Heparin-coated stents.

Stenting has revolutionized the field of interventional cardiology. However, early studies revealed a high incidence of thrombotic occlusion of the stent and significant bleeding complications resulting from the use of intensive anticoagulation after implantation. One of the strategies used to reduce stent thrombosis and hemorrhagic complications has been to decrease the thrombogenicity of the stent surface. For this purpose, a unique heparin surface patented by Carmeda AB (Stockholm, Sweden)--the Carmeda BioActive Surface (CBAS)--has been coated onto Cordis (Johnson & Johnson) stents. Preclinical evaluations of the thromboresistance of heparin-coated stents and clinical studies of heparin-coated stents in a variety of clinical settings are discussed.

Low-molecular-weight heparin (Fragmin) versus heparin for anticoagulation during cardiopulmonary bypass in open heart surgery, using a pig model.

Fragmin and heparin were studied in pigs during 120 min of cardiopulmonary bypass (CPB) and up to 240 min postoperatively, with respect to clotting, bleeding and the effects of protamine. Thirty-three pigs received bolus injections of 300 IU/kg with or without additional dosage during CPB and with or without subsequent protamine sulphate. Doses of Fragmin 60% higher were necessary to prevent clotting. These had 100% higher anti-FXa levels but about 50% shorter activated coagulation time (ACT) compared with heparin. Anti-FXa increased with cumulative doses of heparin and Fragmin but ACT and activated partial thromboplastin time (aPTT) did not, indicating a larger loss of thrombin inhibition compared with anti-FXa in both drugs during CPB. Thrombin inhibition was crucial for prevention of clotting. Protamine efficiently normalized ACT in the Fragmin group but left a residual 20% anti-FXa, which did not increase the bleeding tendency. Fragmin could adequately be monitored with ACT and would be a safe alternative to heparin in CPB.

Fragmin (LMWH) vs heparin for anticoagulation during in vitro recycling of human blood in cardiopulmonary bypass circuits: dose-dependence and mechanisms of clotting.

Low-molecular-weight heparin (LMWH) (Fragmin) vs heparin was studied in vitro in order to investigate its antithrombotic efficacy in the isolated thrombogenic link of cardiopulmonary bypass (CPB). Fresh human blood (400 ml) with various dosages of the anticoagulant was recycled in a CPB circuit for 120 min. The standard dosage of heparin (1,500 IU, n = 6) was compared with a lower dosage (1,000 IU, n = 3) and several dosages of Fragmin (IU anti-FXa): 750 (n = 1), 1,500 (n = 3), 2,100 (n = 4) and 2,500 (n = 3). Clotting occurred in three Fragmin experiments at dosages of 750, 1,500 and 2,100 IU. This was associated with short activated clotting time (ACT) and activated partial thromboplastin time (aPTT) but was independent of the levels of anti-FXa, FVIII, von Willebrand factor and prothrombin complex. It was concluded that at least twice the dose of Fragmin (anti-FXa), compared with heparin, was required, suggesting that thrombin inhibition is crucial for the antithrombotic efficacy of heparin in CPB circuits. Absence of fibrinolytic markers suggests that the well known enhancement of fibrinolysis often seen during CPB, is not due to heparin interaction with normally circulating blood components, but rather to interaction with the vessel walls or to the surgical trauma itself.

The influence of surgical trauma on factor XaI and IIaI activity and heparin concentration after a single dose of low-molecular-weight heparin.

To study the influence of surgical trauma on the XaI and IIaI activity after injection of a low-molecular-weight heparin (LMWH) 24 patients undergoing elective cholecystectomy received one subcutaneous injection of the LMWH Fragmin. Each group of eight patients received either 2,500 or 5,000 XaI U 2 h before operation or 5,000 XaI U 10 h before surgery. For comparison an additional eight patients received 5,000 IU unfragmented heparin (UH) before operation. Laboratory analyses included amidolytically measured XaI- and IIaI-activities and direct measurements of heparin. Dose-dependent increase in the XaI- and IIaI-activity with maximal levels about 3-4 h after injection was seen. Patients given the LMWH 2 h before operation had lower levels of XaI-activity 2 h after injection than those receiving it 10 h before surgery, despite the same dose given. This correlated with the heparin concentrations, where the highest concentration was measured in patients given the LMWH 10 h before surgery. In conclusion, the surgical trauma of a cholecystectomy does not seem to have any major influence on the XaI- or IIaI-activity after administration of the studied LMWH. Alterations of the absorption and/or elimination rates cannot, however, be ruled out, but are related to factors other than the operative trauma per se, such as effects of premedication or circadian rhythmic variations.

Antithrombotic effects of heparin oligosaccharides.

Size homogeneous heparin oligosaccharides were prepared from nitrous acid depolymerized heparin by means of repeated gel filtration chromatography. These oligosaccharides were then further separated with respect to affinity for antithrombin by means of affinity chromatography. All the high-affinity oligosaccharides thus obtained had a strong ability to potentiate factor Xa inhibition while their ability to inhibit factor IIa abruptly dropped below a chain length of 20 monosaccharides. In a rabbit stasis model, high-affinity oligosaccharides below a chain length of 20 units also showed a continuous decrease in antithrombotic effect with increasing degree of depolymerization. However, there was no distinct drop paralleling the thrombin inhibiting capacity. Low-affinity oligosaccharides also exhibited a weak antithrombotic effect, although they did not always contribute to an increased anti-factor Xa activity ex vivo. This was the case whether or not they were administered alone or in combination with high-affinity oligosaccharides. Low-affinity oligosaccharides may therefore exert an antithrombotic effect per se with a mechanism of action that is independent of antithrombin III.

Heparin and its low molecular weight derivatives: anticoagulant and antithrombotic properties.

The anticoagulant and antithrombotic effect of heparin has been known for many decades. Nevertheless, the knowledge of structure-activity relationships and its mechanisms of action has been rather limited. However, in recent years important progress has been made and our understanding of how heparin works has increased significantly. Based on this information, attempts have been made to modify heparin to get improved pharmacological properties. The present communication summarizes the recent development. It is shown that certain heparin derivatives of low molecular weight are highly interesting compounds from a clinical point of view. Biochemical and pharmacological properties of such a preparation named Fragmin are presented.

Antithrombotic properties in rabbits of heparin and heparin fragments covalently coupled to human antithrombin III.

Clinical grade heparin is a very heterogeneous mucopolysaccharide, containing molecules with Mr ranging from 6,000 to 30,000 that have either a high affinity or a low affinity for antithrombin III (AT). In this study, the antithrombotic properties of intact high-affinity heparin (Mr = 15,000) and of two heparin fragments (h16, a 16-monosaccharide fragment, with Mr = 4,300, and h12, a 12-monosaccharide fragment, with Mr = 3,200) and of their functional covalent stoichiometric complexes with human AT were compared in a venous thrombosis stasis model in rabbits. Thrombosis was induced by injection of glass-activated human plasma and measured in a segment of the jugular vein that was isolated between two vascular clamps for 10 min. Injections of 55 micrograms/kg resulted in a clear antithrombotic effect for intact heparin, but not for the two fragments. Equivalent amounts (carbohydrate moiety) of covalent complexes of heparin or of both heparin fragments with human AT resulted in an antithrombotic effect lasting for 45-60 min. Injection of 110 micrograms/kg of heparin and of the heparin fragments yielded an antithrombotic effect, lasting 45-60 min; the corresponding amounts of covalent complexes caused an anti-thrombotic effect for 60-120 min. The free and conjugated fragments produced equal antithrombotic effects at equal plasma levels of anti-Factor Xa activity, but the specific antithrombotic activities of free and complexed intact heparin, on a molar basis, were 10-20-fold greater than those of the free and complexed heparin fragments. The plasma half-life of the covalent complexes of the heparin fragments with AT is, however, 10 times longer than that of the complex between intact heparin and AT and 30 times longer than that of free intact heparin. Covalent complexes between AT and heparin fragments could, therefore, be useful to maintain more stable levels of antithrombotic activity in plasma.

Elimination of intravenously administered radiolabelled antithrombin III and heparin in humans.

Antithrombin III was purified from normal plasma by DEAE-Sephadex chromatography and heparin affinity chromatography; the protein was subsequently radiolabelled with 125I. 125I-antithrombin III alone and 125I-antithrombin III in the presence of high affinity 35S-heparin fractions were injected into normal humans. 125I-radiolabel and protein bound 35S-radioactivity were followed separately. In semilogarithmic plots 125I-antithrombin III disappeared according to a double exponential curve with a half-life in the second phase of 56.8 hr in the absence of heparin and of 33.7 hr in the presence of heparin. Protein bound 35S-radioactivity disappeared much faster than the 125I-radiolabel. These data support the concept that heparin disappears as free heparin from the equilibrium heparin - antithrombin III in equilibrium heparin + antithrombin III. Immuno-reactive antithrombin III decreased from 100% to 85-90% immediately after injection of 125I-antithrombin III in the presence of heparin and returned to normal values within 30 min. This suggests that antithrombin III is transiently sequestered, possibly in trimolecular complexes consisting of antithrombin III, heparin and either lipases or other vascular bound proteins.

Elimination of high affinity heparin fractions and their anticoagulant and lipase activity.

High and low affinity heparin (HA and LA heparin) were prepared from commercial heparin by affinity chromatography to insolubilized antithrombin III. HA heparin was radiolabeled with 35S and subdivided by gel chromatography into high molecular weight (HMW, average 17,000-26,000 daltons), intermediate molecular weight (MMW, average 12,000-13,000 daltons), low molecular weight (LMW, average 5,000-7,000 daltons), and very low molecular weight (VLMW, average 4,600 daltons) fractions. The kinetics of lipolytic and anticoagulant activity and protein-bound radioactivity were studied after intravenous injection of these fractions. LA heparin failed to induce anticoagulant activity but released the hepatic triglyceride lipase (H-TGL) and lipoprotein lipase (LPL) activities normally. VLMW and LMW heparin failed to release both lipolytic enzymes and did not induce anticoagulant activity measurable by the activated partial thromboplastin time (APTT). A powerful anticoagulant effect was found in the anti-Xa assay, which disappeared according to a continuously concave curve in semilogarithmic plots, with elimination rates similar to those of the protein-bound radiolabel. The other heparin preparations induced all activities measured. Heparin anticoagulant activity estimated by the two assays disappeared following a convex curve, preceded by a rapid initial elimination phase in semilogarithmic plots. The disappearance rates of plasma protein-bound heparin radioactivity and heparin anticoagulant activity estimated by factor Xa inactivation were similar. Peak values of the two lipolytic activities were attained rapidly. H- TGL activity, as well as LPL activity, disappeared following convex curves in semilogarithmic plots, with elimination rates similar to those of plasma protein-bound heparin radioactivity. On the basis of these kinetics, we suggest that, after intravenous administration of heparin, the two lipolytic enzymes present in plasma are complexed with heparin, analogous to the heparin-antithrombin III complex. Finally, the kinetic data indicate that elimination of these activities is determined by the heparin part of the complexes, probably by removal of free heparin.

Anticoagulant effects of two types of low molecular weight heparin administered subcutaneously.

Two types of LMW heparin were prepared by gel filtration of standard heparin (LMW fraction) and by degradation of heparin by nitrous acid (LMW fragment), respectively. The effects on factor Xa inhibition (XaI), APTT, platelet aggregation and AT III level of these preparations were studied after subcutaneous administration to humans and compared with those of standard heparin. At a dose of 5000 IU (XaI) the LMW fraction and LMW fragment induced peak plasma XaI activity of 0.32 IU/ml and 0.41 IU/ml respectively, compared to 0.07 IU/ml for heparin. Still 11.5 h after administration both LMW preparations gave higher activities than heparin ever induced. Following administration of 10,000 IU (XaI) of the LMW fragment the plasma peak XaI activity was 0.81 IU/ml. This prolonged the APTT from 36 sec to 46 sec only. The half-lives of the XaI activity in plasma were between 3 and 4 hours. No effect on platelet aggregation or AT-III level was demonstrated.

Covalent complexes between low molecular weight heparin fragments and antithrombin III - inhibition kinetics and turnover parameters.

Two high affinity heparin fragments (Mr 4,300 and Mr 3,200) were covalently coupled to antithrombin III (J. Biol. Chem. 1982; 257: 3401--3408) with an apparent 1:1 stoichiometry and a 30--35% yield. The purified covalent complexes inhibited factor Xa with second order rate constants very similar to those obtained for antithrombin III saturated with these heparin fragments and to that obtained for the covalent complex between antithrombin III and native high affinity heparin. The disappearance rates from plasma in rabbits of both low molecular weight heparin fragments and their complexes could adequately be represented by two-compartment mammillary models. The plasma half-life (t1/2) of both low Mr-heparin fragments was approximately 2.4 hr. Covalent coupling of the fragments to antithrombin III increased this half-life about 3.5 fold (t1/2 congruent to 7.7 hr), approaching that of free antithrombin III (t1/2 congruent to 11 +/- 0.4 hr) and resulting in a 30 fold longer life time of factor Xa inhibitory activity in plasma as compared to that of free intact heparin (t1/2 congruent to 0.25 +/- 0.04 hr).

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.

Anticoagulant and antithrombotic effects of heparin and low molecular weight heparin fragments in rabbits.

Heparin and heparin fragments of different molecular weight and with different anti-factor Xa/APTT activity ratios were studied with respect to their ability to inhibit thrombus formation in an animal model. It is concluded that: a) Neither anti-factor Xa nor the APTT activity alone is a good reflector of the antithrombotic activity. b) Anti-factor Xa active fragments must have a minimum molecular weight in order to elicit good antithrombotic activity. c) High affinity for antithrombin III is important for good antithrombotic activity. d) A heparin fragment of molecular weight 4 000 has the same antithrombotic activity as heparin but less effect on the clotting time.

The molecular-weight dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor XIIa and kallikrein by antithrombin.

Heparin fractions of different molecular weight and with high affinity for antithrombin were studied with respect to their ability to potentiate the inhibition of activated clotting factors by antithrombin. Inhibition of thrombin, Factor IXa and Factor XIa showed similarities in the dependence on the molecular weight of heparin and was found to decrease with decreasing molecular weight. Inactivation of Factor Xa, Factor XIIa and kallikrein was, however, less dependent on the size of the polysaccharide and, to a great extent, was potentiated even by low-molecular-weight heparin fractions that had virtually no effect on the inhibition of thrombin, Factor IXa and Factor XIa.