Thrombosis

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Chapter 10 Thrombosis

Elisabeth M. Battinelli, Jane E. Freedman, Joseph Loscalzo

Overview of Thrombosis

Abnormal hemostasis leads to thrombus formation. Thrombosis in either the arterial or venous system is a leading cause of significant morbidity and mortality. When thrombosis occurs in the arterial system, myocardial infarction (MI) and stroke may result, whereas thrombosis in the venous system leads to venous thromboembolic disease. Thrombosis and thrombotic-related events are among the most common causes of mortality in the Western World. It is estimated that 785,000 people in the United States had new thrombotic events within the coronary circulation in 2009, and that over 450,000 had recurrent events. Stroke also accounts for significant morbidity, with 795,000 people per year suffering from a thrombotic event within the cerebral circulation. There are 200,000 new cases of venous thromboembolism each year, 30% of which result in death in the first 30 days after diagnosis, the majority of deaths being sudden in the setting of a pulmonary embolism (PE).

The pathogenesis of thrombosis was elucidated as early as 1856 when Virchow first described its major determinants, including abnormalities in the vessel wall, platelets, and coagulation proteins as essential for establishing a thrombus. The composition of arterial thrombi is distinct from that of thrombi that form in the venous circulation. Arterial thrombi are composed mainly of platelets and occur in areas of vascular wall injury; venous thrombi, by contrast, are rich in fibrin and dependent on a hypercoagulable response associated with individual coagulation factor abnormalities or mechanical issues related to blood flow limitation. Under normal circumstances, the endothelial lining is not a thrombotic surface, with endothelial cells (ECs) constantly interacting with other cell types, including platelets, to directly inhibit thrombus formation through release of antithrombotic factors such as thrombomodulin, tissue factor pathway inhibitor (TFPI), plasmin, and antithrombin systems. At the same time, platelet aggregation is inhibited through prostacyclins and nitric oxide (NO) released directly from platelets.

When the endothelial surface becomes damaged, however, release of many procoagulant proteins (especially tissue factor) and activation of platelets result in uncontrolled hemostasis at the site of vascular injury.¹ As the thrombus begins to form, it recruits additional platelets to the area, leading to further platelet activation. Initially, tethering of platelets is dependent upon exposure of glycoprotein (Gp)Ib-V-IX in damaged collagen, which binds to von Willebrand factor (vWF), resulting in adhesion of platelets to the area of injury. Further recruitment of platelets is mediated through activation of the GPIIb-IIa platelet receptor, which undergoes a conformational change leading to increased affinity for fibrinogen. These events culminate with further platelet activation that results in release of many essential components for thrombus formation, including adenosine diphosphate (ADP), serotonin, and thromboxane A ₂ (TxA₂).

Exposure of vascular collagen also leads to activation of the normal mechanisms of hemostasis—including the coagulation cascade—through exposure of tissue factor, leading to “hemostasis in the wrong place.” The coagulation regulatory system is outlined in Figure 10-1 and discussed in detail in Chapter 5. Briefly, both the tissue factor–mediated pathway (extrinsic) and the contact-mediated pathway (instrinsic pathway) rely on activation of inactive enzyme precursors of serine proteases, which then reflexively lead to activation of another protein within the cascade. The ultimate step results in cross-linking of fibrin to stabilize a platelet plug, leading to thrombus formation. The tissue factor–initiated pathway is essential for thrombus formation. When tissue factor is released during cellular injury, factor VII is activated and complexes. This complex next activates factors X and XI. Activation of factor X is essential for conversion of prothrombin (factor II) to thrombin through the prothrombinase complex on activated platelets. This cascade of coagulation proteins is essential for hemostasis but also can have deleterious affects when it occurs unregulated, leading to unwanted thrombotic complications.

Figure 10-1 Physiological regulation of blood coagulation. α₂MG, α₂-macroglobin; α₁AT, α₁-antitrypsin; PCI, protein-C inhibitor.

Platelets, Thrombosis, and Vascular Disease

Venous Thrombosis

It is estimated that between 100,000 and 180,000 deaths due to a venous thromboembolic event occur annually. These events occur mainly in the vasculature at the area of the vessel sinus where stasis can lead to a hypercoagulable microenvironment. The hemostatic process is activated when tissue factor is exposed at the site of vascular injury; initiation of the coagulation cascade follows, with subsequent formation of thrombin and conversion of fibrinogen to fibrin. This process evolves at the same time platelets are actively being recruited to the area of injury through collagen exposure, leading to platelet and fibrin thrombus formation. A number of physiological anticoagulants are also present and modulate this response: antithrombin, TFPI, and activated protein C (APC) and its cofactor protein S. Defects in these hemostatic proteins can lead to disorders that elevate the risk of thrombus formation.

Risk factors for venous thromboembolism are associated with venous stasis or acquired and congenital hypercoagulable states and include obesity, smoking, malignancy, pregnancy, hormone therapy, and recent trauma or surgery. Immobilization due to prolonged hospitalization following surgical intervention, and during long-distance air travel also contribute to the risk of VTE.

Genetic risk factors associated with increased risk of VTE include mutations in factor V (Leiden) and prothrombin 20210, as well as mutations leading to deficiencies in antithrombin, protein C, and protein S. Approximately 5% of the Caucasian population has at least one mutation for factor V Leiden, and 15% to 20% of patients who present with a VTE carry the mutation.²^–⁵ Approximately 2% of the population carry the prothrombin gene mutation, but it may be present in approximately 5% to 15% of persons with VTE.⁶ The population frequencies of mutations in other genes responsible for other coagulation factors (e.g., protein C) are estimated to be 1 in 500 individuals. Antithrombin III deficiency is associated with a frequency of 1 in 300 in the general population, and in 3% to 5% of those with thrombotic events. Previously it was thought that genetic mutations in genes important for methylene tetrahydrofolate reductase and hyperhomocysteinemia increased the risk of VTEs; however, recently this association has been shown to be less likely.⁷

One of the acquired risk factors known to be important in both venous and arterial thrombosis is acquisition of antiphospholipid antibodies, which represent a family of antibodies against phospholipids (e.g., cardiolipins) and phospholipid binding proteins (e.g., GpI β₂). Mechanisms responsible for thrombosis are still speculative but may include inhibition of protein C, antithrombin, and annexin A₅ expression; binding and activation of platelets; enhanced EC tissue factor expression; and activation of the complement cascade.⁸ Criteria for diagnosis of the associated disorder, antiphospholipid syndrome, includes the presence of both clinical events and laboratory evidence for the presence of antiphospholipid antibodies.⁹

Arterial Thrombosis

Arterial thrombosis, which accounts for MI and thrombotic cerebrovascular events, initiates from damage to the vessel wall, leading to a cascade of platelet-mediated cellular interactions. Endothelial injury resulting from plaque rupture with exposure of subendothelial collagen and vWF is the core event that leads to arterial thrombosis. Platelet activation through direct interaction with exposed collagen or thrombin generated by tissue factor leads to thrombus formation. Important in this process are platelet factors that are released with activation, including ADP, serotonin, and TxA. Recruitment of additional platelets leads to thrombus growth as platelets aggregate through bridges formed from the binding of fibrinogen to the platelet receptor GpIIb-IIIa. Recruitment and activation of platelets is modulated by a tightly regulated process that involves factors released from the platelet as well as the endothelium, including prostacyclin, NO, and ecto-AD (T)Pase. Prostacyclin, which is generated from arachidonic acid by the endothelium works through cyclic adenosine monophosphate (cAMP) to inhibit platelet function. Nitric oxide, through stimulation of the soluble guanylyl cyclase to produce cyclic guanosine monophosphate (cGMP), directly inhibits platelet activation and prevents thrombosis; cGMP signaling cascades lead to a decrease in fibrinogen binding to GpIIb-IIIa and inhibits the phospholipase A₂ and C pathways.

A primary mechanism of arterial thrombosis is rupture of atherosclerotic plaques, precipitating platelet-rich aggregates. Arterial thrombosis can have catastrophic consequences when it occurs in the coronary or carotid artery circulation. Factors that can exacerbate these types of thrombotic events include smoking, diabetes, hypertension, and hyperlipidemia. Thrombosis generally occurs when there is disruption in the hemostatic balance that results when pro- and anticoagulant molecules are at disequilibrium. Endothelial damage shifts this balance towards a more procoagulant force, leading to exposure of collagen and tissue factor. Collagen that is now exposed can activate platelets in the blood flowing through the vessel, and concomitantly thrombin is generated as the coagulation cascade is initiated in the presence of tissue factor. Genetic modifications of proteins important in coagulation can alter this process, creating a propensity to form thrombi in the arterial system (Box 10-1). These mutations affect platelet function, leading to increased propensity to aggregation.

Polymorphisms in the endothelial nitric oxide synthase (eNOS) gene, which is essential for NO production by ECs, have been described. The 894-G/T polymorphism I exon 7 results in a glutamate-to-aspartate change at position 298. This polymorphism is associated with increased levels of nitrogen oxides that increase risk of hypertension, MI, and stroke in patients who are homozygous for the abnormality.^10,¹¹ A unique polymorphism in the promoter of the glutathione peroxidase-3 gene has been associated with thrombotic strokes in children. Genome-wide associations have identified other loci associated with cardiovascular thrombotic disease.¹²

Increased levels of fibrinogen have been associated with an increased risk of MI, ischemic stroke, and peripheral artery disease.¹³ Age, elevated lipids, and smoking increase the risk associated with fibrinogen. β-Chain variants, such as Arg448Lys, BclI, -148 C/T, -455 G/A, and -854 G/A, with the -455 G/A polymorphism, is present in 10% to 20% of the population and is associated with a significant rise in fibrinogen levels.¹⁴ Studies, however, have not been consistent, and the association between this polymorphism and risk of arterial thrombosis is not established.¹⁵ Another site of potential polymorphisms in the fibrinogen gene is the Thr312Ala substitution in the α chain. When this polymorphism is present, the fibrin stranding is thicker, and there is increased cross-linking that may predispose to an increased thrombotic risk.¹⁶

Other potential associations between arterial thrombosis and increased risk include hyperhomocysteinemia, elevated C-reactive protein (CRP), factor VII polymorphisms, increased plasminogen activator inhibitor (PAI)-1, and platelet hyperreactivity. Wald et al. performed a meta-analysis of 72 prospective cohort studies focusing on a mutation in the methylenetetrahydrofolate reductase (MTHFR) C677T gene and the occurrence of various thrombotic events, including stroke and cardiovascular disease, and found a mild association between the mutation and risk of arterial events.¹⁷ Other factors including CRP, factor VII, and PAI-1 have shown even less promising results.¹⁴

Drugs that modulate arterial thrombosis

Since arterial thrombi are mainly composed of platelets, and platelet activation is key to their formation, manipulation of platelet function is central to preventing arterial thrombosis. The most commonly used drugs that effect platelet function include aspirin, thienopyridines (clopidogrel and ticlopidine), dipyridamole, and GPIIb-IIIa antagonists.

Aspirin’s mechanism of action is to irreversibly inhibit acetylation at serine-529 in the active site of platelet cyclooxygenase (COX)-1. In this manner, the COX-1 enzyme is unable to interact with arachidonic acid, so generation of prostaglandin H₂ and TxA are inhibited. Thromboxane A₂ plays an essential role in regulating platelet activation and aggregation upon stimulation with platelet agonists, including collagen, thrombin, and ADP. When TxA binds to its receptor, phospholipase C is activated, and intracellular calcium increases; this leads to amplification of platelet aggregation and feedback-dependent release of more ADP and TxA. Aspirin functions by producing a dose-dependent inhibition of COX, which prevents the described feedback loop leading to platelet aggregation.

Use of aspirin to prevent arterial thrombosis was first established in the ISIS-2 trial in which aspirin was shown to reduce the mortality rate associated with MI. Other studies showed that aspirin resulted in a 25% relative risk reduction from all vascular-associated events, including MI and stroke, and the benefit occurred with treatment with low-dose aspirin.¹⁸ Although the half-life of aspirin is only 20 minutes, and inhibitory effects of aspirin on COX occur as quickly as 5 minutes after administration, irreversible inhibition of COX ensures that its effects are preserved for the lifespan of the platelet (7-10 days) such that COX activity does not return to normal levels until a new generation of platelets is produced. Interestingly, aspirin’s inhibitory properties appear to be most effective when exposed to weak platelet agonists (e.g., TxA, ADP), whereas exposure to stronger agonists (e.g., thrombin) leaves platelet function, as measured by aggregation, intact. For this reason, many of the essential functions of platelets, such as platelet adhesion to vWF or activation by thrombin, are not inhibited by aspirin. Aspirin resistance may explain some of the clinical failure seen with its use. Two recent meta-analyses regarding aspirin resistance have shown that laboratory evidence of unresponsiveness to aspirin may be associated with a high risk of recurrent thrombotic cardiovascular events.^19,²⁰

Another important modulator of platelet function is ADP, which acts as a weak platelet agonist through two different platelet membrane receptors, P2Y1 and P2Y12.²¹ When ADP stimulates the P2Y1 receptor, the platelet undergoes a shape change and Ca^2 + is mobilized through activation of phospholipase C to initiate platelet aggregation in a reversible manner. The P2Y12 receptor is essential for secretion and stabilization of platelet aggregation by lowering cAMP levels.

There are two available thienopyridine derivatives that act as inhibitors of ADP-induced platelet aggregation: ticlopidine and clopidogrel. Clopidogrel is metabolized by cytochrome P450 (CYP450) into an active metabolite that irreversibly blocks the P2Y12 receptor. Clopidogrel reduces recurrent thrombotic events in patients with cardiovascular disease.²²^–²⁴ The Clopidogrel vs. Aspirin in Patients at Risk of Ischemic Events (CAPRIE) trial found that clopidogrel is more effective than aspirin in reducing the risk of cardiovascular events in patients with recent MI, recent ischemic stroke, and established peripheral artery disease. Ticlopidine is also metabolized by CYP450 to an active metabolite that functions to block the PGY12 receptor. Although it functions in a similar manner to clopidogrel, it is associated with a higher degree of neutropenia and thrombotic thrombocytopenic purpura and is therefore not used as readily as clopidogrel.^25,²⁶ Other antiplatelet drugs in clinical development include ticagrelor, cangrelor, and elinogrel, all of which reversibly inhibit the P2Y12 receptor.²⁷

Another class of drugs in the armamentarium of antithrombotic agents is that which specifically targets the GpIIb-IIIa receptor to prevent the binding of fibrinogen essential for platelet aggregation. This approach blocks platelet aggregation independently of the platelet agonist; GPIIb-IIIa activation is a final common pathway for almost all platelet agonists. The GPIIb-IIIa receptor is the most abundant receptor on platelets and is also found on the surface of megakaryocytes. Under normal circumstances, this Gp receptor is inactive, but when platelets become activated, a signal transduction cascade is initiated that leads to a conformational change and activation of the receptor, allowing it to bind to fibrinogen or, if high-shear conditions are in place, vWF. Binding is mediated by the Arg-Gly-Asp (RGD) and the Lys-Gly-Asp (KGD) sequences in both macromolecules. Drugs that inhibit GPIIb-IIIa include abciximab, eptifibatide, and tirofiban. Abciximab is derived from a murine monoclonal antibody and was one of the first GpIIb-IIIa receptor antagonists to be developed. It functions as a high-affinity antibody to inhibit platelet function through binding to the GpIIb-IIIa receptor, thereby blocking fibrinogen from interacting with its binding site. Abciximab also binds to integrins in the vitronectin receptor, as well as MAC1 and CD11b-CD18, although the clinical significance of these interactions is not understood. Eptifibatide is a cyclic heptapeptide which binds to GPIIb-IIIa through the KGD motif. Tirofiban is a tyrosine derivative that functions as an RGD mimetic.

These drugs work on the final common pathway of platelet aggregation to inhibit binding to fibrinogen in a similar manner to abciximab. They also appear to have anticoagulant activity because there is evidence of prolongation of the activated clotting time. These actions are thought to be regulated by inhibition of thrombin generation via tissue factor and a decrease in microparticle formation. Multiple clinical trials have shown that inhibition of GpIIb-IIIa is effective in preventing recurrent thrombotic events. Use of these drugs leads to a 35% decrease in acute ischemic events and a 26% decrease in recurrent events within 6 months. Long-term use of these drugs was shown to be efficacious in the EPILOG (Evaluation of PTCA to Improve Long-Term Outcome by c7E3 GpIIb-IIIa Receptor Blockade) trial, with a reduction in the incidence of death. Other trials have supported use of GpIIb-IIIa blockade in management of acute coronary syndromes (ACSs).^28,²⁹ Their use, however, has been reserved for high-risk circumstances such as percutaneous coronary intervention (PCI) in ACSs, owing to the recent finding of long-term benefits of ADP receptor antagonists. ³⁰

Oral GpIIb-IIIa inhibitors have not been shown to limit cardiovascular events to date.³¹ This may be due to conformational changes in the GpIIb-IIIa receptor after antagonist dissociation from it. In this case, the receptor remains active and increases binding to fibrinogen and vWF, leading to a paradoxical thrombotic effect. Novel GpIIb-IIIa antagonists that do cause such conformational changes are under development. In one study, RUC-1, which is a novel compound discovered through high-throughput screening, induced partial exposure of the binding site yet still led to decreased platelet aggregation without enhanced fibrinogen binding. RUC-1 may represent a prototype molecule for these types of derivative drugs.³²

Other antiplatelet agents that may inhibit thrombus formation yet preserve hemostasis so bleeding complications do not result are under investigation. One potential new therapeutic target is the GPIb-V-IX complex. Initial studies of patients who have Bernard-Soulier’s syndrome identified a deficiency of the GPIb complex. The GPIb complex is important for building a platelet bridge through vWF at areas of endothelial damage. Drugs under development act as antagonists for the GPIb-vWF interaction, including specific monoclonal antibodies, the GpIb complex antagonists isolated from snake venoms,^33,³⁴ and the Fab fragment of 6B4, which is a murine monoclonal antibody that targets human GPIb and prevents binding to vWF. Early nonhuman primate studies have suggested that thrombus formation can be attenuated using these drugs.³⁵

Inflammation and Thrombosis

Recent evidence has clearly established a role for inflammation in the atherothrombotic process. Patients with ACSs have increased interactions between platelets and leukocytes forming detectable aggregates. The process of inflammation involves a variety of cell types, including leukocytes, ECs, and platelets. The endothelium becomes activated, and multiple cell-adhesion molecules are released, including P-selectin, which essentially has two important roles in inflammation: recruitment of proinflammatory cells and establishing signaling cascades leading to increased expression of CD11b/CD18 (MAC-1).³⁶

Platelets are instrumental in the inflammatory aspects of atherosclerosis. Thrombin activation of platelets leads to release of many procoagulant molecules and release of inflammatory molecules, including platelet factor 4 (PF4), platelet-derived growth factor (PDGF), and RANTES, which is regulated upon activation of normal T-cell expression. In addition, platelets that have been activated in the presence of thrombin secrete CD40 ligand (CD40L). This chemoattractant is key to the recruitment of ECs, smooth muscle cells (SMCs), and macrophages. CD40 ligand also recruits a variety of proinflammatory cytokines (e.g., interleukin [IL]-1, IL-6, and IL-8) and increases expression of the adhesion molecules intercellular adhesion molecule (ICAM)-1, vascular (V)CAM-1, and P-selectin.³⁷ CD40 ligand is also important for release of matrix metalloproteinases (MMPs), which are needed for plaque progression, neovascularization, and plaque rupture. CD40 ligand also initiates release of tissue factor, which then interacts with other cells to create a thrombogenic microenvironment. T lymphocytes orchestrate an inflammatory cascade that begins by binding to VCAM-1 through signaling regulated by interferon (IFN)-γ-inducible chemokine ligands (CXCLs), protein-10, and chemoattractant (I-TAC). Through this binding, a number of inflammatory cytokines are released, including the CD40 ligand; CD154, which leads to metalloproteinase generation; and tissue factor expression, which initiates the coagulation cascade. Mice that lack the CD40L have less atherothrombosis. Patients with ACSs have elevated levels of CD40L, and plasma levels of CD40L predict the risk of future cardiovascular events.³⁸^–⁴⁰ It has been shown that elevated soluble CD40L levels can be decreased by treatment with abciximab.⁴¹

Our understanding of the role of inflammation in the thrombotic response has been expanded by many clinical studies that have shown an association between bacterial infections and increased risk of MI or stroke, although more studies are needed to prove this association.^42,⁴³ One possible mechanism is through Toll-like receptors (TLRs), which are present in platelets. Platelet activation through stimulation of TLR2 activates signaling mechanisms responsible for both thrombotic and inflammatory responses. These effects are responsible for the mechanism by which bacteria induce a proinflammatory cascade in platelets, suggesting that bacteria can directly activate platelet-dependent thrombotic responses.⁴⁴ Recently this process was further refined by demonstrating that TLR2 stimulation leads to platelet activation through PI3-kinase, which is known to be important in platelet activation–associated shape change, calcium release, and granular content secretion.⁴⁵

In summary, as our understanding of hemostasis and thrombosis continues to evolve, so does development of novel agents directed at treating thromboembolic diseases. This development continues to be driven by scientific discovery, growing numbers of patients, and indications for antithrombotics. Targets for antiplatelet drugs continue to be defined, leading to novel therapies as well as development of additional agents in already successful classes.

References

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3 Rosendaal F.R. Venous thrombosis: the role of genes, environment, and behavior. Hematology Am Soc Hematol Educ Program. 2005:1–12.

4 Cushman M. Inherited risk factors for venous thrombosis. Hematology Am Soc Hematol Educ Program. 2005:452–457.

5 Vossen C.Y., Conard J., Fontcuberta J., et al. Risk of a first venous thrombotic event in carriers of a familial thrombophilic defect. The European Prospective Cohort on Thrombophilia (EPCOT). J Thromb Haemost. 2005;3(3):459–464.

6 Kottke-Marchant K. Genetic polymorphisms associated with venous and arterial thrombosis: an overview. Arch Pathol Lab Med. 2002;126(3):295–304.

7 Ray J.G. Hyperhomocysteinemia: no longer a consideration in the management of venous thromboembolism. Curr Opin Pulm Med. 2008;14(5):369–373.

8 Lim W. Antiphospholipid antibody syndrome. Hematology Am Soc Hematol Educ Program. 2009:233–239.

9 Cohen D., Berger S.P., Steup-Beekman G.M., et al. Diagnosis and management of the antiphospholipid syndrome. BMJ. 2010;340:c2541.

10 Elbaz A., Poirier O., Moulin T., et al. Association between the Glu298Asp polymorphism in the endothelial constitutive nitric oxide synthase gene and brain infarction. The GENIC Investigators. Stroke. 2000;31(7):1634–1639.