Coagulation Pathway and Physiology Flashcards
Heparin Sulfate
-glycosaminoglycan -attached to the luminal surface of the endothelium -acts as a cofactor for Antithrombin (main direct inhibitor of many coagulation factors)
Antithrombin
-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)
Platelets
- discoid, anucleate fragments that can be up to 3 microns in size - complex structure including actin and myosin filaments
Zymogen forms of Vitamin K dependent serine proteases
-Factors II, VII, IX, and X -Proteins C, S, and Z
Why is Vitamin K so important for these dependent factors?
-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
How does Warfarin affect this system?
-inhibits the carboxyl addition to the glutamic acid -this effect is monitored by the clot-based PT assay
Fibrinogen (Factor I)
-Adhesive protein that forms the fibrin clot
Prothrombin (Factor II)
-vitamin K dependent -activated form is main enzyme of coagulation
Tissue Factor (Factor III)
-Lipoprotein initiator of extrinsic pathway
Calcium ions (Factor IV)
-necessary for all reactions -metal cation
Factor V (Labile factor)
-Cofactor for activation of prothrombin to thrombin
Factor VII (Proconvertin)
-Vitamin K dependent -With Tissue Factor, initiates the intrinsic pathway
Factor VIII (Antihemophilic Factor)
-Cofactor for intrinsic activation of Factor X
Factor IX (Christmas Factor)
-Vitamin K dependent factor -Activated form is enzyme for intrinsic activation of Factor X
Factor X (Stuart-Power Factor)
-Vitamin K dependent factor -Activated form is enzyme for final common pathway activation of prothrombin
Factor XI (Plasma thromboplastin antecedent)
-serine protease -Activated form is intrinsic activator of Factor IX
Factor XII (Hageman Factor)
-Serine protease -Factor that nominally start aPTT-based intrinsic pathway
Factor XII (Fibrin-stabilizing factor)
-Transamidase that cross-links fibrin clot
Prekallikrein (Fletcher factor)
-Serine protease -Activated form that participates at beginning of aPTT-based intrinsic pathway
PT is a measure of which pathway?
-Extrinsic pathway (Tissue factor and Factor VII)
aPTT is a measure of which pathway?
-Intrinsic pathway
History suggestive of vWD bleeding
-Menorrhagia -Bleeding from minor wounds -Surgical bleeding -tooth extraction -epistaxis
History suggestive of non vWD related bleeding
-postpartum hemorrhage -oral and GI bleeding -hemarthrosis -hematomas -CNS bleeding
Blue top tube
-Citrate anticoagulant -Use for: PT, PTT, TT, Fibrinogen, XDP, FDP, Factor assays, thrombosis assays, and vWD assays
Purple top tube
-used for CBC and platelet counts
PT (Extrinsic pathway) what it is sensitive to
-7, 10, 5, 2, Fibrinogen
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)
aPTT (Intrinsic pathway) what it is sensitive to
-8, 9, 10, 11, 5, 2, Fibrinogen, Heparin
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
Thrombin time (TT) sensitive to
-Final step, fibrinogen -Heparin and fibrinogen
TT prolonged in what conditions
-Heparin therapy or contamination -Severe fibrin(ogen) degradation (DIC) -Dysfibrinogen -Thrombin inhibitor (Leperudin, drugs, antibodies)
Fibrinogen assay
-kinetic method of Clauss -sensitive to Fibrinogen
Cross-linked fibrin degradation products (D-dimer)
-assay is a quantitative latex immunoassay (rapid and available stat)
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
Platelet count
-assay: automated cell counter
Platelet count is decreased in
-Leukemia and cancer therapy -ITP, TTP
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
What increases the platelet function assay time/screen
-Thrombocytopenia -Anemia -Antiplatelet drugs -vWD -other hereditary and acquired platelet dysfunction
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
TEG sensitive to the following
-platelet count -coag factors -fibrinogen -fibrinolysis
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
Antithrombin activity is decreased in what conditions
-Heparin therapy -ongoing extensive thrombosis -systemic coagulation activation (DIC) -Nephrotic syndrome -Hereditary deficiency -Asparaginase therapy
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
Protein C activity is decreased in what conditions
-Warfarin therapy -Vitamin K deficiency -Liver disease -Ongoing extensive thrombosis -systemic coagulation activation (DIC) -Hereditary deficiency
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)
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
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
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
von Willebrand Factor Antigen
-assay: quantitative automated latex immunoassay
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
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
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
Factor VIII Activity
-assay: clot based single stage assay
What is primary hemostasis ?
- Formation of the platelet plus
What is secondary hemostasis ?
- activation of the coagulation proteins
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
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
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)
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
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
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
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
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
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.
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
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
Where is tissue plasminogen activator (tPA) secreted from ?
- endothelium
- main enzymatic activator of the potent fibrinolyitic plasmin
- resting endothelium secretes tPA at low levels
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
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
Why are newborns susceptible to Vitamin K deficiency ?
- lack the acquired GI flora responsible for the production of Vitamin K
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)
What two factors are not essential to the
Coagulation cascade ?
- Factor XII and prekallikrein
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
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
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
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
- 60% is bound to complement inhibitor protein, C4b
- Protein S also a cofactor
- inhibition of factor Xa by TFPI
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
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
