Erik Yngve Holmer

Molecular weight dependency of the heparin potentiated inhibition of thrombin and activated factor X. Effect of heparin neutralization in plasma

Abstract Heparin purified by affinity chromatography on antithrombin III-Sepharose was fractionated according to molecular size by gel filtration. The various fractions obtained were studied with respect to their ability to potentiate the inhibition of thrombin and of Factor Xa in plasma and in a purified system containing antithrombin III. Clotting assays and chromogenic peptide substrate assays were used in parallel. The inhibition of thrombin showed a dependency on the molecular size of the heparin that was similar to that shown earlier for the APTT assay. The inhibition of Factor Xa showed a different dependency, indicating that the mechanisms for heparin-potentiated inhibition of thrombin and Factor Xa by antithrombin III are not entirely the same. The inhibition of thrombin and Factor Xa in plasma differed from that in the pure system and this difference was shown to be dependent on heparin neutralization effects.

Anticoagulant activities and effects on platelets of a heparin fragment with high affinity for antithrombin

Abstract A fragment of heparin containing 10–16 sugar units and retaining ability to bind to antithrombin III has been prepared by degrading standard heparin with nitrous acid. This fragment greatly potentiated the inhibition of factor X a by antithrombin III but had virtually no effect on the inhibition of thrombin. Studies on heparin neutralization showed that the fragment was affected to a much lesser extent than standard heparin by heparin neutralizing components in plasma. The heparin-potentiated aggregation of platelets by low concentrations of ADP was measured for a number of heparin fractions and the fragment. The molecular weight of the heparin was found to be the most important factor determining the platelet aggregation activity, low molecular weight fractions including the fragment being much less active than high molecular weight ones.

A new method for the measurement of dissociation rates for complexes between small ligands and proteins as applied to the palmitate and bilirubin complexes with serum albumin

Abstract Rate constants for the dissociation of ligands bound to proteins can be measured by using insolubilized (matrix-bound) protein as adsorbent for the low-molecular weight component bound to the free protein in solution. The complexes of palmitic acid and bilirubin with human serum albumin have been studied by this method. Rate constants of 4.2 · 10 −2 s −1 and 8.8 · 10 −3 s −1 were obtained, respectively, for the dissociation of palmitate and bilirubin complexes with albumin at 25 °C.

Properties of antithrombin III depleted plasma. I. Effect of heparin.

Abstract Antithrombin III depleted plasma was prepared by immunosorption. The plasma was shown to retain all factors necessary for normal coagulation and the level of the protease inhibitors α 1 -antitrypsin and α 2 -macroglobulin was not affected. The addition of heparin to this plasma had no effect on the inhibition of thrombin and factor X a and only slightly prolonged the coagulation time as measured by the APTT test. When purified antithrombin III was added to the depleted plasma the effect of heparin on the inhibition of thrombin and factor X a and on the over-all clotting time was restored. The formation of antithrombin III-heparin complex in heparinized plasma was shown using an immunosorption technique.

Studies on the blood-anticoagulant activity of sulphated polysaccharides with different uronic acid content

The anticoagulant activities of sulphated alginic acids, amylose, cellulose, chitosan, curdlan, dextran, guaran, laminaran and locust bean gum have been studied. The alginic acids have been partially reduced and some of the neutral polysaccharides have been partially oxidised at C-6 of the glycopyranosyl residues. The activities of sulphated polymers containing different proportions of uronic acid and neutral sugar residues have been compared. The results suggest that polysaccharides with an average of at least one sulphate group on each monomeric unit, a molecular weight higher than 10 000 and a high proportion of sulphated primary hydroxyl functions will display activity in the activated, partial thromboplastin time assay (APTT). Sulphated guaran and locust bean gum had the highest activities of the polymers investigated (70 IU/mg, heparin has 130-50 IU/mg). In the concentration range investigated, none of the sulphated polymers showed any significant activity in an anti-factor Xa assay.

Mechanisms of anticoagulant effects of some sulphated polysaccharides.

The anticoagulant activity of a partially reduced sulphated alginic acid, a partially reduced aminated and sulphated alginic acid and sulphated guaran have been studied. The anticoagulant activities in the APTT assay were 28, 39 and 70 IU/mg respectively. None showed any activity in anti-factor Xa assay. Studies on binding to Antithrombin III - Sepharose showed that sulphated guaran and a fraction of the aminated and sulphated alginic acid was bound, whereas no binding occurred with sulphated alginic acid. The inhibition of thrombin activity by these polysaccharides was studied in purified systems with or without added Antithrombin III, using both fibrinogen clotting and chromogenic peptide substrate assays. The two alginic acid preparations showed Antithrombin III-dependent inhibition of thrombin, whereas the sulphated guaran inhibits both by Antithrombin III-dependent and independent mechanisms.