Platelets and coagulation (haemostasis) Flashcards
what is haemostasis
the normal response of an injured blood vessel where a clot is formed that serves to limit a haemorrhage.
what are the 5 steps of haemostasis
- vascular spasm
- platelet plug formation
- coagulation
- clot retraction and repair
- fibrinolysis
what are the 2 stages of haemostasis
primary & secondary stage
they occur simultaneously !
what happens in the primary stage of haemostasis
- Platelets seal an area of damaged vessel wall through formation of platelet aggregation to stop it from bleeding.
- This occurs within 5 minutes of bleeding.
what happens in the secondary stage of haemostasis
- Platelet aggregate is stabilised to produce a stable fibrin clot.
- This occurs within 10 minutes of bleeding.
what are defects in primary haemostasis characterised by
- purpura (skin haemorrhages - purple coloured spots)
- Petechiae (non-raised patched on skin due to bleeding under skin)
- easy bruising
- epistaxis
- gingival bleeding
- menorrhagia (heavy menstrual bleeding)
- gastrointestinal bleeding
- Defect in patients in primary haemostasis results in excessive bleeding after trauma, surgical procedures, dental surgery
what is thrombosis
pathological clot formation that results when Haemostasis is excessively activated in the absence of bleeding.
where can thrombosis occur
- arteries
- veins
what can venous thrombosis lead to
congestion of affected part of the body
what is congestion
accumulation of excessive blood within the blood vessel of an organ or tissue
what can arterial thrombosis lead to
affects the blood supply and leads to damage of the tissue supplied by the artery ⇒ MI or stroke
what is an emboli
travelling clot, emboli can be formed in either venous or arterial thrombosis and these travel in the circulation causing thromboembolism
describe the difference between the arterial and venous system (2)
- arterial:
- high shear system
- platelets are critical in this system
(opposite is true for venous system)
how is an arterial vs venous thrombosis treated
- arterial is treated using anti-platelet drugs e.g. aspirin, clopidogrel, tricagrelor
- venous is treated using heparin, warfarin, novel oral anti-coagulants (NOACs) e.g. rivaroxaban, apixaban
how does heparin work
it is an anti-coagulant drug → inhibits clotting
works by enhancing anti-thrombin and stimulating production of prostacyclin which inhibits platelets
describe role of platelets in arteries and drugs used in thrombosis
- high sheer system so platelets critical
- arterial thrombosis → anti platelet drugs → aspirin
describe role of platelets in veins and drugs used in thrombosis
- low shear system
- venous thrombosis → anticoagulant → heparin or warfarin
define primary and secondary haemostasis and how quick they occur
- Primary: Formation of the platelet aggregate within 5 minutes of bleeding
- Secondary: Formation of a stable fibre clot within 10 minutes of bleeding
how can we distinguish between platelet and coagulation disorders?
- district pattern of bleeding in platelet and coagulation disorders
- platelet disorder:
- immediate bleeding, petechia, epistaxis, no bleeding in joints (haemathrosis)
- coagulation disorder:
- delayed bleeding, no petechia or epistaxis, haemathrosis
name a platelet and coagulation disorder
- platelet → thrombocytopenia
- coagulation → haemophilia
where is bleeding immediate - in platelet or coagulation disorders?
platelet disorders
in which disorder are petechiae found
platelet disorders (petechiae is small bleeds under the skin)
where is haemarthrosis found
coagulation disorders
in which disorders are haemotomas common
coagulation disorders
in which disorders are epistaxis common
platelet disorders
in which disorders are Menorrhagia common
platelet disorders
describe the structure of platelets
small discs, 1-2 micrometers (much smaller than RBC)
anucleate (no nucelus) → therefore has a short life span
what is the function of platelets
blood clotting
they circulate as quiescent cells surveying the integrity of the vasculature → dramatic change in morphology on activation
which cells do platelets come from
megakaryocytes.
describe how platelets are made
- Megakaryocytes have a a polyploid nucleus which undergoes endomitosis.
- Megakaryocytes interact with sinusoidal endothelial cells in bone marrow and when they are ready to release platelets, proplatelets extend into blood and platelets bud off into blood stream
how is platelet production controlled
Platelet production controlled by the cytokine thrombopoietin (TPO), which is produced in the liver
feedback mechanism for TPO levels
Feedback system: TPO concentration in blood (free TPO) is controlled by platelet binding to its receptor, thereby self- regulating platelet production
where is TPO produced
liver
what initially causes release of proteins from platelets ??
thrombin
what type of granules are found in platelets and what do they contain
alpha and dense granules
- dense granules
- ATP and ADP → activate platelets
- 5-HT (serotonin) → vasoconstriction → reduce blood flow → decrease in bleeding
- polyphosphate → -ve charge → “dense granule” → activates intrinsic clotting system
- calcium + catecholamines
- alpha granules
- VWF
- fibrinogen
- chemokines and cytokines
- membrane proteins → only expressed once platelet is activated
what are some specialised organelles in platelets (5)
- alpha granules
- contains many proteins
- dense granules
- contain ADP & ATP (feedback molecules for platelet activation)
- 5-HT (serotonin) - regulates vascular tone (promotes vasoconstriction)
- polyphosphate - activates intrinsic clotting cascade
- catecholamines
- calcium
- open canicular system
- allow platelet to spread
- microtubules
- form round structure in cytoplasm to give platelets their round shape
- dense tubular system
- where intracellular calcium is stored. This calcium is released into the cytoplasm when platelets are activated.
role of pits and channels in platelets
open canicular system → increase SA for spread
what is released form platelets that has an important role in platelet activation
TxA2 → thromboxane A2
what happens when a platelet comes across vessel wall damage
degranulation occurs to restrict bleeding
what happens when platelets are activated
When platelets are activated, they undergo de novo synthesis of TxA2 (powerful platelet agonist that stimulate platelets).
what regulates normal platelet process
- Vascular endothelium which lines blood vessels.
- It produces substances that keep platelets crescent.
- Platelets, because of their small disc shape, get marginated towards the vessel wall.
- This means they are constantly communicating with endothelium.
- If endothelium is intact the platelets remain crescent.
layers of bvs
endothelium, bm, collagen fibres, smooth muscle,
what do endothelial cells secrete and why (3)
- switches platelets off → secrete regulators that help keep cells quiescent under normal conditions
- PGs and NO
- inhibits coagulation
- eg tissue factor inhibitor
- activating factors
- VWF
- TF
- adhesion molecules
what happens when a blood vessel is injured
- Injury to the blood vessel exposes subendothelial collagen.
- VWF (from endothelial cells) will bind to the exposed collagen
- VWF provides a bridge to which platelets can bind to the vessel wall.
- This results in activation of platelets and the platelets come to a full stop.
- They degranulate and begin forming platelet aggregates which seal the area of vessel wall damage to limit bleeding.
- The Platelet Plug then forms within minutes
- Platelets provide surface for which thrombin is generated. From thrombin, fibrin is formed which allows for stabilisation of the platelet plug (as thrombin converts soluble fibrin into fibrinogen)
what is VWF
- von willebrand factor
- protein found in blood and also synthesised and released by platelets and endothelium
- anchors platelets to sub-epithelium → acts a bridge
via what does VWF bind the platelets
GP1B
what is the mechanism of VWF
- In a damaged blood vessel wall, VWF binds to the exposed collagen.
- VWF undergoes a conformation change (under shear conditions) and exposes binding sites for the receptor GP1b found on platelets.
- When the platelets bind to the biding sites of VWF they slow down and roll across area of damage. This then promotes aggregation.
how is VWF regulated
- It is regulated by an enzyme (VWF protease) which cleaves the molecule and breaks it down into classic Multimer forms.
- It is the biggest forms of VWF that are most effective in promoting platelet adhesion.
- VWF has to be cleaved as if not it will keep unrolling and activate too many platelets → thrombosis
what happens if VWF protease is absent
- Some patients have defect in protease (TTP defect).
- VWF protease cleaves VWF (breaks it down)
- If enzyme is absent then we get no cleavage of VWF so we just get big forms.
- This spontaneously causes platelet aggregation and spontaneous aggregates in organs and skin.
- (the big form is the one which platelets aggregate to)
what happens during direct platelet adhesion
- Once the platelets start rolling, slowing down and interacting with VWF other receptors come into play.
- GP6 and α2β1 (integrin) receptor on platelets allow the platelets to directly bind to the collagen.
- Mechanism:
- The binding of the platelet GP6 receptor to the collage causes platelet activation to occur.
- This activation allows the α2β1 molecule to open its binding site for collagen and allow for firm adhesion.
describe the amplification mechanism
- Signal transduction, occurring through membrane receptors on platelets, allows for the liberation of calcium from dense tubular system.
- This promotes platelet activation, secretion and changes in cytoskeleton of platelets allowing them to change shape
what do dense granules & alpha granules secrete
- Dense granules which release ADP, ATP 5-HT.
- Alpha granules. Which release many proteins including VWF.
- Platelets also make Devona TXA2 via a chemical pathway and release this as well.
how do these ADP, ATP, 5-HT, TXA2 work
- ADP, ATP, 5-HT, and TXA2 have their own GPCR on the platelet cell surface.
- These act in a positive feedback loop to the original and other platelets.
- This allows for amplification which attracts other platelets to the vicinity and allows for the formation of a platelet plug.
describe thromboxane formation
- When platelets are activated you get release of intracellular calcium from dense tubular system activates PLA2 enzyme.
- This enzyme liberates arachidonic acid from membrane bound phospholipids.
- COX1 converts the arachidonic acid to cyclic endoperoxides and then TX-synthases converts them into TxA2.
- TXA2 is released and it binds to its own receptor on the platelet (TXA2 receptor).
- NB: Defect in any of the enzymes above will result in mild bleeding disorders.
- COX 1 – Enzyme:
- COX 1 is the target for aspirin and other NSAIDS (ibuprofen).
- When taking aspirin, the drug binds to cox 1 and irreversible inhibits it.
- So taking low dose aspirin every day can prevent all platelets in system from producing TXA2 (why important anti-thrombotic).
describe ADP-signalling (LOL → only pic given)
- ATP receptors are called P2X receptors.
- ADP receptors are called P2Y receptors.
- There are 2 P2Y receptors → P2Y12 and P2Y1
- These are GPCR.
- Binding of ADP to P2Y1 receptor allows for activation of PLC enzyme.
- Binding of ADP to P2Y12 allows for inhibition of Camp production.
- As a result we are promoting platelet aggregation and shape change simultaneously.
- ATP promotes liberation of intracellular calcium which is important for downstream biochemical processes.
- Drugs such as clopidogrel, prasugrel, ticagrelor, cangrelor target P2Y12 and then inhabit platelets in patients in high risk of thrombosis. These stop ADP interacting with P2Y12
what is GP2B3A
- protein receptor found on the surface of platelets in resting state
- once platelet is activates → due to release of calcium → causes receptor to be active and so binding site revealed TXA2
- fibrin and fibrinogen interact with receptor
how is the clotting cascade controlled
- The clotting cascade is a very controlled amplification system.
- Small amounts of certain molecules allow for the generation of large amounts of thrombin.
- Concentration of molecules as you go down cascade will increase.
- Bottom red box:
- Thrombin (serine proteases) converts soluble fibrinogen into insoluble fibrin.
- It also activates factor 13 to factor 13a which cross links fibrin polymers being formed.
- Fibrin will crosslink and form polymers. Factor 13 mediates the cross linking.
- This creates stable fibrin clot (end product of clotting system).
- Thrombin (serine proteases) converts soluble fibrinogen into insoluble fibrin.
- We have a surface contact (intrinsic - in blood) pathway and extrinsic (tissue damage - out blood) pathway.
- Both of these pathways converge on factor 10 to convert it into factor 10a.
- Extrinsic pathway is activated via release of tissue factor. This activates factor 7 which becomes part of a complex to activate factor 10 to 10a.
- Intrinsic pathway: factor 12 → 12 a. 12 a converts 11 to 11a. 11 a converts 9 to 9a. Factor 9a, factor 8 and PL, form the 10a complex on the platelet surface and this produces factor 10a
- Both of these pathways converge on factor 10 to convert it into factor 10a.
- Factor 10 A convert’s prothrombin to thrombin.
- In order to achieve this factor 10a needs calcium, phospholipids. and cofactor 5 to drive it. This is called a prothrombinase complex
what mechanisms prevent intravascular clot formation (3
- Circulating natural anticoagulant proteins e.g. Anti-thrombin, protein C & protein S
- Unobstructed, non-turbulent blood flow
- Intact vascular endothelium
describe protein C activation
- Thrombin interacts with vessel wall via thrombomodulin receptor.
- This activates protein c to form activated protein C
- Protein C works with its cofactor – protein S.
- These form a complex which is really important is regulating clotting.
- Activated protein C will bind to factor 5a and 8a and break them down.
- These are cofactors in the prothrombinase and 10ase complexes so these complexes no longer form.
- If you deactivate these clotting molecules then you stop clotting via stopping generation of fibrin.
describe different ways the the coagulation cascade can be inhibited (4
- Tissue factor can be expressed on circulating monocytes and endothelial cells when they are activated.
- TF pathway inhibitor (TFPI) stops factor 7a from activating factor 10 and thrombin generation.
- Antithrombin (in presence of heparans) inhibits thrombin.
- It also inhibit factor mostly 10a, 9a, 7a and 12a and 2a.
- Protein c and s attack the cofactors 5 and 8 in the prothrombinase and 10ase complex.
- Finally when fibrin is formed it is insoluble but can be broken down via fibrinolysis and this releases fibrin degradation product.
describe fibrinolysis
- Fibrinolysis: the enzymatic breakdown of the fibrin in blood clots.
- If there is a lot of thrombin around it will activate the vessel wall and release TPA which binds to fibrin and help in the conversion of plasminogen to plasmin.
- Plasmin breaks down the fibrin which breaks down the clot and you get release of FDPs.
what are some disorders predisposing to venous thrombosis (Hereditary)
- Factor V Leiden mutation – stop ability of activated protein c to breakdown factor 5
- Prothrombin gene mutation
- Antithrombin deficiency
- Protein C deficiency
- Protein S deficiency
what are some disorders predisposing to venous thrombosis Acquired
The lupus anticoagulant
what are some anticoagulants drugs (4)
- Heparins and LMWH: target thrombin and 10 a.
- Vitamin k antagonist (Warfarin): targe vitamin K clotting factors 2 7 9 10.
- Direct thrombin inhibitors
- Direct factor 10a inhibitors.
what are some lab tests used to investigate the haemostasis system
- Platelet Count
- Prothrombin Time
- Activated Partial Thromboplastin Time
- Fibrinogen level
- Coagulation factor assays
- Platelet Aggregation studies
- Molecular Biology
Normal platelet count & pathology if over and under
- Normal Platelet Count - 150 - 400 x 10 9/L
- Above 40:
- Spontaneous bleeding uncommon
- Bleeding only occurs after trauma/lesion
- If spontaneous bleeding apparent then there may be an associated platelet function/coagulation defect
- Below 40: Bleeding is common but not always present
- Below 10: Severe Bleeding
- Platelet transfusion threshold now set at 10 x 109/L
- Be Aware of Counting Inaccuracies in thrombocytopenia
what causes Thrombocytopenia
- INHERITED forms
- DRUG INDUCED forms
- Caused by BONE MARROW FAILURE
- HYPERSPLENISM
- OTHER CAUSES
- Lymphoma
- HIV Virus
- Idiopathic Thrombocytopenia Purpura (ITP)
Congenital Platelet Disorders
- Disorders of Adhesion:
- Bernard-Soulier Syndrome: defect in GP1B – molecule that binds platelets to VWF
- Disorder of Aggregation:
- Glanzmann thrombosthenia: defect in GP2B3A.
- Disorders of Granules:
- Grey Platelet Syndrome: defect in alpha granules
- Storage Pool deficiency: defect in ability to store molecules in dense granules
- Hermansky-Pudlak syndrome: defect in dense bodies.
- .Chediak-Higashi syndrome
test of clotting pathway
- We use prothrombin time (PT) as a measure of extrinsic pathway.
- The PT is used to evaluate clotting factors 7, 10, 5, 2, 1
- We use aPTT as a measure of intrinsic pathway.
- APTT is used to evaluate clotting factors 9, 11, 8, 10, 5, 2
- What is their use:
- Finding the fault in the system
- Monitoring response to replacement of clotting factors (e.g. after FFP)
- Monitoring effect of anticoagulants
How the fault in the pathway can be identified
- If APTT is prolonged then clotting factor 8, 9, 11 and 12 may be reduced.
- If PT is prolonged it will be factor 7 that is reduced.
- If both are prolonged, the cause is a reduction in clotting factor 2 5 10 and fibrinogen.
What are some hereditary clotting factor deficiencies
- Clotting factor deficiencies
- sex linked:
- Factor VIII – Haemophilia A
- Factor IX- Haemophilia B
- autosomal recessive (rare)
- Fibrinogen, Prothrombin, FV, FVII, FX, FXI, (FXII), FXIII
- Factor XIII deficiency is associated with bleeding and impaired wound healing PT/APTT normal; clot solubility abnormal
- Factor XII, prekallikrein, HMWK deficiencies do not cause bleeding
- sex linked:
What are some hereditary platelet disorders
- Glanzmann Thrombasthenia
- Bernard – Soulier syndrome
What are the clinical features of VWD
- Synthesized in endothelium and megakaryocytes
- It forms large multimers > 20 million Daltons
- It is a carrier of factor VIII
- It acts to anchors platelets to sub endothelium
- It also forms bridge between platelets.
- Von Willebrand Disease:
- Inheritance is autosomal dominant (mostly, Type 3 is recessive whoever) so affect male and females.
- Incidence - ~1% (most common bleeding disorder)
- Mucocutaneous bleeding pattern
how is VW disease classified (type 1 vs 2 vs 3)
- T- ype 1: Partial quantitative deficiency
- Type 2: Qualitative deficiency
- Qualitative deficiency: loss of high molecular weight Multimer so molecule becomes less efficient at mediating hemostasis.
- Type 3: Total quantitative deficiency
- Type 3 everything is absent
- Type 2: antigen is normal but activity is low because largest Multimer are missing
- Type 1: things are low but not absent
what are the family of drugs with proven clinical efficacy
- COX-1 inhibitors e.g. aspirin
- ADP receptor antagonists e.g. clopidogrel, prasugrel, ticagrelor
- GpIIb/IIIa antagonists e.g. abciximab, eptifibatide, tirofiban
- PAR-1 antagonists e.g. vorapaxar, atopaxar
what is mono therapy used for
used for secondary prevention of MI/Stroke
what is combination therapy
Ticagrelor + aspirin – Acute coronary syndrome patients undergoing percutaneous coronary interventions (PCI) and Atrial fibrillation.
