Haemostasis Flashcards
Outline the structure of platelets
Also known as thrombocytes
Very small
No nucleus
Lifespan ~10 days
Stored in spleen
Membrane-bound fragments of cytoplasm from megakaryocytes
-Surface glycoproteins (GPs)
-Phosphatidyl serine (PS)
-Rich in microfilaments and microtubules - allows contraction
Organelles
-Alpha granules (a)
- Dense granules/bodies (d)
- g and l granules
Outline the importance of alpha granules in platelets
Growth factors
-Insulin-like growth factor
-Platelet-derived growth factor
-Transforming growth factor beta (TGFb)
von Willebrand factor
P-selectin, thrombospondin, fibronectin
Platelet factor 4 (heparin-binding chemokine)
List other granules found in platelets and outline their importance
δ (delta or dense granules)
-contain ADP or ATP
-calcium
-serotonin
γ (gamma granules)
-similar to lysosomes
-contain hydrolytic enzymes
λ (lambda granules)
-contents involved in clot resorption
Outline the function of platelets
Primary:
Initial response to endothelial damage
Local vasoconstriction
Formation of the platelet plug
Secondary:
Strengthening and reinforcement of the platelet plug
Formation of a stable fibrin clot
Tertiary
Removal of fibrin by fibrinolysis
Restore vessel patency (i.e. open / unblocked)
Discuss the process of Thrombopoiesis
Stem cells respond to Thrombopoietin (TPO) which is a growth factor that is produced primarily in liver (and kidney).
The stem cells differentiate into the megakaryocyte precursor cell, which then differentiates into the megakaryocyte.
Megakaryocytes undergo endomitosis until growth limit is reached and the cell membrane begins to bud off to form the thrombocytes
Describe how thrombopoiesis is regulated
Regulated by a negative feed back system where growth factor thrombopoietin binds to TPO receptors on both the megakaryocytes and platelets; if these are in high concentrations in the blood plasma then the level of TPO is kept low, reducing the maturation of CFU-Meg and thrombocyte production.
Describe the basic mechanism of blood clotting
Define haemostasis?
Stopping the flow of blood
Balancing between hypercoagulability (thrombosis) and bleeding (haemorrhagic diathesis)
List the steps involved in primary haemostasis
Initial (transient) response which influences vasoconstriction to reduced blood flow
Platelet adhesion
Platelet activation
Platelet aggregration
Describe the initial transient primary response
Damage to blood vessel endothelium
-Local neural reflexes (pain receptors)
-Muscle contraction (direct mechanical impact on smooth muscle cells)
Platelet activation
-thromboxane A produced
-serotonin released (δ granules)
Describe the primary response of platelet adhesion
Platelets bind to von Willebrand factor on exposed sub-endothelium via GPIb-IX-V receptor
Describe the primary response of platelet activation
-Platelets change shape
-GPIIb/IIIa receptor activation which binds collagen
-Release of agonists
a, b granule contents
ADP
serotonin
platelet activating factor
thromboxane A2
Describe the primary response of platelet aggregation
Primarily mediated by fibrinogen binding to GPIIb/IIIa on adjacent platelets
Enhanced by generation of thrombin (link with secondary haemostasis)
Leads to formation of the platelet plug
Explain the process of haemostasis in the healthy animal
Healthy endothelial cells & platelets repel each other (negatively charged)
Healthy endothelial cells produce prostacyclin (prostaglandin 2, PGl2)
-inhibits platelet activation
-effective vasodilator
Provide an overview of secondary haemostasis
Strengthening and reinforcement of the platelet plug
Involves coagulation factors (e.g. factor VIII)
Formation of a stable fibrin clot
Describe the coagulation cascade
Initiation (extrinsic pathway)
-Injury exposes TF-bearing cell to flowing blood
-small amount of FIXa and thrombin generated
Amplification
-Thrombin activates platelets (exposing PS)
-vWF released (tethers plts to site of injury by binding to GPIb/FIX receptor on one hand and sub-endothelial collagen on the other)
-Leads to activation of FXI, FVIII, FV
Propagation
-Factors assemble on the activated platelet to form intrinsic tenase (FIXa, FVIIIa and Ca2+)
-Results in FXa generation on the platelet surface
-Prothrombinase complex (FXa, FVa, Ca2+) forms and results in a burst of thrombin generation directly on the platelet (stabilisation)
Explain and interpret laboratory tests with regards to haemostasis
Describe the coagulation cascade
Initiation (extrinsic pathway)
-Injury exposes TF-bearing cell to flowing blood
-small amount of FIXa and thrombin generated
Amplification
-Thrombin activates platelets (exposing PS)
-vWF released (tethers plts to site of injury by binding to GPIb/FIX receptor on one hand and sub-endothelial collagen on the other)
-Leads to activation of FXI, FVIII, FV
Propagation
-Factors assemble on the activated platelet to form intrinsic tenase (FIXa, FVIIIa and Ca2+)
-Results in FXa generation on the platelet surface
-Prothrombinase complex (FXa, FVa, Ca2+) forms and results in a burst of thrombin generation directly on the platelet (stabilisation)
List the inhibitors of the coagulation cascade and outline their inhibition process
Antithrombin - main inhibitor of thrombin. Binds and inactivates TF Ixa, Xa, Xia & XIIIa. The enzymatic activity of AT is enhanced by heparin
TFP1 - polypeptide produced by endothelial cells. Natural inhibitor of extrinsic pathway by inhibing TF-VIIa complex. Protein S enhances the interaction of TF Xa in the presence of calcium and phospholipids
Protein C- Inhibits propagation phase of coagulation pathway. Activated by thrombin to form activated protein C, inhibiting TF V & VIII
Outline the process of tertiary haemostasis
Fibrin clot removal
-Tissue plasminogen activator (tPA) activates -plasminogen to form plasmin
-Breaks down fibrin fibres to fibrinogen degradation products (FDPs)
-Smallest = D-dimers
-FDPs act as anticoagulants by interfering with platelet aggregation and fibrin polymerisation
How is haemostasis assessed
Thrombocytopenia
abnormally low platelet count
Decreased production
Increased destruction / consumption
Thrombocytopathy
Abnormal platelet function
e.g. von Willebrand’s disease
Vascular defects
Outline the normal reference values for a dog undergoing haemostasis
Reference: 150–400 x109/L (dogs)
<100 : thrombocytopenia
50 : haemorrhage may be induced <30 : spontaneous haemorrhage
Lists the tests used to measure primary haemostasis
Buccal mucosal bleeding time (BMBT)
Simplate device
Platelet Count
Describe the Buccal mucosal bleeding time (BMBT)
test
Time taken for bleeding to stop after making a small incision
A quick test to perform in practice
Requires practice and does not determine cause of abnormality
Healthy dog: 1.7 – 4.2 min Healthy cat: 1.0 – 2.4 min
This time may be prolonged if disorder of primary haemostasis
Describe how the Simplate device is used
A device used to measure buccal mucosal bleeding time
It is applied around the dog muzzle and two prongs puncture the mucosa. A stopwatch is then used to calculate the time for haemostasis. Dogs aren’t typically anaesthetised but cats are
Describe how platelet counting can be done
EDTA sample
Counts should be performed as soon as possible to avoid clumping
Three methods can be used:
1. Haemocytometer
- Automated cell count
More accurate BUT cannot see what is happening
(e.g. cat platelets tend to clump; CKCS dogs have larger platelets) therefore must check on a smear - Blood smear
Estimated count from a blood smear
= No. x 15 x 109/L
healthy dog/cat >10 platelets / 100x oil immersion field
Check for clumping in feathered region
(pseudo-thrombocytopenia)
Lists the tests used to measure secondary haemostasis
‘Group’ coagulation tests
Coumarin toxicity (rat poisoning)
Activated coagulation time
Thromboelastography (TEG) / ROTEM
Describe the ‘Group’ coagulation tests
Measure the different coagulation factors
Prothrombin time (PT) or OSPT1
-add thromboplastin (@ TF) and Ca2+ ions to activate the extrinsic pathway
Activated partial thromboplastin time (aPTT)
-add reagent to activate the intrinsic system (surface activator & phospholipid) and Ca2+ ions – note no thromboplastin
-Time to clot formation prolonged by alterations of factors (<30% normal)
Thrombin clot time
-assesses the ability of added thrombin to convert fibrinogen g fibrin
Describe how Coumarin toxicity (rat poisoning) affects coagulation
Vitamin K triggers carboxylase which is key in the activation of factors, warfarin which is found in rat poison blocks the action of vitamin K, leading to a reduction of tissue factors. It can take up to 1-2 to start to have an effect and TF VII affected first
Describe how Activated coagulation time is measured
2ml blood in ACT tube containing contact activator
Warm (37°C)
Check for clot formation every 5–10 seconds
Record time that clot forms
Check reference range for the tube type
Describe how Thromboelastography (TEG) / ROTEM is used
Measure process of blood clotting. Machines stir blood when coagulation is added the paddle will slow
Describe the latex agglutination test (FDPs/D-dimers) and how it measures tertiary haemostasis
Measuring D dimers. Antibodies detect d dimers in the wells, if d dimer is present a speckly effect occurs
Outline other specialist tests that can be used in haemostasis
-Platelet function tests
aggregometry, adhesion assays, flow cytometry (expression of surface molecules)
-vWF assays
ELISA for vWF antigen
vWf-dependent platelet aggregation to measure function
Protein immunoelectrophoresis to measure vWf multimer distribution
-Fibrinogen concentration
-Antithrombin concentration
