Coagulation Pathway and Physiology Flashcards
(78 cards)
1
Q
Heparin Sulfate
A
-glycosaminoglycan -attached to the luminal surface of the endothelium -acts as a cofactor for Antithrombin (main direct inhibitor of many coagulation factors)
2
Q
Antithrombin
A
-serine protease inhibitor -5 stranded central beta-sheet (A-sheet) -conformational changes by binding of Heparin to arginine residues increases inhibition effect of antithrombin by 1000 fold (exposes the reactive center)
3
Q
Platelets
A
- discoid, anucleate fragments that can be up to 3 microns in size - complex structure including actin and myosin filaments
4
Q
Zymogen forms of Vitamin K dependent serine proteases
A
-Factors II, VII, IX, and X -Proteins C, S, and Z
5
Q
Why is Vitamin K so important for these dependent factors?
A
-It is necessary cofactor for post-translational modification that adds a carboxyl group to 10-12 glutamic acid residues in the amino terminal portion of the factors and proteins -The factors and proteins utilize this addition to adhere to adhere to phospholipid membranes and assemble multimolecular coagulation complexes -Without this happening there is ineffective fibrin formation
6
Q
How does Warfarin affect this system?
A
-inhibits the carboxyl addition to the glutamic acid -this effect is monitored by the clot-based PT assay
7
Q
Fibrinogen (Factor I)
A
-Adhesive protein that forms the fibrin clot
8
Q
Prothrombin (Factor II)
A
-vitamin K dependent -activated form is main enzyme of coagulation
9
Q
Tissue Factor (Factor III)
A
-Lipoprotein initiator of extrinsic pathway
10
Q
Calcium ions (Factor IV)
A
-necessary for all reactions -metal cation
11
Q
Factor V (Labile factor)
A
-Cofactor for activation of prothrombin to thrombin
12
Q
Factor VII (Proconvertin)
A
-Vitamin K dependent -With Tissue Factor, initiates the intrinsic pathway
13
Q
Factor VIII (Antihemophilic Factor)
A
-Cofactor for intrinsic activation of Factor X
14
Q
Factor IX (Christmas Factor)
A
-Vitamin K dependent factor -Activated form is enzyme for intrinsic activation of Factor X
15
Q
Factor X (Stuart-Power Factor)
A
-Vitamin K dependent factor -Activated form is enzyme for final common pathway activation of prothrombin
16
Q
Factor XI (Plasma thromboplastin antecedent)
A
-serine protease -Activated form is intrinsic activator of Factor IX
17
Q
Factor XII (Hageman Factor)
A
-Serine protease -Factor that nominally start aPTT-based intrinsic pathway
18
Q
Factor XII (Fibrin-stabilizing factor)
A
-Transamidase that cross-links fibrin clot
19
Q
Prekallikrein (Fletcher factor)
A
-Serine protease -Activated form that participates at beginning of aPTT-based intrinsic pathway
20
Q
PT is a measure of which pathway?
A
-Extrinsic pathway (Tissue factor and Factor VII)
21
Q
aPTT is a measure of which pathway?
A
-Intrinsic pathway
22
Q
History suggestive of vWD bleeding
A
-Menorrhagia -Bleeding from minor wounds -Surgical bleeding -tooth extraction -epistaxis
23
Q
History suggestive of non vWD related bleeding
A
-postpartum hemorrhage -oral and GI bleeding -hemarthrosis -hematomas -CNS bleeding
24
Q
Blue top tube
A
-Citrate anticoagulant -Use for: PT, PTT, TT, Fibrinogen, XDP, FDP, Factor assays, thrombosis assays, and vWD assays
25
Purple top tube
-used for CBC and platelet counts
26
PT (Extrinsic pathway) what it is sensitive to
-7, 10, 5, 2, Fibrinogen
27
What conditions prolong PT
-Warfarin therapy or Vitamin K deficiency -Liver disease -Acquired or hereditary deficiency of any of the factors (7, 10, 5, 2, and Fibrinogen) -Factor or assay inhibitors (drugs, antibodies)
28
aPTT (Intrinsic pathway) what it is sensitive to
-8, 9, 10, 11, 5, 2, Fibrinogen, Heparin
29
aPTT is prolonged in what conditions
-Heparin therapy or sample contamination (Hep lock, IV line) -Lupus anticoagulant -Hereditary or acquired deficiency (hemophilia) -Severe liver disease -Factor or assay inhibitors (drugs, antibodies) -Contact system deficiency (not associated with bleeding) --\> Factor 12, HMWK, Pre-Kallikrein
30
Thrombin time (TT) sensitive to
-Final step, fibrinogen -Heparin and fibrinogen
31
TT prolonged in what conditions
-Heparin therapy or contamination -Severe fibrin(ogen) degradation (DIC) -Dysfibrinogen -Thrombin inhibitor (Leperudin, drugs, antibodies)
32
Fibrinogen assay
-kinetic method of Clauss -sensitive to Fibrinogen
33
Cross-linked fibrin degradation products (D-dimer)
-assay is a quantitative latex immunoassay (rapid and available stat)
34
D-dimer is increased in what conditions
-Thrombosis (DVT, PE, extensive arterial thrombosis) IMP: quantitative NPV \>95% for PE and proximal DVT -DIC (\>8 ug/mL suggests DIC) -Thrombolytic therapy
35
Platelet count
-assay: automated cell counter
36
Platelet count is decreased in
-Leukemia and cancer therapy -ITP, TTP
37
Platelet function screen (assay)
-platelet function analyzer -whole blood is drawn through a collagen-epinephrine or collagen-ADP coated orifice -measure the time of occlusion of the orifice
38
What increases the platelet function assay time/screen
-Thrombocytopenia -Anemia -Antiplatelet drugs -vWD -other hereditary and acquired platelet dysfunction
39
Thromboelastograph (TEG)
-assay: measurement of elastic shear modules (ESM) of whole blood as it clots (5 indices) -R= time to clot onset -K= time to 2 cm ESM amplitude -Angle = angle from clot onset tangential to ESM curve -Maximum amplitude = Maximum ESM -Lysis % = % drop amplitude at the end from fibrinolysis
40
TEG sensitive to the following
-platelet count -coag factors -fibrinogen -fibrinolysis
41
Antithrombin activity
-assay: chromogenic substrate anti-IIa asay -Thrombin is Factor IIa -plasma is diluted in heparin buffer, excess thrombin is added -AT/Hep complex binds to and inhibits thrombin -Residual thrombin is measure with chromogenic substrate -Difference orginal-final thrombin equals AT activity
42
Antithrombin activity is decreased in what conditions
-Heparin therapy -ongoing extensive thrombosis -systemic coagulation activation (DIC) -Nephrotic syndrome -Hereditary deficiency -Asparaginase therapy
43
Protein C activity (assay)
-Chromogenic assay, plasma diluted in buffer -snake venom Protein C activator is added -Protein C activity is measured with chromogenic substrate -measures carboxylated and non-carboxylated protein C molecules
44
Protein C activity is decreased in what conditions
-Warfarin therapy -Vitamin K deficiency -Liver disease -Ongoing extensive thrombosis -systemic coagulation activation (DIC) -Hereditary deficiency
45
Protein S (assay)
-Free protein S antigen (preferred screening assay) quantitative latex immunoassay (LIA test) -Protein S activity -conditions affecting this assay: interference from elevated levels of Factor VIII (false low) thrombin inhibitors (false high, Lepirudin)
46
Free protein S antigen and Protein S activity decreased in what conditions
-Warfarin therapy -Vitamin K deficiency -Liver disease -Ongoing or extensive thrombosis -systemic coagulation activation (DIC) -Pregnancy -Inflammatory syndromes with increase C4B binding protein -Hereditary deficiency
47
Lupus inhibitor assay (statclot assay)
-assay: lupus sensitive PTT with and without addition of excess hexagonal phase phospholipid -calculate the difference in clotting time with and without hexagonal phospholipid
48
Activated Protein C resistance
-PT based assay -Factor V activation and inhibition by APC -calculate the ratio with and without APC added -Reduced ratio indicates APC resistance--\> Factor V leiden
49
von Willebrand Factor Antigen
-assay: quantitative automated latex immunoassay
50
von Willebrand Factor Function
-assay: enzyme immunoassay- antibody directed to functional site on vWF -highly correlated with Ristocetin cofactor assay -Type 1: reduction in parallel with vWF antigen -Type 2: greater reduction than vWF antigen
51
Collagen binding- vWF function
-assay: collagen coated plate, plasma vWF binds to collagen, bound vWF detected with anti-vWF antibody -Type 1: reduction in parallel with vWF antigen -Type 2: greater reduction than vWF antigen
52
von Willebrand Factor Multimers
-assay: Western Blot of von Willebrand Factor -Type 2A: loss of intermediate and high MW multimers, abnormal forms -Type 2B: loss of high MW multimers, normal pattern
53
Factor VIII Activity
-assay: clot based single stage assay
54
What is primary hemostasis ?
* Formation of the platelet plus
55
What is secondary hemostasis ?
* activation of the coagulation proteins
56
What is the role of endothelium in the
coagulation process?
* physical barrier with pro-coagulant properties when the wall is breached
* subendothelial matrix proteins:
* fibronectin
* collagen
* thrombospondin
* cause platlet adhesion
* Adventitia of vessel contains Tissue Factor
* activates the extrinsic pathway of the Coagulation cascade
57
What is Tissue Factor and what is its role in coagulation ?
* intrinsic membrane protein
* constituitively expressed on the plasma membrane of adventitial cells of vessel
* activates Extrinsic Pathway
* must be activated by endothelial injury, not normally present in active form
58
What are the prothrombotic properties of the endothelium ?
* major synthetic and storage site for von Willebrand factor
* protects sites of injury from pre-mature fibrinolysis
* Thrombin-activatable fibrinolysis inhibitor
* Plasminogen Activator inhibitor-1 (PAI-1)
59
What is von Willebrand Factor ?
* large multimeric protein that acts as the intercellular glue binding platelets to one another and to the subendothelial matrix at the site of injury
* during synthesis in the endothelium large monomers are dimerized
* they become large multimers of various lengths
* before storage and secretion: vWF propeptides are cleaved from the multimers
* VWF multmers are stored in Weibel Palade bodies along with ADAMTS13
60
How does VWF lead to formation of a clot?
* Note: major role in platelet function as well as carrier protein for Factor VIII
* secreted as ultra-large vWF multimers that are stretched out by shear stress and remain anchored to the endothelium
* have increased platelet binding activity
* require cleavage by ADAMTS13 to prevent pathologic platelet thrombi
* cleaves at specific sites
* creates shorter multimers that form globular structures
61
What is the role of Thrombin-activatable fibrinolysis inhibitor (TAFI)
in thrombosis ?
* a protein that is found on the endothelium that promotes thrombosis
* protects from premature clot dissolution at the site of injury
* cleaved to its active form by Thrombin-Thrombomodulin complex
* Thrombomodulin is found on the endothelial surface
* only known ligand of Thrombin
* when bound to thrombomodulin, thrombin activity focuses on TAFI
* TAFI catalyzes the removal of lysine residues from the fibrin clot
* becomes less recognizeable as a substrate for plasmin
* so the clot persists
62
What is the role of plasminogen activator inhibitor type I
(PAI-1) in promoting thrombosis ?
* PAI-1 is produced by the endothelium and is only activated by inflammatory cytokines in this setting
* other sources: platelets, adipose tissue and liver
* PAI-1 blocks the activity of plasminogen to activate plasmin
* plasmin is the primary enzyme of fibrinolysis
63
What are the antithrombotic properties of
the endothelium ?
* Secretion of tissue factor pathway inhibitor
* assembly of the protein C activation complex
* via thrombomodulin and endothelial protein C receptor
* acceleration of antithrombin activity on the endothelial survace by Heparin sulfate
* secretion of prostacyclin and nitric oxide
* secretion of tissue plasminogen activator
64
How does activated protein C downregulate
the formation of thrombin ?
* activated protein C causes proteolysis of Factor Va and VIIIa to prevent the formation of thrombin.
65
How does Heparin-sulfate contribute to
thrombin degeneration ?
* Heparin sulfate is a cofactor for antithrombin
* this is one of the main inhibitors of coagulation enzymes
* Note: the thrombin-antithrombin complex is removed by hepatocytes from circulation
66
How are platelets inactivated by the endothelium
to prevent thrombosis ?
* by production of PGI2 (prostacyclin) and NO (nitric oxide)
* release of mediators induced by excess thrombin from adjacent vessel injury
* thrombin binds and activates protease activated receptor type 1 (PAR-1)
* activates G coupled proteins
* causes production of PGI2 and NO
* both are potent platelet inhibitors
67
Where is tissue plasminogen activator (tPA) secreted from ?
* endothelium
* main enzymatic activator of the potent fibrinolyitic plasmin
* resting endothelium secretes tPA at low levels
68
What are the Vitamin K dependent factors
and what enzyme is key in their production ?
* Factors II, VII, IX, X, proteins C and S
* zymogen forms of Vitamin K dependent serine proteases
* modification occurs via: Gamma Glutamyl Carboxylase
* adds a carboxyl group to the proteins
* allows the proteins to adhere to phospholipid surfaces and assemble macromolecular coagulation complexes
69
What is the enzyme that Warfarin intereferes with ?
* Vitamin K Epoxide reductase
* interferes with the recycling of reduced Vitamin K
* Note: dietary or medicinal Vitamin K bypasses Warfarin
70
Why are newborns susceptible to Vitamin K deficiency ?
* lack the acquired GI flora responsible for the production of Vitamin K
71
What enzymatic variations are most common in
the individual variation in response to Warfarin ?
* VKORC1 - vitamin K epoxide reductase 1
* CYP2C9 (responsible for breakdown of Warfarin)
72
What two factors are not essential to the
Coagulation cascade ?
* Factor XII and prekallikrein
73
In vivo, where is tissue factor normally seen?
* constituitively present on adventitial fibroblasts
* induced expression seen on monocytes or endothelial cells
* these can deliver Tissue factor to sites of injury
74
What molecule is the sole amplifier of the
coagulation cascade ?
* Thrombin
* greatest instigator of clot formation and maintenance (thrombin burst)
* Amplifies the intrinsic pathway via:
* Factor XI
* Factor V
* Factor VIII
* Thrombin also affects:
* coagulation proteins, platelets and endothelial cells
75
What are some other functions of Thrombin ?
* converts fibrinogen to fibrin
* activates Factors XI, V, VIII and XIII
* activates protein C on the endothelium (inhibits coagulation)
* activates protease-activated receptor type I (PAR-1) on endothelium
* promotes fibrinolysis and blocks platelet activation
* activates TAFI on endothelium
* inhibits fibrinolysis
* activates PAR-1 and PAR-4 on platelets
* promotes platelet activation
76
What are the normal levels of Protein S,
what affects those levels, and what other regulatory process
is protein S involved in ?
* Functional protein S levels are normally around 40% circulating free in plasma
* 60% is bound to complement inhibitor protein, C4b
* C4b binding protein increases in pregnancy and acute phase reponses
* can decrease the fraction of free protein S
* Protein S also a cofactor
* inhibition of factor Xa by TFPI
77
What is the role of Antithrombin ?
* major serpin anticoagulant
* protease inhibitor produced in the liver
* has a Heparan binding helix
* inhibition is accelerated 1000 fold by binding of heparin to argining residues
* IMP: inhibition is not limited to thrombin
* also factor Xa, IXa, XIa and VIIa
* protease gets covalently bound to antithrombin once trapped
78
What is the role of Heparin cofactor II ?
* serpin with activity against thrombin
* produced in the liver, specific only to thrombin and not other factors
* 40% of it colocalizes to extravascular tissues
