have normal levels of platelet lysosomal enzymes and but some have altered secretion. At the same time the lysosomal content and secretion in other tissues (e.g. spleen, liver and kidney) may or may not parallel the platelet defect (Table 1).106–113 Some dense SPD mice (e.g. cocoa and subtle gray) have normal concentration of lysosomal enzymes in all tissues examined.112, 113 The gunmetal mouse had normal lysosomal secretion but defective alpha and dense granules suggesting that the RabGGTase defect may specifically target Rabs involved in alpha and dense granule pathways, but not in the lysosomal pathway. Taken together, the data on the mouse mutants suggest there are important differences between platelet lysosomes and lysosomes in other tissues. In some mutants there is a divergence between lysosomal content and secretion in platelets and other cells. Moreover, in other mutants, there is abnormal platelet secretion despite normal lysosomal content which may imply defects in lysosomal secretory machinery or signaling mechanisms.

functionally distinct organelles that have related but unique ontogenies. There is also abundant data, particularly for alpha granules, that these platelet granules are derived from biosynthetic pathways from the trans-Golgi network, as well as from endocytic pathways that traffic proteins from the plasma membrane surface. In megakaryocytes, the multivesicular body appears to play a key role in endocytic sorting of molecules to both the alpha and dense granule, and perhaps lysosomes. Indeed the multivesicular body, like alpha granules, has an aggregate structure, in which different granule molecules are segregated and stored. Two different working models for granule biogenesis may be proposed (Figure 1). Model 1 postulates that platelet granule contents develop through direct biosynthetic sorting from the trans-Golgi network and through endocytic sorting through the MVB. Model 2 posits that platelet granules develop primarily through biosynthetic and endocytic sorting through the MVB. In both models the dense granule may form through direct sorting from other organelles or primarily through sorting from the lysosome. There are several exciting areas for future research into the development of platelet granules. One promising area is the investigation of the unique molecular mechanisms responsible for alpha granule biogenesis. Current data suggests that the platelet alpha granule, despite its similarity to the Weibel–Palade body of endothelial cells, has different molecular mechanisms or pathways for formation. The MVB appears to be a key sorting organelle for the dense and alpha granule, but the molecular mechanisms that mediate this sorting are not understood. Another important goal is to define the nature of the relationship between the ontogeny of the dense granule and the lysosome in platelets. Despite common properties, studies of dense SPD reveal that platelet dense granules and lysosomes are often differentially affected, suggesting they have related but distinct developmental pathways. Finally, the relationship between coat color abnormalities and dense SPDs is intriguing because when dense SPD mice are crossed into strains of mice with different coat colors the phenotype is moderated despite the presence of the same gene defect. The convergence of new insights into megakaryocyte development and the identification of genes responsible for platelet SPDs is likely to help uncover the molecular pathways that lead to platelet granule biogenesis and define the ontological relationships of these bodies to other secretory organelles.

Summary and future perspectives—models of platelet granule biogenesis There is very strong evidence that lysosomes, alpha and dense granules are morphologically and 297

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Figure 1. Working models for biogenesis of platelet granules in the megakaryocyte. Model I proposes that granule biogenesis occurs through two pathways: (1) a direct biosynthetic pathway from the trans-Golgi network to the granules and (2) an endosomal pathway where endocytosed molecules are sorted and targeted in the MVB. The growing evidence from SPD in humans and mice suggests that adaptor protein (AP) complexes are involved in cargo selection and packaging in these pathways. Specifically, AP-3 may be involved in trafficking to the lysosome and/or dense granule. In some cells AP-1 is implicated in the sorting of proteins through an indirect pathway to the plasma membrane and may function similarly in megakaryocytes and platelets. Other unidentified AP-complexes might function also in trafficking from the trans-Golgi network to alpha granules as well as in trafficking from the MVB. Model II, granule biogenesis results from biosynthetic and endocytic sorting through the MVB. The MVB functions as the major sorting organelle in this model. There is some evidence (see text) that the direct biosynthetic pathway contributes to alpha granule formation (Model I) but this pathway is not yet clearly understood. In both models, the dense granule may or may not arise from sorting through the lysosome.

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Acknowledgements

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The authors gratefully acknowledge discussions with Drs. Janos Polgár and Marianne Wessling–Resnick. This work was supported in part by NIH grants (HL-64057) to G.L.R. and by a graduate student training grant (2T32 CA09078) for S.M.K.

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