TY - JOUR
T1 - Partial purification and characterization of extrinsic pathway inhibitor (the factor xa-dependent plasma inhibitor of factor VIIa/tissue factor)
AU - Warn-Cramei, B. J.
AU - Maki, S. L.
AU - Zivelin, A.
AU - Rapaport, S. I.
N1 - Funding Information:
We thank Dr. Linda Curtiss of Scripps Clinic and Research Foundation for her generous gift of the rabbit antiserum to human apoprotein E. We also wish to acknowledge the technical assistance of Mr. Steven Gunberg. This research was supported by NIH grant HL 27234. Dr. Warn-Cramer was supported in part by training grant HL 07107.
PY - 1987/10/1
Y1 - 1987/10/1
N2 - We report a procedure to purify partially from plasma (1200 fold) the factor Xa-dependent inhibitor of factor VIIa/tissue factor (i.e., the extrinsic pathway inhibitor or EPI) and describe some of its properties. An assay for EPI was developed based upon inhibition of factor VIIa/tissue factor induced release of activation peptide from tritiated factor IX by a test sample in the presence but not in the absence of factor Xa. Approximately 50% of the total EPI activity in plasma was found in the lipoprotein fraction, which was used as the starting material for purification. Total lipoproteins (isolated by density ultracentrifugation) were delipidated and the urea soluble apoproteins gel filtered on Sephacryl S-200. The inhibitory activity co-eluted with the major protein peak, which primarily contained apoprotein A-I. Inhibitory activity was separated from apoprotein A-I by anion-exchange chromatography on Q-Sepharose and was further resolved from higher and lower molecular weight contaminating proteins by polypreparative disc gel electrophoresis in the presence of 0.1% SDS. Functional inhibitory activity eluted from the polypreparative disc gel in two discrete pools of different molecular weights (∼34,000 D and ∼43,000 D). Apoprotein E was identified by immunological techniques as the major protein present in both of these pools. However, incubation with a monospecific polyclonal antibody to human apoprotein E did not decrease EPI activity either in plasma or in the partially purified polypreparative disc gel fractions. A rabbit antiserum was prepared against material from the polypreparative disc gel. The IgG fraction neutralized ∼95% of the total inhibitory activity present in plasma. Therefore, EPI in the lipoprotein fraction and in the non-lipoprotein fraction of plasma appears to be antigenically similar. kw/]blood coagulation, factor VIIa, thromboplastin, factor Xa.
AB - We report a procedure to purify partially from plasma (1200 fold) the factor Xa-dependent inhibitor of factor VIIa/tissue factor (i.e., the extrinsic pathway inhibitor or EPI) and describe some of its properties. An assay for EPI was developed based upon inhibition of factor VIIa/tissue factor induced release of activation peptide from tritiated factor IX by a test sample in the presence but not in the absence of factor Xa. Approximately 50% of the total EPI activity in plasma was found in the lipoprotein fraction, which was used as the starting material for purification. Total lipoproteins (isolated by density ultracentrifugation) were delipidated and the urea soluble apoproteins gel filtered on Sephacryl S-200. The inhibitory activity co-eluted with the major protein peak, which primarily contained apoprotein A-I. Inhibitory activity was separated from apoprotein A-I by anion-exchange chromatography on Q-Sepharose and was further resolved from higher and lower molecular weight contaminating proteins by polypreparative disc gel electrophoresis in the presence of 0.1% SDS. Functional inhibitory activity eluted from the polypreparative disc gel in two discrete pools of different molecular weights (∼34,000 D and ∼43,000 D). Apoprotein E was identified by immunological techniques as the major protein present in both of these pools. However, incubation with a monospecific polyclonal antibody to human apoprotein E did not decrease EPI activity either in plasma or in the partially purified polypreparative disc gel fractions. A rabbit antiserum was prepared against material from the polypreparative disc gel. The IgG fraction neutralized ∼95% of the total inhibitory activity present in plasma. Therefore, EPI in the lipoprotein fraction and in the non-lipoprotein fraction of plasma appears to be antigenically similar. kw/]blood coagulation, factor VIIa, thromboplastin, factor Xa.
UR - http://www.scopus.com/inward/record.url?scp=0023578524&partnerID=8YFLogxK
U2 - 10.1016/0049-3848(87)90341-0
DO - 10.1016/0049-3848(87)90341-0
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AN - SCOPUS:0023578524
SN - 0049-3848
VL - 48
SP - 11
EP - 22
JO - Thrombosis Research
JF - Thrombosis Research
IS - 1
ER -