Hemostasis I Flashcards
What is Hemostasis?
PHYSIOLOGICAL blood clotting
( the cessation of bleeding from a cut or severed vessel)
What is thrombosis?
PATHOLOGICAL blood clotting
(When the endothelium lining blood vessels is damaged or removed (eg, upon rupture of an atherosclerotic plaque).
What are the factors involving in hemostasis and thrombosis?
- Blood vessels
- Platelet aggregation
- Plasma proteins that cause formation of dissolution of platelet aggregates and fibrin
Physiological hemostasis is an extravascular process
-> Name 4 processes
1, primary hemostasis (seconds)
2, coagulation cascade (minutes)
3, clot maturation (hours, days)
4, clot removal– tissue regeneration
The role of platelets
Platelets bind to collagen at the site of vessel wall injury, form thromboxane A2, and release ADP, which activate other platelets flowing by the vicinity of the injury.
The role of thrombin
Thrombin, formed during coagulation at the same site, causes further platelet activation
Upon activation, platelets change shape and, in the presence of ____, aggregate to form the hemostatic plug (in hemostasis) or thrombus (in thrombosis).
fibrinogen and/or von Willebrand factor
The role of primary hemostasis (seconds)
- vasoconstriction
- platelet adhesion and aggregation
The role of coagulation cascade (minutes)
Formation of a fibrin mesh that binds to the platelet aggregate, forming a more stable hemostatic plug or thrombus.
The role of clot maturation (hours, days)
fibrin stabilization, cellular infiltration
The role of clot removal– tissue regeneration
fibrinolysis – a parallel process
2 characteristics of Thrombi on atherosclerotic plaques are intravascular
1, changes in the vessel wall – atherosclerotic plaque
2, intravascular thrombosis due to plaque injury
What type of protein is thrombin?
a serine-protease
What are the 3 AAs in the active site of thrombin?
Ser-His-Asp
Structural characteristics of thrombin?
Trypsin-like: cleaves by Arg (Lys)
Where is thrombin produced?
in the liver
Thrombin is formed by __
limited proteolysis from prothrombin
The role of „Exosite I, II“
binding sites for regulators, substrates („Cofactors “)
the serine-proteases
-> What do “serine” and “protease” mean?
„Protease” : cleaves peptide bonds with hydrolysis
“Serine” : Ser in the active site plays an important role in catalysis
What is the Mechanism of action of the serine-proteases?
The peptide bond is cleaved in 2 steps, a covalent acyl-enzyme intermediate is formed
Substrate specificity of trypsin
-> Characteristics? What are the AAs?
Substrate specificity of chymotrypsin
-> Characteristics? What are the AAs?
Substrate specificity of elastase
Structure of fibrinogen
2X3 polypeptide chains (Aa, Bb, g), N-terminals in the middle, aC domains curling back => intramolecular interaction
Fibrinopeptides A and B : N-terminal 14, 16 AAs (Thrombin removes them)
Describe Fibrin formation
- Thrombin cleaves the water soluble fibrinogen
- Fibrin monomers with poor water solubility are formed
=> they polymerize spontaneously into a fibrin
polymer
Formation of the fibrin network
-> What are the 4 steps?
1, thrombin cleaves the 2 Fibrinopeptide As
2, protofibril formation (Oligomers, polymers), 2-layered, long, half-staggered
-hydrophobic interactions between knobs- (in E), and holes –(in D)
3, thrombin cleaves the 2 Fibrinopeptide Bs, lateral aggregation of the protofibrils, aC-aC-connection of 2 adjacent protofibrils - fibrin fiber
4, branching points: network
What is the role of FXIIIa?
Activated by thrombin; factor VIIIa is a cofactor in the activation of factor X by factor IXa
-> stabilizes the fibrin polymer with covalent bonds
FXIIIa stabilizes the fibrin polymer with covalent bonds
-> What is the role of thrombin?
activates plasma FXIII by the removal of 2 small peptides
FXIIIa stabilizes the fibrin polymer with covalent bonds
-> Thrombin activates FXIII to ___
FXIIIa
FXIIIa stabilizes the fibrin polymer with covalent bonds.
- Thrombin activates plasma FXIII by the removal of 2 small peptides
-> Characteristics of this activation process?
the activation process is Ca-dependent and is accelerated in the presence of
FXIIIa stabilizes the fibrin polymer with covalent bonds
-> What is the role of XIIIa?
XIIIa is a transglutaminase: Activated by thrombin; stabilizes fibrin clot by covalent cross-linkings
FXIIIa stabilizes the fibrin polymer with covalent bonds
-> Characteristics of The covalently stabilized fibrin structure ?
The covalently stabilized fibrin structure is mechanically more stable and fibrinolytic enzymes dissolve it slower
What are FXIIIa; A; B; Plasma FXIII; Platelet FXIII?
FXIIIa is a transglutaminase, Cys–in active site
A: catalytic subunit, B: regulatory subunit
Plasma FXIII (A2B2) and platelet FXIII (A2 form, no B)
Formation of isopeptide bonds by ___ between
Formation of isopeptide bonds by FXIIIa between fibrin monomers
Formation of isopeptide bonds by FXIIIa between fibrin monomers
-> How does stabilization work biochemically and mechanically?
Biochemically - Against proteases– + protease inhibitors are also cross-linked to a – chains
Mechanical stability against shear forces– covalent g-g- Dimers, a-a-Polymers and a-g- Hibrids
Transglutaminase family
-> They all function in which space?
function in the extracellular space, on many protein substrates
Transglutaminase family
-> They all form peptide bonds between __ and ____
a Lys e-amino group and a Gln g- amide group (just like FXIIIa)
Transglutaminase family
-> Which group is in the active site?
Cys-SH-group
Transglutaminase family
-> Does it depend on Ca2+
YES!
Transglutaminase family
-> The cross-linked protein structure are resistant against ___
proteolysis
Transglutaminase family
-> The role of tissue transglutaminase (TGM2) (pathology)
In ECM, plays a role in inflammation, tumors, Alzheimer (stabilizes protein aggregates), etc.
Where is Prothrombin synthesized?
Synthesized in the liver as a secretory protein
In the liver, how is prothrombin post-translationally modified?
In the liver Prothrombin is post- translationally modified–gamma- carboxylation of Glu side chains, a vitamin K-dependent reaction - Gla-domains are formed
(The amino acid gamma-carboxyglutamic acid (Gla))
In the blood, prothrombin is proteolytically activated to thrombin by __
FXa
Is Thrombin soluble?
Thrombin is soluble, no more Gla- domain
(The amino acid gamma-carboxyglutamic acid (Gla))
What is the role of Fragment 1.2?
A laboratory marker of prothrombin activation
Identify these substances
What does a a prothrombinase complex consist of?
consisting of Ca2+, factor Va, and factor Xa
Steps of Prothrombin activation in the prothrombinase complex
When activated to factor Va by traces of thrombin, it binds specifically to the platelet membrane (Figure 55–3) and forms a complex with factor Xa and pro- thrombin.
-> It is subsequently inactivated by activated protein C
-> providing a means of limiting the activation of prothrombin to thrombin
How is protein C activated? What is its role?
Activated to activated protein C (APC) by thrombin bound to thrombomodulin
-> then degrades factors VIIIa and Va
What are the Gla-domains good for?
Gla= gamma-carboxy-glutamate
Binds Ca2+, which stabilize the membrane binding of such proteins
Regulation of prothrombin activation
-> Is this physiological or pathological blood clotting?
PHYSIOLOGICAL
Regulation of prothrombin activation
-> Is this physiological or pathological blood clotting?
PATHOLOGICAL
What are the 3 steps of Physiological hemostasis?
- Initiation of the cascade occurs on the surface of TF- bearing cells, first thrombin is formed here
- Amplification: thrombin accelerates its own formation by positive feed-back reactions
- Activation complexes assemble on the surface of activated platelets, which results in a burst of thrombin generation
Describe tissue factor physiologically? (1st step of Physiological hemostasis)
- Cells normally in contact with blood do not express TF on their surfaces - endothelial cells, platelets, leukocytes, RBCs
- Every other cells express TF on the cell surface, membrane-bound (under the endothelium, and other locations in the body)
- When vessel wall is injured, blood flows out of the vessel into the wall layers, hence, FVIIa of blood meets TF in the vessel wall and the coagulation cascade is initiated
In this TF-complex, FXa activates FVII to FVIIa
Describe tissue factor pathologically?
- Endothelial cells, monocytes express TF on their surface, because of previous activation due to inflammation, hypoxia, etc – this leads to circulating TF and initiation of microthrombosis at several locations.
- Microparticles (derived by certain cells, eg. activated platelets or tumor cells), may also carry TF on their surface leading to microthrombosis
Characteristics of Deficiency of TF or FVII
Deficiency of TF or FVII is lethal in utero, very rare bleeding disorder among living patients
Physiological hemostasis
-> How does amplification occur in 2nd step?
The little thrombin:
- Activates platelets – GpIb receptor:
Binds to exosite II, which increases FXI and further increases platelet activation by thrombin
- FXI activation on platelets
- Activation of cofactors: FVIII (delivered by von Willebrand factor) and FV (secreted by activated platelets)
Physiological hemostasis
-> What is the Role of platelets in thrombin generation in the 3rd step?
In the course of platelet activation phosphatidyl-serine is exposed in the outer lipid leaflet on the surface, this lipid is necessary for the assembly of the activation complexes
Describe Synthesis of vitamin-K-dependent factors in the liver
Gla –Domains bind Ca2+, and hence, stabilize the membrane binding of the factors
(The amino acid gamma-carboxyglutamic acid (Gla))
Synthesis of vitamin-K-dependent factors in the liver: Gla –Domains bind Ca2+, and hence, stabilize the membrane binding of the factors
=> What are procoagulants?
FII, FVII, FIX, FX
Synthesis of vitamin-K-dependent factors in the liver: Gla –Domains bind Ca2+, and hence, stabilize the membrane binding of the factors
=> What are anticoagulants?
Protein C, S, (Z)
What is the role of protein S
Acts as a cofactor of protein C
Synthesis of vitamin-K-dependent factors in the liver: Gla –Domains bind Ca2+, and hence, stabilize the membrane binding of the factors
=> The role of Ca2+
1/ stabilizes the hydrophobic helices (middle 4 Ca2+)
2/ interacts with the head of PS (peripheral Ca2+)
Similarity between Protein C and Protein S
=> The role of Ca2+
both proteins contain Gla (γ-carboxyglutamate) residues
Gla-Domains are localized at ____ of the VKD- Proteins
the N-terminus
FXIIa (a serine-protease) initializes 4 systems:
=> What are they?
What is Factor XII structure?
Factor XII structure
-> The role of Conformational change on „negative surfaces” – pre-activation – protease domain
Conformational change on „negative surfaces” – pre-activation – protease domain activates prekallikrein to kallikrein, and XI to XIa, HK (high molecular weight kininogen) serves as a cofactor.
What are „negative surface” in vitro of Factor XII?
glass, kaolin, long chain fatty acids, uric acid kristalls
Factor XIIa activates ___ (which process?)
Factor XIIa activates the coagulation cascade
What is the positive feedback when it comes to Factor XIIa?
Positive feed-back: Prekallikrein/Kallikrein (PK/Kal), on the surface of the cofactor high molecular weight kininogen (HK)
What is the positive feedback when it comes to Factor XIIa?
Positive feed-back: Prekallikrein/Kallikrein (PK/Kal), on the surface of the cofactor high molecular weight kininogen (HK)
Factor XIIa activates the coagulation cascade
-> Positive feed-back: Prekallikrein/Kallikrein (PK/Kal), on the surface of the cofactor high molecular weight kininogen (HK)
-> What is the role of Kallikrein (Ser-protease) ?
It splits FXII, the resulting alphaXIIa activates XI, the complement system, and the PK.
-> splits XII further: betaXIIa, surface-binding domains lost, no further XI activation, but the complement system, and PK activation remains
What is Thrombomodulin? its role?
Protein on the surface of endothelial cells; binds thrombin, which then activates protein C
