Lecture 3 - Haemostasis Flashcards
Define and describe Haemostasis.
Haemostasis - the sequence of events to stop bleeding to prevent a haemorrhage
There are three components
1. Vessel spasm - After a breech in the blood vessel wall the vessels spasm to reduce blood flow, initiated and aided by thromboxane A2 and serotonin
2. Platelet adhesion, Activation and aggregation - to form a platelet plug
3. Blood clotting - Via the coagulation cascade
Describe platelet structure.
Platelets are pinched of from megakaryocytes & secreted into circulation. They last for 7-10 days.
Structure
* Large surface area (involutions) initiated by complex cascade
* α-granules contain fibrinogen, factor V, von Willebrand factor (vWF) and fibronectin
* Electron dense granules contain ADP, calcium and serotonin
During activation the contents of the granules are released.
Describe platelet adhesion during platelet plug formation.
Platelet adhesion - Endothelial cells are anchored to the vessel wall via the basement membrane (aka basal lamina). The BM is composed of a mesh of proteins including collagen. Damage to the endothelium exposes the collagen to the platelets and soluble vWF in the blood.
Describe platelt adhesion reaction during platelet plug formation
When platelets recognise collagen it signals them to anchor to the collagen and begin forming the platelet plug.
○ GPIa/IIa receptors on the surface of the platelets attach directly to exposed collagen
○ vWF that is circulation in blood attaches to exposed collagen
○ GPIb/IX/V binds to vWF which induces the exposure of the GPIIb/IIIa complex which also attaches to vWF but with a much higher affinity
Describe platelet activation as part of the platelet plug formation.
Platelet activation - platelet binding induces activation which changes the shape and increases the surface area of the platelets.
*Activated platelets release granule contents into the circulation
*Thrombin is produced via the coagulation cascade on the surface of the platelet which causes recruitment and activation of naïve platelets
*Platelets are held together via fibrinogen and vWF bridges that are formed by binding to GPIIb/IIIa on platelets
Name some lab tests for platelet function
- Bleeding time - An incision is made in the forearm, the time taken for the bleeding to stop is measured (<9 mins)
- PFA-100 (platelet function analysis) - Blood is passed through agonist coated hole and the blocking time is measured
3.Light transmission aggregometry - Add platelet agonist to blood and measure the light transmission through the blood - increases as platelets aggregate
What is the coagulation cascade?
Clot formation at the site of an injury must be rapid and localised. The coagulation cascade consists of a series of proteolytic events involving the activation of circulating coagulation factors (usually serine proteases), eventually causing the production of thrombin, leading to the production and deposition of fibrin, which stabilises the platelet plug at the site of injury. The induction of a proteolytic cascade ensures the amplification of the initial clotting signal.
Describe the initiation phase of the co-agulation cascade.
The initiation phase (extrinsic pathway) occurs rapidly (a few seconds). The cascade begins when plasma comes into contact with membrane-bound tissue factor (TF), through injury to a blood vessel. This causes factor VII to bind to TF, in turn causing activation of factor VII (VIIa). TF-VIIa then binds to and causes activation of factor X (to Xa) (and a little production of IXa). Xa can convert II to IIa (thrombin), albeit in very small amounts. This pathway is quickly inhibited by Tissue Factor Pathway Inhibitor (TFPI).
Describe the amplification and propagation phases of the coagulation cascade.
The amplification and propagation phases (intrinsic pathway) occur more slowly than the extrinsic pathway – usually a few minutes. The extrinsic pathway described above produces small amounts of thrombin. Thrombin can cause activation of factors XI, V and VIII and platelets.
Activation of Factor XII: The intrinsic pathway begins when factor XII, comes into contact with exposed collagen.Upon contact with collagen, factor XII becomes activated to factor XIIa.
Activation of Factor XI: Activated factor XII (XIIa) then converts factor XI into its active form, factor XIa, through proteolytic cleavage.
Activation of Factor IX: Factor XIa, in turn, activates factor IX to factor IXa by cleaving it.
Formation of the Tenase Complex: Factor IXa, along with its cofactor factor VIIIa, forms a complex known as the tenase complex on activated platelet surfaces.
Activation of Factor X: Within the tenase complex, factor IXa proteolytically cleaves factor X, converting it to its active form, factor Xa.
Prothrombin Activation: Factor Xa, in combination with factor Va (which is generated through the action of thrombin on factor V), forms the prothrombinase complex on activated platelets. This complex then converts prothrombin (factor II) into thrombin (factor IIa) through proteolytic cleavage.
Thrombin Generation: Thrombin is a central enzyme in the coagulation cascade. It has multiple roles, including converting fibrinogen into fibrin, which forms the structural basis of the blood clot. Thrombin also activates factor XIII, which stabilizes the fibrin clot by cross-linking fibrin strands.
Formation of Fibrin Clot: Fibrin strands polymerize to form a mesh-like structure, entrapping platelets, red blood cells, and plasma proteins, leading to the formation of a stable blood clot.
What is the roll of positive feedback in the coagulation cascade.
Positive feedback in coagulation cascade encourages clot formation but this must be limited to site of injury. Therefore, regulators of clotting are required.
Antithrombin is produced by endothelial cells and blocks action of factor IX, X, XI and II (inhibits serine proteases). Heparin enhances the effect of antithrombin.
Protein C (a serine protease) and protein S cofactor are activated by thrombin binding to its receptor (thrombomodulin) on endothelial cells. Protein C+S inactivates Va, VIIIa; simulates fibrinolysis
Tissue factor pathway inhibitor (TFPI) – made in endothelial cells. Inhibits VIIa and Xa and tissue factor.
Tissue Plasminogen Activator
Most of the factors are generated in the liver therefore damage to the liver can lead to issues with the coagulation cascade.
Describe the common pathway in the coagulation cascade.
This marks the start of the COMMON PATHWAY. XaVa then converts factor II (prothrombin) to factor Iia (thrombin). Thrombin has several roles: generation of fibrin from fibrinogen (thus forming a clot), activation of factors V, VIII, XI and XIII; activation of protein C, and others. Production of XIIIa leads to the formation of a cross-linked fibrin clot.
Describe the plasma coagulation tests.
Activated partial thromboplastin time - (APTT)
* Tests the intrinsic and common pathway
* Reagents added to blood include:
○ Phospholipid - to mimic the surface of the plasma
○ Kaolin - provides a surface for activation of contact-dependent factor XII
○ Calcium
○ Citrate - binds to the calcium and therefore blocks any endogenous coagulation
* The clotting time is measured
* If elevated this indicates
○ Deficit: XII, XI, IX, VIII, X, V, II, I
○ Vitamin K deficiency
○ Liver disease
○ Von Willebrand disease (carries VIII)
Prothrombin time (PT)
* Tests EXTRINSIC and COMMON pathway
* Reagent: tissue factor/calcium, Thromboplastin that provides tissue factor and phospholipid
* Plasma is citrated
* Measure clotting time
* Calculate the international normalised ratio (INR) = PT of the individual/PT normal population
* If clotting time elevated:
○ Deficit: VII, X, V, II, I.
○ Vitamin K deficiency
○ Liver disease…
Thrombin Time (TT)
* Tests COMMON pathway
* Reagent: thrombin/calcium
* Citrated plasma
* Measure clotting time
If clotting time elevated: Low fibrinogen (e.g. DIC)
Overall
If PT ONLY elevated: Defect in extrinsic pathway
If APTT ONLY elevated: defect in intrinsic pathway
If BOTH PT and APTT raised – defect in common pathway
Describe fibrinolysis
Breakdown of clot - Fibrinolysis
Inactive plasminogen is incorporated into the clot as it forms. The plasminogen can be converted to plasmin (breaks down fibrin) via:
* Tissue plasminogen activator (t-PA) released from endothelial cells
* Factor XIIa in the platelet
Fibrin degradation products (FDPs)
* Anticoagulant interferes with fibrin polymerisation
Present in FFP used to treat disseminated intravascular coagulation (DIC)
What medication can be used to prevent and dissolve clots?
Medication to prevent clotting
* Anticoagulants
○ heparin - activates antithrombin
○ warfarin - vitamin K antagonist (vitamin K needed for activation of II, VII, IX, and X),
○ aspirin - inhibits formation of thromboxane A2 (released from platelets)
Medication to dissolve a clot
* Thrombolytic agents, convert plasminogen to plasmin
○ Streptokinase
tissue plasminogen activator (t-PA)
