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).
Images of thrombosis—from naked eye observation to immunohistochemistry (green, platelets; red, fibrinogen) and electronic microscopy (top, scanning; bottom, transmission).
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.
Mechanisms and agonists involved in platelet adhesion, activation, and aggregation. ADP, adenosine diphosphate; ATP, adenosine triphosphate; GP, glycoprotein; 5-HT, 5-hydroxytryptamine; PAR, protease-activated receptor; Rc, receptor; TP, thromboxane receptor, TXA2, thromboxane A2; vWF, von Willebrand factor. Reproduced with permission from Ibanez B, Vilahur G, Badimon J: Pharmacology of thienopyridines: rationale for dual pathway inhibition. Eur Heart J Suppl. 2006;8(Suppl G):G3-G9.
Signal transduction mechanisms of platelet activation and aggregation. AA, arachidonic acid; AC, adenylyl cyclase; ADP, adenosine diphosphate AMP, adenosine 3′5′-cyclic monophosphate; ATP, adenosine triphosphate; DAG, diacylglycerol; Gs, Gi, Gp, Gq, guanine nucleotide-binding regulatory proteins; GP, glycoprotein; 5HT, 5-hydroxytryptamine; IP3, inositol 1,4,5-triphosphate; PGG2, prostaglandin G2; PGH3, prostaglandin H3; PGI2, prostacyclin; PIP2, phosphoinositol diphosphate; PKA, protein kinase A; PKCi and PKCa, protein kinase C, inactivated and activated; PLA2, phospholipase A2; PLC, phospholipase C; IIb/IIIa, receptor glycoprotein for adhesive protein ligands (mainly fibrinogen and vWF); TP, thromboxane receptor; TXA2, thromboxane A2. Reproduced with permission from Badimon L, Vilahur G: Thrombosis formation on atherosclerotic lesions and plaque rupture. J Intern Med. 2014 Dec;276(6):618-632.17
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).
TABLE 33–1.Platelet Membrane Glycoprotein Receptors ||Download (.pdf) TABLE 33–1. Platelet Membrane Glycoprotein Receptors
|Glycoprotein Receptor ||Function ||Ligand |
|GP IIb/IIIa ||Aggregation, adhesion at high shear rate ||Fg, vWF, Fn, Ts, Vn |
|Receptor Vn ||Adhesion ||Vn, vWF, Fn, Fg, Ts |
|GP Ia/IIa ||Adhesion ||C |
|GP Ic/IIa ||Adhesion ||Fn |
|GP IcN/IIa ||Adhesion ||Ln |
|GP Ib/IX ||Adhesion ||vWF, T |
|GP V ||Unknown ||Substrate T |
|GP IV (GP IIIb) ||Adhesion ||Ts, C |
|GMP-140 (PADGEM) ||Interaction with leukocytes ||Unknown |
|PECAM-1 (GP IIa) ||Unknown ||Unknown |
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).
Mechanism involved in thrombus formation. Healthy endothelium (left) presents antithrombotic properties because it is able to release vascular protective substances such as nitric oxide (NO), prostacyclin (PGI2), tissue plasminogen activator (tPA), and tissue factor pathway inhibitor (TFPi). On the contrary, dysfunctional endothelium (right) not only favors platelet adhesion, activation, and aggregation but also promotes vascular lipid deposition, macrophage migration, and tissue factor (TF) expression (activation of the coagulation cascade). After platelet adhesion, activation is characterized by platelet shape change. Activated platelets secrete different agonists, prompting activation of circulating platelets and a procoagulant environment. This prothrombotic milieu favors thrombus formation and the subsequent clinical manifestations. ADP, adenosine diphosphate; RBC, red blood cell; TXA2, thromboxane A2. Reproduced with permission from Ibanez B, Vilahur G, Badimon J: Pharmacology of thienopyridines: rationale for dual pathway inhibition. Eur Heart J Suppl. 2006;8(Suppl G):G3-G9.
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
P2Y platelet receptors. P2Y receptors can be clearly divided into two subgroups: the Gq-coupled subtype P2Y1 and the Gi-coupled P2Y12. P2Y1 is responsible for platelet shape change and calcium mobilization. It is coupled to Gq and activates phospholipase Cβ (PLCβ), leading to the formation of inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG), an activator of protein kinase C (PKC). IP3 causes calcium mobilization from internal stores. The P2Y12 receptor couples primarily to Gαι2 inhibition of adenylyl cyclase (AC). The subsequent decrease in cAMP production leads, in turn, to a reduction in the activation of specific protein kinases (PKA), which can no longer phosphorylate the vasodilator-stimulated phosphoprotein (VASP); VASP phosphorylation is crucial for glycoprotein (GP) IIb/IIIa receptor inhibition. The subunit βγ activates phosphatidylinositol 3-kinase (PI3K), which is an important signaling molecule for P2Y12-mediated platelet-dense granule secretion and GP IIb/IIIa receptor activation. Finally, P2X1 is a gated cation channel protein activated by adenosine triphosphate (ATP). This activation leads to increased intraplatelet calcium, platelet shape change, and a transient and weak platelet aggregation response. ADP, adenosine diphosphate; PGE1, prostaglandin E1. Reproduced with permission from Ibanez B, Vilahur G, Badimon J: Pharmacology of thienopyridines: rationale for dual pathway inhibition. Eur Heart J Suppl. 2006;8(Suppl G):G3-G9.
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
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).
Simplified diagram of the tissue factor pathway with specific inhibitor at the different steps of the cascade. The underlined compounds are those approved by the Food and Drug Administration. APC, antigen-presenting cell; LMWH, low-molecular-weight heparin; TF, tissue factor; TFPI, tissue factor pathway inhibitor.
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).
Simplified diagram of the physiologic anticoagulation system. AT, antithrombin; CV, cardiovascular; NO, nitrous oxide; PGI2, prostacyclin; tPA, tissue plasminogen activator.
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
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.
Role of thrombin in different pathways of coagulation. Thrombin plays a critical role in the maintenance of physiological fibrinolysis system but also in thrombosis. Fg, fibrinogen; FPs, fibrinopeptides; PAR1, protease-activated receptor 1; PTase, prothrombinase; tPA, tissue plasminogen activator; VSMC, vascular smooth muscle cell.
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.