Venous and Arterial Thrombosis

Introduction

The pathogenesis of venous and arterial thrombosis is broad and at times disparate. Virchow originally described venous thrombosis under low flow (shear) with red clots, occurring around and propagating through venous valves, and consisting of red cells and fibrin strands. Arterial thrombosis occurs under high shear stress in large (carotids, coronary, femoral arteries) and small vessels and initially consists of platelets and leukocytes with few red cells and little fibrin (white clot). However, the pathogenesis of these vessel occlusions (thrombosis) may be variations on basically similar mechanistic schema. A developing concept is that venous thromboembolism (VTE) is part of a pan-cardiovascular syndrome that includes coronary artery disease, peripheral artery disease, and cerebrovascular disease. Classically, venous thrombosis has been associated with cancer, surgery, immobilization, fractures, thrombophilia, pregnancy, and estrogens. Arterial thrombosis (atherothrombosis) is associated with smoking, hypertension, diabetes, obesity, and hyperlipidemia. Risk factors common to both venous and arterial thrombosis include aging, obesity, dyslipidemia, hypertension, diabetes, smoking, thrombophilia (antiphospholipid antibody syndrome, elevations of homocysteine, fibrinogen, factor VII, factor VIII, lipoprotein(a), factor V Leiden, and prothrombin 20210 polymorphism), and hormonal therapy (estrogens). In the sections that follow, the mechanism(s) contributing to venous and arterial thrombosis are discussed in the context of clinical disease, with an emphasis on the structural differences between the vessels and how these anatomical differences impact on the complexity of processes that lead to the occlusive events. In both the venous and arterial sections, the anatomy and normal physiology of the vessel are discussed, so as to set the stage for the various elements involved in the pathophysiology of venous and arterial thrombosis, respectively. A brief discussion of the diagnosis and principles of management based upon the mechanistic basis of the disorder concludes each section.

Venous Thrombosis

Normal Venous Anatomy and Physiology

The venous wall is composed of three layers: the intima, media, and adventitia. In contrast to their arterial counterparts, veins have less smooth muscle and elastin. The venous intima consists of an endothelial cell layer resting upon a basement membrane. The adjacent media is composed of smooth muscle cells and elastin connective tissue, while the outer adventitia contains adrenergic neuronal fibers, particularly in the cutaneous veins. Central sympathetic discharge and brainstem thermoregulatory centers alter venous tone, as do other stimuli, such as temperature changes, pain, and volume changes. The histologic features of veins vary, depending on their caliber. Venules are the smallest veins, ranging from 0.1–1 mm and contain mostly smooth muscle cells, whereas larger extremity veins contain relatively few smooth muscle cells. Larger veins have limited contractile capacity. The venous valves prevent retrograde flow; it is their failure or valvular incompetence that leads to reflux and symptoms associated with venous stasis. Venous valves are most prevalent in the distal lower extremity, whereas as one proceeds proximally, the number of valves decreases to the point that no valves are present in the superior vena cava and inferior vena cava (IVC). Most of the capacitance of the vascular tree is in the venous system. Since veins do not have significant amounts of elastin, they can withstand large volume shifts with comparatively small changes in pressure. A vein has a normal elliptical configuration until the limit of its capacitance is reached, at which point the vein assumes a round configuration.

Pathophysiology of Venous Thrombosis

Venous thrombosis results from the sum of risk factors in an individual, including inflammation, age, immobility, and inheritance. The triad of venous stasis, endothelial injury, and hypercoagulable state, first posited by Virchow in 1856 as increasing the chances of thrombosis, has essentially held true more than a century and a half later. A venous clot usually arises in a low-flow portion of a vessel behind a venous valve and propagates proximally to occlude the vein. On gross section, a venous clot is a reddish gelatinous material consisting primarily of red cells and fibrin. While arterial and venous thrombi contain platelets and fibrin, the proportions differ. Venous thrombi, which form under low shear, contain relatively few platelets and consist mostly of fibrin and trapped red blood cells. Venous thrombi appear red because of their high red blood cell content. Unlike an arterial thrombus, a leading edge of leukocytes and platelets that most probably contributed to the initiation of the thrombus is usually absent.

Our current understanding of the cellular basis of venous thrombosis is distinctly different from that classically taught. However, the cellular and molecular events leading to impaired venous blood flow and thrombogenesis remain poorly characterized. In contrast to arterial thrombi, venous thrombi rarely form at sites of vascular disruption. Instead, they usually originate around the valves or in muscular sinuses, where stasis (slow blood flow) is more prominent, resulting in diminished oxygen supply to the lining of affected vein segments. Experimentally, hypoxemia recapitulates the induction of thrombosis in the lung vasculature of mice. The underlying mechanism is hypothesized to be mediated by tissue factor induction in macrophages following activation of the transcription factor early-growth-response (Egr-1) gene. In vitro, these responses lead to pro-inflammatory and pro-adhesive changes. The finding that localized hypoxemia within valve sinuses triggers local activation of endothelial cells, represents a new concept in our understanding of venous thrombosis.

Recent findings also suggest a novel role for innate immune cells in the pathogenesis of venous thrombosis. This contribution by the innate immune cells has been coined in a term to describe thrombosis as a thromboinflammatory state. When fluorescently labeled microparticles derived from mouse monocytes are infused into a mouse before laser injury, the monocytes accumulate within the leading edge of a developing thrombus. These monocyte-derived microparticles express tissue factor and fail to accumulate into thrombi when infused into P-selectin-null mice, demonstrating that the accumulation of monocyte microparticles bearing tissue factor in the thrombus is dependent on the interaction of platelet P-selectin and its counter-receptor, P-selectin glycoprotein ligand 1 (PSGL-1), expressed on the surface of the monocyte microparticles.

In addition to monocytes, neutrophils also contribute to venous thrombosis by their role in the formation of neutrophil extracellular traps (NETs). Originally described in 2004, NETs are comprised of DNA, histones, and antimicrobial proteases such as elastase. NETs are released as a defensive mechanism by activated neutrophils in response to pathogens in a process known as NETosis, and it complements other defensive mechanisms such as phagocytosis and oxidative burst. NETs also have a second role in the pathogenesis of venous thrombosis. They provide a focal point for the activation of coagulation by inflammatory cells of innate immunity. Upon venous constriction, but not complete occlusion, a carpet of neutrophils and monocytes adhere and grow over 6–24 hours as a venous thrombosis develops. It is not yet clear whether neutrophil NETs or platelet thrombus initiates venous thrombus. Experimentally, the initiator of thrombosis may be model-dependent. It is believed that NETS are not an epiphenomenon because peptidylarginine deiminase 4 (PAD4) deficiency protects mice from venous thrombosis. PAD4 is a nuclear enzyme that converts specific arginine residues to citrulline (citrullinization) on histones. The level of plasma NETs is a risk factor for thrombosis in cancer and other conditions. NETs released by adherent neutrophils promote further platelet as well as red cell binding. In experimental murine models of deep vein thrombosis (DVT) induced by partial occlusion/stenosis of the inferior vena cava, endothelial von Willebrand factor also is released and leads to platelet recruitment. P-selectin expression on activated endothelium and platelets then mediates the recruitment and binding of neutrophils.

NETs also promote thrombin generation via autoactivation of factor XII, thereby implicating the ‘intrinsic’ coagulation pathway in thrombus propagation. Factor XII has been known to be a participant in Gram-negative sepsis for decades. Understanding its relation to NETs provides a mechanistic basis for this interaction. Depletion of platelets or neutrophils – or the genetic absence of P-selectin, von Willebrand factor, or PAD4 – is protective against the development of thrombosis. Similarly, systemic administration of DNase I, which degrades the chromatin backbone of NETs, prevents DVT formation in mice. Activated platelets are well known to contribute to thrombin generation through the exposure of negatively charged phospholipids essential for coagulation enzymatic complex assembly. More recently, activated cells and bacteria have been reported to release long-chain polyphosphates that provide a co-factor for activation of factor XII and factor XI by thrombin. Finally, factor-XII-deficient mice are protected from venous thrombosis, which has been proposed to be due to the reduction in NETs’ contribution to autoactivation. Recent studies indicate that leukocyte factor XII deficiency is associated with both neutrophil and macrophage adhesion and chemotaxis defects as well.

These studies illustrate a novel role for innate immune cells in the development of venous thrombosis. These early reports implicating NETs in human thromboembolic disease provide a promising target by which thrombosis risk can be modified by adjuvant, non-anticoagulant-based treatments. Nucleosomes – consisting of a DNA segment coiled around four histone protein cores – are derived from released NETs, and may be detected in the circulation. A recent case–control study demonstrated that plasma levels of nucleosomes and complexes of elastase-α1-antitrypsin were associated with a three-fold increased risk of DVT, with an apparent dose–response relationship. Finally, the dilemma of why some venous thrombi embolize might be a function of nucleosome-mediated stabilization of the thrombus through factor XII activation.

Specific Disorders Contributing to Venous Thrombosis

Venous thrombosis occurs in individuals with both acquired and inherited risk factors ( Table 15.1 ). The majority of risk factors for venous thrombosis are acquired, such as advanced age, obesity, or cancer, which are accentuated when an individual becomes immobile. In all series of patients, over 50% of individuals with VTE are obese. This observation suggests that VTE will be an increasing problem in the developed world. The number of cases of thromboembolism for which an identifiable etiology can be recognized is about 20% of individuals without a family history, and 40% if a family history is present. However, often there is no single etiology for venous thrombosis; the presence of thrombosis is the summation of multiple risk factors. Inherited and acquired factors combine to establish one’s intrinsic risk of thrombosis. Superimposed trigger factors, such as surgery, pregnancy, or hormonal therapy modify these risks and thrombosis occurs when the combination of genetic, acquired, and trigger factors exceeds a critical threshold. It is a challenge for clinical laboratory medicine to develop assays to assess ‘critical thresholds’ for thrombosis risk to guide intensity of prophylaxis and, perhaps, treatment for thromboembolic risk. Figure 15.1 is an integrated presentation of some of the inherited genes and factors that influence the biochemistry of blood coagulation, anticoagulation, and fibrinolytic systems. Despite the interplay between acquired states and inherited traits, common familial (genetic) disorders that contribute to venous thrombosis have been described and are discussed next.

TABLE 15.1

Etiologies of Venous Thrombosis

Inherited	Acquired
Factor V Leiden	Antiphospholipid antibody syndrome
Prothrombin 20210	Disseminated intravascular coagulation
Protein C deficiency	Heparin-induced thrombocytopenia
Protein S deficiency	Pregnancy
Antithrombin deficiency	Malignancy
Lipoprotein(a)	Myeloproliferative disorders
Dysfibrinogenemias	Paroxysmal nocturnal hemoglobinuria

FIGURE 15.1, Biochemistry of venous thrombosis. This figure is a juxtaposition of the proteins involved in the coagulation (green), fibrinolytic (blue), and anticoagulation (red) systems. The boxes around proteins indicate that known defects in that protein are associated with venous thrombosis. PK, prekallikrein; HK, high-molecular-weight kininogen; TF, an abbreviation for tissue factor; FVL, an abbreviation for factor V Leiden; scuPA, single chain urokinase plasminogen activator; tcuPA, two-chain urokinase plasminogen activator; tPA, tissue plasminogen activator; AT, antithrombin; APC, activated protein C; C4bBP, C4b-binding protein; PAI-1, plasminogen activator inhibitor-1. Coagulation factors XII, XI, IX, X, VII and V are represented by their roman numeral alone. The presence of an ‘a’ after the roman numeral represents an ‘activated’ protein.

Inherited Prothrombotic States

Factor V Leiden

The factor V Leiden mutation (a polymorphism in factor V R506Q, FVL) accounts for most cases of activated protein C resistance (APCR) (due to resistance to proteolyze (i.e. inactivate) factor V by changing an arginine to a glutamine) and is inherited in an autosomal dominant fashion. A diagnosis of APCR is established using a functional assay that actually indicates the mechanistic basis of the disorder based on the ratio of the activated partial thromboplastin time (aPTT) of the patient versus control after activated protein C addition. The prevalence of the mutation ranges from 2% to 5% in Caucasians, but is rare in Asians and Africans. In Caucasians, it may account for 20–60% of recognized etiologies for thrombosis. The prevalence of factor V Leiden homozygous states is about 1 in 2500. Patients with factor V Leiden have thrombotic complications but are at a lower risk than those with deficiencies of antithrombin, protein C, or protein S (see below). Factor V Leiden heterozygotes have an annual risk of thrombosis of 0.5–0.7%, but this is a 5–7-fold increased risk over the normal risk. The presence of heterozygous FVL dramatically increases the risk of thrombosis with pregnancy or with the use of estrogen-containing oral contraceptives (35-fold). Homozygous FVL has an 80-fold increased risk for venous thromboembolism (VTE).

Prothrombin Gene Mutation

A G to A nucleotide transition at position 20210 in the 3’-untranslated region of the prothrombin gene (FIIG20210A) results in elevated levels of prothrombin due to slower clearance from plasma. This defect is not structural, rather it is only associated with increased levels of plasma prothrombin. It occurs in 2–4% of the population, but in 6–8% of patients with primary venous thrombosis. Increased prothrombin, in turn, increases the risk of thrombosis by amplifying the generation of thrombin or by inhibiting factor Va inactivation by activated protein C (APC). FIIG20210A is found in 1–6% of Caucasians and may segregate with factor V Leiden, in which case the risk for venous thromboembolic phenomena summates.

Homocysteinemia

Homocysteinemia is a hypercoagulable state that occurs due to the combination of inherited and acquired factors. Its elevation is associated with both venous and arterial thrombosis. Homocysteine is remethylated to methionine or catabolized to cystathionine ( Figure 15.2 ). The major remethylation pathway requires folate and cobalamin (vitamin B12) and involves the action of methylenetetrahydrofolate reductase (MTHFR); a minor remethylation pathway is mediated by betaine-homocysteine methyltransferase. Vitamin B12 deficiency is associated with hyperhomocysteinemia and can present with venous thromboembolism before there are hematologic or neurologic defects. Paradoxically, polymorphisms in MTHFR are usually not associated with hyperhomocysteinemia or VTE. Alternatively, homocysteine is converted to cystathionine in a trans-sulfuration pathway catalyzed by cystathionine β-synthase (CBS), with pyridoxine (B6) used as a cofactor. As will be discussed below with respect to arterial thrombosis, deficiencies or defects in CBS are associated with severe venous and arterial thrombosis and mental retardation. Like vitamin B12, nutritional deficiency of folate or vitamin B6 is associated with thrombosis. Replacement with folate, vitamin B12, and vitamin B6 reduces homocysteine levels, but most trials to date, however, have not shown that such therapy reduces the risk of recurrent cardiovascular events in patients with coronary artery disease, stroke or venous thromboembolic events.

FIGURE 15.2, Homocysteine metabolism. Homocysteine is metabolized by two enzymes, methionine synthase and cystathionine β-synthase. N 5 ,N 10 methylene tetrahydrofolate reductase makes an essential cofactor (MethylTHF) for methionine synthesis. B12 is a cofactor for methionine synthase and B6 is a cofactor for cystathionine β-synthase.

Protein C Deficiency

Activated protein C is a natural anticoagulant that becomes operative when thrombin is generated. Protein C, a vitamin-K-dependent zymogen, is activated by thrombin when both bind to endothelial cell thrombomodulin (TM). Initial thrombin formation results in an anticoagulant effect through protein C activation because the affinity for thrombin to activate protein C when bound to TM is tighter than to proteolyze fibrinogen and other coagulation proteins. The limiting factor in protein C activation and the anticoagulation effect of thrombin is the finite number of thrombomodulin-binding sites on endothelium. The endothelial protein C receptor (EPCR) also binds protein C and contributes to the localization of thrombomodulin-bound thrombin where it generates activated protein C (APC). With its cell membrane localizing cofactor, protein S (PS), APC binds to endothelium and activated platelet membranes functioning as an anticoagulant by proteolytically degrading thrombin-activated factor Va and VIIIa, thus attenuating further thrombin formation. Protein C deficiency can be inherited or acquired. Inherited protein C deficiency is usually divided into two subtypes. The most common form of hereditary protein C deficiency is the classic or type I deficiency state. This disorder is the result of reduced synthesis of a normal protein, and is characterized by a parallel reduction in protein C antigen and activity. Type II protein C deficiency results from production of a dysfunctional protein and is characterized by normal protein C antigen with reduced functional activity. Acquired protein C deficiency can be caused by decreased synthesis as in liver disease, increased consumption as in disseminated intravascular coagulation and loss as seen in nephrotic syndrome (see below). Functional deficiencies of protein C primarily cause venous thrombosis. The risk of thrombosis ranges from 0.5–2.5%/year. Protein C deficiencies are found in <5% of hypercoagulable patients. It is a serious prothrombotic risk disorder. Seventy-five percent of individuals with heterozygous protein C deficiency will suffer venous thromboembolism in their lifetime. When VTE occurs, 70% are spontaneous or non-provoked and 30% are provoked by pregnancy, oral contraceptives, inflammation or infection, surgery, trauma, etc. Homozygous protein C deficiency is associated with neonatal purpura fulminans, which leads to death if not recognized within three days of birth.

Protein S Deficiency

Protein S, a vitamin-K-dependent protein, serves as a cofactor for APC and enhances its capacity to inactivate factors Va and VIIIa. In plasma, it circulates in complex with C4b-binding protein. C4b-binding protein inversely regulates the availability of protein S to serve as a cofactor for APC function. Protein S deficiency can be inherited or acquired. Three types of inherited protein S deficiency have been identified. Type I or classic deficiency results from decreased synthesis of a normal protein, and is characterized by reduced levels of total and free protein S antigen together with reduced protein S functional activity. Type II protein S deficiency is characterized by normal levels of total and free protein S, associated with reduced protein S activity. This type of protein S defect is usually associated with a genetic polymorphism in the exons of the gene. Type III protein S deficiency is characterized by normal levels of total protein S, but low levels of free protein S associated with reduced protein S activity. This kind of protein S deficiency is due to the excessive binding of protein S to C4b-binding protein, which occurs in inflammatory states such as systemic lupus erythematosus or inflammatory bowel disease. Acquired protein S deficiency can be caused by decreased synthesis (liver disease), increased consumption (DIC), shift of free protein S to the C4b-bound form (inflammation), or true loss as seen in nephrotic syndrome. The risk of thrombosis from protein S deficiency may be up to 3.5%/year. It is primarily associated with venous thrombosis. Protein S deficiency is found in <5% of patients with hypercoagulable states and has an incidence of 1 in 2500 in the general population. Heterozygous protein S deficiency is also a serious disorder associated with a 74% lifetime risk of having VTE.

Antithrombin Deficiency

Antithrombin, a member of the serine protease inhibitor (serpin) superfamily synthesized in the liver, plays a critical role in regulating coagulation by forming a covalent complex with thrombin, factor Xa, and all the other activated coagulation factors. Homozygous antithrombin deficiency is incompatible with mammalian gestation and delivery, so the inherited form is found in heterozygosity. The rate of antithrombin interaction with its target proteases is accelerated by heparin. The frequency of symptomatic antithrombin deficiency in the general population has been estimated to be between one in 2000 and one in 5000. Among all patients seen with venous thromboembolism (VTE), antithrombin deficiency is detected in less than 1–2%.

Two forms of antithrombin deficiency exist: an inherited and an acquired form. Patients with type I antithrombin deficiency have proportionately reduced plasma levels of antigenic and functional antithrombin that result from a quantitative deficiency of the normal protein. Impaired synthesis (liver disease), defective secretion, or instability of antithrombin in type I antithrombin-deficient individuals is caused by major gene deletions, single nucleotide changes, or short insertions or deletions in the antithrombin gene. Patients with type II antithrombin deficiency have normal or nearly normal plasma antigen accompanied by low activity levels, characteristics indicative of a functionally defective molecule. Type II deficiency is usually caused by specific point mutations leading to single amino acid substitutions that produce a dysfunctional protein. Acquired antithrombin deficiency can reflect decreased antithrombin synthesis (i.e. liver disease), increased consumption (major surgery, acute thrombosis, sepsis, disseminated intravascular coagulation [DIC], malignancy) or enhanced clearance (heparin use, nephrotic range proteinuria). Antithrombin deficiency is a serious risk factor for VTE with 50% lifetime risk in heterozygous individuals. Onset of thrombosis is usually seen after puberty.

Lipoprotein(a)

Lipoprotein(a) [LP(a)] consists of low-density lipoprotein (LDL) held together with a disulfide bond to apolipoprotein(a), a protein that has 85% sequence homology to kringle IV of plasminogen. LP(a) binds to fibrin and cells where plasminogen and α-2-antiplasmin bind. LP(a) inhibits plasminogen binding and activation, and brings LDL to endothelial cell surfaces to contribute to atherogenesis. It is commonly elevated in certain ethnic groups (e.g. South Asians). Epidemiologically, its elevation is associated with venous thromboembolism and ischemic cardiovascular disease. However, it is not considered a serious risk factor for thrombosis. At present, there is no known means to lower its levels or if lowering it will ameliorate cardiovascular disease risk.

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