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Platelet glycoprotein IIb/IIIa receptors are major platelet membrane constituents. They are integral to the formation of the surface fibrinogen receptor on activated platelets, in which 73% of platelet-derived microparticles are positive for the glycoprotein IIa/IIIb receptor. Activated platelets can shed platelet-derived microparticles, especially during the course of an acute coronary syndrome. Data have shown that platelet-derived microparticles can bind to the endothelium, to leukocytes, and to the submatrix of vascular walls, and launch some signal-transduction pathways, such as the pertussis-toxin-sensitive G protein, extracellular signal-regulated kinase, and phosphoinositide 3-kinase pathways. One research group found that platelet-derived microparticles transfer glycoprotein IIb/IIIa receptors to isolated and whole-blood neutrophils. The receptors can co-localize with β2-integrins and cooperate in the activation of nuclear factor κB (NF-κB), which can be inhibited by glycoprotein IIb/IIIa receptor antagonists. Accordingly, it is possible that glycoprotein IIb/IIIa receptor antagonists produce a direct and marked effect on endothelial cells, smooth-muscle cells, and leukocytes through a platelet-derived microparticle pathway that will lead to a potential treatment for acute coronary syndrome.
Herein, we review the medical literature and discuss the potential application of platelet-derived microparticles toward the treatment of acute coronary syndrome.
Platelet glycoprotein (GP) IIb/IIIa receptors, which are major constituents of platelet membranes, are integral to the formation of the surface fibrinogen receptor on activated platelets. The GP IIb/IIIa receptors are present in a preponderance of platelet-derived microparticles (PMPs). Activated platelets can shed PMPs, especially during an acute coronary syndrome. Platelet-derived microparticles can bind to vessel walls and launch signal-transduction pathways, such as the pertussis-toxin-sensitive G protein, extracellular signal-regulated kinase, and the phosphoinositide 3-kinase (PI3-kinase) pathways. Here, we review the medical literature and discuss how GP IIb/IIIa receptor antagonists, acting through a PMP pathway, suggest a research focus toward the treatment of acute coronary syndrome.
The term microparticles usually refers to particles larger than 100 nm in diameter that are derived from the plasma membrane among the various membrane vesicles that cells release. Smaller vesicles (40–100 nm) that originate from endoplasmic membranes are referred to as exosomes, and larger particles (>1.5 μm) that contain nuclear material are known as apoptotic bodies.1
In 1967, Wolf2 described the membrane fragments that are shed from activated platelets as “platelet dust,” or “platelet vesicles.” After having been observed in electron micrographs, the particles were characterized as procoagulative in 1985.3 These are the particles now widely referred to as PMPs.
All microparticles harbor cell-surface proteins and contain cytoplasmic components of their original cells. They exhibit negatively charged phospholipids, chiefly phosphatidylserine (PS), at their surface, which accounts for the procoagulative character and proinflammatory properties of microparticles, including the alteration of vascular function. The membranes of PMPs contain platelet GP Ib, IIb, IIIa, P-selectin, and thrombospondin,4,5 in addition to other platelet membrane receptors, such as chemokine (C-X-C motif) receptor 4 and protease-activated receptor 1.6,7 It has been reported that arachidonic acid released from PMPs directly activates GP Mac-1 and the intercellular adhesion molecule-1 on monocytes and the P- and E-selectins on endothelial cells.6,7 Bode and colleagues8 found that 73% of PMPs were positive for the GP IIb/IIIa receptor, which is a Ca2+-dependent heterodimer on activated platelets that can bind 1 of 4 different adhesive proteins (fibrinogen, fibronectin, von Willebrand factor, and vitronectin). The binding of fibrinogen primarily enables platelet aggregation; fibronectin and the von Willebrand factor may also enable adhesion and aggregation on the subendothelium.9
Platelet-derived microparticles have been observed in vivo in clinical conditions that are associated with platelet activation, including idiopathic thrombocytopenia purpura, transient ischemic attacks, and during cardiopulmonary bypass. Increased concentrations of circulating PMPs are also found during aging, and further increases are encountered in peripheral arterial disease and myocardial infarction.10 The biological function of PMPs remains speculative, but the tenase and prothrombinase activity that includes factor Va, high-affinity-factor Xa, and factor-VIII activity11 is concentrated on these particles. In addition, PMPs display anticoagulant activity, since they inactivate prothrombinase by means of activated protein C. These observations suggest that PMPs play a role in modulating hemostasis and thrombosis.12
The erosion, fissure, or rupture of an atherosclerotic plaque is the signaling event in acute coronary syndrome, and rupture can also occur during percutaneous coronary intervention. When plaque rupture occurs, the subendothelial protein matrix is immediately disrupted, which allows platelet-adhesion molecules such as von Willebrand factor and collagen to interact with circulating platelets. Platelets adhere to collagen and von Willebrand factor at the site of injury by means of specific GP receptors. This results in platelet activation, with a change in the platelets' shape, the release of storage granules that contain platelet agonists such as adenosine diphosphate and thromboxane A2, and a conformational change in the platelet fibrinogen receptor GP IIb/IIIa. Although platelet deposition is restricted by circulating blood, already-activated platelets (with PMPs released) provide a new prothrombotic interface for fibrin, circulating blood, and a growing thrombus. This results in the growth of thrombus and narrowing of the vessel. Increases in shear stress, associated with vascular narrowing, favor this process by further promoting new platelet activation and the release of PMPs. An occlusive thrombus forms, and patients experience catastrophic events.
When platelets are activated by agonists such as collagen or thrombin, several responses occur: shape change, secretion, aggregation, phosphorylation of specific platelet proteins,13 exposure of anionic phospholipid on the extracellular face of the platelet membrane,14 and release of microparticles that are rich in procoagulant activity.15 These microparticles possess platelet–subendothelium attachment receptors (GP IIb/IIIa, Ib, Ia, and IIa),4,16,17 and P-selectin,16 a receptor that is involved in platelet–leukocyte interactions18–20 and in inflammatory response.20
Siljander and colleagues21 found that PMPs are associated with developed fibrin fibrils. Moreover, the investigators showed in vitro that PMPs, when separated from platelet remnants, did bind to fibrin, where they were able to act as procoagulants in the presence of plasma and tissue factor. Finally, granular GP IIb/IIIa and P-selectin-positive material were seen to decorate fresh, embolectomized thromboemboli in a fibrin-strand-like pattern. The PMPs were shown to bind to the forming thrombus, and specifically to fibrin.
Glycoprotein IIb/IIIa antagonists cannot only inhibit the GP IIb/IIIa receptors on platelets; they also have an effect on PMPs.22 However, few studies have probed this effect.
Different biological effects have been attributed to PMPs, including their possible participation in the pathogenesis of atherosclerosis and vascular injury during inflammation,14 and in the promotion of bone-cell proliferation.23 The attachment of isolated PMPs on subendothelia24 has suggested a hemostatic function for PMPs. Glycoprotein IIb/IIIa-positive PMPs appear to be promising prognostic indicators in patients who have chest pain, but whose cardiac troponin levels are within normal range and whose electrocardiograms are nondiagnostic.25
Platelet glycoprotein IIb/IIIa receptors are major platelet-membrane constituents that are integral to the formation of the surface fibrinogen receptor on activated platelets. Approaches to achieve more profound platelet inhibition at the site of injured coronary plaque have focused on the integrin GP IIb/III receptor on the platelet surface membrane, which binds circulating fibrinogen or von Willebrand factor and cross-link platelets as the final common pathway to platelet aggregation.
The GP IIb/IIIa receptor is largely confined to platelets and megakaryocytes. It is also found on some melanoma cells,26 where, by linking the stromal connective tissue to the M3Dau melanoma cells, the receptor may enable the stromal matrix to regulate tumor growth and differentiation in vivo.26,27 However, recent studies have shown that some phagocytes may acquire the GP IIb/IIIa receptor from PMPs. Salanova and coworkers28 found that GP IIb/IIIa receptors are transferred to isolated and whole-blood neutrophils via PMPs. Using specific antibodies in neutrophils that were treated with granulocyte macrophage colony-stimulating factor (GM-CSF), the investigators observed that acquired GP IIb/IIIa receptors co-localized with β2-integrins and cooperated in the activation of nuclear factor κB (NF-κB). The Src and Syk nonreceptor tyrosine kinases, in addition to the actin cytoskeleton, controlled NF-κB activation. These acquired receptors are functional, and they enable NF-κB activation in GM-CSF–stimulated neutrophils that interact with fibronectin.28 It was not determined exactly how acquired GP IIb/IIIa receptors link to the neutrophil-signaling machinery. However, similar to GP IIb/IIIa receptor signaling in platelets, Src and Syk kinases (and the actin cytoskeleton) seem to be involved.28
A large body of data suggests that macrophages can recognize PS specifically. Several research groups have found that human and rodent macrophages, and insect phagocytes, preferentially take up negatively charged liposomes, particularly those that contain PS.29–34 In addition, human and rodent macrophages (including freshly isolated human alveolar and splenic macrophages, human bone-marrow–derived macrophages cultured for 10 ± 14 days, cultured human monocytes, and resident and thioglycolate-elicited mouse peritoneal macrophages) can bind to and engulf symmetric red-cell ghosts, red cells with PS inserted externally, oxidized red cells, or sickled red cells, all of which express PS externally.35–41 Accordingly, PS plays a key role in signaling phagocytes to perform. These phagocytes can be professional (the leukocytes) or amateur (including fibroblasts, epithelial cells, and vascular smooth-muscle cells). Exposure of PS on the external leaflet of the plasma cell membrane appears to be common to many apoptotic cells,42–50 and this phospholipid appears to be recognized in a stereospecific fashion by subsets of macrophages,42,43,51 by melanoma cells,52 by vascular smooth-muscle cells,47 and by Sertoli cells.50 As mentioned above, on the surface of PMPs there is plenty of PS, which serves as a cofactor for the coagulation cascade. Therefore, PMPs can probably be engulfed by the phagocytes, suggesting also that GP IIb/IIIa antagonists have an effect on the phagocytes through their acquired GP IIb/IIIa receptors via phagocytosis. However, other than the report by Salanova and coworkers,28 studies are few.
The binding of PMPs to cells can modify the cells' functional properties. The PMPs can bind hematopoietic progenitors and stimulate their engraftment.53 The binding of PMPs to neutrophils induces a significant increase in both CD11b expression and phagocytic activity in a concentration-dependent manner. These findings suggest a possible role for PMPs in addition to providing platelet factors: specifically, as an activator and mediator of neutrophils in ischemic injury, thrombosis, and inflammation.54 Janowska-Wieczorek and associates53 found, rather surprisingly, that mobilized-peripheral-blood (mPB) CD341 cells expressed a significantly higher level of GP IIb/IIIa(CD41 antigen) than did CD341 cells that were isolated from either non-mPB or bone marrow. Hence, the investigators hypothesized that the presence of the CD41 antigen on mPB CD341 cells results from the binding of PMPs to their surfaces.53
Platelet microparticles bind to the subendothelial matrix in vitro and in vivo and can act as a substrate forfurther platelet binding. This interaction may play a sub- stantial role in the adhesion of platelets to the site of endothelial injury.55 Platelet-derived microparticles provide a catalytic surface that accelerates coagulation: they can bind to neutrophils54 to mediate leukocyte–leukocyte interaction, and elevated levels of PMPs may amplify leukocyte-mediated tissue injury in thrombotic and inflammatory disorders.56 Therefore, PMPs can bind neutrophils, mediate their aggregation, and activate their phagocytic properties.54
Some data provide evidence that PMPs can transfer biological information between cells, acting as veritable vectors of signal molecules. Even though PMPs can act on hematopoietic and circulating cells, most of the exchange of information from PMPs takes place at the level of the endothelium and contributes to the physiologic and pathophysiologic role of microparticles. Accordingly, PMPs can affect vasodilation and the antithrombotic and antiadhesive properties of the vascular wall. Also, they may be involved in the regulation of vascular permeability and the proliferation of smooth muscle cells. In addition to their role in the regulation of hemostasis and thrombosis, PMPs evoke monocyte adhesion to endothelial cells (ECs) by inducing adhesion-molecule exposure, stimulating the proliferation, survival, adhesion, and chemotaxis of hematopoietic cells, and increasing the engraftment of hematopoietic stem cells.53 Also, PMPs induce angiogenesis in vitro,57 probably through activation of ECs.58,59
How do PMPs work to cause the effects? Nomura and co-authors60 thought that cytoskeleton served as a bridge to signal paths so that the GP IIb/IIIa complex could perform its function. Platelet-derived microparticles can be viewed as a pathway that can be used by cells to exchange information in addition to the transduction linked to the activation of classically known receptors or transporters. Platelet-derived microparticles taken from patients who were experiencing acute myocardial infarction caused severe endothelial dysfunction in rat aortas by affecting the endothelial nitric oxide transduction pathway, but not the endothelial nitric oxide synthase expression.61 Paradoxically, it has been observed that PMPs affect ECs by protecting them from apoptosis and by inducing the proliferation and formation of tubule-like structures.57 On the other hand, PMPs can inflict damage on ECs by inducing an inflammatory response and diminishing endothelium-dependent vessel dilation.62
The PMP-stimulated proliferation, chemotaxis, and tube formation of ECs has been mediated via the pertussis toxin-sensitive G protein, extracellular signal-regulated kinase, and the PI3-kinase pathway.57 Pertussis toxin, a G-protein inhibitor, blocks the effects of PMPs on GP IIb/IIIa.63 Therefore, the G proteins, which regulate (for example) the activity of adrenergic receptors, may be involved in coupling agonist interaction to the receptor function of GP IIb/IIIa.9 The PI3-kinase plays a pivotal role in mediating EC survival, proliferation, cytoskeletal reorganization, and cellular motility, which are all crucially important for vessel growth.64 The PI3-kinase is activated by angiogenesis-related cytokines, such as vascular endothelial growth factor and basic fibroblast growth factor.65
As stated above, PMPs can bind to at least the endothelium, the leukocyte, and the submatrix of a vascular wall, and probably be swallowed by leukocytic and smooth-cell phagocytes to pass the GP IIb/IIIa receptors to them. Salanova and coworkers28 also found that therapeutic GP IIb/IIIa inhibitory compounds such as abciximab, eptifibatide, and tirofiban prevent NF-κB activation through acquired GP IIb/IIIa receptors and may have novel implications in anti-inflammatory treatment protocols. This suggests that the advantageous effect of GP IIb/IIIa antagonists results not only from its platelet inhibition, but partly and probably from its influence on PMPs through GP IIb/IIIa receptors that have originated from platelets.
It is a novel and exciting finding that PMPs can transfer GP IIb/IIIa receptors to other cells, and the presence and consequential effect of PMPs and their receptors in human cells invite further investigation. If GP IIb/IIIa receptor antagonists indeed produce a direct and marked effect on ECs, smooth-muscle cells, and leukocytes through a PMP pathway, investigators have a potential focal point for treatment of acute coronary syndrome.
Address for reprints: Hongliang Cong, PhD, MD, Department of Cardiology, Tianjin Chest Hospital, Xian Road 93#, Heping District, Tianjin 300051, PRC. E-mail: moc.621@gnoCgnailgnoH