Haemostasis: Coagulation/Fibrinolysis Flashcards
What is the process of platelet plug formation?
- Platelet adhesion to the vessel wall
- Platelet activation
- Amplification of activation
- Platelet aggregation and stabilization
- Control mechanisms
What is the process of platelet activation?
- Adhesion
- Activation is mediated by different signal transduction pathways
- Ultimate result is an increased cytosolic calcium
- Amplification of activation
- Aggregation
- Control Mechanisms
Describe the adhesion process in platelet activation.
Receptors on the platelet surface membrane can directly or indirectly bind with collagen to promote adhesion.
Low shear rates:
1. Initial binding is through GPIa/IIa
2. Further binding is through the GPVI receptor and integrin α2β1
High shear rates:
3. Von Willebrand factor via GP1b
Describe the activation process in platelet activation.
- Collagen and VWF mediate activation through tyrosine kinase signalling
- Other agonists bind to receptors that mediate activation through G proteins
- Agonists: Thromboxane A2 (TxA2),
fibrinogen, thrombin, ADP, and adrenaline
- Agonists: Thromboxane A2 (TxA2),
Platelet activation ultimately results in increased cytosolic calcium. This results in: (5)
- Shape change
- Activation of phospholipase A2, releasing arachidonic acid (AA) from membrane
- Granule secretion
- Increased negative charge on platelet membrane
- Activation of αIIbβ3 (GPIIbIIIa), which strengthens adhesion and ultimately leads to aggregation
What is another way of platelet activation?
Activation via G-protein pathways
Describe what happens during the amplification of activation step in platelet activation.
Fusion of platelet granules with membrane and controlled release of contents
2 types of granules:
1. Alpha granules
- Secrete HMW VMF fibrinogen which ↑ cross-linking of platelets and outside inactivation of αIIbβ3
- Increased number of receptors
- Contain coagulation factors V, XI, XIII, II & HMW kininogens
- Dense granules
- House ADP which is a major platelet activator; when binds to receptors
leads to maximal Ca(3+) levels - Houses serotonin: relatively weak effect
- House ADP which is a major platelet activator; when binds to receptors
Generation of thromboxane A2 from arachidonic acid
Describe the aggregation step in platelet hemostasis.
Low shear rate:
Mediated by fibrinogen binding to αIIbβ3
Intermediate shear rate:
Fibrinogen, fibronectin, and VWF binding to αIIbβ3 contribute to aggregation
Highest shear rate:
VWF binding to GP1b critical
Platelet thrombus is stabilized by a fibrin meshwork
What are the control mechanisms in platelet hemostasis?
- Formation of PG12 in endothelial cells is up-regulated by TxA2
- PG12 binds to platelets and increases cytoplasmic cAMP, resulting in decreased Ca++ levels and inhibition of shape change and activation
Which glycoproteins bind to which ligands?
GP IIb & IIIa - Fibrinogen & von Willebrand Factor
GP Ib & IX - von Willebrand Factor
GP Ia & IIa - Collagen
GP V - Thrombin
Describe the assembly and release of VWF.
- Synthesis of pre-pro peptide, which consists of 22 aa signal peptide, 741 aa propeptide and 2050 aa subunit
- Exceptionally high number of cysteine residues which is important in both intra- and inter-molecule bridging required for normal function
- Extensive modification of the peptide
- Storage of multimers and propeptide in the Weibel-Palade bodies (WPBs) of endothelial cells; a granules in megakaryocytes and platelets
What are some examples of extensive modification of the peptide during the assembly and release of VFW?
- Removal of signal peptide
- Formation of C-terminal dimers of pro-VWF in the ER
- Cleavage of the propeptide followed by N-linked multimemrization in the Golgi
** multimer formation requires the presence of propeptide ** - Extensive glycosylation
What is required for the regulated storage of multimers and propeptide in the WPBs of endothelial cells and α granules in megakaryocytes and platelets?
Propeptide
What are the VWF domains and what are their functional properties?
D’ - binding sites for factor VIII and heparin
A1 - platelet glycoprotein 1b (GPIb)*
A2 - target site for ADAMTS13*
A3 - collagens
C1 - integrins, such as s αIIbβ3 (GPIIb/IIIa)
*Hidden in the globular conformation
What is the role of VWF in primary hemostasis?
- In high shear stress situations, these multimers (VWF) unfold into strings and they cross-links platelets to damaged endothelium by binding both to collagen and platelet GP1b
- Platelets bind to VWF strings like “beads on a string” (Reversible rxn)
- Platelets move slowly in the direction of flow
- Formation of additional bonds stabilize the adhesion (between VWF and glycoprotein GPIIb/IIIa)
- The interaction is strengthened by reversible association of plasma VWF with immobilized VWF
Coagulation factors are present in large amounts. T/F?
F. Coagulation factors are present in small amounts.
How is the ideal local site for clot generation found?
Due to the negative charges on activated platelets
What is the key enzyme in the cascade?
Thrombin
What are the forms of the clotting factors that circulate in the blood?
Zymogens (Inactive forms)
How are zymogens activated?
Proteases activate zymogens by cleaving 1-2 bonds of zymogen, hence activation is very rapid
List down the 3 zymogens and their respective activators.
Prothrombin (zymogen) -> thrombin (protease)
Factor IX (zymogen) -> factor IXa (protease)
Factor VIII (zymogen) -> factor VIIIa (binding protein)
What are the different coagulation factors?
Factor I - Fibrinogen (soluble)
Factor II - Prothrombin (soluble)
Factor III - Tissue factor
present in all cells of the body; released by trauma - combines w/ FVII to form the VIIa/TF compplex that initiates fast pathway
Factor IV - Calcium ions (KEY for coagulation)
Factor V - Labile factor, actually a cofactor activated by thrombin
Factor VII - Proconvertin (Vit K dependent factor that is a key component of the extrinsic or fast pathway)
Factor VIII - Antihemophilic factor (HA factor, cofactor similar to FV)
Factor IX - Christmas factor (HB factor), activated by FXIa
Factor X - Stuart-Prower factor, 1st factor in common pathway, activated by FIXa
Factor XI - Plasma thromboplastin antecedent
in vivo (at the start of the slow pathway): activated by thrombin (IIa)
In vitro: activated by factor XIIa (as measured by aPTT)
Factor XII - Hageman factor
Factor XIII - fibrin-stabilizing factor; a transglutaminase cross links the newly formed but unstable fibrin clot; activated by thrombin (IIa)
In vit-K dependent clotting factors, what are carboxylated? State the product.
Some glutamic acid residues are carboxylated to γ-carboxyglutamic acid (Gla)
What happens after Gla proteins bind to calcium ions?
- The binding of Gla to calcium leads to a conformation change
- Ca(2+)-Gla-proteins can bind to phospholipid membranes (supplied by activated platelets at the site of injury)
Why do Gla proteins change conformation (i.e what does it ensure)?
Ensures that fibrin formation occurs at the injury site instead of in the flowing blood
What is absolutely required for membrane binding?
Gla proteins
*Non-carboxylated vitamin K-dependent proteins are…?
Biologically inactive
What is the tenase complex made out of? What is the function?
Factor VIIIa and IXa
What is prothrombinase made out of? What is the function of prothrombinase?
Factors Va and Xa
*What is the effect of membrane complex on factor Xa activity?
Prothrombinase component - Relative rate of thrombin generation
factor Xa - 1
factor Xa + anionic PL - 1
factor Xa + calcium - 2
factor Xa + calcium + anionic PL - 20
factor Xa + calcium + factor Va - 360
factor Xa + calcium + anionic PL + factor Va - 280,000
*Explain the process of which Gla-proteins bind to the Platelet Plug.
- Only Ca(2+)-Gla proteins bind: factor VII, X, IX, prothrombin (called the vitamin K proteins)
- Factors Va and VIIIa also bind (not via Gla though)
- ***These complexes allow:
- Concentration
- Orientation
- Localization
*What factors depend on vitamin K for the formation of their calcium binding sites?
Factors II, VII, IX and X
*There is a slow and fast pathway in coagulation. What is the fast pathway regualted by?
Tissue factor pathway inhibitor (TFPI)
*Describe the process of factor XIII activation.
- Activated by thrombin and Ca(2+) (and other serine proteases)
- Thrombin splits off a piece from the N-terminus of the A subunits, which weakens the association of the A and B subunits
- Binding of Ca leads to the dissociation of the B subunits, a requirement for the truncated A subunits to change conformation to an enzymatically active state
What increases the activation of Factor XIII? Where is FXIII activated in the plasma? Where does it go after it is activated?
The presence of fibrin monomers increases the activation by ~100x
What is the structure of Factor XIII?
Circulates as a tetramer: FXIII-A2B2
A subunits - catalytic activity
- synthesized by hematopoietic cells
B subunits - non-enzymatic, present in excess in plasma, functions as carrier, possible inhibitor
- synthesized by hepatocytes
How is factor XIII different from other coagulation enzymes?
- Pro-transglutaminase that forms ε(γ-glutamyl)lysyl cross- links between two polypeptide chains
- Works on fibrin, an insoluble substrate, which makes it
difficult to analyze using traditional coagulation assays - The A subunit is synthesized by and is present in the cytoplasm of bone marrow cells, including megakaryocytes, platelets (at much higher concentration than in plasma), monocytes/macrophages and dendritic cells
- Also present in chondrocytes, osteoblasts, and osteocytes
- It has multiple functions in addition to its role in cross-linking fibrin
What are the primary physiological substrates of FXIIIa?
Fibrin and α₂-plasminogen inhibitor (α₂-PI)
What is the function of FXIIIa?
- Crosslinks fibrin γ- and α-chains into γ-chain dimers and α-chain polymers
- Binds to α₂-PI ti the α chains of fibrin early in thrombosis, preventing plasmin from breaking down the newly-formed clot
- Wound healing
- crosslinking a number of ECM proteins
- migration of fibroblasts and macrophages
- stimulating angiogenesis - Maintenance of pregnancy
- Decreasing vascular permeability
- Protection against cardiac rupture (mice)
- Bone metabolism
- Intracellular activities in platelets (ex. spreading, clot retraction)
How is FXIIIa inactivated?
No specific plasma protein inhibitor of FXIIIa has been identified
Enzymes released from granulocytes may play a role
Describe the fibrinolysis process.
- Activated by coagulation/Tissue damage
- Endothelium secretes tissue plasminogen activator (tPA) and plasminogen actiator inhibitor 1 (PAI-1)
- Fibrin clot binds plasminogen, tPA, and α2-antiplasmin
- tPA binds lysine-rich site on fibrin, cleaves plasminogen into plasmin, which in turn cleaves fibrin -> fibrin degradation product (FDP)
- Plasmin neutralized by α2-antiplasmin
*Give an example of an anti-platelet agent.
Aspirin
*Give 2 examples of anticoagulants that are calcium ion chelators.
Citrate
EDTA
*Give an example of anticoagulants that accelrate the inhibition of thrombin and factor XA by antithrombin (a serpin).
Heparin
*Give an example of an anticoagulant that inhibits liver carboxylation of Gla proteins.
Coumarol drugs such as warfarin
Give an example of an anticoagulant that directly inhibits thrombin and factor Xa.
New oral anticoagulants
What is antithrombin?
Antithrombin is a serine protease inhibitor (serpin). It irreversibly binds and inactivates thrombin, Xa.
Its activity considerably enhanced by
- Endothelial heparan sulfate GAGs
- UFH, LMWH
Give an example of a synthetic heparin pentasaccharide.
Fondaparinux
Does the size of the heparin chain matter for the accelerating the inhibition of factor Xa by antithrombin? What is the requirement for heparin to accelerate the inhibition of thrombin by antithrombin?
No, any size of heparin chain can accelerate the inhibition of factor Xa by antithrombin. Only heparin chains >18 sugars (unfractionated heparin) can accelerate the inhibition of thrombin by antithrombin (heparin must bind to AT and thrombin)
What is the PC/S Thrombomodulin system?
- Thrombin binds to endothelial thrombomodulin
- Complex cleaves/activates PC to APC
- Free PS binds to endothelium and acts cofactor for APC proteolytic cleavage of Va and VIIIa
How is warfarin metabolized? What is the antidote? How is warfarin monitored?
Hepatic metabolism by cytochrome P450 (CYP450)
Vitamin K
INR
- frequently associated with serious ADE resulting in emergency room visits or hospitalizations
- [Patient PT/MN PT (sec)]^ISI
What are the aPTT reagents?
Partial thromboplastin: contains phospholipids but no TF
- Phospholipids is a substitute for the membrane of activated platelets
- There are different phospholipids in different reagents
What are the uses of aPTT testing?
- Screening test
- Monitor anticoagulation therapy
- Detection of inhibitors
