Physiology of haemostasis

Physiology of haemostasis

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  Physiology of Haemostasis Haemostasisis a complex and sophisticated process that requires the interplay of multiple physiological pathways. Cellular and molecular mechanisms interact to seal damaged blood vessels with localized clot formation preventing significant bleeding. Disruption of the vascular endothelium triggers this interplay of physiological processes, which include formation of an initial platelet plug (primary haemostasis), activation of coagulation to form a fibrin mesh (secondary haemostasis), fibrinolysis and vessel repair. Vascular endothelium The vascular endothelium plays an important role in hemostasis in that quiescent endothelial cells act as a barrier separating the flowing blood from subendothelial components such as tissue factor (activation of coagulation) and collagen (activation of platelets). More than just a passive barrier, the endothelium also produces a variety of substances that modulate platelet, coagulation, fibrinolytic and vascular contraction processes. The functional interactions of endothelial cells can be either procoagulant or anticoagulant in nature, as summarized in the following. Antithrombotic actions of endothelium: 1. Release of prostaglandin derivatives to control platelet activation 2. Synthesis and release of tissue factor pathway inhibitor (TFPI) to control coagulation activation 3. Regulation of thrombin function through thrombomodulin 4. Release of fibrinolytic mediators to regulate the fibrinolytic system 5. Release of nitric oxide to promote vascular dilatation 6. Presence of antithrombotic glycosaminoglycans (heparin-like molecules) Prothrombotic actions of endothelium: 1. Release of tissue factor to initiate the clotting process 2. Release of plasminogen activator inhibitor-1 (PAI-1) to inhibit the fibrinolytic response 3. Generation of procoagulant proteins 4. Expression of von Willebrand factor to promote platelet adhesion
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  Platelets Platelets normally circulatein a non-activated state in the blood but are extremely reactive to changes in their environment. Platelet membranes contain receptors for a variety of agonists including ADP, thromboxane A2, platelet activating factor, immune complexes and thrombin. Serotonin and epinephrine synergistically promote aggregation induced by other agents. Platelet activation Upon activation the expression of cell receptors and procoagulant phospholipids on the platelet surface is upregulated. A number of glycoproteins (GP) present on the membrane serve as receptors for collagen (GPIa/ IIa), fibrinogen (GPIIb/IIIa), and von Willebrand factor (GPIb). These receptors belong to the superfamily of adhesive protein receptors known as integrins as they integrate cell–cell and cell– matrix interactions. Stimulation of these processes allows for bidirectional signaling between the intracellular and extracellular compartments of the platelet. Of the platelet-associated integrins, GPIIb/IIIa is the most abundant. Lack of GPIIb/IIIa receptors leads to the congenital bleeding disorder known as Glanzmann’s thrombasthenia. Platelet GPIb binding to von Willebrand factor, which acts as a bridge to collagen binding in the blood vessel, is important as it serves to anchor the platelets to the blood vessel. Lack of the GPIb receptor leads to the congenital bleeding disorder known as Bernard– Soulier syndrome. Platelet aggregation is another of the fundamental platelet functions. Fibrin(ogen) binding to platelet GPIIb/IIIa receptors is important as it serves as a bridge that links individual platelets together to form a large platelet aggregate. During the activation process, there is a morphologic shape change in the overall platelet structure as pseudopods are formed. This facilitates the platelet aggregation process. Platelet aggregates serve to plug the damage to the vascular wall and their granule release products aid to vasoconstrict the blood vessel decreaseing blood loss. Platelet granule contents The α storage granules contain platelet factor 4 (PF4), β-thromboglobulin, platelet-derived growth factor, fibrinogen, factor V, von Willebrand factor, and PAI-1. The dense or β-granules contain ATP, ADP, and serotonin. The release of platelet granule contents leads to further platelet activation and aggregation as well as coagulation activation.
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  The Coagulation System Theplasma proteins that comprise the coagulation system are referred to as coagulation factors. Most coagulation proteins are zymogens (nonactivated enzymes) that upon activation are converted into active serine proteases. Several of the coagulation factors are dependent on vitamin K for structural formation required for activity. In the intrinsic pathway of the coagulation system, activation occurs when the complex of factor XII, factor XI, prekallikrein, and high molecular weight kininogen come together on a negatively charged surface. This is referred to as contact activation. Factor XII is converted to its active form, factor XIIa, which in turn converts prekallikrein to kallikrein. Kallikrein can convert factor XII to its active form, thereby setting up a positive feedback loop that amplifies the activation of the coagulation system. Factor XIIa converts factor XI to factor XIa, which, in turn, activates factor IX. Factor IXa bound to the negatively charged phospholipid (on activated platelet membranes) along with its cofactor factor VIIIa and calcium ions form the “tenase” complex. Through this complex, factor X is converted to factor Xa initiating activation of the common pathway of the coagulation system. The extrinsic pathway of coagulation is activated when circulating factor VII comes into contact with tissue factor. Tissue factor is a transmembrane glycoprotein that is expressed by subendothelial cells that surround the blood vessel. Tissue factor expression can also be induced on activated monocytes and activated endothelial cells. Both factor VII and factor VIIa bind to tissue factor with equal affinity. The factor VIIa– tissue factor complex can then activate factor X. The tissue factor–factor VIIa complex also activates factor IX to factor IXa. The common pathway of coagulation The small amounts of factor Xa initially generated are sufficient to cleave prothrombin and generate a small amount of thrombin. In a feedback loop, thrombin activates factors V, VIII, and possibly XI, thereby sustaining continued activation of the coagulation cascade. Factors V and VIII are activated through direct proteolytic cleavage by factor Xa or thrombin; they are not active proteases as are the other coagulation factors. The majority of factor Xa joins with its cofactor factor Va, calcium ions and phospholipid (on surface membranes of activated platelets) to form the “prothrombinase” complex. The
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  prothrombinase complex actsto convert prothrombin (factor II) into the active enzyme thrombin. Thrombin (factor IIa) serves many functions in coagulation as well as in various physiological processes. In the coagulation cascade, thrombin holds the key position in that it cleaves soluble fibrinogen to generate an insoluble fibrin clot (thrombus). Fibrinogen circulates as a disulfide-linked dimer containing two A-α chains, two B-β chains and two γ chains. Cleavage of fibrinogen by thrombin results in the release of fibrinopeptides A and B and the exposure of charged domains at opposite ends of the molecule. Exposure of these charged domains leads to polymerization of the fibrin monomers. These monomers are cross- linked by the transaminase factor XIIIa and calcium to form the physical meshwork of the fibrin clot. Thrombin augments its own generation through several feedback loops in the coagulation cascade activating factors XII, XI, VIII, and V. Thrombin also activates platelets, it activates the coagulation inhibitor protein C through binding with thrombomodulin, and it stimulates activated endothelial cells to release the profibrinolytic enzyme tissue plasminogen activator. Natural Inhibitors of Coagulation: 1- Antithrombin (AT) is a single chain glycoprotein with a molecular weight of approximately 58,000 Da. Normal plasma levels of AT are approximately 2–3 μM. AT is the primary inhibitor of coagulation and targets most coagulation factors as well as trypsin, plasmin, and kallikrein. Inhibition takes place when a stoichiometric complex between the active site serine of the enzyme and the Arg393–Ser394 bond of AT forms. The efficient inhibition of proteases by AT requires heparin as a cofactor. In the presence of heparin, the inhibition rate constants for thrombin and factor Xa have been estimated to be accelerated 1000-fold. Deficiency of AT due to low protein levels or to functionally abnormal molecules predisposes an individual to thrombotic complications. 2- Heparin cofactor II (HCII) is another plasma inhibitor that resembles AT in that it is activatable by glycosaminoglycan binding. HCII has a molecular weight of about 68,000 Da. The normal plasma level of HCII is approximately 1.0– 1.4 μM. HCII has higher protease specificity than AT. Of the coagulation enzymes, it only inhibits thrombin. However, it has also been shown to inhibit chymotrypsin and leukocyte cathepsin G. Like AT, HCII inhibits proteases by forming a 1:1 stoichiometric
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  complex with theenzyme. Although the inhibition of protease activity by HCII is promoted by glycosaminoglycan binding, it can be activated by a wide variety of agents unlike AT which is dependent on the presence of a specific heparin chain sequence. Heparins, heparans, and dermatan sulfate all bind to HCII and promote thrombin inhibition. 3- Tissue factor pathway inhibitor (TFPI) is a 42 kDa inhibitor that contains three Kunitz domains tandemly linked between a negatively charged amino terminus and a positively charged carboxy-terminus. It serves an important function to control coagulation activation. The active site of the first Kunitz domain binds to the active site of the VIIa-tissue factor complex; the active site of the second Kunitz domain binds to the active site of factor Xa. The second domain appears to facilitate the inhibitory action of the first domain, and the carboxy-terminus appears to facilitate the action of the second domain. The third Kunitz domain has been shown to contain a heparin-binding site. TFPI is produced by megakaryocytes and the endothelium. Small amounts of TFPI are stored in platelets and can be released upon platelet activation. Plasma TFPI accounts for 10–50 % of the total pool. Most plasma TFPI is bound to lipoproteins, only about 5 % of the plasma pool of TFPI circulates in the free form. The largest pool of TFPI is bound to the endothelial surface. TFPI bound to the endothelium can be released into the plasma by heparin and low molecular weight heparin treatment. 4- Protein C is another important natural anticoagulant. Circulating thrombin can bind to a high affinity receptor on the endothelium known as thrombomodulin. The complex of thrombin bound to thrombomodulin is a 20,000 fold better activator of protein C than is free thrombin. Thrombomodulin-bound thrombin no longer cleaves fibrinogen, is not able to activate other coagulation proteases such as factors V and VIII and does not activate platelets. Protein C is a vitamin K-dependent zymogen. It is made up of disulfide linked heavy and light chains and has a molecular weight of approximately 62,000 Da. Protein C derives its anticoagulant properties from its ability to cleave and inactivate membrane bound forms of factors Va and VIIIa. Protein C requires two cofactors to express its anticoagulant activity, protein S and factor V.
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  The Fibrinolytic System Thefibrinolytic system keeps the formation of blood clots in check. Like the coagulation cascade, this system consists of a number of serine protease activators and inhibitors. Two endogenous activators of plasminogen, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), are produced primarily by the endothelium. tPA and uPA convert plasminogen to the active fibrinolytic enzyme plasmin . Plasmin ultimately cleaves fibrin into smaller fibrin degradation products. Regulation of the fibrinolytic pathway occurs at the level of several inhibitors. PAI-1 inhibits the enzymatic activity of the activators tPA and uPA. PAI-1 covalently binds to the active site of these plasminogen activators, thereby preventing the generation of plasmin. Activated platelets are an important source of PAI 1. Secondly, plasmin can be directly inhibited by the serine protease inhibitor α 2 -antiplasmin. TAFI is a third recently identified inhibitor that has a different type of inhibitory function. TAFI is a procarboxypeptidase that is activated by the thrombin–thrombomodulin complex. Activated TAFI (TAFIa) catalyzes the cleavage of carboxyterminal basic amino acids (such as arginine and lysine) from fibrin, plasmin, and other proteins. Without these end structures plasmin loses its ability to digest fibrin. Thus, fibrinolytic activity is suppressed leaving procoagulant activity to proceed unopposed. Leukocytes Studies have indicated that leukocytes, alone or bound to platelets, play a role in coagulation activation. Cytokines elicit the expression of tissue factor (extrinsic coagulation system activator) on mononuclear cells, and procoagulant activity associated with leukocytes is not limited to the expression of tissue factor. Several monocyte/macrophage derived procoagulant activities have been characterized including factor VII, factor XIII, factor V/Va, and binding sites for factor X and for the factor IXa– VIII complex. Prothrombin can be activated on the cell surface of monocytes and lymphocytes. Monocyte procoagulant activity is also induced by endotoxin, complement and prostaglandins. Coagulation that takes place on the surface of endothelial cells is affected by inflammatory process. Cytokines released from activated leukocytes, such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF),
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  upregulate the procoagulantand downregulate the fibrinolytic nature of endothelial cells. In addition, products of the coagulation process such as thrombin, fibrinopeptides, and fibrin degradation products have chemotactic and mitogenic properties. Autonomic Nervous System The autonomic nervous system may impart control on the regulation of hemostasis and activation mechanisms leading to thrombogenesis. Circadian variations with peak incidences of coronary events in the morning hours have been known. This has been shown to be associated with an increase in blood pressure, heart rate, platelet aggregability, and a decrease in fibrinolytic activity. These physiological responses reflect sympathetic activity largely induced by increased levels of plasma noradrenaline. In combination with an increase in sympathetic mediated vasoconstriction, these factors can lead to atherosclerotic plaque rupture. During hemorrhage the hemostatic mechanisms controlling hemostasis are also partly controlled by the autonomic nervous system.