A novel degradation pathway of tissue factor pathway inhibitor: incorporation into fibrin clot and degradation by thrombin.
Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor with three tandem inhibitory domains (K1, K2, and K3) that regulates the initial reactions of the extrinsic blood coagulation pathway through K1 and K2. In the present study, the effect of thrombin on TFPI in a purified system was first examined using recombinant TFPI from Chinese hamster ovary (CHO) cells. TFPI was inactivated by thrombin with cleavage of three peptide bonds, Lys 254-Thr 255 in the C-terminal basic region, Arg 107-Gly 108 (reactive site toward factor Xa in K2), and Lys 86-Thr 87 between K1 and K2. Then, degradation of radiolabeled TFPI by thrombin was examined in two systems: (1) mixed with plasma and then tissue factor (TF) and calcium ion, and (2) mixed with fibrinogen and then thrombin. TFPI degradation was detected in serum from normal plasma and more extensively from anti-thrombin (AT)-depleted plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Significant radioactivity was found in the clot after coagulation of the plasma, which decreased after 20 hours' incubation. These changes were more prominent in AT-depleted plasma than in normal plasma. When TFPI lacking the C-terminal basic region was used instead of full-length TFPI, most of the radioactivity was found in serum rather than in fibrin clots. Incorporation of TFPI into the fibrin clot was prevented by a synthetic C-terminal peptide of TFPI. Similar results were obtained after mixing radiolabeled TFPI with fibrinogen and then thrombin in the presence of calcium ion or EDTA. These results demonstrate a novel degradation pathway of TFPI, ie, incorporation into fibrin via the C-terminal basic region and degradation by thrombin (possibly fibrin-bound thrombin).
Ohkura
N
N
National Cardiovascular Center Research Institute, Suita, Osaka, Japan.
Enjyoji
K
K
Kamikubo
Y
Y
Kato
H
H
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D008074:Lipoproteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism*
D008074:Lipoproteins; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D005337:Fibrin; D013917:Thrombin