Merton Re

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.

Resistance of normal endothelium to damage by thrombin.

We examined the effect of locally infused thrombin on rabbit neck veins, using autologous [111In]indium‐labelled platelets as a marker of platelet deposition. Purified thrombin at concentrations which clotted the blood in isolated veins did not lead to the deposition of labelled platelets on the vessel wall. However, when the animals were pre‐treated with aspirin (10 mg/kg), there was a marked change in the ratio of radioactive counts between control and treated segments, consistent with platelet deposition on the walls of thrombin‐treated segments.

Studies of human serum amyloid P-component (sap) in relation to coagulation

Abstract Serum amyloid P-component (SAP) undergoes calcium-dependent binding to certain polysaccharides and polyanions. It has been claimed that it acts as a heparin antagonist and that plasma SAP levels are low in patients treated with vitamin K antagonist anti-coagulant drugs. However, in the present study depletion of SAP from plasma had no effect on subsequent coagulation, indicating that it is not a necessary procoagulant. SAP levels in male patients receiving warfarin were the same as in normal controls; in a group of female patients on warfarin, SAP levels were slightly higher than normal, probably in response to the underlying condition for which they were anticoagulated. The SAP from warfarin-treated individuals was indistinguishable from normal SAP antigenically, electrophoretically and in its calcium-dependent ligand binding. Isolated SAP did not inhibit the anti-coagulant activity of heparin, on the contrary, addition of supraphysiological amounts of isolated SAP to citrated plasma or to mixtures of fibrinogen and thrombin inhibited clotting, possibly by interfering with fibrin polymerisation. C-reactive protein (CRP), the classical acute phase reactant, which is closely related to SAP, did interfere with the neutralisation of factor Xa by heparin. This effect was produced by concentrations of CRP within the range commonly seen in disease states, suggesting that it may be the counterpart of an in vivo role for CRP in the acute phase response.

Anticoagulant Actions of Sulphated Polysaccharides

The sulphated polysaccharides to be covered in this review can be divided into two main groups: the glycosaminoglycans, comprising heparin, heparan sulphate and dermatan sulphate, which occur naturally and may contribute towards physiological anticoagulation; artificially sulphated polysaccharides, such as pentosan polysulphate — these are naturally occurring polysaccharides of animal or plant origin which have been artificially sulphated in an attempt to confer heparin like anticoagulant properties.