Week 8 Coagulation Lecture 5&6 - Thrombotic Disorders

Question 1

Venous Thrombosis

Answer 1

Most commonly occurs in the legs
Thrombophlebitis
- thrombosis in the superficial veins
- inflammation or stasis
- self-resolving
Deep vein thrombosis
- most common site is in the deep veins of the legs
- pulmonary embolism is a serious complication
Thrombi commonly form around the valves of the leg veins
VT initiates after endothelial cell activation → fibrin formation at the endothelial surface
Fibrin traps RBCs and platelets

Question 2

Venous Thrombosis - Pathogenesis

Answer 2

Mechanisms of endothelial activation
- inflammation
- stasis at the back of the valve → hypoxia → EC activation
Activated ECs
- express TF
- express P-selectin (allows leukocytes to bind to the endothelium)
- release vWF (allows platelets and leukocytes to bind to the endothelium)
Stasis
- accumulation of prothrombotic factors
- hypoxia in leukocytes, platelets, (and ECs) => expression of TF in monocytes and production of TF-bearing microparticles
- anticoagulant function of the endothelium is decreased in large vessels

Question 3

Diagnosis of Venous Thrombosis

Answer 3

Symptomatic patient
Presence of risk factors
D-dimer
- elevated in VTE
- not diagnostic, but can be used to exclude VTE
Imaging

Question 4

Treatment of Deep Vein Thrombosis

Answer 4

Initially, heparin
- usually LMWH
- UFH only in certain patient populations
Then warfarin or other orally administered anticoagulant
- continues for 3-6 months, to years

Question 5

Risk Factors for Venous Thrombosis

Answer 5

Hospitalisation
Lupus anticoagulant
Cancer
Trauma/fracture
Pregnancy
Oral contraceptives/oestrogen
Obesity

Question 6

Hereditary Thrombophilia

Answer 6

Reduced levels of the naturally occurring anticoagulants:
- antithrombin (AT)
- protein C (PC)
- protein S (PS)
AT and PC have type I and type II deficiencies
- type I: decreased antigenic levels of functional protein
- type II: normal antigenic levels of protein with decreased function
Factor V Leiden (FVL)
Prothrombin Gene Mutation

Question 7

Antithrombin

Answer 7

AT inhibits serine proteases
Main targets are IIa (thrombin), IXa, Xa, XIa, XIIa
Activity is greatly enhanced by heparin/heparan sulphate
Contains two major functional regions
- reactive site
- heparin/heparan binding

Question 8

Antithrombin Testing

Answer 8

Both functional and antigenic assays used for the measurement of AT
Functional - heparin co-factor assay
- patient plasma is mixed with heparin and thrombin
- residual thrombin activity is determined with the addition of a thrombin-specific chromogenic substrate
Antigenic - ELISA, latex-agglutination

Question 9

Congenital AT Deficiency

Answer 9

Affected individuals experience their first thrombotic episode in the first 25 years of life
Heterozygous deficiency confers a 10-20 fold increased thrombotic risk
Homozygous deficiency is incompatible with life
- except for mutations in the heparin-binding site

Question 10

The Protein C/Protein S System

Answer 10

Contains Protein C (PC), Protein S (PS), Thrombomodulin (TM), and the Endothelial Protein C Receptor (EPCR)
PC and PS are vitamin-K dependent proteins produced by the liver
TM is expressed on the EC surface
- binds “non-clot bound” thrombin
- changes thrombin’s substrate specificity
EPCR is expressed on the endothelial surface, binds PC

Question 11

Protein C - Activity Assays

Answer 11

Should be used to measure PC
Chromogenic or clot-based (aPTT)
Both use Protac, which activates Protein C
In the clot-based assay, normal plasma has a prolonged time (< 100s), whereas PC ↓ plasma has a normal time (30-40s)
Chromogenic assays do not detect PC that has abnormal PL or Ca2+ binding, but are preferred over clot-based assays
Clot-based assays may underestimate PC activity in the presence of FVL, ↑ FVIII, hyperlipidaemia, and may give unreliable results in the presence of lupus anticoagulants

Question 12

Protein S

Answer 12

Two pools of PS are present in plasma
- ~60% is bound to C4bp (no co-factor function)
- ~40% circulates unbound
Free vs total PS
Assays:
- total PS antigenic assay
- free PS antigenic assay
- PS functional (activity) assay
- low levels found in the functional assay should be confirmed with free PS antigenic assay

Question 13

Protein S - 3 Types of Deficiency

Answer 13

Type I
- normal PS protein is produced at a reduced level
- decreased antigenic and functional PS
Type II
- normal levels of PS with decreased functional activity
- normal antigenic levels, decreased functional levels
Type III
- decreased free PS antigen, normal total PS antigen

Question 14

The Prothrombin Gene Mutation

Answer 14

A.k.a the Prothrombin G20210A mutation
Mutation in the 3’ UTR of the F2 gene
Increases the stability of the F2 mRNA
Heterozygotes have elevated plasma Prothrombin relative to homozygous WT (~30% increase)
Elevated Prothrombin → increased thrombin generation
Can only be identified by molecular testing

Question 15

Factor V Leiden

Answer 15

The most common thrombophilia
Occurs almost exclusively in Caucasians
Heterozygosity increases thrombotic risk by ~ 2-fold
Homozygosity increases thrombotic risk by ~10-fold
A mutation in F5 results in an arginine to glutamine change at position 506 of the fV protein
In FVL, aPC doesn’t recognise the Gln at position 506, so it doesn’t cleave at position 506
Therefore, in the absence of Protein S, activated FVL is inactivated by aPC at a much slower rate than fVa

Question 16

Factor V Activation

Answer 16

Structure of factor V is a line with A1, A2, B, A3, C1 and C2 regions in that order
For factor V activation:
- thrombin or FXa cleave FV at multiple positions within the B-domain, resulting in the removal of the B-domain
- the resulting pieces of FV (the Heavy Chain and the Light Chain, respectively) are joined by a calcium ion
This is the structure of FVa

Question 17

Factor V Inactivation

Answer 17

Activated factor V (FVa) is inactivated by activated Protein C
Protein C initially cleaves FVa at aa 506
- FVa retains 40% activity after this
Protein C next cleaves at FVa at aa 306
- FVa is inactive after this
Protein C can cleave at aa 306 without previously cleaving at aa 506, but the reaction occurs much slower
This reaction is enhanced by Protein S by approximately 20-fold
Reaction is highly PL-dependent
Both PS and FV (but not FVa) are aPC co-factors that allow it to inhibit factor VIII
For this activity, FV must 1) be cleaved at aa 506, and; 2) must retain the C-terminal portion of the B-domain

Question 18

Factor V Leiden - Testing

Answer 18

Detected in the laboratory using the aPC resistance test
Sample is diluted in fV deficient plasma
Tested in an aPTT, with and without the addition of aPC
In an individual with wild-type FV, the clotting time is prolonged with the addition of aPC
In an individual with fVL, the clotting time is not as prolonged because the fVL is more slowly inactivated by aPC
Ratio of clotting times with and without the addition of aPC is determined
Ratio of test plasma can be divided by the ratio of normal plasma

Question 19

Hereditary Risk Factors for Venous Thrombosis

Answer 19

Blood Group
Dysfibrinoginaemia
Elevated levels of other procoagulant factors (VIII, IX, VII, XI)
Decreased TFPI
Hypofibrinolysis
FVL-independent Activated Protein C Resistance

Question 20

FVL-Independent Activated Protein C Resistance

Answer 20

aPC resistance in the absence of FVL
10-15% of individuals
2-3 fold increased risk of thrombosis
Caused by:
- factor V mutations other than FVL
- other modifiers of the aPC resistance assay
Detected only when patient plasma is not diluted with FV ↓ plasma

Question 21

Thrombophilia Testing

Answer 21

Should not be performed until at least 3 months after an acute thrombotic episode
Functional assays should not be performed while the individual is on anticoagulant therapy
Assays for AT, PC, and PS should be repeated on more than one occasion
Molecular testing can be performed at any time
Family studies

Question 22

Arterial Thrombosis - Risk Factors

Answer 22

Age
Gender
Family history
Blood lipids
Obesity
Hypertension
Cigarette Smoking
Diabetes
Diet
Exercise

Question 23

Heparin Induced Thrombocytopaenia

Answer 23

Thrombocytopaenia after the administration of heparin, due to thrombus formation
More common with UFH than LMWH administration
Begins 5-10 days after heparin exposure
Diagnosis:
- 50% or more decrease in platelet count, or thrombus development, within 5-10 days of initiation of heparin therapy
- decrease is relative to the highest platelet count after the initiation of heparin
- count rapidly decreases

Question 24

Heparin Induced Thrombocytopaenia - Testing

Answer 24

Platelet Count
Immunoassays to detect antibodies against heparin-PF4 complexes
- excellent negative predictive value
- poor positive predictive value
Functional platelet studies
- more specific than immunoassays for the detection of clinically significant antibodies
- serotonin Release Assay (gold standard)
- aggregometry
- flow cytometry

Question 25

Thrombotic Thrombocytopenic Purpura

Answer 25

Microangiopathic haemolytic anaemia w/ schistocytes and thrombocytopenia
Caused by a deficiency in ADAMTS13
ADAMTS13
- A Disintegrin and Metalloprotease with a Thrombospondin Type I motif, member 13
- cleaves ultra-large von Willebrand Factor (UL-VWF) multimers

Question 26

Thrombotic Thrombocytopenic Purpura - Diagnosis

Answer 26

Anaemia and thrombocytopenia in the absence of leukopenia
Schistocytes and polychromatophilic RBCs
Increased LDH, bilirubin, decreased haptoglobin
Negative DAT
N PT and aPTT
Increased UL-VWF
ADAMTS13 assays
- < 5% activity

Question 27

Thrombotic Thrombocytopenic Purpura - Treatment

Answer 27

Plasmapheresis
Cryoprecipitate-depleted plasma
Should be started ASAP, because TTP can fatal and is easily treatable