Barrowcliffe Tw

Low-affinity heparin potentiates the action of high-affinity heparin oligosaccharides

Previous studies have shown that high-affinity (HA) heparin oligosaccharides, with molecular weights of 3,000-5,000, were less effective than unfractionated heparin in preventing serum-induced venous thrombosis in rabbits, using a Wessler stasis model. In the present study, a larger high-affinity fragment (M.Wt. 6,000-6,500) was also found to be less effective than unfractionated heparin as an antithrombotic agent. However, addition of 80 micrograms/kg low affinity (LA) heparin to 80 micrograms/kg of this HA fragment significantly potentiated its antithrombotic activity, and the antithrombotic action of the mixture was equivalent to that of unfractionated heparin. Significant potentiation of antithrombotic activity was also observed on the addition of LA heparin to a HA decasaccharide (M.Wt. 3,000-3,500) with anticoagulant activity only against Factor Xa. The LA heparin content of low molecular weight heparin fractions appears to be an important determinant of their antithrombotic activity.

Inhibition of antithrombin III by lipid peroxides

A study was made of the inhibition of antithrombin III (At III) activity by lipid peroxides prepared from autoxidation of unsaturated fatty acids. Lipid peroxides markedly reduced the thrombin neutralising activity of plasma and purified At III with or without albumin carrier. Heparin Sepharose chromatography and heparin cofactor assays suggested that the primary target of lipid peroxides on the At III molecule may be the heparin binding site. Results from electrophoretic studies suggested that interaction between lipid peroxides and At III increased the negative charge of the At III molecule; however, no aggregation of the At III molecule was observed. Lipid peroxidation is being increasingly recognised as a factor in the pathogenesis of several disease states, and it is possible that local inhibition of At III by lipid peroxides could contribute towards the development of a thrombotic event.

The anticoagulant activity of heparin: Measurement and relationship to chemical structure

For many years the anticoagulant activity of heparin has been estimated by coagulation assays, in which the prolongation of clotting times by heparin is measured under various conditions. More recently, assays have been developed which measure the inhibitory action of heparin on isolated coagulation enzymes, notably Factor Xa and thrombin, using specific amidolytic peptide substrates. The anticoagulant activity of heparin arises primarily from its ability to bind to antithrombin III (AT III), altering the conformation and enhancing the activity of this major protease inhibitor. Passage of heparin through an immobilised AT III column yields two fractions: a high affinity fraction with 300-350 iu mg-1 anticoagulant activity, comprising one-third of the total, and a low affinity fraction with an activity of less than 10 iu mg-1, comprising the remaining two-thirds. Studies in several laboratories have demonstrated that a specific pentasaccharide sequence is required for AT III binding. The authors have shown that the presence or absence of this sequence can be detected by high-field proton NMR, thus providing a semi-quantitative method for a functionally important group. A second major influence on anticoagulant activity is molecular weight distribution. Studies in the authors laboratory on a series of fractions of 5000-35,000 showed that whereas anticoagulant activity in APTT clotting assays decreased with decreasing molecular weight (Mr), activity in anti-Xa assays was maintained or increased in the low Mr fractions. However, in vivo studies showed that high affinity fragments with anti-Xa activity only were poor antithrombotic agents. It appears that the presence of the AT III binding site alone is not sufficient for full antithrombotic activity; an extra length of polysaccharide chain of at least 15 residues is required. Molecular weight distribution is readily assessed by HPLC, although the lack of suitable reference materials hampers assignment of absolute molecular weights. Important determinants of anticoagulant activity can now be assessed by physicochemical techniques but, at present, these techniques are not precise enough to replace anticoagulant assays as predictors of in vivo behaviour.

Phospholipid binding of factor VIII in different therapeutic concentrates

Binding to anionic phospholipid (PL) is essential for the biological function of factor VIII (FVIII). We have developed a method to study the level of PL binding of FVIII in a variety of therapeutic concentrates, using the BIACORETM system which utilizes the Surface Plasmon Resonance (SPR) phenomenon. A HPA sensor chip was employed on to which synthetic phospholipid unilamellar vesicles were adsorbed to form a 3:1 phosphatidylcholine: phosphatidylserine lipid monolayer. Using this surface the interaction of unlabelled FVIII in concentrates was observed from which direct kinetic data (kon, koff and KD values) were obtained in real‐time. Marked differences in the binding to PL, as measured by KD values, between different products were observed. These fell into three categories: two recombinant FVIII products showed high affinities for PL with KD values around 0.05–0.14u2003nm; four high‐purity plasma derived products, two prepared by monoclonal antibody and two prepared by ion‐exchange chromatography, had 6–8‐fold lower affinities, and two intermediate‐purity products had 34–60‐fold lower affinities with KD values in the nm region. Measurements of kon and koff values for each product showed that the differences in the KD values expressed were primarily due to the differences in their respective kon values, although the recombinant products showed changes in the koff values. The study showed that the assessment of binding to PL by FVIII in concentrates was possible without prior purification and gave KD values in the range reported previously for other methods. The difference between the products requires further investigation but may be partly due to other proteins present, in particular the content and quality of von Willebrand factor which is known to affect PL binding of FVIII.

In vivo release of anti-Xa clotting activity by a heparin analogue

Abstract The parenteral injection of a semi-synthetic heparin analogue (SSHA) releases anti-Xa clotting activity, lipoprotein lipase activity and PF 4 antigen. The increased anti-Xa activity is not neutralized by PF 4 or protamine sulphate. A second injection of the drug after 90 minutes, or an increase in dose, does not increase the level of induced anti-Xa clotting activity. Possible mechanisms of action include the release by SSHA of endogenous glycosaminoglycans with anti-Xa activity, and interference by released lipoprotein lipase of a modulator of anti-Xa activity. It is concluded that a drug with weak anticoagulant activity in vitro may nevertheless have significant antithrombotic potential.

Oxygen radicals, lipid peroxidation and the coagulation system

Lipid peroxidation is an oxidative fragmentation of unsaturated lipids which can proceed by controlled enzymic steps or as an uncontrolled radical chain reaction. Products of lipid peroxidation include numerous primary peroxides which, in the presence of suitable transition metal catalysts, decompose to give oxygen radicals and secondary carbonyl compounds. Oxygen radicals can cause molecular damage and both peroxides and aldehydes, in nanomolar concentrations, possess potent biological activities. Tissue damage leading to degenerative disease processes can be caused by increased peroxide formation and decomposition. However, in most cases increased peroxide formation and decomposition may only reflect the consequences of enhanced tissue damage. Products resulting from the autoxidation of polyunsaturated fatty acids have been shown to promote thrombin generation in platelet-poor plasma [1], an activity enhanced by their inhibitory action on antithrombin III [2]. These autoxidation products act on plasma triglyceride-rich lipoproteins, especially chylomicra of dietary origin, to produce the procoagulant activity [3]. Thus, the products of lipid peroxidation, which may arise as a consequence of tissue damage, can themselves lead to the initiation of degenerative disease processes. In the present study, we have focussed on the procoagulant activity of the phospholipid fraction of chylomicra. Injections of heparin or pentosan polysulphate, drugs which release lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL), into healthy volunteers, resulted in a 50% fall in lipid peroxide-induced thrombin generation. In vitro addition of purified HTGL gave a similar reduction, but LPL had little effect. HTGL has a strong phospholipase activity, and incubation of fatty plasma or isolated chylomicra with phospholipase A2 or phospholipase C markedly reduced their procoagulant activity. These results suggest that the phospholipid fraction of chylomicra is responsible for the procoagulant activity, and this was confirmed by isolation of the phospholipids from chylomicra by organic solvent extraction or freeze/thawing. Table 1 shows the results of an experiment in which the surface coat was separated by centrifugation from the core material after repeated freeze/thawing. The surface coat fraction contained virtually all the phospholipid and all the procoagulant activity.

Assay Discrepancies with Highly Purified Factor VIII Concentrates

We have assayed two different monoclonal-antibody-purified concentrates (A and B) and one conventional concentrate (C), against the 3rd International Standard for factor VIII concentrate, using one-stage, two-stage and chromogenic methods. One-stage assays performed with immunodepleted plasmas gave lower potencies than with haemophilic plasma for all concentrates, though the discrepancies were most marked for the two highly purified products. The absence of von Willebrand factor in one of the immunodepleted plasmas appeared to contribute towards the low potencies observed. In addition, potencies of product A were 50% higher by one-stage assays (haemophilic plasma) than by two-stage or chromogenic methods. These results indicate the need for careful evaluation of assay methodologies for assessment of factor VIII:C activity in highly purified concentrates.

Relative Efficacy of Heparin and Related Glycosaminoglycans as Antithrombotic Drugs

In a standardized animal model, unfractionated heparin (UFH) prevents venous thrombogenesis at a dose of 80 micrograms/kg. Oligosaccharide fragments of heparin, with very high anti-Xa activity both in vitro and in ex vivo plasma samples were less effective than UFH in preventing thrombosis. A decasaccharide fragment was virtually inactive in impairing thrombosis at this dose, although a 20-22 monosaccharide fragment showed some impairment. Dermatan sulfate, which has no anti-factor Xa activity, partially impairs both thrombin generation and stasis thrombosis. However, dermatan sulfate could not suppress thrombin generation below about 35% of control at the doses studied. Neither oligosaccharides nor dermatan sulfate were as effective on a weight basis as UFH in impairing thrombosis, particularly after 20 minutes stasus. Maximal antithrombotic effects are achieved when both factor Xa and thrombin are inhibited. Drugs which act primarily on factor Xa (oligosaccharides) or thrombin by non-ATIII pathways (dermatan sulfate) are less efficient than UFH as antithrombotic drugs.

Variability in factor VIII concentrate measurement: results from SSC field collaborative studies

Summary.u2002 Seven ‘field’ collaborative studies on factor (F)VIII concentrate potency measurements were carried out, using local routine methodology, standards and calculation of results. Data from five of the 12 different concentrates studied are described in detail. These studies revealed that, for the intermediate‐purity and the recombinant FVIII concentrates, one‐stage potencies were significantly lower than chromogenic potencies, whilst for the two high‐purity FVIII concentrates one‐stage potencies were significantly greater than chromogenic potencies. On comparing predilution methods for the intermediate‐purity concentrate, equivalent potencies were obtained using either buffer or FVIII‐deficient plasma as prediluent. For the two high‐purity and the recombinant concentrates, potencies obtained using buffer as prediluent were significantly greater and lower, respectively, than potencies obtained using FVIII‐deficient plasma as prediluent. Interlaboratory variabilities were compared over all 12 concentrates studied and coefficients of variation (CVs) for one‐stage assays were found to be much greater than for chromogenic assays. This was true for all concentrates except for the intermediate‐purity concentrate and samples A and B from the first study, where the reverse was true. Furthermore, much better CVs were obtained when using FVIII‐deficient plasma than when using buffer as prediluent, for all FVIII concentrates except for the intermediate‐purity concentrate where the reverse was true, and sample B where CVs were equivalent. Overall, CVs were far worse than those obtained in controlled collaborative studies. Generally, however, CVs were better with chromogenic assays and predilution in FVIII‐deficient plasma, as is recommended by the International Society on Thrombosis and Haemostasis/Scientific and Standardization Committee, particularly for higher purity and recombinant concentrates.

In vitro and in vivo studies of the anti-Xa activity of heparin

Abstract Several heparin preparations have been assayed using the Denson and Bonnar anti-Xa assay. The heat defibrination step involved in this assay has been shown to introduce discrepancies into the results, due mainly to co-precipitation of the heparin with fibrinogen. The amount of heparin lost is dependant on the molecular weight. The anti-Xa activity of ex vivo samples from subjects given heparin was much more resistant to loss during defibrination than in vitro samples, resulting in artificially high potency estimates. A modified assay has been proposed, omitting the defibrination step. The results provide further evidence that the anti-Xa activity observed after subcutaneous injection of heparin differs from that measured when the drug is added to plasma in vitro.