HEMOSTASIS AND THROMBOSIS

Normal hemostasis is a consequence of tightly regulated processes that maintain blood in a fluid, clot-free state in normal vessels while inducing the rapid formation of a localized hemostatic plug at the site of vascular injury. The pathologic form of hemostasis is thrombosis; it involves blood clot (thrombus) formation in uninjured vessels or thrombotic occlusion of a vessel after relatively minor injury. Both hemostasis and thrombosis involve three components: the vascular wall, platelets, and the coagulation cascade. We begin our discussion with the process of normal hemostasis and a description of its regulation.

Normal Hemostasis

The sequence of events in hemostasis at a site of vascular injury is shown in Figure 4-6. After initial injury a brief period of arteriolar vasoconstriction occurs mostly as a result of reflex neurogenic mechanisms and is augmented by the local secretion of factors such as endothelin (a potent endothelium-derived vasoconstrictor; Fig. 4-6A). The effect is transient, and bleeding would resume were it not for activation of the platelet and coagulation systems.

Endothelial injury also exposes highly thrombogenic subendothelial extracellular matrix, allowing platelets to adhere and be activated. Activation of platelets results in a dramatic shape change (from small rounded disks to flat plates with markedly increased surface area) and release of secretory granules. Within minutes the secreted products have recruited additional platelets (aggregation) to form a hemostatic plug; this is the process of primary hemostasis (Fig. 4-6B).

Figure 4-6 Normal hemostasis. A, After vascular injury, local neurohumoral factors induce a transient vasoconstriction. B, Platelets adhere (via GpIb receptors) to exposed extracellular matrix (ECM) by binding to von Willebrand factor (vWF) and are activated, undergoing a shape change and granule release. Released adenosine diphosphate (ADP) and thromboxane A2 (TXA2) lead to further platelet aggregation (via binding of fibrinogen to platelet GpIIb-IIIa receptors), to form the primary hemostatic plug. C, Local activation of the coagulation cascade (involving tissue factor and platelet phospholipids) results in fibrin polymerization, "cementing" the platelets into a definitive secondary hemostatic plug. D,

Counter-regulatory mechanisms, such as release of t-PA (tissue plasminogen activator, a fibrinolytic product) and thrombomodulin (interfering with the coagulation cascade), limit the hemostatic process to the site of injury.

Tissue factor is also exposed at the site of injury. Also known as factor III and thromboplastin, tissue factor is a membrane-bound procoagulant glycoprotein synthesized by endothelium. It acts in conjunction with factor VII (see below) as the major in vivo pathway to activate the coagulation cascade, eventually culminating in thrombinRx generation. ThrombinRx cleaves circulating fibrinogen into insoluble fibrin, creating a fibrin meshwork deposition. Thrombin also induces further platelet recruitment and granule release. This secondary hemostasis sequence (Fig. 4-6C) lasts longer than the initial platelet plug.

Polymerized fibrin and platelet aggregates form a solid permanent plug to prevent any additional hemorrhage. At this stage counter-regulatory mechanisms (e.g., tissue plasminogen activator, t-PA) are set into motion to limit the hemostatic plug to the site of injury (see Fig. 4-6D).

The following sections discuss these events in greater detail.

Endothelium

Endothelial cells modulate several (and frequently opposing) aspects of normal hemostasis. The balance between endothelial anti- and prothrombotic activities determines whether thrombus formation, propagation, or dissolution occurs. At baseline, endothelial cells exhibit antiplatelet, anticoagulant, and fibrinolytic properties; however, they are capable (after injury or activation) of exhibiting numerous procoagulant activities (Fig. 4-7). It should also be remembered that endothelium can be activated by infectious agents, by hemodynamic factors, by plasma mediators, and (most significantly) by cytokines (Chapter 2).

Antithrombotic Properties

Under most circumstances, endothelial cells maintain an environment that promotes liquid blood flow by blocking platelet adhesion and aggregation, by inhibiting the coagulation cascade, and by lysing blood clots.

Antiplatelet Effects

Figure 4-7 Pro- and anticoagulant activities of endothelium. Not shown are pro- and antifibrinolytic properties of endothelium (see text). NO, nitric oxide; PGI2, prostacyclin; t-PA, tissue plasminogen activator; vWF, von Willebrand factor. The thrombin receptor is also called a protease-activated receptor (PAR; see text).

An intact endothelium prevents platelets (and plasma coagulation factors) from interacting with the highly thrombogenic subendothelial ECM. Nonactivated platelets do not adhere to the endothelium, a property intrinsic to the plasma membrane of endothelium. Moreover, if platelets are activated (e.g., after focal endothelial injury), they are inhibited from adhering to the surrounding uninjured endothelium by endothelial prostacyclin (PGI2) and nitric oxide (Chapter 2). Both mediators are potent vasodilators and inhibitors of platelet aggregation; their synthesis by endothelial cells is stimulated by several factors (e.g., thrombin and cytokines) produced during coagulation. Endothelial cells also elaborate adenosine diphosphatase, which degrades adenosine diphosphate (ADP) and further inhibits platelet aggregation (see below).

Anticoagulant Effects

Anticoagulant effects are mediated by membrane-associated, heparin-like molecules and thrombomodulin (see Fig. 4-7). The heparin-like molecules act indirectly; they are cofactors that allow antithrombin III to inactivate thrombin, factor Xa, and several other coagulation factors (see later). Thrombomodulin also acts indirectly; it binds to thrombin, converting it from a procoagulant to an anticoagulant capable of activating the anticoagulant protein C. Activated protein C, in turn, inhibits clotting by proteolytic cleavage of factors Va and VIIIa; it requires protein S, synthesized by endothelial cells, as a cofactor.

Fibrinolytic Properties

Endothelial cells synthesize tissue plasminogen activator (t-PA), promoting fibrinolytic activity to clear fibrin deposits from endothelial surfaces (see Fig. 4-6D).

Prothrombotic Properties

While endothelial cells exhibit properties that usually limit blood clotting, they can also become prothrombotic, with activities that affect platelets, coagulation proteins, and the fibrinolytic system. Endothelial injury results in platelet adhesion to subendothelial collagen; this occurs through von Willebrand factor (vWF), an essential cofactor for binding platelets to collagen and other surfaces. vWF (both circulating and collagen bound) is synthesized largely by normal endothelium. Loss of endothelium exposes previously deposited vWF and allows circulating vWF to also bind to the basement membrane; in quick order, platelets adhere via their glycoprotein Ib (GpIb) receptors (Fig. 4-8).

Figure 4-8 Platelet adhesion and aggregation. Von Willebrand factor functions as an adhesion bridge between subendothelial collagen and the glycoprotein Ib (GpIb) platelet receptor. Aggregation is accomplished by binding of fibrinogen to platelet GpIIb-IIIa receptors and bridging many platelets together. Congenital deficiencies in the various receptors or bridging molecules lead to the diseases indicated in the colored boxes. ADP, adenosine diphosphate.

Cytokines such as tumor necrosis factor (TNF) or interleukin-1 (IL-1) as well as bacterial endotoxin all induce endothelial cell production of tissue factor; as we will see below, tissue factor activates the extrinsic clotting pathway. By binding activated IXa and Xa (see below), endothelial cells augment the catalytic activities of these coagulation factors. Finally, endothelial cells also secrete plasminogen activator inhibitors (PAIs), which depress fibrinolysis (not shown in Fig. 4-7).

SUMMARY

Contribution of Endothelial Cells to Coagulation

Intact endothelial cells maintain liquid blood flow by actively inhibiting platelet adherence, preventing coagulation factor activation, and lysing blood clots that may form.Endothelial cells can be stimulated by direct injury or by various cytokines that are produced during inflammation. Such stimulation results in expression of procoagulant proteins (e.g., tissue factor and vWF) that contribute to local thrombus formation.Loss of endothelial integrity exposes underlying vWF and basement membrane collagen, both substrates for platelet aggregation and thrombus formation.

Platelets

Platelets play a critical role in normal hemostasis. When circulating and nonactivated they are membrane-bound smooth disks expressing several glycoprotein receptors of the integrin family and containing two types of granules:

α-Granules express the adhesion molecule P-selectin on their membranes (Chapter 2) and contain fibrinogen, fibronectin, factors V and VIII, platelet factor 4 (a heparin-binding chemokine), platelet-derived growth factor (PDGF), and transforming growth factor α (TGF-α).Dense bodies, or δ granules, contain adenine nucleotides (ADP and ATP), ionized calcium, histamine, serotonin, and epinephrine.

After vascular injury, platelets encounter ECM constituents (of which collagen is the most important) and additional proteins (vWF being critical) that are normally not exposed when the endothelial layer is intact. Upon contact with these proteins, platelets undergo three reactions: (1) adhesion and shape change, (2) secretion (release reaction), and (3) aggregation (see Fig. 4-6B).

Platelet Adhesion

Adhesion to ECM is mediated largely via interactions with vWF acting as a bridge between platelet surface receptors (e.g., GpIb) and exposed collagen (see Fig. 4-8). Although platelets can adhere directly to ECM, vWF-GpIb associations are required to overcome the high shear forces of flowing blood. Genetic deficiencies of vWF (von Willebrand disease; Chapter 12) or its receptors result in bleeding disorders, highlighting the importance of these interactions. Conversely, failure of the normal proteolytic processing of vWF from high-molecular-weight multimers to smaller forms leads to aberrant platelet aggregation in the circulation; this defect in vWF processing causes thrombotic thrombocytopenic purpura, one of the so-called thrombotic microangiopathies (see Chapter 12).

Secretion (Release Reaction)

Secretion of both granule types occurs soon after adhesion. Various agonists can bind specific platelet surface receptors and initiate an intracellular phosphorylation cascade that leads to degranulation. Release of dense body contents is especially important, since calcium is required in the coagulation cascade and ADP is a potent mediator of platelet aggregation (platelets adhering to other platelets-discussed next). ADP also begets additional platelet ADP release, amplifying the aggregation process. Finally, platelet activation increases surface expression of phospholipid complexes, which provide a critical nucleation and binding site for calcium and coagulation factors in the intrinsic clotting pathway (see later).

Platelet Aggregation

Aggregation follows platelet adhesion and granule release. In addition to ADP, platelet-synthesized thromboxane A2 (TXA2; Chapter 2) is also an important stimulus for platelet aggregation. ADP and TXA2 together drive an autocatalytic process that promotes formation of an enlarging platelet aggregate, the primary hemostatic plug. This primary aggregation is reversible. However, with activation of the coagulation cascade, the generation of thrombinRx results in two processes that make an irreversible hemostatic plug. Thrombin binds to a platelet surface receptor (protease-activated receptor, or PAR, see below); in association with ADP and TXA2, this interaction induces further platelet aggregation. Platelet contraction follows, creating an irreversibly fused mass of platelets ("viscous metamorphosis") constituting the definitive secondary hemostatic plug. Concurrently, thrombin converts fibrinogen to fibrin within and about the platelet plug, contributing to the overall stability of the clot (see below).

Both erythrocytes and leukocytes are also found in hemostatic plugs; leukocytes adhere to platelets and endothelium via adhesion molecules and contribute to the inflammatory response that accompanies thrombosis. ThrombinRx also contributes by directly stimulating neutrophil and monocyte adhesion and by generating chemotactic fibrin split products from the cleavage of fibrinogen.

Importance of Fibrinogen in Platelet Aggregation

The binding of ADP to its platelet receptor induces a conformational change of the GpIIb-IIIa receptors, allowing them to bind fibrinogen. Fibrinogen then acts to connect many platelets together to form large aggregates (see Fig. 4-8). The importance of these interactions is amply demonstrated by the bleeding disorders that occur in patients with congenitally deficient or inactive GpIIb-IIIa proteins. Moreover, the clinical recognition of the central role of these GpIIb-IIIa receptors in platelet cross-linking led to the development of antagonists that can potently block platelet aggregation-either by interfering with ADP binding, as with clopidogrel, or by binding to the GpIIb-IIIa receptors, as with monoclonal antibodies.

Interaction of Platelets and Endothelium

The interplay of platelets and endothelium has a profound impact on the formation of a clot. Prostaglandin PGI2 (synthesized by endothelium) is a vasodilator and inhibits platelet aggregation, whereas TXA2 is a platelet-derived prostaglandin that activates platelet aggregation and is a potent vasoconstrictor. Effects mediated by PGI2 and TXA2 constitute exquisitely balanced pathways for modulating human platelet function: in the normal state, intravascular platelet aggregation is prevented, whereas endothelial injury favors the formation of hemostatic plugs. The clinical use of aspirinRx (a cyclooxygenase inhibitor) in patients at risk for coronary thrombosis is related to its ability to inhibit the synthesis of TXA2. In a manner similar to that of PGI2, nitric oxide also acts as a vasodilator and inhibitor of platelet aggregation (see Fig. 4-7).

SUMMARY

Platelet Aggregation

Endothelial injury exposes the underlying basement membrane ECM; platelets adhere to the ECM and become activated by binding to vWF through GpIb platelet receptors.Upon activation, platelets secrete granule products that include calcium (activates coagulation proteins) and ADP (mediates further platelet aggregation and degranulation). Activated platelets also synthesize TXA2 (increases platelet activation and causes vasoconstriction).Activated platelets expose phospholipid complexes that provide an important surface for coagulation-protein activation (see below).Released ADP stimulates formation of a primary hemostatic plug by activating platelet GpIIb-IIIa receptors that in turn facilitate fibrinogen binding and cross-linking.The formation of the definitive secondary hemostatic plug requires the activation of thrombin to cleave fibrinogen and form polymerized fibrin via the coagulation cascade (see below).

Coagulation Cascade

The coagulation cascade constitutes the third component of the hemostatic process and is a major contributor to thrombosis. The pathways are schematically presented in Figure 4-9; only general principles are discussed here.

The coagulation cascade is essentially an amplifying series of enzymatic conversions; each step in the process proteolytically cleaves an inactive proenzyme into an activated enzyme, eventually culminating in thrombin formation; thrombin is the most important enzyme regulating the coagulation process. Thrombin converts the soluble plasma protein fibrinogen into fibrin monomers that polymerize into an insoluble gel; this gel encases platelets and other circulating cells in the definitive secondary hemostatic

plug. Fibrin polymers are stabilized by the transglutaminase cross-linking activity of factor XIIIa.Each reaction in the pathway results from the assembly of a complex composed of an enzyme (activated coagulation factor), a substrate (proenzyme form of coagulation factor), and a cofactor (reaction accelerator). These components are assembled on a phospholipid complex and held together by calcium ions. Thus, clotting tends to remain localized to phospholipid-rich sites where such an assembly can occur, for example, on the surface of activated platelets. Two such reactions are the sequential conversion of factor X to Xa and then factor II (prothrombin) to IIa (thrombin) are illustrated in Figure 4-10. Parenthetically, the ability of coagulation factors II, XII, IX, and X to bind to calcium requires that additional γ-carboxyl groups be enzymatically appended to certain glutamic acid residues on these proteins. This reaction requires vitamin K as a cofactor and is antagonized by drugs such as coumadin, which is therefore useful for patients who require anticoagulation on a chronic basis-or such as warfarin, which can be used as a rodenticide to cause exsanguination.The blood coagulation scheme has been traditionally classified into extrinsic and intrinsic pathways that converge with the activation of factor X (see Fig. 4-9). The extrinsic pathway was so designated because it required the addition of an exogenous trigger (originally provided by tissue extracts); the intrinsic pathway required only exposing factor XII (Hageman factor) to a thrombogenic surface (even glass would suffice). However, this classification, although useful for clinical testing (see below), is largely an artifact of in vitro testing, since several interconnections exist between the two pathways. The extrinsic pathway is the most physiologically relevant of the two in driving coagulation after vascular damage; it is activated by tissue factor (also known as thromboplastin or factor III), a membrane-bound lipoprotein expressed at sites of injury (see Fig. 4-9).The clinical pathology lab assesses the two pathways using two standard assays: prothrombin time (PT) and partial thromboplastin time (PPT).

Figure 4-9 The classical coagulation cascade. Note the common link between the intrinsic and extrinsic pathways at the level of factor IX activation. Factors in red boxes represent inactive molecules; activated factors are indicated with a lower-case a and a green box. HMWK, high-molecular-weight kininogen. Not shown are the inhibitory anticoagulant pathways (see Figs. 4-7 and 4-12).

The PT assay screens for the activity of the proteins in the extrinsic pathway (factors VII, X, II, V, and fibrinogen) by adding phospholipids and tissue factor to a patient's citrated plasma (sodium citrate chelates any calcium present and prevents spontaneous clotting). The clotting reaction is started by adding exogenous calcium, and the time to fibrin clot formation (usually 11-13 seconds) is recorded. Typically, this is expressed as ratio of the patient's PT to the mean PT for a group of normal patients, othewise known as the International Normalized Ratio (INR). In addition to its value as a screening assay for the normal activity of the extrinsic pathway factors, the PT is also sensitive to the effects of coumadin. It is therefore used to monitor the efficacy of coumadin anticoagulation therapy; ideally, the INR is maintained between 2 and 3 in patients receiving coumadin.

The PTT assay screens for the activity of the proteins in the intrinsic pathway (factors XII, XI, IX, VIII, X, V, II, and fibrinogen) by adding first an appropriate surface (e.g., ground glass) and phospholipids to a patient's citrated plasma, and then exogenous calcium. The time to clot formation (usually 28-35 seconds) is recorded. In addition to its value in screening for the normal activity of intrinsic pathway factors, the PTT assay's sensitivity to the effects of heparin makes it useful to monitor the efficacy of heparin therapy for acute thrombosis or embolism.

In addition to catalyzing the final steps in the coagulation cascade, thrombin exerts a wide variety of effects on the local vasculature and inflammatory milieu; it even actively participates in limiting the extent of the hemostatic process (Fig. 4-11). Most of these thrombin-mediated effects occur through protease activated receptors belonging to a family of seven transmembrane proteins coupled to G proteins (see Fig. 4-7).Once activated, the coagulation cascade must be restricted to the local site of vascular injury to prevent runaway clotting of the entire vascular tree. In addition to the restriction of factor activation to sites of exposed phospholipids, three categories of natural anticoagulants function to control clotting: antithrombins, proteins C and S, and tissue factor pathway inhibitor (TFPI).

Antithrombins (e.g., antithrombin III) inhibit the activity of thrombin and other serine proteases, factors IXa, Xa, XIa, and XIIa. Antithrombin III is activated by binding to heparin-like molecules on endothelial cells-hence the usefulness of administering heparin in clinical situations to reduce thrombotic activity (see Fig. 4-7).Proteins C and S are two vitamin K-dependent proteins that inactivate the cofactors Va and VIIIa. Protein C activation by thrombomodulin was described earlier; protein S is a cofactor for protein C activity (see Fig. 4-7).TFPI is a protein secreted by endothelium (and other cell types) that inactivates factor Xa and tissue factor-VIIa complexes (see Fig. 4-7).

Activation of the clotting cascade also sets into motion a fibrinolytic cascade that moderates the size of the ultimate clot. Fibrinolysis is largely accomplished by the enzymatic activity of plasmin, which breaks down fibrin and interferes with its polymerization (Fig. 4-12). The resulting fibrin split products (FSPs, or fibrin degradation products) can also act as weak anticoagulants. As a clinical correlate, elevated levels of FSPs (clinical laboratories most frequently measure the fibrin d-dimer) are helpful in diagnosing abnormal thrombotic states including disseminated intra-vascular coagulation (DIC), deep venous thrombosis, or pulmonary thromboembolism (described in detail later).

Plasmin is generated by enzymatic degradation of the inactive circulating precursor plasminogen either by a factor XII-dependent pathway or by plasminogen activators (PAs; see Fig. 4-12). The most important of the PAs is t-PA, which is synthesized principally by endothelial cells and is most active when attached to fibrin. The affinity for fibrin makes t-PA a useful therapeutic agent, since it largely confines

fibrinolytic activity to sites of recent thrombosis. Urokinase-like PA (u-PA) is another PA present in plasma and in various tissues; it can activate plasmin in the fluid phase. Finally, plasminogen can be cleaved to its active form by the bacterial product streptokinase, an activity that may be clinically significant in various bacterial infections. As with any potent regulatory component, the activity of plasmin is also tightly restricted. To prevent excess plasmin from lysing thrombi indiscriminately elsewhere in the body, free plasmin rapidly forms a complex with circulating α2-antiplasmin and is inactivated (see Fig. 4-12).Endothelial cells further modulate the coagulation/anticoagulation balance by releasing PAIs, which block fibrinolysis and confer an overall procoagulation effect (see Fig. 4-12). The PAIs are increased by certain cytokines and probably play a role in the intravascular thrombosis accompanying severe inflammation.

Figure 4-10 Sequential conversion of factor X to factor Xa, followed by factor II (prothrombin) to factor IIa (thrombin). The initial reaction complex consists of an enzyme (factor IXa), a substrate (factor X), and a reaction accelerator (factor VIIIa), all assembled on a platelet phospholipid surface. Calcium ions hold the assembled components together and are essential for the reaction. Activated factor Xa becomes the enzyme part of the second adjacent complex in the coagulation cascade, converting the prothrombin substrate to IIa using factor Va as the reaction accelerator. (Modified from Mann KG: The biochemistry of coagulation. Clin Lab Med 4:217, 1984.)

Figure 4-11 Role of thrombin in hemostasis and cellular activation. Thrombin plays a critical role in generating cross-linked fibrin via cleavage of fibrinogen to fibrin and activation of factor XIII. Through protease-activated receptors (PARs, see text), thrombin also modulates several cellular activities. It directly induces platelet aggregation and TXA2 secretion and can activate endothelium to generate leukocyte adhesion molecule and a variety of fibrinolytic (t-PA), vasoactive (NO, PGI2), and cytokine (PDGF) mediators. Thrombin also directly activates leukocytes. ECM, extracellular matrix; NO, nitric oxide; PDGF, platelet-derived growth factor; PGI2, prostacyclin; TXA2, thromboxane A2; t-PA, tissue plasminogen activator. See Figure 4-7 for additional anticoagulant modulators of thrombin activity, such as antithrombin III and thrombomodulin. (Courtesy of Shaun Coughlin, MD, PhD, Cardiovascular Research Institute, University of California at San Francisco; modified with permission.)

SUMMARY

Coagulation Factors

Coagulation occurs via the sequential enzymatic conversion of a cascade of circulating and locally synthesized proteins. Tissue factor elaborated at sites of injury is the most important initiator of the coagulation cascade; at the final stage of coagulation, thrombin converts fibrinogen into insoluble fibrin, which helps to form the definitive hemostatic plug.Coagulation is normally constrained to sites of vascular injury by:

Limiting enzymatic activation to phospholipid complexes provided by activated plateletsNatural anticoagulants elaborated at sites of endothelial injury or during activation of the coagulation cascadeInduction of fibrinolytic pathways involving plasmin through the activities of various Pas

Thrombosis

Having discussed the process of normal hemostasis, we can now turn our attention to the dysregulation that underlies thrombus formation.

Pathogenesis

There are three primary influences on thrombus formation (called Virchow's triad): (1) endothelial injury, (2) stasis or turbulence of blood flow, and (3) blood hypercoagulability (Fig. 4-13).

Endothelial Injury

This is a dominant influence, since endothelial loss by itself can lead to thrombosis. It is particularly important for thrombus formation occurring in the heart or in the arterial circulation, where the normally high flow rates might otherwise hamper clotting by preventing platelet adhesion or diluting coagulation factors. Thus, thrombus formation within the cardiac chambers (e.g., after endocardial injury due to myocardial infarction), over ulcerated plaques in atherosclerotic arteries, or at sites of traumatic or inflammatory vascular injury (vasculitis) is largely a function of endothelial injury. Clearly, physical loss of endothelium leads to exposure of subendothelial ECM, adhesion of platelets, release of tissue factor, and local depletion of PGI2 and plasminogen activators. However, it is important to note that endothelium need not be denuded or physically disrupted to contribute to the development of thrombosis; any perturbation in the dynamic balance of the prothrombotic and antithrombotic activities of endothelium can influence local clotting events (see Fig. 4-7). Thus, dysfunctional endothelium may elaborate greater amounts of procoagulant factors (e.g., platelet adhesion molecules, tissue factor, plasminogen activator inhibitors) or may synthesize fewer anticoagulant effectors (e.g., thrombomodulin, PGI2, t-PA). Significant endothelial dysfunction (in the absence of endothelial cell loss) may occur with hypertension, turbulent flow over scarred valves, or by the action of bacterial endotoxins. Even relatively subtle influences, such as homocystinuria, hypercholesterolemia, radiation, or products absorbed from cigarette smoke, may be sources of endothelial dysfunction.

Figure 4-13 Virchow's triad in thrombosis. Integrity of endothelium is the most important factor. Injury to endothelial cells can also alter local blood flow and affect coagulability. Abnormal blood flow (stasis or turbulence), in turn, can cause endothelial injury. The factors may act independently or may combine to promote thrombus formation.

Alterations in Normal Blood Flow

Turbulence contributes to arterial and cardiac thrombosis by causing endothelial injury or dysfunction, as well as by forming countercurrents and local pockets of stasis; stasis is a major contributor to the development of venous thrombi. Normal blood flow is laminar, such that platelets flow centrally in the vessel lumen, separated from the endothelium by a slower moving clear zone of plasma. Stasis and turbulence therefore:

Disrupt laminar flow and bring platelets into contact with the endothelium.

Prevent dilution of activated clotting factors by fresh-flowing blood.

Retard the inflow of clotting factor inhibitors and permit the buildup of thrombi

Promote endothelial cell activation, resulting in local thrombosis, leukocyte adhesion, etc.

Turbulence and stasis contribute to thrombosis in several clinical settings. Ulcerated atherosclerotic plaques not only expose subendothelial ECM but also cause turbulence. Abnormal aortic and arterial dilations, called aneurysms, create local stasis and consequently a fertile site for thrombosis (Chapter 10). Acute myocardial infarction results in focally noncontractile myocardium; ventricular remodeling after more remote infarction can lead to aneurysm formation. In both cases cardiac mural thrombi form more easily because of the local blood stasis (Chapter 11). Mitral valve stenosis (e.g., after rheumatic heart disease) results in left atrial dilation. In conjunction with atrial fibrillation, a dilated atrium is a site of profound stasis and a prime location for development of thrombi. Hyperviscosity syndromes (such as polycythemia; Chapter 12) increase resistance to flow and cause small vessel stasis; the deformed red cells in sickle cell anemia (Chapter 12) cause vascular occlusions, with the resultant stasis also predisposing to thrombosis.

Hypercoagulability

Hypercoagulability generally contributes less frequently to thrombotic states but is nevertheless an important component in the equation. It is loosely defined as any alteration of the coagulation pathways that predisposes to thrombosis, and it can be divided into primary (genetic) and secondary (acquired) disorders (Table 4-2).

Primary (inherited) hypercoagulable states. Of the inherited causes of hypercoagulability, mutations in the factor V gene and the prothrombin gene are the most common: Secondary (acquired) hypercoagulable states. Unlike the hereditary disorders, the pathogenesis of acquired thrombotic diatheses is frequently multifactorial and is therefore more complicated (see Table 4-2). In some situations (e.g., cardiac failure or trauma), stasis or vascular injury may be most important. Hypercoagulability is associated with oral contraceptive use and the hyperestrogenic state of pregnancy, probably related to increased hepatic synthesis of coagulation factors and reduced synthesis of antithrombin III. In disseminated cancers, release of procoagulant tumor products predisposes to thrombosis. The hypercoagulability seen with advancing age has been attributed to increasing platelet aggregation and reduced endothelial PGI2 release. Smoking and obesity promote hypercoagulability by unknown mechanisms.

Table 4-2. Hypercoagulable States

Primary (Genetic)Common Mutation in factor V gene (factor V Leiden) Mutation in prothrombin gene Mutation in methyltetrahydrofolate geneRare Antithrombin III deficiency Protein C deficiency Protein S deficiencyVery rare Fibrinolysis defectsSecondary (Acquired)High risk for thrombosis Prolonged bedrest or immobilization Myocardial infarction Atrial fibrillation Tissue damage (surgery, fracture, burns) Cancer Prosthetic cardiac valves Disseminated intravascular coagulation Heparin-induced thrombocytopenia Antiphospholipid antibody syndrome (lupus anticoagulant syndrome)Lower risk for thrombosis Cardiomyopathy Nephrotic syndrome Hyperestrogenic states (pregnancy) Oral contraceptive use Sickle cell anemia Smoking

Among the acquired causes of thrombotic diathesis, the heparin-induced thrombocytopenia (HIT) syndrome and antiphospholipid antibody syndrome (previously called the lupus anticoagulant syndrome) deserve special mention.

Seen in as many as 5% of the population, the HIT syndrome occurs when administration of unfractionated heparin (for therapeutic anticoagulation) induces autoantibodies to complexes of heparin and a platelet membrane protein (platelet factor 4) (Chapter 12). This antibody binds to similar complexes present on platelet and endothelial surfaces, resulting in platelet activation and endothelial cell injury, and a net prothrombotic state. The occurrence of HIT syndrome can be reduced by using low-molecular-weight heparin preparations that retain anticoagulant activity but do not interact with platelets; these preparations have the additional advantage of a prolonged serum half-life.Antiphospholipid antibody syndrome has protean manifestations, including recurrent thrombosis, repeated miscarriages, cardiac valve vegetations, and thrombocytopenia; it is associated with autoantibodies directed against anionic phospholipids (e.g., cardiolipin) or-more accurately-plasma protein antigens that are unveiled by binding to such phospholipids (e.g., prothrombin). In vivo these antibodies induce a hypercoagulable state, by inducing direct platelet activation or by interfering with endothelial cell production of PGI2. However, in vitro (in the absence of platelets and endothelium) the antibodies merely interfere with phospholipid complex assembly and thus inhibit coagulation (hence the designation lupus anticoagulant). Patients with antibodies to cardiolipins also have a false-positive serologic test for syphilis, because the antigen in the standard tests is embedded in cardiolipin.

There are two types of antiphospholipid antibody syndrome. Many patients have secondary antiphospholipid syndrome due to a well-defined autoimmune disease, such as systemic lupus erythematosus (Chapter 5). In contrast, those who exhibit only the manifestations of a hypercoagulable state without evidence of other autoimmune disorder are designated as having primary antiphospholipid syndrome. Patients with antiphospholipid antibody syndrome are at increased risk of a fatal event (as many as 7% in one series). Therapy involves anticoagulation, with immunosuppression in refractory cases. Although antiphospholipid antibodies are associated with thrombotic diatheses, they have also been identified in 5% to 15% of apparently normal individuals, implying that they may be necessary but not sufficient to cause full-blown antiphospholipid antibody syndrome.

Morphology

Thrombi can develop anywhere in the cardiovascular system (e.g., in cardiac chambers, on valves, or in arteries, veins, or capillaries). The size and shape of a thrombus depend on the site of origin and the cause. Arterial or cardiac thrombi typically begin at sites of endothelial injury or turbulence; venous thrombi characteristically occur at sites of stasis. Thrombi are focally attached to the underlying vascular surface; arterial thrombi tend to grow in a retrograde direction from the point of attachment, while venous thrombi extend in the direction of blood flow (thus both tend to propagate toward the heart). The propagating portion of a thrombus tends to be poorly attached and therefore prone to fragmentation, generating an embolus.

Thrombi can have grossly (and microscopically) apparent laminations called lines of Zahn; these represent pale platelet and fibrin layers alternating with darker erythrocyte-rich layers. Such lines are

significant only in that they represent thrombosis in the setting of flowing blood; their presence can therefore potentially distinguish antemortem thrombosis from the bland nonlaminated clots that occur in the postmortem state (see also below). Although such lines are typically not as apparent in veins or smaller arteries (thrombi formed in sluggish venous flow usually resemble statically coagulated blood), careful evaluation generally reveals ill-defined laminations.

Thrombi occurring in heart chambers or in the aortic lumen are designated mural thrombi. Abnormal myocardial contraction (resulting from arrhythmias, dilated cardiomyopathy, or myocardial infarction) or endomyocardial injury (caused by myocarditis, catheter trauma) promotes cardiac mural thrombi (Fig. 4-14A), while ulcerated atherosclerotic plaques and aneurysmal dilation promote aortic thrombosis (Fig. 4-14B).

Arterial thrombi are frequently occlusive and are produced by platelet and coagulation activation; they are typically a friable meshwork of platelets, fibrin, erythrocytes, and degenerating leukocytes. Although arterial thrombi are usually superimposed on an atherosclerotic plaque, other vascular injury (vasculitis, trauma) can be involved.

Venous thrombosis (phlebothrombosis) is almost invariably occlusive, and the thrombus can create a long cast of the lumen; venous thrombosis is largely the result of activation of the coagulation cascade, and platelets play a secondary role. Because these thrombi form in the sluggish venous circulation, they also tend to contain more enmeshed erythrocytes and are therefore called red, or stasis, thrombi. The veins of the lower extremities are most commonly affected (90% of venous thromboses); however, venous thrombi can occur in the upper extremities, periprostatic plexus, or ovarian and periuterine veins; under special circumstances they may be found in the dural sinuses, portal vein, or hepatic vein.

Postmortem clots can sometimes be mistaken at autopsy for venous thrombi. However, postmortem "thrombi" are gelatinous, with a dark red dependent portion where red cells have settled by gravity, and a yellow "chicken fat" supernatant, and they are usually not attached to the underlying wall. In contrast, red thrombi are firmer and are focally attached, and sectioning reveals strands of gray fibrin.

Thrombi on heart valves are called vegetations. Bacterial or fungal blood-borne infections can cause valve damage, subsequently leading to large thrombotic masses (infective endocarditis, Chapter 11). Sterile vegetations can also develop on noninfected valves in hypercoagulable states, so-called nonbacterial thrombotic endocarditis (Chapter 11). Less commonly, sterile, verrucous endocarditis (Libman-Sacks endocarditis) can occur in the setting of systemic lupus erythematosus (Chapter 5).

Figure 4-14 Mural thrombi. A, Thrombus in the left and right ventricular apices, overlying white fibrous scar. B, Laminated thrombus in a dilated abdominal aortic aneurysm. Numerous friable mural thrombi are also superimposed on advanced atherosclerotic lesions of the more proximal aorta (left side of picture).

Fate of the Thrombus

If a patient survives the initial thrombosis, in the ensuing days or weeks thrombi undergo some combination of the following four events:

Propagation. Thrombi accumulate additional platelets and fibrin, eventually causing vessel obstruction.

Embolization. Thrombi dislodge or fragment and are transported elsewhere in the vasculature.

Dissolution. Thrombi are removed by fibrinolytic activity.Organization and recanalization. Thrombi induce inflammation and fibrosis (organization). These can eventually recanalize (re-establishing some degree of flow), or they can be incorporated into a thickened vessel wall.

Propagation was discussed above, and embolization is covered in greater detail below. Dissolution is the result of fibrinolytic activation, which leads to rapid shrinkage and even total lysis of recent thrombi. With older thrombi, extensive fibrin polymerization renders the thrombus substantially more resistant to proteolysis, and lysis is ineffectual. This is clinically significant because therapeutic administration of fibrinolytic agents (e.g., t-PA in the setting of acute coronary thrombosis) is generally effective only within a few hours of thrombus formation.

Figure 4-15 Low-power view of an artery with an old thrombus. A, H&E-stained section. B, Stain for elastic tissue. The original lumen is delineated by the internal elastic lamina (arrows) and is totally filled with organized thrombus, now punctuated by a number of recanalized channels (white spaces).

Older thrombi become organized by the ingrowth of endothelial cells, smooth muscle cells, and fibroblasts into the fibrin-rich clot (Fig. 4-15). Capillary channels are eventually formed that, to a limited extent, can create conduits along the length of the thrombus and thereby re-establish the continuity of the original lumen. Although the channels may not successfully restore significant flow to many obstructed vessels, recanalization can potentially convert a thrombus into a vascularized mass of connective tissue that is eventually incorporated into the vessel wall and remains as a subendothelial swelling. Eventually, with contraction of the mesenchymal cells only a fibrous lump may remain to mark the original thrombus site. Occasionally, instead of organizing, the center of a thrombus undergoes enzymatic digestion, presumably because of the release of lysosomal enzymes from trapped leukocytes and platelets.

Clinical Correlations: Venous versus Arterial Thrombosis

Thrombi are significant because they cause obstruction of arteries and veins and are potential sources of emboli. Which effect is most important depends on the site of thrombosis. Venous thrombi can cause congestion and edema in vascular beds distal to an obstruction, but they are most worrisome for their capacity to embolize to the lungs and cause death (see below). Conversely, while arterial thrombi can embolize and even cause downstream tissue infarction (see below), their role in vascular obstruction at critical sites (e.g., coronary and cerebral vessels) is much more significant clinically.

Venous Thrombosis (Phlebothrombosis)

Most venous thrombi occur in the superficial or deep veins of the leg. Superficial venous thrombi usually occur in the saphenous system, particularly when there are varicosities. Such superficial thrombi can cause local congestion, swelling, pain, and tenderness along the course of the involved vein, but they rarely embolize. Nevertheless, the local edema and impaired venous drainage do predispose the overlying skin to infections from minor trauma and to the development of varicose ulcers. Deep thrombi in the larger leg veins at or above the knee joint (e.g., popliteal, femoral, and iliac veins) are more serious because they may embolize. Although they may cause local pain and edema, the venous obstruction may be rapidly offset by collateral bypass channels. Consequently, deep venous thromboses are entirely asymptomatic in approximately 50% of patients and are recognized in retrospect only after they have embolized.

Deep venous thrombosis can occur with stasis or in a variety of hypercoagulable states, as described earlier (see Table 4-2). Cardiac failure is an obvious reason for stasis in the venous circulation. Trauma, surgery, and burns usually result in reduced physical activity, injury to vessels, release of procoagulant substances from tissues, and/or reduced t-PA activity. There are many influences contributing to the thrombotic propensity of peripartum and postpartum states; in addition to the potential for amniotic fluid infusion into the circulation during parturition (see below), late pregnancy and the postpartum period are associated with hypercoagulability. Tumor-associated procoagulant release is largely responsible for the increased risk of thromboembolic phenomena seen in disseminated cancers (called migratory thrombophlebitis, or Trousseau's syndrome). Regardless of the specific clinical setting, advanced age, bedrest, and immobilization increase the risk of deep venous thrombosis because reduced physical activity diminishes the milking action of muscles in the lower leg and so slows venous return.

Cardiac and Arterial Thrombosis

Atherosclerosis is a major initiator of thromboses, because it is associated with loss of endothelial integrity and abnormal vascular flow (see Fig. 4-14B). Cardiac mural thrombi can occur in the setting of myocardial infarction related to dyskinetic myocardial contraction as well as damage to the adjacent endocardium (see Fig. 4-14A). Rheumatic heart disease (Chapter 11) can cause atrial mural thrombi due to mitral valve stenosis, followed by left atrial dilation and concurrent atrial fibrillation. In addition to the obstructive consequences, cardiac and aortic mural thrombi can also embolize peripherally. Virtually any tissue can be affected, but brain, kidneys, and spleen are prime targets because of their large volume of blood flow.

SUMMARY

Thrombosis

Thrombus development depends on the relative contribution of the components of Virchow's triad:

Endothelial injury (e.g., by toxins, hypertension, inflammation, or metabolic products)Abnormal blood flow - stasis or turbulence (e.g., due to aneurysms, atherosclerotic plaque)Hypercoagulability, which can be either primary (e.g., factor V Leiden, increased prothrombin synthesis, antithrombin III deficiency) or secondary (e.g., bedrest, tissue damage, malignancy)

Thrombi may propagate, resolve, become organized, or embolize. Thrombosis causes tissue injury by local vascular occlusion or by distal embolization.