Lecture - Blood (Intro to Haemostasis) Flashcards
Clinical big picture of haemostasis:
What are the three things that happen to stop bleeding (thus haemostasis is achieved)
- Vessel constriction
- Platelets activate and you get platelet plug
- Coagulation cascade is activated and fibrin mesh formed to reinforce platelets
So blood remains fluid inside blood vessels yet is able to clot when a vessel is damaged (process is rapid but localised). Repair to a damaged vessel is rapidly initiated.
But increased bleeding or thrombosis may occur in presence of what 3 things?
- Vessel wall disease
- Abnormal numbers or function of platelets
- Reduced or abnormal coagulation proteins or
their inhibitors
What does a low concentration of platelets result in?
=A risk of bleeding
- Bleeding into the skin due to reduced platelet count – purpura
- Large purport are called ecchymoses.
- Small pinpoint sites of bleeding from capillaries are called petechiae
What are 5 requirements for normal haemostasis?
- Normal vessel walls
- Physical integrity
- Control of blood pressure - Normal endothelium
- To prevent activation of platelets and coagulation factors
- Endotelium plays imp regualtor role that damps down excessive activation of haemostatic system - Normal platelet numbers and function
- Normal amounts of functionally normal coagulation factors
- Risk of clotting if not normal - Normal blood flow and normal inhibitors of
coagulation and platelet activation
Circulating platelets:
- What’s their diameter?
- They have a flattened disk shape
- shape maintained by?
- have _____ receptors
- have two types of granules - what are they?
- have a canalicular system - what does it do?
- have the general mitochondria, glycogen etc - What are some platelet activation receptors?
- 2-4 micron
- Flattened disk shape
• shape maintained by coiled microtubule
• activation receptors
• granules: alpha granules and dense granules (dense have Ca2+)
• canalicular system – opens to outside environment
• mitochondria, glycogen, - Platelet activation receptors:
- Collagen triggers them. Receptor for collagen is Glycoproteinla (GPla) and receptor for vWF is GPlb
- Thrombin
- ADP
- Serotonin
- TXA2
- Adrenalin
- others
Platelet response to vascular damage
So you have platelet adhesion
- You have receptor glycoproteins in platelet membrane such as? What do they bind to?
- Also have other adhesion proteins
= GPIa binds to collagen in basement membrane and
tissues
-Collagen imp protein - lots in endothelium and platelets stick to it and get activated
= GPIb binds von Willebrand factor - an important
cell adhesion ligand produced by endothelial cells
- Secreted both under the endothelial cell for adhesion to the basement membrane and into plasma
-They’re also activated by vWF - comes out as a huge long chain and an enzyme chops it up to functional units
Platelet activation
What are the 4 steps to this?
- Shape change - become sphere and pseudopods form
- They undergo change in shape where they aren’t flattened discs anymore and shoot out these long pseudopods and they start to secrete the alpha and beta granules - empties them out rapidly in the the outside environment quicker
Why do we want that? Bc in the granules, you have ADP and that will activate more platelets. So we fire the trigger with collagen and now we are releasing ADP and that will activate more platelets in the environment.
They also release serotonin - helps to constrict BV
Also secrete vWF etc
Heparin is an anti-clotting factor so mopping up any of that by secreting anti-heparin
- Granules secreted - into canalicular system which empties outside environment (look above for what’s in the granules: ADP, serotonin, vWF and anti-heparin))
3. Phospholipase Arachidonic acid COX pathway Thromboxane A2 -Platets have multiple pathways for getting haemostatic system underway
The phospholipase system is activated as well. So phospholipase is released and it strips arach acid out of the membrane within cell and goes down cyclo-oxigenase pathway, we dont produce an ordinary prostaglandin - we produce specialised prostaglanin thromboxane A2. This is only made by platelts and it is the most potent activator of platelets and is a v potent vasoconstrictor.
To control TXA2 (more platelets activated by TXA2) so it doesnt cause excessive effect; it has a short half life. Either it contacts platelets or vessel walls or it decays away quickly. It is hitting the thromboxane receptor
- GPIIb & GPIIIa
activated
-During this activation process, another thing occurs - that is to modify a couple of important receptor proteins on cell membrane. These are inactive, waiting to go. When platelet gets activated, they are altered sterically so they suddenly become tru receptor for fibrinogen or vWF. Now we can cross-link platelets in a meshwork like in the diagram on slide 9. Platelets are aggregating - being bound together by the GP2b/3a recep with fibrinogen or vWF as the cross-linking agent
Fibrinogen or vWF
bind to GPIIb/IIIa and
platelets aggregate
Platelet activation initially reversible:
- Platelet activation and aggregation depends on what?
- weaker stimuli results in what? - Shearing stress from turbulent blood flow
- may particularly what? how?
- Depends on strength of activation stimuli
= Weaker stimuli result in partial release reaction and
reversible aggregation
- Platelets have impaired function subsequently - May partially activate platelets, eg hypertension,
atherosclerosis, branching of arteries
- Alters von Willebrand Factor (vWF) in plasma
- Activates platelets
- Reversible activation of GPIIb/IIIa – aggregates may form
- Secretion of some granule contents
- Production of some thromboxane A2
Platelet granules:
Release reaction results in secretion of granule contents
-what two granules are there and what do they contain?
- Dense granules
- Calcium
- ADP et al nucleotides
- Serotonin – vasoconstriction & platelet activation - Alpha granules contain coagulation factors and other proteins
- Fibrinogen, vWF, Factor V, Factor VIII
- Platelet specific: platelet factor 4 (an anti-heparin),
- beta-Thromboglobulin
- Variety of mediators of inflammation and tissue repair (example: platelet-derived growth factor (PDGF) - acts on
fibroblasts and smooth muscle cells to stimulate healing)
Platelet and haemostasis - summary
- What 6 responses are there to a damaged vessel wall?
- Platelet plugs hv little strength but they’re essential for stoping bleeding. Just read what I’ve written
- Adhesion
- Activation
- Release reaction
- More platelets induced to aggregate
- Platelets provide phospholipid – Platelet Factor 3
-Clot retraction: Contraction of platelet actin-myosin, slow ~1 hr (actin and myosin will ensure clot contracts so you have dense clot - squeezes out serum bc platets densifying whole mass) - Platelet plugs aren’t v strong. Some ppl have normal platelet count but defective clotting will have normal bleeding time (since platelets stop bleeding) but, the clot is easily dislodged by moving the tissues a little. As a result, bleeding will stop, start, stop, start etc so we need to reinforce the plug with coagnualtion and a good solid fibrin meshwork
Bleeding time test
This is a standardised assessment of microvascular bleeding - what are the five steps? What’s the reference range?
Under what 5 circumstances is the bleeding time test not attempted?
- Sphygmomanometer
- Maintain pressure in capillaries to overcome actin-myosin contraction - Alcohol swab (clean skin)
- Template cutting device
-Standardised incision – depth
& length - Filter paper - to remove blood
- Mop it every minute or so dont disturb platelet plug
Bleeding stopped by platelets - not by clotting. It’s stopped by platets preventing small vessels bleeding
- Stopwatch
= Reference range: 2-8m
___________________________
- Uncooperative patient (children)
- Elderly patients with thin skin
- Too thin to make cut to right depth - Abnormal skin
- like moles etc - Areas other than volar aspect of forearm
- Taking anti-platelet medicines
-Aspirin, Clopidogrel, (but ~10% non responders)
- Many others NSAIDS, antihistamines, etc,
- Some foods / herbs: e.g. chocolate!, garlic, ginger, etc,
in large amounts
Bleeding time test is now largely replaced by the in vitro what test?
- method
- three clinical issues that affect the bleeding time and PFA-100
PFA-100 (Platelet Function Analyser) test
Laboratory method for testing platelet function
It doesn’t produce identical information to the bleeding time test. This one is where you see how long it takes for platelets to plug up the flow
Cartridge containing a capillary coated with activators: -collagen-adrenalin (N = 82-160s) or - collagen-ADP (N = 62-100s) Method: - Fresh citrated blood - Blood drawn through capillary - Time for capillary occlusion by a platelet plug is measured
Clinical issues:
- Platelet number
- Bleeding time increases as platelet count falls below 100 x109/L - Platelet function abnormalities
- Congenital or acquired - including drug effects on platelets - Bleeding Time vs PFA
-Fewer sources of technical errors with PFA-100 test
Mainly specimen age, haematocrit, activation of sample - shaking
-Factors affecting both tests:
Some medicines, diet factors: garlic, ginger, chocolate, alcohol
What are 4 questions you can ask to find out about inherited platelet function abnormalities
cause and increased bleeding & bruising?
Are these questions reliable for excluding a carrier state in parents of affected individuals?
Do you bleed from small cuts for longer than other people? – (>5 min)
Do you get spontaneous nosebleeds?
How often & how long to they last? (- typically >5min)
Do you bruise more easily than others
How often do you get a bruise – (often monthly or more)
How large are the bruises? - (often >2.5 cm)
—- But these questions are not reliable for excluding a carrier state
in parents of affected individuals. Carriers of some single gene
abnormalities may or may not have a bleeding history.
What cell produces platelets?
- what divides and what doesn’t?
- granules are formed - what two types?
- how do platelets form?
Megakaryocytes get their name from their large nuclei formed by the fusion of multiple nuclei: These cells undergo mitotic divisions multiple times with no cytoplasmic separation. Thus, the nuclei often have two (4N), four (8N), or eight (16N) nuclear lobes and abundant cytoplasm
(OHHHHH so like in one cell, you have multiple nuclei that keep forming but the cells stays 1)
Forms alpha and dense granules
Platelet strings budded off
- How many platelets produced each day? What’s their life span?
- Aprrox how many platelets sequestered in spleen at anyone time?
- After splenectomy, what happens to platelet count?
- What happens to platelets in older and younger people?
- 1 x 10^11 annnnnnd circulating platelet’s life span is 7-10 days
- Approximately 25% of platelets are sequestered in the spleen at anyone time
- After splenectomy, the platelet count will be approximately 20% higher.
- Older platelets - reduced granules, smaller
- Younger platelets – greater haemostatic effectiveness
Thrombopoesis is the formation of platelets. Thrombocytopenia is reduced platelet count. What’s the normal reference range and then what is mild, moderate and severe thrombocytopenia like?
Reference range: 150-400 x109/L, ~12% replaced each day
Mild 50-150x109/L:
- increased bleeding with trauma / surgery if <100x109/L
- slow oozing from all cut surfaces
Moderate 20-50x109/L:
- Bruising common
Severe <20x109/L:
- Bruising with minimal trauma, petechiae, may be
spontaneous if <5x109/L
- Life-threatening bleeding into brain, from gut, retroperitoneal
Bleeding time in thrombocytpenia
- What prolongs the bleeding time and by how much?
- The bleeding time prolongs in a roughly ______ pattern as the platelet count falls below
_______
- Aspirin prolongs the bleeding
time ~3m in most people, but
- non-responders &
- hyper-responders, exist - The bleeding time prolongs in a roughly linear pattern as the platelet count falls below ~100x109/L
Causes of thrombocytopenia : what 4 process abnormalities can reduce the platelet count?
- Reduced production
- Hereditary - rare
- Acquired - common (Can come as secondary consequence of diseases like leukemia or viral infections or drugs affecting arrow) - Shortened survival: acquired disorders are
common
- Activation / consumption of platelets by coagulation
- Antibodies
- Other mechanisms - Dilutional – massive transfusion
- Usually requires 5-10+ L volume replacement
- Normal blood volume ~5-6L in an adult (70mL/kg)
- Treated with transfusion of platelet concentrates - Sequestration in the spleen
-Pooling of 30-80+% platelets in a pathologically enlarged
spleen - uncommon
Cause #1 of thrombocytopenia
Reduced production of platelets - what 5 things can cause this?
- Global marrow depression
- Chemotherapy, radiotherapy, some chemicals &
drugs - Marrow failure due to marrow replacement
= Cancer of bone marrow cells
- Leukaemia, Plasma cell myeloma, Lymphoma
= Secondary marrow infiltration –
- Cancer, fibrosis, disseminated tuberculosis, sarcoidosis, etc - Selective depression of megakaryocytes
- Some viral infections, eg chicken pox
- Many drugs and chemicals - Aplastic anaemia
= Marrow failure due to aplasia - rare
- Usually autoimmune suppression of haemopoietic stem cells - Hereditary (genetic) – rare
Cause #2 of thrombocytopenia
Shortened platelet survival due to 2 reasons - what are they?
- Increased destruction of platelets
=Autoimmune – common : ‘ITP’ - immune thrombocytopenia
- autoantibody binds to platelets – cleared by spleen
- CD8+ cytotoxic T cell destruction of platelets in some cases)
=Drug-induced antibodies
- Immune complex of: plasma protein + drug + antibody – binds to
platelet which is cleared by spleen - Activation/consumption
= Immune
- circulating immune complexes (viral infections, systemic allergic
reactions including some allergic drug reactions)
-activate platelets via Fc receptor for IgG
=Disseminated intravascular coagulation
-Gram negative septicaemia **
-any severe form of clinical shock
=Others: Not common
Acquired platelet function defects
- What are some anti-platelet drugs (long acting agents)?
- What are other drugs or food that are anti-platelet? (short acting)
Inherited platelet function defects
- Mild platelet defects cause what (3)?
- What are they caused by?
- What are three diagnostic features?
- Severe defects are rare (mild more common). They can be major defects of receptors or storage granules - what three things can be affected?
- Aspirin which targets COX so don’t get TXA2 and then platelet activation is reduced - get slower, smaller platelet plug
-Platelet COX is a
Cyclooxygenase type 1
Clopidogrel are ADP receptor blockers (antagonist) so reduced activation
GPIIb/IIIa inhibitors - so don’t get vWF and fibrinogen binding
- = Non-steroidal anti-inflammatory drugs (NSAIDS)
- Large group of agents used for musculoskeletal pain and
inflammatory disorders
- But not paracetamol, opiates and tramadol
=Many other drugs produce mild platelet impairment
_______________________
- Increased bruising, bleeding time, dental/surgical bleeding
- Heavy periods, often recurrent nose bleeding
- Iron deficiency is common due to recurrent bleeding - Caused by a wide range of genetic conditions:
- Platelet receptors / metabolic pathways
- Specific defect not usually identifiable - - History from patient
- Abnormal platelet function test
- Normal von Willebrand & coagulation factor levels - =GP1b – von Willebrand factor receptor
- Reduced platelet adhesion
- Reduced numbers of larger platelets
=GPIIb-IIIa
-‘Essential thrombasthenia’
- A family of genetic disorders with defects in one of the
2 peptides of the GPIIb & GPIIIa receptor
=Granule defects - e.g.
-Gray platelet syndrome / absence of dense granules
Normal blood flow, platelets &
the role of endothelium
- Are platelets normally able to adhere to endothelium?
- NO and Prostacyclin are secreted by endothelial cells - what do they do? (2)
- How does blood flow in the vessel?
- What clears the ADp released by activated platelets?
- Nope
- - Dilate blood vessels,
- Inhibit platelet activation
3. Velocity of flow faster in centre of blood vessels – lower shear stress at endothelial cell surface - Central red cell-rich zone - Peripheral plasma & platelet-rich zone
- Endothelial cells clear ADP
released by activated platelets
Vascular injury, endothelium & platelets
- Endothelium is activated - what two things happen?
- Endothelial cells lost - what 4 things happen?
- - P-selectin expressed: platelets may adhere
- Endothelin produced: enhances vasoconstriction - = Vessel constriction
- Nerve reflexes
= Platelets adhere to connective tissue
-Collagen & subendothelial vWF
= Platelet activation & release reaction
- Release of ADP
- Release of serotonin: vessel constriction
- TXA2 synthesised
= More platelets activated & aggregated
- Plug forms: bleeding from small vessels stops
