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Platelets and plateletdisorders in Africa E. M. ESSIEN

The blood platelet is functionally a very dynamic cell. Although its role in haemostasis has received the greatest attention, it is becoming increasingly clear that the circulating platelet plays significant roles in many other aspects of vertebrate biology. These include its involvement in the regulation of growth, angiogenesis and determination of vasomotor tone, among other functions. This chapter will concentrate, however, on the haemostatic aspects of the circulating platelet in man in the tropical African environment and the response to some prevalent infections. For its role in other diseases , for example cardiovascular diseases , diabetes mellitus , malignancy and different haematological disorders, the reader is referred to several existing excellent texts and reviews. Platelets are derived from mature megakaryocytes by a process of cytoplasmic fragmentation following formation and subsequent widening of the demarcation membrane system (DMS) during megakaryocyte maturation. The platelets are SUbsequentlyshed by a mechanism finally involving constriction of the DMS stalk attached to the megakaryocyte cytoplasm, and up to 4000 platelets may be released from each mature megakaryocyte (Federeko and Lichtman, 1982). The circulating blood platelet has a normal lifespan of 8-10 days, including its period of temporary rest in the splenic platelet pool. The normal spleen may accommodate up to about one -third of the total viable platelets in the pool (Shulman and Jordan, 1982); the size of the pool increases in splenomegaly and may give rise to thrombocytopenia, in some instances without concomitant hypersplenism . Platelet structure The circulating platelet is a small, disc-shaped non-nucleated blood cell, whose volume varies, depending on the method of its isolation, in the range 6± 0.2 ~m3 (Karpatkin, 1969). Viable platelets usually exist in either a non-activated (Figure 1) or an activated state (Figures 2 and 3). In the non-activated state , the platelet surface appears smooth, although closer study reveals tiny indentations which are openings from the interior of the platelet to its surface . These openings , unique to the platelet among blood cells, are connected to the open canalicular system (OCS) within the cell (Hovic, 1968; White , 1971). Bailliere's ClinicalHaematology-« Vol. 5, No.2, April 1992 ISBN 0-7020-1627-6

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Copyright© 1992,by Bailli~re Tindall All rights of reproduction in any form reserved

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Figure 1. D iagram of a resting platelet showing internal structures . DB , dense bod y; G, glycogen granul es; M, mitoch ondria ; MT , circumfer ent ial band of rnicrotubules: OC, o uter coal ; oes, open surface canalicular connecting system; SMZ , submembrane zone ; TS , tubular system. (Mod ified from White and Gerrard , 1982).

Peripheral zone of the platelet A cross-sectional vie w of the resting platelet (see Figure 1) at the equator shows from the outside inwards: (1) a wall con sisting of an ext erior coat (glycocal yx) of about 2 x 10- 7 urn thick; (2) a typical unit membrane; a nd (3) an immediate subme mbra ne zone made up of 8-24 filaments , each of which is composed of short actin fibres cross-linked by actin-binding protein (White, 1969; Gerrard and White , 1976). The filaments , although associated with th e membrane glycoproteins (Gp ) or integrins, such as Gplb-IX complex and GpIa-IIa receptors, ar e responsible for keeping other structures away from direct contact with the internal platelet cell wall. The glycocalyx , the unit membrane and the sub-membrane filaments, together with the imm ediate layer of blood pla sma surrounding each platelet (sometime s called th e 'atmosphe re plasmatique' (R oskam, 1923», cons titute the platelet periph eral zone . The plasma environment surro unding each circul ating platelet contains other blood cells with which platelet s ma y interact , and several chemi cal substances such as fibronectin , fibrinogen , other blood coagulation facto rs

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Figure2. Transmission electron micrograph showing part of a platelet lying on and adapting to the shape of the inner surface of a damaged rabbit thoracic aorta. Arrows show points of contact between the platelet and the damaged surface. (Magnification x 8250.)

as well as other plasma proteins . These frequently exert significant immediate or potential effects on the platelet. If activated platelets are present in the vicinity, their secreted proteins and other chemical substances (see below) will also be present in this environment. Therefore, the plasma region immediately surrounding the glycocalyx is usually regarded as an extension of the platelet external wall .

Internal structure Resting platelets. Deep to the platelet wall. as defined above, is a ring of 8-24 circumferential microtubules, each 2 .5 x 1O-7f.Lm in diameter and made up of 12-15 sub-filaments. the latter composed of tubulin protein (see Figure 1) (White and Gerrard, 1982). These microtubules are cross-linked in a network pattern by actin-binding protein (ABP), and form part of the platelet cytoskeleton . Another major component of the cytoskeleton is actin, which forms 15-20% of the platelet total protein. Other important components

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Figure 3. Electron micrograph of platelets on the inner surface of a damaged rabbit thoracic aorta. Note the shape change manifested by the platelets, and the pseudopodia with which the platelets adhere to the surface and to eaeh other. (Magnification x 24000; reduced to 75% on reproduction. )

include: o-actrrun, tropomyosin, profilin, gelsolin, talin, vinculin and spectrin (Fox, 1987). Dispersed within the cytoplasm of each platelet are a number of structures and substances which are vital to platelet function and survival (see Figure 1). These include a-granules, dense bodies, mitochondria, glycogen storage granules and the network of OCS, whose lining and structure are similar to, and continuous with the components of the platelet cell wall. There is, in addition, the dense tubular system (DTS), a distinct system of channels derived from the rough endoplasmic reticulum of the megakaryocyte. The DTS is closely associated with the circumferential microtubules. However, both communication systems, the OCS and DTS, though distinct are in close apposition to each other and are involved in ionic (especially Ca 2 +) transfer to the exterior.

Activated platelets. When platelets are activated, either on interaction with agonists (including thrombin, adenosine diphosphate (ADP), adrenaline, ionophore A23187, hirudin, collagen, serotonin or arachidonate and its products such as thromboxane A 2 (TXA 2 ) ) , or on contact with damaged vascular endothelium or sub-endothelium, there follows a sequence of both morphological and biochemical changes. These usually manifest as shape change, adhesion and aggregation (see Figure 2). A result ofthe activation is that the platelets secrete their contents into the surrounding medium (Tables 1 and 2).

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Table 1. Summary of features of resting platelet and changes on its activation. Feature

Resting platelet

Activated platelet

Shape Actin polymerization Actin filament content (%) Distribution Periphery Centre Profilactin Filopodia Actin in filopodia Myosin distribution

Discoid

.,

Discoid shape lost Burst

~50

100

Loose network Few Loose network Associated Nil Scanty Uniform

+++

Myosin phosphorylation (%) Tropomyosin distribution Actin-binding protein a-Actinin Protein 4.1

10 Diffuse Diffuse Diffuse

Membrane skeleton

Intact

.,

? Rings around centralized granules Dissociated ++ in bundles

++

Concentrated around centralized granules

100 Concentrated in filopodia Concentrated in filopodia Concentrated in filopodia Concentrated in submembranous arrays in filopodia Dispersed

Table2. Some important secretory proteins from activated platelets.

Thrombogenic promotingproteins Adenosine diphosphate (ADP) Adenosine triphosphate (ATP) Thromboxane A 2 (TXA 2) von Willebrand's factor (vWF) Platelet factor 4 (PF 4 ) Thrombospondin Platelet activating factor (PAF) Elastase Endoglycosidase

Proteins whichincrease vasomotor tone Platelet derived growth factor (PDGF)* Serotonin (5-HT) Vasopressin PAP TXA 2 (in human coronary arteries)

Growthmodulators PDOFt Transforming growth factor f3 (TOF-f3)t Some substances are cell adherence promoters, e.g. PDOF, PAP, TOF-P, and are also produced by vascular endothelial cells. *Also promote cell adherence. t Also produced by vascular endothelial cells.

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Platelet shape change and centralization of internal structures and contents are the major morphological features characteristic of an activated platelet. The details and mechanisms of these changes have been extensively reviewed (Hovic, 1968; Mustard and Packham, 1970; White and Gerrard, 1982; Lasslo, 1984) (see Table 1). The normal platelet count in different populations The normal platelet count in adult European populations has been established at 150-400 x 109/1 (Dacie and Lewis, 1975). However, the lower limit of the normal platelet count in adult African populations has been established at 100 x 109/1 , with a normal range of 100-300 x 109/1 (Essien et al, 1973; Dupuy et al, 1978; Gill et al, 1979; Ukaejiofor et al, 1979; Mukiibi et al, 1981; Onwukeme and Uguru, 1990; O. Akinyanju, personal communication; A. A. Lawal et al, personal communication). In Nigerian neonates, a normal platelet count of 59-490 x 109/l was obtained (Effiong et al, 1976) and was reported as being comparable to the values obtained in neonates from other populations (Aballi et al, 1968). The reasons for these relatively lower normal figures in adults are not clear. A suggestion was that the finding might be related to the factors of poor nutrition and high prevalence of infection due to the socioeconomic circumstances of the subjects studied (Essien et al, 1973): this was disputed (0. Akinyanju, personal communication). Another suggestion was that the high prevalence of relative 'splenomegaly' due to recurrent malaria, with a resultant enlarged splenic platelet pool, might account for the observation. These suggestions, amongst others, have not, to my knowledge, been resolved. Platelet function The circulating platelet plays a critical role in haemostasis and significant roles in tissue repair. It also contributes to body defence. The critical role of platelets in providing balanced haemostasis becomes evident when purpura manifests in thrombocytopenic states, irrespective of their causes. Thrombocytopenia is defined as a state of reduced circulating platelet count in blood below the normal level (see above). It may be mild, moderate or severe; typical purpuric bleeding, with or without ecchymosis, very frequently occurs in severe thrombocytopenia, and may occur, usually without ecchymosis, in moderate thrombocytopenia. The mechanism of bleeding in thrombocytopenia is fairly well known. It has been suggested that breaks in the vascular endothelial lining frequently occur in vivo, but blood seepage through such damaged sites is readily prevented by prompt platelet responses, which include platelet shape change, adhesion, secretion, aggregation and the formation of an unstable platelet plug (Packham and Mustard, 1984), subsequently stabilized by fibrin strands formed around the plug through platelet coagulant activity (Walsh, 1974). These changes are initiated by platelet contact with the damaged site where, as a result of the damage, the normal electrostatic forces between the platelet and the vascular endothelium are disrupted, and

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direct contact between the circulating platelet and the damaged site occur. As a result of such contact, the platelet is activated, changes its shape to assume that of the damaged site and adheres through exposed receptors (see Table 3 and Figure 2). Within 30 seconds of such contact and activation, there is rapid centralization of platelet contents and secretion of some of them into the platelet environment (see Table 2). Other platelets within the vicinity react in a similar fashion to the chemical substances (such as ADP, serotonin and TXA2), or to the exposed submembrane structures (e.g. collagen), and form the platelet aggregates and plug on the damaged site: the haemorrhage is thus arrested. Later, clot lysis removes the plug. In thrombocytopenia, the number of circulating platelets is inadequate to perform these functions. Table J. Some platelet and platelet-related adhesion proteins . Platelet glyooproteins GplblIX GpIV GpIa-I1a GpIc-I1a GpIe-I1a GpII~IIIa GpII~IIIa

Integrins ? ? a2131 asl3l 0;\131 (l11613J

a.133

Extracellular protein substrate von Willebrand factor (vWF) Thrombospondin (TSP) Collagen Fibronectin Laminin F6/FN/vWFNNrrsP/Coll F6IFN/vWFrrSp

Repair of the damaged vasculature is promoted by, among other substances, the platelet secretory proteins, platelet derived growth factor (PDGF) (Ross et al, 1986; Schwartz et aI, 1986; Sjolund et al, 1988), which is a powerful mitogen, and transforming growth factor-B (TGF-J3) (Roberts et al, 1985; Assoian and Sporn, 1986; Majack, 1987). PDGF and TGF-J3 are also released from activated vascular endothelial cells (Dicorleto and Bowen-Pope, 1983) and smooth muscle cells (Rabinovitch and TurnerGomes, 1989). TGF-J3 also inhibits vascular smooth muscle cell proliferation in appropriate circumstances (Roberts et al, 1985), thus exhibiting modelling and modulating roles in tissue repair. These latter actions have longer-term effects and are designed to secure not only vascular repair but also its recovery from accidents (Barrett and Benditt, 1987). As summarized above, the role of platelets in health involves not only vital normal haemostatic functions, but also promotion of tissue growth and angiogenesis, roles that can all be deranged. CIRCULATING PLATELETS IN AFRICANS IN THE TROPICAL ENVIRONMENT In the tropical African environment, the scanty evidence available indicates that environmental factors frequently affect some aspects of normal platelet function in man. These factors include infections, such as malaria, a situation that is now complicated by the recent onset of the phenomenon of

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chloroquine resistance in Plasmodiumfalciparum: This has exacerbated the burden of malaria . There is also the more recent onset of the human immunodeficiency virus (HIV) pandemic. Another important factor is the abject poverty and malnutrition that are widespread in the continent, made worse in some areas by unfavourable climatic conditions like drought or flooding, and unstable sociopolitical situations. These factors and their effects have been exacerbated by the downturn in the world economy of the last one and a half decades (The Challenge to the South, 1990). Some effects of the latter situation include worsening malnutrition, with the attendant disturbances in the immune system and resultant increased susceptibility to different infections of the population in general. HIV infection at present appears to affect some regions of Africa and black Africans more severely than other world regions and other populations. The reason for this include poverty, malnutrition and their associated health and social effects (Konotey-Ahulu, 1989). Therefore, this section will examine in greater detail the respective effects of the interactions between the circulating blood platelet and the malaria parasite, as well as those between the platelet and HIV infection, including the acquired immunodeficiency syndrome (AIDS). The circulating platelet and malaria parasite

Following Maslova's early observation that platelets were reduced in the peripheral blood of patients with acute malaria (Maslova, 1924), it is now generally accepted that the circulating platelet is regularly affected during acute malaria infection in man or in experimental infections in animals, (Essien et al, 1979; Inyang et al, 1987a; Essien, 1989). Splenomegaly is a regular feature in chronic malaria infection. It may be complicated by hypersplenism, but it frequently occurs without such complication. Nonhypersplenic splenomegaly may sometimes result in severe thrombocytopenia, as a result of the enlarged splenic platelet pool; however, thrombocytopenia occurs more frequently in hypersplenism. The question of detailed platelet changes in chronic malaria infection with splenomegaly has, as far as I am aware, not been fully addressed . The discussions below, however, refer to platelet dysfunction in acute malaria infection . Platelet count in acute malaria

During acute malaria infection in man, the blood platelet count is regularly reduced and reaches thrombocytopenic levels (i.e. < 100x 109/1) in only a minority of cases of people normally resident in holoendemic areas, including infants aged 2 years and below (Essien and Oruamabo, 1976; Essien et al, 1979; E. M. Essien and M. I. Ebhota, unpublished observations; A. A. Lawai, unpublished observations). Other reports, however, usually based on studies involving small numbers of cases (25-40 cases) of non-immune subjects who visited malarial zones, have stated that thrombocytopenia, often severe, occurs regularly in acute malaria infection in man (Dennis et

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al, 1967; Beale et al, 1972; Horstmann et ai, 1981; Kueh and Yeo, 1982; Perrin et al, 1982). In a study of 202 cases, including 105 children aged 4 months to 12 years, the mean platelet count of 132.0 X 109/l ± 61.6 x 103 was significantly lower than the count of234.0 x 109/l ±94.0 x UP (t= 6.496; P

Platelets and platelet disorders in Africa.

Blood platelets, which are known to play important roles in normal vertebrate biology, are influenced by a variety of factors, the majority of which a...
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