Biomedical subjects
Publications and source records attributed to B Bednar.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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BACKGROUND: Thrombin inhibitors have been shown to be efficacious in animal models of thrombosis and in initial human clinical trials. It is unknown if their efficacy is due to their prevention of thrombin-mediated fibrin formation or to an inhibitory effect on thrombin-stimulated platelet activation. Appropriate tools to address this question have not been available. Therefore, to evaluate the role of the platelet thrombin receptor in intravascular thrombus formation, a polyclonal antibody was raised against a peptide derived from the thrombin-binding exosite region of the cloned human thrombin receptor. This antibody serves as a selective inhibitor of the thrombin receptor for in vivo evaluation. METHODS AND RESULTS: The immune IgG (IgG 9600) inhibited thrombin-stimulated aggregation and secretion of human platelets. In contrast, it had no effect on platelet activation induced by other agonists including ADP, collagen, or the thrombin receptor-derived peptide SFLLR-NH2. IgG 9600 also inhibited thrombin-induced aggregation of African Green monkey (AGM) platelets. By Western blot analysis, the IgG identified a protein of approximately 64 kD in homogenates of both human and AGM platelets. The effect of thrombin receptor blockade by this antibody on arterial thrombosis was evaluated in an in vivo model of platelet-dependent cyclic flow reductions (CFRs) in the carotid artery of the AGM. The intravenous administration of IgG 9600 (10 mg/kg) abolished CFRs in three monkeys and reduced CFR frequency by 50% in a fourth monkey. Ex vivo platelet aggregation in response to up to 100 nmol/L thrombin was completely inhibited during the 120-minute postbolus observation period in all four animals. There was a twofold increase in bleeding time, which was not statistically different from baseline, and ex vivo clotting time (APTT) was not changed. The glycoprotein IIb/IIIa receptor antagonist MK-0852 and the thrombin inhibitor recombinant hirudin also demonstrated inhibitory effects on CFRs at doses that did not significantly prolong template bleeding time. Control IgG had no effect on CFRs, ex vivo platelet aggregation, bleeding time, or APTT. CONCLUSIONS: These results demonstrate that blockade of the platelet thrombin receptor can prevent arterial thrombosis in this animal model without significantly altering hemostatic parameters and suggest that the thrombin receptor is an attractive antithrombotic target.
Optimal conditions for a method to simultaneously measure aggregation in 96 samples using a microplate reader were developed. The temperature of the assay was set at 25 degrees C, the optimal platelet concentration range was determined to be from 1-3 x 10(8) per mL, the assay volume was determined to be best at 100 microL and an agitation rate of setting #5 on the vortex was found to yield the most reliable aggregation response. After these initial assay parameters were established, EC50 values for standard platelet agonists including ADP, thrombin, collagen and thrombin receptor activating peptides were determined using the plate assay and compared to those obtained by measuring light transmittance in an aggregometer. The results were quantitatively similar, and qualitatively the shapes of the aggregations as monitored by both methods were characteristic of those expected for each agonist. The use of this assay was then extended to quantitate the inhibition of aggregation by antagonists of the fibrinogen receptor as well as by an inactive thrombin receptor peptide and by antibodies against the thrombin receptor. This method provided useful data for characterization of both platelet agonists and antagonists and should be useful for future platelet aggregation studies.
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