WK08L1 - Platelets + Endothelium (Ben) Flashcards by Leiel Lalo

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.

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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….)

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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)

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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)

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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)

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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

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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

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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)

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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)

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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

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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)

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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

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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

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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 (K_off) decreases
means platelets can bind vWF efficiently even in high shear areas

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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

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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

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What integrin, which is activated by purinergic platelet membrane receptors, serves to enhance platelet adhesion + aggregation?

How?

integrin α_IIbβ₃

(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 are the contents of alpha granules released by platelets after activation?

7 items, 4 of which have yet to be discussed

What are the basic effects of these 4?

Various proteins which amplify coagulation and aggregation…

Fibrinogen
Factor V
vWF
P-selectin - serves interaction of platelets + leukocytes
β-thromboglobulin + platelet factor 4 - neutralize heparin-like substances (anionic polysacchs.) to support coagulation
substance P - activates other platelets

What do the dense bodies (AKA delta granules) released by platelets after activation contain?

4 items

small molecules…

Ca++ - activates platelet factor XIII
ADP - activates purinergic receptors –> GP IIb/IIIa
Serotonin (5HT) - activates more platelets
poly(Pi) - activate factor XII

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…)

How can PAR activation be stopped?

cleavage of PAR by thrombin is irreversible

so to stop PAR activation, degradation of PAR or its cleaved N-terminal peptide ligand is necessary

What other calcium-dependent path for platelet activation exists, based on a molecule that the platelet itself produces? How does it produce this molecule?

Calcium signals also trigger activation of ... **Phospholipase A2 --\> arachidonic acid --\> thromboxane A2** - TXA2 can then activate other plateles

What 4 processes of platelet activation are stimulated by Gq-mediated intraplatelet calcium increase? 2 already mentioned, and 2 new process

1. **TXA2** formation via PLA2/arach. acid 2. **integrin activation** for binding of fibrin/vWF 3. **MLCK activation** for platelet contraction (similar to smooth muscle mechanism) 4. **Membrane rearrangement** - increasing neg.-charged binding sites for complex formation

Describe the activation of **GP IIb/IIIa** by intracellular calcium increase.

1. **Ca-calmodulin-dependent GEFI** (guanine nucleotide exchange factor) exchanges GDP for GTP on monomeric g-protein **Rap1**, activating it. 2. Active Rap1 binds **RIAM** (Rap1-interacting Adaptor Molecule) and RIAM then binds **Talin** 3. Talin and another protein, **Kindlin**, can then bind the _intracellular domains_ of **GP IIb/IIIa** to activate it. ## Footnote (this leads to the aforementioned vWF-fibrinogen bridge formation btwn platelets with these active GP receptors + platelet contraction stabilizes the aggregate plug)

How do membranes rearrange on _activated platelets_ to increase the efficiency of blood coagulation?

* in basal state, platelet membranes contain more **phosphatidylcholine** than **phosphatidylserine** in their outer layer * P-serine's _negative charge_ is necessary for _binding + formation of pro-coagulatory membrane complexes_ * **_scramblase_** exchanges P-serine and P-choline if _activated by calcium_ which is increased during platelet activation

Aside from ADP receptor antagonists and COX inhibitors like aspirin, what other _three_ kinds of anti-platelet drugs are there?

1. **PAR antagonists** - small peptides that antagonize natural ligand peptide of PAR 2. **GP IIb/IIIa inhibitors** * ****only in situations with _very high_ risk for platelet agg. * in acute MCI, mechanical removal of coronary clot is preceded or immediately followed by GP inhibitors * can be either _antibodies_ (**abciximab**) or _peptides_ (**eptifibatide**) 3. **PDE3 inhibitors** - block PDE3 and thus increase cAMP

Which _endothelially-produced_ _eicosanoid_ inhibits platelets? How?

**PGI2** or **prostacyclin** - raises cAMP which prevents activation + contraction of platelets

Which _endothelial enzyme_ affects platelet activation by acting on a nucleotide? Where is it located? How does it act?

**ectoADPase** - _membrane protein_ that _breaks down ADP_ to inhibit platelet activation

What _normally extravascular_ molecule is expressed by endothelial cells under certain conditions? Which conditions?

**Tissue Factor** - expressed on endothelial cell membranes in _infection_ and _hypoxia_

The function of endothelial cells in different parts of the circulation differs greatly. What endothelial molecules are expressed highly in veins? Arteries? Capillaries?

* **Veins** - thrombomodulin (TM), vWF, endothelial protein C receptor (EPCR) * **Arteries** - tPA, TM, EPCR * **Capillaries** - TM and TFPI

What protein is responsible for controlling LDL receptor "turnover"? How does it affect the receptor? What does this mean for mutations of this protein?

**PCSK9** - it binds to LDLRs and initiates their degradation - mutations of this protein slow down LDLR turnover, increasing the number of receptors available for LDL uptake, thus _decreasing atherosclerotic risk_

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

How does flow-dependent bending of an endothelial cell's _primary cilium_ result in intracellular signals?

* transmembrane proteins **PC1 + PC2** ("polycystin") on the cilium membrane act as _mechanosensors_ * **PC2** is a _calcium channel_ and interactions btwn both PCs _as a complex_ allows Ca++ entry

What happens with the PC1-PC2 complex under conditions of _no flow_?

* **PC1** has _nuclear factors_ attached to its _C-terminal_ * under no flow, the PC complex interactions change + _C-terminal proteolysis_ of PC1 allows these nuclear factors + a small piece of PC1 to be released * these factors _translocate_ to the nucleus and _alter transcription of certain genes_

What is an important target of the calcium which enters endothelial cells via _PC2_ upon bending of the primary cilium? Where does the product of this target act and how?

**NO synthase** - under flow, Ca++ enters and _activates_ NO synthase, producing NO - NO acts on _smooth muscle cells_ via its receptor **soluble guanylyl cyclase** to induce relaxation/vasodilation (guanylyl cyclase activation also leads to platelet inhibition... more on that later)

Describe the sequence of events following **guanylyl cyclase** activation by NO which will affect _smooth muscle_ activity.

1. **guanylyl cyclase** converts **GTP** to **cGMP** 2. cGMP activates **PKG** * PKG _activates_ **myosin-light-chain phosphatase** * PKG _deactivates_ **phospholipase C** 3. MLCP dephosphorylates **myosin light chain** resulting in _smooth muscle relaxation_ 4. inactive PLC can not create **IP3** and thus calcium remains stored in the SR (also inducing relaxation)

Describe the sequence of events following **guanylyl cyclase** activation by NO which will affect _platelet_ activity.

- same PLC inhibiton/MLCKP activation mechanisms from the smooth muscle card, PLUS... 1. **cGMP** (made by g. cyclase from GTP) _directly inhibits_ **phosphodiesterase 3** 2. without PDE3 to degrade cAMP, [cAMP] increases 3. high [cAMP] inhibits platelets

NO affects one of the numbered plasma factors. Which one and how?

**Factor XIIIa** - NO modifies _sulfhydryl groups_ via formation of _nitrosylated proteins_ - sulfhydryl groups are present in the active site of FXIIIa (on Cys residues) and this nitrosylation **inactivates** factor XIIIa