Week 6 Coagulation Lecture 4 - Inhibition of Haemostasis

Question 1

Coagulation Pathway Revision

Answer 1

The most crucial step is the generation of thrombin
Thrombin generation is initiated when damage to a vessel wall exposes the blood to tissue factor (TF) in the sub-endothelium
TF forms a complex with FVIIa and activates FX
Together, FVa & FXa form the prothrombinase complex, which then cleaves a small amount of prothrombin (FII) to thrombin (FIIa)
This small amount of thrombin activates platelets, FV, FVIII and FXI, feeding back into the cycle to increase thrombin formation
Factor IXa, previously activated by either TF-VIIa or by FXIa on the platelet surface, and FVIIIa in the presence of calcium, complex on the platelet surface to form the platelet tenase complex
Platelet tenase activates more FX, which with FVa, generates a thrombin burst
It is this burst of thrombin rather than the initial thrombin activation that is crucial for the formation of a stable haemostatic plug

Question 2

Physiological Inhibitors of Coagulation

Answer 2

Several mechanisms:
- tissue factor pathway inhibitor (TFPI)
- antithrombins
- protein C/S system
- protein Z/ZPI system
Act upon factors within the coagulation cascade
Typically act to inactivate factors that have been activated

Question 3

Tissue Factor Pathway Inhibitor (TFPI)

Answer 3

Major inhibitor of blood coagulation
Produced by predominantly by endothelial cells & megakaryocytes
There are two isoforms resulting from alternative splicing
- TFPIα, TFPIβ
- both present in endothelial cells
- platelets only have TFPIα
TFPIα contains three Kunitz domains (K1, K2 and K3) followed by a positively charged C-terminal tail
TFPIβ which consist of K1 and K2 followed by a unique C-terminus encoding for a glycosyl-phosphatidylinositol (GPI) anchor
The K1 and K2 domains of TFPIα bind & inhibit FVIIa
The K1 and K2 domains of TFPIβ bind & inhibit FXa
The K3 domain is known to bind protein S => Protein S enhances FXa inhibition by TFPIα

Question 4

Antithrombin-Glycosaminoglycan Pathway

Answer 4

Antithrombin is:
- produced by the liver
- not Vitamin K dependent
- serine protease inhibitor (SERPIN)
- important physiological inhibitor of thrombin
- inhibits FIIa, FXa (& also FIXa, FVIIa)
Importantly AT when binds to heparin sulphate
- activity increase x1000
- prostacyclin (PGI2) synthesis, vasodilation,
- inhibits platelet aggregation

Question 5

Protein C/S System - Protein C

Answer 5

Circulating Vitamin K dependent protein when activated significantly prolongs the clotting time of normal plasma
Activated via thrombin (& thrombomodulin)
Endothelial Protein C receptor (EPCR) binds Protein C & presents to thrombin
Results in activated Protein C (APC)
Activated Protein C inhibits FVa & FVIIIa
Cofactors: Protein S, phospholipid
Limited by protein C inhibitor, α2-macroglobulin, α1- antitrypsin

Question 6

Protein C/S System - Protein S

Answer 6

Vitamin K dependent glycoprotein
Produced by liver & endothelial cells
Exists in plasma as free & bound forms (to C4b-binding protein)
Free form only has anticoagulant activity
Co-factor to APC in FVa & FVIIIa inactivation
Also has anticoagulant activity independent of APC on FVaFXa complex

Question 7

Protein Z/Z Dependent Protease Inhibitor

Answer 7

Protein Z dependent protease inhibitor (ZPI) =>
inhibits FXa
Requires presence of Protein Z & calcium ions
Protein Z is a vitamin K dependent glycoprotein that acts as a cofactor of ZPI
ZPI also inhibits FIXa, FXIa independent of Protein Z

Question 8

Disorders of Anticoagulants

Answer 8

Deficiency of anticoagulant typically predisposes to venous thrombosis
Typically comprise 5-10% of patients with clinical venous thromboembolism (VTE)
Include
- AT deficiency
- TFPIα deficiency
- APC resistance
- protein C deficiency
- protein S deficiency

Question 9

Antithrombin Deficiency May Result From…

Answer 9

1. Decreased production
   - liver cirrhosis
2. Increased loss
   - nephrotic syndrome
   - protein losing enteropathy
3. Enhanced consumption
   - sepsis
   - burns
   - disseminated intravascular coagulation (DIC)
   - prolonged unfractionated heparin therapy

Question 10

Pathological Inhibitors of Coagulation

Answer 10

Substances that inhibit the normal functioning of coagulation cascade
Result in haemorrhagic diathesis
Include:
- auto-antibodies to coagulation cascade factors
- antiphospholipid syndrome

Question 11

Haemophiliac vs Non-Haemophiliac

Answer 11

Haemophiliac
- ‘inhibitors’ = antibodies to coagulation factors
- e.g. Haemophilia A; inhibitors form in response to transfusion FVIII concentrate
Non-haemophiliacs
- ‘acquired’ haemophilia
- e.g. formation antibodies to FVIII without known cause
- initially recognised with ↑ aPTT

Question 12

Definition of Inhibitors

Answer 12

An inhibitor is a high affinity IgG antibody that specifically neutralises the procoagulant activity of the relevant clotting factor
High responders = >5 (BU/mL)
Low responders = <5 (BU/mL)

Question 13

Principle of the Bethesda Titre

Answer 13

FVIII inhibitors may quantified by mixing plasma with a known amount of FVIII
Incubated @ 37°C for 2h
Amount of inhibitor calculated as the difference from control mixture containing pooled plasma & buffer
1 Bethesda unit = inhibitor that will inactivate half the factor present in mixture of equal volumes of patient plasma & pooled normal plasma after 2h of incubation

Question 14

Acquired Haemophilia A (AHA)

Answer 14

Caused by autoantibodies against FVIII
Clinically, AHA is similar to severe haemophilia A with an increased risk of spontaneous bleeds, but the bleeding is usually mucocutaneous, soft tissue or gastrointestinal
Aetiology:
- 50% of cases have FVIII autoantibodies
- the remaining cases may be associated with postpartum period, autoimmune diseases, cancers, infections

Question 15

Diagnosis of AHA

Answer 15

Identified ↑ aPTT
Mixing test (rule in inhibitor, rule out deficiency)
Test for anti-phospholipid antibodies
Confirm with Bethesda Assay for anti-FVIII antibodies

Question 16

Antiphospholipid Syndrome

Answer 16

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder where different prothrombotic factors interact to induce arterial & venous thrombosis
It is defined by a combination of at least one clinical feature characterised by vascular thrombosis or pregnancy morbidity & raised titres of antiphospholipid antibodies (aPL)
aPL include lupus anticoagulant (LA), anticardiolipin antibodies (aCL) and/or anti-ß2 glycoprotein-I antibodies

Question 17

Lupus Anticoagulant

Answer 17

LA is apparent as a prolongation of a phospholipid-dependent coagulation test that is not due to an inhibitor specific to a coagulation factor
Classical findings for a LA are:
- prolongation of a phospholipid-dependent clotting test
- demonstration of the phospholipid dependence of the inhibitor
Test using a Dilute Russell Viper Venom Time and aPTT

Question 18

Anticoagulant Therapy

Answer 18

Used for:
- venous thromboembolism
- pulmonary embolism
- atrial fibrillation
- valvular heart disease (including prosthetic heart valves)
- myocardial infarct/cardiomyopathy
For each class of anticoagulant there are medical situations where the anticoagulant is:
- indicated
- not indicated/contraindicated

Question 19

Warfarin

Answer 19

Warfarin and other coumarin anticoagulants act by inhibiting the synthesis of functional vitamin K-dependent coagulation factors II, VII, IX and X
Vitamin K is required to effect carboxylation of precursor coagulation proteins synthesised by hepatocytes
Without γ-carboxylation the Ca2+ binding sites are non-functional & the proteins are not able to be ‘activated’
The effective half-life of warfarin ranges from 20 to 60 hours
The duration of effect is 2-5 days
The drug is completely absorbed after oral administration, and peak concentrations occur within 4 hours
The elimination of warfarin is almost entirely by metabolism

Question 20

Venous Thromboembolism Treatment

Answer 20

First episodes of acute VTE should be treated with warfarin along with a parenteral anticoagulant, such as unfractionated heparin (UFH), low molecular weight heparin (LMWH) or fondaparinux
Treat for at least 5d and until the INR is ≥2.0 for at least 24h
For the initial period of treatment the target INR should be 2.5
Recurrent VTE whilst anticoagulated and within the therapeutic range should be managed by increasing the INR target to 3.5

Question 21

Heparin

Answer 21

Heparin is a naturally occurring glycosaminoglycan produced by the mast cells
The pharmaceutical drug is extracted from porcine or bovine mucosa
All anticoagulant properties of UFH & LMWH depend on the presence of a specific pentasaccharide sequence, which binds with high affinity to antithrombin & potentiates its activity
Several proteins interact strongly with heparin to antagonise its anticoagulant activity, the most important being platelet factor 4 (PF4) and protamine
There is unfractionated heparin (UFH) and low molecular weight heparin (LMWH)

Question 22

Heparin Charactersitics

Answer 22

In all LMWH preparations the anti-Xa activity exceeds the anti-IIa activity
The ratio of anti-Xa to anti-IIa activity varies between 1.6 to 9.6
Inhibition of factor Xa requires only the pentasaccharide sequence (approximate MW 1700 Da)
Whereas thrombin inhibition requires a minimum total chain length of 18 saccharides
LMWH has longer half life than UFH
Unlike subcutaneous UFH, which has a bioavailability of <50%, all LMWHs have a bioavailability after subcutaneous injection of 90– 100%

Question 23

Monitoring of Heparin Treatment - UFH

Answer 23

The APTT has been used most widely for monitoring of therapeutic doses of UFH in VTE
A target ratio versus midpoint of normal range of 1.5–2.5 is employed
The great sensitivity to heparin of the standard thrombin time test renders monitoring and dosage adjustment using this test difficult

Question 24

Monitoring of Heparin Treatment - LMWH

Answer 24

The APTT is generally insensitive to LMWHs
The anti-Xa assay is more informative but there are significant limitations

Question 25

Fondaparinux

Answer 25

A synthetic analog of the AT-binding pentasaccharide found in LMWH
Its specific anti-Xa activity is higher than that of LMWH (about 700 units/mg cf 100 units/mg)
Its half-life after subcutaneous injection is longer than that of LMWH (17h cf about 4h)

Question 26

Direct Oral Anticoagulants (DOACs)

Answer 26

FXa inhibitors
- Rivaroxaban, Apixaban, Edoxaban, Betrixaban
- inhibits FXa & prothrombinase complex activity
- => inhibits thrombin generation
- e.g. Rivaroxaban binds directly and reversibly to the active site of free and prothrombinase complex-bound FXa
Thrombin inhibitors
- Dabigatran, Argatroban
- inhibit action of thrombin
- e.g. Dabigatran reversibly inhibits free and fibrin-bound thrombin by binding to the thrombin active site

Question 27

Measurement of Oral anti-Xa Agents Rivaroxaban & Apixaban

Answer 27

Anti-Xa chromogenic assays should be used to determine plasma concentration of direct FXa inhibitors
Product-specific calibrator should be used
LMWH reference standards should not be used a calibrators for direct FXa inhibitors
PT and APTT should not be used to measure the plasma concentration of Xa inhibitors

Question 28

Measurement of Dabigatran and Argatroban

Answer 28

Dilute thrombin-based assays, ecarin-based assays or chromogenic anti-IIa assays (in the absence of heparin)
Assays to determine anticoagulant concentration should be calibrated with drug-specific calibrators
PT and APTT should not be used to measure the plasma concentration of dabigatran