Leslie R. Berry

Mechanisms responsible for the failure of protamine to inactivate low‐molecular‐weight heparin

Summary. Protamine is unable to completely reverse the anticoagulant effect of the low‐molecular‐weight heparins (LMWH), a fact of clinical importance given the rapid increase in use of LMWH in clinical practice. This investigation sought to determine the mechanism by which LMWH were able to resist protamine‐mediated inactivation. Affinity fractionation of LMWH by passage through a protamine column, with subsequent determination of molecular mass and sulphate charge density, demonstrated that the protamine‐resistant fraction in LMWH is an ultra‐low‐molecular‐weight fraction with low sulphate charge density. This group of molecules was not found in unfractionated heparin, even when species of similar molecular mass were compared. We then determined that different commercially available LMWH varied in their ability to be neutralized by protamine, and that this variability correlated with the total sulphate content of the LMWH. We conclude that reduced sulphate charge, not molecular mass, is the principle reason that protamine is unable to fully inactivate LMWH. Furthermore, different LMWH vary in their ability to be neutralized by protamine, suggesting that product‐specific recommendations for neutralization might be developed.

Inhibition of thrombin by antithrombin III in the presence of certain glycosaminoglycans found in the mammalian aorta

Abstract Chondroitin-4-sulphate, chondroitin-6-sulphate, dermatan sulphate, heparan sulphate and hyaluronic acid were compared with heparin in their abilities to influence the inactivation of bovine thrombin by rabbit antithrombin III. The effect of the glycosaminoglycans on the enzymic activity of thrombin was examined using the substrate α-N-benzoyl arginine ethyl ester. Heparin, dermatan sulphate, heparan sulphate and, to a smaller extent, chondroitin-6-sulphate increased the esterase activity, whereas chondroitin-4-sulphate and hyaluronic acid had a negligible effect. Heparan sulphate and dermatan sulphate markedly accelerated the inactivation of thrombin by antithrombin III, but chondroitin-4-sulphate, chondroitin-6-sulphate and hyaluronic acid did not significantly affect the reaction. The glycosaminoglycans were adsorbed on Sepharose-lysine or polyacrylamide-ethylenediamine to test their ability to bind thrombin or antithrombin III under conditions where neither protein could directly bind to the conjugates. The binding of thrombin to immobilised heparan sulphate or dermatan sulphate compared well with that to heparin. On immobilised chondroitin-6-sulphate, thrombin was retarded and on chondroitin-4-sulphate no thrombin binding was observed. In contrast to thrombin, antithrombin III only bound to heparin: antithrombin III did not bind to dermatan sulphate, heparan sulphate or the other glycosaminoglycans. Antithrombin III inactivation of 125 I-thrombin adsorbed to either heparan sulphate or dermatan sulphate produced a thrombin-antithrombin III complex which was spontaneously released from the glycosaminoglycan. However, no complex was recovered from immobilised heparin unless the column was eluted by 1M NaCl. These results support the view that heparan sulphate and dermatan sulphate could play a role similar to that of heparin in the inactivation of thrombin by antithrombin III.

Blood-compatible biomaterials by surface coating with a novel antithrombin–heparin covalent complex

Covalent antithrombin-heparin complex (ATH) was covalently grafted to a polycarbonate urethane (Corethane) endoluminal graft (a kind gift of Corvita Corporation) after being activated using 0.3% m/m NaOCl in 0.15 M phosphate pH 6.0. ATH graft density (1.98 x 10(-7) mol/m2) was 6 times the maximum amount of unfractionated heparin (UFH) that could be bound to polycarbonate urethane surfaces. Surface-bound ATH could be stored in sterile 0.15 M NaCl at 4 degrees C for at least 2 months with good antithrombotic activity before being implanted into rabbits. Analysis of ATH-coated tubing showed that it contained significant direct thrombin inhibitory activity. In vivo testing in a rabbit model was compared to non-activated non-coated surfaces, activated-non-coated surfaces, hirudin-coated surfaces and antithrombin (AT)-coated surfaces. The weight of the clot generated in the ATH-coated graft tubing was significantly less than the weight of the clot generated within the hirudin-coated graft (p = 0.03 with a 1-tailed Students t test). The anticoagulant nature of ATH grafts in vivo was shown to be due to bound ATH because boththe AT-coated surfaces and non-coated but activated surfaces showed similar thromboresistant efficacy to that of untreated material (ANOVA; p < 0.05). Apart from the direct antithrombin activity that contributed to much of the prolonged patency in vivo, surface-bound ATH likely catalyzed AT inhibition of thrombin, as evidenced by a significant number of 125I-AT binding sites (> or = 1.5 x 10(-8) mol/m2). Thus, ATH appears to be a good candidate for coating cardiovascular devices, such as endoluminal grafts, with high levels of substitution and significant long-term blood-compatibility.

Tritiation of Commercial Heparins by Reaction with NaB3H4: Chemical Analysis and Biological Properties of the Product

Abstract A simple method to tritiate commercial samples of heparin is described. Heparin is reacted with NaB3H4 at pH 8.0 for 3 h at room temperature, after which the 3H-labeled mucopolysaccharide is isolated by gel filtration. Using NaB3H4 of low specific radioactivity (227–555 mCi/mmol), [3H]heparins containing 3.7–8.0 × 105 dpm/mg are made whereas with NaB3H4 of higher specific activity (5–10 Ci/mmol), preparations of 1.2–1.8 × 107 dpm/mg are obtained. From analysis of [3H]heparin using several techniques (periodate oxidation; mild acid hydrolysis accompanied by ion-exchange chromatography and high-voltage electrophoresis), the labeling procedure largely relies on a small proportion (approximately 4–6%) of heparin molecules possessing a terminal monosaccharide which, on reaction with NaB3H4, is reduced to yield an alditol. Consequently, 3H is incorporated on C1 of this modified terminus. The product, [3H]heparin as the Na salt, is very stable under normal conditions of treatment and storage. The behavior of [3H]heparin when chromatographed on Sepharose-antithrombin III and on Sepharose-thrombin compares closely with that of unlabeled heparin. However, gel filtration on Sephadex G-200 reveals that [3H]heparin ( V e V 0 = 2.19 ) chromatographs more slowly than native heparin ( V e V 0 = 2.08 ), a feature which may reflect the true nature of the constituent molecules present in the heparin sample.

Anticoagulant Dermatan Sulfate Proteoglycan (Decorin) in the Term Human Placenta

Normal pregnancy is characterized by increased in vivo thrombin generation. A greater proportion of endogenously generated thrombin is complexed to heparin cofactor II in plasma from pregnant women compared to plasma from nonpregnant ones. The increase in thrombin-heparin cofactor II complexes suggests that the site of the additional thrombin generation is relatively rich in dermatan sulfate. We postulated that the site of thrombin generation may be the placenta since endogenous thrombin generation returns rapidly to normal after delivery. This report describes the isolation and characterization of placental dermatan sulfate proteoglycan from villous tissue of the term human placenta. Placental dermatan sulfate was isolated by guanidine HCI extraction and anion exchange chromatography. The isolated material was found to have anticoagulant activity with a relative activity of approximately 40% of that of a porcine mucosal dermatan sulfate which is undergoing clinical trial as an antithrombotic agent. The dermatan sulfate was present as a proteoglycan with a molecular mass of 90-150 kD. Upon degradation with chondroitin ABC lyase, the core protein was demonstrated to be a doublet with molecular masses of 42 and 44 kD. This core protein reacted with antiserum against the core protein of decorin on Western analysis. The role of this placental dermatan sulfate in local regulation of thrombin in the placenta warrants further study.

|[alpha]|2-Macroglobulin Remains as Important as Antithrombin III for Thrombin Regulation in Cord Plasma in the Presence of Endothelial Cell Surfaces

ABSTRACT: Infants and children rarely develop thrombotic complications compared with adults, suggesting that there are protective mechanisms in place for the young. Because endothelial cell surfaces regulate thrombin formation and inhibition, we compared thrombin regulation by human umbilical vein endothelial cell surfaces exposed to defibrinated cord and adult plasmas. After activation by either 10% activated partial thromboplastin reagent (strong activator) or coagulant phospholipids (weak activator) the following were measured: free thrombin, thrombin bound to antithrombin III (ATIII), heparin cofactor II, α2-macroglobulin (α2M), and prothrombin concentration. Free thrombin activity was expressed as remaining activity, after subtraction of thrombin-α2M activity. After 10% activated partial thromboplastin reagent, 100% of prothrombin was consumed and significant amounts of thrombin generated by 2 min. Cord plasma generated significantly less thrombin than adult plasma, reflecting the lower initial plasma concentration of prothrombin. Correspondingly, concentrations of thrombin inhibitor complexes were significantly greater in adult plasma than in cord plasma. After coagulant phospholipids, 50% of prothrombin was consumed and negligible thrombin activity measured for both adult and cord plasma. Similar amounts of thrombin inhibitor complexes were formed. ATIII was the predominant inhibitor of thrombin in adult plasma, whereas α2M was as important as ATIII in cord plasma for both activators. When cord plasma concentrations of ATIII were increased to adult values, the proportion complexed to α2M decreased. We conclude that on human umbilical vein endothelial cells, the capacity to generate thrombin is decreased in adult and cord plasmas. Furthermore, α2M is at least as important an inhibitor as ATIII in cord plasma, even in the presence of endothelium.

Galactose-specific elimination of human asialotransferrin by the bone marrow in the rabbit

Abstract Rabbit bone marrow accretes and degrades human asialotransferrin in vivo through a mechanism that recognizes the exposed galactose groups in this glycoprotein. After the liver, rabbit bone marrow is the second mammalian tissue now being identified as possessing a galactose-specific pathway for the elimination of a plasma protein. However, comparative studies with desialylated glycoproteins containing bi-, tri-, and tetraantennary glycans (asialofetuin, asialoorosomucoid, chicken α 1 -acid glycoprotein, and rabbit transferrin) indicate that the bone marrow recognizes fewer glycan structures than does the liver. Optimal uptake and degradation of an asialoglycoprotein by the bone marrow requires the presence of biantennary glycans.

Antithrombin-Heparin Covalent Complex A Possible Alternative to Heparin for Arterial Thrombosis Prevention

Background—The anticoagulant effect of heparinoids is attributed to their cofactor activity for antithrombin (AT) and heparin cofactor II. In patients with thrombosis, however, thrombin is often protected from AT-dependent, heparin-mediated inactivation. The purpose of this study was to compare the properties of unfractionated/standard heparin (UFH/SH) and those of a novel covalent AT-heparin complex (ATH) in a rabbit arterial thrombosis prevention and bleeding model. Methods and Results—Thrombosis in the distal aorta was triggered by vessel wall injury and critical stenosis. Blood flow in the damaged arterial segment was monitored with a flow probe placed distal to the constrictor. Rabbits were given doses of SH (62.5 to 187.5 IU · kg−1 · 90 min−1) or ATH (16 to 65 IU · kg−1 · 90 min−1). Cumulative blood loss from skin incisions was used to assess drug safety. The antithrombotic effects of ATH were greater than those of SH as measured by clot weight, blood flow, and vessel patency; eg, complete thrombus resolution was achieved with ATH (33 to 65 IU/kg), but not SH (125.0 to 187.5 IU/kg). At doses that produced equivalent vessel patency (50% to 60%), blood loss induced by ATH (60.2 &mgr;L) was 2.6-fold lower (P <0.05) than that induced by SH (154.6 &mgr;L). Conclusions—In our experimental system, ATH was able to control arterial thrombosis more effectively than its SH precursor, without pronounced bleeding.

In vivo rabbit acute model tests of polyurethane catheters coated with a novel antithrombin-heparin covalent complex

Catheter use has been associated with an increased risk of thrombotic complications. The objective was to make catheters less thrombogenic with the use of antithrombin-heparin covalent complex (ATH). The antithrombotic activity of ATH-coated catheters was compared to uncoated (control) and heparincoated catheters in an acute rabbit model of accelerated occluding clot formation. Anaesthetized rabbits were pre-injected with rabbit (125)I-fibrinogen, followed by insertion of test catheters into the jugular vein. Blood was drawn and held in a syringe, reinjected, then flushed with saline. The experiment was terminated when blood could no longer be withdrawn (occluding clot). Efficacy was defined as the ability of catheters to remain unoccluded. Clot formation, determined by measuring deposition of radiolabeled fibrin, was a secondary endpoint. ATH-coated catheters were resistant to clotting for the full 240-minute duration, while uncoated and heparin-coated catheters had an average clotting time of 78 and 56 minutes, respectively. The patency of ATH coating was dependant on intact heparin pentasaccharide sequences, rather than the chemistries of the basecoat, the PEO spacer arm, or the antithrombin (AT) protein. The ATH coating was stable to ethylene oxide sterilization, modest abrasion, protease attack, and the coating did not appear to leach off the catheter. Surface tension measurements showed that the ATH modified surface was more hydrophilic than uncoated control catheters or heparin-coated catheters. Thus, ATH-coated catheters are better at preventing clots than uncoated or heparin-coated catheters and show promise as an alternative to the currently available catheters in reducing thrombotic complications associated with its use.

Immobilization of an antithrombin–heparin complex on gold: Anticoagulant properties and platelet interactions

The anticoagulant properties and platelet interactions of gold surfaces modified with an antithrombin-heparin (ATH) complex are reported. ATH was attached to gold through either a short disulfide (linker) or a thiol-terminated polyethylene oxide (PEO) (linker, spacer). Analogous surfaces were prepared with uncomplexed heparin. Antithrombin (AT) uptake was measured before and after selectively destroying the active pentasaccharide sequence of the heparin moiety, and was found to be predominantly through the active sequence on all of the surfaces. AT binding was higher on the ATH surfaces than on the corresponding heparin surfaces. Heparin activity was assessed by an anti-factor Xa assay. The ratio of active heparin density (from the anti-FXa assay) to total heparin density was taken as a measure of heparin bioactivity. The ratio was greater on the ATH- than on the heparin-modified surfaces, i.e. the PEO-ATH surfaces showed the greater proportion of active heparin. Platelet adhesion from flowing whole blood was found to be reduced on PEO- and ATH-modified surfaces compared to bare gold. The PEO-ATH modified surfaces, but not the heparinized surfaces, were shown to prolong the clotting time of recalcified plasma.