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Development of a method to separate lipoprotein-bound and lipoprotein-depleted tissue factor pathway inhibitor. Measurement of free tissue factor 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 fractions. This was performed by ultracentrifugation of plasma, after increasing its density to 1.21 g/ml with potassium bromide. Blood was taken from nine volunteers before and after an injection of low-molecular-weight heparin. The ultracentrifugation procedure was adequate, since the mean cholesterol recovery was 91% and only 2% of the cholesterol remained in the lipoprotein-depleted fraction. No free TFPI protein was found in the lipoprotein-rich fraction. Moreover, the amount of free TFPI in the lipoprotein-depleted fraction was close to that found in plasma. Using this method, we confirmed that heparin does not induce an increase in bound TFPI and that the moderate increase in total TFPI antigen in plasma is entirely caused by the enhancement of free TFPI. We then applied the TFPI chromogenic assay to the lipoprotein-depleted fraction 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 filtration for measuring free TFPI activity, and might be of value in the search for TFPI abnormalities in patients with thrombosis.