Experimental studies on a recombinant hirudin, CGP 39393.

The in vitro anticoagulant activities of recombinant desulphatohirudin (r-hirudin) were studied in the activated partial thromboplastin time (APTT) and the thrombin generation test systems. In the APTT, at concentrations below 5 micrograms/ml, r-hirudin showed a dose-response curve. At concentrations above 5 micrograms/ml, the plasma became unclottable, but in the thrombin generation test, at least 10 micrograms/ml of r-hirudin was required for full inhibition of thrombin generation. The antithrombotic effect was assessed using a rabbit venous stasis model; 150 micrograms/kg r-hirudin completely prevented thrombus formation at 10 and 20 min stasis. At this full antithrombotic dose, the mean bleeding time ratio measured in a rabbit ear template model, was not prolonged over control values. At higher doses, the bleeding time ratios were higher than those observed for the same dosage of heparin. These data indicate that while r-hirudin is an effective antithrombotic agent, antithrombotic doses have to be carefully titrated to avoid excessive bleeding.

Low anticoagulant heparin retains anti-HIV type 1 activity in vitro.

Heparin is a potent inhibitor of HIV-1 replication, in addition to being a well-established inhibitor of blood coagulation. The major anticoagulant activity of heparin results from binding to the plasma protein antithrombin (AT). The high-affinity binding site for AT is a specific pentasaccharide sequence that is of low abundance and completely absent from the majority of heparin chains. We have examined the anti-HIV-1 activity of both conventional and low molecular weight heparins fractionated according to affinity for AT. The high- and low-affinity fractions, despite differing markedly in anticoagulant activity, are identical in their ability to bind to the envelope glycoprotein of HIV-1, and in their inhibitory effect on HIV-1 replication in vitro (EC50 1 and 8 micrograms/ml for conventional and low molecular weight fractions, respectively). Our study shows that the anti-HIV activity of heparin is independent of its antithrombin-mediated inhibition of coagulation proteases. Therefore, heparin preparations retaining full anti-HIV-1 activity in vitro but with greatly reduced anticoagulant activity may be readily produced for further clinical investigation in the prophylaxis and therapy of HIV infection.

Anti-Xa clotting activities in different hepatic-triglyceride lipase preparations from post-heparin plasma.

Two different preparations of hepatic triglyceride lipase (HTGL) with comparable lipolytic activities, purified from post-heparin human plasma, were assessed for their anti-Xa activities by two clotting and one chromogenic method. Preparation 1, prepared by heparin affinity followed by ion exchange chromatography, did not contain antithrombin III and exhibited no anti-Xa activity in any of the assay systems. Preparation 2, prepared by two consecutive heparin affinity chromatography steps, was active in all three assay systems, and was shown to contain antithrombin III (AT III). Addition of purified AT III to preparation 1 did not result in the anti-factor Xa activity of preparation 2, and monoclonal antibodies to AT III did not antagonize the activity of preparation 2. These results show that the anti-Xa activity of some HTGL preparations is neither due to the lipase itself nor to the content of AT III, but suggest, that it could be due to contamination with another protein, which binds to heparin sepharose columns but is removed during ion exchange chromatography. Most likely the effect is due to the extrinsic pathway inhibitor (EPI), also called lipoprotein-associated coagulation inhibitor (LACI), which has recently been shown to be released by heparin.

Monoclonal antibodies against antithrombin III and their application in an immunoradiometric assay.

Monoclonal antibodies (MAbs) to antithrombin III (AT III) have been produced and characterized, and a two-site immunoradiometric assay (IRMA) developed, using one of the MAbs as a 'catcher' antibody and a radiolabelled affinity-purified rabbit antibody for detection. The IRMA could be performed in one day and the optimum concentration range of AT III was 1-100 ng/ml. Assays on AT III concentrates by the IRMA method showed a good correlation with AT III antigen values measured by the immunoelectrophoresis method (Laurell), the IRMA results averaging 94.3% of the Laurell values. Two of the MAbs (11 and 16) inhibited heparin cofactor but not AT III progressive activity, and the binding of AT III to MAb 11, used for the IRMA, was blocked by heparin. These results indicate that MAb 11 is directed at or near to the heparin binding site, and could therefore be useful in the study of structural aspects of this site in normal and genetically abnormal AT III.

Simulation of coronary artery revascularization.

Simulation of the commonly constructed geometries of aorto-coronary bypass anastomoses was carried out using especially fabricated distensible tubes and a pulsatile pump. The system pressure was maintained between 80 and 120 mmHg. The total mean flow was set at 250 ml min-1 (Reynolds number of 200) and the pulsatile frequency was varied from 0 to 2 Hz. A water-glycerine mixture having a density and viscosity similar to that of blood was used throughout. A 16 mm film of the front of black dye injected proximal to the anastomosis was made as the dye approached and passed through the anastomosis. Anastomotic geometries consisted of: end to side, parallel, 45 degree angle, and 90 degree angle. Stenoses, located in the tube representing the coronary artery, were simulated using a bevelled insert which represented an 80-85% area reduction. Flow visualization revealed that distensible tubes gave more realistic flow patterns than rigid tubes, a result particularly evident when a stenosis was present. Pulsatile flow demonstrated considerably more mixing than steady flow. The use of pulsatile flow in distensible tubing with a partial stenosis showed retrograde flow through the stenosis which was not evident for either steady flow or for flow in rigid tubing. The flow at the anastomatic site of the graft having an angle of 0 degrees showed a jetting action with a zone of recirculating fluid being present whereas for a 90 degree graft a distinct helical flow was formed distal to the anastomosis.

The development and evaluation of a 125I radioimmunoassay for the measurement of LSD in body fluids.

A radioimmunoassay (RIA) has been developed for the direct detection of LSD in biological fluids. The radiotracer, (+)-2-[125I]iodo-LSD, allows the use of gamma-counting rather than the liquid scintillation counting currently used for existing 3H radioimmunoassays. The assay is specific for LSD and very closely related compounds. It is inexpensive, sensitive, simple to use and small volumes of samples (50 microliter) can be assayed directly without the need for any time-consuming extraction procedures. The cut-off levels are 1.2 ng/ml in blood and 3.0 ng/ml in urine. The results obtained using the 125I assay described in this work compare very favourably with those obtained using the 3H assay currently used by Home Office Forensic Science Laboratories. The advantages of the assay make it a most appropriate method for the routine screening of LSD in biological samples of forensic interest.

Vesicle sizing: Number distributions by dynamic light scattering.

A procedure is described which optimizes nonnegative least squares and exponential sampling fitting methods for analysis of dynamic light scattering (DLS) data from aqueous suspensions of vesicle/liposome systems. This approach utilizes a Rayleigh-Gans-Debye form factor for a coated sphere and yields number distributions which can be compared directly to distributions obtained by freeze-fracture electron microscopy (EM). Excellent agreement between the DLS and EM results are obtained for vesicle size distributions in the 100-200-nm range.

Role of N- and C-terminal amino acids in antithrombin binding to pentasaccharide.

We have used a monoclonal antibody-based binding procedure to determine the dissociation constants of the interactions between the essential antithrombin-binding pentasaccharide and a series of 13 distinct N- and C-terminal antithrombin substitution mutation variants with defective binding interaction with heparin. The reduction in binding affinity of the pentasaccharide with the N-terminal variants (with substitution mutations Pro-41-->Leu, Arg-47-->Cys and His, Leu-99-->Val and Phe, Gln-118-->Pro, Arg-129-->Gln) compared to normal antithrombin, Kd 200 nM, ranged from 15-984-fold and was generally less than 150-fold. Reduced binding affinity is assumed to arise mostly by perturbation, direct or indirect, of the initial contact of pentasaccharide with basic residues of antithrombin. Surprisingly, the binding interaction of the pentasaccharide with the C-terminal variants (with substitution mutations in or near strand 1C/4B, Phe-402-->Leu, Cys, and Ser, Ala-404-->Thr, Pro-407-->Thr, Pro-429-->Leu) was more uniformly and yet substantially (135-482-fold) decreased, despite the spatial separation between the site of mutation and the proposed primary contact site of the pentasaccharide. These results demonstrate that strand 1C/4B region integrity is required for optimum interaction with the pentasaccharide, suggesting its involvement in transmission of the induced conformation change required for high affinity binding.

Heparin binding affinity of normal and genetically modified antithrombin III measured using a monoclonal antibody to the heparin binding site of antithrombin III.

The inhibitory activity of the plasma serine proteinase inhibitor antithrombin III (AT III) is enhanced about 1000-fold upon binding to heparin. We have determined the dissociation constants, Kd, of 48.8 nM for the heparin-AT III interaction, of 175 nM for the specific pentasaccharide-AT III interaction, and of 13 microM for the low-affinity heparin-AT III interaction, using a binding assay based on a monoclonal antibody (MAb) that recognizes an epitope at or close to the heparin binding site of AT III. The heparin binding affinities and proportions of normal and variant AT III in plasma from patients with mutations of AT III have been quantitated for the first time using the binding assay. Substitution mutations in three regions of AT III have been investigated: (i) mutations in the reactive site loop affecting Ala382, Arg393, and Ser394 have no discernible effect on heparin binding; (ii) mutations in the previously identified N-terminal heparin binding region, affecting Arg47, Leu99, and Arg129, produce variant AT III molecules with heparin affinities reduced 11-924-fold, the largest reduction being observed for the substitution mutation Arg47-Cys in Padua 2, which has an affinity of 65.6 microM; (iii) mutations in the hydrophobic regions around strand 1C of the C terminus, affecting Phe402, Ala404, Asn405, Pro407, and Pro429, have pleiotropic effects that include the production of reduced amounts of low-affinity AT III with dissociation constants from 6 to 43 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombins Southport (Leu 99 to Val) and Vienna (Gln 118 to Pro): two novel antithrombin variants with abnormal heparin binding.

We report the characterization of three variant antithrombins with reduced heparin binding as the primary abnormality. Two of these variants, antithrombin Southport (Leu 99 to Val, 2759 C to G) and antithrombin Vienna (Gln 118 to Pro, 5349 A to C) were novel, whereas the third, Pro 41 to Leu, has been previously described as antithrombin Basel. All three variants exhibited reduced binding for heparin on crossed immunoelectrophoresis and in a quantitative monoclonal antibody-based assay. The mutations were characterized by direct sequence analysis of enzymatically amplified genomic DNA and all affected individuals were heterozygous for the mutations. These three mutations do not occur at the sites of the basic amino acids directly involved in heparin binding nor do they result in a change in charge of the affected residue. It seems probable that they reduce heparin affinity either by perturbing the initial contact site involved in the heparin-binding domain (Arg 47, Arg 129 and possibly Arg 24), or by preventing the subsequent heparin-induced conformational change.