Lecture 7.2: Regulation of Protein Activity

Question 1

Proteolytic Cleavage

Answer 1

The enzymatic hydrolysis of a peptide bond in a peptide or protein substrate by a family of specialised enzymes termed proteases

Question 2

What are zymogens?

Answer 2

Digestive enzymes synthesised as zymogens (inactive precursors) stomach/pancreas

Question 3

Acute Respiratory Distress Syndrome

Answer 3

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

Question 4

Incretins

Answer 4

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

Question 5

Amplification of Enzyme Cascades

Answer 5

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

Question 6

How much blood does an adult have?

Answer 6

5-6 litres of blood

Question 7

Blood Loss

Answer 7

Loss of 0.5 litre is usually without harmful effects

Loss of 1 litre may cause shock

Question 8

What is the Coagulation Pathway?

Answer 8

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)

Question 9

The 3 phases of Haemostasis

Answer 9

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

Question 10

The Clotting Cascade (3) [blood vessel, clotting factors, clot reversal]

Answer 10

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

Question 11

What enzyme is responsible for the proteolytic cleavage of fibrin?

Answer 11

Plasmin

Question 12

What is the product of the proteolytic cleavage of fibrinogen?

Question 13

Intrinsic Pathway of the Blood Clotting Cascade

Answer 13

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

Question 14

Extrinsic Pathway of the Blood Clotting Cascade

Answer 14

Trauma releases tissue factor III (3)

FACTOR X (10) ACTIVATION Common endpoint for both pathways

THROMBIN ACTIVATION

FORMATION OF FIBRIN CLOT

Question 15

Pneumonic to remember Proteins/Cofactors of Blood Coagulation

Answer 15

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

Question 16

The role of γ-carboxyglutamate (Gla) residues

Answer 16

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

Question 17

Calcium-binding region of prothrombin

Answer 17

• 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

Question 18

The Extrinsic Pathway/Tissue Factor Pathway

Answer 18

• 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

Question 19

Structure of Fibrinogen

Answer 19

• 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

Question 20

Formation of a ‘Soft’ Fibrin Clot

Answer 20

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

Question 21

Formation of a ‘Hard’ Fibrin Clot

Answer 21

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

Question 22

Stopping the Clotting Process: Localisation of (pro)thrombin

Answer 22

Dilution of clotting factors by blood, removal by liver

Question 23

Stopping the Clotting Process: Specific inhibitors

Answer 23

• 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

Question 24

Stopping the Clotting Process: Digestion by Proteases

Answer 24

• 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

Question 25

Clot Removal: Fibrinolysis

Answer 25

Via enzyme Plasmin

Question 26

Anticoagulant Sites of Action

Answer 26

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

Question 27

Classic Haemophilia (Defect in Factor VIII)

Answer 27

• 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

Question 28

Anti-Coagulants and Dementia

Answer 28

Dementia currently has no cure, new research shows anti-coagulants are linked to reduction in dementia risk

Question 29

What are the 7 key control points in blood clotting?

Answer 29

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