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Anticoagulant Activities of High and Low Molecular Weight Heparin Fractions

Summary. The anticoagulant activities of high and low molecular weight heparin fractions were measured by three assay methods, both in vitro, and after intravenous injection in volunteers. The low molecular weight (LMW) fraction had similar anti‐Xa activity in vitro to the high molecular weight (HMW) fraction, but in APTT assays the HMW fraction was about twice as potent. After intravenous injection, the two fractions gave equal heparin levels by anti‐Xa assays, but in APTT assays the LMW fraction had only half the activity of the HMW fraction. Anti‐Xa assays using synthetic substrate S‐222 gave about 20% lower levels than anti‐Xa clotting assays for both heparins. Complete protamine neutralization of the post‐injection heparin activity was found in APTT and synthetic substrate assays, but about 20% of the clotting anti‐Xa effect could not be neutralized. Complete neutralization of the fractions by protamine was shown by all three in vitro. This non‐neutralizable activity probably accounts for the difference between the anti‐Xa clotting and synthetic substrate assays. Studies by crossed immunoclectrophoresis and affinity chromatography indicated that the antithrombin III binding properties of the two fractions were similar.

Anticoagulant activities of lung and mucous heparins

Abstract It is generally assumed that heparins prepared from different tissues have equivalent actions. However, significant differences were found between lung and mucosal heparins when they were examined in an anti-Factor Xa assay, both in vivo and in vitro . These differences were not seen when the heparins were assayed by an APTT assay. With both types of heparin, anti-Xa activity increased with low molecular weight fractions, while the APTT activity decreased. However, with lung heparin the specific activities by anti-Xa assay were much lower at all molecular weights examined. It is believed that these observations have important implications for the assay and clinical use of heparin.

A low molecular weight heparin compared with unfractionated heparin

A 6000 daltons low molecular weight heparin (LMWH) was compared with unfractionated mucosal heparin in vitro and in vivo. Despite unimpressive specifications by clotting assays in vitro, the LMWH gave high and sustained activity in vivo by anti-Factor Xa assays, following subcutaneous injection. However, activity measured by APTT and calcium thrombin time assays was at least as high as occurred following unfractionated heparin. On the basis of clotting assays, there seems no reason to expect a lower incidence of haemorrhagic side-effects following the clinical use of this LMWH. The study also strikingly demonstrates the inadequacy of in vitro clotting assays for assessing the in vivo behaviour of LMWH.

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.

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.

The relationship between vessel wall injury and venous thrombosis: an experimental study

Summary The relative importance of stasis, vessel wall damage and hypercoagulability in the pathogenesis of venous thrombosis remains disputed. While the combination of local vascular stasis and systemic hypercoagulability can be shown to produce experimental thrombi within a few minutes, it has been claimed that vessel wall damage is also a necessary component of venous thrombogenesis. In this experimental study, mechanical crushing of the jugular veins produced patchy areas of denuded endothelium, with underlying vessel wall oedema, as seen by ultrastructural examination. While the exposed subendothelium became covered with activated platelets following restored blood flow, there was no fibrin formation after 5 min. When blood flow was restored for 60 min following the crush injury, white cells could be seen adhering to and migrating through the vessel wall, although there was still no visible fibrin. The addition of venous stasis for 20 min did not lead to the formation of stasis thrombi in association with the damaged areas. The present experiments demonstrate that, far from there being subtle endothelial damage contributing to acute venous thrombosis, even readily demonstrable damage is a poor stimulus to fibrin formation at local sites of vessel wall injury.

The effect of stasis on the venous endothelium: an ultrastructural study

In carefully dissected neck veins, no evidence was found of platelet adherence to the vessel wall or leucocyte migration. However, 30–60 min of total stasis led to polymorphonuclear leucocytes sticking to the endothelium and their subsequent migration. This migration across the vessel wall resulted from stasis and not the trauma of dissection. Adherence and migration of leucocytes did not cause gross endothelial cell damage or desquamation within the observed period of stasis and there was no associated platelet adherence following restoration of blood flow. Thus leucocyte migration does not impair the non‐thrombogenicity of the endothelium in acute experiments.

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.

HEPARIN, LOW MOLECULAR WEIGHT HEPARIN, AND HEPARIN ANALOGUES

In 1976, Johnson and colleagues first showed that a low molecular weight heparin, when given subcutaneously to man, produced higher anti-Xa blood levels than did unfractionated heparin. Shortly afterwards a heparin analogue, called SSHA (semi-synthetic heparin analogue), was reported to produce blood levels comparable to heparin, as measured by an anti-Xa clotting assay, despite low anticoagulant activity in vitro and evidence that the analogue did not bind to antithrombin 111 (Thomas et al, 19 7 7; Lane et al, 19 7 7). It is now clear that some low molecular weight heparins and heparin analogues with unremarkable in vitro specifications by clotting assays, nevertheless have significant effects in vivo. Conversely, certain oligosaccharide heparin fragments, with very high anti-Xa activity in vitro, are not as effective as might be expected in preventing experimental thrombosis (Thomas et al, 1982). In contrast to unfractionated heparin, these low molecular weight materials demonstrate a lack of correlation between in vitro assays and in vivo events, thus complicating the assessment of their anti-thrombotic effectiveness. While understanding at the molecular level of the mode of action of heparin is now quite sophisticated, the relationship between in vitro kinetics and the anti-thrombotic action of heparin is still far from being fully understood.

Anticoagulant Activities of Heparin and Fragments

Interest in low-molecular-weight (LMW) fractions and fragments of heparin was stimulated by observations some 10-12 years ago that, whereas anticoagulant activity measured by traditional assays such as APTT decreased with decreasing molecular weight (MW), activity measured by anti-Xa assays was retained or even increased in the LMW fractions.14 It was also shown that a LMW fraction gave much higher blood levels than a high molecular weight (HMW) fraction when both were injected subcutaneously in equal amounts.s Following these observations, there has been intensive interest in the manufacture of LMW heparins for clinical In order to get a satisfactory yield, most manufacturers have used chemical or enzymatic depolymerization methods and the resulting products are mixtures of oligosaccharide fragments with mean MW mostly between 4000 and 6OOO but encompassing quite a wide range of molecular weights. For studies of the mechanism of their anticoagulant actions, fragments of more precisely defined MW are preferable. Such fragments can be produced by nitrous acid depolymerization and gel filtration,8 and more recently, the pentasaccharide fragment, which represents the smallest heparin chain capable of binding to antithrombin I11 (ATIII), has been chemically synthe~ized .~ In this review, the comparative inhibitory actions of unfractionated heparin (UFH) and its fragments on isolated coagulation enzymes will be discussed and attention will be focused on the relevance of inhibitory actions against purified enzymes to overall anticoagulant activity in plasma. In this context, “anticoagulant” activity is defined in its classical sense, i .e., against clotting. Since clotting only occurs in the presence of thrombin, anticoagulant activity is defined as any activity which enhances thrombin inhibition, or inhibits, delays or prevents the generation of thrombin. To distinguish between in vitro and in vivo events, the term “anticoagulant” is reserved for in uitro activities in plasma and “antithrombotic” is used to describe in uivo inhibition of clotting.