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The anticoagulant activities of high and low molecular weight heparin fractions were measured by three assay methods, both in vitro, and after intravenous injection in volunteers. The low molecular weight (LMW) fraction had similar anti-Xa activity in vitro to the high molecular weight (HMW) fraction, but in APTT assays the HMW fraction was about twice as potent. After intravenous injection, the two fractions gave equal heparin levels by anti-Xa assays, but in APTT assays using synthetic substrate S-2222 gave about 20% lower levels than anti-Xa clotting assays for both heparins. Complete protamine neutralization of the post-injection heparin activity was found in APTT and synthetic substrate assays, but about 20% of the clotting anti-Xa effect could not be neutralized. Complete neutralization of the fractions by protamine was shown by all three assays in vitro. This non-neutralizable activity probably accounts for the difference between the anti-Xa clotting and synthetic substrate assays. Studies by crossed immunoelectrophoresis and affinity chromatography indicated that the antithrombin III binding properties of the two fractions were similar.
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