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Normal Coagulation

Normal coagulation

 

Clot formation

Initiation

1.      TF-VIIa complex catalyses Xa formation (and some IXa)

2.      Xa converts prothrombin to small quantity of thrombin

3.      Thrombin activates V, VIII and XI

 

Amplification / thrombin propagation

1.      VIIIa-IXa complex catalyses formation of Xa strongly

2.      Xa-Va complex then strongly catalyses the conversion of prothrombin to thrombin

 

Fibrin formation

1.      Thrombin converts fibrinogen to fibrin

2.      Thrombin also activates XIIIa

3.      XIIIa crosslinks fibrin to form a strong clot

 

Negative feedback

TF-VII

§   Rapidly bound and inactivated by TFPI (tissue factor pathway inhibitor)

§   Thus need for secondary propogation independent of TF-VIIa

 

Antithrombin

§   Generally damps down coagulation by forming inactive complexes with IX, X, XI and thrombin

 

Activated protein C

1.      Thrombin bound by thrombomodulin which changes thrombins activity from V, VIII and XI activation to activation of PC (particularly when this is bound to the membrane by the endothelial protein C receptor EPCR)

2.      APC binds Cofactor Protein S

3.      APC inactivates Va and VIIIa by proteolysis

 

 

Fibrinolysis

1.      Tissue plasminogen activator (tPA) binds to fibrin which then increases its activity to convert plasminogen to plasmin.

2.      Plasmin then degrades fibrin at the site of clot formation

 

 

Endothelial interactions

 

Platelet inhibition

§   NO and PGI2 normally released by endothelial cells to prevent platelet aggregation.

§   PGI2 has a half life of a few minutes and can be measured in the circulation but is probably only half the concentration required to cause platelet inhibition (unless severe trauma perhaps).

§   Ecto-ATPase (CD39) also expressed on platelet surface which metabolises ATP and ADP released by platelets thereby reducing platelet recruitment and aggregation

 

Platelet activation

Endothelin 1 (ET1) causes vasodilation and platelet aggregation

 

High molecular weight vWF secreted by endothelium

 

Thrombospondin 1

§   Abundant constituent of alpha granules of platelets and Weibel-Palade bodies on endothelial cells

§   Binds to platelet membrane which results in platelet aggregation

 

Anticoagulant properties of endothelial cells

§   Thrombomodulin constitutively expressed on all endothelial cells except brain.

§   Endothelial protein C receptor expressed on endothelial cells in the heart and lung but less expressed in capillaries in the skin, kidney and liver.

§   TFPI is synthesized and secreted by endothelial cells.

 

Vessel injury

1.      Tissue factor exposed – binds VIIa which activates coagulation cascade and leads to fibrin generation

2.      Collagen exposed which binds circulating vWF

3.      This leads to platelet aggregation and release of the platelet alpha granules further increasing the local concentration of vWF

4.      vWF leads to activation of endothelial cells which leads to:

a.      Release of Weibel-Palade bodies (further increasing vWF)

b.      Expression of P-selectin which further recruits platelets

c.      Secretion of endothelin 1 which causes vasoconstriction and platelet activation

 

Fibrinolytic factors

§   tPA and plasminogen activator inhibitor are both synthesized by endothelial cells but are not stored in granules (but synthesis can be increased in some situations).

 

Platelet physiology

§        Megakaryopoiesis is regulated by TPO (synthesised by liver)

§        TPO binds receptor, c-mpl on circulating platelets and BM megas and is then internalised and degraded

§        Total mass of cells thus determines free TPO levels and regulates megakaryopoiesis and platelet production

§        Mean life span of 10 days

§        Marginal band of microtubules found below outer surface – which depolymerise on platelet activation

§        Three types of platelet granules

§        Delta (ATP, ADP, Ca, Serotonin, Pyrophosphate, P selectin, TGF-beta, adrenaline / noradrenaline, GDP/GTP)

§        Alpha (PF4, Beta-thromboglobulin, Fibrinogen, Factor V, Thrombospondin, Fibronectin, PDGF, PAI-1, P selectin)

§        Lysosomal (Galactosidases, fusocidases, hexoaminidase, glucuronidase, cathepsin)

§        Platelet membrane is asymmetric with sphingomyelin and phosphatidylcholine on the outer surface and phosphatidyl-ethanolamine, -inositol and –serine on the inner surface

§        Contains at lease nine glycoproteins – GpI-GpIX

§        GpIa – collagen receptor

§        GP1b/X/V – vWF receptor (reduced in Bernard Soulier)

§        GPIIb/IIIa – (reduced in Glanzmann’s thrombasthenia)

§        Upon vessel wall damage

§        Adhesion

§        vWF facilitates initial adhesion via GP1b/IX/V complex

§        slows down platelets and allows binding via GPIIb/IIIa and GPIa/IIa

§        Activates the platelet via a G-protein mechanism results in the depolymerisation of the microtubule network and the platelet changes from a disc shape into a sphere with numerous projecting pseudopods

§        Aggregation and secretion

§        Two main pathways

§        Activation of PLA2 releases free AA from membrane phospholipids which is then converted to leucotienes which are important chemoattractants of white cells and TXA2.  TXA2 causes secondary granule release and local vasoconstriction as well as further local platelet aggregation.

§        Platelet agonists including thrombin, collagen, TXA2 bind receptors and activate PLC, which generates PLC – this results in the generation of IP3 and DAG, which elevate Cai and activate PKC respectively.  Raised Cai results in activation of myosin and actin. 

§        Receptor stimulation results in G protein interactions that trigger intracellular metabolic pathways

§        Shape change via cytoskeletal changes

§        Release of alpha and dense granule contents

§        Stimulation of phospholipase A2, liberation of thromboxane A2 and activation of GP IIb/IIIa receptors

 

Inhibitors of coagulation

 

1. TFPI

§        Largely expressed by endothelial cells

 

2. Endogenous heparin like molecules

§        Heparan sulphate, dermatan sulphate – expressed by endothelial cells

 

3. Protein Z and protein Z inhibitor

 

Fibrinolysis

tPA

§        Synthesised in endothelial cells

§        Cleared by the liver (increases in liver disease)

§        Binds to polymerized fibrin and promotes conversion of plasminogen to plasmin

§        Inhibitied by PAI-1 (endothelial cells) and PAI-2 (placenta)

 

Plasmin

§        Catalyses the degredation of fibrin to FDPs

§        Inhibited by alpha 2 antiplasmin (deficiency of this leads to a bleeding disorder – Miyasato disease)

 

Thrombin activated fibrinolytic inhibitor (TAFI)

§        This is activated by the thombin-thrombomodulin complex – to produce TAFIa

§        This removes the C-terminal lysine residues on fibrin reducing binding of tPA to fibrin.

 

FDPs

§        Inhibit thrombin activity

§        Will increase TT

§        Inhibit platelet function

§        Bound by protamine which will correct TT in the presence of high FDPs

 


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