WK06L1 - Hemostasis (Ben) Flashcards
What is the difference between “hemostasis” and “thrombosis”?
(According to simplified definitions of the two terms.)
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Hemostasis - physiological avoidance of bleeding
- formation of a “hemostatic plug” in/around exposed extra-endothelial tissue in order to close off damaged endothelium
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Thrombosis - pathological formation of blood clots
- clot forms within the lumen of the vessel
What are three general steps of hemostasis?
And what is the time frame during which each occurs?
(More of a physiological distinction of steps than a biochemical one, according to Kolev.)
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Primary Hemostasis - platelet activation/aggregation and vasoconstriction at the point of vessel injury.
- occurs within seconds of injury
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Blood Coagulation - formation of a solid fibrin matrix
- occurs within minutes of injury
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Clot Maturation - cellular infiltration/inflammation, slow covalent cross-linking of clot
- occurs within hours to days of injury
What is the general importance of RBCs and platelets in blood clotting?
(As in, why would clotting not be effective if only fibrin networks were formed without the presence/help of these cell types?)
How do these cells and fibrin connect?
- cells fill in the spaces between the fibrin strands in order to further impede blood flow
- without the cells to “plug” these spaces, free plasma flow through the strands would be practically unchanged
- cells interact with fibrin via surface receptors
What is the importance of neutrophils in thrombus formation?
- They release NETs or Neutrophil Extracellular Traps.
- provoked by phagocytosis of bacteria and/or cytokines
- special type of apoptosis
- cell releases a fine mesh of its DNA and proteins (mostly histones) which contributes to clot formation
What are the other names for factor I / Ia?
Where is factor I formed and secreted to?
Briefly, what activates it to Ia?
(Just what activates it… not how… yet.)
Factor I = fibrinogen, Factor Ia = fibrin
- fibrinogen is formed in the liver and secreted into the blood
- thrombin activates fibrinogen into fibrin
What is the general structure of fibrinogen molecules?
How are the N and C ends of each of their chains placed within the molecule?
- 6 polypeptide chains: 2 alpha, 2 beta, 2 gamma
- N terminals of ALL chains are in the center of the molecule
- C terminals of beta and gamma form distal regions of molecule
- C terminals of alpha chains turn back and form a dimer in the center of the molecule
- central N terminals of alpha chains stabilize the C terminal dimer
What is the significance of the C terminals of the gamma prime chain of fibrinogen in binding other molecules during clot formation?
How many AAs of the C terminal are involved and what’s important about them?
Gamma prime chains of fibrinogen are longer than normal gamma chains (due to alternative splicing)…
… they have 20 extra C-terminal AAs with negative charges on the gamma chain which will bind…
…thrombin and factor XIII during clot formation
Via what part of their structure do fibrinogen molecules interact with each other?
C terminal ends of the gamma chains of fibrinogen interact with each other.
What is the consequence of increases or decreases in the amount of gamma prime chains in fibrinogen molecules?
Why?
- increased gamma prime (above 20%) = increased arterial thrombosis
- more FXIII bound to fibrin means more covalent stabilization of clots
- decreased gamma prime (below 5%) = increased venous thrombosis
- less gamma prime to bind free thrombin means more clot formation
In what form does most fibrinogen circulate in the plasma?
What structural features keep fibrinogen in this form?
Most fibrinogen circulates as a dimer of two fibrinogen molecules.
Hydrogen bonds between the C terminals of gamma chains (red part of picture) connects the two molecules.
(Specifically Tyr-Arg and Arg-Ser H bonds)
Why doesn’t fibrinogen circulate as longer polymers, rather than just dimers?
Because sheer forces of blood flow and the presence of cells in the blood would break up longer molecules.
What does thrombin do to fibrinogen alpha chains to initiate fibrin network formation?
How does this change how fibrin molecules can now interact with each other?
(Be specific about the AA sequence with which fibrins interact.)
Fibrin cleaves a 16 AA N terminal peptide from the alpha chains, leading to…
…disassembly of the C terminal dimer of the alpha chain…
…and binding of newly exposed N-terminal Gly-Pro-Arg** sequences of **alpha chains to…
C-terminals of other fibrin molecules’ gamma chains
all of this results in formation of a double stranded protofibril
What does thrombin do to beta chains of fibrin to facilitate fibrin network formation?
Thrombin cleaves N-terminals of beta chains to expose binding sites for lateral association of fibrin protofibrils.
(Protofibrils = already associated via alpha-gamma chain interactions mentioned before. Multiple ~45 nm diameter protofibrils associate laterally to form thicker ~200 nm fibers.)
How are C terminals of fibrin’s alpha chains affected by thrombin and what does this result in?
Alpha chain C terminal dimers of fibrinogen disassemble when thrombin cleaves N-terminal peptides.
This conformational change of the C terminals allows further interaction of fibrin protofibrils to form networks.
To summarize, in what 3 ways do fibrin molecules interact to form networks after being acted on by thrombin?
- N-terminal alpha chain to C-terminal gamma chain interactions form double-stranded protofibrils
- After N-terminal cleavage, beta chains allow lateral association of protofibrils
- Dissociated C-terminal alpha chain dimers can now further connect the laterally associated protofibrils
Generally, what kinds of molecular interactions are involved in fibrin polymerization?
(And what does this mean for the initial integrity of fibrin networks, before they are acted on by other factors?)
H-bonds (NOT covalent bonds) between different parts of the various chains of fibrin are responsible for its initial polymerization.
Its structural integrity is fairly low before covalent cross-linkages are formed, and sheer forces of blood flow can degrade it.
What is another name for factor XIII / XIIIa?
Fibrin Stabilizing Factor
- because it performs the covalent cross-linking of fibrin strands which keeps fibrin polymers from degrading via sheer forces of blood
What is the general structure of factor XIII and where do its different parts come from?
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2 catalytic A subunits - formed by megakaryocytes in the bone marrow
- has N-terminal activation peptides
- 2 carrier B subunits - formed by Kupfer cells in the liver
- the two types of subunits meet in the blood
How is factor XIII activated?
By what molecule and via what steps?
- Thrombin cleaves N-terminal activation peptides from the catalytic A subunits
- Physiological concentrations of Ca2+ trigger dissociation of the carrier B subunits
- A subunit catalytic site is now exposed (with cysteine sulfhydryl group important later in its inactivation)
What exactly does factor XIIIa do to stabilize fibrin networks?
(Its exact molecular-level action, including which AAs are involved.)
Factor XIIIa is a transglutaminase which covalently bonds glutamine and lysine side chains of two adjacent gamma or alpha carboxy terminals.
(This process releases ammonia.)
Describe the cascade that results in activation of prothrombin to thrombin.
This process is labeled as the initiation phase of hemostatic clotting (on the slides anyways).
Initiation
- Factor X is activated to Factor Xa by membrane-bound Factor VIIa in complex with Tissue Factor (TF)
- Factor Xa cleaves the B domain of Factor V, activating it to Factor Va.
- FXa and FVa together for the prothrombinase complex which activates prothrombin (FII) to thrombin (FIIa)
What molecule can inhibit the acceleration of clot formation which occurs specifically when blood comes in contact with cells it does not normally touch?
Mention its structure and direct actions.
Tissue Factor Pathway Inhibitor (TFPI)
- binds the active site of Ser proteases
- has 3 Kunitz (K) domains
- K-3 binds protein S which binds the TFPI to a membrane
- K-2 binds/inactivates factor Xa
- K-1 binds/inactivates factor VIIa
(binding of FXa to K-2 changes K-1 conformation to allow FVIIa binding)
What happens to platelets when they are exposed to the extracellular matrix beyond the endothelium?
What then binds to them?
Priming
They adhere to the surface of the exposed extracellular space and activate by the exposure of more negatively-charged phosphatidylserines on their membrane.
They then become a binding site for factors Va, VIIIa and XIa
(5, 8 and 11)
How can the substrate affinity of thrombin be changed by molecules other than fibrin?
And if this happens, what other substrates does thrombin then affect?
- A platelet membrane receptor, Gp1b-alpha, can bind to exosite II on a thrombin molecule
- With exosite II bound, thrombin then cleaves other platelet membrane receptors such as PAR-1 and GpV, as well as cleaving/activating factor XI (a liver-made plasma protein)
Describe the positive feedback mechanism known as propagation, which contributes to high generation of thrombin at platelet surfaces independent of tissue factor.
(Hint: it includes a numbered plasma factor that has not been mentioned in detail in the cards thus far.)
- As mentioned before… exosite II-bound thrombin –> XIa –> IXa –> Xa
- The relatively small amounts of Xa that are now formed by this cascade will activate more thrombin (IIa)
- As a positive feedback mechanism this thrombin activates plasma factor VIII to VIIIa
- VIIIa complexes with IXa to enhance its activation of X, thus increasing the rate of this entire cascade.
What general element of plasma factor activity can increase the rates of prothrombotic reactions manyfold?
What is the clinical significance of this?
Membrane-binding and complex formation by plasma factors increases their action by many orders of magnitude.
Clinical/pharmacological inhibition of membrane-binding can help decrease thrombotic risk.
What is the structure of gamma carboxy glutamate?
Simply a glutamate with another -COOH group on the gamma carbon.
How do Gla domains on plasma clotting factors influence the membrane-binding ability of these factors?
(HINT: 1 co-factor and 2 ways that presence of this co-factor influences membrane-binding.)
Gla domains (yellow) bind Ca++ (pink)
- 4 core Ca++ bind and help maintain a hydrophilic loop that can insert into the membrane
- More peripheral Ca++ ions bind and have direct interactions with negatively-charged phosphatidylserine in the membrane
- Without Ca++, disordered structure of clotting factors allows no membrane binding to occur.
What enzyme converts glutamate residues of certain clotting factor proteins in to Gla domains?
What does it use to do this?
What is the general name for the clotting factor proteins which are substrates of this enzyme?
Vitamin K-dependent Carboxylase (a gamma carboxylase)
- uses CO2 and O2 to carboxylate
- its subtrates are called vitamin K-dependent (VKD) proteins
What cofactor does the gamma carboxylase enzyme which creates Gla domains on clotting factors use?
What are the steps of the cycle that regenerates this cofactor after its use?
Vitamin K
- Hydroquinone form VKH2 enters the gamma carboxylase reaction.
- Epoxide derivative form VKO leaves rxn.
- VKOR (vit-k oxidoreductase) enzyme reconverts VKO to hydroquinone form for re-use.
