WK08L1 - Platelets + Endothelium (Ben) Flashcards
Describe generally what happens with regards to platelet adhesion in a vessel displaying stenosis.
- In a narrowed vessel, the same volume of blood must pass through a smaller diameter. This is achieved by acceleration of flow which results in increased shear stress (up to 30x normal value)
- Attraction of platelets to endothelium as well as endothelial binding sites for platelets will increase with increased shear stress.
- Platelet attaches to membrane + as flow pushes it away, a tether forms which holds it in place.
- On the downstream side of the stenosis, flow/shear stress is decreased, so this is the most favorable site for adhesion.
What is the difference between the main factor on which thrombosis is dependent in arteries vs. veins?
- In arteries - thrombosis is platelet-dependent due to the shear microgradients in arteries
- In veins - thrombosis is coagulation-dependent
(not really sure what the significance of this is… but he red-boxed it soooo….)
What is the process which contributes to the flow rate-dependence of platelet adhesion?
What factor other than flow rate affects this process?
-
Margination - displacement of platelets to the periphery of vessel due to high speed of central RBC flow increasing the diffusivity of platelets
- higher flow rate = more margination = more platelets contacting vessel wall = higher chance of platelet collision with binding site
- Depends also on hematocrit
- higher hematocrit means more RBCs to knock platelets to the side = more margination + adhesion = higher risk of thrombosis
(this is how i understand it… not sure if the RBCs are actually deflecting the platelets away from the center or if its just the plasma flow itself doing that)
What is the molecular trigger for the interaction of platelets with an injured vessel wall?
Collagen
- if the endothelium detaches/is injured, type IV collagen is exposed just below and platelets bind to it
(other types of collagen also exist further into the vessel wall)
What are 3 receptors on platelet membranes involved in platelet interaction with collagen?
- Glycoprotein 6 (GPVI) - always present on platelet but has low affinity for collagen
- α2β1 - an integrin which is inactive in the basal/resting state of the platelet, but can be activated by intracellular signals
- Glycoprotein 1bα - interacts with vWF to stimulate platelet adhesion (not sure how yet… will come back to it)
What is the large pro-thrombotic molecule which bridges platelets and collagen ?
Describe its structure.
What is its source?
von Willebrand Factor
- very large (10 mil Da) monomer which is inactive alone but active as a polymer (>20 but <40 monomers)
- has Cys residues at N and C-terminals for disulfide bridging w/ other monomers to form longitudinal polymers
- made and secreted by endothelium, made and retained in granules by megakaryocytes
What are the two most important binding sites on vWF?
How do they work together?
How is their action restricted?
- GP1bα binding site - in the A1 domain, binds the GP1bα platelet receptor
- Collagen binding site - in the A3 domain, binds collagen
- work together as a bridge between platelets and collagen
- restricted by their proximity on the monomer; a single monomer can’t bind both simultaneously; requires poly-merization of vWF and binding of platelets/collagen to separate monomers
vWF interacts with a numbered plasma coagulation factor.
Which one and how?
Factor VIII
- binds to D3 domain of vWF
- vWF “carries” VIII in blood and prolongs its half-life (2 hrs without, 12 hours with)
What important hemostatic molecule other than fVIII, GP1bα and collagen can vWF bind?
With what affinity and under what conditions?
GpIIb/IIIa
- another platelet membrane integrin
- binds vWF more strongly than GP1bα
- requires activation before it can bind
(not sure if this means platelet activation, or just activation of the Gp itself)
What enzyme controls the activity of vWF?
How does it do this and why must vWF be “controlled”?
ADAMTS13
- a zinc-containing metalloprotease and disintegrin
- if vWF is too large (>40 monomers), it will attract platelets even when not necessary
- ADAMTS13 cleaves vWF at its A2 domain in order to shorten vWF polymers
Where is vWF mostly produced and in what form?
How must it be altered to be better functional in other parts of the circulation?
- preferentially produced by venous endothelium in a very large form
- must be cleaved by ADAMTS13 in order to function efficiently in arteries (otherwise would overattract platelets)
What happens if ADAMTS13 continues to act on vWF after it is at optimal length for arterial function?
- ADAMTS13 can shorten vWF fragments to inactive forms (<20 monomers) and is thus an activator AND inactivator of vWF
How does shear stress affect the structure + function of vWF molecules?
- at low shear stress, vWF remains in globular form and its binding sites are masked
- at higher shear stress, vWF is stretched into linear form and multiple GP1b__α binding sites are revealed
How does shear stress affect GP1bα in relation to vWF?
What is the consequence of this for platelet adhesion?
GP1bα binding is mechanical force-dependent
- if shear stress is present, GP1bα undergoes a conformational change which increases vWF binding affinity and so-called “catch bonds” form btwn the two
- in other words, the vWF dissociaton constant (Koff) decreases
- means platelets can bind vWF efficiently even in high shear areas
Other than being a mechanical anchor for vWF, what effect does the GP1bα on platelet function?
- it produces a strong intracellular calcium signal
- this triggers platelet activation which results in granule release
What important content of platelet granules serves to enhance adhesion of platelets?
How?
(Hint: It is a common metabolic byproduct.)
ADP
- released from granules and stimulates purinergic receptors on platelet surface (P2Y1 and P2Y12)
- activation of purinergic receptors increases intracellular calcium signalling which activates integrins
What integrin, which is activated by purinergic platelet membrane receptors, serves to enhance platelet adhesion + aggregation?
How?
integrin αIIbβ3
(AKA GP IIb/IIIa)
After intraplatelet Ca++ increase via ADP-mediated activation of P2Y1 and P2Y12…
… this integrin is in active conformation and increases vWF binding strength of the platelet, as well as…
… binding fibrinogen AND vWF to form fibgen/vWF bridges which serve as attachment sites for other platelets, stimulating aggregation.
Besides ADP, what are some other activators of platelets?
5 items
- Thrombin
- Collagen
- Serotonin
- TXA2
- PAF
(this slide was barely talked about, don’t think all details of img are important, but might as well know these 5)
What anti-platelet drugs work by increasing cAMP and decreasing Ca++ in platelets?
How?
Purinergic Receptor Antagonists
- P2Y12 antagonists such as clopidogrel** and **ticlopidine block this receptor’s Gi-coupled function
- P2Y1 antagonists block this receptor’s Gq-coupled function
What endogenous receptor/ligand combination blocks platelet function by increasing cAMP?
PGI2 (prostacyclin) via the IP receptor
- Gs-coupled, increases cAMP and thus inhibits platelets
(right side of img)
How does thrombin activate platelets?
- thrombin binds to GP1bα
- once it is bound, it can cleave PAR1 or PAR4 (protease-activated receptors) at their N-terminals
- cleaved N-terminal peptides release from the receptors and become ligands of the receptors
- PAR receptors then increase calcium and block adenylate cyclase to activate platelets
(thrombin is represented by the scissors in the img below…)
Describe the activation of GP IIb/IIIa by intracellular calcium increase.
- Ca-calmodulin-dependent GEFI (guanine nucleotide exchange factor) exchanges GDP for GTP on monomeric g-protein Rap1, activating it.
- Active Rap1 binds RIAM (Rap1-interacting Adaptor Molecule) and RIAM then binds Talin
- Talin and another protein, Kindlin, can then bind the intracellular domains of GP IIb/IIIa to activate it.
(this leads to the aforementioned vWF-fibrinogen bridge formation btwn platelets with these active GP receptors + platelet contraction stabilizes the aggregate plug)
Describe the general structure of the primary cilium of an endothelial cell.
it is a membrane-covered cilium containing 9 double microtubules formed form tubulin protein, a globular protein which polymerizes to form the microtubules
