Recombinant human extrinsic pathway inhibitor. Production, isolation, and characterization of its inhibitory activity on tissue factor-initiated coagulation reactions.
Previous studies have shown that extrinsic pathway inhibitor (EPI) is an effective inhibitor of factor Xa alone or factor VIIa-tissue factor complex in the presence of factor Xa. Since tissue factor exposure is implicated in thrombogenesis, we hypothesized that EPI may be valuable in the treatment of some thromboembolic episodes. Furthermore, EPI may be an important factor in bleeding complications in hemophiliacs. In the present study, human EPI was expressed in baby hamster kidney cells using a mammalian expression vector. Transfected cells expressed 1-2 micrograms/ml of recombinant EPI (rEPI) which was purified to homogeneity by heparin-Sepharose chromatography, ion-exchange chromatography, and reverse phase high performance liquid chromatography. Purified rEPI exhibited a specific activity of 30,000 units/mg and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 42,000. In addition, the NH2-terminal sequence of rEPI was identical to that of HepG2 EPI and HeLa EPI. The ability of rEPI to inhibit factor X activation by a complex of factor VIIa-tissue factor was then examined in the presence and absence of plasma concentrations of human factors VIII and IX. Using relipidated human brain tissue factor apoprotein, rEPI inhibited the factor VIIa-mediated activation of factor X half-maximally at 2.5 and 1 nM in the presence and absence of factors VIII and IX, respectively. Using monolayers of a human bladder carcinoma cell line (J82) as the source of tissue factor, the activation of factor X by cell-bound factor VIIa was inhibited half-maximally by 5 nM rEPI in the presence of factors VIII and IX. The proteolytic activity of J82 cell-bound factor Xa toward prothrombin was inhibited half-maximally at approximately 5 nM rEPI, while the amidolytic activity of factor Xa in solution was inhibited by rEPI with a Ki of 130 pM. Recombinant EPI also inhibited the amidolytic activity of factor VIIa half-maximally at 10 nM rEPI in the presence of relipidated tissue factor apoprotein and calcium. These results indicate that, in the presence of plasma concentrations of factors VIII and IX, at least 10 times the plasma concentration of EPI is required to reduce factor VIIa-dependent factor X activation one order of magnitude in vitro. In the absence of functional factor VIII and IX, rEPI at plasma levels was a potent inhibitor of factor VIIa-mediated factor X activation, and this activity presumably accounts for the inability of hemophiliacs to initiate hemostasis via the extrinsic pathway.
Department of Pathology, University of New Mexico School of Medicine, Albuquerque 87131.
A H
AH
Pedersen
O
O
Nordfang
F
F
Norris
F C
FC
Wiberg
P M
PM
Christensen
K B
KB
Moeller
J
J
Meidahl-Pedersen
T C
TC
Beck
K
K
Norris
U
U
Hedner
D000595:Amino Acid Sequence; D000906:Antibodies; D001483:Base Sequence; D001777:Blood Coagulation; D002460:Cell Line; D003001:Cloning, Molecular; D005167:Factor VII / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D015942:Factor VIIa / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D010455:Peptides / Q000138:chemical synthesis; D011516:Prothrombin / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D013925:Thromboplastin / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
D000906:Antibodies; D008074:Lipoproteins; D015345:Oligonucleotide Probes; D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D005167:Factor VII; D011516:Prothrombin; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa