10 - Clotting Issues and the Diagnosis of Disease

Question 1

Gla domain

Answer 1

amino acids that bring coagulation cascade to the correct place

Question 2

Coagulation cascade activation

Answer 2

• platelets activated with negative charge on surface
• binds to Ca and then binds to Gla residues located on a protein in coagulation. (Gla residues serve as Ca2+ binding site to attach polypeptide to phospholipid. Residues generated by carboxylation process with vitamin K).
• allow coagulation enzymes to bind to platelets so cascade at right place.

Question 3

What are Gla domains used for?

Answer 3

To localised to the site of the injury

• Factors VII, VIII, IX, X and prothrombin all rely on the presence of Gla domains

Question 4

Vitamin K

Answer 4

cofactor for Gla residues (gamma-carboxyglutamate).

• If deficient, cannot coagulate properly, Ca2+ not chelated.
• Inhibitors of this reaction if thrombosis risk e.g. Warfarin.

Question 5

Gla Domains and the need for Vitamin K

Answer 5

• Factors VII, VIII, IX, X and prothrombin contain specialised Gla domain which contain10 y-carboxyglutamate (Gla) residues which serve as Ca2+-binding sites to attach the protein to phospholipids found on the outside of the platelets.
• This causes the above factors to localise to the site of injury where the platelet plug has formed. This is a critical process in the formation of the clot.

Question 6

Thrombin

Answer 6

central enzyme to form fibrin clot

Question 7

Prothrombin (aka FII)

Answer 7

• Prothrombin contains 10-12 glutamic acid (Glu) residues which in the presence of Vitamin K are carboxylated to gamma-carboxyglutamic acid residues (Gla).

• made and released when factor 10 is made after the phospholipid is activated and then binds calcium.
• Brought to the correct site by Gla domains.
• Cleaved by active factor 10.

Question 8

What can go wrong?

Answer 8

Clotting must occur in the correct place and at the correct time.
Deficiency or excess of any component could cause pathology.
• Platelets
• Vitamin K
• Coagulation factors

Question 9

Haemophilia

Answer 9

• Type A: most common, severe, Factor VII deficiency
• Type B: 2nd most common, moderate, Factor IX deficiency
• Type C: rare, mild, Factor XI deficiency

Question 10

Haemophilia A

Answer 10

• most common, hereditary disease where patient has tendency to bleed, cannot coagulate properly.
• Deficiency in factor 8 - in intrinsic pathway so extrinsic and common still occur, but coagulation is not quick.
  • The severity of haemophilia emphasises the importance of the intrinsic pathway

Question 11

Haemophilia A transmission

Answer 11

Genetically transmitted as sex-linked recessive characteristic, on X chromosome.

Question 12

Factor V Leiden

Answer 12

Risk factor for thrombosis
- single point mutation in factor V, Arg506 replaced with Gln.
- now resistant to degredation
- some present venous thrombosis
- gene frequency of 5-10%
• The mutation manifests itself in the form of abnormal blood clots in the legs (DVTs) and lungs.

Question 13

Platelet adhesion

Answer 13

Following an injury to blood vessels, the process of primary haemostasis is initiated, involving the release of molecules such as serotonin, causing vasoconstriction. at the same time collagen is exposed, causing platelets to be attracted and attached

Question 14

Stopping the coagulation cascade

Answer 14

plug forms, platelet activation has stopped, enzymes and cofactors are broken down due to their half life

Question 15

vonWillebrand disease

Answer 15

coagulation is impaired

Question 16

Mechanism of platelet adhesion

Answer 16

Activation, and the change in shape that occurs is triggered by GP1b receptors for vWF and collagen.
These are only found inside the blood vessel wall.
Therefore, they are only exposed if the vessel is damaged.

Question 17

Main symptoms of a deficiency of vWF

Answer 17

Large bruises or bruising easily
Frequent or long lasting nose bleeds
Bleeding gums 
Heavy or long lasting bleeding from cuts
Heavy periods/ parturition haemorrhage 
Heavy or long lasting bleeding after tooth extraction

Question 18

Adhesion leads to Activation which leads to

Aggregation

Answer 18

• Activated platelets release granules that help coagulation, platelet activation and vasoconstriction.
• Activated platelets will express surface glycoproteins required for aggregation and fibrin attachment.
• Activated platelets are flatter and have pseudopodia

Question 19

Platelet Activation and Role of Gla Domains

Answer 19

• Gla modules on coagulation factors interact, via Ca2+, with the activated platelets.
• This allows coagulation to happen only at the site of damage; the only place platelets are activated.
• The binding of factors Xa and Va to the platelets enhances the production of Thrombin at the site of injury

Question 20

platelet number decrease

Answer 20

can accommodate a large drop, but if significant then clotting time rapidly increases - even if coagulation pathway is fine

Question 21

thrombocytopaenia - platelet deficiency

Answer 21

bleeding time increases

produces faecal occult blood due to lack of clotting within the body

Question 22

Laboratory Investigations

Answer 22

• Prothrombin Time (PT)
• Activated Partial Thromboplastin Time (aPTT)
• Thrombin Time (TT)
• Mixing Studies

Question 23

Prothrombin Time (PT)

Answer 23

If elevated, problem in the extrinsic pathway.
Blood sample mixed with components and time measured for coagulation to occur.
- add recombinant TF, phospholipid, calcium

Question 24

INR (International Normalised Ratio)

Answer 24

ratio of a patient’s prothrombin time to a control.
This takes account of differences between manufacturers products.
It is the preferred test of choice for patients taking vitamin K antagonists such as warfarin.

Question 25

Activated Partial Thromboplastin Time (aPTT)

Answer 25

An elevated aPTT tells us that there is a problem with the intrinsic pathway or the common pathway.
Doesn’t tell us where, but we can discount a number of components and factors.
- add contact activator (collagen), phospholipid (forms membrane), calcium (to activate factors and adhere them to the membrane)

Question 26

Thrombin time (TT)

Answer 26

If elevated, problem in the common pathway.
Add excess thrombin to blood sample and monitor coagulation time. Determine efficiency of fibrin clot formation.
Determines if there is a deficiency in factor 13.

Question 27

Factor 13 deficiency

Answer 27

no fibrin clot produced, regardless of thrombin added.

Question 28

Mixing studies

Answer 28

• Take a sample which is not coagulating and add something to it – if this brings about normal coagulation, what you added brought about (rescued/corrected) the end so the problem in the original sample is a component you’ve added.
• Clotting factor deficiencies may be diagnosed via mixing studies.

Question 29

Mix sample with normal blood

Answer 29

if no coagulation, sample may have an inhibitor as normal blood has all factors and cofactors sufficient in the coagulation cascade

Question 30

Mixing studies

Answer 30

• Take a sample which is not coagulating and add something to it – if this brings about normal coagulation, what you added brought about (rescued/corrected) the end so the problem in the original sample is a component you’ve added.
• Clotting factor deficiencies may be diagnosed via mixing studies.

Question 31

Mix sample with barium sulphate absorbed plasma

Answer 31

Lacks factors II, VII, IX and X.
Contains factors I, V, VIII and XIII.
If no coagulation, then plasma doesn’t contain the factors that were absent.
Can then perform thrombin test to test for factor 13

Question 32

Mix sample with serum

Answer 32

lacks factors I, V, VII and XIII.
Contains factors II, VII, IX and X.
If no coagulation then plasma doesn’t contain the absent factors.

Question 33

Protein C and Protein S

Answer 33

• Anti-coagulation proteins
- control extent of coagulation
- inactivate factor Va and VIIIa in the coagulation cascade
• Deficiency leads to a hyper-coagulable state
- Patient at risk of developing a thrombus
- Deep vein thrombosis
- Pulmonary embolism
• Prevents excessive fibrin formation (secondary haemostasis)

Question 34

Protein C and Protein S deficiencies

Answer 34

• Autosomal dominant inheritance.
• Type 1 – quantitative issue (not enough)
• Type 2 – qualitative issue (proteins are made but are non-functional)
• The thrombomodulin-thrombin-C/S complexes can not form.
• Leads to increases in active Factors V and VIII.
- increases risk of DT and pulmonary emboli

Question 35

Thrombomodulin and Protein C activation

Answer 35

• Thrombomodulin is a protein attached to the endothelial cells lining the vessel.
• It binds thrombin and the proteins C and S to form a complex.
• Protein C now gains proteolytic activity and acts on Factors V and VIII thereby reducing the activity of Factors X and IX, respectively.

Question 36

Acquired Protein C and S deficiencies

Answer 36

These occur:
• in patients with liver disease
• those taking Warfarin
• patients with nephrotic syndrome - Proteins lost in the urine