Platelet Dense Granules

Thrombosis Research 95 (1999) 1–18

MINI-REVIEW

Platelet Dense Granules: Structure,Function and Implications for HaemostasisArchibald McNicol1,2 and Sara J. Israels3,4

1Department of Oral Biology, 2Department of Pharmacology and Therapeutics and 3Department ofPediatrics and the 4Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada.

(Received 3 August 1998 by J.S. Bennett; revised/accepted 15 December 1998)

Key Words: Dense granule; Exocytosis; Granule defi- structural, pathophysiological, and genetic obser-ciency; Membrane composition; Granule contents vations.

Platelets play pivotal roles in the haemostatic1. Structureprocess including detection of vascular le-

sions, adherence at sites of injury, recruit-1.1. Dense Granule Contentment of additional platelets, and consolidation into

a haemostatic plug [1]. Although the presence ofNormal human platelets contain three to eightsmall circulating elements that formed haemostaticdense granules per platelet [7,8] with a luminal pHplugs was initially reported in 1882 by both Bizzoz-of 6.1 [9,10]. The granules are highly osmophilicero and Hayem [2,3], it was not until the adventwhen viewed by transmission electron microscopyof electron microscopy and the subsequent devel-and are inherently electron dense when viewed inopment of relatively sophisticated fixation andunstained whole mount preparations (Figure 1).staining techniques, that a full appreciation of theThese granules contain serotonin, a nonmetabolicintracellular architecture of these anucleate cellsadenine nucleotide pool of ATP and ADP, calcium,was reached. Numerous studies have now definedand pyrophosphate [9,10]. Holmsen and Weiss cal-the role that the intracellular organelles contributeculated the intragranular concentrations to be 65to the multifunctional nature of platelets, includingmM serotonin, 436 mM ATP, 653 mM ADP, 2.2haemostasis, thrombosis, allergic inflammation,M calcium, and 326 mM pyrophosphate [9]. Thenonallergic responses, and tumour progressiongranular adenine nucleotide pool, which is likely in[1,4]. Of particular importance is the process ofthe form of insoluble calcium complexes, is distinctexocytosis from three types of secretory granule:from the cytoplasmic nucleotides, and these twoalpha granules, lysosomes, and dense granulespools are not readily exchangeable. Studies in pa-(Table 1). Alpha granules and lysosomes have beentients with dense granule deficiencies indicate thatreviewed in depth [5,6]. In the present review, thedeficiencies in serotonin and ATP, and of ADPstructure, function, and dysfunction of plateletand calcium, are highly correlative [9]. In general,dense granules will be discussed in light of recentdense granules from other species tend to containserotonin, calcium, and adenine nucleotides, al-

Abbreviations: PSGL-1, P-selectin glycoprotein ligand-1; HPS,though absolute levels vary significantly [11]. Fur-Hermansky-Pudlak Syndrome; CHS, Chediak-Higashi Syndrome;thermore porcine platelets have been shown to alsoNSF, N-ethylmaleimide-sensitive factor; SNAPs, synaptosomal-

associated proteins; SPD, storage pool deficiency. contain histamine [12].Corresponding author: S.J. Israels, Manitoba Institute of Cell Biol- After exocytosis, ADP acts as a platelet agonist,ogy, 100 Olivia Street, Winnipeg, Manitoba, R3E 0V9, Canada.

potentially via multiple distinct P2 receptors [13],Tel: 11 (204) 787 4141; Fax: 11 (204) 787 2507; E-mail: ,[email protected].. and is important to the activation of additional

0049-3848/99 $–see front matter 1999 Elsevier Science Ltd. All rights reserved.PII S0049-3848(99)00015-8

2 A. McNicol et al./Thrombosis Research 95 (1999) 1–18

Table 1. Platelet granule contents

Alpha granulesa Dense granules Lysosomal granulesb

Albumin Serotonin Cathepsin DFibrinogen ATP Cathepsin EFibronectin ADP Carboxypeptidase AVitronectin Calcium Carboxypeptidase BOsteonectin Pyrophosphate Proline carboxypeptidasevon Willebrand factor b-N-acetyl-d-hexosaminidasevon Willebrand antigen II b-d-glucuronidaseThrombospondin b-d-galactosidasePlatelet factor 4 a-d-mannosidaseIgG, IgA, IgM a-l-arabinofuranosidaseC1 inhibitor a-d-galactosidasePlasminogen a-l-fucosidasePlasminogen activator inhibitor-1 b-d-fucosidasePlatelet-derived collagenase inhibitor b-d-glucosidaseHigh molecular weight kininogen a-d-glucosidaseProtein S Acid phosphatasea2-antitrypsin Arylsulphatasea2-macroglobulina2-antiplasminMultimerinPlatelet basic proteinb-thromboglobulinHistidine-rich glycoproteinConnective tissue-activating protein IIINeutrophil-activating protein IIPlatelet-derived growth factorTransforming growth factor b-Endothelial cell growth factorCoagulation factor VCoagulation factor VIII

aFrom reference [5].bFrom reference [6].

circulating platelets and, hence, to their recruit- However, the role of platelet-derived ATP on vas-cular patency remains largely unknown.ment to the site of injury [1]. The significance of

this effect is seen in the compromised haemostatic Serotonin is not synthesised in platelets but isactively taken up from the plasma and accumulatedcapacity of patients with dense granule defi-

ciencies. in dense granules where it is likely complexed withATP and potentially with calcium [16]. The seroto-Dense granules contain a nonmetabolic pool of

ATP, which is released during exocytosis. ATP is nin released by exocytosis is relatively stable andfunctions as a weak platelet agonist on 5HT2 recep-rapidly removed from the plasma by conversion to

AMP and adenosine [9]. An ATP receptor is pres- tors [16]. Dense granule derived serotonin, there-fore, acts to activate additional platelets and thusent in platelets [14], and a role in activation has

been proposed [15]. recruit them into the aggregate [1], although it isprobably less important in this respect than ADP.Interestingly, ATP also acts on P2Y receptors on

endothelial cells to release prostacyclin and nitric The positive feedback effect of serotonin may beof secondary importance to its vasoconstrictive ac-oxide, which in turn cause vasodilation. It is possi-

ble that, in pathological situations where the vascu- tion, which reduces flow at the site of injury andthereby limits blood loss [17].lar endothelium has been damaged or removed,

ATP, via a direct action on vascular smooth mus- Calcium imparts an electron dense property tohuman dense granules allowing them to be viewedcle purine receptors, causes vasoconstriction [15].

3A. McNicol et al./Thrombosis Research 95 (1999) 1–18

Fig. 1. Transmission electron micrographs of platelet whole mounts (a and b) and thin sections of glutaraldehyde-osmiumfixed plastic embedded platelets (c and d), from a normal human control (a and c) and an individual with Hermansky-Pudlak Syndrome (b and d). Arrowheads identify dense granules. The patient with Hermansky-Pudlak Syndrome hasbeen previously described in detail (references [45,70]).

directly by electron microscopy with no additional be important for the binding of adhesive proteinsto their platelet receptors.staining (Figure 1). Dense granule calcium theoret-

ically accounts for 60–70% of the total plateletcalcium [9]. The function, if any, of released cal- 1.2. Dense Granule Membranescium remains elusive, although it is postulated thatsecretion will produce elevated local levels at the Progress has been made in the identification and

cataloguing of proteins which are inherent in theexternal surface of activated platelets, which may


dense granule membrane. A H1-pumping ATPase, including fibrinogen, fibronectin, and von Wille-brand factor becomes associated with the plateletdistinct from that at the plasma membrane, has

been identified that acts to maintain the low pH cytoskeleton through an attachment to talin andvinculin [35–37].of the granule lumen [18]. The ionic pumps respon-

sible for the acidic granule lumen also may play a Dense granules also contain P-selectin (GMP-140) [38], a leukocyte binding protein, which wasrole in serotonin accumulation [19]. Fishkes and

Rudnick have reported that at least two H1 ions initially localised to platelet alpha granules [39].P-selectin is a transmembrane protein with luminalare countertransported per serotonin cation [20].

The mechanism of granule accumulation of seroto- “lectin” and “EGF” domains and nine tandem con-sensus repeats related to complement-binding pro-nin differs from that responsible for transport

across plasma membranes [21]; a platelet serotonin teins, followed by a transmembrane region and thecytoplasmic domain [40]. After activation, the lumi-transporter has been cloned and its structure de-

duced [22]. nal region of P-selectin is expressed on the surface ofactivated platelets and mediates platelet-leukocyteRendu et al. localised the tyrosine kinase src to

dense granules [23]. However, this observation has interaction through P-selectin glycoprotein ligand-1(PSGL-1), a mucin-like transmembrane proteinbeen challenged by two groups who have reported

that src is present on plasma, rather than on inter- constitutively expressed on leukocytes [41], and isfound at the sites of platelet-platelet contact [38].nal, membranes [24,25].

There are several reports of the presence of low Blockade of P-selectin attenuates aggregation, sug-gesting an additional role in platelet-platelet inter-molecular mass monomeric GTP binding proteins

in platelets including: rab1, rab3B, rab4, rab6, rab8, action [42].CD63 (LIMP-CD63, PTLGP40) [43] was initiallyrap1, ralA, and ralB [26–31]. These proteins have

been implicated in the exocytosis process and likely identified as a lysosomal granule membrane protein[44]. However, immunological studies have shownplay a role in the docking of granules to the plasma

membrane that precedes, and is required for, exo- that CD63 is also present in the membranes ofdense granules [38,45]. Thus CD63, like P-selectin,cytosis. Although most subcellular localisation

studies in platelets have focused on alpha granules, is present in the membranes of two of the granularcompartments in platelets. CD63 has four trans-Mark et al. have demonstrated that ral is associated

with the membranes of dense granules [31]. membrane domains with three N-linked glycosyla-tion sites clustered on one of the luminal regionsPlatelet plasma membranes have a variety of

adhesive receptors, including GPIb and the aIIb/ [46]. It is identical to the malignant melanoma anti-gen ME491 [47] as well as to MLA1, NGA, andb3, aV/b3, a5/b1, a6/b1, and a2/b1 integrins, which

function to bind platelets to a number of adhesive AD1 [43] and is a member of the tetraspan superfamily of proteins [43]. The specific function(s) offactors present in plasma, on circulating cells and

on exposed subendothelium. Interestingly, dense the tetraspans, including CD63, remains elusivealthough a role as a mediator of cell surface proteingranule membranes also have been shown to con-

tain adhesive receptors whose binding domains are complexes facilitating signaling has been pro-posed [43].luminal but that are expressed on the surface of

platelets after exocytosis and thereby expand the LAMP-1 (CD107a) and LAMP-2 (CD107b) area pair of proteins that have been identified as com-pool of surface adhesive receptors on activated

platelets. Elegant immunohistochemical studies ponents of lysosomal granule membranes [48,49].Immunological analysis have shown that LAMP-2,have demonstrated that both GPIb and the aIIb/b3

integrin are components of dense granule mem- but not LAMP-1, is also present in platelet densegranule membranes [50]. LAMP-2 therefore pro-branes [32]. GPIb is the major von Willebrand fac-

tor receptor in platelets, and when present on the vides the second example of co-localisation of aprotein to lysosomal and dense granule mem-plasma membrane its cytoplasmic tail is associated

with the cytoskeleton principally by an interaction branes. This is consistent with the murine modelsreported by Novak et al., who suggested a closewith actin binding protein [33,34]. Similarly, after

exocytosis, the cytoplasmic portion of granular aIIb/ relationship between these two granule popula-tions [51,52]. Structurally LAMP-2 has a large lu-b3, the receptor for a number of adhesive proteins


minal domain containing four disulphide loops and characterized phospholipase C-mediated hydrolysisof phosphatidylinositol 4,5-bisphosphate (PIP2) re-a large number of N- and O-glycans [53]. The pres-

ence of carbohydrates on the luminal regions of sulting in the bifurcating signal transduction path-ways mediated by elevated cytosolic calcium levelsLAMP-2, and of CD63, may provide a continuous

carbohydrate coating on the inner surface of the and protein kinase C activity [59,60]. Studies byGerrard et al. suggest that the synergistic activationgranular membrane. Although this coating would

protect the membrane from enzymatic degradation of both pathways is necessary for granule release[61]. This in turn led to a proposed mechanism byin lysosomal granules, its role in dense granules is

unclear. The intragranular domain of LAMP-2 is which protein kinase C–mediated events act to fusethe granule and plasma membranes, and the in-expressed on the surface of activated platelets [50]creased intracellular calcium, with consequent cy-and may play a role in protecting the plasma mem-toskeletal contraction, expel the granular contentsbrane from hydrolysis [53]. LAMP-2 also is ex-[62]. Consistent with this model, Morimoto andpressed on the surface of peripheral blood mono-Ogihara demonstrated that ATP is required fornuclear cells where it mediates adhesion to vasculardense granule exocytosis both as a cofactor forendothelium and may contribute to lymphocyteprotein phosphorylation and by priming the gran-migration during inflammation [54].ules prior to release [63]. Parallels can be drawnMolecular analysis of patients with a pair ofwith other exocytotic processes, notably chromaf-dense granule defects, Hermansky-Pudlak Syn-fin granule release. A two-step model of exocytosisdrome (HPS) and Chediak-Higashi Syndromehas been proposed that, although consistent with(CHS) (see below), has led to the deduced struc-studies specifically in dense granules [61–63], in-ture of two additional dense granule membranecludes observations from other exocytotic cellsproteins. The HPS protein has two transmembrane[64]. This model involves (1) docking of the gran-domains with a cytoplasmic loop [55,56] and ap-ules to the inner leaflet of the plasma membrane,pears to have a unique structure. In contrast theand (2) fusion of granule and plasma membranes.CHS protein has no transmembrane domains but

a motif consistent with membrane association[56,57]. It is likely that the CHS protein is not 2.1.1. Dockinginherent in the granule membranes but is inti- The model involves the initial docking of the gran-mately associated with the its cytosolic surface. Al- ules with the inner wall of the external plateletthough no function can be implied from the struc- membrane. Low molecular mass GTP binding pro-ture of either protein, the CHS protein has similar teins, notably rab3, have been implicated in thearchitecture to a yeast protein, which plays a role docking process. Rab3 is present in platelet

a-granules and is phosphorylated in response toin vacuolar sorting [56].agonists [27], although to date its presence in densegranules has not been reported. Rab3 is likely im-portant in the formation of complexes with2. FunctionSNARE proteins [64]. SNARE proteins are inher-ent components of both granule and plasma mem-2.1. Dense Granule Exocytosisbranes that recognise each other and, through theformation of a complex termed the 7S dockingThe mechanism underlying dense granule exocytosis

is not clearly defined. Secretion accompanies plate- complex, facilitate the docking of the granule tothe membrane. Lemons et al. demonstrated thatlet activation induced by a number of agonists, in-

cluding thrombin, thrombin receptor-activating pep- platelet membranes contain two members of theSNARE family: syntaxins 2 and 4 [65]. This initialtide, and thromboxane analogues. In addition, ADP

and collagen both cause dense granule release, al- docking of the dense granule to the plasma mem-brane is not ATP dependent [63].though in both cases this secretion is aspirin sensitive

indicating that the exocytosis occurs secondary to A series of priming reactions have been hypoth-esised to prepare the docked granules for mem-the release of thromboxane A2 [58]. Thrombin,

thrombin receptor-activating peptide, and throm- brane fusion. This process includes the ATP-dependent binding of the 7S docking complex toboxane analogues each activate platelets by the well-


other proteins, such as the cytoplasmic proteins deficiency (to differentiate it from a-storage poolN-ethylmaleimide-sensitive factor (NSF) and syn- deficiency), or dense body deficiency, and canaptosomal-associated proteins (SNAPs), to form occur either in isolation or as part of other congeni-the 20S fusion complex. NSF and two forms of tal syndromes. The clinical presentation is one ofSNAP, a-SNAP and g-SNAP, are present in plate- a moderate bleeding diathesis, although a minoritylets [65]. In addition SNARE activity is detectible of individuals may manifest more severe, life-in platelet homogenates [65]. This activity depends threatening, bleeding. Typical manifestations in-on the SNARE-dependent association of SNAP clude: easy bruising, epistaxis, menorrhagia, post-with NSF and therefore demonstrates that the partum hemorrhage, and bleeding after surgical orthree components are both present and functional dental procedures.in platelets. The 20S fusion complex is important The pathogenesis of SPD is variable. In SPDfor ATP-dependent priming of the platelet for exo- associated with congenital syndromes of pigmentcytosis. Similarly roles for enzymes that synthesise abnormality, evidence suggests abnormal organellePIP2 [65] and for the cytoskeleton, specifically myo- development. In some nonalbino-associated vari-sin light chain kinase [61,66], have been proposed ants, the primary defect appears to be one of up-in the priming process. take or storage of biogenic amines [71].

Definitive laboratory evidence for the diagnosis2.1.2. Membrane Fusion of SPD is the finding of decreased ADP and seroto-The next stage in the exocytosis pathway is the nin content of platelets and an elevated ATP/ADPfusion of granule membrane to the plasma mem-

ratio [72,73]. Clinically the diagnosis is often basedbrane with the resultant secretion of granular con-on a prolonged bleeding time and abnormal plate-tents. The mechanism of this fusion is not under-let aggregation studies, typically with poor re-stood although some evidence suggests that SNAPsponses to collagen and no secondary wave in re-binding to NSF stimulates the ATPase activity ofsponse to ADP or epinephrine [72,74]. However,NSF causing the 20S fusion complex to disrupt andin vitro aggregation patterns may be normal, espe-SNAREs to be released. These SNAREs then maycially in cases of partial deficiency. Nieuwenhuisplay an integral part in the fusion process [64].et al. reported 106 patients with confirmed SPDCalcium is believed to be the trigger for exocytosis.(as determined by decreased platelet content ofSynaptotagamin is the calcium receptor thought toserotonin and adenine nucleotides), of whom onlymediate these effects [64]; however, synaptotag-33% had the characteristic abnormal aggregationamin I is not present in platelets, although thepattern and 23% had completely normal aggrega-presence of other isoforms has not been addressed.

It is proposed that the formation of fusion pores tion profiles [75]. Abnormalities in dense granulebetween the granule and plasma membranes is the secretion can be identified by measuring the releasefinal step in the exocytosis pathway [64]. This pro- of 14C- serotonin from labelled platelets [61] or bycess is not well defined, although roles for integral the release of adenine nucleotides detected usinggranule proteins, such as synaptophysin and synap- a luciferin/luciferase assay [76].togyrin, have been suggested [64,67,68]. However, Electron microscopy can be particularly helpfulalthough synaptophysin is present in rabbit plate- in differentiating dense granule deficiency fromlets [69], immunological evidence suggests that it release defects and identifying individuals with par-is not present in human platelets [70]. To date, tial deficiency where aggregation studies may notthe identity of the dense granule protein(s) that be sufficiently sensitive [8,71]. This is particularlyperform fusogenic functions during exocytosis re- true of a rapid whole mount method, originallymains unknown. described by White and Witkop et al. [7,77], in

which platelet-rich plasma is placed on a formvar-coated electron microscopy grid, the platelets are3. Clinical Correlatesallowed to settle, and the dense granules arecounted. Normal dense granule numbers per plate-3.1. Storage Pool Deficiencylet approximate four to eight (Figure 1). Densegranules can be identified by the uranaffin reaction,Congenital deficiency of dense granules is referred

to as storage pool deficiency (SPD), d-storage pool specific for 59 phosphonucleotides [78] or by the


Table 2. Storage pool deficiency syndromesfluorescent dye quinacrine (mepacrine), which isactively and specifically taken up by dense gran- I Inherited SPDules. Quinacrine uptake can be quantitated by fluo-

IA SPD associated with pigment abnormalitiesrescent microscopy [79] or by flow cytometry Hermansky-Pudlak Syndrome[80,81]. Chediak-Higashi Syndrome

More recently, Weiss et al. have demonstrated IB SPD associated with other inherited disordersTAR syndromeother functional abnormalities of dense granule-Wiskott-Aldrich Syndromedeficient platelets. These defects include an adhe-Familial leukemiasion defect at high shear stress [82] and impaired

IC Isolated SPDprothrombinase activity [83], both of which appear Autosominol-dominant inheritanceto be secondary to a deficiency of secreted ADP. Empty sack syndromeIn addition, the same group has demonstrated that Giant dense body disorder

ID Combined a,d-SPDdense granule-deficient platelets show increasedII Aquired SPDsensitivity to endogenous ADP, as manifest by en-

In vivo activationhanced ADP-induced calcium influx [73] Myelodysplasia and myeloproliferative disordersClinically, patients with mucocutaneous bleed-

ing and a prolonged bleeding time, should bescreened for SPD by methods that evaluate boththe presence of dense granules and the release of

Two families have been described with giantgranular contents [8,50,75].

dense body disorder [74,89], associated with variableclinical bleeding and in vitro aggregation responses.

3.2. Variant Storage Pool Deficiency The dense granules are larger than normal (indeedlarger than a-granules) with abnormal structure, as

A combined deficiency of a-granules and dense evaluated by transmission electron microscopy.granules, termed a,d-SPD, has been described byWeiss et al. [84]. Individuals with either partial or

3.3. Syndromes Associatedcomplete absence of a-granules, in conjunction withwith Storage Pool Deficiencya dense granule deficiency, have been described.

These platelets have decreased numbers of denseThere are a number of well-described syndromes ingranules, as evaluated by electron microscopy, aswhich SPD is associated with abnormalities in awell as decreased a-granule contents, including fi-variety of tissues (Table 2). Many of these syn-brinogen, platelet factor 4, b-thromboglobulin, anddromes share abnormalities of melanocytes and ly-platelet-derived growth factor. Some patients withsosomes, as well as platelet dense granules, sug-a,d-SPD also have decreased levels, and expression,gesting a common origin of these intracellularof P-selectin [85].organelles although the specific biochemical abnor-The empty sack syndrome was described bymalities leading to defects in multiple subcellularMcNicol et al. in two sisters with a moderate bleed-organelles remains unknown [90]. This constellationing diathesis [86]. Electron microscopy revealed de-of abnormalities is parallelled by a number of mu-creased numbers of typical dense granules, althoughrine mutations affecting pigmentation, platelet func-the granule membranes, as demonstrated by immu-tion, and lysosomal enzyme content (see below) [91].nodetection of specific dense granule membrane

proteins, were present. In addition, the granular con-3.3.1. Hermansky-Pudlak Syndrometents, both serotonin and adenine nucleotides, wereHPS is an autosomal recessive disorder with tyrosi-absent. These patients appear similar to patientsnase-positive oculocutaneous albinism, SPD, and ly-previously reported by Lorez et al. where uranaffin-sosomal accumulation of ceroid lipofuscin [92,93].detected granules were more numerous than quina-The ceroid lipofuscin accumulation is associatedcrine-containing organelles [87]. These syndromeswith pulmonary fibrosis and granulomatous colitis.may represent a defect in the uptake or retentionIndividuals with HPS often have profound denseof serotonin and adenine nucleotides, as suggested

by Lages et al. [88]. granule deficiency and associated severe, repeated


mucocutaneous bleeding (epistaxis, menorrhagia) tion of the mouse beige gene on chromosome 13[108,109].and postoperative bleeding requiring platelet and

The CHS1 protein is expressed in the cytosol ofred cell transfusions [94].a variety of tissues [109] but has no homology toIt has been presumed that the fundamental defectother known proteins although it does contain awould involve a common protein required for thenumber of protein motifs similar to a yeast serine/structure or function of membranes of three organ-threonine protein kinase, Vps 15, associated withelles: melanosomes, platelet dense granules, and ly-vacuolar protein sorting [55]. These studies suggestsosomes. The recently cloned HPS gene encodes athat the defect in CHS may be an abnormality of79.3-kD protein of unknown function predicted tosubcellular organelle protein trafficking [56,100].be a granule membrane component on the basis of

its transmembrane structure [55,56]. The gene is3.3.3. Association withmapped to chromosome 10q23 by linkage analysisOther Congenital Syndromesof patients from northwest Puerto Rico, where thereSPD has been described in individuals with TARis a high prevalence of HPS [95–97]. The Puerto(thrombocytopenia with absent radii) syndromeRican patients have been found to have a 16-bp[110] and Wiskott-Aldrich syndrome [111,112], al-duplication in exon 15 of the HPS gene, which isthough it is not present in all patients [113,114].not found in other individuals with HPS [94]. TwoThe relationship between SPD and the other anom-different point mutations have been identified inalies in these syndromes is not understood, butnon-Puerto Rican populations [55].these syndromes are associated with abnormalitiesof megakaryocytopoiesis, including ultrastructural3.3.2. Chediak-Higashi Syndrome anomalies of the megakaryocytes and platelets that

CHS is an autosomal recessive disorder with hypo- may be related to the deficiency of dense granules.pigmentation, immune deficiency, SPD, and neuro- Gerrard et al. have described a large, multigenera-logical abnormalities [98,99]. The most important tional kindred with SPD and a high incidence ofclinical manifestations are recurrent severe pyogenic myeloid leukemia [115]. Unlike the acquired forminfections. Individuals may present various other of SPD, which occurs in individuals with myelopro-immunologic abnormalities, including neutropenia liferative disease and leukemia, the SPD in thisand neutrophil dysfunction and lack of natural killer family preceded the onset of leukemia in severalcell cytotoxicity [100]. The majority of the patients family members, and SPD was present in membersenter an accelerated phase, which is manifested by without malignancy at the time of investigation.a lymphoproliferative syndrome with lymphohistio- Other families with an association between an in-cytic infiltration of lymph nodes, liver and spleen, creased incidence of leukemia and platelet dys-pancytopenia and die unless treated by bone mar- function have been described, although the plateletrow transplantation [101–104]. The bleeding mani- defects have been less well characterized [116,117].festations, due to SPD, are mild to moderate andprimarily mucocutaneous [105,106]. 3.4. Animal Models of SPD

The histological characteristics of CHS are giantgranular inclusion bodies, giant lysosomes, and SPD has been identified in several animal species,giant melanosomes. The giant inclusion bodies are including mice, cattle, cats, dogs, mink, and pigs,found in all leukocytes of peripheral blood and and some have been advanced as models for indi-bone marrow. Platelets, however, do not have giant vidual forms of SPD in man.granules but are dense granule deficient [74]. Mela- Novak, Swank, and colleagues have extensivelynocytes have giant melanosomes, and Schwann catalogued, and partially characterized, a numbercells have characteristic giant granules. Similar dis- of murine models of SPD (Table 3). Many of theorders have been described in other mammals in- models have “classic” SPD phenotypes, such ascluding the beige mouse, which has a clinical and decreased dense granule numbers, decreased levelshistological phenotype similar to human CHS but of serotonin and ADP, and elevated ATP/ADPlacks the accelerated phase. The human CHS1 gene ratios. In addition, many have associated pigment,

inner ear. and lysosomal defects [51,52,91,118–maps to 1q42-43 [96,107], homologous to the posi-


122]. As outlined above, the only definitive correla-tion between a murine model and human SPD isthe beige mouse model of CHS [52,103,104]. TheCHS gene maps to chromosome 1q43 in humansand the corresponding chromosome 13 in the mu-rine model.

To date, the murine equivalent of HPS has notbeen definitively identified, although two candidatemodels have been proposed. Ruby eye and paleear both have phenotypic characteristics similar tothose of HPS [52,91,122], and both map to murinechromosome 19; HPS is located on the correspond-ing human chromosome 10q24.1-q25.1 [91,96,97].

Although of interest, none of the other murinestrains have been advanced as models for humanSPD. Cocoa, muted, and mocha mutations are densegranule deficient as reported by whole mount tech-niques, serotonin levels, and ATP release [119,120].However, quinacrine-labelled granules were nu-merically normal suggesting that the granules arepresent but have reduced contents [119,120]. Thesestrains are similar, but not identical, to empty sacksyndrome patients who have deficiencies in wholemount dense granule numbers, serotonin levels,and ATP release but also have reduced quinacrine-labelled granules [86].

In addition to humans and mice, CHS has beenreported to occur in cattle, cats, Aleutian mink, andkiller whales. To date, however, these comparisonshave been based on phenotypic, rather than geno-typic, analysis. Myers et al. have reported that bo-vine CHS platelets contain decreased numbers ofgranules, and this decrease is associated with re-duced granular levels of serotonin, ADP, and ATP[123–125]. Interestingly, and in contrast to humanCHS and the beige mouse, there was no lysosomalabnormality observed in the platelets of CHS cattle[123]. The same group also has studied the pro-posed feline CHS counterpart and indicated thatthese cats also have reduced granular levels of sero-tonin, ADP, and ATP, with no associated deficien-cies of lysosomal enzymes. These cats do have ocu-lar and dermatological abnormalities characteristicof CHS [126]. Studies in CHS Aleutian mink alsohave indicated reduced granular levels of seroto-nin, ADP, and ATP, as well as ocular and dermato-logical abnormalities [127].

Two groups of SPD pigs have been identified invon Willebrand disease swine colonies [128,129].T

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;2,a

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.

In each case, the SPD seems to be unrelated to


the von Willebrand disease. These pigs have fewer the synthetic vasopressin analogue, 1-desamino-8-dense granules by electron microscopy and quina- d-arginine vasopressin (desmopressin, DDAVP),crine fluorescence, as well as reduced levels of is frequently effective in SPD [148–151]. DespiteADP, ATP, and serotonin as compared with non- the lack of evidence that desmopressin has a directSPD porcine controls. A human equivalent of the effect on platelet function, it is often effective inporcine SPD/von Willebrand disease association shortening the bleeding time, improving clinicalhas been reported where patients with combined haemostasis, and allowing successful surgical pro-SPD/von Willebrand disease have more severe cedures without excess bleeding. These effectsclinical bleeding than individuals with either abnor- probably result from the desmopressin-induced in-mality alone [130]. creases in plasma levels of Factor VIII and vonA study of a group of American Cocker Spaniels

Willebrand Factor. The rise in von Willebrand Fac-provides an interesting canine variant of SPD. Thetor, in particular, enhances platelet adhesion to theplatelets from these dogs contain seemingly normalvessel wall, improving primary haemostasis.numbers of dense granules associated with normal

Desmopressin is usually administered intrave-handling and storage of serotonin. However, thenously during episodes of severe bleeding or preop-platelet ADP levels are low, and ATP/ADP ratio iseratively but for mild bleeding may be given eitherincreased. It has been suggested that these plateletssubcutaneously or intranasally, with good effect,have an inherent, isolated abnormality in adeninemaking it available for self administration by somenucleotide storage [131].patients [152]. Desmopressin is not universally ef-

3.5. Acquired Storage Pool Deficiency fective in SPD, and a test dose should be given toidentify response, particularly if surgical haemosta-

SPD can present in association with several other sis is required. For nonresponders, platelet transfu-acquired platelet dysfunction abnormalities, as a sion may be required for severe bleeding episodesresult of two different mechanisms: The first type or in preparation for major surgery [153].of acquired SPD is due to in vivo activation of Adjunctive therapies such as oral and topical anti-platelets and partial release of granule contents fibrinolytic agents (tranexamic acid and e-amino-(alpha as well as dense granule), often in the con- caproic acid) are useful for epistaxis and oropharyn-text of abnormal or damaged vasculature. This has geal bleeding. Avoidance of aspirin and other drugsbeen described in the setting of consumptive coagu-

affecting platelet function is essential.lopathy [132], cavernous haemangioma [133], val-vular heart disease [134], preeclampsia [135,136],diabetes mellitus [137], coronary artery disease

4. Summary[138], and stenting and angioplasty [139]. Plateletactivation can be detected by increased expressionof the dense granule membrane proteins CD63 and The advances that have been made over the lastP-selectin on the platelet surface [140]. decade in microscopic, biochemical, molecular, and

The second type of acquired SPD is associated genetic techniques have led to substantial improve-with myelodysplasia, myeloproliferative disease, ment in our understanding of platelet dense granuleand the chronic leukemias, including chronic my- structure and function, and the implications of denseelocytic leukemia and chronic lymphocytic leuke- granule deficiencies for haemostasis. However, muchmia [141–146]. The dense granule deficiency is has still to be learned. For example, what is theprobably a result of abnormal megakaryocy- specific mechanism of docking and fusion that occurstopoeisis associated with myelodysplasia or malig-

during dense granule exocytosis? What are the rolesnancy. Treatment of chronic myelocytic leukemiaof dense granule membrane proteins during exo-with a-interferon has been reported to improvecytosis or after expression on the surface of activatedthe SPD associated with the primary disease [147].platelets? Finally, how do the genetic defects identi-fied in HPS and CHS result in the clinical phenotype3.6. Therapyof these diseases, and what does this tell us aboutthe origin and function of the affected subcellularAs treatment for episodes of mucosal bleeding and

as prophylaxis for surgical and dental procedures, organelles?


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