Natural anticoagulants

Question 1

Features of protein C

Answer 1

Serine protease synthesised in the liver (requires vitamin K)
Secreted to circulate in plasma as inactive precursor (zymogen)
Activated through proteolytic cleavage by the thrombin/thrombomodulin complex
Regulates activity of coagulation FVa and FVIIIa

Question 2

Features of protein S

Answer 2

Co-factor for activated protein C
Vitamin K dependent plasma protein
Increases APC activity
Regulates complement (30-40% bound to C4b)
Only free protein S binds APC

Question 3

Features of Antithrombin

Answer 3

Serine protease inhibitor (SERPIN), plasma protein synthesised in Liver
Protects circulation from free pro-coagulant enzymes and limit the coagulation process to sites of vascular injury
Regulates activity of thrombin, FIXa, FXa, FXIa, FXIIa
Formation of stable covalent complex of Antithrombin-Thrombin
Enhanced by heparin

Question 4

What is thrombomodulin?

Answer 4

Cell surface receptor on endothelial cells
Binds thrombin
Coverts thrombin from procoagulant to anticoagulant enzyme

Question 5

What happens when thrombin is converted from procoagulant to anticoagulant?

Answer 5

Thrombin loses ability to cleave fibrinogen
Thrombin gains ability to activate protein C

Question 6

How is protein C activated?

Answer 6

EPCR binds protein C, presents to thrombin/thrombomodulin complex to enhance its activation x20 by partial proteolytic cleavage

Question 7

Features of APC

Answer 7

Inactivates FVa and FVIIIa by proteolytic cleavage
Cofactors in tenase and prothrombinase complexes activation
Reduces production of thrombin and fibrin

Question 8

Features of protein C deficiency

Answer 8

Inherited disorder; Autosomal dominant inheritance
Genetic risk factor for DVT
Type I mutation -> affects reduced protein C synthesis
Type II mutation -> affects biological activity of protein C
=> Purpura fulimans🏥

Question 9

How is protein S deficiency acquired and what are the risk associated?

Answer 9

Inherited disorder; autosomal dominant inheritance
Genetic risk factor for DVT

Question 10

Explain how activated protein C resistance is acquired and its affect on clotting

Answer 10

Inherited trait
95% of APC-R caused by FV Leiden mutation
Mutated FV resistant to inactivation
Prolonged FV activity => increased tendency to clot

Question 11

How is FV inactivated?

Answer 11

APC destroys FV in series of sequential cleavages
The first cleavage at Arg506 exposes additional cleavage sites at Arg306 and Arg679

Question 12

Name and explain the laboratory tests for APC-R

Answer 12

Activated Partial Thromboplastin Time (APTT) Assay
If positive, test for FVLeiden using PCR

Question 13

Explain the laboratory test for Factor V Leiden

Answer 13

Collect blood sample -> DNA -> PCR FVgene -> Fluorescent melting curve analysis

Question 14

Explain how fluorescent melting is measured

Answer 14

1️⃣ Light cycler instrument measures fluorescence as temperature increases
2️⃣ Increasing temperature -> mutation probe dissociates
3️⃣ Physical separation of anchor probe and mutation probe prevents fluorescence transfer
4️⃣ As temperature increases, fluorescence detected falls
5️⃣ LC identifies peak rate of decrease in fluorescence
6️⃣ If mutation is present melting temp is reduced

Question 15

Explain the APTT test

Answer 15

APC added to plasma -> FV and FVIII inactivated
APPT increases (+/-ratio >2.5)
Effect less pronounced in some thrombosis patients

Question 16

Explain the role of heparin

Answer 16

Stimulates ability of AT to inhibit thrombin x1000
Glycosaminoglycan
Present on surface of endothelial cells
Binds antithrombin

Question 17

How is antithrombin deficiency acquired and what risks does this pose?

Answer 17

Inherited, autosomal dominant
Genetic risk of DVT
Mutations of SERPINC1 gene
Homozygous mutations => death

Question 18

What are the two outcomes of SERPINC1 mutations

Answer 18

1. Mutation results in reduced AT synthesis (type 1)
2. Mutation affects biological activity of AT(type 2)
   2a. Defect in the active site
   2b Defect in the AT heparin binding site

Question 19

Type 1 Thrombophilia

Answer 19

Low expression & low activity of anticoagulant proteins

Question 20

Type 2 Thrombophilia

Answer 20

Normal expression & low activity of anticoagulant proteins due to missense mutations