Tissue factor pathway inhibitor TFPI

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Tissue factor pathway inhibitor (TFPI)

• Role in hemostasis
• Methodology

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Characteristics of TFPI

• MW 42 kDa
• 75-90 in endothelial cells
• 10-25 in plasma of which the major
• part is bound to lipoproteins
• Plasma concentration of TFPI about
• 2.5 nM (1.3-3.6 nM (2 SD) 50-140 ng/mL) of
  which only 5-10 is free TFPI
• Heparin induces release of
• intracellular TFPI
• No gender difference, slight increase
• with age

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Action of TFPI

• Direct inhibitor of FXa
• TFPI/FXa complex
• inhibits TF/FVIIa

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Schematic structure of TFPI and its interactions


FXa
Binding of heparin and lipoproteins

Kunitz-2
TF FVIIa
Kunitz-3
Kunitz-1
NH2
COOH
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Is TFPI an important physiological regulator of
hemostasis?

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Yes!

• Antibody-blocked TFPI activity in rabbits
  challenged with endotoxin results in massive
  DIC
• Addition of rTFPI prevents DIC
• Simultaneous presence of physiological levels
  of AT and TFPI effectively prevents explosive
  thrombin generation
• Severe TFPI deficiency not found - not
  compatible with life?

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TFPI levels and disease

• Increased levels in patients with
• Hyperlipidemia
• malignancy
• sepsis TFPI poor prognosis
• poorly regulated diabetes
• uremia

TFPI increase seems to be associated with
inflammation
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TFPI levels and disease, cont.

• Decreased basal level of TFPI
• Case report of moderately decreased TFPI in
  familial thrombosis with no other known genetic
  abnormality
• Two studies indicate increased thrombotic risk
  in FVQ506 carriers with moderately decreased
  TFPI

Abnormal release after heparin infusion may
provide valuable information
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Methodology

• A. Antigen methods
• Determination of total TFPI
• Determination of free TFPI requires binding
  to 3rd Kunitz domain
• B. Activity methods
• Chromogenic, FXa-generation
• Clotting, prothrombin time-based

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Chromogenic method anno 2000

• Main improvement over earlier methods
• - No heat inactivation step (56C, 15 min) for
  removal of FVII and for defibrination

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Assay procedure

• Plasma dilution 140 25 mL
• Combined reagent (CR) 100 mL
• Incubation 30 min, 37C
• FX S-2222 50 mL
• Hydrolysis 30 min, 37C
• Acetic acid, 50 50 mL

CR rTF (Innovin) 1320 FVIIa 12
nM FXa 1.4 nM Ca2 13 mM I-2882
0.12 mg/mL
Ref Bendz B, Andersen TO, Sandset PM. Thromb
Res 97, 463-472 (2000)
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TFPI standard curve
1,6
1,4
1,2
1
A405
0,8
0,6
0,4
0,2
0
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
1,1
TFPI ( plasma)
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Performance of new chromogenic method

• Correlation to other methods
• Old CS Actichrome Total TFPI ELISA
• 0.82 0.90 0.96

Precision, CV Intra-assay Inter-assay Norma
l TFPI 1.7 9.9 High TFPI 10
19.7
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