The exon encoding the fibronectin type III-9 repeat is constitutively included in the mRNA from chick limb mesenchyme and cartilage.

The fibronectin monomer is comprised of three types of homologous repeating units, the types I, II, and III elements. Each type III repeat is encoded by two exons except for the two type III repeats involved in alternative splicing (IIIB and IIIA) and the type III-9 repeat which are all encoded by one exon. The fact that the type III-9 repeat is the only other type III repeat encoded by one exon has led to speculation that this exon may also be alternatively spliced. However, no evidence exists for alternative splicing of this exon in any tissues examined to date. The recent localization of a cell adhesion synergy site within the type III-9 repeat increases the likelihood of functional ramifications if the exon encoding this repeat is alternatively spliced in specific cells or tissues. We have shown previously that chick cartilage contains an unusual fibronectin mRNA splicing pattern and that the pattern changes during chondrogenesis from B+A+V+ to B+A-V+. In order to completely characterize the fibronectin mRNA in cartilage and other mesenchymal tissues for all possible alternative splicing events, we have determined whether or not the exon encoding the type III-9 repeat is alternatively spliced in these tissues. RNase protection and RT/PCR assays indicate that the fibronectin mRNA in all of these tissues, including cartilage, contains the type III-9 repeat as a constitutively included exon. Thus the exon encoding the type III-9 repeat will serve as a useful control exon for examining the regulation of tissue-specific alternative splicing during chondrogenesis.

Catalytically competent human and bovine zeta-thrombin and chimeras generated from unfolded polypeptide chains.

Human and bovine alpha-thrombin cleaved at the B-chain by chymotrypsin generates catalytically competent zeta-thrombins, which are comprised of two noncovalently linked fragments: a 36-(human) or 49-(bovine) residue A-chain linked by a disulfide to B-chain residues B1-148 (zeta 1-thrombin) and B-chain residues B149-259 (zeta 2-thrombin). Human and bovine D-Phe-Pro-Arg-CH2-zeta- and PhMeSO2-zeta-thrombins were prepared by reaction of the active-site histidine (H-B43) and serine (S-B205) with PPACK and PMSF, respectively. Unfolding and dissociation of the noncovalently linked polypeptide chains of either human or bovine D-Phe-Pro-Arg-CH2-zeta- and PhMeSO2-zeta-thrombins in 4.5 M guanidine-HCl and refolding upon 30-fold dilution in 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 0.1% PEG resulted in biphasic generation of catalytic activity. The slow phase was eliminated in the presence of the competitive inhibitor benzamidine-HCl. Unfolding and refolding mixtures of the appropriate inactive precursors generated the active chimeric thrombins bovine zeta 1-thrombin:human zeta 2-thrombin and human zeta 1-thrombin:bovine zeta 2-thrombin. Human zeta 1-thrombin and zeta 2-thrombin were isolated, and, upon recombining, the isolated fragments refolded to generate catalytically competent zeta-thrombin with an active-site content, specific activity toward Chromozym-TH, and a specificity constant (kcat/Km) for FPA release from fibrinogen that were all within 60% of those of native alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Design of novel, potent, noncovalent inhibitors of thrombin with nonbasic P-1 substructures: rapid structure-activity studies by solid-phase synthesis.

Study of surface representations of the inhibitor-bound thrombin P-1 pocket revealed a lipophilic recess in this pocket which is not occupied by any known inhibitor. Solid-phase synthesis was used to generate benzylamides of D-diphenylAlaPro by aminolysis of Boc dipeptide Kaiser resin. The resulting amides inhibited thrombin in the range IC50 = 3-13,000 nM, and the structure-activity relationships and molecular modeling suggest a unique fit of the benzyl side chain into P-1 with the meta substituent occupying the recess.

Potent noncovalent thrombin inhibitors that utilize the unique amino acid D-dicyclohexylalanine in the P3 position. Implications on oral bioavailability and antithrombotic efficacy.

In an effort to prepare orally bioavailable analogs of our previously reported thrombin inhibitor 1, we have synthesized a series of compounds that utilize the unique amino acid D-dicyclohexylalanine as a P3 ligand. The resulting compounds are extremely potent and selective thrombin inhibitors, and the N-terminal Boc derivative 8 exhibited excellent oral bioavailability and pharmacokinetics in both rats and dogs. The des-Boc analog 6 was not orally bioavailable in rats. The high level of oral bioavailability observed with 8 appears to be a direct function of its increased lipophilicity versus other close analogs. Although increased lipophilicity may serve to increase the oral absorption of tripeptide thrombin inhibitors, it also appears to have detrimental effects on the antithrombotic properties observed with the compounds. Compound 6 performed extremely well in our in vivo antithrombotic assay, while the much more lipophilic but essentially equipotent analog 8 performed poorly. We have found that in general with this series of thrombin inhibitors as well as with other unreported series, increased lipophilicity and the associated increases in plasma protein binding have detrimental effects on 2X APTT values and subsequent performance in in vivo antithrombotic models.

Discovery of a novel, selective, and orally bioavailable class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position.

A novel class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position has been discovered. Four of these thrombin inhibitors (13b,c,e and 14d) showed nanomolar potency (Ki 0.8-12 nM), 300-1500-fold selectivity for thrombin compared with trypsin, and good oral bioavailability (F = 40-76%) in rats or dogs. The neutral P1 was expected to increase metabolic stability and oral absorption. Identification of this novel aminopyridyl group at P1 was a key step in our search for a clinical candidate.

Synthesis of a series of potent and orally bioavailable thrombin inhibitors that utilize 3,3-disubstituted propionic acid derivatives in the P3 position.

As part of an effort to prepare efficacious and orally bioavailable analogs of the previously reported thrombin inhibitors 1a, b, we have synthesized a series of compounds that utilize 3,3-disubstituted propionic acid derivatives as P3 ligands. By removing the N-terminal amino group, the general oral bioavailability of this class of compounds was enhanced without excessively increasing the lipophilicity of the compounds. The overall properties of the molecules could be drastically altered depending on the nature of the groups substituted onto the 3-position of the P3 propionic acid moiety. A number of the compounds exhibited good oral bioavailability in rats and dogs, and numerous compounds were efficacious in a rat FeCl3-induced model of arterial thrombosis. Compound 7, the 3,3-diphenylpropionic acid derivative, showed the best overall profile of in vivo and in vitro activity. Molecular modeling studies suggest that these compounds bind in the thrombin active site in a manner essentially identical to that previously reported for compound 1a.

Discovery and development of the novel potent orally active thrombin inhibitor N-(9-hydroxy-9-fluorenecarboxy)prolyl trans-4-aminocyclohexylmethyl amide (L-372,460): coapplication of structure-based design and rapid multiple analogue synthesis on solid support.

Early studies in these laboratories of peptidomimetic structures containing a basic P1 moiety led to the highly potent and selective thrombin inhibitors 2 (Ki = 5.0 nM) and 3 (Ki = 0.1 nM). However, neither attains significant blood levels upon oral administration to rats and dogs. With the aim of improving pharmacokinetic properties via a more diverse database, we devised a resin-based route for the synthesis of analogues of these structures in which the P3 residue is replaced with a range of lipophilic carboxylic amides. Assembly proceeds from the common P2-P1 template 7 linked via an acid-labile carbamate to a polystyrene support. Application of the methodology in a repetitive fashion afforded several interesting analogues out of a collection of some 200 compounds. Among the most potent of the group, N-(9-hydroxy-9-fluorenecarboxy)-prolyl trans-4-aminocyclohexylmethyl amide (L-372,460 8, Ki = 1.5 nM), in addition to being fully efficacious in a rat model of arterial thrombosis at an infusion rate of 10 micrograms/kg/min, exhibits oral bioavailability of 74% in dogs, and oral bioavailability of 39% in monkeys with a serum half-life of just under 4 h. On the basis of its favorable biological properties, inhibitor 8 has been subject to further evaluation as a possible treatment for thrombogenic disorders.

Design and synthesis of a series of potent and orally bioavailable noncovalent thrombin inhibitors that utilize nonbasic groups in the P1 position.

As part of an ongoing effort to prepare therapeutically useful orally active thrombin inhibitors, we have synthesized a series of compounds that utilize nonbasic groups in the P1 position. The work is based on our previously reported lead structure, compound 1, which was discovered via a resin-based approach to varying P1. By minimizing the size and lipophilicity of the P3 group and by incorporating hydrogen-bonding groups on the N-terminus or on the 2-position of the P1 aromatic ring, we have prepared a number of derivatives in this series that exhibit subnanomolar enzyme potency combined with good in vivo antithrombotic and bioavailability profiles. The oxyacetic amide compound 14b exhibited the best overall profile of in vitro and in vivo activity, and crystallographic studies indicate a unique mode of binding in the thrombin active site.

Efficacious, orally bioavailable thrombin inhibitors based on 3-aminopyridinone or 3-aminopyrazinone acetamide peptidomimetic templates.

We have addressed the key deficiency of noncovalent pyridinone acetamide thrombin inhibitor L-374,087 (1), namely, its modest half-lives in animals, by making a chemically stable 3-alkylaminopyrazinone bioisostere for its 3-sulfonylaminopyridinone core. Compound 3 (L-375,378), the closest aminopyrazinone analogue of 1, has comparable selectivity and slightly decreased efficacy but significantly improved pharmacokinetics in rats, dogs, and monkeys to 1. We have developed an efficient and versatile synthesis of 3, and this compound has been chosen for further preclinical and clinical development.

A kinetic method for characterization of heterogenous fibrinogen and its application to fibrinogen Grand Rapids, a congenital dysfibrinogenemia.

A kinetic analysis was developed to determine the steady state kinetic parameter Kcat/KM for the thrombin-catalyzed release of FPA from abnormal and normal fibrinogen in mixtures of the two. Such mixtures are likely to comprise the fibrinogen of individuals with congenital dysfibrinogenemia. The analysis was used to characterize fibrinogen Grand Rapids a new congenital dysfibrinogenemia. It indicated that fibrinogen from affected individuals was composed of normal and abnormal fibrinogen in roughly equal amounts, and that the value of kcat/KM for the thrombin-catalyzed release of FPA from the fibrinogen variant was 77-fold lower than that for the release of FPA from the normal fibrinogen. In separate studies, fibrinogen Grand Rapids was found to exhibit a reduced clottability. Additionally, affected individuals appeared to have plasma fibrinogen concentrations which were about one-third the normal value.

Assessment of thrombin inhibitor efficacy in a novel rabbit model of simultaneous arterial and venous thrombosis.

The importance of thrombin in arterial and venous thrombosis renders thrombin inhibition an important therapeutic target. Identification of novel inhibitors requires an appropriate animal model. We modified a previously reported rat arterial thrombosis model to allow simultaneous assessment of the arterial and venous antithrombotic efficacies of heparin, hirudin, hirulog, a novel thrombin inhibitor H-(N-Me-D-Phe)-Pro-L-trans-4-aminocyclohexyl-Gly-[CO-CO]-NHCH3+ ++ (L-370,518) and the factor Xa inhibitor tick anticoagulant peptide in rabbits. Thrombosis was induced through application of 70% ferric chloride to the femoral artery and jugular vein. Incidence of occlusion, thrombus weight, aPTT and plasma inhibitor concentrations were determined. Heparin was efficacious in preventing arterial and venous occlusive thrombosis but at a dose that profoundly elevated aPTT. On a molar dosing basis, the approximate order of potency of the thrombin and factor Xa inhibitors was similar in artery and vein: hirudin>tick anticoagulant peptide>hirulog> or =L-370,518. Data suggested that compounds tended to be more potent in preventing venous thrombosis than arterial. This thrombin-dependent model is an economical and efficient approach to arterial and venous antithrombotic efficacy screening that eliminates variabilities encountered when multiple model/multiple animal strategies are employed.

Inhibition of thrombin by peptides containing Lysyl-alpha-keto carbonyl derivatives.

Several H-N-Me-D-Phe-Pro-Lysyl-alpha-keto carbonyl derivatives were shown to be potent thrombin inhibitors (Ki 0.2 to 27 nM). The inhibitory potencies of these compounds toward tissue plasminogen activator, plasmin and factor Xa were minimal; however, substantial cross-reactivity versus trypsin was observed (Ki values from 0.5 to 1500 nM). Inhibition of thrombin by alpha-keto carbonyl compounds appeared to occur via a one-step reversible reaction. The alpha-keto carbonyl inhibitors bound thrombin with a second order rate constant (k1 1-4 microM-1s-1) that was 10-100-fold slower than that expected for a diffusion-controlled reaction. Certain alpha-keto carbonyl inhibitors were as potent (on a weight basis) as hirudin when evaluated in a rat arterial thrombosis model. The modest oral bioavailability (10-19%) in rats demonstrated for three of the alpha-keto carbonyl thrombin inhibitors suggests the possibility that alpha-keto amide containing thrombin inhibitors may have utility as orally-active antithrombotic agents.

A thrombin assay based upon the release of fibrinopeptide A from fibrinogen: definition of a new thrombin unit.

An assay for thrombin is presented wherein thrombin-catalyzed hydrolysis at Arg-A alpha-16 to release fibrinopeptide A (FPA) from fibrinogen is measured using high-performance liquid chromatography (HPLC). In this assay one thrombin unit (TU) is defined as that amount of thrombin that will release half of the FPA in one min from one ml of a solution of greater than 90% clottable normal human fibrinogen (less than or equal to 0.35 microM) at 37 degrees C, pH 7.4, /2 0.15. One TU is equivalent to approximately 0.1 NIH unit of thrombin and approximately 1 pmol of pure human thrombin. At 37 degrees C, pH 7.4, and plasma levels of fibrinogen of 3 mg/ml, one TU will catalyze the release of 3.6 nmol FPA min-1. Variability in fibrinogen samples which produce dramatic differences in clotting time assays with the same sample of thrombin, produce little or no variation in the catalytic assay for TU. The assay for TU obviates the need for maintenance of a thrombin reference standard.

Inhibition of thrombin and other trypsin-like serine proteinases by cyclotheonamide A.

Cyclotheonamide A (CA), a cyclic peptide isolated from the marine sponge of the genus Theonella was shown to be a slow-binding inhibitor of several trypsin-like serine proteinases. Values of 4.6 x 10(4), 4.8 x 10(4), 9.3 x 10(3), 2.1 x 10(3) and 2.7 x 10(2) M-1 s-1 were determined for the second-order rate constants for formation of CA complexes with thrombin, trypsin, plasmin, 2-chain t-PA and factor Xa, respectively. The equilibrium constant (Ki) was measured for dissociation of CA from the CA complex with human thrombin (Ki = 1.0 nM), bovine trypsin (Ki = 0.2 nM), human plasmin (Ki = 12 nM), human factor Xa (Ki = 50 nM) and human 2-chain tissue plasminogen activator (t-PA) (Ki = 40 nM). CA produces dose dependent increases in clotting time assays. The clotting time in the thrombin time, activated partial thromboplastin time and prothrombin time assays, were doubled by 1.5, 0.9 and 48 microM CA, respectively. A model for the binding of CA to the active site of thrombin is proposed.

Initial report of rotational ablation: the Toronto Hospital experience.

OBJECTIVE: To describe the initial experience of rotational ablation (using the Rotablator device), in terms of safety and the effectiveness as a proportion of final angiographic outcome when combined with adjunctive balloon angioplasty. DESIGN: Retrospective analysis of single-centre experience, including operator learning curve. SETTING: Tertiary care hospital, Cardiac Catheterization Laboratory. PATIENTS: Consecutive subjects (14 men, 11 women) selected for rotational ablation based on ostial/bifurcation lesions (n = 10), 'long' (more than 10 mm) stenoses (n = 11) or extensive dystrophic calcification (n = 4). INTERVENTIONS: Rotational ablation (Rotablator) with routine adjunctive balloon angioplasty. Quantitative coronary arteriography using the Cardiac Measurement System. RESULTS: Rotational ablation reduced coronary obstruction, as demonstrated by minimal lumen diameter (preprocedure, 0.57 =/- 0.28 to 1.17 +/- 0.32 mm, P<0.05), with further improvements following adjunctive balloon angioplasty (1.93 +/- 0.35 mm). Similar changes were observed in relative stenosis after Rotablator (preprocedure, 79.7 +/- 7.6 to 56.1 +/- 13.1% diameter), with typical post-angioplasty residual narrowings (29.7 +/- 8.2% diameter). Estimated stenotic flow reserve was improved by the interventional procedures (preprocedure, 0.94 +/- 0.70; rotational ablation 3.07 +/- 1.14; and angioplasty, 4.73 +/- 0.25 times baseline). Complications were acceptable, and included three acute occlusions requiring balloon angioplasty recanalization and three non-Q wave myocardial infarctions (with creatine phosphokinase levels of 270, 417 and 602 IU, respectively). CONCLUSIONS: The Rotablator is a relatively user-friendly device with a reasonable safety profile, accounting for approximately 50% of minimum lumen diameter gains when used in conjunction with routine balloon angioplasty. The precise role of rotational ablation, particularly in the context of preselected lesion specific uses (bifurcations, long lesions, dystrophic calcification), requires prospective, randomized studies.

Pet therapy program for antepartum high-risk pregnancies: a pilot study.

OBJECTIVE: This pilot study evaluated the potential benefits of pet therapy on symptoms of anxiety and depression in antepartum hospitalized women with high-risk pregnancies. STUDY DESIGN: Eighty-two women in a hospital-based setting completed the State-Trait Anxiety Inventory and the Beck Depression Inventory before and after the pet therapy visit. For both questionnaires, paired t-test was used and adjusted P-values were obtained using the Hochberg step-up Bonferroni method. RESULT: The mean scores for depressive symptoms significantly improved from the pre-pet therapy (10.1 ± 6.3) compared with the post-pet therapy (6.3 ± 5.9) (P<0.0001). Likewise mean scores of the state anxiety significantly improved from the pre-pet therapy test (44.8 ± 11.7) compared with the post-pet therapy (34.5 ± 10.5) (P<0.001). CONCLUSION: Pet therapy significantly reduced anxiety and depression in antepartum hospitalized women with high-risk pregnancies.