Test 6: Inhibition of Preactivated Xa by TFPI - 10/08/2008

Test 6: Inhibition of preactivated Xa by TFPI - 10/08/2008

Objective:

1. Test TFPI activity with preactivated Xa.

2. In each well different concentrations of Xa, TFPI and chromogenic substrate of Xa.

Results Summary:

1. TFPI does not seem to have an inhibiting effect at all.

2. Conditions similar to those reported in articles.

3. Maybe TFPI does not effect the site that activates the color?

Next steps:

1. Consult with American Diagnostica about the procedure.

2. Purchase new TFPI and reconstitute with BSA.

3. ???

Results:
Experiment 1: TFPI 0-5nM, Xa 70nM.
No notable effect.
Rate taken from first two points of absorbance (when reaction still linear)
Rate seems to decrease with rising TFPI until a concentration of 2.4nM. At 5nM something happens?!
Repeat test with lower concentration of Xa.

Experiment 2: 30nM of Xa
Still not clear effect. Next step reduce Xa to 3nM

Experiment 3: Xa 3nM
something went wrong. need to repeat.

Experiment 4: Xa 3nM - repeat experiment 3
still seems that TFPI has no effect. Let's try to raise TFPI concentration.

Rate - linear fit:

Experiment 5: TFPI at 25-37nM
Taking points 2,3,4 (seconds 10 to 30) and fitting a linear regression, we get something that starts to look normal. Let's reduce concentration of Xa with these TFPI concentrations

Experiment 6: wait 5 minutes before putting color. Let the XaTFPI complex form for 5 minutes.
Doesn't seem to help.
Repeat with lower concentration of Xa.

exactly opposite of what we would expect.

Experiment 7: wait 5 minutes before putting color. Let the XaTFPI complex form for 5 minutes.
Xa 3nM. TFPI 12-37nM.
Still seems that TFPI does not have an effect.

Reagents:

· Chromogenic substrate: American Diagnostica Spectrozyme® FXa

· Xa: HTI factor Xa (#HCXA-0060)

· TFPI: American Diagnostica (no. ???)

· Tris Buffer

Reagent preparation:

1. Tris buffer: Tris 20mM, NaCl 100mM pH 7.3, 10mM Ca2+.

2. Color - X: Spectrozyme® FXa: Reconstitute with 1.0 mL of filtered deionized water to obtain a concentration of 5 mM. Required concentration 0.5-1 mM (insert solution as 10% of well)
Used frozen aliquots.

3. Xa: Arrives in 50% glycerol/H20. Concentration is 6.7 mg/ml, at molecular weight of 46000, this is 145uM.
Dilute by factor 100 - 1ul Xa (145uM) and 99ul buffer to reach concentration of 1.45uM.
Inserting 10μl into well will bring a concentration of 145nM.
This is a very high concentration - as if all X was activated.
For experiment 3, used further dilution of 1:50 - stock concentration of 30nM.

4. TFPI: Add 200μL of filtered deionized water. 5 μg of purified TFPI. which is equivalent to 0.025mg/ml. The molecular weight is approximately 35,000. so the concentration is 700nM. Dilute 3ul with 97ul buffer to achieve stock solution of 24nM. Add as 10% of well to achieve 2.4nM concentration which is close to natural concentration.
Used frozen aliquots.
Higher concentration: (experiment 5) - Dilute 10ul of 700nM with 20ul buffer to achieve stock solution of 240nM.

Assay Procedure

1. Add buffer to wells

2. Add chromogenic substrate X

3. Add factor Xa

4. Add TFPI

5. Measure the absorbances at 405 nm using a microwell plate reader. At 37°.

Plate well contents:
Experiment 1: Xa=70nM

1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
A / control
Buffer: 80
C-Xa: 10
Xa: 5μl
TFPI: 0μl / TFPI-1.5nM
Buffer: 75
C-Xa: 10
Xa: 5μl
TFPI: 5μl / TFPI-2.5nM
Buffer: 70
C-Xa: 10
Xa: 5μl
TFPI: 10μl / TFPI-5nM
Buffer: 60
C-Xa: 10
Xa: 5μl
TFPI: 20μl
B
C
D
E
F
G
H

Experiment 2: Xa=35nM

1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
A
B / control
Buffer: 80
C-Xa: 10
Xa: 2μl
TFPI: 0μl / TFPI-1.5nM
Buffer: 75
C-Xa: 10
Xa: 2μl
TFPI: 5μl / TFPI-2.5nM
Buffer: 70
C-Xa: 10
Xa: 2μl
TFPI: 10μl / TFPI-5nM
Buffer: 60
C-Xa: 10
Xa: 2μl
TFPI: 20μl
C
D
E
F
G
H

Experiment 3: Xa=3nM

1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
A
B
C / control
Buffer: 80
C-Xa: 10
Xa: 10μl
TFPI: 0μl / TFPI-1.5nM
Buffer: 75
C-Xa: 10
Xa: 10μl
TFPI: 5μl / TFPI-2.5nM
Buffer: 70
C-Xa: 10
Xa: 10μl
TFPI: 10μl / TFPI-5nM
Buffer: 60
C-Xa: 10
Xa: 10μl
TFPI: 20μl
D
E
F
G
H

Experiment 4: Xa=3nM (repeat experiment 3)

1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
A
B
C
D / control
Buffer: 80
C-Xa: 10
Xa: 10μl
TFPI: 0μl / TFPI-1.5nM
Buffer: 75
C-Xa: 10
Xa: 10μl
TFPI: 5μl / TFPI-2.5nM
Buffer: 70
C-Xa: 10
Xa: 10μl
TFPI: 10μl / TFPI-5nM
Buffer: 60
C-Xa: 10
Xa: 10μl
TFPI: 20μl
E
F
G
H

Experiment 5: Xa=30nM, TFPI - 12,24,34 nM

1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
A / control
Buffer: 88
C-Xa: 10
Xa: 2μl
TFPI: 0μl / TFPI-15nM
Buffer: 83
C-Xa: 10
Xa: 2μl
TFPI: 5μl / TFPI-25nM
Buffer: 78
C-Xa: 10
Xa: 2μl
TFPI: 10μl / TFPI-36nM
Buffer: 73
C-Xa: 10
Xa: 2μl
TFPI: 15μl
B
C
D
E
F
G
H

Experiment 6: same as exp5. Add Buffer, TFPI and Xa to wells and wait 5 minutes before adding color and measurement.
Xa=30nM, TFPI - 12,24,34 nM

1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
A
B
C
D
E
F / control
Buffer: 88
C-Xa: 10
Xa: 2μl
TFPI: 0μl / TFPI-15nM
Buffer: 83
C-Xa: 10
Xa: 2μl
TFPI: 5μl / TFPI-25nM
Buffer: 78
C-Xa: 10
Xa: 2μl
TFPI: 10μl / TFPI-36nM
Buffer: 73
C-Xa: 10
Xa: 2μl
TFPI: 15μl
G
H

Experiment 7: same as exp6 with lower Xa.
Xa=3nM, TFPI - 12,24,34 nM
Add Buffer, TFPI and Xa to wells and wait 5 minutes before adding color and measurement.