Ratios of anti‐factor Xa to antithrombin activities of heparins as determined in recalcified human plasma

Abstract

Summary. Anti-factor Xa and anti-thrombin activities of unfractionated (UF) and low molecular weight (tMW) heparins have been measured in human plasma and with purified human antithrombin III (ATIII) in the absence and presence of 1'5 mu calcium. The anti-factor Xa and antithrombin activities were measured directly, by assessing the heparin-dependent pseudo-first order rate constants of inactivation of human factor Xa or thrombin. These activities were studied with the 4th International Standard for UF heparin, the lst International Standard for LMW heparin, C'1216, enoxaparin, Cy222, and the synthetic pentasaccharide' In plasma, calcium equally well increased the specific antifactor Xa catalytic activities as compared to purified ATIII' That is, 1 '5 mu calcium stimulated the UF standard heparincatalysed inactivation of factor Xa 2'L-2'4 times' In the presence of the LMW heparins the effect of calcium was smaller (1 '3-1 '7 times), and in plasma there was no effect of calcium on the pentasaccharide-catalysed inactivation of factor Xa. Thus, the largest effects of calcium in the inactivation reaction of factor Xa is seen with UF standard heparin. Calcium reduced the anti-thrombin activities of all the heparin preparations studied about 1'5 times when purified ATIII was used, although in plasma this effect was less clear. Consequently, in the presence of 1'5 mlu calcium the ratio of the anti-factor Xa to the anti-thrombin activities of UF standard heparin approximated those of the LMW heparins. The only exception was CY222, which under all conditions retained anti-factor Xa/anti-thrombin ratios significantly higher than those ofUF standard heparin' One of the regulatory mechanisms of blood coagulation is the inhibition of enzymes of the coagulation system by the plasma protein antithrombin III (ATIII)' its main targets in clotting plasma being thrombin and factor Xa (Harpel' 1987). Inactivation ofthese serine proteases is catalysed by heparin, and thus the heparin-dependent anti-factor Xa and anti-thrombin activities have become main points of interest in studying the mode of action and the standardization of heparin. It should also be noted here, however, that the ATlll-dependent inactivation of free factor fXa can contribute significantly to the heparin effect in clotting plasma (B6guin et al, l99l).Since 1976 it is known that low molecular weight (LMW) heparin fractions have little effect on tests of overall clotting' such as the activated partial thromboplastin time (APTT)' but + Present address:

Inhibition of Prothrombinase at Macroscopic Lipid Membranes: Competition between Antithrombin and Prothrombin

“…For inhibition by antithrombin, we observed a true second-order rate constant of 0.06 x lo6 M-l min-I, which is only 50% lower than the value, 0.12 x lo6 M-I min-l , reported for free factor Xa (Olson, 1992). The value k i n h = 130 x lo6 M-' min-l for antithrombinheparin (UFH) is even closer to the value k i n h = 180 x lo6 M-' min-' calculated by interpolation from the rate constants k i n h = 260 x lo6 M-l min-I at 1.5 mM CaC12 and 120 x lo6 M-' min-l at 4 mM CaC12 found for free factor Xa (Schoen et al, 1992).…”

Comparison of the Non-Specific Binding of Unfractionated Heparin and Low Molecular Weight Heparin (Enoxaparin) to Plasma Proteins

“…Human factor Xa (25 pl, Enzyme Research Laboratories) was added to each well to a final concentration of 4.6 nM. After 5 min incubation at 37" C, 25 pl of chrornogenic substrate (3.43 mM solution of Chromozym X, Boehringer Mannheim, Laval, Canada) was added and the linear change in optical density in each well was monitored kinetically at 405 nm over 60 s (A OD/ min) at 37" C using a Thermomax microplate reader (Molecular Devices Corp., Menlo Park, CA). Each assay was performed in duplicate and the absorbance of a blank solution containing all reagents except for factor Xa was subtracted from each data point.…”

Fatal Ovarian Hemorrhage Associated With Anticoagulation Therapy in a Yucatan Mini-Pig Following Venous Stent Implantation

“…The anticoagulant effects and pharmacological properties vary with the size of the molecules and it has limited bioavailability because it binds to plasma proteins, platelets (platelet factor 4), macrophages, and endothelial cells, resulting in a highly variable anticoagulant response. UFH potentiates the effects of antithrombin III on factor Xa and thrombin (23,24). Heparin is cleared from the blood via two principle mechanisms, and its clearance is dependent upon dosage and molecular weight.…”