Lecture 7.2: Regulation of Protein Activity Flashcards
Proteolytic Cleavage
The enzymatic hydrolysis of a peptide bond in a peptide or protein substrate by a family of specialised enzymes termed proteases
What are zymogens?
Digestive enzymes synthesised as zymogens (inactive precursors) stomach/pancreas
Acute Respiratory Distress Syndrome
A secondary complication of pancreatitis
Inflammation within the pancreas spreads to other organs causing protein dysregulation due to proteolytic cleavage
Seen as widespread opacities in the lungs
Incretins
Incretins are hormones that decrease blood glucose levels
Incretins (GLP-1 & GIP) are released after eating
Regulate the secretion of insulin by a blood glucose-dependent mechanism
Require proteolytic cleavage for activation and inactivation
Amplification of Enzyme Cascades
When enzymes activate enzymes, the number of affected molecules increases geometrically in an enzyme cascade
Amplification of signals by the cascade allows amplification of the initial signal several orders of magnitude within a few milli seconds
How much blood does an adult have?
5-6 litres of blood
Blood Loss
Loss of 0.5 litre is usually without harmful effects
Loss of 1 litre may cause shock
What is the Coagulation Pathway?
A cascade of events that leads to haemostasis (body’s normal physiological response for the prevention and stopping of bleeding/haemorrhage)
Allows for rapid healing and prevention of spontaneous bleeding:
• Vascular spasm
• Platelet plug formation
• Blood clotting (coagulation cascade)
The 3 phases of Haemostasis
1) Vascular Spasm: smooth muscle contracts causing vasoconstriction
2) Platelet Plug Formation: injury to lining of vessel exposes collagen fibres, platelets adhere, platelets release chemicals that make nearby platelets sticky, platelet plug forms
3) Coagulation: fibrin forms a mesh that traps RBCs & platelets forming a clot
The Clotting Cascade (3) [blood vessel, clotting factors, clot reversal]
Injury to a blood vessel, activates platelets, initiates a cascade of clotting factors resulting in a blood clot
Clotting factors are proteases or cofactors needed to activate the next step, proteolytic cleavage of fibrinogen forms fibrin and aggregation to form clots
Clot formation is tightly controlled, reversed by proteolytic cleavage of fibrin by the enzyme plasmin
What enzyme is responsible for the proteolytic cleavage of fibrin?
Plasmin
What is the product of the proteolytic cleavage of fibrinogen?
Intrinsic Pathway of the Blood Clotting Cascade
Damaged endothelial lining of blood cells promotes binding of factor XII (12)
FACTOR X (10) ACTIVATION Common endpoint for both pathways
THROMBIN ACTIVATION
FORMATION OF FIBRIN CLOT
Extrinsic Pathway of the Blood Clotting Cascade
Trauma releases tissue factor III (3)
FACTOR X (10) ACTIVATION Common endpoint for both pathways
THROMBIN ACTIVATION
FORMATION OF FIBRIN CLOT
Pneumonic to remember Proteins/Cofactors of Blood Coagulation
Foolish: Fibrinogen I
People: Prothrombin II
Try: Tissue Factor III
Climbing: Calcium IV
Long: Proaccelerin V
Slopes: Proconvertin VII
After: Antihemophilia factor B VIII
Christmas: Christmas factor IX
Some: Stuart-Prower factor X
People: PTA (Thromboplastin antecedent) XI
Have: Hageman factor XII
Fallen: Transglutaminase XIII
The role of γ-carboxyglutamate (Gla) residues
Post-translational modification (carboxylation) of factors II, VII, IX, X in the liver
Vitamin K dependent
Addition of COOH groups to glutamate residues to form carboxyglutamate (Gla)
Allows interaction with sites of damage and brings together clotting factors
Calcium-binding region of prothrombin
• Prothrombin binds calcium ions via Gla residues
• Only prothrombin next to site of damage will be activated
• Clots are localised to the site of damage
The Extrinsic Pathway/Tissue Factor Pathway
• Activated by external trauma
• Results in blood loss from vascular system
• More rapidly activated than the intrinsic pathway
• Membrane damage exposes extracelluar domain of tissue factor III
• Autocatalytic activation of Factor VII
Structure of Fibrinogen
• 340 kDa protein
• 2 sets of tripeptides α, β, γ, joined at the N-termini by disulphide bonds
• 4 globular domains linked by rods
• N-terminal regions of A and B chains are highly negatively charged and
prevent aggregation of fibrinogen
Formation of a ‘Soft’ Fibrin Clot
Thrombin cleaves fibrinopeptides A and B from the central globular domain of fibrinogen
Globular domains at the C-terminal of the β and γ chains interact with exposed sequences at the N-termini of the cleaved β and α chains to form a fibrin mesh or soft clot
Formation of a ‘Hard’ Fibrin Clot
Soft clot stabilised through covalent crosslinks-amide bonds between side chains of lysine and glutamine residues in different monomers
Cross-linking is catalysed by transglutaminase, activated from protransglutaminase by thrombin
Stopping the Clotting Process: Localisation of (pro)thrombin
Dilution of clotting factors by blood, removal by liver
Stopping the Clotting Process: Specific inhibitors
• Anti-thrombin III (AT3)
• Anti-thrombin inactivates Factor IIa, IXa and Xa
• Enhanced by heparin binding
• AT3-heparin does not act on thrombomodulin bound thrombin
Stopping the Clotting Process: Digestion by Proteases
• FVa and VIIIa are degraded by Protein C.
• Protein C activated by thrombin binding to endothelial receptor,
thrombomodulin
• Defects in Protein C cause thrombotic disease
Clot Removal: Fibrinolysis
Via enzyme Plasmin
Anticoagulant Sites of Action
Vitamin K Antagonists:
• e.g. Warfarin
• Carboxylation of clotting factors II, VII, IX and
X all depend on Vitamin K
Heparin:
• Activates Antithombin III
• → Inactivates Factor IIa & Xa
Oral Anticoagulant Drugs:
• Newer drug class
• Inhibit either IIa or Xa
Classic Haemophilia (Defect in Factor VIII)
• FVIII (‘anti-haemophilic factor’) not a protease, but stimulates the activity of
FIXa, a serine protease.
• Activity of FVIII increased by proteolysis by thrombin and FXa
• Positive feedback amplifies clotting signal, accelerates clot formation
• Reduced/absent FVIII results in reduced/absent clotting and prolonged
bleeding
• Treatment with recombinant FVIII
Anti-Coagulants and Dementia
Dementia currently has no cure, new research shows anti-coagulants are linked to reduction in dementia risk
What are the 7 key control points in blood clotting?
1) Inactive zymogens present at low concentration
2) Undergo proteolytic activation
3) Amplification of initial signal by cascade mechanism
4) Clustering of clotting factors at site of damage
5) Feedback activation by thrombin ensures continuation of clotting
6) Termination of clotting by multiple mechanisms
7) Clot breakdown controlled by proteolytic activation
