Inflammation and platelets

Question 1

what is the role of platelets?

Answer 1

At site of vascular injury e.g. clot, trauma, platelets adhere and stop bleeding in arteries
- Platelets stick to damage and become activated – release granular molecules to recruit more platelets for clot formation – productive response to prevent fatal bleeding

Question 2

what happens if platelets are too occlusive?

Answer 2

If platelet is too occlusive: - stop oxygen delivery to tissues – ischaemia and thrombosis
- Arterial thrombosis – myocardial infarction – heart attack
- Stroke – clot in brain – ischaemia
- Uncontrolled platelet activation and aggregation leads to pathogenic thrombosis

Question 3

how do anti-platelet drugs work?

Answer 3

Anti-platelet drugs stops two platelet receptors
- e.g. aspirin stops recruitment phase of platelet formation
- Not many drugs available for stopping the initial phase
- can also inhibit platelet aggregation - but can cause drastic effects and cause bleeding, so not used anymore

Question 4

how does aspirin work?

Answer 4

Aspirin blocks cox1 – prevents amplification and further recruitment - stops recruitment/amplification phase

Question 5

are there multiple ways to activate platelets?

Answer 5

yes there are multiple receptors and ligands which can activate and recruit platelets

Question 6

what receptors are involved in initial recruitment of platelets?

Answer 6

e.g. GPVI, a2b1 integrins
- these are at sites of vascular injury where platelets can adhere

Question 7

what is P2Y12?

Answer 7

an inhibitor that blocks mediators that platelets release to prevent further recruitment

Question 8

what is thromboinflammation?

Answer 8

where the immune system drives a clot in response to infection
- therefore, anti-platelet drugs against this don’t work, because the immune system is the driver

Question 9

how can immune control of platelets be modulated?

Answer 9

Platelets encode immune receptors e.g. CLEC-2 which interacts with M1 macrophages – influences inflammatory function and migration of macrophages

Question 10

what roles and pathologies are platelets involved in?

Answer 10

• haemostasis - prevent bleeding
• thrombosis
• cancer - metastasis
• immunity
• thromboinflammation
• infection immunothrombosis
• inflammation
• vascular integrity

Question 11

what is inflammatory haemostasis?

Answer 11

• occurs in absence of vascular injury
• triggered by inflammatory reaction at sites of innate immune cell trafficking, which leaves gaps in endothelium
• occurs in absence of platelets - only a single platelet adheres
• platelet aims to seal the damage inflicted by immune transmigration

Question 12

how is inflammatory haemostasis induced?

Answer 12

• Neutrophils transmigrate to tissue site of inflammation
• Platelets see inflammatory damage by the immune cells – no injury, but platelets recruited – seal small bleeds
• GPIIb/IIIa Integrin-independent – only one or two platelets needed at each site – no need for aggregation
• No vascular injury, just inflammatory response e.g. cytokines/cells disrupting endothelium – transmigration damages endothelium – platelet fixes this

Question 13

would an anti-aggregation drug be useful in inflammatory haemostasis?

Answer 13

Drug which inhibits aggregation would be futile as only couple platelets are used - there is no aggregation to block

Question 14

how is inflammatory haemostasis organ-dependent?

Answer 14

• specific receptor at the tissue site will be responsible e.g. distinct in brain, lung, skin – correcting one organ could disrupt other organs
• need to identify one receptor present in this process for all organs
• Inflammation is organ-dependent and depends on subendothelial matrix and stromal cells – these affect the response to the clot

Question 15

how can we investigate the role of platelets in inflammatory haemostasis?

Answer 15

• reverse arthus reaction: inject immune complexes into skin of mice to trigger local inflammation
• WT mouse should be protected from bleeding - control
• mouse deficient in platelets will have persistent bleeding at those sites, despite there being no injury
• can measure haemoglobin to measure bleeding
• can inject drugs and see their effects

Question 16

what other model can be used to look at inflammatory haemostasis?

Answer 16

lung inflammation induced by LPS (gram-negative bacteria component)
- LPS applied to normal mouse, no bleeding in lungs
- LPS applied to platelet-deficient mouse = bleeding in absence of injury in lungs
- measure haemoglobin to look at bleeding

Question 17

what cells/factors are involved in a platelet clot?

Answer 17

platelets
immune cells
coagulation factors
- lots of crosstalk and meshwork
- fine balance needed

Question 18

does blocking GPiibiiia lead to bleeding in inflammatory haemostasis?

Answer 18

Block integrins to prevent aggregation:
- platelets still can inhibit bleeding as they don’t need to aggregate to deal with inflammatory haemostasis
- integrins are not needed in inflammatory haemostasis

Question 19

what happens when neutrophil transmigration is blocked in inflammatory haemostasis?

Answer 19

• Induce immune complexes in skin and block neutrophil transmigration with PTX
• No bleeding when neutrophil migration is inhibited
• Inflammation/immune response causes bleeding

Question 20

what are the features of classical haemostasis?

Answer 20

• platelet activation and aggregation
• depends on platelet granule secretion
• activated and procoagulant platelets
• protects from injury-induced bleeding
• blocked by anti-platelet drugs
• similar mechanism in different organs
• protective mechanism

Question 21

what are the features of inflammatory haemostasis?

Answer 21

• absence of platelet aggregation
• independent of platelet granule secretion
• key role for procoagulant platelets
• protects from inflammation-induced bleeding
• resistant to anti-platelet drugs - actually makes it worse
• organ-dependent mechanisms and involvement of distinct receptors in various vascular beds
• protective mechanism

Question 22

what immune receptors do platelets express?

Answer 22

TLRs, CLECs, NODs
- Platelets can modulate immune response independent of leukocytes
- Can bind immune cells and amplify their function – improves neutrophil function, makes monocytes more inflammatory

Question 23

how can platelets act as immunomodulators?

Answer 23

• improves neutrophil function, makes monocytes more inflammatory
• Patients with higher platelet-leukocyte complexes had higher inflammatory response
• Platelets can induce NETosis in neutrophils
• Platelets can release microvesicles – membrane fragments containing inflammatory contents which can disseminate quickly through organs as they are small – move from local to systemic inflammation

Question 24

what are the main functions of platelets and their outcomes?

Answer 24

Platelet aggregation
= thrombosis

Platelet-leukocyte aggregates = Inflammation

Platelet-induces NETosis
= immunothrombosis

Microvesicle release
= dissemination of inflammation and thrombosis

Question 25

how do platelets regulate immune cell trafficking?

Answer 25

Platelets can affect immune cell trafficking at every step - Inflamed epithelium – platelets adhere to endothelium and attract immune cells - Platelets can release CXCR4 – indirect recruitment for immune cells - Inflammatory haemostasis at site of transmigration – platelets protect vascular integrity – don’t want to block this function of platelets

Question 26

how can platelets modulate macrophage function?

Answer 26

Platelets can also enter tissue from tissue to reprogramme macrophages – controls innate immunity - CXCL4/PF4 and CXCL12 help macrophage differentiation to M1 or M2 – platelets can shift pro/anti-inflammatory responses depending on the organ environment - highly complex

Question 27

what factors can platelets secrete to modulate the immune system?

Answer 27

If platelets activated, many immune molecules can be released - e.g. coagulation factors, anti-microbials, chemokines, growthfactors - Need to avoid exacerbating the system

Question 28

how do platelets induce NETosis? why is this a problem?

Answer 28

activated platelet can bind to neutrophil: - Release a mesh clot covered in DAMPs which can then activate platelets - Positive feedback loop of NETs and platelets – amplifies the response – high inflamed and proteolytic - vicious cycle where NET can recruit more platelets - Need to act and block early enough in this pathway to avoid this excessive situation

Question 29

are platelet interactions with the immune system pro or anti-inflammatory?

Answer 29

- Platelets tend to induce pro-inflammatory state in immune cells in blood - In tissues, platelet influence over immune phenotype depends on the state of those cells in the tissue - if tissue is inflammatory, platelets induce anti-inflammatory macrophages, and vice versa - they can directly bind immune cells

Question 30

how do platelets regulate neutrophils?

Answer 30

increase: - adhesion and transmigration - NET formation - phagocytosis - ROS production - TF-driven coagulation

Question 31

how do platelets regulate monocytes?

Answer 31

increase: - adhesion and transmigration - inflammatory cytokine release - TF-driven coagulation - chemotaxis - differentiation to macrophage

Question 32

how do platelets regulate macrophages?

Answer 32

increase: - thrombosis - bacterial trapping - organ- and stimuli-specific increase in inflammation but can shift macrophage from pro to anti-inflammatory, depending on the environment - If organ is inflammatory, platelets dampen macrophage inflammation, and vice versa

Question 33

how can we investigate the role of platelets using human cells?

Answer 33

need to take cells from patients - but to study immune cells, immunologists tend to deplete platelets first to avoid contamination - but this doesn't fully represent what happens in vivo

Question 34

how are platelets isolated from blood?

Answer 34

Isolate immune cells via centrifugation of patient blood Platelets in serum - platelet-rich plasma - Neutrophils surrounded by platelets too - Take into consideration that immune cell isolates contain platelets which can impact immune response - Different results due to platelet contamination which may alter results of assay - immunologists sometimes ignore the fact that platelets are present

Question 35

how is an experiment looking at platelets and NETosis conducted?

Answer 35

- Isolate neutrophils from blood - stimulate with PMA and uric acid - DNA stain for netosis net of chromatin - Positive control – induces netosis in 3 hours - if neutrophils cultured with platelets, netosis is amplified – this is what happens in patients

Question 36

how can platelet adhesion on inflamed endothelial cells be investigated?

Answer 36

ex vivo in real time: - Immobilised endothelial cells to mimic vessel and perfuse blood over it - Add TNF and TGFb inflammatory cytokines to stimulate platelets and test drugs - Can take blood from patients, perfuse onto inflammatory endothelial cell and see effect of drug - Dose of drug matters – these assays should assess efficacy of drug in patients with reprogrammed platelets, shouldn’t just use healthy donors as their platelets are different

Question 37

how can endothelial cell and platetet cooperation be seen in patients?

Answer 37

Blood perfusion over endothelial cells (HUVECs) at 1000s-1 - stain platelets with GFP - Blood with haemolysis and inflammatory cells induces more clots – replicates patient pathology Endothelial cell and platelet activation cooperate to support arterial thrombosis under haemolytic conditions

Question 38

how can platelets affect inflammatory macrophage cytoskeletal remodelling?

Answer 38

Bone-marrow derived macrophages: - IM macrophage are round and don’t migrate – stay in tissues to induce inflammation - Live microscopy – label actin green – active but doesn’t move If platelets added to M1 BMDM: - Platelet labelled in orange - IM becomes less round and become elongated – form filopodia to traffic – moves longer distances until platelets are fully phagocytosed, and then returns to anti-inflammatory state platelets can shift inflammatory state of macrophage

Question 39

how can live imaging be used to see effect of platelets on macrophage phagocytosis?

Answer 39

Phagocytosis of E.Coli beads by macrophages - Label bacterium and see endocytosis – when phagocytosed turned green - If IM is added with platelets, enhanced phagocytosis for 30 mins, which then reduces

Question 40

how can the contribution of platelets to inflammation be assessed in animal models?

Answer 40

intravital imaging: - Put mouse under microscope, label cells and image lung microcirculation e.g. in sickle cell mice, label platelets and neutrophils and see how drug affects their interaction

Question 41

how do platelets and neutrophils interact in sickle cell mice ?

Answer 41

In control mice after hour of imaging: - Neutrophil adheres to microcapillary In sickle mice, there is already a high baseline of neutrophil adherence - Pulmonary embolism via platelets which isn’t stable – need to stabilise inflammatory clot rather than remove it – removal could lead to migration of clot to other sites which could cause death

Question 42

what are the affects of a tyrosine kinase inhibitor on sickle cell mice?

Answer 42

Tyrosine kinase inhibitor (immune drug to treat leukaemia) – drug repurposing - treatment reduces clot formation - shows the need to lower immune system activity rather than block platelets - Drug blocks the platelet clot completely by inhibiting the immune system to improve vascular function

Question 43

should platelets and immune system be studied separately?

Answer 43

no - there is so much cross-talk, so need to study in parallel, not isolation