Platelets in Inflammation and Atherothrombosis

 

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The present issue of Seminars in Thrombosis and Hemostasis on platelets in inflammation and atherothrombosis by Meinrad Gawaz and his team at the University Hospital, Tübingen, Germany, reviews new topics regarding platelets, inflammation, and atherothrombosis in acute coronary syndromes. This issue covers various established and novel concepts, and provides extensive knowledge on platelet physiology and pathophysiology, as well as platelet-leukocyte-monocyte-endothelial and -subendothelial interactions for a much better understanding of the etiology of atherothrombosis, atheroprogression, and restenosis after percutaneous interventions (PCIs). Understanding the role of platelets and inflammation as interactive multicellular processes at sites of vascular injury in patients with cardiovascular atherosclerotic disease has important diagnostic and therapeutic implications.

During the adhesion to subendothelium, platelets become activated and release an arsenal of potent inflammatory and mitogen substances into the local microenvironment of the damaged vessel wall secondary to trauma, atherosclerosis, or inflammatory diseases, thereby altering the chemotactic, adhesive, and proteolytic properties of endothelial cells, leukocytes, and smooth muscle cells. These platelet-induced alterations of the endothelial-subendothelial phenotype support chemotaxis, adhesion, and transmigration of monocytes to the site of inflammation and induce platelet-mediated thrombotic processes at sites of atherosclerotic vessel wall injury. Platelets provide the adherent surface for monocyte adhesion as bridging cells. Adherent and/or circulating activated platelets express P-selectin and interact with monocyte P-selectin glycoprotein ligand 1 (PSGL-1), and monocyte Mac-1. Stimulated platelets interact via activated glycoprotein (GP) IIb/IIIa (α_IIbβ₃) with von Willebrand and fibrinogen bridges between platelets as the final endpoint of aggregation. The von Willebrand receptor GPIbβ mediates platelet adhesion to subendothelium, and subsequent induction of activated platelets initiates monocyte secretion of chemokines, cytokines, procoagulant tissue factor; upregulates and activates adhesion receptors; and induces monocyte differentiation into macrophages. Similarly, platelets become stimulated by monocytes with regard to the secretion of chemokines, growth factors, cytokine-like factors, and the expression of adhesion receptors. These processes provide an atherogenic milieu vasculaire pathologique, which allows monocyte recruitment, foam cell transformation, and subsequent plaque formation and rupture as the underlying multicellular mechanisms of acute coronary syndromes. Interestingly, platelet-leukocyte aggregates as detected by flow cytometry appear to be more sensitive markers of in vivo platelet activation than platelet surface P-selectin expression.

Platelets and endothelial cells (ECs) interact intensively in health and disease. Every week about 1 kg of new platelets interacts with ECs, which cover an area measuring 300-fold the skin surface of a healthy individual. After initial stimulation of platelets by whatever mechanism, increased production of thromboxane A2 (TxA2) and prostaglandin endoperoxide H2 (PGH2) by platelet cyclooxygenase 1 (COX-1) will activate the TxA2/PGH2 receptor. This is an important receptor for autocrine amplification of platelet activation in vitro and in vivo. Inhibition of platelet COX-1 by one loading dose of aspirin (200 to 250 mg) followed by a daily maintenance dose of 40 to 100 mg is able to control the platelet function and is effective in reducing recurrences of vascular events in high-risk patients with cardiovascular disease. Extensive cross-talk between platelets and ECs occurs continuously over a distance (paracrine signaling), via transient interactions (so-called touch-to-touch and give-to-go mechanisms), or through receptor-mediated cell-cell adhesion at sites of endothelial cell dysfunction or damage. The interaction between circulating von Willebrand factor (vWF) and platelet GPIb at high shear in the microcirculation is rapidly reversible in the presence of a normal endothelial cell layer. Platelets coordinate indirectly via the endothelium changes in coagulation (tissue factor [TF], tissue-type plasminogen activator, etc.), leukocyte trafficking, and extracellular matrix modeling/turnover (matrix metalloproteinase [MMP] and so on). Activated endothelial cells express P-selectin. Platelet tethering to ECs is mediated by platelet surface PSGL-1 and endothelial P-selectin interaction, and platelet rolling involves transient interactions between platelet GPIb and vWF expressed on ECs. Even activated platelet GPIIb-IIIa may adhere firmly to intercellular adhesion molecule expression on activated ECs. The subendothelium extracellular matrix at sites of atherosclerotic lesion interacts with platelets through its collagen receptor, GPVI. Platelets may even adhere much better to the subendothelium when covered with vWF through its GPIb receptor. Subsequent outside-inside signaling will activate the platelet GPIIb-IIIa to form vWF and/or fibrinogen bridges between platelet aggregates followed by clot formation. EC-stimulated production of nitric oxide and prostacyclin cause vasodilatation, whereas TxA2 secretion of adhered platelets on damaged ECs (subendothelial) will cause vasoconstriction, which is a possible cause of angina. A number of additional genes belonging to the group of transcription factors, growth factors (vascular endothelial growth factor [VEGF], transforming growth factor beta; interleukin-1-beta [IL-1β], platelet-derived growth factor [PDGF], or thrombospondin), and adhesion receptors (P-selectin) for extracellular matrix proteins (integrins) are upregulated by activated platelets to induce EC and smooth muscle cell proliferation to initiate wound healing in health and to contribute to the formation of atherosclerotic plaque in hyperlipidemic disease states.

Platelets play a critical role in homing of endothelial progenitor cells (EPCs) from the bone marrow to vascular lesions in promoting neo-endothelialization and smooth muscle cell proliferation. Platelets adhere through GPIb and GPIIb-IIIa receptors to VWF covered subendothelium (collagen type 1), secrete stromal cell-derived factor 1 (SDF-1) to stimulate EPC release from the bone marrow, promote PDGF-mediated proliferation of smooth muscle cells (intimal proliferation), thereby keeping the PSGL-1 receptor free for homing EPC to the platelet layer on subendothelium and subsequent VEGF-mediated induction of one layer of neo-endothelial cells. In cover slip experiments where arterial shear conditions can be simulated, EPCs do not adhere to collagen type 1 or vWF but to platelet adhered to subendothelial cells, which can be inhibited by blocking P-selectin or PSGL-1; these results indicate the pivotal role of adhered platelets for attraction of EPCs for neo-endothelialization at sites of vascular injury.

Circulating megakaryocytes are released from the bone marrow by still-unknown mechanisms; there is substantial evidence that most platelets are generated in the lung capillary system. Platelets carry mRNA for protein synthesis and expression of canalicular system, contractile elements, and membrane receptors on the surface. Platelets also carry mRNA of nuclear protein. The pre-mRNA for IL-1β, TF, are spliced into mature mRNA in a signal-dependent fashion, demonstrating that platelets contain functional nuclear components that have biological significance. Platelet proteonomics rapidly change depending on different states of platelet activation, adhesion aggregation, and clot formation in the context of multicellular processes, and therefore are difficult to interpret. Upon activation of platelets, a transcriptional machinery starts, in which eukaryotic initiation factor 4E binds to the methyl-7 mRNA cap. This complex is able to bind the monosome. The active monosomes on an mRNA strand are called polysomes, which fabricate the peptide-protein, and by reaching the end of the mRNA strand, the peptide-protein is released to do the work at the proper place in platelet function. B-cell lymphoma (BCL)-3 is essential for clot formation. Platelet IL-1β is synthesized in microvesicles and promotes adherence of neutrophils to inflamed endothelial cells.

SDF-1 binds to CXCR4 and the SDF-CXCR4. This complex is of vital importance to human development, including hematopoiesis, angiogenesis, atherosclerosis, and cancer growth and metastasis. SDF-1 is produced constitutively in many organs, including bone marrow, spleen, and lung, and plays a vital role in organ homeostasis by creating cellular niches and retaining hematopoietic stem cells and progenitors within the bone marrow. SDF-1 knock-out mice cannot survive due to multiple organ defects including impaired hematopoiesis. SDF-1 regulates primitive hematopoiesis by suppressing apoptosis and enhancing proliferation and differentiation of immobilized CD34⁺ blood cells. Platelets express SDF-1 mRNA and platelet adhesion to cause subendothelial release of SDF-1, which promotes EPC mobilization from the bone marrow to the circulation. Circulating EPCs are attracted by and home on adhered platelets by ligand interaction in health and disease, as discussed above. Similarly, SDF-1 plays a critical role in angiogenesis, vascular repair in wound healing, tumor growth, and atherosclerosis.

The evil in atherosclerosis is the induction of monocyte transformation into foam cells by adherent platelets and oxidized low-density lipoprotein (LDL). Platelet adherence at sites of vascular injury regulates recruitment and differentiation of CD34⁺ progenitor cells into ECs and foam cells via specific adherence receptors, including P-selectin, PSGL-1, and β1 and β2 integrins. In vitro studies showed that CD34 cells do not adhere to collagen but adhere to attached platelets on vWF-covered subendothelium (collagen type 1) by P-selectin and integrin receptor ligand interactions, and subsequent induction of platelet-induced transformation of CD34⁺ cells to mature foam cells. In this process platelets and ox-LDL loaded platelets are phagocytosed by macrophages and involved in foam cell generation derived from CD34⁺ cells. In these in vitro experiments, interactions between monocytes and platelets induce MMP-9 expression. Statins inhibit foam cell degeneration and reduce MMP-9 secretion by macrophages, and therefore will have an important role in plaque stabilization because of their pronounced LDL-lowering effect.

Peroxisome proliferation-activated receptors (PPARs) are ligand-activated intracellular transcription factors regulating lipid and glucose metabolism. Three different PPARs (α, δ, and γ) are encoded by a separate gene. Both PPAR α and γ are expressed in fully differentiated human macrophages. PPAR-α (fenofibrate, WY 14643) and PPAR-γ (troglitazone, GW 1929) agonists reduce foam cell generation in vitro and in vivo. In platelet/CD34⁺ coculture experiments, PPAR-γ agonist was more potent and PPAR-α agonist was less potent in platelet-mediated foam cell generation.

Surface expression of platelet activation markers P-selectin, GPIbα, and GPVI (measured using flow cytometry) are elevated in patients with stable angina compared with controls. Preliminary data in patients with acute coronary syndrome (ACS) show that GPVI is already elevated several hours before the increase in troponin and creatinine kinase occurs; therefore, GPVI is a potent platelet-specific thrombosis marker to identify patients at high risk of myocardial infarction. Increased GPVI expression and soluble GPVI very likely are the consequence of platelet activation at sites of vascular injury where components of endothelial cellular matrix (ECM), including collagen, are exposed to trigger GPVI-mediated platelet adhesion, activation, and aggregation. At high shear in small arteries (coronary arteries), the interaction between platelet GPIb and vWF immobilized on subendothelium (collagen type 1) is crucial for the initial rolling, tethering, and firm adhesion of platelets followed by GPIIb-IIIa-mediated aggregation and thrombus formation in ACS. Three potential therapeutic options for the intervention of the primary event of platelet-induced thrombosis at sites of vascular injury where ECM components are exposed (including humanized monoclonal antibodies against GPVI, GPI, and the A1 domain of the vWF) have been shown clearly to prevent thrombosis in arterial and coronary animal models. The development of such new humanized monoclonal antibodies is of great importance because the current type of GPIIb-IIIa blockade by the humanized monoclonal antibody abciximab is effective in the setting of ACS and primary PCI, but has a narrow therapeutic window and may be accompanied by several side effects and major bleeding during PCI.

Noninvasive molecular imaging of vulnerable plaques prone to rupture is under intense basic research investigations. Several methods to image components involved in plaque formation are used in the experimental setting by basic researchers and biologists in the laboratory, and contribute significantly to elucidation of plaque formation; however, none are suitable for detection and clinical diagnosis of vulnerable plaque.

Noninvasive angiographic imaging of the coronary arteries with magnetic resonance imaging (MRI) and computed tomography (CT), multislice CT (MCT) in particular, have significantly improved the assessment of coronary artery morphology and plaques. MCT high X-ray doses and the lack of sensitivity to soft tissue variation and contrast agent aggregation limit its clinical application. The application of MRI appears to be much more attractive due to the versatile image contrast and its possible high sensitivity to contrast agent aggregation. The major limiting factor is the long acquisition time required in MRI for providing sufficient spatial and contrast resolution. Electrocardiogram-triggered data acquisition gated to the mid- or end-diastolic cardiac time avoids cardiac motion disturbances of imaging. Arrhythmias impair imaging quality and diagnostic accuracy. Suppression of breathing artifacts can be achieved by the so-called navigator techniques. Optimal MRI imaging is dependent on signal-to-noise ratio, coil selection, and parallel imaging, and also depends on the skill of technicians and engineers.

The most popular clinical application with the best scientific evidence from MCT is the noninvasive detection and quantification of coronary calcifications. The coronary calcium scoring has been incorporated in the current guidelines, indicating that risk calculations in the presence or absence of coronary calcification differ in terms of clinical significance.

Restenosis is the main limitation of percutaneous transluminal coronary angioplasty and occurs in 20 to 50% of all cases. Stent implantation has reduced the percentage of restenosis to a limited extent, but the clinical problem still exists. Drug-eluting stents, which release an antiproliferative compound from a stent coating, have reduced the incidence on in-stent restenosis dramatically. Local drug delivery using special application catheters are an alternative approach for intracoronary pharmacotherapy. In animal models local catheter-based delivery of paclitaxel reduced restenosis formation by synergistic effects on proliferation and remodeling mediated by structural changes of the vascular smooth muscle cells. Local catheter-based application of GPVI to injured collagen-exposed subendothelium prevents thrombosis in animal models. The main advantage of this concept is the fact that the problems of drug-eluting stents can be avoided.

Randomized clinical trial studies in patients undergoing elective PCI showed that dual antiplatelet therapy with aspirin and clopidogrel is as effective as triple antiplatelet therapy with abciximab plus aspirin and clopidogrel in terms of thrombotic events, whereas triple antiplatelet therapy was associated with increased bleeding complications. Variable responses to clopidogrel in terms of the degree of adenosine diphosphate (ADP) -induced platelet aggregation inhibition and clinical outcome have been reported in patients with ACS. Clopidogrel is a prodrug that requires conversion into its active form by hepatic cytochrome P450 and 3A5. The active form leads to an irreversible blockade of the G1 protein coupled to P2Y12 receptor, and this action causes inhibition of cyclic adenosine monophosphate-mediated phosphorylation of vasodilator-stimulated phosphoprotein, which is known to inhibit GPIIb-IIIa receptor. Four to 30% do not respond adequately to clopidogrel loading and treatment dose. Low response to clopidogrel is associated with an increased risk of major cardiovascular events and increased risk of cardiovascular death after 3 months. Patients with ACS may have a 75-fold risk to be low responder to clopidogrel compared with patients with stable angina. A high variability of response to clopidogrel as measured by ADP-induced platelet aggregation may be explained by intraindividual variability in enteral absorption, metabolism of inactive to active metabolites in the liver, and genetic variants of cytochrome P450 and ADP receptor. Single-nucleated polymorphism in cytochrome P3A5 and ADP-receptor P2Y12 genotype have been found to be associated with decreased response to clopidogrel and clinical events in small study populations.

Aspirin irreversibly inhibits platelet COX-1. Aspirin sensitivity can be measured easily by its inhibition of arachidonic acid (AA) -induced platelet aggregation. Aspirin resistance has to be defined by its inability to inhibit COX-1. By using this definition, aspirin resistance very likely does not exist. A specific rapid laboratory test of either AA-induced platelet aggregation or AA-induced malondialdehyde and TxB2 production in platelet-rich plasma is needed to test aspirin sensitivity. The reports on so-called aspirin resistance are usually due to drug-drug interactions (such as ibuprofen and others), noncompliance of aspirin intake, or consumption of inadequate dose of aspirin. In addition, data generated from using nonspecific platelet function tests have also added confusion to this observed phenomenon of aspirin resistance. Those who are considered responders to the aspirin therapy as defined by nonspecific or clinical outcome may need additional protection. COX-1-inhibited platelets by low-dose aspirin are still sensitive or hypersensitive to other agonists such as collagen exposed on subendothelium after endothelial damage or plaque rupture, or have a hypercoagulation pathway, indicating the need of dual or triple antiplatelet therapy during primary and elective PCI in patients with cardiovascular disease.