Hemostasis II Flashcards
- Describe how antithrombin functions as a regulator of coagulation and explain how heparin affects its function.
Antithrombin is a_______.
Serine protease inhibitors, or serpins, are a large and diverse group of proteins which share the feature of being able to bind to *chymotrypsin‐like serine proteases at their active‐site serine, generally for purposes of protease inactivation, though they can perform other functions as well, such as storage or transport of the target protease.
-For proper functioning, serine proteases require the presence of a serine residue in their catalytic domain. Serpins contain what’s called a variable reactive site loop, which binds with specificity to the catalytic groove of the target serine protease. With protein‐protein interactions between the serpin and its target, sometimes involving participation of a cofactor, structural changes occur that allow exposure of the reactive site loop to the serine residue of the protease, leading to formation of a covalent bond between the two, along with significant structural changes in both the protease and the serpin, leading to complete inactivation of both (why serpins are sometimes called “suicide” protease inhibitors).
- Describe how antithrombin functions as a regulator of coagulation and explain how heparin affects its function.
What two factors in the coagulation cascade do antithrombin inhibit?
- Describe how antithrombin functions as a regulator of coagulation and explain how heparin affects its function.
What acts as a key cofactor for antithrombin?
- Describe how antithrombin functions as a regulator of coagulation and explain how heparin affects its function.
The shortened version of heparin is able to speed to process of inactivation of ____1____.
The longer form of heparin is required for acceleration of thrombin inactivation by _____2_____.
1- Factor Xa
2- Thrombin
- Explain how protein C is activated and how the protein C - protein S system regulates coagulation.
Protein C is a vitamin _____-dependent serine protease.
What activates protein C?
Once thrombin cleaves protein C, it is referred to as_________.
What factors do activated protein C inactivate?
With what factor does protein C interacts to enhance its activity?
The protein C system
As mentioned in part 1 of this introduction, protein C, along with the procoagulant factors II, VII, IX, and X, is a vitamin K‐dependent serine protease.
It circulates as a zymogen. When thrombin is generated from prothrombin, in addition to its role as a procoagulant, it binds to thrombomodulin, a transmembrane protein constitutively expressed on endothelial cells. Once bound to thrombomodulin, thrombin’s procoagulant activity is neutralized, but the thrombin‐thrombomodulin complex on the cell surface can bind to and activate protein C.
-Thrombomodulin is a cofactor in this reaction, accelerating the reaction about 1000‐fold, in a situation analogous to the tenase and prothrombinase complexes that are part of the coagulation cascade. In fact, it is sometimes referred to as the “protein C‐ase” complex. Thrombomodulin expression can be down‐regulated with exposure of the endothelial cells to proinflammatory agents, likely contributing to the hypercoagulability that can be seen with inflammation.
Thrombin cleavage of protein C leads to formation of activated protein C, sometimes referred to as “APC.” Once generated, APC goes on to cleave and inactivate the cofactors Va and VIIIa, leading to decreased generation of thrombin.
This reaction is enhanced by interaction of protein C with its cofactor, protein S, on an anionic phospholipid surface (i.e., the activated platelet surface). Interaction of protein S with APC alters the structure of APC and moves the APC active site closer to the membrane surface.
- Explain how protein C is activated and how the protein C - protein S system regulates coagulation.
Is protein S a serine protease?
60% of protein S is found circulating bound to what?
Protein S is the only vitamin K‐dependent factor that is not a serine protease.
Only about 40% of protein S circulates in the blood in the free form, whereas the remaining 60% circulates bound to C4b‐binding protein (C4bBP), a regulator of the complement pathway. This becomes important when measuring protein S levels in plasma, since protein S bound to C4bBP is inactive. Protein S probably has other roles in inhibiting coagulation outside of its role as a cofactor for protein C. Though its precise function still isn’t fully clear, it undoubtedly plays an important role as an anticoagulant as evidenced by the pro‐thrombotic risk associated with deficiency.
- Explain what factor V Leiden is.
¨A mutation of factor V that makes it resistant to inactivation by protein C.
Sometimes called “APC resistance.”
¨Common risk factor for venous thromboembolism
¨3-8% of Caucasian population
- Describe how tissue factor pathway inhibitor (TFPI) functions in regulating coagulation.
TFPI is expressed by _______. It inhibits the _____ arm of the coagulation pathway.
The quaternary complex has___. ___. ____. ____
Tissue factor pathway inhibitor (TFPI), expressed constitutively by endothelial cells, is an important inhibitor of the extrinsic arm of the coagulation pathway and thus the initiation phase of thrombin generation.
How this happens?
As factor Xa is generated by the extrinsic Xase complex (factor VIIa‐tissue factor), TFPI can bind to Xa’s active site. Once surface bound, the factor Xa‐TFPI complex rapidly binds and inactivates tissue factor‐factor VIIa, forming a stable quaternary complex of tissue factor, factor VIIa, TFPI, and factor Xa.
Factor IXa‐TFPI can also bind to and inhibit factor VIIa‐tissue factor, though TFPI binds to factor IXa with significantly less affinity, making this less likely to be of physiologic relevance. Ninety percent of circulating TFPI is found in association with lipoproteins, and TFPI has been implicated, in addition to its role in coagulation, to play a role in protection from atherosclerosis. Recombinant TFPI is currently being studied to see if may play a therapeutic role in the treatment of hypercoagulable states.
The combination of TFPI inhibition of the initiation phase of thrombin generation and protein C pathway inhibition of the amplification phase of thrombin generation leads to a situation where, under physiologic conditions, a sufficient signal needs to be generated to reach the threshold necessary for thrombin generation to proceed to the propagation phase, serving as a mechanism to protect the body from uncontrolled clot formation.
- Explain the role of plasmin in fibrinolysis. Explain how plasminogen is activated to plasmin.
Plasmin can cleave both____ and _____.
Fibrinolysis is the process of clot breakdown that occurs following clot formation, allowing eventual repair of the damaged blood vessel following injury.
It begins as soon as the clot begins forming. The key enzyme is plasmin, a serine protease which is cleaved from its zymogen precursor, plasminogen, to its active form.
Plasmin can cleave both fibrinogen (fibrinogenolysis) and fibrin (fibrinolysis) to form innumerable different types and sizes of fragments which can be detected in the blood (collectively called fibrin degradation products or FDPs).
Plasmin can also break down extracellular matrix proteins, aiding in the remodeling process involved with repair of the damaged vessel. Plasminogen is synthesized in the liver and circulates in the plasma as well as being present in a wide variety of extravascular tissues and body fluids.
- Explain the role of plasmin in fibrinolysis. Explain how plasminogen is activated to plasmin.
What is the primary activator of plasminogen in tissue? Where is produced?
Whats is its half-life and what is an inactivator of it?
Plasminogen activation
The primary activator of plasminogen in vivo is tissue plasminogen activator (t‐PA). t‐PA is a serine protease produced predominantly in endothelial cells. Its secretion from the endothelium is regulated by numerous important mediators of blood clotting and inflammation, such as thrombin, histamine, bradykinin, epinephrine, serotonin, and interleukins.
Once released, t‐PA has a very short half‐life of about 2.5 minutes. It is rapidly cleared in a receptor‐mediated fashion by the liver and by endothelial cells, as well as inactivated by various inhibitors, the most important being plasminogen activator inhibitor‐1 (PAI‐1).
t‐PA is a poor activator of plasmin in the absence of fibrin but efficiently activates plasminogen to plasmin upon binding to fibrin, with catalytic efficiency enhanced several hundredfold.
t‐PA itself can be cleaved by plasmin or other enzymes into a more active form. As a clot forms, t‐PA and plasminogen bind to the fibrin being generated, localizing plasmin generation to the site of the clot. Initial plasmin degradation of fibrin increases the number of plasminogen‐binding sites in the clot, further amplifying plasmin generation. Recombinant t‐PA is extensively used today therapeutically for clot lysis.
- Explain the role of plasmin in fibrinolysis. Explain how plasminogen is activated to plasmin.
The other main activator of plasminogen is urokinase plasminogen activator (u‐PA). It was first identified in the urine in the 1940’s and was subsequently found to be synthesized by kidney cells as well as endothelial cells. It also can be secreted by tumors and is thought to contribute to metastasis by breaking down extracellular matrix to facilitate tumor invasion. It is a serine protease that is synthesized and released as prourokinase or single‐chain u‐PA (scu‐PA). Similar to t‐PA, it has a very short half‐life of about 5 minutes. The prourokinase becomes bound to the clot and is then cleaved, primarily by already generated plasmin, into its active form which can then contribute to further clot lysis.
- Describe how plasminogen activation inhibitor 1 (PAI-1), alpha2-antiplasmin, and thrombin-activatable fibrinolysis inhibitor (TAFI) regulate fibrinolysis.
Where is TAFI synthesized?
What cleaves it to its active form?
What kind of cleavage enzyme it is?
Thrombin‐activatable fibrinolysis inhibitor (TAFI) is a zymogen that is synthesized in the liver and circulates in the blood in complex with plasminogen.
Factor XIIIa can covalently attach it to fibrin. Like protein C, it is cleaved to its active form through binding to the thrombin‐thrombomodulin complex.
-Once activated, it is an exopeptidase that removes basic amino acids (arginine, lysine) from the C‐terminal of proteins. It targets the C‐terminal of fibrin molecules and FDPs. Removal of these amino acids reduces the number of plasminogen‐binding sites on fibrin, decreasing the amount of plasminogen available to t‐PA or u‐PA, thus down‐regulating plasmin generation and slowing clot lysis.
- Describe how plasminogen activation inhibitor 1 (PAI-1), alpha2-antiplasmin, and thrombin-activatable fibrinolysis inhibitor (TAFI) regulate fibrinolysis.
In blood, a major fraction of PAI-1 in blood is present in the ______ granules of platelets.
Deficiency of PAI-1 leads to what?
The primary physiologic inhibitor of plasminogen activation, targeting t‐PA and u‐PA, is plasminogen activator inhibitor‐1 (PAI‐1).
PAI‐1 is produced in multiple different cell types and has a half‐life of less than 10 minutes in the blood. A major fraction of PAI‐1 in blood is present in the α‐granules of platelets.
PAI‐1 is a serpin that binds to and inactivates t‐PA. Circulating levels of PAI‐1 are in several‐fold excess to levels of t‐PA. Thus, any t‐PA circulating in the blood is rapidly bound to and inactivated by PAI‐1. For u‐PA, PAI‐1 only binds to the activated form.
Deficiency of PAI‐1 leads to excessive bleeding, and higher levels of PAI‐1 lead to slower clot breakdown and a more thrombotic state.
- Describe how plasminogen activation inhibitor 1 (PAI-1), alpha2-antiplasmin, and thrombin-activatable fibrinolysis inhibitor (TAFI) regulate fibrinolysis.
Plasmin bound to ____ is somehow protected from alpha2-antiplasmin.
α2‐Antiplasmin is the primary plasmin inhibitor in blood. It is another member of the serpin family.
It acts by binding to and inactivating plasmin in a 1:1 fashion.
α2‐Antiplasmin rapidly inhibits plasmin free in the circulation, preventing systemic fibrinogen degradation.
By contrast, plasmin bound to fibrin is somewhat protected from inactivation by circulating α2‐antiplasmin, leading to localization of fibrinolysis to the site of the clot. As with TAFI, factor XIIIa can covalently link α2‐antiplasmin to fibrin, leading to stabilization of the fibrin scaffold in the developing clot. Individuals with deficiency show a bleeding disorder, indicating its important physiologic role in preventing fibrinolysis.
- List some of the mechanisms the endothelial cell lining uses to prevent clot formation in the resting state.
Anticoagulation mechanisms include:
Expression of heparan sulfate and dermatan sulfate, which act as cofactors for antithrombin and heparin cofactor II, respectively.
Expression of thrombomodulin, which acts as a cofactor with thrombin for activation of protein C.
Expression of TFPI to inhibit the extrinsic Xase complex.
****Fibrinolytic mechanisms include synthesis and release of t‐PA and u‐PA.
Antiplatelet mechanisms include:
Synthesis and secretion of prostacyclin (PGI2) and nitric oxide (NO), which prevent adhesion of activated platelets and cause vasodilation.
Expression of an enzyme that rapidly metabolizes ADP (a potent platelet agonist) to AMP and adenosine (a potent inhibitor of platelet function).
What happens when vessel damage occurs?
- Explain the terms primary hemostasis and secondary hemostasis
Let us now attempt to put all of these pieces of the hemostatic process together to describe what happens when a blood vessel is damaged or disrupted.
With exposure of the subendothelium, von Willebrand factor (VWF) binds to the subendothelial matrix. Circulating platelets adhere to the subendothelium through platelet integrin receptor interactions with VWF, collagen, and other subendothelial components. Binding leads to platelet shape change and activation as well as platelet aggregation.
This leads to release of platelet granules containingmany of the coagulation proteins as well as platelet agonists and vasoactive substances that cause vasoconstriction to help stem blood loss.
In addition, phosphatidylserine, normally kept on inner leaflet of the cell membrane, is translocated to the outer leaflet, providing a surface for the coagulation reactions to take place. This part of the hemostatic process, involving platelet adhesion, aggregation, and activation, is sometimes referred to as “primary hemostasis.”
- Explain the terms primary hemostasis and secondary hemostasis
At the same time, tissue factor, either constitutively expressed on cells in the extravascular space or exposed on damaged endothelial cells in the area of the injury, becomes exposed to the small amount of factor VIIa circulating in the plasma, leading to initiation of the extrinsic coagulation pathway. This leads to activation of factors IX and X. A prothrombinase complex made up of factor Xa and factor Va, on the anionic phospholipid surface of the platelet and in the presence of calcium, binds and activates prothrombin to thrombin. The generated thrombin activates multiple factors, including factor XI, factor VIII, and factor V, leading to amplification of the coagulation process. This then leads to the propagation phase of coagulation, with generation of a burst of thrombin. Thrombin cleaves fibrinogen to fibrin to form a fibrin network around the activated platelets in the area of injury. Thrombin also cleaves and activates factor XIII, which covalently cross‐links the fibrin, stabilizing the clot. This process, involving activation of the coagulation cascade and formation of the fibrin clot, is sometimes referred to as “secondary hemostasis.”
