Explain the mechanism of action of Heparin
Heparin has an anticoaguland and antithrombotic properties. It can inhibit thrombosis by inactivating factor Xa and thrombin factor IIa.
What is the role of haemostasis?
- In a physiological state, it maintains fluid blood flow within the vasculature.
- On a vessel injury/trauma, it limits/arrests bleeding by forming a clot at the site of injury while maintaining blood flow in the rest of the vessel.
- Results in removal of blood clot following wound healing.
What is Thrombosis?
- Thrombosis is the pathological manifestation of haemostasis.
- It can cause restriction/blockage of blood vessel, causing hypoxia and tissue damage.
- Thrombi can dislodge, leading to embolisation.
Broadly, what are the three stages of haemostasis?
1. Vascular Spasm - damaged blood vessels constrict, reducing blood flow to the damaged area.
2. Platelet Plug Formation - (primary haemostasis) Platelets bind to the damaged vessel wall and form a platelet plug.
3. Coagulation - (secondary haemostasis) A stable clot forms by converting fibrinogen to fibrin.
VPA = vascular platelet plug and coagulation.
Coagulation = conversion of fibrinogen to fibrin.
What are platelets?
- Platelets are small (2-3nm) fragments of megakaryocyte cytoplasm.
A healthy adult produces around 1011 platelets a day. The physiological range is 150-400x109 L/blood.
What is the lifespan of a platelet?
How are platelets destroyed?
By Kupfer cells in the liver or by phagocytosis in the spleen.
Describe the ultrastructure of a platelet.
What are the critical components of platelets?
- Membrane proteins
- Secretory granules
- Surface-connected open cannalicular systems (SCOCS).
What is the role of alpha-granules in platelets?
- Most priminent and numerous (around 50-80 per platelet)
- Around 200-500nm in size.
Alpha Granules Contain:
- Adhesive proteins (fibrinogen, fibronectin, vWF)
- Platelet-specific proteins (PF4, PDGF)
- Alpha-Granule Specific Proteins (P-selectin)
What is contained in the Dense-Granules of platelets?
- 2-7 dense granules/platelet, 2-300nm in diameter.
- Electron dense when stained with osmium tetroxide.
- Dense core with a halo.
- Vasoconstrictive Agents (serotonin)
- Platelet Agonist (ADP, ATP)
- Calcium and Magnesium
Basically, the dense core contains vasoconstrictive agents (i.e. serotonin) and also platelet agonists.
So when a platelet is releasing these bad boys it can start to attract other platelets.
What is the role of GPIIb3a?
It interacts with fibrinogen which has been released and tethers to molecules inside the platelets (makes the platelets do various things)
- Collegen receptors are exposed following an injury.
Explain the process of Platelet Adhesion.
1. All cells in blood vessels have a sub-endothelial layer
2. When you damage the blood vessel you have an exposure of the sub-endothelial layer.
3. Platelets will adhere to VWF using their GPIB receptors.
4. They will roll and roll and roll until they get more adherence.
5. This adhesion causes platelets to release granules and shit which attract more platelets.
6. This is a positive feedback mechanism.
Platelet activation is an example of primary haemostasis. Give an example of some platelet agonists.
- Thromboxane A2
- Adhesive proteins
Explain how platelet plugs are formed
- A platelet monolayer forms on the exposed sub-endothelial layer.
- The activated platelet monolayer release platelet agonists such as ADP, thromboxane A2 and adhesive proteins, such as fibrinogen, which recruits further platelets to the developing plug.
- As the newly-recruited platelets are attracted to the plug, they also change shape, allowing for greater platelet-platelet interactions, especially via fibrinogen cross-bridges, which bind to GPIIaIIIb
What is the coagulation cascade?
When coagulation factors (which are groups of zymogens - inactive precursors of enzymes) which cooperate in an integrated system of enzyme activation and inactivation.
- The process is sometimes characterised as a cascade because one event promts the next, as in a multi-level waterfall.
- Your serum tends to have a high concentration of inactive coagulation cascade proteins or zymogens.
Describe the instrinsic coagulation cascade
- Intrinsic pathway begins with damage to the blood vessel wall (like atherosclerosis)
- Happens in the bloodstream
- Results in production of clot made up of a mesh of fibrin, an insoluble filamentous protein derived from fibrinogen, in which platelets and blood cells are trapped.
1. Factor 12 (XIIa activated version)
2. Leads to activation of factor 11
3. Leads to activation of 9
4. Leads to activation of 8 which eventually activates factor 10.
Describe the extrinsic pathway in coagulation
- Caused by trauma to extravascular cells
- Activation of factor III
- Activation of factor 7 etc.
How do anticoagulants work?
- They come in and block the interaction between factor 7 and Xa
How does anti-thrombin work?
- It comes in at factor 11, 5, 7.
- Can have a huge role in stopping clotting.
What is the difference between antiplatelet drugs and anticoagulant drugs?
- Antiplatelet drugs interfere with platelet aggregation
- Anticoagulation drugs interfere with the coagulation cascade.
What is platelet Aggregometry?
How does a coagulation test work?
Coagulometer measures time taken for metal ball to become suspended in solution as a result in fibrinous clot in plasma.
What is: Prothrombin time (PT)
Prothrombin Time can identify issues with the extrinsic pathway:
- Factor VII
- Factor X
- Favtor V
- Factor II
- Fibrinogen/Fibrin degradation products
- Lupud Anticoagulant
- Liver Disease
What is the Thromboplastin Time? (PTT)
- These will focus more on the pathways of the intrinsic area (factors 9, 11 & 12).
What could PT and PTT results give you?
What is Immune Thrombocytopenia?
Thrombocytopenia is the term used to describe a condition of low circulating platelets (<100,000). Primary (or idiopathic) thrombocytopenia describes a condition where low-platelet counts occur spontaneously for no reason.
Immune Thrombocytopenia - causes antibodies to platelet proteins, usually GP-IIbIIIa or Ib/IX, are found in plasma. The condition is then termed Immune Thrombocytopenia (ITP).
The IgG-coated platelets are then cleared either by splenic macrophages or Kulfer cells in the liver. This can cause extreme ITP, with platelets count as low as 5,000/ul.
Patients with milt ITP can develop small haemorrhages called petechia, which appears as a rash. Severe ITP can present with extensive hematomas and cerebral haemorrhage.
What is Glanzmann's Thromboasthenia?
- There are also a number of disorders which affect platelet function.
- Glanzsmann's Thrombasthenia (GT) is an autosomal recessive bleeding syndrome affecting the megakaryocyte lineage and characterised by a lack of platelet aggregations.
GpIIb/3a are essential to platelet function and mutations can explain this disease.
Considering the large number and range of mutations which can cause GT, the pathogenesis is also very broad, from bleeding gums to severe hemorrhage.
Describe the pathophysiology of Haemophilia.
In addition to platelet number/function, disorders, there are a large number of bleeding disorders characterised by changes in clotting proteins.
Haemophilia is a recessive sex-linked X-chromosome disorder, meaning the vast majority of sufferers are male.
Haemophilia A - deficiency in FVIII
Haemophilia B - less common - FIX deficiency.
Describe the role of Thromboxane A2
Thromboxane A2 is a type of thromboxane produced by activated platelets and has prothrombotic properties.
It stimulates the activation of new platelets as well as increasing platelet aggregation.
Aspirin is a thromboxane A2 inhibitor.
How doe aspirin work?
- Inhibits platelet cyclooxygenase, a key enzyme in thromboxane A@ generation (TXA2)
- Thromboxane A2 triggers reactions that lead to platelet activation and aggregation, aspirin acts as a potent antiplatelet agent by inhibiting generation of this mediator.
This effect lasts for the life of the platelet, about 7-10 days.
Aspirin also inhibits COX-1 from arachidonic acid.
But also inhibits COX-2 which is produced in response to inflammation and is also a driver of platelet aggregation.
What are the side-effects of Aspirin?
How do ADP receptor antagonists (Clopidogrel) work? -
Inhibits the binding of ADP, preventing platelet aggregation.
- Used to reduce the chance of thrombotic events.
- Some people have mutations in the CYP2C19 enzyme, so can give different ones: prasugrel and ticagreor.
Clopidogrel is a pro-drug. It gets metabolised into its active form by hepatic CYP2C19 (a form of the P450 enzyme).
- They are generally well-tolerated. Side effects similar to those seen in aspirin treatment.
- Primary side effects of all ADP receptor inhibitor drugs is bleeding, although incidences of GI bleeding and intracranial hemorrhage are actually lower than with aspirin.
- One potentially fatal side effect of clopidogrel is thrombotic thrombocytopenic purpura (TTP) - a total inactivation of platelets.
Explain the mechanism of action of Heparin
- Heparin is a heterogenous natural polysaccharide
- Unfractionated herpain (UFH) is isolated from mammalian tissues, usually porcine intestinal mucosa or bovine lung.
- Various procedures are used to fractionate UFH to low molecular weight heparins.
What is unfractionated Heparin (UFH)?
- Unfractionated Herparin binds to AT causing a conformational change which increases its binding affinity for Fxa and thrombin.
- Thie inactivation of thrombin is achieved by UFH and AT complex forming a ternary structure with the thrombin.
What is the role of anti-coagulants?
- Primary role is to reduce the formation of fibrin.
Two primary mechanisms of action
1. Inhibit the synthesis of clotting factors.
2. Inhibit the activity of clotting factors.
Can be further seperated into 2 groups.
1. Direct thrombin inhibitors.
2. Direct Factor Xa inhibitors.
How does low molecular weight heparin work?
LMW heparin has the same pentasachharide complex as UFH but can only inactivate Fxa. Most molecules of LMWH cannot form a ternary complex with thrombin.
What are the side effects of Warfarin and Heparin?