Elimination of intravenously administered radiolabelled antithrombin III and heparin in humans.

Antithrombin III was purified from normal plasma by DEAE-Sephadex chromatography and heparin affinity chromatography; the protein was subsequently radiolabelled with 125I. 125I-antithrombin III alone and 125I-antithrombin III in the presence of high affinity 35S-heparin fractions were injected into normal humans. 125I-radiolabel and protein bound 35S-radioactivity were followed separately. In semilogarithmic plots 125I-antithrombin III disappeared according to a double exponential curve with a half-life in the second phase of 56.8 hr in the absence of heparin and of 33.7 hr in the presence of heparin. Protein bound 35S-radioactivity disappeared much faster than the 125I-radiolabel. These data support the concept that heparin disappears as free heparin from the equilibrium heparin - antithrombin III in equilibrium heparin + antithrombin III. Immuno-reactive antithrombin III decreased from 100% to 85-90% immediately after injection of 125I-antithrombin III in the presence of heparin and returned to normal values within 30 min. This suggests that antithrombin III is transiently sequestered, possibly in trimolecular complexes consisting of antithrombin III, heparin and either lipases or other vascular bound proteins.

Elimination of high affinity heparin fractions and their anticoagulant and lipase activity.

High and low affinity heparin (HA and LA heparin) were prepared from commercial heparin by affinity chromatography to insolubilized antithrombin III. HA heparin was radiolabeled with 35S and subdivided by gel chromatography into high molecular weight (HMW, average 17,000-26,000 daltons), intermediate molecular weight (MMW, average 12,000-13,000 daltons), low molecular weight (LMW, average 5,000-7,000 daltons), and very low molecular weight (VLMW, average 4,600 daltons) fractions. The kinetics of lipolytic and anticoagulant activity and protein-bound radioactivity were studied after intravenous injection of these fractions. LA heparin failed to induce anticoagulant activity but released the hepatic triglyceride lipase (H-TGL) and lipoprotein lipase (LPL) activities normally. VLMW and LMW heparin failed to release both lipolytic enzymes and did not induce anticoagulant activity measurable by the activated partial thromboplastin time (APTT). A powerful anticoagulant effect was found in the anti-Xa assay, which disappeared according to a continuously concave curve in semilogarithmic plots, with elimination rates similar to those of the protein-bound radiolabel. The other heparin preparations induced all activities measured. Heparin anticoagulant activity estimated by the two assays disappeared following a convex curve, preceded by a rapid initial elimination phase in semilogarithmic plots. The disappearance rates of plasma protein-bound heparin radioactivity and heparin anticoagulant activity estimated by factor Xa inactivation were similar. Peak values of the two lipolytic activities were attained rapidly. H- TGL activity, as well as LPL activity, disappeared following convex curves in semilogarithmic plots, with elimination rates similar to those of plasma protein-bound heparin radioactivity. On the basis of these kinetics, we suggest that, after intravenous administration of heparin, the two lipolytic enzymes present in plasma are complexed with heparin, analogous to the heparin-antithrombin III complex. Finally, the kinetic data indicate that elimination of these activities is determined by the heparin part of the complexes, probably by removal of free heparin.

Kinetics of intravenously administered heparin in normal humans.

Heparin of five commercially available brands was used to study the disappearance of heparin anticoagulant activity in normal humans. The drug was administered intravenously by bolus injection and by continuous infusion. Heparin anticoagulant activity was determined by two assays: a diluted activated partial thromboplastin time (APTT) and an assay based on inactivation of bovine factor Xa, using a clotting system. After a bolus injection, the data fitted neither single exponential nor zero-order clearance. In semilogarithmic plots, heparin anticoagulant activity disappeared according to a slightly convex curve almost always preceded by a rapid initial loss of heparin anticoagulant activity. This disappearance profile was observed with all heparin regardless of the brand or assay system. Heparin anticoagulant activity estimated by the APTT disappeared faster than heparin anticoagulant activity estimated by the anti-Xa activity in the first phase. As expected, higher anticoagulant levels with the anti-Xa assay than with the APTT were also found on continuous infusion in normals as well as in patients treated for deep vein thrombosis or pulmonary embolism. The experimental data suggested a model based on the combination of a saturable and a linear clearance mechanism. These experimental data provide reliable guidelines for adjustment of the dose of heparin in single patients.