The Anticoagulant Effect of Heparin + Antithrombin III Using Purified Fibrinogen as Substrate

Abstract

The anticoagulant effect of heparin in test systems composed of purified factors [antithrombin III (AT-III), fibrinogen and thrombin] was studied. As expected, the sensitivity to heparin depended on the concentrations of AT-III and thrombin, whereas the fibrinogen level was less decisive. In addition, qualitative differences proved important. Thus, the sensitivity to heparin was greater with crude bovine thrombin than with highly purified thrombin from the same species. Further, the sensitivity to heparin increased following removal of cold-insoluble material from the fibrinogen preparation. Finally, during storage of purified AT-III at + 4 °C for more than 4 weeks, the sensitivity to heparin decreased more rapidly than expected from the amidolytic AT-III assays. Smaller amounts of heparm were required to give a prolonged thrombin clotting time than in whole plasma, indicating that components of normal platelet-poor plasma (besides AT-III and fibrinogen) interfere with the anticoagulant effect of heparin. The present test systems may prove suitable for the detection and evaluation of factors of importance for the so-called heparin tolerance.

The Antiheparin Effect of α₁-Acid Glycoprotein and Platelet Material, Evaluated by the Heparin Thrombin Clotting Time

“…The experimental conditions and the composition of the test systems were described in a previous paper [2]. If nothing else is stated, the final concentrations of fibrinogen and AT-III were 2.5 mg/ml and 1 U/ml, respectively.…”

“…Actually, a positive correlation between increased heparin tolerance and the plasma content of a r acid GP has been demonstrated [5], whereas haptoglobin apparently had no similar effect [5]. Also fibrinogen, within a wide range of concentrations, does not seem to affect heparin tolerance [2].…”

“…Recently, test systems composed of purified antithrombin III (AT-III), fibrinogen and thrombin proved to be much more sensitive to small amounts o f heparin than was whole plasma, indicating that compo nents o f normal platelet-poor plasma (besides AT-III and fibrinogen) inter fere with the anticoagulant effect of heparin [2], Below, these test systems were utilized to study the influence of o^-acid GP and platelet material on the combined anticoagulant effect of heparin and AT-III.…”

The Antiheparin Effect of α₁-Acid Glycoprotein, Evaluated by the Activated Partial Thromboplastin Time and by a Factor Xa Assay for Heparin

“…o.¡-Acid glycoprotein and antithrombin III were isolated from human plasma and handled as previously described (1,2,11,16). Citrate saline buffer (ph 7.4, ionic strength 0.20) contained 1 part 0.11 mol/1 of trisodium-citrate, 1 part veronal buffer (12) and 3 parts 0.15 mol/1 of saline.…”

Thrombin‐, Epinephrine‐ and Collagen‐Induced Platelet Aggregation Inhibited by α1‐Acid Glycoprotein

“…At this temperature the activity (assayed amidolytically) remained unchanged for about 5-6 weeks. However, as a gradual loss of activity was observed earlier when tested by the heparin thrombin clotting time (Andersen & Godal 1977a), only freshly purified AT-I11 preparations were used.…”