To investigate AT-III affinity dependence on heparin's actions, heparin (UH) was fractionated on an AT-III-Sepharose column into a high (HAH) and a low affinity (LAH) fraction. Molecular profiling revealed a molecular weight of 11.8 kDa for (UH), 12.6 kDa for HAH, and 10.6 kDa for LAH. The USP anticoagulant potencies were found to be: UH = 160 U/mg, HAH = 198 U/mg, and LAH = 42 U/mg. The anticoagulant effects of each of these fractions were proportionate to the USP potencies. However, protease generation inhibitory activities did not follow the same order. All fractions were also tested for their interactions with tissue factor pathway inhibitor (TFPI). No significant differences were noted on the anti-Xa effects of TFPI with these fractions. Administration of each fraction to primates resulted in equivalent release of TFPI. In a model of jugular vein clamping induced venous occlusion, all agents produced a dose-dependent antithrombotic action. HAH produced a 60 to 70% stronger antithrombotic effect than LAH or UH. Simultaneous administration of TFPI markedly augmented the antithrombotic actions of both UFH and LAH. The effect of TFPI on the antithrombotic activity of HAH was weaker than that on LAH. These observations suggest that AT-III affinity is not the sole determinant of the antithrombotic actions of heparin. The endogenous release of TFPI may contribute to the antithrombotic actions of heparin and related glycosaminoglycans. Furthermore, TFPI is capable of AT-III independent amplification of the antithrombotic actions of both UH and LAH, suggesting a crucial role of this polyvalent inhibitor in the control of thrombogenesis.