Week 12 - Factor V East Texas and TFPI

Question 1

Clinical History Summary of Factor V East Texas

Answer 1

Moderately severe bleeding disorder
Symptoms:
- easy bruising
- epistaxis
- bleeding after trauma or surgery
- menorrhagia

Question 2

Mixing Studies

Answer 2

Performed to differentiate factor deficiency from presence of an inhibitor
Mix patient plasma with normal plasma and perform aPTT
If the clotting time normalises (corrects) → factor deficiency
If the clotting time does not correct → Inhibitor

Question 3

PT and aPTT with Factor V East Texas

Answer 3

Both prolonged

Question 4

AT3

Answer 4

Antithrombin 3
AT3 close to the gene for Factor V (F5)
Sequencing of F5 revealed a novel mutation found in affected individuals only
- A→G at nt2440 (Exon 13 of the Factor V gene)
- Ser→Gly at a.a. 756
Remember, [FV] is normal in affected individuals

Question 5

Ser→Gly at a.a. 756 Mutation - Factor V Activation

Answer 5

Only the Heavy and Light Chains are required for factor V procoagulant activity
The B-domain is not required for factor V procoagulant activity
The basic and acidic regions bind to keep Factor V in an inactive state
Removal of either region activates Factor V
Factor V is activated by thrombin or factor Xa
Thrombin cleaves the B-domain at aa’s 709, 1018, and 1545
Factor Xa cleaves the B-domain at aa’s 709 and 1018, but is very inefficient at cleaving at aa 1545
Both proteins separate the basic region from the acidic region, producing an active form of factor V

Question 6

What is TFPI

Answer 6

Tissue factor pathway inhibitor
Anticoagulant protein found circulating in plasma
Binds factor V

Question 7

TFPIα Structure

Answer 7

Kunitz domain 1 - fVIIa
Kunitz domain 2 - fXa
Kunitz domain 3 - protein S
Positively charged C-terminus - fV

Question 8

TFPIα Function - fXa and TF:fVIIa

Answer 8

TFPI inhibits fXa and TF:fVIIa
At normal physiological concentrations, TFPI must bind to fXa before it can inhibit TF:fVIIa
At elevated concentrations, TFPI can inhibit TF:fVIIa independently of fXa

Question 9

TFPIα Function - fVa

Answer 9

TFPI binds to forms of fVa that retain the acidic region
These are sometimes contained in the prothrombinase complex
The binding of TFPI to fVa allows it to inhibit fXa present in the prothrombinase complex
These forms are also found in platelets

Question 10

How does TFPIα Bind Factor V?

Answer 10

Basic regions of fV and TFPIα are ~homologous (LIKTRKKKK vs LIKTKRKRKK)
Basic region of TFPIα binds to acidic region of fV

Question 11

Factor V2440 Mutation

Answer 11

All family members that have the A→G mutation at nt 2440 have a shortened form of FV protein in their plasma
This suggests that the FV mRNA may be shortened

Question 12

Identification of Shortened Exon 13

Answer 12

RNA is isolated from leukocytes of all family members
cDNA is produced
PCR performed using a F primer in exon 12 and R primer in exon 14

Question 13

How is Exon 13 Shortened?

Answer 13

The A→G mutation produces a novel splicing donor site that causes an in-frame deletion of 702 aa

Question 14

FV Short Protein and its Association with fXa-activated FV

Answer 14

The short protein resembles the activated form

Question 15

Calibrated Automated Thrombography

Answer 15

A.k.a CAT assay
Used to measure thrombin generation in plasma
Plasma is incubated with a low [TF] (1 pM)
Coagulation is initiated with the addition of Ca2+
Thrombin generation is monitored in real time by measuring cleavage of a fluorogenic thrombin substrate

Question 16

East Texas CAT Results

Answer 16

Thrombin generation (TG) is decreased in individuals w/ FV East Texas

Question 17

Decreased TG in East Texas Patients

Answer 17

Decreased TG is not inherent to fV-short
itself
Use anti-fV antibody to deplete unaffected and affected plasma of fV
Add recombinant full-length fV or fV-short to plasma
Perform CAT assays

Question 18

TFPIα Levels in East Texas Patients

Answer 18

Plasma TFPIα is increased
ELISAs demonstrate a ~10 fold increase in TFPIα

Question 19

Association between TFPIα and Thrombin Generation

Answer 19

Increased plasma TFPIα is the cause of decreased TG
Add anti-TFPI antibody to affected and unaffected plasma
Perform CAT assay
Subsequent studies demonstrate that fV-Short binds TFPIα

Question 20

Summary of East Texas - fV-Short Protein Formation

Answer 20

Individuals with fV East Texas have an A→G mutation at nt 2440 of the F5 gene
This mutation results in use of novel splice donor and acceptor sites within exon 13 of the F5 gene
The resulting fV mRNA is shortened
The shortened fV mRNA encodes a shortened fV protein (fV-Short)
- this protein is missing 702 aa from the B-domain
- the basic region is contained within this missing region
- therefore, fV-short is missing the basic region, but retains the acidic region

Question 21

Summary of East Texas - Role of TFPIα

Answer 21

TFPIα binds fV-Short, resulting in ↑ plasma [TFPIα]
- TFPIα binds fV-Short because it doesn’t have to compete with the FV basic region
- it is hypothesised that the clearance of TFPIα is prevented after its
binding to fV-Short
↑ plasma [TFPIα] is why individuals with fV East Texas have a bleeding phenotype

Question 22

fV Amsterdam

Answer 22

Affected family members have ↓ TG that is corrected by anti-TFPI antibody
Have a shortened fV protein
Like fV East Texas, fV Amsterdam is due to a novel mutation in exon 13 of F5
C→G mutation at nt 2588, producing a novel splicing donor site
Causes an in-frame deletion of 623 aa within the B-domain
- again, the basic region of fV is removed while the acidic region is retained
→ Markedly increased plasma TFPIα

Question 23

fV Atlanta

Answer 23

832 bp deletion in exon 13
Predicted:
- omission of 277 aa’s
- frameshift mutation
- translation of 12 non-native aa’s
- premature STOP codon (Null allele)
* Predicted Null allele should → decrease
plasma FV, but it doesn’t
F5-ATL is heterozygous, may not show up on electrophoresis

Question 24

Transfection of HEK-293 Cells w/ full Length F5 cDNAs

Answer 24

Evidence confirming that a significant amount of F5-ET cDNA is produced from F5-ATL
Suggests that splicing regulators [silencer(s)] sit within the region deleted in F5-ATL

Question 25

WATCH LECTURE FOR DIAGRAM ANALYSIS