Clotting Flashcards
Draw a diagram of haemostasis
What are the two phases of haemostasis
Primary
Secondary
Primary haemostasis involves?
Formation of a primary platelet plug via adherence, activation and aggregation
- Adherence as with exposure of subendothelium platelrtys adhere to vWF and collagen
- Activation due to binding and local thrombin production
- Degranulation and conformatinal changes
- Binds soluble fibrinogen GP2B3a
Vasoconstriction
What are the 3 phases of a primary paltelet plug being formed
Adhesion, activation –> aggregation
How does platelet adhesion occur? What phase of haemostasis does this represent?
Primary haemostasis
- Adherence as with exposure of subendothelium platelrtys adhere to vWF and collagen
- Activation due to binding and local thrombin production
- Degranulation and conformatinal changes
- Binds soluble fibrinogen GP2B3a
Secondary haemostasis involves?
Deposition of insoluble fibrin from the coagulation cascade forming a mesh strengthening and stabilising the clot
This occurs via 3 phases
- Initiation
- Amplification
- Propogation
What are the 3 phases of secondary haemostasis
deposition of insoluble fibrin generated from the coagulation cascade forming a mesh strengthening and stabilising the clot
* Initiation phase - tissue factor exposed on subendothelial surfaces bonding with factor VIIa activating IX and X –> small amounts of thrombin –> activation of plt, V and VIII
* Amplification - amplification occurs with plt degranulation and activated surfaces with cofactor availability propogating the coagulation cascade reaction as both tissue factor-factor VIIa complex and XIa activate IX –> IXa - VIIIa (tenase) generates more Xa
* Propogation - Tenase generates large amounts of Xa –> interact with Va forming prothrombinase converting prothrombin to thrombin
◦ Thrombin propogates the reaction further while also converting fibrinogen to fibrin which polymerises to form a stable clot
What is included in the intiaition phase of secondary haemostasis
deposition of insoluble fibrin generated from the coagulation cascade forming a mesh strengthening and stabilising the clot
* Initiation phase - tissue factor exposed on subendothelial surfaces bonding with factor VIIa activating IX and X –> small amounts of thrombin –> activation of plt, V and VIII
* Amplification - amplification occurs with plt degranulation and activated surfaces with cofactor availability propogating the coagulation cascade reaction as both tissue factor-factor VIIa complex and XIa activate IX –> IXa - VIIIa (tenase) generates more Xa
* Propogation - Tenase generates large amounts of Xa –> interact with Va forming prothrombinase converting prothrombin to thrombin
◦ Thrombin propogates the reaction further while also converting fibrinogen to fibrin which polymerises to form a stable clot
What is included in the amplification phase of secondary haemostasis?
deposition of insoluble fibrin generated from the coagulation cascade forming a mesh strengthening and stabilising the clot
* Initiation phase - tissue factor exposed on subendothelial surfaces bonding with factor VIIa activating IX and X –> small amounts of thrombin –> activation of plt, V and VIII
* Amplification - amplification occurs with plt degranulation and activated surfaces with cofactor availability propogating the coagulation cascade reaction as both tissue factor-factor VIIa complex and XIa activate IX –> IXa - VIIIa (tenase) generates more Xa
* Propogation - Tenase generates large amounts of Xa –> interact with Va forming prothrombinase converting prothrombin to thrombin
◦ Thrombin propogates the reaction further while also converting fibrinogen to fibrin which polymerises to form a stable clot
What is included in the propogation phase of secondary haemostasis
deposition of insoluble fibrin generated from the coagulation cascade forming a mesh strengthening and stabilising the clot
* Initiation phase - tissue factor exposed on subendothelial surfaces bonding with factor VIIa activating IX and X –> small amounts of thrombin –> activation of plt, V and VIII
* Amplification - amplification occurs with plt degranulation and activated surfaces with cofactor availability propogating the coagulation cascade reaction as both tissue factor-factor VIIa complex and XIa activate IX –> IXa - VIIIa (tenase) generates more Xa
* Propogation - Tenase generates large amounts of Xa –> interact with Va forming prothrombinase converting prothrombin to thrombin
◦ Thrombin propogates the reaction further while also converting fibrinogen to fibrin which polymerises to form a stable clot
What are the 5 naturally occuring anticoagulants
Thrombomodulin
Portein C and S
Heparin sulfate
Antithrombin 3
What is thrombomodulin? What does it do? Where do you find it
- Thrombomodulin- cofactor for thrombin converting it from a procoagulant to an anticoagulant by activating protein C
◦ Glycoprotein present on endothelial cells
What are protein C and S? What is their production dpendent on? Whata re they regulated by? What are they activated by? What do they act on?
- Protein C and S - factor Va and VIIIa regulated, vitamin K dependent serine proteases
◦ Protein C favours destroying V and VIII
◦ Protein S enhances C by binding C to platelet surfac
protein C activated by thrombomodulin bound to thrombin
What does protein C do>
- Protein C and S - factor Va and VIIIa regulated, vitamin K dependent serine proteases
◦ Protein C favours destroying V and VIII
◦ Protein S enhances C by binding C to platelet surfac
protein C activated by thrombomodulin bound to thrombin
What does protein S do
- Protein C and S - factor Va and VIIIa regulated, vitamin K dependent serine proteases
◦ Protein C favours destroying V and VIII
◦ Protein S enhances C by binding C to platelet surfac
protein C activated by thrombomodulin bound to thrombin
What is heparin sulfate?
- Heparin sulfate - stimulates the activation of antithrombin which inactivates thrombin and factor Xa
Antithrombin 3 acts on?
- Antithrombin 3 - serine protease inhibitors inhibiting activated thrombin and activated factor X
What is vWF? How does it bind to platelets?
◦ VWF - large multimeric protein secreted by endothelial cells is present in an immobilised state in sub endothelial matrix and soluble form in plasma (does not bind), damage exposes it
‣ Adherence via glycoprotein receptor GP 1b/IX complex binds to VWF attached to collagen
What factors can activate a platelet?
‣ ADP
‣ Serotonin
‣ VWF
‣ Thrombin - protease activated receptors (PAR) initiate cell signalling pathways result in platelet granule secretion and remodellling of platelet cytoskeleton
What factors does the endothelium have to avoid clotting in the first place when healthy?
◦ Prevents exposure to collagen, tissue factor, VWF
◦ Glycocalyx layer contains mucopolysacchardies repealing PLT and coagulation factors
◦ Thrombomodulin activates protein C
◦ Heparin sulphate activating antithrombin 3 is secreted
◦ Vasodilators -NO, PGI2 enhance blood flow
◦ Tissue factor pathway inhibitor enhances protein C effect
How does platelet adhesion occur to the subendothelial matrix
Exposure to collagen in subendothelium of damaged vessels leading to ADHESION as platelets bind to vWF attached to collagen via glycoprotein receptor GP 1b/IX
When a platelet is activated what occurs?
Degranulation
Change in cellular surface proteins
- GP2B/3a
- Phsophatidylserine exposure for clotting cascade
Confirmational change
Aggregation promoting factors such as thromboxane A2 production
In the initiation phase of secondary haemostasis what ist he limiting step
‣ rate of reaction limited by lack of cofactors, and lack of cofactor bearing surfaces
◦ Thrombin activation causes local activation of platelets, and activates available cofactors V and VIII
What is Tenase composed fo
Factor 9 and factor 8complex
Prothrombinase composed of
Xa and Va
What does thrombin activate
Platelets
Factors 9/10/11/5/8
What is dividing line between amplification and propogation
Tenase signals the start of proopgation
Draw the coagulation cascade
What is HMWK? What role does it have in haemostasis?
High moelcular weight kinogen - on platelets or subendothelial tissue and required for XI –> XIa
What steps require Calcium in the coagulation cascade?
Formation of factor 9a, and formation of factor Xa, tissue factor/VIIa complex
What is the intrinsic pathway
XII, XI, IX, XIII
What is the common pathway
X, V, thrombin, fibrinogen
What is the extrinsic pathway
VII, X
How is fibrinogen turning to fibrin?
Hydrlyses arginine-glycine bonds to creatine fibrinopeptide A and B which are monomers and link via hydrogen bonds to form a loose insoluble polymer
How is fibrinogen stabilised
Ca and factor XIII - covalent cross links
Where is VIII found?
Prouduced by the liver and inactivated by protein C and S
Bound to vWF in endothelium and platelets circulating as a high moelcualr weight multimers
Where do IX and X interact with paltelets
Alpha carboxyglutamic acid residues via Ca bridges to phsopholipid and VIII bound to the surface
Draw a diagram of thrombolysis? Indicate where drugs influence this process>
How does TXA act
How does tPA act
How does a platelet bidn to collagen
GP 1a/2a
How does a platelet bind to vWF
GP 1b/IX
Describe the intracellular process of activation
◦ Activation process —> membrane phospholipase A2 and C —> convert inositol biphosphate to diacycl glycerol and IP3 —> protein phosphorylation —> increased calcium —> reactions leading to below
‣ Phospholipase A2 actins on phospholipids to produce Thromboxane A2 —> lowers platelet cAMP initiating release reaction, promoting aggregation and vasoconstriction
◦ Activation leads to
‣ Degranulation - releases platelet activating factors creating a exponentially increasing reaction, vasoconstrictors (seratonin/thromboxane A2)
* Within 30 seconds dense granules release aggregation factors
* Further 30 seconds later alpha granules release other factors mediating and reinforcing aggregation and adhesion
‣ Confirmational change - sphere with long projections (pseudopods)
‣ Surface proteins facilitating coagulation are exposed - GP 2b/3a
‣ Aggregation promoting factors - thromboxane A2, ADP
Plasminogen is what type of protein
beta globulin
Turns into a serine protease when activated by vessel wall activators, extrinsically by tissue activators
What are the two mechanisms without drugs being given when plasmin might be activated
◦ Tissue plasminogen activator - serine protease found on all endothelial cells plus circulating soluble form, activates plasminogen in the presence of fibrin as a cofactor. Released from damaged vessels
◦ Urokinase - produced by monocytes, macrophages,urinary epithelium also an activator of plasminogen. Affinity is lower than that of tPa but does not require fibrin as a cofactor
What inhibits fibrinolysis
◦ Plasminogen activator inhibitor 1 and 2 - PAI - 1/2 - produced by the liver, inhibits tPA and urokinase
◦ Alpha 2 anti plasmin - produced by the liver, circulating serine protease inhibitor which inhibits plasmin directly by binding in a covalent manner
◦ Thrombin activatable fibrinolysis inhibitor - TAFI - circulating inhibition activated by thrombin/Thrombomodulin or plasmin cleaves lysine residues on fibrin preventing binding of plasminogen to fibrin
How does the fibrinolysis mechanism interact with the clotting apthway
XIIa, Xa and thrombin prompt serine protease inhibitors to be released from endothelial cells
PLasmin cleaves fibrin the end product of the coagulation pathway
Thrombomodulin activating protein C which itself inactviates TAFI and PAI
How does TXA act?
Molecules resmeble lysine - lysine residues on fibrin are the molecular target of plasminogen. Therefore TXA binds to plasminogen preventing fibrin binding, and tPA can only activate plasminogen if its is bound to fibrin.
Why is the classical model not used as much anymore
Depletion in factor XII does not affect bleeding, even XI doesn’t matter that much
Cell based model more closely replicates coagulation because
More emphasis on interactoin between cell surfaces and clotting factors
How do the cell based model correlate with TEG?
CFT/K value -> amplification phase (fibrinogen problem); alpha angle -> propagation phase (thrombin burst; fibrinogen problem); MCF/MA-> dependent on platelet (80%) and fibrin (20%) interacting via GP2b/3a. CL/A30 or LY30 -> fibrinolysis phase.
Describe the phases of platelet activation
platelet activation, adhesion, aggregation, secretion
What does TEG not differenitate between that could be causing coagulation issues? It is not something you will necessarily find later in FBC/Coags/fibrinogen tests
Specifically, TEG/ROTEM doesn’t differentiate thrombocytopenia from platelet dysfunction
ROTEM doesn’t tell you if the patient is not clotting due to hypocalcemia, hypothermia, or acidosis - the lethal triad
How does thromboxane A2 act
Gq GPCR –> vasoconstriction via phospholipase C –> PIP2 hydrolysis to DAG and IP3 which increase Ca and protein kinase C
Also stimulates platelet activation and aggregation by binding thromboxane receptors to cause paltelet shape change and degranulation, with increased GP2b/3a expression
