CHAPTER 21: ARTERIAL AND VENOUS THROMBOSIS

GENERAL OVERVIEW

Thrombosis is defined as "hemostasis in the wrong place,"¹ and it is a major cause of morbidity and mortality in a wide range of arterial and venous diseases and patient populations. In 2009 in the United States, an estimated 785,000 people had a new coronary thrombotic event, and about 470,000 had a recurrent ischemic episode. Each year, approximately 795,000 people have a new or recurrent stroke. Annually, more than 200,000 new cases of venous thromboembolism are found; 30% of these individuals die within 30 days, with one-fifth having sudden death due to pulmonary embolism.

In the nondiseased state, physiologic hemostasis reflects a delicate interplay between factors that promote and inhibit blood clotting, favoring the former. This response is crucial as it prevents uncontrolled hemorrhage and exsanguination following injury. In specific settings, the same processes that regulate normal hemostasis can cause pathological thrombosis, leading to arterial or venous occlusion. Importantly, many commonly used therapeutic interventions may also alter the thrombotic–hemostatic balance adversely.

Hemostasis and thrombosis primarily involve the interplay among three factors: the vessel wall, coagulation proteins, and platelets. Many prevalent acute vascular diseases are due to thrombus formation within a vessel, including myocardial infarction, thrombotic cerebrovascular events, and venous thrombosis. Although the end result is vessel occlusion and tissue ischemia, the pathophysiologic processes governing these pathologies have similarities as well as distinct differences. While many of the pathways regulating thrombus formation are similar to those that regulate hemostasis, the processes triggering thrombosis and, often, perpetuating the thrombus are distinct. In venous thrombosis, primary hypercoagulable states reflecting defects in the proteins governing coagulation and/or fibrinolysis or secondary hypercoagulable states involving abnormalities of blood vessels and blood flow lead to thrombosis. By contrast, arterial thrombosis is highly dependent on the state of the vessel wall, the platelet, and factors related to blood flow.

OVERVIEW OF ARTERIAL THROMBOSIS

In arterial thrombosis, the platelet 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. The regulation of platelet adhesion, activation, aggregation, and recruitment will be described in detail later in the chapter. In addition, while the primary function of platelets is regulation of hemostasis, our understanding of their role in other processes, such as immunity and inflammation, continues to grow.

ARTERIAL THROMBOSIS AND VASCULAR DISEASE

Arterial thrombosis is a major cause of morbidity and mortality both in the United States and, increasingly, worldwide. Coronary heart disease is estimated to cause about one of every five 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. 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.

THE PLATELET

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 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. While 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.

Platelet adhesion

(See Fig. 21-1) 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. 21-1). 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.

Platelet activation

The activation of platelets is controlled by a variety of surface receptors that regulate various functions in the activation process. Platelet receptors 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.

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 PARs 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 (Fig. 21-2). The activation of both the P2Y₁₂ and P2Y₁ receptors is essential for ADP-induced platelet aggregation. The thienopyridine derivatives clopidogrel and prasugrel are clinically utilized inhibitors of ADP-induced platelet aggregation.

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. 21-1). 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. 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 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 directly 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 21-2A); however, 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 21-1). 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 drug effect, drug compliance, and drug metabolism. Many candidate platelet genes have been studied for their interaction with antiplatelet and antithrombotic agents.

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 cyclooxygenase 1 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. As these are common genetic variants, this observation has been shown to be clinically relevant in large studies.

OVERVIEW OF VENOUS THROMBOSIS

Coagulation is the process by which thrombin is activated and soluble plasma fibrinogen is converted into insoluble fibrin. These steps account for both normal hemostasis and the pathophysiologic processes influencing the development of venous thrombosis. The primary forms of venous thrombosis are deep-vein thrombosis (DVT) in the extremities and the subsequent embolization to the lungs (pulmonary embolism), referred to together as venous thromboembolic disease. Venous thrombosis occurs due to heritable causes (Table 21-2B) and acquired causes (Table 21-3).

DEEP-VEIN THROMBOSIS AND PULMONARY EMBOLISM

More than 200,000 new cases of venous thromboembolism occur each year. Of these patients, 30% die within 30 days and one-fifth experience sudden death owing to pulmonary embolism; 30% go on to develop recurrent venous thromboembolism within 10 years. Data from the Athereroscerosis Risk in Communities study reported a 9% 28-day fatality rate from DVT and a 15% fatality rate from pulmonary embolism. Pulmonary embolism in the setting of cancer has a 25% fatality rate. The mean incidence of first DVT in the general population is 5 per 10,000 person-years; the incidence is similar in males and females and increases dramatically with age from 2 to 3 per 10,000 person-years at 30–49 years of age to 20 at 70–79 years of age.

OVERVIEW OF THE COAGULATION CASCADE AND ITS ROLE IN VENOUS THROMBOSIS

Coagulation is defined as the formation of fibrin by a series of linked enzymatic reactions in which each reaction product converts the subsequent inactive zymogen into an active serine protease (Fig. 21-2). This coordinated sequence is called the coagulation cascade and is a key mechanism for regulating hemostasis. Central to the function of the coagulation cascade is the principle of amplification: owing to a series of linked enzymatic reactions, a small stimulus can lead to much greater quantities of fibrin, the end product that prevents hemorrhage at the site of vascular injury.

The coagulation cascade is primarily initiated by vascular injury, exposing tissue factor to blood components (Fig. 21-2). Tissue factor may also be found in bloodborne cell-derived microparticles and, under pathophysiologic conditions, in leukocytes or platelets. Plasma factor VII (FVII) is the ligand for and is activated (FVIIa) by binding to tissue factor exposed at the site of vessel damage. The binding of FVII/VIIa to tissue factor activates the downstream conversion of factor X (FX) to active FX (FXa). In an alternative reaction, the FVII/FVIIa–tissue factor complex initially converts FIX to FIXa, which then activates FX in conjunction with its cofactor factor VIII (FVIIIa). Factor Xa with its cofactor FVa converts prothrombin to thrombin, which then converts soluble plasma fibrinogen to insoluble fibrin, leading to clot or thrombus formation. Thrombin also activates FXIII to FXIIIa, a transglutaminase that covalently cross-links and stabilizes the fibrin clot.

Several antithrombotic factors also regulate coagulation; these include antithrombin, tissue factor pathway inhibitor (TFPI), heparin cofactor II, and protein C/protein S. Under normal conditions, these factors limit the production of thrombin to prevent the perpetuation of coagulation and thrombus formation. Typically, after the clot has caused occlusion at the damaged site and begins to expand toward adjacent uninjured vessel segments, the anticoagulant reactions governed by the normal endothelium become pivotal in limiting the extent of this hemostatically protective clot.

RISK FACTORS FOR VENOUS THROMBOSIS

The risk factors for venous thrombosis are primarily related to hypercoagulability, which can be genetic (Table 21-2) or acquired or due to immobilization and venous stasis. Independent predictors for recurrence include increasing age, obesity, malignant neoplasm, and acute extremity paresis. Often, multiple risk factors are present in a single individual. Significant risk is incurred by major orthopedic, abdominal, or neurologic surgeries. Moderate risk is promoted by prolonged bedrest; certain types of cancer, pregnancy, hormone replacement therapy, or oral contraceptive use; and other sedentary conditions such as long-distance plane travel. It has been reported that the risk of developing a venous thromboembolic event doubles after air travel lasting 4 h, although the absolute risk remains low (1 in 6000). The relative risk of venous thromboembolism among pregnant or postpartum women is 4.3, and the overall incidence (absolute risk) is 199.7 per 100,000 woman-years.

GENETICS OF VENOUS THROMBOSIS

(See Table 21-2) Less common causes of venous thrombosis are those due to genetic variants. These abnormalities include loss-of-function mutations of endogenous anticoagulants as well as gain-of-function mutations of procoagulant proteins. Heterozygous antithrombin deficiency and homozygosity of the factor V Leiden mutation significantly increase the risk of venous thrombosis. While homozygous protein C or protein S deficiencies are rare and may lead to fatal purpura fulminans, heterozygous deficiencies are associated with a moderate risk of thrombosis. Activated protein C impairs coagulation by proteolytic degradation of FVa. Patients resistant to the activity of activated protein C may have a point mutation in the FV gene located on chromosome 1, a mutant denoted factor V Leiden. Mildly increased risk has been attributed to elevated levels of procoagulant factors, as well as low levels of TFPI. Polymorphisms of methylene tetrahydrofolate reductase as well as hyperhomocysteinemia have been shown to be independent risk factors for venous thrombosis, as well as arterial vascular disease; however, many of the initial descriptions of genetic variants and their associations with thromboembolism are being questioned in larger, more current studies.

FIBRINOLYSIS AND THROMBOSIS

Specific abnormalities in the fibrinolytic system have been associated with enhanced thrombosis. Factors such as elevated levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1) have been associated with decreased fibrinolytic activity and an increased risk of arterial thrombotic disease. Specific genetic variants have been associated with decreased fibrinolytic activity, including the 4G/5G insertion/deletion polymorphism in the (plasminogen activator type 1) PAI-1 gene. Additionally, the 311-bp Alu insertion/deletion in tPA's intron 8 has been associated with enhanced thrombosis; although genetic abnormalities have not been associated consistently with altered function or tPA levels, raising questions about the relevant pathophysiologic mechanism. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase that regulates fibrinolysis; elevated plasma TAFI levels have been associated with an increased risk of both DVT and cardiovascular disease.

The metabolic syndrome also is accompanied by altered fibrinolytic activity. This syndrome, which comprises abdominal fat (central obesity), altered glucose and insulin metabolism, dyslipidemia, and hypertension, has been associated with atherothrombosis. The mechanism for enhanced thrombosis appears to be due both to altered platelet function and to a procoagulant and hypofibrinolytic state. One of the most frequently documented prothrombotic abnormalities reported in this syndrome is an increase in plasma levels of PAI-1.

Although there is overlap, venous and arterial thrombosis are initiated differently, and clot formation progresses by somewhat distinct pathways. In the setting of stasis or states of hypercoagulability, venous thrombosis is activated with the initiation of the coagulation cascade primarily due to exposure of tissue factor; this leads to the formation of thrombin and the subsequent conversion of fibrinogen to fibrin. In the artery, thrombin formation also occurs, but thrombosis is primarily promoted by the adhesion of platelets to an injured vessel and stimulated by exposed extracellular matrix (Figs. 21-1 and 21-2). There is wide variation in individual responses to vascular injury, an important determinant of which is the predisposition an individual has to arterial or venous thrombosis. This concept has been supported indirectly in prothrombotic animal models in which there is poor correlation between the propensity to develop venous versus arterial thrombosis.

Despite considerable progress in understanding the role of hypercoagulable states in venous thromboembolic disease, the contribution of hypercoagulability to arterial vascular disease is much less well understood. While specific thrombophilic conditions, such as factor V Leiden and the prothrombin G20210A mutation, are risk factors for DVT, pulmonary embolism, and other venous thromboembolic events, their contribution to arterial thrombosis is less well defined. In fact, to the contrary, many of these thrombophilic factors have not been found to be clinically important risk factors for arterial thrombotic events, such as acute coronary syndromes.

Clinically, although the pathophysiology is distinct, arterial and venous thrombosis do share common risk factors, including age, obesity, cigarette smoking, diabetes mellitus, arterial hypertension, hyperlipidemia, and metabolic syndrome. Select genetic variants, including those of the glutathione peroxidase gene, have also been associated with arterial and venous thrombo-occlusive disease. Importantly, arterial and venous thrombosis may both be triggered by pathophysiologic stimuli responsible for activating inflammatory and oxidative pathways.

The diagnosis and management of DVT and pulmonary embolus are discussed in Chap. 22.

The authors would like to thank Hannah Iafrati for her assistance with the figures.