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Rumbaut RE, Thiagarajan P. Platelet-Vessel Wall Interactions in Hemostasis and Thrombosis. San Rafael (CA): Morgan & Claypool Life Sciences; 2010.

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Platelet-Vessel Wall Interactions in Hemostasis and Thrombosis.

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Chapter 2General Characteristics of Platelets

2.1. Overview

Platelets were recognized as a distinct blood element in the late 19th century; the seminal work by Bizzozero in 1882 demonstrated that platelets (and not white blood cells) were responsible for formation of “white” clots at the sites of vascular injury in guinea pig microvessels in vivo [2]. As illustrated in those early descriptions, platelets are considerably smaller than the other previously recognized blood elements, erythrocytes and leukocytes. Mammalian platelets are anucleated cells arising from cytoplasmic fragmentation of megakaryocytes in the bone marrow, and have a typical diameter of ~2–3 μm. Platelets circulate in a discoid form (see Figure 2.1) and their average lifespan in humans is ~10 days [3]. However, following activation, they undergo dramatic changes in shape and ultrastructure; the membranes become ruffled with cytoplasmic projections and the granules are centralized and discharged [4,5]. Normal human platelet count is ~150,000–400,000/μl, though spontaneous bleeding resulting from reduced (but functionally normal) platelets is unusual at levels >10,000/μl [6].

Figure 2.1. Transmission electron microscopy image of a resting human platelet, revealing many granules (Gr), several dense granules (DB, or dense bodies), the open canalicular system (OCS), and the dense tubular system (DTS).

Figure 2.1

Transmission electron microscopy image of a resting human platelet, revealing many granules (Gr), several dense granules (DB, or dense bodies), the open canalicular system (OCS), and the dense tubular system (DTS). The image also depicts glycogen (Gly), (more...)

Despite their lack of a nucleus, platelets are actively involved in a broad range of physiologic and pathologic processes. Platelets contain a variety of mediators that regulate hemostasis and thrombosis as well as a myriad of other functions including recruitment of other cells (chemotaxis), vasomotor function, cell growth, and inflammation, among others. Relevant constituents for thrombosis are present both on the cell membrane and in the cytoplasm, mainly within platelet granules. The platelet membrane, which consists of a typical bilayer of phospholipids, contains membrane glycoproteins that interact with various ligands, either soluble ligands that activate the platelets, or fixed ligands within the vessel wall or on other cells through which the platelets adhere to these structures. One unique feature of the platelet is that its plasma membrane contains a network of numerous invaginations into the platelet interior, connected to the exterior through small pores [7,8], known as the open canalicular system (OCS). This feature imparts upon the platelet a much greater surface area than would normally be found on such a small cell. Platelets contain a second channel system, derived from megakaryocyte smooth endoplasmic reticulum, known as the dense tubular system (DTS). The DTS stores calcium and a variety of enzymes involved in platelet activation; in contrast to the OCS, the DTS does not associate with the plasma membrane [9,10].

Platelet granules serve as secretory vesicles, releasing components to the extracellular fluid and also serve to direct molecules to the plasma membrane in a process of exocytosis. Three main populations of granules are evident in unactivated, normal platelets, which differ in their ultrastructure, granule contents, kinetics of exocytosis, and function.

2.2. Platelet Granules

2.2.1. Alpha Granules

Alpha (α) granules are the largest (~200–400 nm) and most prevalent and heterogenous platelet granules [11]. There are about 50–60 granules per platelet, and they are responsible for the granular appearance of the cytoplasm in peripheral blood smears (stained with Romanowsky stains). These granules contain the majority of platelet factors involved in hemostasis and thrombosis. These include large polypeptides such as thrombospondin, P-selectin, platelet factor 4 and beta thromboglobulins as well as several factors involved in coagulation (Factors V, XI, XIII, fibrinogen, von Willebrand factor and high molecular weight kininogens). α-granules also contain a variety of adhesion molecules involved in platelet-vessel wall interaction such as fibronectin and vitronectin. The membrane of α-granules contains several proteins that are also expressed on the platelet cell membrane such as GPIb complex, GPVI, GP IIb/IIIa, and P-selectin. In addition, α-granules contain proteins involved in inflammation, wound healing, mitogenic growth factors (including platelet-derived growth factor, vascular endothelial growth factor, and transforming growth factor-β, among others) and a broad range of chemokines. Our understanding of platelet α-granule constituents is evolving; for example, a recent proteomic analysis of α-granules revealed the presence of 284 non-redundant proteins, of which 44 had not been described previously in these granules [12]. Further, α-granules demonstrate heterogeneity in their constituents and specific sub-populations of these granules may be released in response to various agonists [13,14]. Understanding the mechanisms of differential release of α-granules, and the broad range of effects induced by release of their numerous constituents is an area of active investigation.

Secretion of α-granules during platelet activation is a complex process, involving coalescence in the platelet center, fusion of granules with the OCS and each other, as well as fusion with the plasma membrane [11,1517]. Platelets contain the complex machinery for granule release, including SNARE (soluble NSF [N-ethylmaleimide-sensitive factor] attachment protein receptors) and associated proteins and membrane lipids [17,18]. Some α-granule constituents, such as P-selectin, exert their main physiologic role following their incorporation into the platelet membrane [19]. Other granule constituents exert their role following release from granules and participate in platelet aggregation, thrombosis, platelet adhesive interactions with leukocytes and other substrates, and regulation of cell proliferation via release of various growth factors. Deficiency of platelet α-granules occurs in a rare inherited disease, Gray Platelet syndrome (GPS), associated with quantitative and qualitative platelet dysfunction and a bleeding predisposition [20]. In GPS, proteins endogenously synthesized by megakaryocytes or endocytosed by platelets fail to enter α-granules of platelets due to abnormal formation of α-granules during megakaryocytic differentiation. This results in continued release of α-granule contents such as growth factors and cytokines into the bone marrow resulting in fibrosis (myelofibrosis). Morphologically, the platelets appear gray in peripheral smears.

2.2.2. Dense Granules

Platelet dense granules are the smallest granules (~150 nm) and appear as dense bodies on electron microscopy (see Figure 2.1), due to their high calcium and phosphate content [9]. There are about 3–8 dense granules per platelet. In addition they contain high concentrations of adenine nucleotides and serotonin. Dense granules also contain small GTP-binding proteins and have been reported to contain relevant adhesion molecules present primarily on other platelet compartments including GPIb, GPIIb/IIIa, and P-selectin (discussed in separate sections). During platelet activation, dense granule membrane proteins incorporate with the platelet plasma membrane and granule contents are released into the extracellular environment. The released constituents contribute to recruit other platelets (aggregation) and also contribute to local vasoconstriction (e.g., serotonin). The ADP contained in dense granules is primarily involved in hemostasis and does not equilibrate with the metabolic pool of ADP, it is said to belong to the storage pool. Release of dense granules involves mechanisms comparable to those identified for α-granules, although the role of certain SNARE proteins have been reported to differ between the two granule populations [21]. The importance of dense granules to normal hemostasis is shown by the bleeding disorder in patients with deficiency of these granules. Platelet dense granule deficiency has been identified in two rare human conditions associated with predisposition to bleeding, Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome [22]. HPS is defined by pigment dilution (affecting skin, hair, and eyes), resulting in oculocutaneous albinism, and platelet storage pool deficiency due to deficiency of dense granules. HPS is due to mutations in genes that mostly function in membrane and protein trafficking. There are eight known human HPS genes, each resulting in specific clinical variants of HPS [23]. Mouse strains that are deficient in orthologous genes also have been characterized and have a bleeding diathesis [24].

Chediak-Higashi syndrome is a rare autosomal recessive disorder characterized by oculocutaneous albinism, lymph node enlargement, hepatosplenomegaly (liver and spleen enlargement), and recurrent infections [22]. The CHS1/LYST gene on chromosome 1 affects the synthesis and maintenance of secretory granules within cells [25]. Lysosomes of fibroblasts, melanocytes, and hematopoietic cells, grossly enlarged to form giant lysosomes or vesicles [22]. In platelets, electron microscopy shows a large reduction in the number of dense granules but normal amounts of α-granules [26]. Platelet aggregation studies are consistent with deficiency in the storage pool of dense granule substances and suggest that this granule defect has an influence on the release mechanism of other granule constituents.

2.2.3. Lysosomes

Lysosomes represent the third category of platelet granules, with a size intermediate between α- and dense granules (~200–250 nm); they contain an intralumenal acidic pH with hydrolytic enzymes active towards a number of substrates including constituents of the extracellular matrix [9,27]. Due to similar electron density, lysosomes cannot be distinguished from α-granules in routine electron microscopy images such as those shown in Figure 2.1. However, they can be identified under electron microscopy with the use of cytochemical stains directed at enzymes contained in lysosomes such as acid phosphatase or arylsulfatase [28]. Platelet lysosome contents can be released upon activation, although their release requires greater stimulation than needed for release of α- and dense granules. Release of lysosomes involves mechanisms analogous to those involved in release of the other platelet granules [29,30]. While the functional role of platelet lysosomes is less well understood than that of α- and dense granules, lysosome release has been postulated to contribute to regulation of thrombus formation and remodeling of the extracellular matrix [9,27]. As in the case of α- and dense granules, lysosome release results in incorporation of lysosome proteins to the platelet plasma membrane; for example, platelet surface expression of lysosomal integral membrane protein (LIMP-1, or CD63) is used as a marker of significant (or “strong”) platelet activation [31].

2.3. Platelet Adhesion Molecules

Platelets contain a number of adhesion molecules both on the plasma membrane and within granules that are relevant for hemostasis and thrombosis, as well as cell-cell and cell-subendothelial matrix interactions (see Figure 2.2). The cellular localization and activation state of these molecules vary according to the state of platelet activation. The main adhesion molecules involved in hemostasis and thrombosis will be reviewed individually.

Figure 2.2. Schematic of the major platelet adhesion molecules and G-protein-coupled receptors and their signaling pathways; see text for details on individual molecules.

Figure 2.2

Schematic of the major platelet adhesion molecules and G-protein-coupled receptors and their signaling pathways; see text for details on individual molecules.

2.3.1. P-Selectin

P-selectin (CD62P, ~140 kd) is the largest of the selectin family of adhesion molecules. It is contained primarily on platelet α-granules, though has also been described on dense granules [32]; it is also present in the Weibel-Palade bodies of endothelial cells. Under resting, unstimulated conditions, little P-selectin is evident on the surface of platelets. However, following activation of platelets (or endothelial cells), the fusion of granule membranes with the cell membrane results in rapid expression of P-selectin on the cell surface. While the kinetics of this response varies according to agonist and dose, maximal surface expression of P-selectin has been reported to range from ~30 seconds to 10 minutes following stimulation [33,34]. P-selectin surface expression is commonly used as a marker of platelet activation [19], as illustrated in Figure 2.3. The ligands for P-selectin include P-selectin glycoprotein ligand-1 (PSGL-1), which is expressed primarily on leukocytes [35], von Willebrand factor [36], glycoprotein Ibα [37] and sulfatides [38]. Platelet P-selectin contributes to hemostasis and thrombosis, as well as interactions between platelets, leukocytes and endothelial cells in inflammation [39]. In addition, a soluble form of P-selectin present in plasma may contribute to thrombosis [40].

Figure 2.3. Flow cytometry of P-selectin expression on mouse platelets.

Figure 2.3

Flow cytometry of P-selectin expression on mouse platelets. Resting platelets reveal limited expression of P-selectin, whereas platelets incubated with thrombin (1 U/ml for 10 minutes) demonstrate significant P-selectin expression.

2.3.2. Glycoprotein Ib/IX/V (GPIb/IX/V)

This large glycoprotein receptor complex is the main platelet receptor for von Willebrand factor (vWF) and is composed of four distinct molecules. They include GPIbα (~145 kd), which is the main site for binding vWF, as well as GPIbβ (~22 kd), GPIX (~17 kd), and GPV (~82 kd). GPIbα is linked to two GPIbβ subunits through membrane-proximal disulfide bonds. The α/β2 complex, also known as GPIb, is non-covalently associated with GPIX (GPV is more loosely associated with two GPIb-IX complexes) [41]. Binding of vWF to GPIb initiates signal transduction events that lead to the activation of the platelet integrin GPIIb/IIIa (αIIb3), which becomes competent to bind vWF or fibrinogen to mediate platelet aggregation. In addition to being the main vWF receptor on platelets, GPIbα has also been reported to bind a large number of ligands, including thrombospondin [42], counter-receptors such as P-selectin [37], the integrin Mac-1 on leukocytes [43], thrombin [44], coagulation factors XII, XI, and VIIa [4547], and kininogen [48]. However, binding of GPIbα to these other ligands is less well characterized than its interaction with vWF. The cytoplasmic C-terminal tails of GPIb contain several serine phosphorylation sites and interacts with filamin, calmodulin, 14-3-3, and the regulatory p85 subunit of the phosphoinositide 3-kinase [41]. Interaction with filamin links GPIb-IX-V to the membrane cytoskeleton; all three subunits are required for efficient expression of the GPIb-IX complex on the plasma membrane of transfected cells. Deficiency or dysfunction of the GPIb complex results in a bleeding disorder known as the Bernard–Soulier Syndrome [49], and a number of disease-causing mutations have been mapped to the genes encoding GPIbα, GPIbβ, and GPIX [50]. While this receptor is present constitutively on the platelet plasma membrane, and vWF is normally present in plasma, binding of the receptor with its ligand involves a conformational change in either or both components. These changes are induced by alterations in blood flow and the resultant shear stress, a concept discussed in greater detail in Chapter 3. The antibiotic ristocetin induces these changes in the absence of shear stress and is used to assess this interaction in vitro [51].

2.3.3. Glycoprotein IIb/IIIa (GP IIb/IIIa)

Platelet GP IIb/IIIa (αIIbβ3) plays an essential role in platelet aggregation, and thus it has been studied most intensively. Like other integrins, it is a heterodimer with an alpha (αIIb, ~136 kd) and a beta (β3, ~92 kd) subunit. There are approximately 80,000 copies of GP IIb/IIIa on the surface of unstimulated human platelets, and additional molecules in the membranes of platelet granules are translocated to the platelet surface during platelet secretion [52]. This molecule is expressed constitutively on the plasma membrane as an inactive form in resting platelets, though undergoes conformational changes during activation. As depicted in Figure 2.4, this integrin is composed of a large extracellular nodular head with its ligand-binding site and flexible stalks containing its transmembrane (TM) and cytoplasmic domains [53,54]. The nodular head of the alpha subunit is folded into a β-propeller configuration, followed by a “thigh” and 2 “calf” domains, constituting the extracellular portion of the αIIb stalk. The β3 head consists of a βA-domain containing a metal ion-dependent adhesion site (MIDAS) motif, as well as a hybrid domain whose fold is similar to that of I-set Immunoglobulin domains. The β3 stalk consists of a PSI (plexin, semaphorin, integrin) domain, 4 tandem epidermal growth factor (EGF) repeats, and a unique carboxyterminal βTD domain. In the resting stage, the head region is severely bent over in a compact “V” shape (Figure 2.4). Activation induces a change in the shape of the headpiece, shifting the αIIb and β3 domains from a closed conformation with adjacent stalks to an open conformation with stalks separated, exposing the ligand binding site, consisting of a β3βA-domain and forming a “cap” composed of 4 loops on the upper surface of the αIIbβ-propeller domain.

Figure 2.4. Model of platelet integrin αIIbβ3 (GPIIb/IIIa), and conformational changes induced following activation.

Figure 2.4

Model of platelet integrin αIIbβ3 (GPIIb/IIIa), and conformational changes induced following activation. The inactive “V”-shaped compact form is shown on the left. Binding of a ligand results in extension of the molecule (more...)

Platelet GPIIb/IIIa can bind to fibrinogen, as well as other ligands such as vWF, fibronectin and vitronectin [55]. The key role of GP IIb/IIIa in platelet aggregation is discussed in Chapter 4; this molecule represents a major target for directed therapy in patients with thrombotic disorders [56].

2.3.4. Collagen Receptors

As reviewed in Chapter 3, platelet interaction with subendothelial collagen is an essential step in primary hemostasis. The α2β1 integrin and glycoprotein VI (GP VI, ~65 kd) are the primary collagen receptors; both play a prominent role in hemostasis. These receptors bind to specific sequences on collagen with different affinities [57]. α2β1 binds to various sequences containing the GER (glycine-glutamic acid-arginine) triplet, while GPVI binds to sequences containing at least two GPO (glycine-proline-hydroxyproline) triplets [58,59]. Platelet adhesion promoted by integrin α2β1 induces activation of platelet GPIIb/IIIa through the phospholipase C (PLC)-dependent stimulation of the small GTPase Rap1b [60]. Platelet GPVI is expressed constitutively on the platelet plasma membrane, and is also expressed on α-granules [61]. Following platelet activation, surface expression of GPVI increases and intracellular expression decreases, consistent with their release from α-granules and incorporation into the plasma membrane. GPVI belongs to the immunoglobulin superfamily that contains two C2 immunoglobulin-like domains and an arginine residue in the transmembrane region that forms a salt bridge with the aspartic acid residue of the Fc receptor γ (FcRγ)-chain [62]. Activation by collagen leads to phosphorylation of its immunoreceptor tyrosine-based activation motif (ITAM), leading to a sequence of events involving several adaptor proteins, and resulting in phosphorylation and activation of PLCγ2 [63,64]. GPVI mainly binds to collagen types that can form large collagen fibrils such as collagen type III. GPVI is a key adhesion molecule involved in hemostasis and thrombosis; absence of GPVI in humans is associated with a predisposition to bleeding [65].

2.4. G-Protein-Coupled Receptors

2.4.1. Thrombin

Thrombin is a key component of the blood coagulation cascade and a potent stimulator of platelets. Platelet responses to thrombin are mediated by protease-activated-receptors (PAR). PAR are unique among G protein-coupled receptors in that activation occurs through proteolytic cleavage of the receptor by thrombin and unmasking a specific ligand [66]. Thrombin binds to the extracellular domain of PAR-1 and PAR-4, which are then cleaved to form a new amino terminus with its tethered ligand; the tethered ligand activates the receptor and induces signaling. Synthetic peptides, called thrombin-receptor agonist peptides (TRAPs), mimic the new amino terminus and potently activate the thrombin receptor resulting in platelet activation, secretion, and aggregation independently of receptor cleavage. Both PAR-1 and -4 activation must be inhibited to prevent platelet activation subsequent to thrombin binding to platelets. In mouse platelets, PAR-3 and PAR-4 (as opposed to PAR-1 and PAR-4), mediate the response to thrombin [67,68]. Human platelets also express PAR3, though in contrast to mice, PAR3 does not appear to contribute to platelet responses to thrombin [67,69]. Thrombin signaling via either PAR1 or PAR4 induces platelet activation, shape change, and granule release; PAR1-dependent responses are evident at lower thrombin concentrations than those induced by PAR4 [67]. The signaling pathways downstream of PAR-1 and PAR-4 in human platelets are not entirely clear, though both PAR-1 and PAR-4 couple to Gq and G12/13 G-proteins, resulting in activation of phospholipase C, calcium mobilization, and protein kinase C activation [70]. Thrombin receptor antagonists may represent a novel target for clinical antithrombotic therapy are undergoing large scale clinical trials [71].

2.4.2. Adenosine Diphosphate (ADP) and Adenosine Triphosphate (ATP)

ADP has long been recognized as a stimulus for platelet adhesion and aggregation, though with distinct features than the response to thrombin. Exposure of human platelets to low concentrations of ADP results in an initial reversible aggregation without granule release. Higher concentrations of ADP, induce release of granules and synthesis of prostaglandins giving rise to a characteristic biphasic response with irreversible aggregation.

The response to ADP on human platelets is mediated by G-protein coupled P2Y receptor family of G protein-coupled, seven transmembrane domain receptors (P2Y1 and P2Y12). The P2Y1 receptor couples to Gq and mobilizes intracellular calcium ions to mediate platelet shape change and aggregation. The P2Y12 receptor is coupled to the inhibition of adenylyl cyclase through Gi and it is the target of antithrombotic agents, such as ticlopidine, clopidogrel, and prasugrel [72]. Mutations in the P2Y12 receptor are associated with a life-long bleeding disorder [73]. ATP is a less potent stimulus than ADP, but results in platelet activation, shape change, and enhances platelet responses to other agonists such as collagen; ATP-induced signaling occurs via a ligand-gated ion channel (P2X1) [74].

2.4.3. Prostanoids

Thromboxane A2 (TXA2) is a product of arachidonic acid metabolism; two isoforms of thromboxane receptors have been described: TPα and TPβ, though the TPα is the predominant isoform expressed on human platelets [74,75]. TP is also a member of the G protein-coupled receptor (GPCR) family and is shown to be coupled with Gq and G13, resulting in phospholipase C activation and RhoGEF activation, respectively [76]. TXA2 is released by various cells, including platelets, where it acts in an autocrine and paracrine manner. TXA2 results in platelet shape change, aggregation, degranulation and enhancement of response to other agonists, thus amplifying platelet activation. Inhibition of TXA2 production (by inhibition of cyclooxygenase-1) is one of the mechanisms of action of aspirin, a commonly used antiplatelet agent [56,77,78], and direct inhibitors of TP-α are undergoing evaluation for potential clinical use [79]. Platelets express receptors for other prostanoids, including prostacyclin (which mediates inhibition of platelet aggregation) and prostaglandin E2 (which has a biphasic effect on platelets).

Copyright © 2010 by Morgan & Claypool Life Sciences.
Bookshelf ID: NBK53455

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