Inhibitory effects of LDL-associated tissue factor pathway inhibitor.
BACKGROUND
Tissue factor pathway inhibitor (TFPI) is present in plasma as full-length free TFPI (TFPIα) and as C-terminally degraded forms mainly associated with low-density lipoprotein particles (LDL-TFPI). Addition of TFPIα to plasma induces a prolongation of the clotting time when tested in a diluted prothrombin time (dPT) assay, whereas no prolongation is observed with LDL-TFPI or truncated recombinant TFPI (TFPI 1-161). The aim was to further characterize kinetic properties of purified LDL-TFPI in thrombin generation and chromogenic activity assays.


METHODS
LDL-TFPI was purified from human plasma by sequential flotation ultracentrifugation. Thrombin generation was measured in human plasma or in FVIII-immunodepleted plasma using either 1 pM tissue factor and 4 μM phospholipids or 0.5 nM factor Xa (FXa) and 4 μM phospholipids, respectively.


RESULTS
TFPIα prolonged the lag-phase and decreased the thrombin peak in tissue factor-induced thrombin generation, whereas LDL-TFPI exclusively decreased the peak height of thrombin without effecting the lag phase. Steady-state and transient kinetics showed that LDL-TFPI was a more potent inhibitor of FXa than TFPIα and TFPI 1-161, indicating that FXa inhibition was not rate determining for the lag phase, whereas it appeared to affect thrombin generation during the propagation phase. This was supported by FXa-induced thrombin generation showing that LDL-TFPI, compared with TFPIα, more actively decreased the peak height.


CONCLUSIONS
Our results suggest that LDL-TFPI affects thrombin generation during the propagation phase, and is kinetically different from TFPI 1-161. It may therefore play a more prominent physiological role in vivo than hereto anticipated from dPT measurements.
Haemophilia, in vitro Biology, Biopharmaceutical Research Unit, Novo Nordisk A/S, Måløv, Denmark.
Cecilia
C
Augustsson
Haemophilia, in vitro Biology, Biopharmaceutical Research Unit, Novo Nordisk A/S, Måløv, Denmark.
Ida
I
Hilden
Haemophilia, in vitro Biology, Biopharmaceutical Research Unit, Novo Nordisk A/S, Måløv, Denmark. Electronic address: lcp@novonordisk.com.
Lars C
LC
Petersen
D000925:Anticoagulants / Q000627:therapeutic use*; D001777:Blood Coagulation / Q000187:drug effects*; D006487:Hemostasis; D006801:Humans; D008074:Lipoproteins / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D013925:Thromboplastin / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D000925:Anticoagulants; D008074:Lipoproteins; D008077:Lipoproteins, LDL; C051928:lipoprotein-associated coagulation inhibitor; D013925:Thromboplastin
Anticoagulant; Haemostasis; lipoprotein; tissue factor