DEVELOPMENT OF A METHOD TO SEPARATE LIPOPROTEIN-BOUND AND LIPOPROTEIN-DEPLETED TISSUE FACTOR PATHWAY INHIBITOR - MEASUREMENT OF FREE TISSUEFACTOR PATHWAY INHIBITOR ACTIVITY

The aim of this study was to develop a method to separate lipoprotein- bound from lipoprotein-free tissue factor pathway inhibitor (TFPI) and to measure the TFPI chromogenic activity and antigen in both fraction s. This was performed by ultracentrifugation of plasma, after increasi ng its density to 1.21 g/ml with potassium bromide. Blood was taken fr om nine volunteers before and after an injection of low-molecular-weig ht heparin. The ultracentrifugation procedure was adequate, since the mean cholesterol recovery was 91% and only 2% of the cholesterol remai ned in the lipoprotein-depleted fraction. No free TFPI protein was fou nd in the lipoprotein-rich fraction. Moreover, the amount of free TFPI in the lipoprotein-depleted fraction was close to that found in plasm a. Using this method, we confirmed that heparin does not induce an inc rease in bound TFPI and that the moderate increase in total TFPI antig en in plasma is entirely caused by the enhancement of free TFPI. We th en applied the TFPI chromogenic assay to the lipoprotein-depleted frac tion to assess the activity of free TFPI. The activity was 0.11 +/- 0. 03 and 0.36 +/- 0.08 U/ml before and after heparin, respectively (a 3. 6-fold increase) while the activity of bound TFPI did not increase at all. We suggest that this method may be an alternative to gel filtrati on for measuring free TFPI activity, and might be of value in the sear ch for TFPI abnormalities patients with thrombosis. (C) 1998 Lippincot t Williams & Wilkins.