Nonpeptide GPIIB/IIIA receptor antagonists. Part 21: C-6 flexibility and amide bond orientation are important factors in determining the affinity of compounds for activated or resting platelet receptors.

Compound affinity for activated and resting GPIIb/IIIa receptors may differ, and comparison of those differences determines selectivity. Structural features that influence selectivity are discussed.

Expression of beta-catenins and cadherins by follicular dendritic cells in human lymph nodes.

In lymph nodes, dendritic cells form a complex meshwork and are linked by intercellular junctions. Intercellular junctions contribute to the integrity of lymphatic follicles and can potentially be affected by malignant processes in neighbouring B cells. We examined whether transmembrane molecules that constitute "adherens junctions" are present in follicular dendritic cells of normal human lymph nodes. We found that follicular dendritic cells but not interdigitating dendritic cells or sinus lining cells expressed cadherin molecules. Follicular dendritic cells also expressed beta-catenin but not vinculin. The cadherin molecules, which were identified in situ with the use of a monoclonal pan-cadherin antibody, were not recognized by antibodies to E-cadherin, N-cadherin or P-cadherin. Intrafollicularly, cadherins were clearly colocalized with beta-catenins, in a dot-like fashion. We also detected intrafollicular expression of desmogleins and desmosomal plaque proteins. These findings indicate the presence of desmosomes within the dendritic meshwork. However, pan-cadherin reactivity was not only colocalized with desmoglein immunoreactivity that was abundantly present. Immunoprecipitation showed that pan-cadherin reactivity was absent in fractions of desmosomal plaque proteins or pan-desmogleins. We speculate that complexes of cadherins of an unknown subclass and beta-catenins form non-desmosomal intercellular junctions in the intrafollicular dendritic meshwork.

The platelet cytoskeleton regulates the affinity of the integrin alpha(IIb)beta(3) for fibrinogen.

Agonist-generated inside-out signals enable the platelet integrin alpha(IIb)beta(3) to bind soluble ligands such as fibrinogen. We found that inhibiting actin polymerization in unstimulated platelets with cytochalasin D or latrunculin A mimics the effects of platelet agonists by inducing fibrinogen binding to alpha(IIb)beta(3). By contrast, stabilizing actin filaments with jasplakinolide prevented cytochalasin D-, latrunculin A-, and ADP-induced fibrinogen binding. Cytochalasin D- and latrunculin A-induced fibrinogen was inhibited by ADP scavengers, suggesting that subthreshold concentrations of ADP provided the stimulus for the actin filament turnover required to see cytochalasin D and latrunculin A effects. Gelsolin, which severs actin filaments, is activated by calcium, whereas the actin disassembly factor cofilin is inhibited by serine phosphorylation. Consistent with a role for these factors in regulating alpha(IIb)beta(3) function, cytochalasin D- and latrunculin A-induced fibrinogen binding was inhibited by the intracellular calcium chelators 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethyl ester and EGTA acetoxymethyl ester and the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A. Our results suggest that the actin cytoskeleton in unstimulated platelets constrains alpha(IIb)beta(3) in a low affinity state. We propose that agonist-stimulated increases in platelet cytosolic calcium initiate actin filament turnover. Increased actin filament turnover then relieves cytoskeletal constraints on alpha(IIb)beta(3), allowing it to assume the high affinity conformation required for soluble ligand binding.

Non-peptide GPIIb/IIIa inhibitors. 20. Centrally constrained thienothiophene alpha-sulfonamides are potent, long acting in vivo inhibitors of platelet aggregation.

The synthesis and pharmacology of 4, a potent thienothiophene non-peptide fibrinogen receptor antagonist, are reported. Compound 4 inhibited the aggregation of human gel-filtered platelets with an IC50 of 8 nM and demonstrated an 8-fold improvement in affinity for isolated GPIIb/IIIa receptors over analogues possessing an isoindolinone backbone. Flow cytometry studies revealed that the binding of 4 to resting platelets is a diffusion-controlled process (kon = 3.3 x 10(6) M-1 s-1) and that 4 binds to dog and human platelets with comparable affinity (Kd = 0.04 and 0.07 nM, respectively). Ex vivo platelet aggregation in dogs was completely inhibited by an iv dose of 5 microg/kg [corrected], and an oral dose of 50-90 microg/kg [corrected] followed by low daily doses of 10 microg/kg [corrected] was sufficient to maintain approximately 80% inhibition of ex vivo platelet aggregation over several days. Inhibition of ADP-induced platelet aggregation in anesthetized dogs at 77 +/- 7% resulted in a moderate 2.5-fold increase in bleeding time, while complete inhibition (100%) resulted in an approximately 10-min bleeding time. Additional doses were required to increase the bleeding time to the maximum time allowed in the protocol (15 min), thus indicating a potentially useful and safe separation of efficacy and bleeding time.

Fibrinogen receptor antagonist-induced thrombocytopenia in chimpanzee and rhesus monkey associated with preexisting drug-dependent antibodies to platelet glycoprotein IIb/IIIa.

Most clinical trials with fibrinogen receptor antagonists (FRAs) have been associated with thrombocytopenia. This report describes the occurrence of thrombocytopenia in one chimpanzee and one rhesus monkey upon administration of potent FRAs. Chimpanzee A-264 experienced profound thrombocytopenia on two occasions immediately upon intravenous administration of two different potent FRAs, L-738, 167 and L-739,758. However, an equally efficacious antiaggregatory dose of another potent antagonist, L-734,217, caused no change in platelet count. These compounds did not affect platelet count in five other chimpanzees or numerous other nonhuman primates. Flow cytometric analysis showed drug-dependent antibodies (DDAbs) in the plasma of chimpanzee A-264 that bound to platelets of chimpanzees, humans, and all other primates tested only in the presence of the compounds that induced thrombocytopenia. Rhesus monkey 94-R021 experienced thrombocytopenia upon administration of a different antagonist, L-767,679, and several prodrugs that are converted into the active form, L-767,679, in the blood. More than 20 other FRAs, including those that induced thrombocytopenia in chimpanzee A-264, had no effect on platelet count in this monkey. Flow cytometric measurements again identified DDAbs that reacted with platelets of all primates tested and required the presence of L-767,679. Screening for DDAbs in the plasma of 1,032 human subjects with L-738, 167 and L-739,758 demonstrated that the incidence of these preexisting antibodies in this population was 0.8% +/- 0.6% and 1.1% +/- 0.6%, respectively.

A reduction in the nursing labor market and its impact on the renal industry.

Nurses, especially those with specialized training such as nephrology, are looking more and more endangered. A cursory review of the classified ads supports the likelihood of another nursing shortage with the reappearance of key words such as, "relocation assistance," "incentive pay," and "hiring bonus." It is now commonplace to see hiring bonuses ranging from $3,000-$6,000. This article will investigate the definition of occupational shortages, possible causes of the nursing shortage, future implications, and strategies to be considered.

[Over a half-century at the Charles University 1st Medical Faculty. 2. Pathology under Professor Sikl]. / Více nez pul století na 1. LF UK: 2. Patologie za Sikla.

Hlava Institute of Pathology was directed in a pleasant climate of broadminded indulgence. Sikl was easy to get in favour being personally attractive and imposing in work. He always sided with the youth and helped us even against authorities which made him more often laugh critically than treat with respect. Hlava Institute under Sikl became a model of progressing narrow specialization and modernizing. Sikl filled it with a programme of perspective development. Thus he prepared the whole discipline for cooperation which was useful for comparison of all findings with clinical picture. He made the biopsy independent and carried out its wide appreciation. He insisted upon the usage of big scale surveying histological cuts which he investigated thoroughly in detail. He did not live to see an explosion of methodological possibilities. Nevertheless, he prepared the structural diagnostics for an effective cooperation of all specialized methods.

[Over a half-century at the Medical School of Charles University: 1. Student years]. / Více nez pul století na 1. LF UK: 1. zákovská léta.

Nearly all student years in Prague Medical School represented a "big class" schooling. Professors and their lectures were in the centre of programme and practical training was very limited. Interruption of studies during the war brought many difficulties but was in a way positive for those who had opportunity to further practical education. We found out that even a very good preparation by the medical school did not do and had to be complemented by the school of practice. Its standardization could be hardly achieved without any connection with a university department.

Identification of low molecular weight GP IIb/IIIa antagonists that bind preferentially to activated platelets.

A critical function of fibrinogen in hemostasis and thrombosis is to mediate platelet aggregation by binding selectively to an activated form of glycoprotein (GP) IIb/IIIa. Although numerous peptide and nonpeptide fibrinogen receptor antagonists have been described, their binding selectivity for resting and activated platelets has not been explored. Therefore, dissociation constants of GP IIb/IIIa antagonists for two biochemically separated forms of purified GP IIb/IIIa and for resting and activated platelets were determined by competitive displacement of the dansyl fluorophore containing GP IIb/IIIa antagonist L-736,622. Also, coating either form of the purified GP IIb/IIIa onto yttrium silicate scintillation proximity assay fluomicrospheres produced an activated form of the receptor, whose binding affinity for GP IIb/IIIa antagonists was measured conveniently by competition with the arginine-glycine-aspartic acid (RGD) containing heptapeptide [125I]L-692,884. In addition, direct binding measurements with radiolabeled GP IIb/IIIa antagonists also were performed on resting and activated platelets. We identified two classes of compounds. One class binds to both forms of GP IIb/IIIa, as well as resting and activated platelets, with similar Kd values (e.g., L-736,622 and Echistatin). The other class of compounds binds with much higher affinity to the activated form of GP IIb/IIIa (purified or on platelets) as compared with the resting form (e.g., L-734,217, MK-852, tirofiban and L-692,884). Selective antagonists, like L-734,217 (KdActivated = 5 nM and KdResting = 620 nM), can effectively inhibit ex vivo platelet aggregation at concentrations of drug that produce low levels of occupancy of the circulating platelet receptors. The potential clinical advantages of selective versus nonselective GP IIb/IIIa antagonists remain to be explored in clinical trials.

A retrospective analysis of employee turnover in the health care setting.

Significant factors implicated in staff turnover include: variables in organizational structure; employee characteristics; needs and values, and the nature of tasks performed. This article will present the causative factors related to turnover and the conceptual models of the motivational theorists Maslow, Herzberg, Adams, and Mobley. No quantitative or qualitative research could be found on the potential causes of turnover in freestanding dialysis clinics. The staff turnover of a for-profit dialysis company for a 12 month period will be reported by job title, tenure, and level of job satisfaction.

[Over a half century at the 1st Medical School of Charles University: development of interdisciplinary cooperation]. / Více nez pul století na 1. LF UK: 3. Rozvoj mezioborové spolupráce.

A vast interdisciplinary and methodological cooperation became a leading feature of pathology. It brought critical evaluation and synthesis of data from several points of view without prevalence of a pure structural empirism. Nevertheless, detailed structural analysis remains an unmatched and effective introductory way of diagnostic algorithms. A wide methodology is not always useful in its capacity but should be open to each of teachers. For the time being we rely more on outstanding personalities. Call for them as well as their reasonable uniting may restore the original sense to the School Scientific Council.

Arginine-glycine-aspartic acid mimics can identify a transitional activation state of recombinant alphaIIb beta3 in human embryonic kidney 293 cells.

The platelet-specific integrin alphaIIb beta3 achieves a high affinity binding state in response to extracellular agonists such as thrombin, ADP, or collagen. During this activation, the receptor undergoes a number of conformational changes. To characterize the different conformations of alphaIIb beta3, we expressed recombinant alphaIIb beta3 in human embryonic kidney (HEK) 293 cells. Antigenic and peptide recognition specificities of the full-length recombinant receptor resembled those of the native receptor in platelets. We used an array of peptidic and nonpeptidic arginine-glycine-aspartic acid (RGD) mimics that specifically bind to human platelet alphaIIb beta3 to determine the affinity state of the receptor. Some of these RGD mimics were previously shown to clearly discriminate between resting and activated alphaIIb beta3. Solution-phase binding of these RGD mimics to the recombinant cells suggested that in HEK 293 cells the full-length alphaIIb beta3 is expressed in a "transitional" activation state. This observation was confirmed by the binding of the activation-specific, monoclonal anti-alphaIIb beta3 antibody PAC1 to cells expressing the full-length recombinant alphaIIb beta3. Deletion of the entire cytoplasmic domain of the beta subunit was sufficient to convert the receptor in HEK 293 cells to a fully active form, as found in activated platelets. In addition, the full-length receptor was capable of mediating agonist-independent aggregation of cells in the presence of fibrinogen. Thus, by using RGD mimics, we have identified a functional transitional activation state of alphaIIb beta3 that is capable of mediating fibrinogen-dependent cell aggregation.

Non-peptide glycoprotein IIb/IIIa inhibitors. 17. Design and synthesis of orally active, long-acting non-peptide fibrinogen receptor antagonists.

The synthesis and pharmacological evaluation of 5 (L-738, 167), a potent, selective non-peptide fibrinogen receptor antagonist is reported. Compound 5 inhibited the aggregation of human gel-filtered platelets with an IC50 value of 8 nM and was found to be > 33000-fold less effective at inhibiting the attachment of human endothelial cells to fibrinogen, fibronectin, and vitronectin than it was at inhibiting platelet aggregation. Ex vivo platelet aggregation was inhibited by > 85% 24 h after the oral administration of 5 to dogs at 100 micrograms/kg. The extended pharmacodynamic profile exhibited by 5 appears to be a consequence of its high-affinity binding to GPIIb/IIIa on circulating platelets and suggests that 5 is suitable for once-a-day dosing.

Flow cytometric measurement of kinetic and equilibrium binding parameters of arginine-glycine-aspartic acid ligands in binding to glycoprotein IIb/IIIa on platelets.

Antagonists of platelet glycoprotein IIb/IIIa (GPIIb/IIIa) represent a new therapeutic approach in inhibiting platelet aggregation, thus providing a powerful form of antithrombotic therapy. The measurement of binding of arginine-glycine-aspartic acid (RGD) peptidomimetics to GPIIb/IIIa on platelets is a key for the further understanding of ligand-receptor interactions and, thus, the design of new antagonists. The flow cytometric measurement of dynamic and equilibrium binding parameters of two new potent RGD peptidomimetics, L-762,745 and L-769,434, containing a fluorescein moiety is described in this paper. Kinetic binding measurements with these fluorescent ligands indicate a two-step binding mechanism that involves a conformational rearrangement of the receptor-ligand complex. The overall second-order binding constants are for both fluorescent ligands several orders of magnitude slower than for diffusion-controlled processes. The values of k(-1) and K(D) obtained by fitting the kinetic binding data in a two-step model are in good agreement with directly detected values of k(off)(L-762,745) = (1.9 +/- 0.6) 10(-3) s(-1), k(off)(L-769,434) = (5.1 +/- 0.7) 10(-3) s(-1), KD(L-762,745) = 12 +/- 0.5 nM, and K(D)(L-769,434) = 8 +/- 0.3 nM. Equilibrium binding measurements of fluorescent ligands with an orally active nonfluorescent antagonist, L-738,167, provided apparent dissociation binding constant K(D) of this ligand in the range from 0.1 to 0.2 nM. The kinetic dissociation measurement of L-738,167 using the binding of the fluorescent ligand L-762,745 as a reporting method yielded a k(off) for L-738,167 of (4.1 +/- 0.1) x 10(-4) s(-1) (t1/2 = 28 min).

[Diagnostic certainty of lymph node biopsy in malignancies]. / Jistota bioptické diagnostiky malignit z lymfatických uzlin.

Bioptic diagnosis of malignant processes in lymph nodes is a difficult method and is less accurate than sometimes expected. After transfer of haematological patients-as a rule to provide specialized treatment-a diagnostic baseline examination of biopsy is useful. The clinician procures for this "second reading" all basic data to improve the diagnostic certainty and deserves support. A controversial character of the assembled data is not associated with any specially or professional skill. It ensues, however, from the general principle of good care of the patient. In oncology a "second reading" is not a novelty. It is a recommended approach during typing according to the international classification of oncological diseases. Assessment of certainty (C factor) is moreover included in assessment of the grade of malignity in the TNM system.

[Typing of malignant mantle zone lymphomas]. / Typizace maligních lymfomu plást'ové zóny.

Principles derived from a group of 46 ML of the mantle zone are presented: Mantle pattern of a ML and its cytological structure are mostly sufficient for positive basic diagnosis. Diffuse mantle zone ML need detection of BCL-1 and CD5 hyperexpression which are characteristic for small-cell and centrocytoid forms when compared with BCL-2 positive centrofollicular lymphomas. B monocytoid lymphomas from the parafollicular subgroup as well as plasmacytoid ML from the marginal subgroup retain faint BCL-1 positivity but lose CD5 positivity. That may results in attempt of problematic narrowing of mantle zone definition because of existence of the mixed cellularity forms of mantle zone ML. Nodular mantle zone ML are clinically recognized late and are unsensitive to treatment which is opposite to the original idea of their relative benignity. M-coding of mantle zone ML is very defective because the codes do not separate nodular (perifollicular) and diffuse variants.

[PCR analysis of DNA in paraffin sections of malignant lymphomas]. / Analýza DNA z parafínových rezu pomocí metody PCR u maligních lymfomu.

The polymerase chain reaction was used to detect the clonal rearrangement of immunoglobulin heavy chain gene in paraffin embedded samples of human lymph nodes. We developed a sensitive and reliable method of the DNA isolation from 4-5 tissue sections, which enabled us to perform 50-100 PCR reactions. We compared the reactive lymph nodes and non-Hodgkin's malignant lymphomas using framework 3 and J region primers. PCR products were examined by agarose gel electrophoresis. The dominant 80-120 bp amplification product was found in all lymphoma samples. The samples of reactive nodes were negative.