haemostasis/the coagulation cascade

week 3 block 2 ctb

haemostasis

• the process to limit blood loss from damaged vessels

• the precisely orchestrated series of regulatory processes that culminate in the formation of a blood clot that limits bleeding from an injured vessel

haemostasis allows:

• blood to be in a fluid state in normal vessels

• formation of a localised haemostatic clot at sites of vascular injury 

• prevents haemorrhage

  • achieved by a balance between a procoagulant and anticoagulant reactions that occur continuously in the blood

physiological processes

• coagulation: process of formation of a haemostatic plug (clot)

• fibrinolysis: the process of the breakdown of fibrin within a haemostatic plug (clot)

pathological processes

• haemorrhage: the extravasation of blood into the extravascular space

• thrombosis: the formation of a solid mass of blood products in a vessel lumen

main components of haemostasis

• vascular wall (endothelium and subendothelial structures)

• platelets

• coagulation cascade

division of blood vessel wall

1. tunica intima (next to lumen)

2. tunica media

3. tunica adventitia

subendothelial connective tissue under  tunica intima

• endothelium= single layer of squamous cells lining the lumen of the vessel

• subendothelium = layer of connective tissue containing collagen 

role of endothelium

• antiplatelet

  • inhibits platelets/coagulation cascade and promotes the breakdown of clots

• anticoagulant

• fibrinolytic

• expresses factors to prevent thrombosis in undamaged vessels and limit clot formation to site of vascular injury

• act as a barrier between procoagulant subendothelium and coagulation factors in the blood

• damage to endothelial cells cause them to release factors which promote coagulation and exposes the subendothelium

role of platelets

• form the primary haemostatic plug

• provide a surface for recruitment and concentration of coagulation factors and acts as a catalytic membrane

steps in haemostasis

1. vasoconstriction

2. primary haemostasis

3. secondary haemostasis

4. clot stabilisation and resorption

1. vasoconstriction

• mediated by reflex neurogenic mechanisms and release of endothelin from endothelial cells

• minimises blood loss

• maximises interactions between platelets, clotting factors vessel wall

primary platelet plug

3 stages to the formation of the primary platelet plug:

1. platelet adhesion

2. platelet activation

3. platelet aggregation 

platelet adhesion

• blood contains circulating platelets

• endothelium has antiplatelet properties normally, but damage to the vessel wall exposes the subendothelium

• von Willebrand factor circulates in blood/is released by endothelial cells

• acts as an anchor between the first platelets arrive at the site of the injury and damaged the vessel wall

• vWf binds collagen 

• platelets binds to vWf 

  • monolayer of platelets form (within 1-2)

• plts then bind directly to collagen after transient tethering

platelet activation

• plts are activated once they bind to the subendothelium and change shape

• become more spherical and develop projections in their cytoplasm (makes them look spiky)

• change in shape of GPIIb/IIIa (receptors on the plt surface) 

  • allows plt-plt interactions to takes place 

• target for antiplatelet drugs 

• activated platelets release ADP and thromboxane A2: platelet release reaction

  • helps promote haemostasis and activates further platelet activation

  • can be modulated by antiplatelet drugs

• plts secrete fibrinogen (acts as a cross-bridge) and allows plts to bind to each other via their GPIIb/IIIa receptors and vWf receptors

• serotonin promotes vasoconstriction 

• calcium ions released (used in secondary haemostasis)

platelet aggregation 

• further platelets activated 

• activated platelets bind via their GPIIb/IIIa receptors to other platelets

  • form fibrinogen cross-bridges

• more and more platelets are recruited to the area and activated and get stuck together

• cross linking between platelets: platelet aggregation 

• mass of platelets form a primary haemostatic plug

• primary haemostatic plug seals vessel wall and stops bleeding 

  • stabilisation and reinforcement of the plug is necessary

secondary haemostasis: stabilisation and reinforcement of plug

• acts on the fibrinogen cross-bridges between platelets 

fibrin generation 

• damage to vessel wall → exposure of tissue factor on subendothelial cells

• TF binds and activates factor VII → coagulation (clotting) cascade

• thrombin generated → cleaves fibrinogen into fibrin

• fibrin polymerises into long chains

• consolidates primary platelet plug and forms a secondary haemostatic plug

clotting/coagulation cascade

• proteolytic cleavage of pro-enzymes to active enzymes

• amplification system

• proteins involved are called clotting/coagulation factors 

• goal: produce thrombin which converts fibrinogen to fibrin, stabilising the clot (secondary haemostasis)

coagulation cascade requirements

• coagulation factors (pro-enzymes) → activated coagulation factors (enzymes) 

  • factors XII, XI, IX, X, VII and prothrombin (factor II)

  • active form indicated by ‘a’ e.g: FXIa

• cofactors (reaction accelerators) 

  • factors V and VIII 

• negatively charged phospholipid surface

• activated plts

• Ca2+ ions

• vitamin K

  • factors VII, IX, X and prothrombin require are dependent on vit K for correct production

coagulation pathways

• traditional pathways:

  • extrinsic 

  • intrinsic

(separated by laboratory assays)

• both lead to final common pathway

extrinsic pathway

• prothrombin time (PT)

• in lab, initiated by adding tissue factor, phospholipid, calcium to a plasma sample and recording the time for a fibrin clot to form

• called extrinsic factor as the blood isn’t normally exposed to TF (as it’s in the subendothelium)

• TF activates factor VII and forms a complex with calcium ions

• this complex activates factor IX in intrinsic pathway

• and factor X starts the final common pathway

  • the amount of factor Xa produced in extrinsic pathway is small compared to the factor Xa produced in intrinsic pathway

intrinsic pathway

• clinically measured as activated partial thromboplastin time (aPTT)

• initiated in lab by adding negatively charged particle, phospolipids and calcium to a plasma sample and recording the time for a fibrin clot to form

• intrinsic pathway initiated when factor XII comes into contact with a negatively charged surface

  • (this would be the cell membrane of an activated plt in the body)

intrinsic pathway cont

• factor XII activation

• which cleaves factor XI into XIa

• this then cleaves factor IX into factor IXa

• activated factor IX forms a complex with factor VIIIa and calcium ions: the tenase complex

• tenase complex is powerful activatory for factor X (tenase is an enzyme for factor X)

  • therefore large amounts of factor X are produced

final common pathway

• begins with activation of factor X either by tenase complex from intrinsic pathway or tissue factor VIIa calcium complex from extrinsic pathway

• factor Xa forms a complex with factor Va and calciu ions called: prothrombinase complex

• prothrombinase cleaves prothrombin to thrombin

actions of thrombin

• conversion of fibrinogen to fibrin

• amplifies coagulation process by further activating:

  • FXI

  • FVIII

  • FV

• activates FXIII → covalently cross linking fibrin polymers which stabilises the secondary haemostatic plug

• further platelet activation 

• proinflammatory effects: contributes to tissue repair and angiogenesis (process of forming new blood vessels from pre-existing ones)

• anticoagulant effects: when interacting with normal endothelium → helps limit clots to the site of the injury

in vivo pathway

• initiation phase

• amplification phase

initiation phase of in vivo pathway

1. exposure of TF in the sub endothelium which then activates and binds to TF VII

2. form a complex with calcium ions and does 2 functions:

• activates TF IX 

• activates a small amount of factor X

only a small amount of factor Xa is produced

amplification phase of in vivo pathway

3. the surface of an activated plt acts as a catalyst for the conversion of a small amount of prothrombin to thrombin by Xa on its own

   (prothrombin= factor II and thrombin = factor IIa)

4. thrombin then activates factor VIII to factor VIIIa and factor V to factor Va

5. first phase of in vivo pathway = initiation phase

6. factor VIIIa and factor IXa form the tenase complex

7. potent activator for factor X than TF VIIa-calcium complex

8. large amount of factor X is produced and factor Xa and forms a complex with factor Va (prothrombinase complex)#

9. thrombin converts fibrinogen → fibrin 

control mechanisms are needed:

• to ensure restriction of coagulation to the site of injury

• to prevent spontaneous activation of coagulation in the absence of injury 

factors that limit coagulation

• dilution: washes away coagulation factors

• need for a negatively charged surface provided by activated platelets 

• adjacent intact endothelium: antiplatelet, anticoagulant, fibrinolytic

• circulating inhibitors:

  • antithrombin III: actively augmented by heparin-like molecules on intact endothelium → inhibits thrombin, FIXa, FXa, FXIa and FXIIa

• fibrinolytic cascade

  • limits the size of clots amd contributes to their breakdown

• heparin

actions of adjacent intact endothelium

• physical separation of blood from subendothelium

• platelet inhibitory factors

• fibrinolytic effects

  • tissue plasminogen activator (t-PA)

• anticoagulant effects

  • TF pathway inhibitor: inhibits the TF-FVIIa- Ca2+ complexes

  • thrombomodulin and endothelial protein C receptor: activates protein C, protein C/S complex inhibits factors Va and VIIIa

  • heparin-like molecules: binds and activates antithrombin IIIfibrinolytic effects

heparin MOA

• bnds reversibly to antirhombin III and enchances inactivation of thrombin and FXa

  • activates antithrombin III by inducing a conformational change that opens up antithrombin III

• inhibits thrombosis (low dose)

• prevents progression of existing clots (higher dose)

• used in prophylaxis and treatment of venous thromboembolism

unfractioned heparin

• inactivates both FXa and thrombin

• unfractioned heparin contains longer chains that can also stabilise antithrombin III complexes with thrombin, leading to thrombin inactivatioN

low molecular weight heparin

• e.g: dalteparin

• primarily inactivates FXa

• heparin-activated antithrombin III binds FXa directly via the open active site and inactivates FXa

• preferred to UFH in pts (except in severe renal failure)

  • because LMW heparin has a more predictable response

  • more favourable side effects profile

  • doesn’t need routine plasma monitoring or dose adjustments

Fondaparinux

• synthetic pentasaccharide (similar structure to heparin)

• binds irreversibly to antithrombin III

• enhances antithrombin III’s ability to inhibit factor Xa but doesn’t inactivate thrombin (due to shorter chain length)

• treatment indications:

  • VTE prophylaxis for surgical pts

  • treatment of unstable angina and NSTEMI

warfarin

• affects vitamin K metabolism

• inhibits synthesis of vitamin K-dependent coagulation factors (factors VII, IX, X and prothrombin)

• prophylaxis and treatment of venous thromboembolism and prevention of ischaemic stroke in AF

direct oral anticoagulants (DOACs)

• e.g: dabigatran: competitive, reversible thrombin inhibitor

• has a similar/greater efficacy than warfarin, fewer drug interactions and no monitoring at standard doses required

clot stabilisation and resorption

• FXIIIa mediates formation of covalent cross-links between fibrin polymers

• polymerised fibrin and platelet aggregates undergo contraction to form a permanent plug

• counter-regulatory mechanisms limit the coagulation to the site of the injury

• clot reabsorption and tissue repair 

• involves the fibrinolytic system (mechanism by which clots are broken down)

  • important to limit clot size and contributing to their dissolution when they’re no longer needed

D-dimer

• degradation product of crosslinked fibrin (by factor XIIIa) and indicates ongoing activaition of haematostatic system

• non-specific

• can be raise in many physiological/pathological condition

  • infection

  • inflammation

  • malignancy

  • pregnancy

• negative test can be useful but +ve is less useful

fibrinolytic system

• enables clot breakdown

  • removes and limits clot size

• inactive circulating plasminogen is converted to plasmin

• converted either by FXII-dependent pathway or by plasminogen activators (tissue plasminogen activator t-PA)

• plasmin breakd down fibrin polymers

• antifibrinolytic factors oppose fibrinolysis 

haemorrhage

• extravasation of blood into extravascular space due to blood vessel damage 

  • tissues

  • body cavities

  • out of the body

• can result in:

  • purpura (visible haemorrhage into skin/mucous membrane) → non blanching

  • ecchymoses (bruises) 

    • larger, often associated with trauma

  • petichiae (very small haemorrhages, less than 3mm)

mechanisms of haemorrhage

• damage to blood vessel

  • trauma

  • atherosclerosis

  • inflammatory/neoplastic erosion

  • chronically congested tissues

• defective haemostasis: haemorrhagic diatheses

  • inherited (haemophilia A/factor VIII deficiency)

  • acquired (disseminated intravascular coagulation DIC)

factors affecting clinical significance of haemorrhage 

• volume of blood loss

• rate of blood loss

• medical fitness pre blood loss

• site of bleeding

• chronic/recurrent external blood loss