Ian R. MacGregor

Anticoagulant activities of four unfractionated and fractionated heparins

Abstract Four commercial heparins, two of bovine lung and two of porcine mucosal origin, have been fractionated by gel filtration (and one of them also by ion-exchange chromatography) to yield different fractions whose mean MWs lie in the MW range 7,000–26,000. Three plasma anticoagulant assays have been used to measure the specific activities of the heparin fractions, a kaolin-cephalin clotting time method (KCCT), a calcium-thrombin clotting time method (TCT) and a specific anti-factor Xa assay. The two unfractionated mucosal heparins were found to have higher specific activities when measured in the anti-Xa assay compared to the KCCT assay, while the unfractionated beef lung heparins had lower anti-Xa than KCCT activities. An increase with MW of the heparin fractions was found in specific activities as measured by KCCT and TCT assays over most of the MW range. The low MW heparin fractions (less than 10,500) from all of the heparins had specific activity ratios, KCCT:anti-Xa and TCT:anti-Xa, which were always less than unity. These specific activity ratios increased with increasing MW, usually to greater than unity for the highest MW (greater than 22,000) fractions. There was a tendency for these specific activity ratios to be higher for the beef lung than the mucosal heparin fractions at a given MW. The ability of heparin fractions to inhibit the thrombin induced aggregation of platelets in citrated platelet-rich plasma increased with MW over most of the MW range studied and seemed to follow the specific activities of the fractions when measured by TCT or KCCT assays.

An objective study of alternative methods of heparin administration

Abstract The efficacy and safety of two methods of heparin administration was investigated; 100 patients with calf vein thrombosis were randomly allocated to receive either subcutaneous (SC) or intravenous (IV) heparin for seven days. Venography was performed in each patient to confirm the exact size and site of thrombus and was repeated at the end of heparin treatment. IV heparin was administered using a constant infusion pump. The dose of heparin to be administered was determined by daily estimation of KCCT. In SC group, thrombi increased in size in 2%, remained unchanged in 50% and decreased in 48%. In the IV group, they increased in size in 20%, were unchanged in 62% and decreased in 18%. The difference in decreases in size was significant (p

Molecular weight dependence of the anticoagulant properties of heparin: intravenous and subcutaneous administration of fractionated heparins to man.

Abstract A commercial mucosal heparin preparation has been fractionated by gel filtration into five different MW fractions of approximate mean MWs 11500–21500. These five heparins were randomly administered to 5 normal volunteers by intravenous and subcutaneous injection. The anticoagulant effects of the fractions were then determined by KCCT, anti-Xa clotting and anti-Xa chromogenic substrate assays. Following intravenous injection the high MW fraction produced identical elimination curves in all three assays, but with decreasing MW there was a divergence between results obtained by KCCT and anti-Xa assays. The lowest MW fraction produced between 2–3 times greater heparin levels when measured by anti-Xa assay. This divergence in assay results was produced as a consequence of the differences in specific activities seen when fractionated heparins were assayed in vitro by the different assay methods. That is, low MW heparin fractions had a higher anti-Xa specific activity than that determined by the KCCT assay. Conversely, high MW heparin had a greater specific activity when determined by the KCCT assay. When the response to intravenous heparin injection (measured by KCCT and anti-Xa assays) was compared to the dose administered it was found that the anti-Xa response was greater than expected. This increased plasma heparin like activity was not dependant upon the MW of the heparin fractions, and could not be neutralised by PF 4 . A MW dependance was observed for the absorption of the heparin fractions into the circulation following subcutaneous injection, with low MW heparin producing higher heparin levels determined by all the assays.

Inhibition of human factor Xa by various plasma protease inhibitors.

Abstract The inhibitory effects of the plasma protease inhibitors antithrombin III, α2-macroglobulin and α1-antitrypsin on the activity of the human factor Xa have been studied using purified proteins. The rate of inhibition was determined by measuring the residual factor Xa activity at timed intervals utilizing the synthetic peptide substrate Bz-Ile-Glu(piperidyl)-Gly-Arg-pNA. Kinetic analysis with varying molar concentrations of inhibitors demonstrated that the inhibition of factor Xa by antithrombin III, α2-macroglobulin and α1-antitrypsin followed second-order kinetics. Calculated values of the rate constants for the inhibition of factor Xa by antithrombin III, α2-macroglobulin and α1-antitrypsin were 5.8·104, 4.00·104 and 1.36·104 M–1·min–1, respectively. The plasma concentrations of the inhibitors can be used to assess their potential relative effectiveness against factor Xa. In plasma this was found as α1-antitrypsin>antithrombin III>;α2-macroglobulin in the ratio 4.64:2.08:1.0. Cephalin was shown to inhibit the rate of reaction between factor Xa and antithrombin III.

The anti-heparin properties of human low-density lipoprotein

High-density (HDL), low-density (LDL) and very low-density lipoproteins (VLDL) have been purified from normal human plasma by a combination of ultracentrifugation in high-density salt and agarose gel filtration. The ability of these lipoproteins to inhibit different molecular weight heparin fractions has been compared, using incubation mixtures comprised of antithrombin III and factor Xa. Residual factor Xa activity was measured using the chromogenic peptide substrate Bz-Ile-Glu-Gly-Arg-pNA. LDL inhibited the high molecular weight (but not low molecular weight) heparin accelerated neutralisation of factor Xa by antithrombin III. VLDL showed a similar, though much reduced anti-heparin activity, while the addition of HDL to the factor Xa incubation mixture produced no measurable anti-heparin activity. These observations suggest that certain plasma lipoproteins may selectively modulate the inhibitory action of heparin against factor Xa.

Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III.

The ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.

The heparin-accelerated neutralisation of bovine α and β thrombins by antithrombin III

Abstract The heparin-accelerated neutralisation of bovine α and β thrombins has been examined using a peptide substrate H- d -phenylalanyl-pipecolyl-arginine-paranitroanilide-HCl to measure thrombin amidase activity. α and β thrombins were both neutralised by antithrombin III and this neutralisation was further accelerated by the presence of small amounts of heparin. Low and high molecular weight heparin and heparins fractionated by their affinity for antithrombin III were all able to accelerate the neutralisation of α and β thrombin. This work is therefore unabel to confirm reports that α and β thrombins have different heparin sensitives.