9 - Plasma Proteins and Introduction to the Clotting Cascade

Question 1

Why is serum protein separation useful in clinical practice?

Answer 1

• Disease causing production of a particular protein (e.g. Multiple myeloma, tumour markers)
• Disease causing lack of production of a particular protein (e.g. malnutrition (albumin)
• Identification of a particular isoenzyme may indicate affected part of body e.g. MI (creatine kinase)

Question 2

Methods used for separating serum proteins

Answer 2

• Cellulose acetate electrophoresis
• SDS-PAGE
• Native PAGE
• Isoelectric focussing
• 2D PAGE
• Capillary zone electrophoresis

Question 3

Plasma protein electrophoretic profiles

Answer 3

• 7-9% of the plasma, 65-90 g/L are synthesized by the liver (with te exception of y-globulins)
• Fractions
  
  • Albumins-plasma concentration- 45 g/L
  • Globulins (a,b,y) - 27 g/L
  • Fibrinogen - 3 g/L
• Albumins have the smallest molecular mass whereas fibrinogen is the largest

Question 4

Relevance of Blood Clotting Biochemistry

Answer 4

• In the UK ~35% of people die as a result of failures associated with haemostasis.
• Examples:
  
  • blood clots,
  • coronary thromboses,
  • non-haemorrhagic strokes
  • pulmonary emboli.
• By contrast, the proportion of deaths by all cancers is ~26% in the UK.

Question 5

intrinsic pathway of coagulation cascade

Answer 5

long arm, factor 12 to 10

more stages so the effect is greater and more amplified.

Question 6

extrinsic pathway of coagulation cascade

Answer 6

short arm, factor 8 to 10
short and fast, first to reach factor 10 to form common pathway. Generates thrombin with positively feedbacks to speed up intrinsic pathway.

Question 7

Thrombin

Answer 7

enzyme that drives coagulation cascade forward.
Also converts fibrinogen to fibrin to form clot.
- Positive feedback, converts factor 5 to activated form which then makes thrombin

Question 8

Haemorrhage

Answer 8

Inadequate arrest of bleeding

Question 9

Thrombosis

Answer 9

Inappropriate formation of a clot

Question 10

Stages of blood coagulation

Answer 10

When a blood vessel is damaged there are 4 stages of recovery:
1• Vascular constriction – very rapid, occurs in response to serotonin (a neurotransmitter) endothelins, tissue factor (aka Factor III)
2• Platelet activation and plug formation – temporary platelet plug made in seconds.
3• Coagulation Cascade and Fibrin Clot Formation – minutes
4• Clot dissolution – bleeding stopped and clot formed

Question 11

Factor 5 and 8

Answer 11

cofactors of the cascade, combine with other factors to accelerate the activity.

Question 12

The Blood Coagulation Cascade

Answer 12

• The molecular mechanisms in blood coagulation are examples of a key method of enzymatic control in extracellular systems - proteolytic cleavage.
• Many enzymes (e.g. serine proteases) occur in inactive precursor forms, called zymogens. These are activated by proteolytic cleavage of a peptide bond.
• The action of one enzyme upon a zymogen to produce a further enzyme can result in the formation of a cascade, which gives scope for signal amplification.
• There are a lot of positive and negative feedback systems within the cascade.

Question 13

The intrinsic (contact) pathway

Answer 13

• When blood is exposed to collagen released from the surface of the damaged vessel, the intrinsic pathway is activated.
• Exposed collagen activates Factor XII to XIIa
• In the presence of HMW Kininogen, Factor XIIa activates Factor XI to XIa.
• XIa combines with calcium, and activates Factor IX to IXa
• At the same time, platelet factor 3 (PF3) is released from platelets and Factor VIII will be activated to VIIIa.
• Factor IXa, (with the help of PF3) will join together with FactorVIIIa and form Factor X activating factor.

Question 14

The extrinsic (tissue factor) pathway

Answer 14

• Commences in the blood vessel wall.
• Factor III (aka tissue factor, thromboplastin) is released from damaged endothelial cells,
• The amount of Factor III released is directly proportional to the amount of damage.
• In the presence of calcium, Factor III activates Factor VII creating Factor VIIa

Question 15

The common pathway

Answer 15

• Factor X is activated, either by VIIa or Factor X activator complex , to form Xa – aka prothrombinase
• Factor Xa, in the presence of calcium ions will hydrolyse prothrombin into thrombin!
• Thrombin aka Factor IIa
• Thrombin will then hydrolyse and activate fibrinogen to fibrin. Fibrin strands will begin to join together, and with the help of XIIIa, cross-linking of fibrin strands occurs.
• Factor XIII is also activated by thrombin. XIII is also known as fibrin stabilising factor.
• Thrombin can also act as a ‘catalyst’ in other areas of the cascade to speed up the process by activating Factors VII and V

Question 16

coagulation stops

Answer 16

because molecules involved in the cascade are cleared as they reach their half life.