OVERVIEW OF ARTERIAL THROMBOSIS
In arterial thrombosis, the platelets and abnormalities of the vessel wall typically play a key role in vessel occlusion. Arterial thrombus forms via a series of sequential steps in which platelets adhere to the vessel wall, additional platelets are recruited, and thrombin is activated (Fig. 22-1). The regulation of platelet adhesion, activation, aggregation, and recruitment will be described in detail below. In addition, while the primary function of platelets is regulation of hemostasis, our understanding of their role in other processes, such as immunity, wound healing, and inflammation, continues to grow.
Platelet activation and thrombosis. Platelets circulate in an inactive form in the vasculature. Damage to the endothelium and/or external stimuli activates platelets that adhere to the exposed subendothelial von Willebrand factor and collagen. This adhesion leads to activation of the platelet, shape change, and the synthesis and release of thromboxane (TxA2), serotonin (5-HT), and adenosine diphosphate (ADP). Platelet stimuli cause conformational change in the platelet integrin glycoprotein (GP) IIb/IIIa receptor, leading to the high-affinity binding of fibrinogen and the formation of a stable platelet thrombus.
ARTERIAL THROMBOSIS AND VASCULAR DISEASE
Arterial thrombosis is a major cause of morbidity and mortality both in the United States and, increasingly, worldwide. Although the rates have declined in the United States, the overall burden remains high and accounts for approximately 33% of deaths. Overall, coronary heart disease is estimated to cause about 1 of every 5 deaths in the United States. In addition to the 785,000 Americans who will have a new coronary event, an additional 195,000 silent first myocardial infarctions are projected to occur annually. Although the rate of strokes has fallen by a third, each year, about 795,000 people experience a new or recurrent stroke, although not all are caused by thrombotic occlusion of the vessel. Approximately 610,000 strokes are first events and 185,000 are recurrent events; it is estimated that 1 of every 18 deaths in the United States is due to stroke.
Many processes in platelets have parallels with other cell types, such as the presence of specific receptors and signaling pathways; however, unlike most cells, platelets lack a nucleus and are unable to adapt to changing biologic settings by altered gene transcription. Platelets sustain limited protein synthetic capacity from megakaryocyte-derived and intracellularly transported microRNA (miRNA) and messenger RNA (mRNA). Most of the molecules needed to respond to various stimuli, however, are maintained in storage granules and membrane compartments.
Platelets are disc-shaped, very small, anucleate cells (1–5 μm in diameter) that circulate in the blood at concentrations of 200–400,000/μL, with an average lifespan of 7–10 days. Platelets are derived from megakaryocytes, polyploidal hematopoietic cells found in the bone marrow. The primary regulator of platelet formation is thrombopoietin (TPO). The precise mechanism by which megakaryocytes produce and release fully formed platelets is unclear, but the process likely involves formation of proplatelets, pseudopod-like structures generated by the evagination of the cytoplasm from which platelets bud. Platelet granules are synthesized in megakaryocytes before thrombopoiesis and contain an array of prothrombotic, proinflammatory, and antimicrobial mediators. The two major types of platelet granules, alpha and dense, are distinguished by their size, abundance, and content. Alpha-granules contain soluble coagulation proteins, adhesion molecules, growth factors, integrins, cytokines, and inflammatory modulators. Platelet dense-granules are smaller than alpha-granules and less abundant. Whereas alpha-granules contain proteins that may be more important in the inflammatory response, dense-granules contain high concentrations of small molecules, including adenosine diphosphate (ADP) and serotonin, that influence platelet aggregation.
(See Fig. 22-1) The formation of a thrombus is initiated by the adherence of platelets to the damaged vessel wall. Damage exposes subendothelial components responsible for triggering platelet reactivity, including collagen, von Willebrand factor, fibronectin, and other adhesive proteins, such as vitronectin and thrombospondin. The hemostatic response may vary, depending on the extent of damage, the specific proteins exposed, and flow conditions. Certain proteins are expressed on the platelet surface that subsequently regulate collagen-induced platelet adhesion, particularly under flow conditions, and include glycoprotein (GP) IV, GPVI, and the integrin α2β1. The platelet GPIb-IX-V complex adhesive receptor is central both to platelet adhesion and to the initiation of platelet activation. Damage to the blood vessel wall exposes subendothelial von Willebrand factor and collagen to the circulating blood. The GPIb-IX-V complex binds to the exposed von Willebrand factor, causing platelets to adhere (Fig. 22-1). In addition, the engagement of the GPIb-IX-V complex with ligand induces signaling pathways that lead to platelet activation. von Willebrand factor–bound GPIb-IX-V promotes a calcium-dependent conformational change in the GPIIb/IIIa receptor, transforming it from an inactive low-affinity state to an active high-affinity receptor for fibrinogen.
The activation of platelets is controlled by a variety of surface receptors that regulate various functions in the activation process. Platelet receptors control many distinct processes and are stimulated by a wide variety of agonists and adhesive proteins that result in variable degrees of activation. In general terms, the stimulation of platelet receptors triggers two specific processes: (1) activation of internal signaling pathways that lead to further platelet activation and granule release and (2) the capacity of the platelet to bind to other adhesive proteins/platelets. Both of these processes contribute to the formation of a thrombus. Stimulation of nonthrombotic receptors results in platelet adhesion or interaction with other vascular cells including endothelial cells, neutrophils, and mononuclear cells.
Many families and subfamilies of receptors are found on platelets that regulate a variety of platelet functions. These include the seven transmembrane receptor family, which is the main agonist-stimulated receptor family. Several seven transmembrane receptors are found on platelets, including the ADP receptors, prostaglandin receptors, lipid receptors, and chemokine receptors. Receptors for thrombin comprise the major seven transmembrane receptors found on platelets. Among this last group, the first identified was the protease activation receptor 1 (PAR1). The PAR class of receptors has a distinct mechanism of activation that involves specific cleavage of the N-terminus of thrombin, which, in turn, acts as a ligand for the receptor. Other PAR receptors are present on platelets, including PAR2 (not activated by thrombin) and PAR4. Adenosine receptors are responsible for transduction of ADP-induced signaling events, which are initiated by the binding of ADP to purinergic receptors on the platelet surface. There are several distinct ADP receptors, classified as P2X1, P2Y1, and P2Y12. The activation of both the P2Y12 and P2Y1 receptors is essential for ADP-induced platelet aggregation. The thienopyridine derivatives, clopidogrel and prasugrel, are clinically used inhibitors of ADP-induced platelet aggregation.
Activation of platelets results in a rapid series of signal transduction events, including tyrosine kinase, serine/threonine kinase, and lipid kinase activation. In unstimulated platelets, the major platelet integrin GPIIb/IIIa is maintained in an inactive conformation and functions as a low-affinity adhesion receptor for fibrinogen. This integrin is unique as it is only expressed on platelets. After stimulation, the interaction between fibrinogen and GPIIb/IIIa forms intercellular connections between platelets, leading to the formation of a platelet aggregate (Fig. 22-1). A calcium-sensitive conformational change in the extracellular domain of GPIIb/IIIa enables the high-affinity binding of soluble plasma fibrinogen as a result of a complex network of inside-out signaling events. The GPIIb/IIIa receptor serves as a bidirectional conduit with GPIIb/IIIa-mediated signaling (outside-in) occurring immediately after the binding of fibrinogen. This leads to additional intracellular signaling that further stabilizes the platelet aggregate and transforms platelet aggregation from a reversible to an irreversible process (inside-out).
THE ROLE OF PLATELETS AND THROMBOSIS IN INFLAMMATION
Inflammation plays an important role during the acute thrombotic phase of acute coronary syndromes. In the setting of acute upper respiratory infections, people are at higher risk of myocardial infarction and thrombotic stroke. Patients with acute coronary syndromes have not only increased interactions between platelets (homotypic aggregates), but also increased interactions between platelets and leukocytes (heterotypic aggregates) detectable in circulating blood. These latter aggregates form when platelets are activated and adhere to circulating leukocytes. Platelets bind via P-selectin (CD62P) expressed on the surface of activated platelets to the leukocyte receptor, P-selectin glycoprotein ligand 1 (PSGL-1). This association leads to increased expression of CD11b/CD18 (Mac-1) on leukocytes, which itself supports interactions with platelets partially via bivalent fibrinogen linking this integrin with its platelet surface counterpart, GPIIb/IIIa. Platelet surface P-selectin also induces the expression of tissue factor on monocytes, which promotes fibrin formation.
In addition to platelet–monocyte aggregates, the immunomodulator, soluble CD40 ligand (CD40L or CD154), also reflects a link between thrombosis and inflammation. The CD40 ligand is a trimeric transmembrane protein of the tumor necrosis factor family and, with its receptor CD40, is an important contributor to the inflammatory process leading both to thrombosis and atherosclerosis. While many immunologic and vascular cells have been found to express CD40 and/or CD40 ligand, in platelets, CD40 ligand is rapidly translocated to the surface after stimulation and is upregulated in the newly formed thrombus. The surface-expressed CD40 ligand is cleaved from the platelet to generate a soluble fragment (soluble CD40 ligand).
Links have also been established among platelets, infection, immunity, and inflammation. Bacterial and viral infections are associated with a transient increase in the risk of acute thrombotic events, such as acute myocardial infarction and stroke. In addition, platelets contribute significantly to the pathophysiology and high mortality rates of sepsis. The expression, functionality, and signaling pathways of toll-like receptors (TLRs) have been established in platelets. Stimulation of platelet TLR2, TLR3, and TLR4 directly and indirectly activates the platelet’s thrombotic and inflammatory responses, and live bacteria induce a proinflammatory response in platelets in a TLR2-dependent manner, suggesting a mechanism by which specific bacteria and bacterial components can directly activate platelet-dependent thrombosis.
GENETICS OF ARTERIAL THROMBOSIS
Some studies have associated arterial thrombosis with genetic variants (Table 22-1A); however, the associations have been weak and not confirmed in larger series. Platelet count and mean platelet volume have been studied by genome-wide association studies (GWAS), and this approach identified signals located to noncoding regions. Of 15 quantitative trait loci associated with mean platelet volume and platelet count, one located at 12q24 is also a risk locus for coronary artery disease.
TABLE 22-1Heritable Causes of Arterial and Venous Thrombosis ||Download (.pdf) TABLE 22-1 Heritable Causes of Arterial and Venous Thrombosis
|A. Arterial Thrombosis |
|Platelet Receptors |
| β3 and α2 integrins |
| Pl A2 polymorphism |
| Fc(gamma)RIIA |
| GPIV T13254C polymorphism |
| GPIb |
| Thrombin receptor PAR1-5061 → D |
|Redox Enzymes |
| Plasma glutathione peroxidase |
| H2 promoter haplotype |
| Endothelial nitric oxide synthase |
| −786T/C, −922A/G, −1468T/A |
| Paraoxonase |
| −107T allele, 192R allele |
| Cystathionine β-synthase 833T → C |
| 5,10-Methylene tetrahydrofolate reductase (MTHFR) 677C → T |
|B. Venous Thrombosis |
|Procoagulant Proteins |
| Fibrinogen |
| −455G/A, –854G/A |
|Prothrombin (20210G → A) |
|Protein C Anticoagulant Pathway |
| Factor V Leiden: 1691G → A (Arg506Gln) |
| Thrombomodulin 1481C → T (Ala455Val) |
|Fibrinolytic Proteins with Known Polymorphisms |
| Tissue plasminogen activator (tPA) |
| 7351C/T, 20 099T/C in exon 6, 27 445T/A in intron 10 |
| Plasminogen activator inhibitor (PAI-1) |
| 4G/5G insertion/deletion polymorphism at position –675 |
| Cystathionine β-synthase 833T → C |
| 5,10-MTHFR 677C → T |
In the area of genetic variability and platelet function, studies have primarily dealt with pharmacogenetics, the field of pharmacology dealing with the interindividual variability in drug response based on genetic determinants (Table 22-2). This focus has been driven by the wide variability among individuals in terms of response to antithrombotic drugs and the lack of a common explanation for this variance. The best described is the issue of “aspirin resistance,” although heterogeneity for other antithrombotics (e.g., clopidogrel) has also been extensively examined. Primarily, platelet-dependent genetic determinants have been defined at the level of (1) drug effect, (2) drug compliance, and (3) drug metabolism. Many candidate platelet genes have been studied for their interaction with antiplatelet and antithrombotic agents.
TABLE 22-2Genetic Variation and Pharmacogenetic Responses to Platelet Inhibitors ||Download (.pdf) TABLE 22-2 Genetic Variation and Pharmacogenetic Responses to Platelet Inhibitors
|Potential Gene Altered ||Target Therapeutic Class ||Specific Drug |
|P2Y1 and P2Y12 CYP2C19, CYP3A4, CYP3A5 ||ADP receptor inhibitors ||Clopidogrel, prasugrel |
|COX1, COX2 ||Cyclooxygenase inhibitors ||Aspirin |
|PlA1/A2 ||Receptor inhibitors ||Abciximab, eptifibatide, tirofiban |
|INTB3, GPIbA ||Glycoprotein IIb-IIIa receptor inhibitors || |
Many patients have an inadequate response to the inhibitory effects of aspirin. Heritable factors contribute to the variability; however, ex vivo tests of residual platelet responsiveness after aspirin administration have not provided firm evidence for a pharmacogenetic interaction between aspirin and COX1 or other relevant platelet receptors. As such, currently, there is no clinical indication for genotyping to optimize aspirin’s antiplatelet efficiency. For the platelet P2Y12 receptor inhibitor clopidogrel, additional data suggest that genetics may affect the drug’s responsiveness and utility. The responsible genetic variant appears not to be the expected P2Y12 receptor but an enzyme responsible for drug metabolism. Clopidogrel is a prodrug, and liver metabolism by specific cytochrome P450 enzymes is required for activation. The genes encoding the CYP-dependent oxidative steps are polymorphic, and carriers of specific alleles of the CYP2C19 and CYP3A4 loci have increased platelet aggregability. Increased platelet activity has also been specifically associated with the CYP2C19*2 allele, which causes loss of platelet function in select patients. Because these are common genetic variants, this observation has been shown to be clinically relevant in large studies. In summary, although the loss-of-function polymorphisms in CYP2C19 is the strongest individual variable affecting pharmacokinetics and antiplatelet response to clopidogrel, it only accounts for 5–12% of the variability in ADP-induced platelet aggregation on clopidogrel. In addition, genetic variables do not appear to significantly contribute to the clinical outcomes of patients treated with the P2Y12 receptor antagonists prasugrel or ticagrelor.