CHAPTER 33: CORONARY THROMBOSIS: LOCAL AND SYSTEMIC FACTORS

The formation of an acute thrombus on a ruptured coronary atherosclerotic lesion, obstructing coronary blood flow and reducing the oxygen supply to the myocardium, leads to the onset of acute coronary syndromes (ACS). These thrombotic episodes largely occur in response to atherosclerotic lesions that have progressed to a high-risk inflammatory or prothrombotic stage. Although they are distinct from one another, the atherosclerotic and thrombotic processes appear to be closely related, causing ACS through a complex, multifactorial process called atherothrombosis. ACS represents a spectrum of ischemic myocardial events that share similar pathophysiology; these include unstable angina/non–ST-segment elevation myocardial infarction (UA/NSTEMI), ST-segment elevation myocardial infarction (STEMI), and sudden cardiac death.

Atherosclerosis is a systemic disease involving the intima of large and medium-sized arteries, including the aorta, carotids, coronaries, and peripheral arteries, that is characterized by intimal thickening caused by the accumulation of cells and lipids (Fig. 33–1).¹ Lipid accumulation results from an imbalance between the mechanisms responsible for the influx and efflux of lipids into the arterial wall.² Secondary changes may occur in the underlying media and adventitia, particularly in advanced disease stages. The early atherosclerotic lesions might progress without compromising the lumen, because of compensatory vascular enlargement (Glagovian remodeling).³ Importantly, the culprit lesions leading to ACS are usually mildly stenotic and, therefore, barely detected by angiography.⁴ These high-risk, rupture-prone lesions usually have a large lipid core, a thin fibrous cap, and a high density of inflammatory cells (particularly at the shoulder region, where disruptions most often occur).

Recent evidence has highlighted the importance of lesion neovascularization and blood extravasation in plaque destabilization and plaque growth.^5,6,7 Leaky vasa vasorum with the subsequent red blood cell extravasation has been postulated as a major source for intraplaque cholesterol deposition. This change in composition, characterized by increased extracellular cholesterol within the lipid core and excessive macrophage infiltration, increases the vulnerability of the atherosclerotic lesions. In fact, postmortem studies have shown a strong correlation between macrophage infiltration and increased vasa vasorum in human atherosclerotic lesions. Preexisting vasa vasorum in the adventitia is thought to spread into the intima, prompting intimal neovascularization.⁶ A recent study using optical coherence tomography has associated vasa vasorum increase with fibrous plaque volume and intraplaque neovessels with plaque vulnerability.⁸ These observations suggest that imaging for microvasculature could become a new biomarker for plaque vulnerability. On the other hand, inhibition of plaque neovascularization could be seen as a potential new therapeutic intervention to prevent plaque disruption.

Inflammation is another important process that affects plaque progression, vulnerability, and subsequent thrombus formation. Inflammation could be considered as the link between atherosclerosis and thrombosis. In fact, inflammation and atherothrombosis could represent different faces of the same disease. Most of the studies on inflammation and cardiovascular disease have been based on the effects of inflammatory cyto/chemokines without paying the deserved attention to their cellular sources. Recent studies are bringing light to the key role played by leukocytes and platelets as sources of proinflammatory cytokines. Inflammatory cells (monocyte/macrophages, T cells, and mast cells) are present in the core and shoulder of atherosclerotic lesions. Many of the cells exhibit signs of activation and release inflammatory cytokines and matrix metalloproteinases (MMPs) that affect each step of atherosclerosis, from lesion formation, to progression, to disruption and ACS. The circulating monocytes are recruited within the subendothelial space in response to the synthesis and exposure of adhesive proteins (eg, selectins, monocyte chemotactic protein [MCP]-1) triggered by the early accumulation of lipids. The internalized monocytes release inflammatory mediators that maintain the monocyte recruitment, favoring lesion progression. The presence of growth factors, such as the macrophage colony-stimulating factor, facilitates the replication of these cells and their transformation into macrophages. Several factors, such as netrin-1 and vascular cell adhesion molecule-1 (VCAM-1) are responsible for their retention in the lesions. The attenuation of the proatherogenic state, as occurs in conditions characterized by high-density lipoprotein (HDL)-raising or low-density lipoprotein (LDL)-lowering by statins, seems to facilitate the efflux of the wall macrophages. Whether it is due to reduced levels of the retaining factors or systemic lipid lowering is not clear yet.⁹

The monocyte/macrophage system includes the most representative cells of the innate immunity via the scavenger and Toll-like receptors. Innate immunity does not require “previous education” but only recognizes a few hundred antigens. Monocytes, dendritic cells, and mast cells are the major players in the innate immunity (Fig. 33–2). Mast cells are inflammatory by releasing histamine, leukotrienes, interleukin (IL)-6, and interferon (IFN)-gamma. Dendritic cells are responsible for antigen presentation via human leukocyte antigen (HLA) molecules CD80/86 and CD40. Monocytes are divided into two major types according to their expression of CD14 and CD16. CD14+/CD16^– monocytes, also known as “classic” or Mon 1, are the most abundant subtype. They express CCR2 (receptor for MCP-1), CD62L, CD64, and CD115, but not CXCR1, and they perpetuate the inflammatory status by releasing reactive oxygen species, tumor necrosis factor (TNF), IL-1, and other inflammatory cytokines. A second subtype is the CD14+/CD16++, Mon 3, cells, also called “nonclassic” or resident. These do not express CCR2 or CD64 but express CXCR1 (receptor for fractalkine). They release transforming growth factor-beta, CD-36, CD163, and the angiogenic factor vascular endothelial growth factor. They are less inflammatory and responsible for metabolic tissue repair. A recently described subtype is the CD14++/CD16+, Mon 2, monocytes, also called intermediate. These express high levels of CCR2, CX3CR1, and CDS115, but not CD62. Their role is still not well understood, although they may play an inflammatory role.^9,10

The acquired immunity is more modern from an evolutionary view. It is very specific and recognizes millions of antigens but requires “previous education.” This makes it similar to vaccines, which require weeks of prior generating antibodies. It is involved in the interactions between lymphoids with the dendritic and lymphatics of the atherosclerotic lesions.¹¹ Lymphoids, CD4, and the helper T cells are involved in cellular responses while B lymphocytes are responsible for antibody release. Among the T lymphocytes, the Th1 and 17 lineages release IFN-gamma while regulatory T cells (Treg) are anti-inflammatory (Fig. 33–3). The possibility of modulating the adaptive immune system may be seen as a novel therapeutic target to treat atherosclerosis.¹² Studies are ongoing into the development of vaccines that could effectively interfere with the proportion of pro- and anti-inflammatory cells in the lesions and the secretion of protective antibodies while affecting the general host defense.¹³ T cells constitute 10% of all cells in human lesions. Seventy percent of T cells are described to be CD4+ and the remainder CD8+. Most of the CD4+ cells are T helper 1 (Th1) and a major source of INF-gamma and TNF, and thus estimated to be inflammatory.

Inflammation is beginning to be a potential therapeutic target for atherosclerosis, although without significant success so far.^11,12 One of the major problems for anti-inflammatory strategies as treatment for atherosclerosis is that, so far, all the considered “anti-inflammatory” approaches have also affected plasma lipid levels (eg, statins). Recent advances in the understanding of the pathophysiology of the inflammatory pathways may lead to more success. In fact, several ongoing trials with specific anti-inflammatory interventions—the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) (NCT01327846) studying canakinumab and the cardiovascular inflammation reduction trial (CIRT) (NCT 02576067) studying low-dose methotrexate—are testing the effectiveness of this novel approach.

Chronic inflammatory diseases are associated with an increased risk of cardiovascular disease. The role of inflammation as an important proatherosclerotic factor is illustrated by the increased cardiovascular risk exhibited by chronic inflammatory conditions such as rheumatoid arthritis, systemic lupus erythematosus, and psoriasis, as well as with infections such as periodontal disease and human immunodeficiency virus (HIV).^3,14,15 There is an abundance of evidence, mostly from imaging studies, supporting an association between inflammation and calcium deposition, but the question still unanswered is whether inflammation precedes calcification or rather calcification triggers inflammation.¹⁶

Platelets also play an important role in the inflammatory environment. Although formerly platelets were suggested to be passive players because they are organelles lacking nuclei, currently we know that they are very active, being important not only in thrombosis itself but also in perpetuating the inflammatory environment. Platelets secrete various vasoactive chemokines and cytokines. These include CD40L; thrombospondin; platelet-activating factor; regulated on activation, normal T-cell expressed and secreted (RANTES); epithelial-derived neutrophil-activating peptide 78 (ENA-78); macrophage inflammatory protein (MIP); and c hemokine (C-X-C motif) ligand 4 (CXCL4), with autocrine and paracrine effects. Also very important is the notion that free cholesterol, a major component of atherosclerotic plaques, is one the most inflammatory substances triggering the recruitment of more macrophages. Therefore, inflammation plays a dual role in atherothrombosis, both at the vascular and circulating levels.¹⁷

A reliable, noninvasive imaging tool able to detect early atherosclerotic disease and characterize lesion composition would be clinically advantageous. Although magnetic resonance imaging (MRI) has been widely tested for plaque composition analysis, other imaging techniques, such as computed tomography (CT) and radionuclide imaging (positron emission tomography [PET]),^{18,19,20,21,22} have recently been used for this enterprise.

All the molecular knowledge gathered on the biochemical and molecular processes involved in the genesis and progression of atherosclerosis have been incorporated into the imaging techniques generating the concept of molecular imaging for a more specific and better characterization of atherosclerotic lesions. Studies from the multi-ethnic study of atherosclerosis (MESA) trial suggest adding coronary artery calcium to the American College of Cardiology (ACC)/American Heart Association (AHA) Guidelines to improve subjects statin eligibility for statin therapy.²³

Growing thrombi on atherosclerotic vessels may occlude the lumen locally or embolize and be washed away by the blood flow to occlude distal vessels. However, thrombi may be physiologically or spontaneously lysed by mechanisms that block thrombus propagation. Thrombus size, location, and composition are regulated by local fluid dynamic conditions (mechanical effects), the thrombogenicity of exposed substrate (local molecular effects), the relative concentration of fluid phase and cellular blood components (local cellular effects), and the efficiency of the physiologic mechanisms of control of the system, mainly fibrinolysis. Similarly, the size and stability of the thrombus are major modulators of the severity of the ACS.^1,7

Although several decades ago the endothelium was viewed as a simple barrier separating the fluid phase of the blood from the highly thrombogenic smooth muscle vascular wall, today we know that the endothelium is a critical player in the maintenance of the normal function of the arterial bed. The endothelium (endothelial cells) constantly secretes substances (eg, hormones, growth factors) into the vascular lumen to maintain vascular tone and to avoid abnormal platelet adhesion or activation and clot formation. When the endothelium is damaged and cannot perform this crucial task, it is dysfunctional. Endothelial dysfunction, as well as a discontinuity of the endothelial integrity, triggers a series of biochemical and molecular reactions aimed at preventing excessive blood loss and repairing the vessel wall. Vasoconstriction and platelet adhesion at the site of injury combine to form a hemostatic aggregate as the first step in vessel wall repair and the hemostasis. A few platelets may interact with injured and dysfunctional endothelium and release growth factors that stimulate intimal hyperplasia. Several layers of platelets may be deposited on the lesion with mild injury and may or may not evolve to become a mural thrombus (Fig. 33-4). The release of platelet growth factors may contribute significantly to accelerated intimal hyperplasia, as occurs in the coronary vein grafts within the first postoperative year. With severe injury and exposure of components of deeper layers of the vessel, as in spontaneous plaque rupture and in angioplasty, marked platelet aggregation with mural thrombus formation follows. Vascular injury of this magnitude also stimulates thrombin formation through both the intrinsic (surface-activated) and extrinsic (tissue factor [TF]–dependent) coagulation pathways (see Fig. 33–3).

Platelets

Platelets are anucleated cells (2 μm in diameter) that are devoid of genomic DNA but contain messenger RNA and have the capability of synthesizing proteins. They are cytosolic fragments of bone marrow–derived megakaryocytes and they have a life span of 7 to 10 days under normal conditions. Plaque rupture facilitates the interaction of the inner plaque components with the circulating blood.²⁴ Among these components, TF exhibits a potent activating effect on platelets and coagulation. There is now understanding of the biochemical events involved in platelet activation. At the site of vascular lesions, circulating von Willebrand factor (vWF) binds to the exposed collagen that subsequently binds to the glycoprotein (GP) Ib/IX receptor on the platelet membrane. Under pathological conditions and in response to changes in shear stress, vWF can be secreted from the storage organelles in platelets or endothelial cells, reinforcing the activation process. Although GP Ib/IX–vWF interaction is enough to promote binding of platelets to subendothelium, it is highly transient, resulting in rapid dislocation of platelet from the site of injury. GP VI binding to matrix collagen has slower binding kinetics, but when initiated, it promotes a firm adhesion of platelet to the vessel surface.²⁵ Figure 33–5 shows mechanisms and agonists involved in platelet adhesion, activation, and aggregation.²⁶ Finally, both GP Ib/IX and GP VI also regulate platelet-leukocyte adhesion and thereby are implicated in other vascular processes, such as inflammation and atherosclerosis.^27,28,29 Exposed matrix from the vessel wall and thrombin generated by activation of the coagulation cascade, as well as epinephrine and adenosine diphosphate (ADP), are powerful platelet agonists. Each agonist stimulates the discharge of calcium and promotes the subsequent release of its granular content. Platelet-related ADP and 5-hydroxytryptamine (5-HT) stimulate adjacent platelets, further enhancing the process of platelet aggregation. Arachidonate, which is released from the platelet membrane by the stimulatory effect of collagen, thrombin, ADP, and 5-HT, promotes the synthesis of thromboxane A₂ (TXA₂) by the sequential effects of cyclooxygenase (COX) and thromboxane synthetase. TXA₂ not only promotes further platelet aggregation but is also a potent vasoconstrictor (Fig. 33–6; see also Fig. 33–5). The initial recognition of damaged vessel wall by platelets involves (1) adhesion, activation, and adherence to recognition sites on the thromboactive substrate (extracellular matrix [ECM] proteins such as vWF, collagen, fibronectin, vitronectin, and laminin); (2) spreading of the platelet on the surface; and (3) aggregation of platelets to form a platelet plug or white thrombus. Figure 33–5 shows the processes implicated in the formation of a thrombus on a plaque disruption.²⁶ The efficiency of platelet recruitment depends on the underlying substrate and local geometry (local factors). A final step involving the recruitment of other blood cells also occurs; erythrocytes, neutrophils, and occasionally monocytes are found on evolving mixed thrombus.

Platelet function depends on the adhesive interaction of several compounds. Most of the GPs on the platelet membrane surface are receptors for adhesive proteins. Many of these receptors have been identified, cloned, sequenced, and classified within large gene families that mediate a variety of cellular interactions (Table 33–1). The most abundant is the integrin family, which includes GP IIb/IIIa, GP Ic/IIa, the fibronectin receptor, and the vitronectin receptor, in decreasing order of magnitude. Another gene family present in the platelet membrane glycocalyx is the leucine-rich GP family represented by the GP Ib/IX complex, receptor for vWF, on unstimulated platelets that mediates adhesion to subendothelium and GP V. Other genes include the selectins (such as GMP-140) and the immunoglobulin domain protein human leukocyte antigen (HLA) class I antigen and platelet/endothelial cell adhesion molecule 1. Unrelated to any other gene family is GP IV (IIIa) (see Table 33–1).

Randomly distributed on the surface of resting platelets are about 50,000 molecules of GP IIb/IIIa. The complex is composed of one molecule of GP IIb (disulfide-linked large and light chains) and one of GP IIIa (single polypeptide chain). It is a Ca²⁺-dependent heterodimer, noncovalently associated on the platelet membrane. Calcium is required for maintenance of the complex and for binding of adhesive proteins. On activated platelets, the GP IIb/IIIa is a receptor for fibrinogen, fibronectin, vWF, vitronectin, and thrombospondin.

The GP Ib/IX complex consists of two disulfide-linked subunits (GP Ibα and GP Ibβ) tightly (not covalently) complexed with GP IX in a 1:1 heterodimer. GP Ibβ and GP IX are transmembrane GPs and form the larger globular domain. The elongated, protruding part of the receptor corresponds to GP Ibα. The major role of GP Ib/IX is to bind immobilized vWF on the exposed vascular subendothelium and initiate adhesion of platelets. GP Ib does not bind soluble vWF in plasma; apparently, it undergoes a conformational change on binding to the ECM and then exposes a recognition sequence for GP Ib/IX. The cytoplasmic domain of GP Ib/IX has a major function in linking the plasma membrane to the intracellular actin filaments of the cytoskeleton and functions to stabilize the membrane and to maintain the platelet shape.

Thrombin plays an important role in the pathogenesis of arterial thrombosis. It is one of the most potent known agonists for platelet activation and recruitment. In addition, thrombin is critical in the maintenance of the fibrin mesh. The thrombin receptor has 425 amino acids with seven transmembrane domains and a large NH₂-terminal extracellular extension that is cleaved by thrombin to produce a “tethered” ligand that activates the receptor to initiate signal transduction.²⁷ Thrombin is a critical enzyme in early thrombus formation, cleaving fibrinopeptides A and B from fibrinogen to yield insoluble fibrin, which effectively anchors the evolving thrombus. Both free and fibrin-bound thrombin are able to convert fibrinogen to fibrin, allowing propagation of thrombus at the site of injury (Fig. 33-7).

Therefore, platelet activation triggers intracellular signaling and expression of platelet membrane receptors for adhesion and initiation of cell contractile processes that induce shape change and secretion of the granular contents. The expression of the integrin IIb/IIIa (αIIbβ₃) receptors for adhesive GP ligands (mainly fibrinogen and vWF) in the circulation initiates platelet-to-platelet interaction. The process is perpetuated by the arrival of platelets from the circulation. Most of the GPs in the platelet membrane surface are receptors for adhesive proteins or mediate cellular interactions. vWF has been shown to bind to platelet membrane GPs in both adhesion (platelet-substrate interaction) and aggregation (platelet-platelet interaction), leading to thrombus formation, as seen in perfusion studies conducted at high shear rates.²⁸ Ligand binding to the different membrane receptors triggers platelet activation with different relative potencies. Great interest in the platelet ADP receptors (P2Y, P2X) has recently been generated because of available pharmacologic inhibitors. The P2Y₁ receptor is responsible for inositol trisphosphate formation through activation of phospholipase C, leading to transient increase in the concentration of intracellular calcium, platelet shape change, and weak transient platelet aggregation.^29,30 Pharmacologic data have revealed an essential role for the P2Y1 receptor in the initiation of platelet ADP-induced activation, TXA₂ generation, and platelet activation in response to other agonists. The P2Y₁₂ receptor is responsible for completion of the platelet aggregation response to ADP. There are several signaling molecules downstream of P2Y₁₂ activation, including cAMP, vasodilator-stimulated phosphoprotein dephosphorylation, phosphoinositide 3-kinase, and Rap1B. Pharmacologic approaches have shown a role for the P2Y₁₂ receptor in dense granule secretion, fibrinogen-receptor activation, P-selectin expression, and thrombus formation, identifying it as a central mediator of the hemostatic response. Both P2Y₁₂ and P2Y₁ are indirectly involved in platelet P-selectin exposure and formation of platelet-leukocyte conjugates, which leads to leukocyte-TF exposure.^{31,32,33,34,35} Although not activated by ADP, platelets possess a third purinergic receptor (P2X₁), which is a fast adenosine triphosphate (ATP)-gated calcium channel receptor mainly involved in platelet shape change. Figure 33–8 shows in detail the platelet purinergic receptors. The most recent advances in antiplatelet therapy related to the inhibition of the P2Y₁₂ receptors are focused on the availability of faster acting and reversible inhibitors.^36,37

Dual antiplatelet therapy, with aspirin, a platelet COX-1 inhibitor, and P2Y₁₂ receptor blocker, remains a major drug strategy to prevent ischemic event occurrence in patients with ACS and in patients undergoing coronary stenting. The new P2Y₁₂ receptor blockers, prasugrel and ticagrelor, have been shown to have an enhanced benefit on the prevention of atherothrombotic events when compared with clopidogrel in patients with ACS. However, despite these improvements, there are still a significant number of events still taking place. A recent article reported the antithrombotic effects of the synergistic inhibition of both P2Y₁ and P2Y₁₂ ADP receptors; given the intravenous formulation of this agent, the authors suggested that this dual inhibitory agent could serve as a promising strategy in the initial phases of ACS.³⁷ Other strategies focusing on additional targets have been investigated. One of these potential targets is thrombin. Thrombin is not only the most potent agonist of platelet aggregation and degranulation but also plays a major role in the conversion of fibrinogen into fibrin and the maintenance of the formed thrombus. Thrombin activates platelets by cleaving platelet protease activated receptors (PARs). The thrombin effect on platelets is mostly mediated via PAR-1 and PAR-4. PAR-1 receptor blockade by specific inhibitors, such as vorapaxar, results in the inhibition of thrombin-induced platelet signaling and in platelet function inhibition.³⁸

Coagulation System

During plaque rupture, flowing blood interacts with inner components of the lesions, TF being among them. In addition to the TF located in the plaques, there is robust evidence of the existence of a bloodborne circulating TF. Such circulating TF is found in monocytes and small microparticles (MPs). It has been shown that leukocytes may transfer TF-enriched MP platelets by the Cd15 and platelet P-selectin interaction. These interactions contribute to a high local TF concentration in the disrupted area. One issue yet to be clarified is the individual contribution of the TF from the plaque versus the contribution of systemic TF to thrombus formation.^1,7

The blood coagulation system involves a sequence of reactions integrating zymogens (proteins susceptible to activation into enzymes via limited proteolysis) and cofactors (nonproteolytic enzyme activators) into three groups: (1) the contact activation (generation of factor XIa via the Hageman factor), (2) the conversion of factor X to factor Xa in a complex reaction requiring the participation of factors IX and VIII, and (3) the conversion of prothrombin to thrombin and fibrin formation³⁹ (Fig. 33–9).

Citrate is a calcium chelate frequently used in studies of platelet–vessel wall interaction. Its anticoagulant properties are based on its action over calcium. It not only blocks the coagulation cascade—the coagulation reactions do not proceed further than the activation of factor XI because of their dependence on Ca²⁺—but also inhibits platelet activation because of the central role of calcium in platelet activation (see Fig. 33–5). Platelets may provide the membrane requirements for the activation of factor X, although the participation of cells of the vessel wall (in exposed injured vessels) has not been excluded. As such, endothelial cells in culture have been shown to support the activation of factor X. Factor VIII forms a noncovalent complex with vWF in plasma, and its function in coagulation is the acceleration of the effects of IXa on the activation of X to Xa. Absence of factor VIII or IX produces the hemophilic syndromes (see Fig. 33–10).

The TF pathway, previously known as the extrinsic coagulation pathway, through the TF–factor VIIa complex in the presence of Ca²⁺ induces the formation of Xa. A second TF-dependent reaction catalyzes the transformation of IX into IXa. TF is an integral membrane protein that serves to initiate the activation of factors IX and X and to localize the reaction to cells on which TF is expressed. Other cofactors include factor VIIIa, which binds to platelets and forms the binding site for IXa, thereby forming the machinery for the activation of X, and factor Va, which binds to platelets and provides a binding site for Xa. The human genes for these cofactors have been cloned and sequenced. In physiologic conditions, no cells in contact with blood contain active TF, although cells such as monocytes and polymorphonuclear leukocytes can be induced to synthesize and express TF.⁴⁰

Activated Xa converts prothrombin into thrombin. The complex that catalyzes the formation of thrombin consists of factors Xa and Va in a 1:1 complex. This activation results in the cleavage of fragment 1.2 and formation of thrombin from fragment 2. The interaction of the four components of the “prothrombinase complex” (Xa, Va, phospholipid, and Ca²⁺) enhances the efficiency of the reaction.

Activated platelets provide a procoagulant surface for the assembly and expression of both intrinsic Xase and prothrombinase enzymatic complexes.⁴¹ These complexes respectively catalyze the activation of factor X to factor Xa and prothrombin to thrombin. The expression of activity is associated with the binding of both of the proteases factor IXa and factor Xa and the cofactors VIIIa and Va to procoagulant surfaces. The binding of IXa and Xa is promoted by VIIIa and Va, respectively, such that Va and likely VIIIa provide the equivalent of receptors for the proteolytic enzymes. The surface of the platelet expresses the procoagulant phospholipids that bind coagulation factors and contribute to the procoagulant activity of the cell.

Thrombin acts on multiple substrates, including fibrinogen, factor XIII, factors V and VIII, and protein C in addition to its effects on platelets. It plays a central role in hemostasis and thrombosis. The catalytic transformation of fibrinogen into fibrin is essential in the formation of the hemostatic plug and in the formation of arterial thrombi. Thrombin binds to the fibrinogen central domain and cleaves fibrinopeptides A and B, resulting in the formation of fibrin monomer and polymer formation. The fibrin mesh holds the platelets together and contributes to the attachment of the thrombus to the vessel wall.

The new orally active anticoagulants (NOACs) are focused on the inhibition of the TF pathway, clearly proving the importance of TF in the onset of thrombotic events. The NOACs can be divided into two major classes according to their mechanism of action. Direct thrombin inhibitors (DTIs), such as dabigatran, that block thrombin activity but not its generation, and factor Xa inhibitors that block the activation of factor Xa, suppressing the in vivo thrombin generation (however, they are not able to inhibit the thrombin already generated). Rivaroxaban, apixaban and most recently, edoxaban are the major exponents of coagulation factor Xa inhibitors. It is not the scope of this chapter to expand on the clinical efficacy of these antithrombotic agents; they are discussed in other chapters.

New techniques that permit the retrieval of thrombus from ACS patients allow a better characterization of the thrombus formed in vivo on atherosclerotic plaques. Recent data obtained by thromboaspiration of coronary occlusive thrombi (manifested as STEMI) indicate that the recruitment of platelets is an early and fast event as thrombi more than 6 hours old show a significant reduction in the platelet number and an increased in coagulation products. Of interest, older intracoronary thrombi are still capable of recruiting systemic inflammatory cells.⁴²

Spontaneous Fibrinolysis

The control of the coagulation reactions occurs by diverse mechanisms, such as hemodilution and flow effects, proteolytic feedback by thrombin, inhibition by plasma proteins (eg, antithrombin III [ATIII]), endothelial cell–localized activation of an inhibitory enzyme (protein C), and fibrinolysis (Fig. 33–10). Although ATIII readily inactivates thrombin in solution, its catalytic site is inaccessible while bound to fibrin; it may still cleave fibrinopeptides even in the presence of heparin. Thrombin has a specific receptor in endothelial cell surfaces, thrombomodulin, which triggers a physiologic anticoagulation system. The thrombin-thrombomodulin complex serves as a receptor for the vitamin K–dependent protein C, which is activated and released from the endothelial cell surface. Thrombin generated at the site of injury binds to thrombomodulin, an endothelial surface-membrane protein, initiating activation of protein C, which in turn (in the presence of protein S) inactivates factors Va and VIIIa and limits thrombin effects. Loss of Va decreases the role of thrombin formation to negligible levels. Thrombin stimulates successive release of both tissue plasminogen activator (tPA) and plasminogen-activator inhibitor type 1 (PAI-1) from endothelial cells, thus initiating endogenous lysis through plasmin generation from plasminogen by tPA with subsequent modulation through PAI-1. Thrombin therefore plays a pivotal role in maintaining the complex balance of initial prothrombotic reparative events and subsequent endogenous anticoagulant and fibrinolytic pathways. The pivotal role of thrombin in the different mechanisms exposed here is represented in Fig. 33–11.

Endogenous fibrinolysis, a repair mechanism, involves catalytic activation of zymogens, positive and negative feedback control, and inhibitor blockade (see Fig. 33–10). Blood clotting is blocked at the level of the prothrombinase complex by the physiologic anticoagulant-activated protein C and oral anticoagulants. Oral anticoagulants prevent post-translational synthesis of γ-carboxyglutamic acid groups on the vitamin K–dependent clotting factors, preventing binding of prothrombin and Xa to the membrane surface. Activated protein C cleaves factor Va, rendering it functionally inactive.

The cellular and molecular mechanisms of platelet deposition and thrombus formation after vascular damage are modulated by the type of injury, the local geometry at the site of damage (degree of stenosis), and local hemodynamic conditions.^43,44 Similarly, three major factors also determine the vulnerability of the fibrous cap: (1) circumferential wall stress, or cap “fatigue”; (2) lesion characteristics (location, size, and consistency); and (3) blood flow characteristics.

Effects Derived from the Severity of Vessel Wall Damage

Exposure of deendothelialized vessel wall, native fibrillar collagen type I bundles with a rough surface, or atherosclerotic plaque components at similar blood shear rate conditions lead to increasing degrees of platelet deposition. Thromboplastin or TF, readily available in the atherosclerotic intimal space exposed by endothelial loss, contributes to the high thrombogenicity of atherosclerotic plaques.^45,46 Overall, it is likely that when injury to the vessel wall is mild, the thrombogenic stimulus is relatively limited, and the resulting thrombotic occlusion is transient, as occurs in UA. On the other hand, deep vessel injury secondary to plaque rupture or ulceration results in exposure of collagen, TF, and other elements of the vessel matrix, leading to relatively persistent thrombotic occlusion and myocardial infarction (MI).

It is likely that the nature of the substrate exposed after spontaneous or angioplasty-induced plaque rupture determines whether an unstable plaque proceeds rapidly to an occlusive thrombus or persists as nonocclusive mural thrombus. The analysis of the relative contribution of different components of human atherosclerotic plaques (fatty streaks, sclerotic plaques, fibrolipid plaques, atheromatous plaques, hyperplasic cellular plaque, and normal intima) to acute thrombus formation has shown that the atheromatous core is up to six-fold more active than the other substrates in triggering thrombosis.⁴³ The atheromatous core remained the most thrombogenic substrate when the various components were normalized by the degree of irregularity, as defined by the roughness index. Therefore, ruptured plaques with a large atheromatous core are at high risk of leading to ACS. The plaque TF content is directly related to its thrombogenicity.⁴⁶ As proof of concept, we showed that local tissue blockade of TF by treatment with TF pathway inhibitor significantly reduces thrombosis.^46,47 Recently, the use of active site–inhibited recombinant FVIIa (FF-rFVIIa) has been shown to significantly reduce thrombus growth on severely damaged vessels.

Monocytes and macrophages are key to the development of vulnerable plaques. The vulnerable plaques (AHA type IV and Va), commonly composed of an abundant lipid core separated from the lumen by a thin fibrotic cap, are particularly soft and prone to disruption.⁴⁸ A high density of activated inflammatory cells has been detected in the disrupted areas of atherectomy specimens from patients with ACS. These cells are capable of degrading ECM by secreting proteolytic enzymes, such as MMPs.^49,50 In addition, T cells isolated from rupture-prone sites can stimulate macrophages to produce metalloproteinases and may predispose to the disruption of lesions by weakening their fibrous caps. Leukocytes, their enzymes, and their activation, play a critical role in plaque vulnerability and rupture.^51,52 Links between the activity of the leukocyte enzyme myeloperoxidase (MPO) and features of vulnerable plaque have been reported.⁵³ MPO has been implicated in endothelial cell loss or denudation and development of a prothrombotic environment. LDL has been shown to downregulate the expression of lysil-oxidase (LOX) in vascular wall cells. LOX is an enzyme that contributes to the maturation of the elastin and collagen fibrils of the ECM. Its decrease is associated with increased permeability of the vascular wall and hence may contribute to plaque destabilization. Cell apoptosis and MPs with procoagulant activity and postulated apoptotic origin have also been linked to inflammation and thrombosis.^54,55,56 Resident smooth muscle cells also play a role in the thrombogenic status of the atherosclerotic lesions. Aggregated LDL is able to induce TF expression in smooth muscle cells by inhibiting sphingomyelinase. The expression of TF in smooth muscle cells strongly depends on the expression of the LDL receptor–related protein (LRP).⁵⁴

Macrophages are suggested to play a key role in inducing plaque rupture by secreting proteases capable of destroying the ECM that provides physical strength to the fibrous cap. Recently, it has been showed that macrophage-mediated matrix degradation by MMPs can induce plaque rupture. MMP-9 enhances elastin degradation and induces significant plaque disruption when overexpressed by macrophages in advanced atherosclerotic lesions of apolipoprotein E–deficient (apo E^–/–) mice.⁵⁷ Other MMPs have also been implicated in different stages of atherosclerotic plaque progression.⁵⁸ In addition to secreting proteases and increasing the likelihood of plaque rupture, lipid-rich macrophages within the atherosclerotic plaque undergo apoptosis, expressing TF and increasing the thrombogenic status of the vascular lesions.⁵⁹ Plaque vulnerability and thrombogenic status can be reduced by long-term treatment with lipid-lowering agents.⁶⁰

Epidemiological and retrospective studies have suggested an important protective effect of HDL against cardiovascular disease. Preclinical evidence and small human studies suggest that acute treatment with recombinant apo A-I Milano or other HDL-targeting interventions can induce acute plaque stabilization, representing a promising approach for high-risk patients.^61,62,63,64 However, the lack of benefit seen in the most recent randomized clinical trials targeting HDL (eg, AIM-HIGH, ACCORD, HPS-2) has generated a cloud on the postulated benefits effect of HDL. Those failures, despite attaining significant increases in HDL, have initiated an important discussion on the physiological value of the quality versus quantity of HDL. Major criticisms of the design and the selected population for the trials have to be taken into account before totally disregarding the protective benefits of “functional” HDL as those raised by exercise, diet, etc. These failures have supported the existence of a “dysfunctional” HDL. Several cardiovascular risk factors, such as preexistent coronary artery disease, diabetes, inflammation, etc, are associated with this dysfunctional HDL.^65,66,67

Effects Derived from Geometry

Platelet deposition is directly related to the degree of stenosis in the presence of the same degree of injury, indicating a shear-induced platelet activation.⁶⁸ In addition, analysis of the axial distribution of platelet deposition indicates that the apex, not the flow recirculation zone distal to the apex, is the segment of greatest platelet accumulation. These data suggest that the severity of the acute platelet response to plaque disruption partly depends on the sudden changes in geometry after rupture. Interestingly, hemodynamic effects play a role in the regulation of the thrombotic response in different arteries. In the absence of atherosclerotic changes in the porcine normolipemic model, the dilatation of carotid and coronary arteries in the same animal produced significantly different levels of platelet deposition in the arterial beds, with the coronaries triggering a significantly greater deposition than the carotids.⁶⁸ Also, it has been shown in apo E^–/– mice that whereas lowered shear stress induces larger lesions with a vulnerable plaque phenotype, vortices with oscillatory shear stress induce stable lesions.⁶⁹

Spontaneous lysis of thrombus does occur, not only in UA but also in acute MI. In these patients as well as in those undergoing thrombolysis for acute infarction, the presence of a residual mural thrombus predisposes to recurrent thrombotic vessel occlusion. Two main contributing factors for the development of rethrombosis have been identified. First, because platelet deposition increases with increasing degrees of vessel stenosis, residual mural thrombus encroaching into the vessel lumen may result in an increased shear rate, which facilitates the activation and deposition of platelets on the lesion. Second, a fragmented thrombus appears to present one of the most powerful thrombogenic surfaces. A gradual increase in platelet deposition in the area of maximal stenosis may be followed by an abrupt decrease in platelet deposition, probably because of spontaneous embolization of the thrombus or platelet deaggregation.⁷⁰ Such an episode can be immediately followed by a rapid increase in platelet deposition, suggesting that the remaining thrombus is markedly thrombogenic. In fact, platelet deposition is increased two to four times on residual thrombus compared with deeply injured arterial wall, and thrombus continues to grow during heparin therapy. However, it is inhibited by specific antithrombin treatment.

A specific antithrombin such as r-hirudin (a recombinant molecule that blocks both the catalytic site and the anion-exosite of the thrombin molecule) is capable of significantly inhibiting the secondary growth. Thus, after lysis, thrombin becomes exposed to the circulating blood, leading to activation of the platelets, coagulation, and further thrombosis. The antithrombin activity of heparin is limited for three main reasons: (1) a residual thrombus contains active thrombin bound to fibrin, which is thus poorly accessible to the large heparin-ATIII complexes; (2) a platelet-rich arterial thrombus releases large amounts of platelet factor 4, which may inhibit heparin; and (3) fibrin II monomer, formed by the action of thrombin on fibrinogen, may also inhibit heparin. Conversely, molecules of hirudin and other specific antithrombins are at least 10 times smaller than the heparin-ATIII complex and have no natural inhibitors and, therefore, have greater access to thrombin bound to fibrin. These findings clarify the clinical observations in patients with acute MI undergoing thrombolysis, which have shown that residual stenosis is partly related to residual nonlysed thrombus. The effects of different antithrombotic treatment regimens on thrombus formation triggered by a residual mural thrombus have been evaluated, and specific thrombin inhibition has been shown to be the most effective method of slowing the progression of thrombus growth compared with aspirin, heparin, or both.⁷¹

The fact that a clear predilection exists for lesion formation at arterial branch points strongly indicates the important influence of local hemodynamics and rheologic conditions on atherosclerosis. Furthermore, vascular cell gene expression profiles are also modulated by acute changes in flow profiles.^72,73

The severity of coronary thrombosis and associated ACS is modulated by the magnitude or stability of the formed thrombus. In addition, successive events of plaque disruption and asymptomatic thrombus formation have been postulated to be responsible for the rapid progression of disease in certain patients. When a plaque ruptures, in addition to the local factors mentioned above, systemic factors modulate, predispose, or lead to ACS. In fact, current knowledge supports the concept that rupture of atherosclerotic plaques happens more frequently than initially thought. This disruption occurs in an asymptomatic fashion and thus remains clinically unnoticed. Postmortem evidence has demonstrated the existence of repeated and healed thrombotic episodes within the same lesion.⁷⁴ This plaque rupture without a superimposed thrombus suggest that in addition to local factors implicated in coronary thrombosis, other circulating systemic factors modulate coronary thrombosis (Table 33–2). This knowledge led to the concept of vulnerable patient as a composite of vulnerable plaque plus vulnerable blood.^{75,76,77,78,79}

One-third of ACS cases, particularly those involving ischemic sudden cardiac death, develop without plaque disruption but just superficial erosion of a markedly stenotic and fibrotic plaque. Under such conditions, thrombus formation seems to depend on the hyperthrombogenic state triggered by systemic factors (Table 33–3). Indeed, systemic factors—including elevated LDL, decreased HDL, cigarette smoking, diabetes, and dysregulated hemostasis—are associated with increased thrombotic complications. Our group has reported an increased blood thrombogenicity associated with hyperlipemia as well as diabetes; more importantly, the effective management of these risk factors normalized this increased blood thrombogenicity.^80,81

As stated above, TF is a major local player in the vulnerability and thrombogenicity of atherosclerotic plaques. TF is highly expressed in atherosclerotic plaques, and its content has been related to plaque thrombogenicity. More recent is the knowledge of the existence of a bloodborne pool of TF that may play a critical role in the propagation of thrombosis. Moreover, it has been reported that polymorphonuclear leukocytes might be involved in the transport of circulating TF to platelets. High plasma levels of TF antigen and TF-positive MPs with procoagulant activity have been reported in patients with ACS.^81,82 It seems that bloodborne TF plays an important role in the blood thrombogenicity and in the thrombotic complications of plaque disruption and erosion. Our group has found increased levels of circulating TF activity to be associated with cardiovascular risk factors. Our group has found high levels of circulating TF activity in certain pathological conditions characterized by high rate of thrombotic complications (diabetes, hyperlipidemia, and smoking). In addition, we have found that patients with improvement in glycemic control showed a reduction in circulating TF,⁸³ suggesting that circulating TF may be the mechanism of action responsible for the increased thrombotic complications associated with the presence of these cardiovascular risk factors. Figure 33–12 shows the possible cellular sources of circulating bloodborne TF. C-reactive protein (CRP) has been implicated in various aspects of cardiovascular disease. It has been shown that a circulating isoform of CRP (mCRP) enhances platelet deposition and thrombus growth in contrast to the native form of CRP.⁸⁴ In addition, CRP seems to be a direct mediator of neovessel formation of high-risk plaques.⁸⁵

Thrombogenic systemic factors can be modulated by controlling the cardiovascular risk factors and by dietary and pharmacologic strategies. The development of additional novel therapeutic approaches depends on the increasing knowledge of the pathogenesis of ACS.

Atherosclerosis and inflammation could represent different faces of the same disease. Currently, it is well known that inflammatory circulating markers correlate with cardiovascular events and the severity of the disease. Several inflammatory markers are being postulated as having a significant prognostic value for recurrent cardiovascular events. Therefore, given the role of inflammation on atherosclerosis, it is plausible that drugs directed against inflammation (eg, COX-2 inhibitors and nonsteroidal anti-inflammatory drugs [NSAIDs]) could render a positive impact in the prevention of cardiovascular events. However, several studies associated a significant increase in cardiovascular events with the use of COX-2 inhibitors. At that time, it was thought that NSAIDs did not have the same association with cardiovascular events and could even be protective; however, it has been demonstrated that NSAIDs also seem to have a negative impact in patients with cardiovascular disease.^86,87 Therefore, it is clear that the best approach to treating inflammation in patients with cardiovascular disease is an aggressive management of all the cardiovascular risk factors (eg, statins, angiotensin-converting enzyme inhibitors, hypoglycemic agents, antiplatelets). Interestingly, the use of these therapeutic interventions has been demonstrated to not only offer significant benefits but also to reduce the systemic levels of proinflammatory markers. This emphasizes the idea that atherothrombotic disease and inflammation could be two faces of the same disease rather than two different entities. As stated above, the infusion of recombinant Apo A-I Milano is able to stabilize atherosclerotic plaques. Interestingly, this effect is not restricted to the vessel wall because systemic markers of inflammation are downregulated.⁸⁸

The antithrombotic agents presently used in clinical practice can be subdivided into three categories, according to their mechanism of action: fibrinolytics, inhibitors of the intrinsic coagulation cascade, and antiplatelet agents. Among the novel antithrombotic strategies that are reaching clinical research are the inhibitors of the TF:FVIIa pathway, direct factor Xa inhibitors, and direct thrombin inhibitors (DTIs). The newly generated understanding of the role of TF in atherothrombosis has suggested the inhibition of its pathway as a new antithrombotic approach. The biochemistry of TF and the clotting cascade has identified TF:VIIa, factor Xa, and thrombin as potential targets. Several direct and specific antagonists to each of these targets have been developed and are being investigated in the clinical arena. Specific anti-TF antibodies, recombinant forms of endogenous TF pathway inhibitors, inhibitors of factor VIIa, and inhibitors of factor Xa may afford at least a theoretical advantage over therapies that target more “downstream” components of the coagulation cascade.

DTIs inhibit thrombin by directly binding to exosite 1 or the active site of thrombin. DTIs produce a predictable anticoagulant response because they exhibit minimal binding to plasma proteins or cellular elements and inhibit fibrin-bound thrombin as well as fluid-phase thrombin. The intravenous DTIs approved in the United States by the Food and Drug Administration (FDA) for anticoagulation in patients with heparin-induced thrombocytopenia are the recombinant hirudin, lepirudin, and argatroban. An oral DTI, dabigatran, has been tested in a large clinical trial recruiting more than 18,000 patients. The results from the Randomized Evaluation of Long-Term Anticoagulation Therapy (RE-LY) study have been recently reported. Two doses of dabigatran were tested against warfarin in patients with atrial fibrillation. The low dabigatran dose (110 mg) resulted in similar results in thromboembolic event prevention, but it was associated with significantly lower hemorrhagic episodes. The high dose of dabigatran (150 mg) was associated with significantly lower thromboembolic events but similar rates of major hemorrhages.⁹⁰ In addition, the REDEEM (Dabigatran vs Placebo in patients with acute coronary syndromes) Trial studied the safety and indication for efficacy of the combination of dabigatran and dual antiplatelet therapy in patients with a recent MI. The results showed a dose-dependent increase in bleeding events and significant reduction in the coagulation activity.^91,92

Another way to interfere with thrombin generation is the possibility of inhibiting the activation of FX. Selective factor Xa inhibitors effectively block coagulation because factor Xa is positioned at the start of the common pathway of the extrinsic and intrinsic coagulation systems (see Fig. 33–9). The indirect factor Xa inhibitor fondaparinux was the first of the selective factor Xa inhibitors to receive FDA approval for the prevention and treatment of venous thromboembolism. Several direct FXa inhibitors— rivaroxaban, apixaban, and edoxaban—have been approved by the FDA for the prevention of stroke in patients with nonvalvular atrial fibrillation. Their action is exerted selectively toward factor Xa, contrary to the action of unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH), which act on a number of coagulation factors. UFH has equipotent activity against factors IIa and Xa but also acts on factors IXa, XIa, and XIIa in an antithrombin-dependent manner. LMWHs, which are prepared by chemical or enzymatic depolymerization of UFH, have relatively more antifactor Xa than antifactor IIa activity. Figure 33–9 shows the site of action of the different drugs targeting the coagulation cascade.

The effectiveness and safety of the new oral anticoagulants suggesting the possibility of their use in the prevention of ACS, combined with the increasing incidence of atrial fibrillation in today’s aging population, has generated a major clinical dilemma.⁹³ The ATLAS ACS 2–TIMI 51 (NCT 00809965) investigated the effects of rivaroxaban (2.5 and 5.0 mg twice a day) in acute coronary syndromes receiving a low dose of aspirin and randomized based on the investigator's intent to administer a thienopyridine. Rivaroxaban reduced the risk of composite end point (cardiovascular death, MI, and stroke). These benefits were associated with an increased risk of major bleeding and intracranial hemorrhage but not fatal bleeding.⁹⁴ Similarly, apixaban was also studied in ACS patients receiving dual antiplatelet therapy in the Apixaban for Prevention of Acute Ischemic and Safety Events (APPRAISE) trial (NCT 00831441). The results showed increased bleeding complications without reductions in ischemic events.⁹⁵ What is the best therapeutic intervention in those patients with atrial fibrillation undergoing stent deployment? Or what should be done in patients with stents being diagnosed with atrial fibrillation? There is not a clear suggestion from any of the guidelines; simply it is recommended to balance the risk of thrombosis and the risk of bleeding prior making a therapeutic decision. Several ongoing trials are being developed to investigate this question.

Other approaches, still in early preclinical phase, are inhibitors of GP Ib receptor (anti-vWF), inhibitors of vWF-collagen binding (saratin), and inhibitors of the ADP receptor P2T and PAR-1,⁹⁶ among others.

The formation of a thrombus within a coronary artery with obstruction of coronary blood flow and reduction in oxygen supply to the myocardium produce the several types of ACS. These thrombotic episodes largely occur in response to atherosclerotic lesions that have progressed to a high-risk inflammatory or prothrombotic stage by a process modulated by local and systemic factors. Although distinct from each other, these atherosclerotic and thrombotic processes appear to be closely related as the cause of ACS through a complex multifactorial process called atherothrombosis. The cellular and molecular mechanisms at play in the formation, growth, and stabilization of a coronary thrombus are being thoroughly investigated, and many of the activation pathways and receptor-ligand interactions have been identified. Inflammation has been revealed as a great player in the systemic and diffuse pattern of atherothrombotic disease. Strategies combining dietary, pharmacologic-medical, and interventional-surgical therapies have shown considerable success in the prevention and treatment of major cardiovascular events. These regimens focus on inhibiting the various pathways involved in thrombus generation. Novel strategies based on the knowledge of the biochemistry of platelet aggregation and the coagulation processes as well as the geometric conditions encountered in the circulation are presently in different stages of development and clinical trials. Advances in noninvasive imaging techniques will help to identify plaques at risk and reduce the clinical impact of atherothrombosis.