This study was performed in order to separate plasma fractions of tissue factor pathway inhibitor (TFPI) on the basis of TFPI's heparin binding properties. A main goal was to look for differences in anticoagulant effect of the TFPI fractions from plasma. Normal plasma and plasma with increased amounts of TFPI were used; plasma from cancer patients and post-heparin plasma (plasma drawn 5 min after heparin injection). Heparin affinity chromatography separated plasma TFPI into four fractions with increasing heparin affinity: the flow-through fraction, a low affinity fraction eluting at less than 0.3 M NaCl, an intermediate affinity fraction eluting at 0.3-0.55 M NaCl and a high affinity fraction eluting at 0.55-1.0 M NaCl. These fractions corresponded partly to the three TFPI activity fractions obtained by gel filtration. In plasma from cancer patients, the two fractions with intermediate and high heparin affinity were increased three- to four-fold, compared to normal plasma. The TFPI activity in these two fractions eluted with low-molecular-weight (35-60 kD) on gel filtration. In post-heparin plasma an even larger increase in the fraction with high heparin affinity was found; compared to that in normal plasma it was increased 14-fold. TFPI was purified to 0.72 U/mg protein in this fraction (about 43-fold compared to normal plasma TFPI). The anticoagulant effect of TFPI, relative to the chromogenic substrate TFPI activity, was greater in plasma fractions with high heparin affinity than in the other plasma TFPI fractions, and it was five-fold greater than the anticoagulant effect of recombinant TFPI. Thus, plasma TFPI is heterogenous in heparin affinity and in anticoagulant potency.(ABSTRACT TRUNCATED AT 250 WORDS)