Temperature-dependent sex determination in Hd-rR medaka Oryzias latipes: gender sensitivity, thermal threshold, critical period, and DMRT1 expression profile.

The developmental time and thermal threshold for temperature-dependent sex determination (TSD), gender differences in temperature sensitivity, the fertility of thermally sex reversed fish, and the effect of temperature on the expression of two major sex determination/differentiation genes (DMY/DMRT1bY and DMRT1) were examined in the Hd-rR strain of medaka, Oryzias latipes. Fertilized eggs were exposed from either shortly after fertilization (8-16 cells; embryonic stages 5-6) or from middle embryogenesis (heart development stage; stage 36) until hatching to temperatures ranging from 17 degrees C to 34 degrees C. Secondary sexual characteristics, gonadal histology, progeny testing, sex-linked body coloration and gene expression were used to determine phenotypic and genotypic sex. Sex determination was unaffected by low or high temperatures in genotypic (XY) males. In contrast, genotypic (XX) females treated from stages 5-6 showed increasing rates of sex reversal into phenotypic males at temperatures above 27 degrees C up to 100% at 34 degrees C. Thermal manipulation of sex was ineffective after stage 36, indicating that gonadal fate in medaka is determined considerably earlier than histological differentiation (stage 39). High temperature induced DMRT1 expression in genotypic females, which was observed already from stage 36. Sex-reversed males had histologically normal testes, were capable of sexual courtship and, with the exception of fish from 34 degrees C, sired viable progeny when mating with fertile females. These results clarify the pattern of TSD in medaka and provide important clues to understand the mechanism of sex determination in this species. They also suggest that a brief exposure to high temperature early in life could impair the fertility of medaka as adults.

Functional role of alpha-calcitonin gene-related peptide in the regulation of the cardiovascular system.

It remains unknown whether the extent of vasoactive response to exogenous calcitonin gene-related peptide (CGRP) varies among different regional vascular beds. It is also unclear whether endogenous CGRP plays a functional role in regulating basal vascular activity. To address these two issues, experiments were conducted in 27 anesthetized rats instrumented with a carotid flow probe and catheters in a jugular vein, left ventricle (LV), and femoral artery, and in 6 conscious dogs, chronically instrumented with LV pressure gauge, aortic and atrial catheters, and ascending aortic, coronary, carotid, and renal flow probes. In both species, administration of human alpha-CGRP (0.1-0.5 microg/kg, i.v.) induced a dose-dependent peripheral vasodilation that was completely abolished by pretreatment with alpha-CGRP[8-37] (30 microg/kg/min, i.v.), a competitive antagonist of CGRP receptors. Regional blood flow measured by the radioactive microsphere technique in rats showed that the alpha-CGRP (0.3 microg/kg, i.v.)-induced increase in blood flow was greater (p < 0.05) in the heart (+53 +/- 16%) than in the brain (+14 +/- 6%). In the presence of beta-adrenergic receptor blockade with propranolol, however, the increases in blood flow in these two vascular beds were identical. In conscious dogs, alpha-CGRP (0.3 microg/kg, i.v.) produced similar increases in coronary (+24 +/- 6%), carotid (+26 +/- 3%), and renal (+26 +/- 6%) blood flow, which were different from the patterns induced by other vasodilators; at an equivalent level of reduction in mean arterial pressure and total peripheral resistance, alpha-CGRP increased coronary and carotid blood flow significantly less (p < 0.05) than adenosine or nitroprusside. Unlike alpha-CGRP, adenosine and nitroprusside, as expected, induced pronounced differential blood flow changes in these vascular beds. Neither systemic hemodynamics nor regional blood flow distribution was altered by the administration of a pharmacological blocking dose of alpha-CGRP[8-37] in the two species. Thus, we conclude that endogenous alpha-CGRP does not play an important role in cardiovascular regulation under normal, resting conditions, although exogenous alpha-CGRP induces a marked, comparable vasorelaxation in different regional vascular beds.

A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.

Stargardt-like macular dystrophy (STGD3, MIM 600110) and autosomal dominant macular dystrophy (adMD) are inherited forms of macular degeneration characterized by decreased visual acuity, macular atrophy and extensive fundus flecks. Genetic mapping data suggest that mutations in a single gene may be responsible for both conditions, already known to bear clinical resemblance. Here we limit the minimum genetic region for STGD3 and adMD to a 0.6-cM interval by recombination breakpoint mapping and identify a single 5-bp deletion within the protein-coding region of a new retinal photoreceptor-specific gene, ELOVL4, in all affected members of STGD3 and adMD families. Bioinformatic analysis of ELOVL4 revealed that it has homology to a group of yeast proteins that function in the biosynthesis of very long chain fatty acids. Our results are therefore the first to implicate the biosynthesis of fatty acids in the pathogenesis of inherited macular degeneration.

Platelet glycoprotein IIb/IIIa receptor inhibitor preserves coronary flow reserve during progressive coronary arteriostenosis in swine.

Thrombosis resulting from blood platelet aggregation via glycoprotein (GP) IIb/IIIa receptor activation triggers the local release of vasoactive substances. Therefore, inhibition of these receptors could affect coronary vasoactive function during thrombotic coronary arteriostenosis. Twenty pigs were instrumented with an aortic catheter and with hydraulic occluders and flow probes on both the left anterior descending (LAD) and the left circumflex (LCx) coronary arteries. One of these 2 coronary arteries was repeatedly injured by external clamping for 15-second periods at 30-minute intervals while the pigs were given either a GP IIb/IIIa receptor inhibitor (L-739,758) (n=5), heparin (n=5), aspirin (n=3), or saline (n=7). There were no baseline differences between the 4 groups in mean arterial pressure, resting coronary blood flow (CBF), or reactive hyperemic response (RHR), which was induced by brief coronary artery occlusion and expressed as flow debt repayment. After multiple injuries, resting CBF had decreased by 95+/-2% (ie, nearly complete coronary artery occlusion) at 15+/-4 minutes in the control group, whereas in the heparin-, aspirin-, and GP IIb/IIIa inhibitor-treated groups, resting CBF had decreased by only 21+/-7% at 18+/-3 minutes, 15+/-3% at 18+/-5 minutes, and 15+/-7% at 21+/-4 minutes, respectively, suggesting that heparin, aspirin, and the GP IIb/IIIa inhibitor each prevented injury-induced coronary artery occlusion. After the initial injury, the RHR was progressively reduced in the control and heparin- and aspirin-treated groups but not in the GP IIb/IIIa inhibitor-treated group. At a comparable level of resting CBF ( approximately 15% below baseline), the RHR was reduced more in the control (-56+/-9%), heparin-treated (-49+/-9%), and aspirin-treated (-61+/-12) groups (P:<0.05) than in the GP IIb/IIIa inhibitor-treated group (-26+/-6%). When the resting CBF had decreased by approximately 35%, the RHR still was reduced significantly more (P<0.01) in the heparin-treated group (-64+/-9%) than in the GP IIb/IIIa inhibitor-treated group (-21+/-6%). In a separate group of control pigs (n=4) subjected to 2 injuries, coronary perfusion pressure distal to the injury site was reduced by 14+/-1 mm Hg from the arterial pressure, and the RHR was 20+/-6%. When the distal coronary perfusion pressure was reduced similarly (-14+/-1 mm Hg) in a separate group of GP IIb/IIIa inhibitor-treated pigs (n=4) by 2 injuries and the use of a hydraulic occluder, the RHR was 130+/-16% (P<0.01 versus control). Our data demonstrate for the first time that a platelet GP IIb/IIIa receptor inhibitor can preserve the distal coronary vasodilatory response during progressive coronary arteriostenosis.

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.

Adhesion of microbes using 3-aminopropyl triethoxy silane and specimen stabilisation techniques for analytical transmission electron microscopy.

A variety of adhesive support-films were tested for their ability to adhere various biological specimens for transmission electron microscopy. Support films primed with 3-amino-propyl triethoxy silane (APTES), poly-L-lysine, carbon and ultraviolet-B (UV-B)-irradiated carbon were tested for their ability to adhere a variety of biological specimens including axenic cultures of Bacillus subtilis and Escherichia coli and wild-type magnetotactic bacteria. The effects of UV-B irradiation on the support film in the presence of air and electrostatic charge on primer deposition were tested and the stability of adhered specimens on various surfaces was also compared. APTES-primed UV-B-irradiated Pioloform was consistently the best adhesive, especially for large cells, and when adhered specimens were UV-B irradiated they became remarkably stable under an electron beam. This assisted the acquisition of in situ phase-contrast lattice images from a variety of biominerals in magnetotactic bacteria, in particular metastable greigite magnetosomes. Washing tests indicated that specimens adhering to APTES-primed UV-B-irradiated Pioloform were covalently coupled. The electron beam stability was hypothesised to be the result of mechanical strengthening of the specimen and support film and the reduced electrical resistance in the specimen and support film due to their polymerization and covalent coupling.

Defining the role of laparoscopic-assisted sigmoid colectomy for diverticulitis.

PURPOSE: The purpose of this study was to evaluate the safety and efficacy of laparoscopic-assisted sigmoid colectomy for the treatment of diverticulitis. METHODS: The Norfolk Surgical Group Laparoscopic Surgery Registry identified all patients undergoing laparoscopic colon and rectal surgery. Retrospective chart review was performed for all patients undergoing elective sigmoid resection for a final diagnosis of diverticulitis and minimum follow-up of 12 months. Demographic data, indications for surgery, operative data, conversion rate, reason for conversion, complications, postoperative course (days to flatus and regular diet), and length of stay were identified. A telephone survey determined the incidence of recurrent diverticulitis. Statistical analysis was performed to evaluate the frequency of conversion over time, to determine risk factors for conversion, and to compare the laparoscopic-assisted and conversion groups with regard to postoperative days to flatus, regular diet, and discharge. RESULTS: From June 1992 to September 1997, elective laparoscopic-assisted sigmoid colectomy was attempted in 69 patients. Uncomplicated recurrent diverticulitis was the most common indication for surgery, occurring in 51 of 69 patients (75 percent). No deaths occurred. Complications were identified in seven patients (10.1 percent) including one wound infection and one incarcerated port-site hernia with small bowel obstruction. There were no anastomotic leaks or major septic complications. Conversion to laparotomy occurred in 18 of 69 patients (26 percent). Uncomplicated, recurrent diverticulitis was associated with conversion in 7 of 51 patients (14 percent), whereas complicated diverticulitis required conversion in 11 of 18 patients (61 percent). Logistic regression identified fistula and abscess as predictors of conversion (P = 0.0009). Comparison of the laparoscopic-assisted sigmoid colectomy group with the conversion group revealed that postoperative days to regular diet were 3.5 and 5.2 (P = 0.0004), respectively, and lengths of stay were 4.2 and 6.4 days (P < 0.0001), respectively. No difference was noted with regard to operative time or postoperative complications. Median follow-up was 48 (range, 13-76) months, and a single recurrence of diverticulitis has been identified. CONCLUSIONS: Laparoscopic-assisted sigmoid colectomy for diverticulitis can be safely performed. Conversion appears to be associated with complicated diverticulitis (fistula or abscess), which may be better approached by laparotomy. Short-term follow-up indicates that recurrence is rare and suggests that laparoscopic-assisted sigmoid colectomy achieves adequate resection. Laparoscopic-assisted sigmoid colectomy offers benefits of decreased ileus and length of stay and may represent the procedure of choice for elective resection for uncomplicated sigmoid diverticulitis.

EXP3174, the AII antagonist human metabolite of losartan, but not losartan nor the angiotensin-converting enzyme inhibitor captopril, prevents the development of lethal ischemic ventricular arrhythmias in a canine model of recent myocardial infarction.

OBJECTIVES: The antiarrhythmic efficacies of the competitive angiotensin II (AII) antagonist losartan, losartan's more potent noncompetitive AII antagonist human metabolite EXP3174 and the angiotensin-converting enzyme inhibitor captopril were assessed in a canine model of recent myocardial infarction. BACKGROUND: Multiple hemodynamic and electrophysiologic effects of AII may contribute to cardiac electrical instability. In the recent Losartan Heart Failure Study, Evaluation of Losartan in the Elderly (ELITE), a 722-patient trial primarily designed to assess effects on renal function, an unexpected survival benefit was observed with losartan compared with captopril, with the lower mortality using losartan primarily confined to a reduction in sudden cardiac death. METHODS: Intravenous losartan (1 mg/kg + 0.03 mg/kg/min), EXP3174 (0.1 mg/kg + 0.01 mg/kg/min), captopril (1 mg/kg + 0.5 mg/kg/h) or vehicle were infused in anesthetized dogs with recent (8.1 +/- 0.4 days) anterior myocardial infarction. Electrolytic injury of the left circumflex coronary artery to induce thrombotic occlusion and posterolateral ischemia was initiated 1 h after the start of treatment. RESULTS: Losartan, EXP3174 and captopril elevated plasma renin activities and comparably and significantly reduced mean arterial pressure. No significant electrocardiographic or cardiac electrophysiologic effects were noted with any treatment. Incidences of acute posterolateral ischemia-induced lethal arrhythmias were: vehicle, 7/9 (77%); losartan, 6/8 (75%); EXP3174, 2/8 (25%; p < 0.05 vs. vehicle control); captopril, 7/10 (70%). There were no among-group differences in time to onset of acute posterolateral ischemia or underlying anterior infarct size. CONCLUSIONS: EXP3174, but not losartan nor captopril, reduced the incidence of lethal ischemic ventricular arrhythmia in this preparation. The antiarrhythmic efficacy of EXP3174 may be due to an attenuation of deleterious effects of local cardiac AII formed during acute myocardial ischemia or, alternatively, a non-AII-related activity specific to EXP3174. These findings suggest that in humans, metabolic conversion of losartan to EXP3174 may afford antiarrhythmic protection.

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.

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.

Ultrastructural localization of an Echinococcus granulosus laminin-binding protein.

Murine antibodies, raised against a purified recombinant 30 kDa laminin-binding protein from Echinococcus granulosus, were used to investigate the tissue distribution of the native protein in protoscoleces and brood capsules. Immunofluorescence, in combination with confocal microscopy, revealed that the protein was distributed in small annular foci near the peripheral regions of the protoscoleces. Immunoelectron microscopy of thawed cryosections demonstrated that the laminin-binding protein was present in the cytoplasm of tegumentary cytons and myocytons, but not in cells of the excretory system. The protein was associated with amorphous regions of the cytoplasm, and was not expressed at the surfaces of cells. This distribution resembles those of other invertebrate laminin-binding proteins, which are thought to act in the cell cycle and cell proliferation events. A low degree of label was consistently detected in extracellular matrices of the protoscolex.

Nonpeptide glycoprotein IIB/IIIA inhibitors. 19. A new design paradigm employing linearly minimized, centrally constrained, exosite inhibitors.

A new series of potent, linearly-minimized, orally active, selective GPIIb/IIIa inhibitors is identified. Thus 15 (L-750,034) achieves interaction via a constrained, non-turned conformation that maintains the proper distance between its charged termini and full sulfonamide exosite interaction. The diminutive stature and the proposed linear conformation of L-750,034 define a new paradigm for the conceptualization of RGD mimics.

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.

Nonpeptide glycoprotein IIb/IIIa inhibitors: substituted quinazolinediones and quinazolinones as potent fibrinogen receptor antagonists.

The synthesis and biological activity of a series of 3,6-substituted quinazolinediones and quinazolinones are described. The potent activity of these compounds as platelet aggregation inhibitors demonstrates the utility of these structures as central templates for nonpeptide RGD mimics.

Nonpeptide glycoprotein IIb/IIIa inhibitors: 14: oral antithrombotic efficacy of L-738,167 in a conscious canine model of coronary artery electrolytic injury.

BACKGROUND: A conscious dog model of left circumflex coronary artery electrolytic injury was used to assess the oral antithrombotic efficacy of L-738,167, a potent nonpeptide antagonist of platelet GP IIb/IIIa. L-738,167 was administered either as a single oral pretreatment dose 2 hours before initiation of vessel injury or as two oral doses administered 24 hours apart, 12 hours before and after initiation of vessel injury. METHODS AND RESULTS: In untreated controls, electrolytic coronary injury (50 microA, 3 hours) resulted in thrombotic occlusion and myocardial ischemia in 15 of 16 dogs, with 4 developing lethal arrhythmias. Significant reductions in thrombus mass and complete prevention of myocardial ischemia and infarction were achieved with a single 100- to 300-microg/kg dose of L-738,167 pretreatment and with two 100-microg/kg doses administered 12 hours before and after initiation of vessel injury. Delays and/or reductions in incidence of ischemia, thrombus mass, and infarct sizes also were achieved with 10- to 30-microg/kg pretreatment and with two 30-microg/kg doses administered 12 hours before and after initiation of vessel injury. None of the L-738,167-treated animals developed lethal arrhythmias. A single oral 100-microg/kg dose of L-738,167 achieved >90% inhibitions of ADP (extent)- and collagen (rate)-induced ex vivo platelet aggregation and fivefold to sixfold or greater elevations in bleeding time; a single oral 30-microg/kg dose of L-738,167 achieved sustained 40% to 70% inhibitions of ADP- and collagen-induced ex vivo platelet aggregation and modest twofold to threefold elevations in bleeding time. At 12 to 24 hours after single oral 30- and 100-microg/kg doses of L-738,167, a substantially greater L-738,167 concentration was associated with platelets than free in plasma. CONCLUSIONS: These findings are indicative of potent and sustained oral antithrombotic efficacy and suggest that L-738,167 possesses potential for the oral management of chronic thrombotic occlusive disorders.

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).

Nonpeptide glycoprotein IIb/IIIa inhibitors. 15. Antithrombotic efficacy of L-738,167, a long-acting GPIIb/IIIa antagonist, correlates with inhibition of adenosine diphosphate-induced platelet aggregation but not with bleeding time prolongation.

The nonpeptide platelet glycoprotein IIb/IIIa antagonist, L-738, 167, was characterized in dog and nonhuman primate. In an anesthetized canine model of coronary artery electrolytic lesion, L-738,167 elicited dose-dependent (3, 4, 4.5 and 5 micrograms/kg i.v.) decreases in incidence of occlusion, reductions in thrombus mass and elevations in bleeding time. Antithrombotic efficacy correlated with inhibition of adenosine diphosphate-induced platelet aggregation but was dissociated from marked bleeding time elevation. Similarly, suppression of platelet-dependent cyclic flow reductions with L-738,167 in the canine coronary artery (5 micrograms/kg i.v.) and African green monkey carotid artery (10 micrograms/kg i.v.) correlated with inhibition of adenosine diphosphate-induced platelet aggregation but not with inhibition of thrombin-induced platelet aggregation or significant prolongation of bleeding time. In conscious dogs and sedated chimpanzees, single dose intravenous bolus (5-20 micrograms/kg) and oral (25-200 micrograms/kg) administration of L-738,167 exhibited long duration (> or = 8 hr) inhibition of ex vivo platelet aggregation. Once daily oral administration to conscious dogs (10-30 micrograms/kg/day for 15 days) and rhesus monkeys (200-250 micrograms/kg/day for 11 days) maintained significant but submaximal (50-90% inhibition) trough levels of inhibition of adenosine diphosphate-induced ex vivo platelet aggregation. Platelet sensitivity to adenosine diphosphate after multiple days of oral dosing in dogs was similar to pretreatment sensitivity. L-738,167 showed characteristics suitable for chronic oral therapy with a glycoprotein IIb/IIIa inhibitor.

Tirofiban provides "platelet anesthesia" during cardiopulmonary bypass in baboons.

OBJECTIVE: Tirofiban (Aggrastat) is a reversible, nonpeptide inhibitor of platelet glycoprotein II/IIIa receptors. We tested the hypothesis that tirofiban preserves platelet number and function and shortens postoperative bleeding times in baboons after cardiopulmonary bypass. METHODS: Four groups were studied: control, n = 12; low-dose tirofiban (0.1 microg/kg per minute), n = 7; high-dose tirofiban (0.3 microg/kg per minute), n = 7; and bolus tirofiban (15 microg/kg) followed by 0.1 microg/kg per minute during cardiopulmonary bypass, n = 7. After heparin, animals were perfused for 60 minutes at 50 ml/kg per minute and 37 degrees C with a bubble oxygenator, roller pump, and peripheral cannulation. Hemodynamics, platelet count, platelet aggregation to adenosine diphosphate, and release of beta-thromboglobulin were measured before tirofiban infusion, before heparin, after heparin before bypass, after 5 and 55 minutes of bypass, after protamine, and 60 minutes after protamine. Template bleeding times were measured at the same times except during cardiopulmonary bypass and 120 and 180 minutes after protamine administration. Platelet glycoprotein IIIa antigen was measured in Triton X-100 washes (Sigma Chemical Company) of the perfusion circuit after bypass. RESULTS: High-dose tirofiban completely prevents platelet loss during cardiopulmonary bypass. beta-Thromboglobulin release and sensitivity to adenosine diphosphate are significantly less than control at the end of bypass in all tirofiban groups. Template bleeding times return to preoperative values in both the low- and high-dose tirofiban groups 180 minutes after protamine administration and are significantly less than control bleeding times at both 120 and 180 minutes after protamine. Surface glycoprotein IIIa antigen does not significantly differ between groups. CONCLUSION: High-dose tirofiban completely preserves platelet number and improves platelet function during cardiopulmonary bypass in baboons and significantly accelerates restoration of normal template bleeding times after bypass.