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Keywords:

  • anticoagulant;
  • antithrombin;
  • fibrinogen;
  • meizothrombin;
  • protease activated receptors

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structure and Synthesis
  5. Coagulation and Fibrin Clot Formation
  6. Anticoagulation and Fibrinolysis
  7. Thrombin Receptors
  8. Inflammation and Tissue Repair
  9. Platelet and Endothelial Effects
  10. Thrombin and Neoplasia
  11. Thrombin and the Musculoskeletal System
  12. Thrombin and the Nervous System
  13. Thrombin and Disseminated Intravascular Coagulation
  14. Antithrombotics
  15. Conclusion
  16. References

Objectives – To review the role of thrombin in physiology and clinical disease and to discuss the pharmacology of antithrombosis.

Data Sources – Original research articles, scientific reviews, textbooks.

Human Data Synthesis – Thrombin and thrombin receptors are involved in a variety of physiologic and pathologic processes resulting in a great deal of interest in thrombin-related pharmacologic intervention.

Veterinary Data Synthesis – Although there is little clinical research data available on thrombin specifically in veterinary patients, some of the original research on protease activated receptors was performed at veterinary institutions and many of the human molecular biology studies have been done on animals including dogs.

Conclusion – Thrombin plays a significant role in coagulation, anticoagulation, and fibrinolysis. Antithrombotic treatment is focused on preventing thrombosis while maintaining hemostasis. Pharmaceutical agents are selected for the specific component of the coagulation pathway associated with a specific disease process, for a proven prophylactic benefit with procedures that carry a risk of thromboembolism, for rapidity of onset and ease of reversibility, for limited monitoring requirements, and for oral formulation and bioavailablity. Recent insight into other aspects of thrombin physiology presents an opportunity for pharmacologic intervention in a variety of other processes such as inflammation and sepsis, peripheral blood cell activation and chemotaxis, vascular endothelial and smooth muscle activity, cellular development and tissue repair, mitogenesis, neoplasia, and the function of nervous tissue following injury.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structure and Synthesis
  5. Coagulation and Fibrin Clot Formation
  6. Anticoagulation and Fibrinolysis
  7. Thrombin Receptors
  8. Inflammation and Tissue Repair
  9. Platelet and Endothelial Effects
  10. Thrombin and Neoplasia
  11. Thrombin and the Musculoskeletal System
  12. Thrombin and the Nervous System
  13. Thrombin and Disseminated Intravascular Coagulation
  14. Antithrombotics
  15. Conclusion
  16. References

Thrombin is essential for homeostasis in many physiologic systems. Its central role in coagulation is well-known; however, it is also involved in anticoagulation and fibrinolysis, tissue repair and wound healing, platelet and endothelial cell activation, the progression of neoplasia, and inflammation. Virtually every cell type examined experimentally exhibits some physiologic response to thrombin.1

Structure and Synthesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structure and Synthesis
  5. Coagulation and Fibrin Clot Formation
  6. Anticoagulation and Fibrinolysis
  7. Thrombin Receptors
  8. Inflammation and Tissue Repair
  9. Platelet and Endothelial Effects
  10. Thrombin and Neoplasia
  11. Thrombin and the Musculoskeletal System
  12. Thrombin and the Nervous System
  13. Thrombin and Disseminated Intravascular Coagulation
  14. Antithrombotics
  15. Conclusion
  16. References

Thrombin is a 36,000 Da molecule that is formed when the zymogen prothrombin is cleaved by a prothrombinase complex that forms in the presence of calcium from platelet phospholipids, FVa, and FXa. Prothrombin is a 72,000 Da single chain protein produced in the liver and at much lower levels in other tissues such as the brain, diaphragm, kidney, spleen, and intestine.2 Thrombin, also known as FIIa, and prothrombin, known as FII, are serine proteases. They are members of the family of vitamin K-dependent coagulation factors characterized by an NH2-terminal domain of γ-carboxyglutamic acid (Gla). This domain is formed from glutamic acid in the presence of a vitamin K-dependent carboxylase that is required for conversion to Gla. Other similar proteins with Gla domains include FVII, FX, and protein C (PC).3 The Gla domain mediates the mineral and membrane binding capabilities of thrombin and prothrombin. Calcium ion binding induces the conformational changes necessary for prothrombin to bind to the phospholipids of cell membranes. The membrane surface is a structural element of the prothrombinase complex and also functions to position the prothrombin substrate to enhance enzymatic activity.4

Coumarin compounds, including some of the anticoagulant rodenticides, deplete intracellular stores of the reduced form of vitamin K. This limits the γ-carboxylation of glutamic acid to Gla, decreases the affinity of thrombin and prothrombin for calcium and for membrane binding, and thereby interferes with coagulation.5

Thrombin is composed of 2 polypeptide chains covalently linked by a single disulfide bond. The smaller A-chain has no documented functional role; however, the B-chain contains a number of molecular binding sites. Four functional binding sites have been identified: (1) a sodium binding site, (2) an active site, (3) exosite I, and (4) exosite II. The sodium binding site is highly conserved in thrombins of different species6 and plays a major role in determining if thrombin will have a procoagulant or anticoagulant effect. The binding of a sodium ion allosterically modulates the function of thrombin and creates a structure that favors binding with procoagulant substrates including fibrinogen, FV, and FVIII.7 In the absence of sodium, thrombin has increased specificity for PC and the primary activity is anticoagulant.8

Exosite I is an electropositive anion-binding site responsible for binding fibrinogen. This site is also important in that it binds the COOH terminal domain of hirudin that is the most potent natural inhibitor of thrombin. Hirudin was first recognized in the saliva of the medicinal leech, Hirudo medicinalis, and has subsequently become a model for bioengineered anticoagulant medications as it has a very high specificity for thrombin as compared with other serine protease molecules.

Exosite II is also an electropositive anion-binding site found on the opposite side of the thrombin molecule. This is the binding site for highly sulfated polysaccharides such as heparin and for thrombomodulin. The active site works in concert with exosite 1 for the binding of hirudin-like compounds and is the principle site of interaction of substrates such as antithrombin (AT) as well as PC.2

Coagulation and Fibrin Clot Formation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structure and Synthesis
  5. Coagulation and Fibrin Clot Formation
  6. Anticoagulation and Fibrinolysis
  7. Thrombin Receptors
  8. Inflammation and Tissue Repair
  9. Platelet and Endothelial Effects
  10. Thrombin and Neoplasia
  11. Thrombin and the Musculoskeletal System
  12. Thrombin and the Nervous System
  13. Thrombin and Disseminated Intravascular Coagulation
  14. Antithrombotics
  15. Conclusion
  16. References

Normal coagulation and fibrinolysis result when fibrin creation and degradation are properly balanced. This prevents excessive blood loss at the site of vascular disruption while preserving blood flow in normal vasculature. Excessive hemorrhage or thrombosis results from deregulation or depletion of these systems.9

Thrombin plays a central role in the regulation of coagulation and fibrin clot formation (Figure 1). Following endothelial damage, the coagulation cascade is initiated when circulating coagulation FVII contacts tissue factor (TF).10 TF is expressed in relatively high concentrations in the adventitia of the blood vessels, the capsules of organs, and in other vital tissues such as the brain and heart.11 TF is also expressed by subendothelial cells such as fibroblasts and macrophages. These facts support the appealing concept of TF forming a hemostatic envelope surrounding the vascular bed, positioned to respond to vascular injury.12

image

Figure 1.  Thrombin generation. The molecule of thrombin plays a central role within the coagulation cascade. The activation of coagulation proceeds through a stepwise activation of proteases that eventually results in the fibrin framework. After vascular injury, tissue-factor expression by endothelial cells is a critical step in the initial formation of fibrin, whereas the activation of FXI, FIX, and FVIII is important to continue the formation of fibrin. The formation of the clot is highly regulated by natural anticoagulant mechanisms that confine the hemostatic process to the site of the injury to the vessel. Most of these natural anticoagulants are directed against the generation or action of thrombin and include antithrombin and the protein C system. Solid lines denote activation pathways, and dashed lines denote inhibitory pathways. (Adapted from Di Nisio M, Middeldorp S, Buller HR. Direct thrombin inhibitors. N Engl J Med 2005;353:1028–1040. Reprinted with permission.)

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The interaction of TF with FVIIa, Ca2+, and phospholipids culminates in activation of FX to form FXa, and results in the production of a small amount of thrombin and meizothrombin from circulating prothrombin. Meizothrombin is an intermediate that is produced during the conversion of prothrombin to thrombin. It acts on the local adrenergic receptors to cause local vasoconstriction that limits further blood extravasation. Meizothrombin also directly activates platelets and cleaves fibrinogen. The small amount of thrombin produced by FXa feeds back to activate FVIII,13 which results in more FXa production as well as activation of FV. The combination of FVa and FXa, in association with phospholipid and calcium, constitutes the prothrombinase complex that subsequently catalyzes the production of larger quantities of thrombin. The majority of thrombin generation occurs on the surface of activated platelets.7

Blood-borne TF has been identified on microparticles derived from leukocytes and other cell types.14 TF may also be expressed by endothelial cells as well as circulating monocytes and platelets, especially in disease states. The blood-borne TF is involved in thrombus propagation15 as endothelial and subendothelial expressed TF is rapidly incorporated into the clot at the site of vascular injury and is no longer exposed to the vascular lumen. Endothelial and platelet activation by thrombin, histamine and cytokines results in P-selectin adhesion molecule expression by these cells. Subsequently they recruit TF-bearing microparticles expressing the P-selectin glycoprotein ligand-1 and bind them to the region of thrombus formation.16

The most significant role of thrombin in coagulation occurs when it binds soluble fibrinogen at exosite 1 and the active site. Fibrinogen is cleaved to insoluble fibrin monomers that then polymerize spontaneously. The activation of FXIII by thrombin results in the formation of covalent bonds between fibrin glutamate and lysine residues and subsequent cross-linking of the clot. This is why FXIII is also called fibrin-stabilizing factor.

The presence of thrombin has a positive feedback effect on the production of FVa,13 FVIIa,17 FVIIIa,13 and FXIa.18 This positive feedback mechanism results in further thrombin synthesis. In other words, thrombin production results in amplification of the coagulation cascade and additional thrombin production. Thrombin's positive feedback effect on the activation of FXI prevents clinical bleeding in patients with FXII deficiency.19 The absence of clinical bleeding in these patients is attributed to the lack of the dependency of FXIa production on the presence of FXII. In contrast to this example, there is significant risk of hemorrhage associated with FXI deficiency, seen clinically in Kerry Blue Terriers. In patients with sufficient levels of FXI, coagulation initiated by vascular endothelial injury results in TF-VIIa-Ca2+ complex formation with subsequent activation of FIX and FX rapidly followed by inactivation of the TF complex by tissue factor pathway inhibitor (TFPI). The small amount of thrombin produced by this interaction feeds back on the production of FVa, FVIIa, FVIIIa, as well as FXIa, which amplifies the production of thrombin. FXI deficiency results in failure of a portion of this amplification pathway resulting in unstable clots and rebleeding multiple days following injury.

Similarly, patients with hemophilia A (FVIII deficiency) and hemophilia B (FIX deficiency) bleed even though they appear to have an alternative pathway to activation of FX. The explanation for this apparent contradiction is that the catalytic efficiency for FX activation by FVIIIa plus FIXa is far in excess of the catalytic efficiency of activation of FX by TF plus FVIIa20 (Figure 2).

image

Figure 2.  Thrombin activation and inhibition.

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When coagulation is initiated in the presence of a normal endothelium, the activity of thrombin is localized due to its generation being mediated primarily by membrane-associated TF and FVa as well as the membrane-bound Gla domain of prothrombin. Physiologic mechanisms also exist to reduce the risk of systemic activity in the case of endothelial disruption. Thrombin's direct stimulation of vascular smooth muscle and meizothrombin's stimulation of adrenergic receptors causes local vasoconstriction, which limits the risk of widespread thrombosis by reducing blood flow.21 Thrombin also has a negative feedback mechanism on its own production and helps to propagate fibrinolysis.7

Anticoagulation and Fibrinolysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structure and Synthesis
  5. Coagulation and Fibrin Clot Formation
  6. Anticoagulation and Fibrinolysis
  7. Thrombin Receptors
  8. Inflammation and Tissue Repair
  9. Platelet and Endothelial Effects
  10. Thrombin and Neoplasia
  11. Thrombin and the Musculoskeletal System
  12. Thrombin and the Nervous System
  13. Thrombin and Disseminated Intravascular Coagulation
  14. Antithrombotics
  15. Conclusion
  16. References

It is extremely important, once coagulation is triggered at the site of vascular endothelial injury, that there are mechanisms to reduce the advancement of the hemostatic plug into normal vasculature. There are a number of biologic processes mediated by thrombin that promote anticoagulation and fibrinolysis. Thrombomodulin presented by endothelial cells binds to thrombin at exosite 1. This action competitively inhibits fibrinogen binding at exosite 1 and slows fibrin generation resulting in an anticoagulant effect. There is a greater affinity for this reaction when sodium is not present at the sodium-binding site. Thrombin binding to TM also decreases the level of free thrombin (thus inhibiting further platelet and endothelial cell activation), contributes to the activation of PC, and results in the activation of thrombin activatable fibrinolysis inhibitor (TAFI).

Activated protein C (APC) is produced when PC binds to the endothelial PC receptor (EPCR) and interacts with the active site of thrombin-TM that is already bound to the endothelial cell membrane at exosite 12 (Figure 3). APC then associates with Protein S (PS) and catalyzes the inactivation of FVa and FVIIIa. Decreasing FV and FVIII activation by APC and PS directly decreases the rate of thrombin generation and thereby contributes to anticoagulation. APC also promotes fibrinolysis by complexing and inactivating plasminogen activator inhibitor-1 (PAI-1). The role of PAI-1 is the inhibition of plasmin formation and consequent inhibition of clot dissolution or fibrinolysis. Inhibition of this inhibitor of fibrinolysis by APC following thrombin-TM activation, therefore, results in enhanced fibrinolysis or anticoagulation.

image

Figure 3.  Thrombin-thrombomodulin induced activation of the protein C anticoagulation pathway. Thrombin (T) binds to thrombomodulin (TM) and activates protein C (PC), which is reversibly bound to the endothelial cell protein C receptor (EPCR). Activated protein C (APC) binds to protein S and inactivates FVa and FVIIIa. (Adapted from Esmon CT. Coagulation. In: Fink MP AE, Vincent JL, Kochanek PM, eds. Textbook of Critical Care. 5th ed. Philadelphia, PA: Elsevier Saunders; 2005, pp. 165–72. Reprinted with permission.)

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Activation of the APC pathway by thrombin-TM results in accelerated inactivation of FXa and FIXa by AT.22 AT is a vitamin K-dependent glycoprotein and a broad-spectrum inhibitor of thrombin and other coagulation factors including FXa and FIXa. The complexes of FXa-FVa and FIXa-FVIIIa are more resistant to AT inactivation than FXa or FIXa are alone. Activation of the APC pathway leads to the reduced production of FVa and FVIIIa and therefore decreases FXa-FVa and FIXa-FVIIIa complex formation. AT is then free to inactivate the noncomplexed FXa and FIXa. The thrombin-TM complex is inactivated much more quickly than free thrombin by circulating AT, which provides some regulation of this anticoagulant effect.23

The thrombin-TM complex also activates TAFI.24 The activation of TAFI by the thrombin-TM complex couples the phenomenon of coagulation-induced inhibition of fibrinolysis with the profibrinolytic effect of APC, as the primary function of TAFI is to prolong clot lysis (procoagulant). This is accomplished by removing the terminal lysine residues of fibrin that normally facilitate the binding actions of fibrinolytic plasmin and tissue-plasminogen activator.25

Thrombin Receptors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structure and Synthesis
  5. Coagulation and Fibrin Clot Formation
  6. Anticoagulation and Fibrinolysis
  7. Thrombin Receptors
  8. Inflammation and Tissue Repair
  9. Platelet and Endothelial Effects
  10. Thrombin and Neoplasia
  11. Thrombin and the Musculoskeletal System
  12. Thrombin and the Nervous System
  13. Thrombin and Disseminated Intravascular Coagulation
  14. Antithrombotics
  15. Conclusion
  16. References

A great deal of research has been focused on how thrombin exerts its cellular effects. These effects are mediated by protease activated receptors (PARs), a small family of the 7 transmembrane guanine nucleotide binding proteins (G-protein). PARs are highly expressed on platelets. They are also found on endothelial cells, monocytes, fibroblasts, T-lymphocytes, smooth muscle cells, neurons, certain tumor cell lines, and other cell types.26 PAR-1, PAR-3, and PAR-4 are the receptors that are activated by thrombin.21 The unique characteristic of these PARs is they carry their own ligands that remain tethered to the receptor after the receptor is cleaved by proteases including thrombin. The cleavage or activation of the PAR is irreversible. However, the duration of effect is limited by rapid internalization of the receptor and ligand with degradation in cellular lysosomes.27 The PAR is therefore used once and discarded, which may have significant implications when considering pharmacologic intervention (Figure 4).

image

Figure 4.  Scheme of protease-associated receptor-1 (PAR-1) structure and receptor activation. PAR-1 has a seven-transmembrane helix bundle and an additional eighth intracellular C-terminal helix anchored by Cys-palmitoylate to the inner leaflet of the lipid bilayer. Thrombin docks at the extracellular N-terminus of the receptor and irreversibly cleaves the receptor between the Arg and Ser residues (indicated by scissors). Consequently, a new N-terminus is unmasked acting as a tethered ligand. This tethered ligand binds to the second extracellular loop of the receptor, through electrostatic interaction between the basic arginine and the acidic glutamate residue at the second extracellular loop (shown by dotted line), thereby initiating signal transductions. TM, transmembrane helix bundle (Adapted from Luo W, Wang Y, Reiser G. Protease-activated receptors in the brain: receptor expression, activation, and functions in neurodegeneration and neuroprotection. Brain Res Rev 2007;56:331–345. Reprinted with permission.)

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Inflammation and Tissue Repair

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structure and Synthesis
  5. Coagulation and Fibrin Clot Formation
  6. Anticoagulation and Fibrinolysis
  7. Thrombin Receptors
  8. Inflammation and Tissue Repair
  9. Platelet and Endothelial Effects
  10. Thrombin and Neoplasia
  11. Thrombin and the Musculoskeletal System
  12. Thrombin and the Nervous System
  13. Thrombin and Disseminated Intravascular Coagulation
  14. Antithrombotics
  15. Conclusion
  16. References

Thrombin-induced transmembrane signaling occurs via a wide variety of mechanisms, however, the principal mechanism of PAR-1 receptor signaling involves the activation of the Gq G-protein subtype, which activates phospholipase C and the inositol triphosphate and diacylglycerol pathway that leads to an increase in intracytoplasmic calcium and protein activation via phosphorylation. PAR-1 signaling stimulates angiogenesis, cell growth and differentiation, and smooth muscle cell, macrophage, and endothelial cell proliferation.26 These effects are important as they promote healing of injured tissues. Cellular signaling via the PAR and G-protein mechanism is terminated by degradation of the PAR by lysosomal and cell surface proteases as well as by receptor phosphorylation and decoupling from the G-protein.28 The in vitro production of connective tissue growth factor (CTGF) by fibroblasts in response to thrombin via the PAR-1 receptor is one example of this mechanism acting to promote tissue healing. CTGF stimulates fibroblast mitogenesis and chemotaxis and promotes procollagen and fibronectin production by fibroblasts.29

Vascular endothelial growth factor (VEGF) is a specific endothelial cell mitogen that initiates angiogenesis.30 Angiogenesis is enhanced locally in response to tissue injury. Thrombin upregulates VEGF receptors on endothelial cells and potentiates the effects of VEGF.31 Local tissue effects of VEGF include cellular migration, endothelial cell proliferation, vascular tube formation, and positive feedback on the production of additional thrombin.28 Thrombin trapped within a thrombus is protected from inactivation by AT and is slowly released locally during thrombolysis to aid in the process of angiogenesis, tissue repair, and healing.

PAR-1 is the receptor responsible for mediating most of the proinflammatory and profibrotic effects of thrombin including the upregulation of the leukocyte adhesion molecule P-selectin on endothelial cells and platelets. This results in binding, rolling, and eventual attachment of platelets and leukocytes to the endothelial surface. Mitogenesis of lymphocytes, fibroblasts, and smooth muscle cells32 is enhanced by exposure to thrombin. Thrombin-mediated PAR activation has also been linked to the production of chemokines such as platelet activating factor (PAF) and monocyte chemotactic protein (MCP) that recruit inflammatory cells to the site of injury,33 as well as the stimulation of interleukin-6 and interleukin-8 production from monocytes.34,28 Promotion of cellular adhesion and chemotaxis are just a few of the proinflammatory effects of thrombin.

Thrombin affects vasomotor function as well as vascular permeability. Thrombin or PAR-1 agonists will stimulate release of nitric oxide35 from intact vascular endothelium and cause vasodilation; however, with endothelial disruption the result is vasoconstriction.36,37 In addition to this direct effect on the endothelium, thrombin stimulates the release of histamine from mast cells and serotonin from platelets that cause additional vasodilation. These vasoactive substances contribute to the increased vascular permeability, edema, and swelling that are associated with inflammation. Thrombin can enhance this effect directly. In a study evaluating human umbilical vein endothelial cells exposed to thrombin, thrombin caused a rapid contraction of the endothelial cells, exposure of a significant amount of subendothelium, and an increase in vascular permeability.38

Thrombin stimulates production of prostacyclin (PGI2)39 via endothelial cell G-protein induced increase in cytosolic calcium, secondary stimulation of phospholipase A2, and arachadonic acid production with conversion through the prostaglandin H synthase pathway. PGI2 inhibits platelet aggregation and activates protein kinase A. These lead to smooth muscle relaxation and vasodilation by activation of myosin light chain kinase.

Circulation of inflammatory cytokines such as tumor necrosis factor (TNF) has the effect of reducing endothelial expression of TM and EPCR. Neutrophil elastase release from activated neutrophils cleaves TM from the endothelial surface and results in a less active form of TM that is readily damaged by oxidation. This has a procoagulant effect as decreased PC activation leads to decreased inactivation of FVa and FVIIIa. This leads to increased thrombin formation and increased formation of FVa-FXa and FIXa-FVIIIa complexes that are resistant to AT degradation. Analysis of circulating APC in humans with severe sepsis has confirmed the APC levels are reduced.40 This is associated with increased mortality.41

Platelet and Endothelial Effects

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structure and Synthesis
  5. Coagulation and Fibrin Clot Formation
  6. Anticoagulation and Fibrinolysis
  7. Thrombin Receptors
  8. Inflammation and Tissue Repair
  9. Platelet and Endothelial Effects
  10. Thrombin and Neoplasia
  11. Thrombin and the Musculoskeletal System
  12. Thrombin and the Nervous System
  13. Thrombin and Disseminated Intravascular Coagulation
  14. Antithrombotics
  15. Conclusion
  16. References

Thrombin generation and PAR activation regulates endothelial cell and platelet function. The endothelial response to thrombin was some of the first evidence of the relationship between neutrophil adhesion and endothelial cell activation.42 Endothelial stimulation by thrombin results in monocyte and neutrophil chemotaxis by MCP, P-selectin expression on endothelial cells and platelets, and the dysregulated vascular response during critical illness. Exposure of pulmonary artery and microvascular endothelial cells to thrombin in experimental studies results in a dose-dependent intracellular calcium rise and subsequent development of intercellular gap formation.43 Thrombin also signals endothelial cells and vascular smooth muscle cells to control vascular resistance and amplifies the inflammatory response by modulating transendothelial movement of neutrophils and plasma proteins.

Platelets are essential for efficient blood coagulation. Although platelets are activated by many stimuli, thrombin is the most potent of the platelet activators.44 Platelets undergo shape change characterized by spike-like projections in response to thrombin followed by platelet aggregation by means of integrin binding von Willebrand factor and fibrinogen.23 Thrombin also induces synthesis and secretion of adenosine diphosphate, endothelial platelet activating factor, serotonin, and thromboxane A2.

Thrombin stimulates platelet activation through PAR-1 and PAR-4 receptors. The PAR-1 receptor activation results in increased intracellular calcium and upregulation of the glycoprotein IIb/IIIa fibrinogen receptor, as well as mobilization of P-selectin and CD40 ligand to the platelet surface. The result is an increased binding of platelets to fibrinogen and an increase in platelet cross-linking and thrombus formation.27 PAR-4 receptor activation appears to modulate and stabilize interplatelet binding. There is a platelet PAR-3 receptor in primates that has a high affinity for thrombin, but the role of this receptor is uncertain.44

Thrombin and Neoplasia

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structure and Synthesis
  5. Coagulation and Fibrin Clot Formation
  6. Anticoagulation and Fibrinolysis
  7. Thrombin Receptors
  8. Inflammation and Tissue Repair
  9. Platelet and Endothelial Effects
  10. Thrombin and Neoplasia
  11. Thrombin and the Musculoskeletal System
  12. Thrombin and the Nervous System
  13. Thrombin and Disseminated Intravascular Coagulation
  14. Antithrombotics
  15. Conclusion
  16. References

Evidence suggests that thrombin plays an important role in tumor biology and pathogenesis via a coagulation independent mechanism. Thrombin induced proliferation and migration of endothelial cells in response to vascular injury is important for tissue healing. In the context of a neoplastic environment, however, this effect results in angiogenesis within the developing tumor. Thrombin promotes angiogenesis and endothelial proliferation by induction of VEGF secretion and upregulation of the VEGF receptors via PAR activation.30 Treatment of a line of human prostatic cancer cells with thrombin resulted in enhanced VEGF expression.45 Upregulation of the PAR-1 receptor has been documented in a number of cancer cell lines including breast, prostate, and melanoma.46 The increased expression of PAR-1 in breast cancer cells has been correlated with the invasive potential of the tumor.47

Thrombin also induces production of αvβ3 integrin, which is an angiogenic marker in vascular tissue and in tumor cells. The integrins are important membrane proteins that attach cells to the extracellular matrix. They also have ligand binding functions that control cellular attachment or detachment as well as migration, proliferation, and apoptosis. The αvβ3 integrin has been demonstrated to be upregulated by thrombin in human colon adenocarcinoma cells.48 Upregulation of integrins is a mechanism by which thrombin promotes endothelial cell survival following detachment from basement membranes and during migration to the distal sites necessary for angiogenesis associated with tumor progression and metastasis.30,49 Intracellular calcium-sensitive kinases activated by PAR-1 cleavage include mitogen activated protein kinase and extracellular signal regulated kinase. These specific kinases are often expressed in humans with a relatively common genetic mutation of the oncogene Ras.50 These mechanisms suggest a potential role of thrombin inhibitors or PAR blockers in treatment of some forms of neoplasia.

Thrombin and the Musculoskeletal System

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structure and Synthesis
  5. Coagulation and Fibrin Clot Formation
  6. Anticoagulation and Fibrinolysis
  7. Thrombin Receptors
  8. Inflammation and Tissue Repair
  9. Platelet and Endothelial Effects
  10. Thrombin and Neoplasia
  11. Thrombin and the Musculoskeletal System
  12. Thrombin and the Nervous System
  13. Thrombin and Disseminated Intravascular Coagulation
  14. Antithrombotics
  15. Conclusion
  16. References

PARs are expressed in all of the major cell types of bone, cartilage, and muscle. Thrombin-mediated activation of PAR-1 leads to osteoblast, chondrocyte, and myoblast proliferation and there is evidence that these cells, normally quiescent in adults, are activated following trauma or inflammation.51 Thrombin is present in coagulated blood in concentrations adequate to activate their associated PARs and it enhances the establishment of osteogenic cells at the site of bone injury.52 This may be important in the initial stages of fracture repair.

Thrombin and the Nervous System

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structure and Synthesis
  5. Coagulation and Fibrin Clot Formation
  6. Anticoagulation and Fibrinolysis
  7. Thrombin Receptors
  8. Inflammation and Tissue Repair
  9. Platelet and Endothelial Effects
  10. Thrombin and Neoplasia
  11. Thrombin and the Musculoskeletal System
  12. Thrombin and the Nervous System
  13. Thrombin and Disseminated Intravascular Coagulation
  14. Antithrombotics
  15. Conclusion
  16. References

PARs are abundant in the brain and are expressed in neurons, astrocytes, and oligodendrocytes. Recent evidence supports the direct involvement of thrombin-activated PARs in brain development, in brain function including synaptic plasticity and neuroprotection, and in brain pathology such as neurodegeneration.53 Thrombin levels are increased in the brain under certain conditions such as trauma, hemorrhage, and ischemia. Brain endothelial cells are capable of releasing thrombin. The presence of PARs and thrombin in the central nervous sytem suggests regulation of thrombin activity in the future may affect outcome related to CNS disease.

Thrombin is produced in the brain immediately following cerebral hemorrhage or following disruption of the blood brain barrier secondary to inflammation. The source of the thrombin is neurons, astrocytes, resident or migrating inflammatory cells, or from gradual release by hematomas. Protease inhibitors such as TM are present within the CNS and upregulation of these inhibitors by thrombin under normal conditions may serve to control excessive activation of PAR-1 on astrocytes.

Thrombin levels and PAR-1 expression influence cellular survival following insult to astrocytes or neurons. High levels of thrombin have been shown to be detrimental to cell survival in cases of cerebral hemorrhage or ischemia; however, lower thrombin levels can be neuroprotective.54 The pathophysiology of the detrimental effects of high CNS thrombin concentration may be a result of direct cellular toxicity to neural cells, excessive fibroblastic proliferation, potentiation of cerebral edema, exacerbation of glutamate-induced excitotoxicity, or from stimulation of the complement cascade and cytokine release.55 There is debate about the mechanism of protection of low levels of thrombin. Experimentally, intracerebral administration of a low dose of thrombin before inducing intracerebral hemorrhage (thrombin preconditioning) in the rat resulted in reduced brain injury.56 The mechanism of this protective effect may be related to thrombin's ability to control progression of hematoma formation, upregulation of heat shock proteins or thrombin receptors, induced mitogenesis of astrocytes, or induction of thrombin inhibitors such as PAI-1.

Thrombin and Disseminated Intravascular Coagulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structure and Synthesis
  5. Coagulation and Fibrin Clot Formation
  6. Anticoagulation and Fibrinolysis
  7. Thrombin Receptors
  8. Inflammation and Tissue Repair
  9. Platelet and Endothelial Effects
  10. Thrombin and Neoplasia
  11. Thrombin and the Musculoskeletal System
  12. Thrombin and the Nervous System
  13. Thrombin and Disseminated Intravascular Coagulation
  14. Antithrombotics
  15. Conclusion
  16. References

Disseminated intravascular coagulation is a pathologic consumptive thrombo-hemorrhagic disorder characterized by excessive thrombin production variably disseminated throughout the systemic circulation. Activation of endothelial cells and monocytes secondary to a variety of disease processes results in excessive presentation of TF, which stimulates thrombin production via the coagulation cascade. Thrombin induces platelet shape change and aggregation that leads to platelet depletion. Persistent TF expression results in rampant thrombin generation and the coagulopathy seen with disseminated intravascular coagulation.57 PAR-mediated transmembrane signaling results in the exacerbation of systemic inflammatory responses including production of PAF, MCP, TNF, and the interleukins. Fibrinolysis is impeded due to high levels of PAI-1, which reduces the production of plasmin, and high levels of TAFI, which increases the resistance of clots to plasmin-induced fibrinolysis. The anticoagulant mechanisms involving AT, PC, and TFPI are impaired due to the reduced endothelial expression of EPCR and TM secondary to inflammation. The combination of increased formation of fibrin and inadequate removal of fibrin contributes to the disseminated microvascular thrombosis. The subsequent increased concentration of fibrinogen degradation products actively competes with fibrinogen for thrombin exosite I binding and reduces fibrin formation.58 This competitive inhibition of thrombin as well as the consumption of platelets and coagulation factors eventually results in hemorrhagic diathesis.59

Antithrombotics

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structure and Synthesis
  5. Coagulation and Fibrin Clot Formation
  6. Anticoagulation and Fibrinolysis
  7. Thrombin Receptors
  8. Inflammation and Tissue Repair
  9. Platelet and Endothelial Effects
  10. Thrombin and Neoplasia
  11. Thrombin and the Musculoskeletal System
  12. Thrombin and the Nervous System
  13. Thrombin and Disseminated Intravascular Coagulation
  14. Antithrombotics
  15. Conclusion
  16. References

Antithrombotic medications in veterinary medicine have been limited primarily to antiplatelet agents, heparins, and vitamin K antagonists that indirectly antagonize the effects of thrombin. The therapeutic use of vitamin K antagonists and antiplatelet agents are not discussed in this article.

AT inactivates thrombin as well as other factors involved in coagulation. Unfractionated heparin (UFH) induces a conformational change in AT that increases the rate of thrombin inactivation by a factor of a thousand. Circulating thrombin is irreversibly bound in a ternary complex with AT bound to the thrombin active site and heparin bound to AT and the thrombin exosite II. This ternary complex between AT-thrombin-heparin requires a heparin molecule of at least 18 saccharide units.60 UFH has a mean molecular weight of 15,000 Da with a range of 3,000–30,000 Da. AT in the presence of heparin strongly inhibits thrombin, FXa, and FIXa, and weakly inhibits FXIa, FXIIa, and FVIIa as well as trypsin and plasmin. By inactivating thrombin, AT-heparin inhibits thrombin-catalyzed activation of FV and FVIII and reduces fibrin formation and platelet activation. This spectrum of inhibitory activity makes AT a key regulator of coagulation. Heparin clearance is mediated primarily by binding to endothelial and macrophage heparin receptors with a small fraction cleared by the kidneys5 (Figure 5).

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Figure 5.  Mechanism of action of direct thrombin inhibitors (DTIs) as compared with heparin. In the absence of heparin, the rate of thrombin inactivation by antithrombin is relatively low, but after conformational change induced by heparin, antithrombin irreversibly binds to and inhibits the active site of thrombin. Thus, the anticoagulant activity of heparin originates from its ability to generate a ternary heparin-thrombin-antithrombin complex. The activity of DTIs is independent of the presence of antithrombin and is related to the direct interaction of these drugs with the thrombin molecule. Although bivalent DTIs simultaneously bind the exosite 1 and the active site, the univalent drugs in this class interact only with an active site of the enzyme. In the lower panel, the heparin-antithrombin complex cannot bind fibrin-bound thrombin, whereas given their mechanism of action, DTIs can bind to and inhibit the activity of not only soluble thrombin but also thrombin bound to fibrin, as is the case in a blood clot. (Adapted from Di Nisio M, Middeldorp S, Buller HR. Direct thrombin inhibitors. N Engl J Med 2005;353:1028–1040. Reprinted with permission.)

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Low-molecular-weight heparin (LMWH) induces the conformational change necessary to activate AT, but fewer than half of the LMWH molecules are of sufficient size (>18 saccharide units) to bind the exosite II of thrombin while maintaining the AT bond. FXa inhibition is therefore greater than thrombin inhibition with LMWH.61 LMWH has a mean molecular weight of 5,000 Da with a range of 1,000–10,000 Da. The primary advantages of LMWH over UFH include longer half-life permitting less frequent administration, and a more predictable dose response that improves safety and reduces the need for laboratory monitoring.5 The half-life and effective dose differs with different species.62,63

There is some concern with the use of heparin in inflammatory conditions such as sepsis. It has been estimated that there are approximately 500,000 binding sites for AT on each endothelial cell and many of these sites are heparin sulfate proteoglycans. The binding of AT to these sites has been associated with an anti-inflammatory effect mediated by PGI2 release from the endothelium, an effect that is blocked by heparin.64 There have been some sepsis studies that demonstrate improved survival in patients whose AT activity is enhanced with high dose AT therapy instead of heparin administration.65

Newer anticoagulant drugs are designed around several mechanisms: (1) preventing the initiation of coagulation, (2) decreasing thrombin generation, and (3) directly inhibiting thrombin interactions. Newer pharmaceuticals designed to block the initiation of coagulation include tifacogin and NAPc2. Tifacogin is a recombinant TFPI. TFPI inhibits TF-FVIIa complex via a 2 step process. TFPI first binds and inactivates FXa and then complexes with TF-bound FVIIa. This limits the initiation phase of coagulation. The 2 stage process theoretically improves safety because some FXa is produced before factor FVIIa becomes unavailable. In addition, a small amount of thrombin can feed back on FXIa production, which leads to additional FXa production. Thus, the risk of hemorrhage is decreased. The NAPc2 is an anticoagulant peptide found in the hookworm Ancylostoma caninum that is produced in recombinant form by a yeast organism. NAPc2 has a high affinity for FX and FXa and the resulting NAPc2-FXa complex inhibits TF-FVIIa. Tifacogin has not been shown to improve mortality in severe sepsis but is currently being evaluated in patients with community-acquired pneumonia.66 NAPc2 is being evaluated for use in prophylaxis of venous and arterial thromboembolism.67 Synthetic pentasaccharides such as fondaparinux and idraparinux have been developed to target FXa but not thrombin directly.68 These drugs are currently being used for prophylaxis and treatment of venous thromboembolism in high-risk orthopedic patients.

Drugs classified as direct thrombin inhibitors (DTI) bind to thrombin and prevent interaction with its substrates. These compounds have the potential to prevent and treat arterial and venous thromboembolism more effectively because they inactivate fibrin-bound thrombin as well as circulating thrombin. They may also have a more predictable effect than heparins because they are not protein bound and unlike heparins, they act independently from AT. They are also not inactivated by platelet factor-4.69 By reducing thrombin mediated platelet activation, DTI also have antiplatelet effects.70

DTI are classified as univalent or bivalent (Figure 6). The univalent drugs bind only to the active site of thrombin and include argatroban, melagatran, and dabigatran. The bivalent compounds bind to both the active site and exosite I of thrombin and include hirudin, bivalirudin, lepirudin, and desirudin.

image

Figure 6.  Targets of anticoagulant drugs. (Adapted from Mackman N. Triggers, targets and treatments for thrombosis. Nature 2008;451:914–918. Reprinted with permission.)

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The US Food and Drug Administration has approved the use of argatroban in humans for heparin-induced thrombocytopenia as well as in percutaneous coronary artery interventions. Argatroban is a synthetic DTI derived from l-arginine, which reversibly binds the active site of both free and clot-associated thrombin and prevents thrombin reactions with other substrates. Bivalirudin has been approved for use as an alternative to heparin with percutaneous coronary interventions. This is a recombinant hirudin that binds to both the active site and the exosite 1 of thrombin. It produces a transient and reversible inhibition of thrombin. Another recombinant hirudin is desirudin, which is used for prophylaxis of venous thromboembolism following hip replacement.71

DTI have been shown to reduce brain edema near hematomas following intracranial hemorrhage in animals and have been used in human clinical trials to provide safe anticoagulation in acute ischemic stroke.72,73 Ximelagatran initially showed promise because it is an oral drug with a high affinity for the active site of thrombin. Its small molecular mass permits penetration into clots to reach fibrin-bound thrombin and the effects are reversible. The compound was abandoned in 2006 due to risk of hepatopathy. There is a low-molecular-weight DTI, dabigatran etexilate that has recently been approved for use in humans by the European Commission (March 2008). It appears to have many of the biologic advantages of ximelagatran without the risk of hepatopathy as it is primarily excreted by the kidney and not metabolized by the liver to any significant degree.

Another pharmaceutical approach to control thrombosis involves the development of PAR antagonists. A clinically useful PAR antagonist would selectively block the inflammatory and thrombotic effects of thrombin by blocking PAR activation without adversely affecting the APC pathway and without inhibiting fibrin generation. There is a PAR-1 thrombin receptor antagonist SCH530348 that has recently received fast track US FDA approval for a phase III clinical trial.74 A class of compounds called pepducins is being developed to penetrate the cell and bind to the receptor G-protein interface on the inner cell membrane. They can be designed to exhibit agonist or antagonist effects and have shown some benefits in experimental models of neoplasia, inflammation, and sepsis.44

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structure and Synthesis
  5. Coagulation and Fibrin Clot Formation
  6. Anticoagulation and Fibrinolysis
  7. Thrombin Receptors
  8. Inflammation and Tissue Repair
  9. Platelet and Endothelial Effects
  10. Thrombin and Neoplasia
  11. Thrombin and the Musculoskeletal System
  12. Thrombin and the Nervous System
  13. Thrombin and Disseminated Intravascular Coagulation
  14. Antithrombotics
  15. Conclusion
  16. References

Thrombin plays a significant role in coagulation, anticoagulation, and fibrinolysis. Antithrombotic treatment is focused on preventing thrombosis while maintaining hemostasis. Pharmaceutical agents are selected for the specific component of the coagulation pathway associated with a specific disease process, for a proven prophylactic benefit with procedures that carry a risk of thromboembolism, for rapidity of onset and ease of reversibility, for limited monitoring requirements, and for oral formulation and bioavailability. Recent insight into other aspects of thrombin physiology presents an opportunity for pharmacologic intervention in a variety of other processes such as inflammation and sepsis, peripheral blood cell activation and chemotaxis, vascular endothelial and smooth muscle activity, cellular development and tissue repair, mitogenesis, neoplasia, and the function of nervous tissue following injury.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structure and Synthesis
  5. Coagulation and Fibrin Clot Formation
  6. Anticoagulation and Fibrinolysis
  7. Thrombin Receptors
  8. Inflammation and Tissue Repair
  9. Platelet and Endothelial Effects
  10. Thrombin and Neoplasia
  11. Thrombin and the Musculoskeletal System
  12. Thrombin and the Nervous System
  13. Thrombin and Disseminated Intravascular Coagulation
  14. Antithrombotics
  15. Conclusion
  16. References