Coagulation Flashcards
What initiates primary hemostasis?
Exposure of subendothelial collagen following injury to the vessel wall
Anticoagulant properties of intact endothelium
- prostacyclin (PLT inhibitor)
- nitric oxide (vascular relaxing)
- heparin sulfate
- tissue factor pathway inhibitor (suppress extrinsic pathway)
- tissue plasminogen activator (activate fibrinolysis)
- smooth continuous surface (inert)
- expression of endothelial protein C receptors (inactivates Va, VIIIa)
- expression of cell membrane thrombomodulin
Pro-coagulant properties of damaged vasculature:
- exposure of subendothelial connective tissue (collagen binds vWF and PLT)
- vasoconstriction caused by harm
- secretion of vWF from endothelial cells and regulation of PLT and leukocyte receptors
- subendothelial cells (smooth muscle and fibroblasts contain tissue factor and FIII
This is a reversible process where pots binds to subendothelial collagen
Adhesion
- need vWF to promote or initiate activation
The bridge between PLT and collagen
VWF
When PLTs bind each other to form PLT plug
Aggregation
- blocks site of injury
- induced by ADP released from PLTs from adhesion
Function of ADP in aggregation
- induces it
- exposes fibrinogen receptor sites on the PLT membrane (GPIIb/IIIa)
- up-regulates p-selection to surface of PLT = now PLT can absorb fibrinogen from plasma and aggregate with PLTs that have adhered to wall
T or F. Fibrinogen binding is a calcium-dependent process
T!
GPIIb/IIIa deficiency/calcium/ fibrinogen deficiency =
Defective platelet aggregation
granule secretion is simultaneous with this step
aggregation
which granules promote irreversible aggregation?
TxA2, ADP, thrombin
1st wave vs 2nd wave of granule secretion
1st wave = reversible, loose agg
2nd wave = stronger stimulus; irreversble
plug stabilized via fibrin
thromboxane A2 function
Ca2+ release
vasoconstriction
PLT agg
dense plt granules
- fuse with plasma membrane to secrete contents
- contains small molecules: ADP, ATP, Ca, Mgm serotonin
ADP
promotes agg
same with Ca and Mg
prolongs vasoconstriction
serotonin
alpha plt granules
secrete contents into surface-connected canalicular system that releases contents to external environment
- contains large molecules: PF4, PDGF, B thromboglobulin, endothelial GF, transforming GF, factors V, XI, vWF, fibronogen
Inactive form of an enzyme
Zymogen
Serine protease
Active form of clotting factor whose activity depend on the amino acid serine at activation site; serine proteases hydrolyze peptide bonds at specific cleavage site to convert the next factor into another serine protease
The Serine protease are…
Kallikrein
Factor IIa
VIIa
IXaa
Xa
XIa
XIIa
Plasminogen
Protein C
What does vit K do?
Catalyses the carbosylation of the amino-terminal glutamic acids
- carbonyl groups can then bind calcium and allow for the binding of platelet factor 3
Which coat factors are vitamin K dependent?
II (prothrombin)
VII
IX
X
Protein C, S, Z
These laces are particularly rich in tissue factor
Placenta, brain
Lungs
Thymus
TF = protein + phospholipid
What is PF3?
Membrane phospholipid on platelets
Becomes exposed when PLTs are activated
Catalytic surface; localized environment for activation of clotting factors
T or F. PT pathway is really slow
T! Will feedback and activate more of the PTT coat factors (does more of the work)
Role of vWF
- carrier and stabilizes VIIIC
- adhesion of PLTs to subendothelium
- ristocetin agg of PLT
In-Vito hemostasis
TWO phases
- Initiation = on TF bearing cell; FCIIa + TF activates X and IX; Xa/Va activates small amounts of thrombin
- Propagation = large amts of thrombin on PLT; XIa binds activated PLT and IX -> IXa
Thrombin function
Activates PLT, Va, VIIIa, XIa, fibrinogen to fibrin
FVII does not just activate FX, it also activates…
IX and VIII
Without _____, FVIII is very labile
VWF
Which factor dissociates FVIII from vWF
Thrombin
Tenase complex
Active form of IX going to form more factor X; composed of FIX, VIII, PL on platelet surface
Prothrombinase complex
FXa combines to form this; with FV and PL = creates a burst of thrombin = promotes feedback into loop
Inhibitor and regulatory mechanisms of coagulation
- fibrinolytic system
- tissue factor pathway inhibitor
Protein C system
Anti-thrombin - protein Z-dependent protease inhibitor
Components of the fibrinolytic system
Plasminogen
TPA
Urokinase
Plasminogen activator inhibitor 1
Alpha 2 anti plasmin
Thrombin-activatable fibrinolysis inhibitor
Source of tpa
Damaged endothelial cells
What is plasminogen
Breaks down fibrin polymers to FDPs
- can digest both fibrin and fibrinogen
- digests FV, FVIII
- causes 1st fibrinolysis
PREVENTED BY alpha-2-antiplasmin
TPA function
- binds plasminogen and initiates digestion
- circulating TPA bound by inhibitors (PAI-1)
- recombinant TPA used to dissolve clots
Urokinase
Prevents clot formation in extra-renal collecting system
- incorpated into clot along w plasminogen & TPA
- activated in presence of fibrin and plasmin
Plasminogen activator inhibitor 1
- primary inhibitor of plasminogen activation
- inactivates TPA and urokinase
- prevents conversion of plasminogen to plasmin
- present in excess of TPA, binds circulating TPA
Alpha 2 anti-plasmin
- 1ry inhibitor of free plasmin
- rapidly and irreversibly binds free plasmin
-slows down digestion of clot
Primary inhibitor of plasminogen
PAI-1
TAFI
- thrombin-activatable fibrinolysis inhibitor
- inhibits fibrinolysis by cleaving plasminogen/tPA binding sites on fibrin
- prevent plasmin formation and fibrinolysis
Marker of thrombosis and fibrinolysis
D-dimmers
-X-linked fibres cleaved by plasmin
How can fragments inhibit hemostasis and contribute to hemorrhage?
It prevents PLT cit action and also hinders fibrin polymerization
TFPI
TISSUE FACTOR PATHWAY INHIBITOR
- 80%% bound, rest circulates
- inhibits coagulation by inactivating FXa; binds FXa & inactivates TF:FVIIa
- protein S = co-factor
- TF pathway short-lived
Protein C system
- activated thrombin binds thrombomodulin (endothelial membrane protein)
- thrombin + thrombomodulin = conformational change in thrombin = loses pro-coat properties
- this complex activates protein C
- APC combines with protein S
- APC + PS = inactivates FVa, FVIIIa
- APC promotes fibrinolysis (binds PAI-1)
Anti-thrombin
- inhibits FII, IX, X, XI, XII, Kallikrein, plasmin
- activated by heparin from mast cell granules and hep. Sulfate on endo cells
- therapeutic heparin increases activity of AT 1000x = potent inhibitor of coagulation
Protein Z dependent protease inhibitor
- inhibits FXa, FXIa (degrade)
- binds cofactor protein Z and FXa with PL and Ca 2+
= accelerated by heparin - vit K dependent!
Bernard Soulier
- aut recessive
- allows binding of vWF to bring PLT closer to collagen
- deficiency = adhesion problems
- ristocetin induces this process that we can use for aggregation studies in vitro
Bernard Soulier lab test results
- normal PT/PTT
- n to increased PLTs
- bleeding time increased
- PLT agg studies
> neg with ristocetin
> decreased w thrombin
> N w ADP, epi, collagen (don’t use GPIb)
Glanzman’s Thrombasthenia
- aut recessive
- GPIIb/IIIa allows fibrinogen to cross link PLT
- deficiency of the receptor leads to defective aggregation and bleeding issues
Glanzman’s lab test results
- normal PT/PTT and PLT #
- increased bleeding time
- PLT agg studies = normal with ristocetin only; neg with all other
Method to study dense granules
- electron microscopy
- genetic studies
Dense granule deficiency
- non-albinism = granule membrane there but no condense; inability to package; mild bleeding; PLT agg = prolonged
- albinism = Chediak Higashi; profound granule def; no response
Alpha granule deficiency
- auto recessive
- agranular appearance (can see in PBS)
- grey PLT syndrome
- prolonged BT and decreased PLT count and slightly larger in size
VWF disease
- autosomal dominant
- mutations in vWF gene (qual or quant)
- bleeding probs due to abnormal PLT adhesion
- usually accompanied by FVIII:c deficiency
- disease severity varies (depending on homogeneity)
- bleeding usually mucocutaneous (easy bruising, nosebleeds, etc.)
- less vWF = type O blood
VWF disease usually accompanied by a deficiency in
Factor VIII
VWF disease lab test results
- PLT agg = ristocetin variable and others = normal
- PT/PTT = N; PTT may be increased
- PLT - N
- BT = prolonged
- ristocetin co-factor assay = no aggregation
Treatment for vWF disease
Desmopressin
- triggers release of vWF from endothelial cells or give recombinant vWF
Hemophilia A
- F8 def
- X-linked recessive
- may have Abs to FVIII or FVIII:c
- PTT prolonged; TT = normal; inhibitor screen may be abnormal; BT = normal
- FVIII assay to tell severity of disease
F9 deficiency
Hemophilia B
- X-linked recessive
- Christmas
- prolonged PTT
- normal BT and TT
F11 def
Hemophilia C
- aut dominant
- prolonged PTT
- normal BT, TT
- inhibitors = normal
FV Leiden mutation
- aut dom thrmobophilia
- single pt mutation that removes APC binding site on FV (APC cannot inactivate it) = thrombosis/increased clots
- clot-based assay to screen; DNA/genetic studies for confirmation
Prothrombin G20210A
- inherited thrombophilia
- elevated plasma prothrombin concentration
- increase rick of thrombosis
- DNA/genetic studies/PCR to diagnose
Acquired thrombotic disorder - lupus anticoagulant/antiphospholipid
- non-specific inhibitor
- binds protein-phospholipid complex in PTT reagent
- no- in-vivo effects
- patients prone to stroke and DVT
- everything prolonged; but viper venom normal and mixing studies no correction
Causes of a quad factor deficiencies
- liver disease
- vit. K deficiency = no carboxylation and activation
- autoantibodies = inhibits action of coat factors ; FVIII most common (SLE, pregnancy, idiopathic, etc.)
NOTE -results not corrected in mixing studies ; corrected using diluted samples which dilute out inhibitor activity
Treatment for autoantibodies
Immunosuppressants
Sample requirements
- sodium citrate
- filled to capacity
- 1:9 anticoag:sample
- do not heat or freeze
- can freeze plasma
Benefits of sodium citrate
- preserves V, VIII
- stabilizes pH
- does not inhibit PLT function
- easily reversed with calcium
Why isn’t EDTA used in coat testing
- does not preserve FV
- interferes with fibrin polymerization
Why isn’t heparin used in coag testing
- activates anti-thrombin
- prolongs results
Normal results in D-dimmer testing rules out…
DIC, DVT, PE
