Biomedical subjects

Dietmar Seiffert

Publications and source records attributed to Dietmar Seiffert.

10 recordsLinked to original sources

PubMed2003

Effects of the glycoprotein IIb/IIIa antagonist Roxifiban on P-selectin expression, fibrinogen binding, and microaggregate formation in a phase I dose-finding study: no evidence for platelet activation during treatment with a glycoprotein IIb/IIIa antagonist.

The hypothesis that glycoprotein (GP) IIb/IIIa antagonists stimulate platelets is controversial. Here, we report the results of flow cytometric measurements of platelet activation markers in a phase I dose optimization study of Roxifiban, an orally active GP IIb/IIIa antagonist. Whole blood was collected at pre-dose and during the dosing interval directly into citrate fixative so that circulating levels of platelet activation could be assessed. P-selectin expression and fibrinogen binding of single platelets were unchanged at any of the dosing intervals compared to the pre-dose values, whereas microaggregate formation was reduced. Blood was also collected in hirudin to maintain physiological calcium concentrations and stimulated with platelet agonists to test whether GP IIb/IIIa antagonists lower the threshold for platelet activation. After stimulation with a concentration range of ADP and TRAP, P-selectin expression was not altered by Roxifiban administration compared to pre-dose levels. Fibrinogen binding and microaggregate formation were reduced by Roxifiban dosing in a dose-dependent manner. Inhibition of both parameters was retained at trough and no increase above pre-dose values was observed at any time. This study provides evidence for a dose-dependent inhibition of platelet functions by an orally active GP IIb/IIIa antagonist and does not detect paradoxical activation of platelets by a GP IIb/IIIa antagonist in humans.

Adenosine Diphosphate↗

PubMed2003

CD32-dependent platelet activation by a drug-dependent antibody to glycoprotein IIb/IIIa antagonists.

Thrombocytopenia is observed with a frequency of up to 2% in patients treated with glycoprotein (GP) IIb/IIIa antagonists. We recently provided evidence that thrombocytopenia is caused by antibody binding to drug-induced conformational changes in GP IIb/IIIa. Here, we report that a murine monoclonal antibody binds to GP IIb/IIIa in an antagonist-dependent manner and activates platelets. Platelet stimulation is associated with a disruption of the phospholipid asymmetry, resulting in the assembly of catalytic active intrinsic Xase and prothrombinase complexes. Further mechanistic studies revealed that this response is (I) mediated in cis, (II) not associated with the formation of prothrombotic microparticles, and (III) requires intact platelet signaling and (IV) is blocked by increases in cAMP. The prothrombotic response is not observed using F(ab')2 fragments and is blocked by incubation of platelets with neutralizing antibodies to the platelet FcgammaRIIa receptor (CD 32).Taken together, these observations suggest that GPIIb/IIIa antagonist-dependent antibody binding to the platelet fibrinogen receptor has the propensity to lead to CD32-mediated platelet activation and accelerated platelet clearance, leading to thrombocytopenia.

Amidines↗

PubMed2002

Gamma-secretase activity is not involved in presenilin-mediated regulation of beta-catenin.

Presenilins (PS) are involved in gamma-secretase-mediated processing of beta-amyloid precursor protein (APP) and the Notch family of proteins. In addition, presenilin 1 (PS-1) binds to members of the armadillo family of proteins. In this study the relationship between PS-1-mediated proteolytic activity and PS-1-mediated regulation of beta-catenin function was investigated. Incubation of cells with a potent, small molecule gamma-secretase inhibitor did not affect PS-1/beta-catenin interaction as determined by co-immunoprecipitation, or affect the regulation of beta-catenin turnover, as determined by pulse-chase analysis, even at inhibitor concentrations that completely blocked PS-mediated APP processing. Moreover, inhibition of PS-1-mediated proteolytic activity did not affect beta-catenin trafficking, as determined by immunolocalization and immunoblotting, or beta-catenin-mediated transcription. These results indicate that PS-1-mediated regulation of gamma-secretase activity and PS-1-mediated regulation of beta-catenin function can be pharmacologically separated and support the idea that these are distinct functions.

Active Transport, Cell Nucleus↗

PubMed2002

Mutations at the P1' position of Notch1 decrease intracellular domain stability rather than cleavage by gamma-secretase.

Gamma-secretase is a unique protease which cleaves within the transmembrane domain of several substrate proteins. Among gamma-secretase substrates are members of the Notch family of receptors and the amyloid precursor protein. In this study we used a cell-free Notch-cleavage assay and specific gamma-secretase inhibitors to study the cleavage of Notch by gamma-secretase. Using this assay, we found that, in contrast to previous reports, the presence of valine at the P1(') position of Notch1 is not required for gamma-secretase cleavage. Our results suggest that the presence of valine at the N-terminus of the Notch intracellular domain cleavage product is important for its stability. Thus it appears that Notch cleavage is very similar to APP cleavage with respect to the lack of sequence specificity.

Acetylcysteine↗

PubMed2002

Regulation of clot retraction by glycoprotein IIb/IIIa antagonists.

Binding of fibrinogen to platelet glycoprotein (GP) IIb/IIIa induces clot retraction. Significant differences among GP IIb/IIIa antagonists were previously noted to inhibit thromboelastography in whole blood specimens. The relationship between efficacy of these agents and inhibition of clot retraction is unclear. Here, we use a plasma-free clot retraction assay to evaluate potency of GP IIb/IIIa antagonists to inhibit clot retraction and modulate platelet signaling, and to address whether these effects are realized in the clinically relevant dose range. The potencies for inhibition of clot retraction and aggregation are similar for antagonists with high affinity for resting platelets and slow off-rates, whereas lower affinity and fast off-rate antagonists are disproportionately less effective in blocking clot retraction. A positive correlation is observed between inhibition of clot retraction and inhibition of tyrosine dephosphorylation across a number of GP IIb/IIIa antagonist pharmacophores. For lower affinity and fast off-rate antagonists, the concentrations required for inhibition of clot retraction clearly exceed the clinical dose range. Site occupancy studies combined with clot retraction experiments addressed whether high affinity and slow off-rate compounds can alter clot retraction during the dosing interval. Binding studies using [3H] Roxifiban, a high affinity GP IIb/IIIa antagonist, indicate that occupancy of >95% of GP IIb/IIIa sites is required to inhibit clot retraction. This level of occupancy is not routinely achieved in the clinic and is not tolerated, at least for chronic therapy. These results suggest that inhibition of clot retraction is not necessary for efficacy of GP IIb/IIIa antagonists.

Abciximab↗

PubMed2002

Effects of glycoprotein IIb/IIIa antagonists on platelet activation: development of a transfer method to mimic peak to trough receptor occupancy.

Several oral glycoprotein (GP) IIb/IIIa antagonists, Sibrafiban, Orbofiban and Lotrafiban, have been studied in large phase III trials; each has failed to provide efficacy and has been associated with increased mortality. Roxifiban has pharmacokinetic and pharmacodynamic properties believed to be more favorable than the earlier oral agents. Here, we revisit the controversial hypothesis of platelet activation liabilities of GP IIb/IIIa antagonists. The effects of site occupancy by four fibans (Roxifiban, Sibrafiban, Orbofiban and Lotrafiban) on platelet activation was assessed using P-selectin expression, fibrinogen binding and microaggregate formation. All four fibans inhibited ADP and TRAP-stimulated fibrinogen binding and microaggregate formation in a concentration-dependent manner, whereas P-selectin expression was relatively unaltered. To more vigorously test for activation liabilities, the effects of transition from peak to trough receptor occupancy upon platelet stimulation was analyzed. The high affinity of Roxifiban for resting platelets precluded reduction of site occupancy by dialysis or gel filtration. A method was developed that takes advantage of the rapid equilibrium of Roxifiban between platelets and soluble GPIIb/IIIa. The platelet occupancy is controlled by the ratio of platelet GPIIb/IIIa to soluble GPIIb/IIIa. This method allows in vitro investigation of peak/trough transitions on platelet activation. A decrease in site occupancy from peak to trough of Roxifiban or Sibrafiban did not result in increased activation of platelets. The loss of platelet-bound antagonist upon incubation with purified soluble GPIIb/IIIa returned fibrinogen binding/microaggregate formation to no drug levels. In conclusion, these studies do not provide evidence for an activation liability of GPIIb/IIIa antagonists in vitro.

Alanine↗

PubMed2002

Prospective testing for drug-dependent antibodies reduces the incidence of thrombocytopenia observed with the small molecule glycoprotein IIb/IIIa antagonist roxifiban: implications for the etiology of thrombocytopenia.

Thrombocytopenia is a relatively common side effect observed during glycoprotein (GP) IIb/IIIa antagonist therapy. With the oral antagonist roxifiban, we observed thrombocytopenia, defined as 50% reduction of platelets over predose values or below 90 000/microL (9 x 10(10)/L), with a frequency of 2% (8 of 386). Thrombocytopenia occurred either early (days 2 to 4) or delayed (days 11 to 16). No additional cases were observed with up to 6 months of treatment. Retrospective analysis provided evidence for drug-dependent antibodies (DDABs) to GP IIb/IIIa in 5 of 6 subjects, suggestive of an immune etiology of thrombocytopenia. The hypothesis that excluding patients based on positive DDAB reaction would reduce the frequency of thrombocytopenia was tested. Patients were screened for DDABs during the study qualification period and, overall, 3.9% of the patients were excluded based on pre-existing DDAB concentrations above a statistically defined medical decision limit. An additional 2.6% were excluded based on therapy-related antibody production during the first 2 weeks. With antibody testing, 0.2% of patients (2 of 1044) developed immune-mediated thrombocytopenia. One case developed a rapidly increasing antibody concentration and presented with thrombocytopenia despite discontinuation of roxifiban therapy. The second case was related to a false-negative test result. The frequency of thrombocytopenia was statistically significantly reduced from 2% to 0.2% (P =.0007) comparing nonscreened and screened patients. Testing for DDABs can reduce the frequency of thrombocytopenia in patients treated with roxifiban and, by analogy, other GP IIb/IIIa antagonists. Thus, DDAB testing may be employed to increase the safety of GP IIb/IIIa antagonists.

Amidines↗

PubMed2002

Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated.

Glycoprotein (GP) IIb/IIIa antagonists are effective therapeutic agents, but elicit thrombocytopenia with a frequency that approaches 2%. Here, we provide evidence that thrombocytopenia in humans treated with the GP IIb/IIIa antagonist roxifiban is immune mediated. Two patients underwent conversion to a highly positive drug-dependent antibody (DDAB) status temporally associated with thrombocytopenia. Despite the continued presence of DDABs, the fall in platelet count was reversed by discontinuation of drug treatment, pointing to the exquisite drug dependency of the immune response. DDABs appear to bind to neoepitopes in GP IIb/IIIa elicited on antagonist binding. This information was used to develop an enzyme-linked immunosorbent assay (ELISA) for DDAB using solid-phase GP IIb/IIIa. A high level of specificity is indicated by the observation that DDAB binding is dependent on the chemical structure of the GP IIb/IIIa antagonist and that only 2% to 5% of human blood donors and 5% of chimpanzees present with pre-existing DDABs. Furthermore, none of 108 nonthrombocytopenic patients from the phase II roxifiban study showed an increase in antibody titer. Absorption of thrombocytopenia plasma with platelets reduced the DDAB ELISA signal, indicating that the test detects physiologically relevant antibodies. Screening patients for pre-existing or increasing DDAB titer during treatment with GP IIb/IIIa antagonists may reduce the incidence of drug-induced thrombocytopenia.

Administration, Oral↗

PubMed2002

Plasminogen has a broad extrahepatic distribution.

Plasmin is the major enzyme that dissolves fibrin in the vasculature and the predominant source of its zymogen, plasminogen, is liver. However, plasmin has a broad substrate spectrum and, if present in other tissues, may perform additional functions. We tested the hypothesis that plasminogen is expressed broadly extrahepatically. A sensitive and specific isotopic quantitative RT-PCR assay was developed to detect plasminogen mRNA from total RNA isolated from C57BL/6J mice tissues. Plasminogen mRNA was detected in adrenal, kidney, brain, testis, heart, lung, uterus, spleen, thymus and gut. Of these tissues, adrenal had the highest plasminogen mRNA content. In situ hybridization was utilized to localize plasminogen mRNA expressing cell types. Besides hepatocytes, positive cells were identified in both adrenal and kidney medullae and cortexes. Plasminogen mRNA expression was detected in cerebral, hippocampal and cerebellar neurons. Plasminogen mRNA was detected in cells in early stages of spermatogenesis in testis, present in the cortex and medulla of the thymus and in splenic white and red pulps. Our results suggest that the plasminogen gene is expressed broadly in extrahepatic tissues. Thus, tissues separated by local anatomic barriers as well as tissues accessible to circulating plasminogen have the capacity to provide local sources of plasminogen.

Animals↗

PubMed2001

Identification and characterization of presenilin-independent Notch signaling.

Presenilin (PS) proteins control the proteolytic cleavage that precedes nuclear access of the Notch intracellular domain. Here we observe that a partial activation of the HES1 promoter can be detected in PS1/PS2 (PS1/2) double null cells using Notch1 Delta E constructs or following Delta 1 stimulation, despite an apparent abolition of the production and nuclear accumulation of the Notch intracellular domain. PS1/2-independent Notch activation is sensitive to Numblike, a physiological inhibitor of Notch. PS1/2-independent Notch signaling is also inhibited by an active gamma-secretase inhibitor in the low micromolar range and is not inhibited by an inactive analogue, similar to PS-dependent Notch signaling. However, experiments using a Notch1-Gal4-VP16 fusion protein indicate that the PS1/2-independent activity does not release Gal4-VP16 and is therefore unlikely to proceed via an intramembranous cleavage. These data reveal that a novel PS1/2-independent mechanism plays a partial role in Notch signal transduction.

Amyloid Precursor Protein Secretases↗