Secondary Hemostasis Flashcards
Describe the steps of the intrinsic pathway.
• The intrinsic pathway begins by a process termed contact activation involving the
participation of three different procoagulant proteins: prekallikrein (PK), High Molecular
Weight Kininogen (HMWK) and Factor XII.
• Upon exposure of blood to an anionic surface these proteins form a large macromolecular
complex that leads to a conformational change in the Factor XII molecule resulting in its
activation to Factor XIIa.
• Factor XIIa is an active protease which subsequently cleaves Factor XI to form Factor XIa
and also cleaves PK to Kallikrien, which further amplifies contact activation by cleaving
more Factor XII to active products.
• In the final step ofthe intrinsic pathway, Factor XIa converts Factor IX to Factor
Describe the steps of the extrinsic pathway.
• The extrinsic pathway of blood coagulation is initiated by tissue trauma which exposes an
integral membrane protein called Tissue Factor (Factor III).
• Tissue factor is then able to bind circulating Factor VII or small amounts of circulating
Factor VIIa to form an active Tissue Factor-Factor VIla complex.
• The Tissue Factor-Factor VIla complex formed from the extrinsic pathway or the Factor IXa complex formed from the intrinsic pathway is then able to activate a series of reactions that are collectively referred to as the common pathway (first step is to cleave Xa). Tissue Factor-Factor VIIa complex also activates Factor IX to a more significant extent than Factor XIa does.
Where does the common pathway occur? How is this made possible. Describe the initial steps in the common pathway and what factors or cofactors are involved.
• The initial reaction complex consists of an enzyme (IXa), a
substrate (X) and a cofactor or reaction accelerator (VIIIa).
- They are assembled on the phospholipid surface of platelets.
- Calcium ions hold the assembled components together and
are essential for the reaction.
- The result is activation of Factor X to produce Factor Xa.
- Factor X can alternatively be activated by Tissue Factor-Factor
VIIa complex from the extrinsic pathway.
• Factor Xa then becomes the enzyme in the next adjacent
complex in the coagulation cascade, converting prothrombin
(II) to thrombin (IIa), with the cooperation of the cofactor or
reaction accelerator, Factor Va.
Describe the formation and stabilization of Fibrin and the factors involved.
• Thrombin cleaves the precursor molecule,
fibrinogen, to form fibrin monomers.
• The monomers undergo polymerization by hydrogen
bonding interactions, producing polymerized but
unstable fibrin (Fibrin II).
• Thrombin next cleaves Factor XIII to activate it,
producing Factor XIIIa. This step is very important in
clot formation.
• Factor XIIIa catalyzes the covalent cross-linking
between the monomers of the hydrogen-bonded
polymerized fibrin, producing a stable fibrin clot.
Describe how clotting actually occurs in vivo, including regulation of tissue factor-factor VIIa, as well as the role of thrombin in continuing clotting after that regulation.
• Physiological (i.e., in vivo) clotting is initiated by the
extrinsic pathway following vascular injury.
• Exposed Tissue Factor complexes with Factor VII/VIIa
and the complex activates Factors IX and X.
• Tissue Factor-Factor VIIa complex is inhibited by Tissue
Factor Pathway Inhibitor.
• Continuing formation of Factor Xa, leading to formation
of a fibrin clot, now depends on thrombin activation of
Factors XI, VIII and V.
• HMWK, prekallikrein and Factor XII are not required for
in vivo clotting, and their deficiencies cause no bleeding.
What is the role of Vitamin K in clotting? What specifically does it do? Why is it necessary? What happens if it’s deficient?
• Factors II, VII, IX, X, Protein C and Protein S require
modification by vitamin K to be functionally active.
• Vitamin K catalyzes gamma-carboxylation of glutamic acid
residues in these inactive proteins.
• This confers on the proteins the ability to bind to anionic
phospholipids on platelet surfaces in the presence of Ca2+.
• Binding to platelet surfaces concentrates the clotting factors
on platelet surfaces and enhances the clotting process.
• Vitamin K deficiency causes a significant bleeding disorder.
Describe the Antithrombin II system including the clotting factors involved.
Antithrombin III (AT-III) System. The operation of the AT-III system is illustrated in
Figure 4. Antithrombin III binds to acid mucopolysaccharides, in particular, cell surface-
bound heparan sulfate or circulating soluble heparin sulfate. AT-III is a serine protease
inhibitor that forms stoichiometric complexes with activated Factors IXa, Xa, XIa or
thrombin (Factor IIa). AT-III binding functions to irreversibly inhibit the activity of these
procoagulant proteases. It is believed that the AT-III:protease complexes are eventually
removed by reticuloendothelial cells in the liver and spleen. By virtue of its ability to bind to
surface bound heparan sulfates or circulating heparin, AT-III is able to function as a protease
inhibitor of both circulating as well as surface-localized proteases.
Describe the protein C protein S system. What are its two roles?
Protein C/Protein S system. Protein C and Protein S are two Vitamin K-dependent
anticoagulant proteins which are synthesized in the liver and circulate in plasma. The system
is activated when thrombin, formed at sites of vascular injury, diffuses to nearby endothelial
cells where it encounters a surface bound thrombin receptor called thrombomodulin
expressed on the surface of endothelial cells (see Figure 5). Upon binding to
thrombomodulin, the protease activity of thrombin is modified in a way that allows it to
cleave Protein C to form activated Protein C. Activated Protein C binds to its cofactor
protein, Protein S to form a functionally active surface-bound protease that is able to cleave
Factor Va and Factor VIIIa, leading to the formation of inactive fragments.
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Because of the vitally important role of activated Factor V and activated Factor VIII as co-
factors of the surface bound catalytic complexes that lead to the formation of prothrombin or of
Factor X, respectively, the cleavage of Factor Va and Factor VIIIa by activated Protein C and
Protein S provides a very effective means of inhibiting the coagulation process. Thus, the
thrombomodulin, Protein C and Protein S system functions to antagonize hemostasis in two
ways:
1. by thrombomodulin binding to thrombin thus removing thrombin from the system and
converting it to an anticoagulant protein and
- by destroying two important co-factor proteins, Factor Va and Factor VIIIa.
Describe how the Tissue Factor Pathway Inhibitor works. What are its two roles?
Tissue Factor Pathway Inhibitor (TFPI). TFPI binds to Factor Xa generated at cell or
platelet surfaces (see Figure 6). The bound TFPI-Factor Xa complex is now capable of
attacking and inactivating the Tissue Factor-Factor VIIa complex, thus terminating the
activity of this critical complex in the initiation of physiologic hemostasis. Thus, TFPI has a
dual anticoagulant action. First it complexes to and removes Factor Xa from the hemostatic
process and second, the TFPI-Xa complex binds to and inactivates the Tissue Factor-Factor
VIIa complex. Because, according to the current model of hemostasis, the activity of the
Tissue Factor-Factor VIIa complex is rate-controlling for physiologic hemostasis in vivo, the
inhibition of this complex by TFPI means that TFPI is a vitally important regulator of
physiologic hemostasis.
Describe the basic steps of fibrinolysis including which factors are activated, which cytokines activate them, the function, and how it is regulated.
Once a stable hemostatic plug is formed, the thrombus must be remodeled and excess clot
material removed in order to restore vessel patency. This is achieved by the process of
fibrinolysis, which refers to the sequence of steps that lead to the eventual dissolution of the
cross-linked fibrin clot (see Figure 7). Fibrinolysis is localized to the fibrin clot by the reversible
binding of a plasma protein called plasminogen and Tissue Plasminogen Activator (TPA),
released from endothelial cells and other cells adjacent to the growing thrombus. TPA,
plasminogen and fibrin form a ternary complex that leads to conversion of the proenzyme
plasminogen to the active protease plasmin. Plasmin then causes lysis of the clot by hydrolyzing
the fibrin into products referred to as fibrin degradation products (FDP). The fibrinolytic process
is regulated by Plasminogen Activator Inhibitor I (PAl-I), which inhibits TPA and by alpha-2
antiplasmin, which inhibits plasmin released into the fluid phase.
Describe some of the antithrombotic properties of normal endothelial cells and how they work.
Endothelial cells modulate several important steps in normal hemostasis. Resting endothelial
cells possess antiplatelet, anticoagulant and fibrinolytic properties that help to prevent
spontaneous clot formation. The antithrombotic properties of normal endothelium are illustrated
in Figure 8.
Normal endothelial cells provide a thromboresistant coating of blood vessels that precludes contact
of blood with subendothelial matrix proteins. This functions to prevent adherence of nonactivated
platelets and activation of the coagulation cascade. Normal platelets also produce prostacyclin (PGI2)
and nitric oxide (NO), both of which are potent vasodilators and inhibitors of platelet aggregation.
Normal endothelial cells secrete an enzyme, adenosine diphosphatase which hydrolyzes ADP, a
potent platelet aggregating agent. Endothelial cells express the surface membrane protein
thrombomodulin which binds to thrombin and activates the Protein C anticoagulant system, which
leads to cleavage of Factors Va and VIIIa. Endothelial cells also express on their surface heparin-like
mucopolysaccharides which are capable of binding to and activating the protease binding site of
antithrombin III, leading to inhibition of thrombin and other activated proteases such as Factor Xa
and Factor IXa.
What can activate endothelial cells? What do endothelial cells do once activated? Describe various actions.
The normal antithrombotic endothelial cell phenotype may be altered by activation of endothelial
cells by infectious agents, hemodynamic factors, plasma mediators and most importantly by
inflammatory cytokines. This activation converts the endothelial cells from an antithrombotic to a
prothrombotic state where the endothelial cell properties are altered in a way that supports
hemostasis.
As shown in Figure 9, activated endothelial cells also actively secrete von Willebrand factor (vWF)
which binds to exposed subendothelial collagen and promotes platelet adhesion. Activated or
damaged endothelial cells also synthesize tissue factor, an integral membrane protein expressed on
the endothelial cell surface. In addition, the activated endothelial cell surface membrane contains
exposed anionic phospholipids which promote binding of vitamin K-dependent factors as well as
Factor Va, augmenting the catalytic activity of these procoagulant proteins. Activated endothelial
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cells also secrete inhibitors of plasminogen activators such as PAI-1, thus depressing fibrinolysis and
enhancing clot stability locally.
Aside from its role in activating clotting factors, describe 5 roles of thrombin.
- Directly induces platelet aggregation and secretion
- Activates endothelium to generate a variety of fibrinolytic (t-PA), and inhibitory substances
(NO, PGI2). Thus, thrombin not only stimulates thrombus formation, but can also serve to
control and limit the size of the thrombus.
• Activates endothelium to generate leukocyte adhesion molecules. These adhesion molecules
promote the attachment of neutrophils, lymphocytes and monocytes to the endothelial cell
surface. Subsequent emigration of these cells into the subendothelial space is an important
component of the repair process.
- Activates mononuclear inflammatory cells.
- Activates endothelium to generate growth factor (PDGF) mediators. These growth factors
play an important role in repair by stimulating fibroblasts and vascular smooth muscle cells
in the vessel wall.
Thus, thrombin plays a vitally important role in controlling the events of hemostasis, both as a
direct clot-promoting agent, as a hemostatic regulatory protein that limits the size of the clot, and
as a mediator of the repair process.
