Identification and initial structure-activity relationships of a novel class of nonpeptide inhibitors of blood coagulation factor Xa.

The discovery and some of the basic structure-activity relationships of a series of novel nonpeptide inhibitors of blood coagulation Factor Xa is described. These inhibitors are functionalized beta-alanines, exemplified by 2a. Docking experiments placing 2a in the active site of Factor Xa implied that the most expeditious route to enhancing in vitro potency was to modify the group occupying the S3 site of the enzyme.

Anti-thrombotic activity of RG13965, a novel platelet fibrinogen receptor antagonist.

RG13965, a pseudotetrapeptide analogue of Arg-Gly-Asp (RGD), inhibited collagen-induced dog, monkey, human, hamster, mouse, and pig platelet aggregation in vitro with IC50 values of 3.7, 4.6, 6.

Regions of the human neurokinin A receptor involved in the generation of second messengers and in receptor desensitization.

Deletion analysis was used to study sites of human Neurokinin A receptor (HNKAR) necessary for signal transduction in CHO cells. Deletion of 62 and 81 amino acids from the c-terminus of HNKAR forms mutant receptors HNKAR delta 62 and HNKAR delta 81, which bind neurokinin A with high affinity but are functionally different. Wild type HNKAR and HNKAR delta 62 are functionally active whereas HNKAR delta 81 is functionally inactive.

Platelet aggregation on extracellular matrix: effect of a recombinant GPIb-binding fragment of von Willebrand factor.

Platelets in whole blood incubated on extracellular matrix (ECM) produced by bovine corneal endothelial cells under oscillatory flow conditions demonstrate extensive aggregate formation. Since both platelet-subendothelium and platelet-platelet interactions are mediated by von Willebrand factor (vWF), we used this system to examine the effect of a recombinant GPIb-binding fragment of vWF (designated RG12986), comprising residues 445-733 of the native vWF subunit, on platelet reactivity with ECM. The seven cysteines present in the RG12986 fragment were reduced and alkylated in order to achieve a monomeric conformation.

Optimization of a recombinant von Willebrand factor fragment as an antagonist of the platelet glycoprotein Ib receptor.

The binding of von Willebrand factor (vWF) to platelet glycoprotein (GP) Ib receptor is one of the initial events in thrombus formation. Previous studies have shown that RG12986, a reduced and alkylated recombinant fragment of vWF (Ser445-Val733), can inhibit binding of native vWF to GP Ib and offers potential as an anti-thrombotic agent. We have now evaluated a series of deletion mutants of RG12986 and found that reduced and alkylated rvWF508-704 is close to the minimal sequence with optimal RG12986-like activity (IC50 for inhibition of GP Ib-dependent platelet aggregation in the absence of modulators: 0.

Clinical evaluation of an EIA for the sensitive and specific detection of serum antibody to Trypanosoma cruzi (Chagas' disease).

A commercial EIA for the detection of antibody to Trypanosoma cruzi was clinically evaluated. The primary use of this test is in the diagnosis and screening of donated blood in Latin America. When compared with sera positive by xenodiagnosis, the assay had a clinical sensitivity of 100%.

Production and functional characterization of a recombinant fragment of von Willebrand factor (vWF): an antagonist to platelet receptor GP Ib.

We expressed a recombinant peptide fragment (Ser445-Val733) of human von Willebrand factor (vWF), containing the binding domain for the platelet receptor of GP Ib, in E. coli. This 33 kD peptide blocks binding of the intact vWF molecule to GP Ib in the presence of modulators.

Quantitative analysis of the accuracy of linear array transrectal ultrasound in measurement of the prostate.

In 61 prostate autopsy specimens, linear array transrectal ultrasound and electronic vernier calipers were used to measure 3 maximal diameters along the longitudinal, anterior-posterior and transverse axes. The results were comparable. We assessed the reliability of empirical formulae, derived from various combinations of these 3 diameters, for the prediction of prostatic volume and weight.

Absorption-enhanced solid-phase immunoassay method via water-swellable poly(acrylamide) microparticles.

Water-swellable hydrogel microspheres based on cross-linked polyacrylamide were used as solid supports in immunoassay formats. Capture antibody was covalently bound at the surfaces and the pore size of particles was such that these antibodies were excluded from the interiors. Upon contact with a solution containing immunological reactants, water molecules quickly penetrated the microspheres, causing them to swell, thereby concentrating analyte at the surface.

The immunochemistry of sandwich-ELISAs. IV. The antigen capture capacity of antibody covalently attached to bromoacetyl surface-functionalized polystyrene.

The antigen capture capacity of antibodies covalently immobilized on injection-molded polystyrene beads was evaluated. Bromoacetyl groups on the bead surfaces rendered them reactive to protein nucleophilic groups. The bromoacetyl surface exhibited up to a ten-fold greater capacity for protein compared to unmodified polystyrene, with no detectable dissociation such as occurs with simple adsorption.

2,3-Diaminophenazine is the product from the horseradish peroxidase-catalyzed oxidation of o-phenylenediamine.

NMR and mass spectroscopic evidence has been obtained which indicates that the product of the oxidation of o-phenylenediamine by hydrogen peroxide, uncatalyzed or catalyzed by horseradish peroxidase, is 2,3-diaminophenazine. These results settle disparate literature descriptions. The process is most likely free radical in nature starting with the abstraction of a labile amino hydrogen atom.

The fourth component of human complement treated with amines or chaotropes or frozen-thawed (C4b-like C4): interaction with C4 binding protein and cleavage by C3b/C4b inactivator.

We have shown previously that C4 treated with amines or chaotropes, although uncleaved, exhibits properties that are similar to C4b. Studies by other groups suggest that this C4b-like form of C4 is characterized by the lack of an internal thiolester bond that is present in native C4. We report here that C4 treated with N2H4 or KSCN or frozen-thawed, unlike native C4, forms a complex with C4-binding protein (C4-bp) and is cleaved by C3b/C4b inactivator (I).

Chido and Rogers antigenic determinant on the fourth component of human complement.

The blood group substances Chido (Cha) and Rogers (Rga) represent two electrophoretic variants of human C4. Based on the observation that anti-Cha and anti-Rga antisera agglutinated human red blood cells prepared in sucrose-activated autologous serum (LIS cells) at 37 degrees C, it has been assumed that the Cha and Rga antigenic determinants reside in the C4d fragment of C4. Here, we present evidence indicating that C4d is not present on those cells.

Synthesis and antimalarial effects of N2-aryl-N4-[(dialkylamino)alkyl]- and N4-aryl-N2-[(dialkylamino)alkyl]-2,4-quinazolinediamines.

A series of N2(and N4)-aryl-N4(and N2)-[(dialkylamino)alkyl]-2,4-quinazolinediamines has been synthesized for antimalarial evaluation. Condensation of the appropriate 2,4-dichloroquinazoline (IV) with the requisite N,N-dialkylalkylenediamine afforded a series of 2-chloro-N-[(dialkylamino)alkyl]-4-quinazolinamines (V) which were condensed with the appropriate arylamine to provide the corresponding N2-aryl-N4-[(dialkylamino)alkyl]-2,4-quinazolinediamines (VI). Hydrolysis of 2,4-dichloroquinazoline to 2-chloro-4-quinazolinol was followed by condensation with the appropriate N,N-dialkylalkylenediamine to give an array of 2-[[(dialkylamino)alkyl]amino]-4-quinazolinols (IXa).

Metabolism of orally and intravenously administered purines in rats.

One experiment was conducted in which radioactively labeled purine bases (adenine, guanine, hypoxanthine and xanthine) were individually given intravenously to young adult rats and the recovery of radioactivity in urine and gut, gut content and liver was measured at the end of the next 24 hours. The total recovery of radioactivity from orally and intravenously administered adenine was measured in experiment 2. A third experiment measured the recoveries of radioactivity from oral and intravenous adenine in a wider variety of tissues and organs than in experiment 1.

On the reaction of methylmercuric hydroxide with methylbabalamin.

The methylmercury-induced dealkylation of the corrinoid coenzyme methylcobalamin, yielding aquocobalamin and dimethylmercury as products, was studied spectrophotometrically at 350 nm using water as a solvent. Rate data were determined for the pH 7-9 region and also at pH 3.37.