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Sites of action of the four major physiologic antithrombotic pathways: antithrombin (AT); protein C/S (PC/PS); tissue factor pathway inhibitor (TFPI); and the fibrinolytic system, consisting of plasminogen, plasminogen activator (PA), and plasmin. PT, prothrombin; Th, thrombin; FDP, fibrin(ogen) degradation products. (Modified from BA Konkle, AI Schafer, in DP Zipes et al [eds]: Braunwald’s Heart Disease, 7th ed. Philadelphia, Saunders, 2005.)
Antithrombin (AT)
Antithrombin (AT) is a small protein molecule that inactivates several enzymes of the coagulation system. Antithrombin is a glycoprotein produced by the liver and consists of 432 amino acids. It contains three disulfide bonds and a total of four possible glycosylation sites. α-Antithrombin is the dominant form of antithrombin found in blood plasma and has an oligosaccharide occupying each of its four glycosylation sites. A single glycosylation site remains consistently un-occupied in the minor form of antithrombin, β-antithrombin.[3] Its activity is increased manyfold by the anticoagulant drug heparin, which enhances the binding of antithrombin to factor IIa and factor Xa.[4]
Antithrombin (or antithrombin III) is the major plasma protease inhibitor of thrombin and the other clotting factors in coagulation. Antithrombin neutralizes thrombin and other activated coagulation factors by forming a complex between the active site of the enzyme and the reactive center of antithrombin. The rate of formation of these inactivating complexes increases by a factor of several thousand in the presence of heparin. Antithrombin inactivation of thrombin and other activated clotting factors occurs physiologically on vascular surfaces, where glycosoaminoglycans, including heparan sulfates, are present to catalyze these reactions. Inherited quantitative or qualitative deficiencies of antithrombin lead to a lifelong predisposition to venous thromboembolism.
Protein C is a plasma glycoprotein that becomes an anticoagulant when it is activated by thrombin. The thrombin-induced activation of protein C occurs physiologically on thrombomodulin, a transmembrane proteoglycan-binding site for thrombin on endothelial cell surfaces. The binding of protein C to its receptor on endothelial cells places it in proximity to the thrombin-thrombomodulin complex, thereby enhancing its activation efficiency. Activated protein C acts as an anticoagulant by cleaving and inactivating activated factors V and VIII. This reaction is accelerated by a cofactor, protein S, which, like protein C, is a glycoprotein that undergoes vitamin K–dependent posttranslational modification. Quantitative or qualitative deficiencies of protein C or protein S, or resistance to the action of activated protein C by a specific mutation at its target cleavage site in factor Va (factor V Leiden), lead to hypercoagulable states.
Tissue factor pathway inhibitor (TFPI) is a plasma protease inhibitor that regulates the TF-induced extrinsic pathway of coagulation. TFPI inhibits the TF/factor VIIa/factor Xa complex, essentially turning off the TF/factor VIIa initiation of coagulation, which then becomes dependent on the “amplification loop” via factor XI and factor VIII activation by thrombin. TFPI is bound to lipoprotein and can also be released by heparin from endothelial cells, where it is bound to glycosaminoglycans, and from platelets. The heparin-mediated release of TFPI may play a role in the anticoagulant effects of unfractionated and low-molecular-weight heparins.
Fibrinolysis.
None
On vascular injury, platelets adhere to the site of injury, usually the denuded vascular intimal surface. Platelet adhesion is mediated primarily by Von Willebrand factor (VWF), a large multimeric protein present in both plasma and the extracellular matrix of the subendothelial vessel wall, which serves as the primary “molecular glue,” providing sufficient strength to withstand the high levels of shear stress that would tend to detach them with the flow of blood.
Platelet adhesion is also facilitated by direct binding to subendothelial collagen through specific platelet membrane collagen receptors.
Platelet adhesion results in subsequent platelet activation and aggregation. This process is enhanced and amplified by humoral mediators in plasma (e.g., epinephrine, thrombin); mediators released from activated platelets (e.g., adenosine diphosphate, serotonin); and vessel wall extracellular matrix constituents that come in contact with adherent platelets (e.g., collagen, VWF). Activated platelets undergo the release reaction, during which they secrete contents that further promote aggregation and inhibit the naturally anticoagulant endothelial cell factors. During platelet aggregation (platelet-platelet interaction), additional platelets are recruited from the circulation to the site of vascular injury, leading to the formation of an occlusive platelet thrombus. The platelet plug is anchored and stabilized by the developing fibrin mesh.
The platelet glycoprotein (Gp) IIb/IIIa (αIIbβ3) complex is the most abundant receptor on the platelet surface. Platelet activation converts the normally inactive Gp IIb/IIIa receptor into an active receptor, enabling binding to fibrinogen and VWF. Because the surface of each platelet has about 50,000 Gp IIb/IIIa–binding sites, numerous activated platelets recruited to the site of vascular injury can rapidly form an occlusive aggregate by means of a dense network of intercellular fibrinogen bridges. Because this receptor is the key mediator of platelet aggregation, it has become an effective target for antiplatelet therapy.
Thrombin, formed during coagulation at the same site, causes further platelet activation.
In the presence of fibrinogen and/or von Willebrand factor, aggregate to form the hemostatic plug (in hemostasis) or thrombus (in thrombosis).
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- Thromboxane, which causes vasoconstriction and potentiates platelet adhesion and aggregation.
Site of action of antiplatelet drugs. Aspirin inhibits thromboxane A2 (TXA2) synthesis by irreversibly acetylating cyclooxygenase-1 (COX-1). Reduced TXA2 release attenuates platelet activation and recruitment to the site of vascular injury. Ticlopidine, clopidogrel, and prasugrel irreversibly block P2Y12, a key adenosine diphosphate (ADP) receptor on the platelet surface; cangrelor and ticagrelor are reversible inhibitors of P2Y12. Abciximab, eptifibatide, and tirofiban inhibit the final common pathway of platelet aggregation by blocking fibrinogen and von Willebrand factor (vWF) binding to activated glycoprotein (GP) IIb/IIIa. SCH530348 and E5555 inhibit thrombin-mediated platelet activation by targeting protease-activated receptor-1 (PAR-1), the major thrombin receptor on human platelets. (Reproduced with permission from Longo DL, et al. Harrison's Principles of Internal Medicine, 18th ed. New York: McGraw-Hill; 2012.)
Platelet plug formation consists of adhesion, aggregation, and activation of platelets.
Adhesion
On vascular injury exposes von Willebrand Factor (vWF), normally located between the endothelium and the basement membrane.Platelet adhesion is mediated primarily by Von Willebrand factor (VWF), a large multimeric protein present in both plasma and the extracellular matrix of the subendothelial vessel wall, which serves as the primary “molecular glue,” providing sufficient strength to withstand the high levels of shear stress that would tend to detach them with the flow of blood.
Platelet adhesion is also facilitated by direct binding to subendothelial collagen through specific platelet basement membrane collagen receptors.
Platelet adhesion results in subsequent platelet activation and aggregation. This process is enhanced and amplified by humoral mediators in plasma (e.g., epinephrine, thrombin); mediators released from activated platelets (e.g., adenosine diphosphate, serotonin); and vessel wall extracellular matrix constituents that come in contact with adherent platelets (e.g., collagen, VWF). Activated platelets undergo the release reaction, during which they secrete contents that further promote aggregation and inhibit the naturally anticoagulant endothelial cell factors. During platelet aggregation (platelet-platelet interaction), additional platelets are recruited from the circulation to the site of vascular injury, leading to the formation of an occlusive platelet thrombus. The platelet plug is anchored and stabilized by the developing fibrin mesh.
The platelet glycoprotein (Gp) IIb/IIIa (αIIbβ3) complex is the most abundant receptor on the platelet surface. Platelet activation converts the normally inactive Gp IIb/IIIa receptor into an active receptor, enabling binding to fibrinogen and VWF. Because the surface of each platelet has about 50,000 Gp IIb/IIIa–binding sites, numerous activated platelets recruited to the site of vascular injury can rapidly form an occlusive aggregate by means of a dense network of intercellular fibrinogen bridges. Because this receptor is the key mediator of platelet aggregation, it has become an effective target for antiplatelet therapy.
Control of formation of the initial plug is via nitrous oxide and prostacyclins. NO, nitric oxide.
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Adhesion of platelets to the endothelium: Collagen is highly thrombogenic and platelets will adhere to it. vWF, contained within platelets and endothelial cells, enhances platelet adhesion by increasing the number of links between platelets and collagen fibrils.
Following the initial formation of a platelet plug, partly aided by exposed von Willebrand factor (vWF) at the injury site, platelets continue to aggregate via fibrin linking of their Gp IIb/IIIa receptors. Adhesions by these receptors lead to activation of the platelets and degranulation, which releases
adenosine diphosphate (ADP), thromboxane A2 (TXA2), and other factors, all of which further increase the formation of initial platelet plug/clot. Nitric oxide and antiplatelet
prostacyclin (
PGI2) limit plug and clot formation beyond the injury. The clotting cascade noted in the figure is described further below. EC, endothelial cell.
Aggregation
Aggregation of platelets to one another: Platelets stick to one another by fibrin linking the glycoprotein IIb/IIIa (Gp IIb/IIIa) of one platelet to the Gp IIb/IIIa of another.
This adhesion event then triggers activation of platelets by collagen, thrombin generated by tissue factor, or both, allowing them to aggregate and form a tight platelet plug that stanches the further egress of blood from the injured vessel.
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Activation: The adhesion step causes platelets to release their stored granules (activation) that contain the following, which all act to enhance plug formation and limit bleeding:
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Adenosine diphosphate (ADP)—increases expression of Gp IIb/IIIa on platelets and causes them to swell.
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Prostaglandin Thromboxane A2 (TXA2) activates a G-coupled protein receptor (Gq). TXA2 increases vasoconstriction (to decrease blood flow and limit hemorrhage) and the aggregation of platelets.
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PLA2 increases platelet adhesion to fibrin via Gp IIb/IIIa.
- ADP and Ca2+ lead to additional fibrin deposition.
Historical Perspective
Clinical and laboratory investigation of congenital bleeding disorders have been crucial in working out the structure and . Two famous patients with hemophilia are descendants of Queen Victoria. They are shown in the photographs on the right, convalescing from acute bleeding episodes. On the upper right, the queen's son, Prince Leopold of Albany, is attended by the celebrated British physician Sir William Jenner. Below, Prince Alexei, czarevitch of all the Russias and great-grandson of the queen, is attended by his mother, Czarina Alexandra.
Prince Alexei and his mother
[Patients with defects in the formation of the primary platelet plug (defects in primary hemostasis, eg, platelet function defects, von Willebrand dsease) typically bleed from surface sites (gingiva, skin, heavy menses) with injury.
{Platelet rich thrombi tend to occur in arteries.]
Clot formation is the formation of a fibrin mesh that binds to the platelet aggregate, forming a more stable hemostatic plug or thrombus.
The conversion of fibrinogen to fibrin and cross-linking of fibrin by activated factor XIII, stabilizes the clot.
Deficiency or functional abnormality of the factors involved in these reactions causes bleeding disorders.
Natural inhibitors of clotting factors include antithrombin III, protein S, and protein C. When activated, these proteins inactivate specific clotting factors, providing a regulatory mechanism that serves to control the coagulation response and limit the extension of the clot. Physiologic or natural inhibitors should not be confused with acquired inhibitors of coagulation factors, which are discussed in this review. Inhibitors to coagulation factors, also known as circulating anticoagulants, are antibodies that neutralize specific clotting proteins, thereby interfering with their normal function. Antibodies may be directed against isolated clotting factors, as is the case with factor VIII or IX inhibitors. On the other hand, the antiphospholipid antibodies are known to develop against multiple coagulation proteins. In contrast to patients with antibodies against isolated clotting factors, who commonly present with spontaneous bleeding, individuals with antiphospholipid antibodies may be asymptomatic or present with venous or arterial thrombosis. In this article I refer to inhibitors developing in patients with hemophilia A or other congenital factor deficiency as alloantibodies, and to spontaneous formation of antibodies in patients without prior history of hemorrhagic diathesis as autoantibodies. The antiphospholipid antibodies are discussed separately.
The platelets’ secretory substances (intrinsic pathway), along with tissue factor (extrinsic pathway) synthesized in the endothelium, activate the coagulation cascade. This activates thrombin, which converts fibrinogen to fibrin.
Figure below depicts the coagulation proteins involved in clot formation prior to the formation of fibrin.
Proteins in the proximal coagulation cascade. A) Zymogens are converted to active enzymes by proteolytic cleavage. Blue rectangles at left = signal peptides; • • • • • = propeptide; Y = specific sites of glutamic acid carboxylation. The curved arrows to the right show sites of cleavage releasing the C-terminal catalytic domain (long blue rectangle on right), which now functions as an active serine protease. B) Cofactors serve as docking sites for zymogens. Tissue factor is a transmembrane protein. (The lipid bilayer is shown as a pair of vertical rectangles.) Factors VIII and V are activated by proteolytic cleavage at sites shown by the curved arrows. (Modified with permission from Furie B and Furie BC. The molecular basis of blood coagulation. Cell. 1988. 53: 505-518.)
The zymogens, shown in image A (prothrombin and factors VII, IX, X, and XI), are synthesized in the liver. As this figure demonstrates, they have some striking structural similarities. All have a signal peptide at the N-terminus, and except for factor XI, a propeptide is immediately adjacent. These N-terminal portions are cleaved within the hepatocyte prior to the release of the mature zymogen into the circulation. All five zymogens have a large catalytic domain at the C-terminus that, when activated, functions as a serine protease that specifically cleaves and activates the next protein in the coagulation cascade. Four of these zymogens, factors VII, IX, X, and prothrombin, undergo a crucial post-translational modification in the Golgi body prior to secretion into the plasma. Specific glutamic acid residues in the region adjacent to the propeptide undergo vitamin K–dependent carboxylation. This additional negative charge facilitates the binding of calcium ions, an essential cofactor for optimal function.
Protein cofactors play an equally important role in the regulation of the coagulation cascade. They bind to platelet and endothelial cell membranes, serving as docking sites for the zymogens and contributing importantly to the amplification that is essential for the rapid formation of the fibrin clot. As shown in Figure 13-3B, tissue factor is a transmembrane protein on the surface of extravascular cells, cells within the vessel wall, and circulating microparticles. Like the zymogens mentioned earlier, factors VIII and V are actually pro-cofactors—soluble plasma proteins that require proteolytic cleavage for activation.
In the test tube, the formation of the fibrin clot proceeds by the intrinsic pathway depicted in Figure 13-4. Exposure of the plasma to the glass surface of the tube triggers the activation of factor XII to XIIa (the a stands for activated), which in turn, with the participation of high molecular weight kininogen, activates factor XI to factor XIa. Factor XIa, in the presence of calcium ions, catalyzes the activation of factor IX to factor IXa. Factor IXa forms a complex with activated factor VIII (factor VIIIa) on membrane surfaces in the presence of calcium ions to trigger the activation of factor X to factor Xa.
The blood coagulation cascade in vitro. A fibrin clot can be formed by activation of either the intrinsic or extrinsic pathway. In the test tube, surface contacts trigger the intrinsic pathway. Key: diamonds = zymogens; circles = active enzymes and cofactors; pink rectangles = pro-cofactors; green rectangles = bimolecular complexes; HMWK = high molecular weight kininogen; TF = tissue factor; PT = prothrombin; T in small circle = thrombin; FG = fibrinogen; F = fibrin. (Modified with permission from Furie B and Furie BC. The molecular basis of blood coagulation. In Hoffman R, Benz EJ, Shattil SJ, et al, eds. Hematology, Basic Principles and Practice, 3rd Edition, New York USA, Churchill Livingstone, 2001:1784.)
According to conventional wisdom, the extrinsic pathway depicted in Figure 13-4 is responsible for the initiation of the coagulation cascade in vivo. Tissue factor is constitutively expressed on cells within blood vessels. Upon vessel injury, tissue factor is exposed and binds to minute amounts of activated factor VII (factor VIIa) that are normally present in plasma. The procoagulant complex factor VIIa/tissue factor then initiates the coagulation cascade by converting factor X to factor Xa. As Figure 13-4 shows, this is the point at which the extrinsic and intrinsic pathways converge.
However, in vivo coagulation is more complicated. Low concentrations of tissue factor may be concentrated in the thrombus early in its development. The model that best reflects in vivo coagulation in humans is depicted in Figure 13-5. As mentioned earlier, the primary initiating trigger is the exposure of tissue factor on the injured blood vessel. Tissue factor, along with phosphatidylserine on the plasma membrane of platelets and endothelial cells, forms a complex with activated factor VII (factor VIIa), which not only catalyzes the activation of factor X but also the activation of factor IX. The latter event is of little importance in the test tube but is critical in vivo, because individuals with deficiencies of factor IX or its cofactor, factor VIII, suffer from hemophilia (Chapter 15), a severe bleeding disorder.
The blood coagulation cascade in vivo. The primary initiating event is the exposure of tissue factor (TF) on the injured blood vessel. Tissue factor forms a complex with activated factor VII (FVIIa), which not only catalyzes the activation of factor X but also the activation of factor IX. This dual role of the FVIIa/tissue factor complex contributes to the dramatic amplification of blood coagulation in vivo. Activated coagulation factors are shown as circles. (Modified with permission from Furie B and Furie BC. The molecular basis of blood coagulation. In Hoffman R, Benz EJ, Shattil SJ, et al, eds. Hematology, Basic Principles and Practice, 3rd Edition, New York USA, Churchill Livingstone, 2001:1785.)
At each successive step in the bottom of the cascade, the concentration of the respective zymogen increases, thus favoring amplification of clot formation. It follows then that by far the most abundant coagulation protein in the plasma is the structural protein fibrinogen, which is polymerized to form fibrin in the final step in the cascade. As shown in Figure 13-6A, fibrinogen is a heterodimer composed of a pair of three subunits, Aα, Bβ, and γ, linked by disulfide bonds. Upon cleavage by thrombin (factor IIa), small fibrinopeptides, designated fibrinopeptide A and fibrinopeptide B, are released from the Aα and Bβ subunits, respectively. Measurement of fibrinopeptide A reflects the conversion of fibrinogen to fibrin and can be useful in monitoring patients with disseminated intravascular coagulation (Chapter 16). Upon cleavage, fibrinogen undergoes a conformational change that enables it to form noncovalent polymers via end-to-end interactions as well as side-to-side interactions between the linear strands. The strength and durability of the fibrin polymer is then greatly enhanced by the action of factor XIIIa, which catalyzes the formation of covalent crosslinks between the fibrin strands.
Fibrinogen and fibrin. A) Diagram of the dimeric structure of fibrinogen showing its three subunits connected by disulfide bonds. Fibrinopeptides A and B (FPA and FPB) are cleaved by thrombin, enabling fibrinogen to polymerize into fibrin by forming end-to-end as well as side-to-side contacts, as shown in panel B. (Modified with permission from Furie B and Furie BC. The molecular basis of blood coagulation. In Hoffman R, Benz EJ, Shattil SJ, et al, eds. Hematology, Basic Principles and Practice, 3rd Edition, New York USA, Churchill Livingstone: 2001: 1797, 1798.)
Fibrin stabilizes the clot. Activation of the coagulation cascade via the extrinsic pathway results in the deposition of insoluble polymers composed of fibrin leading to a more stable hemostatic plug.
The protagonists are a set of plasma proteins that normally circulate as inactive zymogens. Figure below depicts a prototypical or consensus pathway.
Consensus pathway responsible for the rapid and controlled formation of the fibrin clot. Zymogen activation by limited proteolysis from inactive to active enzyme (left) and inhibition of the enzyme (E) by a specific inhibitor (I).
When the coagulation cascade is activated, either in vivo or in the test tube, a zymogen is converted into an active proteolytic enzyme capable of specifically cleaving the next zymogen in the set, and so on. This highly controlled limited proteolysis leads to the eventual formation of thrombin and generation of the fibrin clot.
The recruitment of specific inhibitors prevents unwanted extension of the clot beyond the site of injury. The final step, fibrinolysis, enables the vessel to be recanalized, thereby restoring blood flow.
Recent studies have shown that these phases are less clearly demarcated in vivo than this idealized scenario would suggest. Indeed, the use of specific fluorescent molecular markers clearly shows that fibrin deposition occurs as the platelet plug is being formed.
Clots consist of platelets, the protein fibrin, and RBCs. Fibrin assumes a netlike matrix that spans the full thickness of the clot, ensnaring RBCs throughout the thickness of the meshwork. Ensnaring the RBCs in the fibrin network creates the clot that stops hemorrhage.
Clotting occurs in a controlled and localized fashion, neither occluding the vessel nor causing clots to form at remote sites that have no need for clot formation. Physiologic anticoagulants such as AT-III and Activated Protein C oppose thrombosis, serving to localize it to sites of vascular injury. Clot formation is balanced by plasmin-mediated fibrinolysis, resulting in the formation of D-dimers and other fibrin degradation products.
A cascading series of limited proteolytic reactions occur. At each step, a clotting factor zymogen undergoes limited proteolysis and becomes an active protease and culminates in the formation of thrombin. Thrombin proteolytically cleaves small peptides from fibrinogen, allowing fibrinogen to polymerize and form a fibrin clot.Thrombin also activates many upstream clotting factors. Thrombin also activates factor XIII, a transaminase that cross-links the fibrin polymer and stabilizes the clot.
Blood coagulates due to the transformation of soluble fibrinogen into insoluble fibrin by the enzyme thrombin.
In contrast, patients with defects in the clotting mechanisms (secondary hemostasis, eg, Hemophilia A) tend to bleed into deep tissues (joints, muscles, retroperitoneum), often with no apparent inciting event and bleeding may recur unpredictably.
Venous thrombi tend to be more fibrin-rich, contain large numbers of trapped red blood cells and are recognized pathologically as red thrombi.
Factor |
Deficiency State | Hemostatic Levels | Half-Life of Infused Factor | Replacement Source |
|---|---|---|---|---|
| 1 | Hypofibrinogenemia | 1 g/dL | ||
Prevention of Coagulation - Antithrombin
Antithrombin (AT) is a small protein molecule that inactivates several enzymes of the coagulation system. α-Antithrombin is the dominant form of antithrombin found in blood plasma and has an oligosaccharide occupying each of its four glycosylation sites. A single glycosylation site remains consistently un-occupied in the minor form of antithrombin, β-antithrombin. Its activity is increased manifold by the anticoagulant drug heparin, which enhances the binding of antithrombin to factor II and factor X.1
Reference(1)
1. http://en.wikipedia.org/wiki/Antithrombin
There Are Three Types of Thrombi
Three types of thrombi or clots are distinguished. All three contain fibrin in various proportions.
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The white thrombus is composed of platelets and fibrin and is relatively poor in erythrocytes. It forms at the site of an injury or abnormal vessel wall, particularly in areas where blood flow is rapid (arteries).
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The red thrombus consists primarily of red cells and fibrin. It morphologically resembles the clot formed in a test tube and may form in vivo in areas of retarded blood flow or stasis (eg, veins) with or without vascular injury, or it may form at a site of injury or in an abnormal vessel in conjunction with an initiating platelet plug.
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A third type is fibrin deposits in very small blood vessels or capillaries.
[whereas thrombosis is the cessation of blood flow inside a blood vessel when the endothelium lining blood vessels is damaged or removed (eg, upon rupture of an atherosclerotic plaque).]