Search PubMed⌕ Search

Biomedical subjects

Robert Flaumenhaft

Publications and source records attributed to Robert Flaumenhaft.

11 recordsLinked to original sources
PubMed2005

Phosphatidylinositol 4,5-bisphosphate regulates activation-induced platelet microparticle formation.

While the role of the cytoskeleton in microparticle formation is well-described, the role of membrane phospholipids in regulating this process is poorly defined. PIP(2) binds many cytoskeletal proteins and may oppose microparticle formation through associations with these proteins. To determine whether PIP(2) effects microparticle formation, PIP(2) was incorporated into platelet membranes prior to activation-induced microparticle formation. Incorporation of PIP(2) into platelet membranes inhibited activation-induced microparticle formation by >or=90%. Inhibition was dose-dependent with an IC(50) of 12-18 microM. A permeabilized platelet system was next used to assess the effect of modulation of endogenous PIP(2) levels on microparticle formation. Infusion of type IIbeta PIP kinase into permeabilized platelets inhibited microparticle formation by 75 +/- 8%. In contrast, incubation of permeabilized platelets with PI-specific phospholipase C augmented microparticle formation by greater than 3-fold. Evaluation of PIP kinases following platelet activation demonstrated that they were lost from platelets in a calpain-dependent manner during microparticle formation. Purified mu-calpain cleaved recombinant type IIbeta PIP kinase and inhibited its ability to phosphorylate PI(5)P. In permeabilized platelets, incubation of purified mu-calpain reduced PIP(2) levels, while exposure to calpeptin increased PIP(2) levels. Calpain has previously been implicated in platelet microparticle formation. These studies show that calpain may help limit PIP(2) formation following platelet activation and that PIP(2) content is an important determinant of platelet microparticle formation.

Blood Platelets↗
PubMed2005

The actin cytoskeleton differentially regulates platelet alpha-granule and dense-granule secretion.

Stimulation of platelets with strong agonists results in centralization of cytoplasmic organelles and secretion of granules. These observations have led to the supposition that cytoskeletal contraction facilitates granule release by promoting the interaction of granules with one another and with membranes of the open canalicular system. Yet, the influence of the actin cytoskeleton in controlling the membrane fusion events that mediate granule secretion remains largely unknown. To evaluate the role of the actin cytoskeleton in platelet granule secretion, we have assessed the effects of latrunculin A and cytochalasin E on granule secretion. Exposure of platelets to low concentrations of these reagents resulted in acceleration and augmentation of agonist-induced alpha-granule secretion with comparatively modest effects on dense granule secretion. In contrast, exposure of platelets to high concentrations of latrunculin A inhibited agonist-induced alpha-granule secretion but stimulated dense granule secretion. Incubation of permeabilized platelets with low concentrations of latrunculin A primed platelets for Ca(2+)- or guanosine triphosphate (GTP)-gamma-S-induced alpha-granule secretion. Latrunculin A-dependent alpha-granule secretion was inhibited by antibodies directed at vesicle-associated membrane protein (VAMP), demonstrating that latrunculin A supports soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent membrane fusion. These results indicate that the actin cytoskeleton interferes with platelet exocytosis and differentially regulates alpha-granule and dense granule secretion.

Actins↗
PubMed2003

Initial accumulation of platelets during arterial thrombus formation in vivo is inhibited by elevation of basal cAMP levels.

Platelet accumulation at sites of vascular injury is the primary event in arterial thrombosis. Initial platelet accrual into thrombi is mediated by interactions of platelet adhesion receptors with ligands on the injured endothelium or in the sub-endothelial matrix. The role of intracellular signals in initial platelet accumulation at sites of endothelial injury, however, is the subject of debate. We have used a newly discovered inhibitor of phosphodiesterase 3A (PDE3A) and the well-characterized PDE3A inhibitor, cilostazol, to modulate 3',5'-cyclic adenosine monophosphate (cAMP) levels in an in vivo model that enables the kinetic analysis of platelet accumulation. These studies demonstrate that elevation of basal cAMP levels results in an overall decline in platelet accumulation at the site of vascular injury. In particular, the initial rate of accumulation of platelets is inhibited by elevation of cAMP. Analysis of the kinetics of individual platelets at injury sites using intravital microscopy demonstrates that cAMP directs the rate at which platelets attach to and detach from thrombi. These studies demonstrate that cAMP in circulating platelets controls attachment to and detachment from sites of arteriolar injury. Thus, the status of the intracellular signaling machinery prior to engagement of platelet receptors influences the rate of platelet accumulation during thrombus formation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2003

Molecular basis of platelet granule secretion.

The energy-dependent release of granule contents from activated platelets is a well-established component of normal hemostasis and thrombosis. A role for membrane fusion in this process has been presumed for decades, but only recently have the mechanisms of platelet membrane fusion been investigated at the molecular level. Such studies have demonstrated that platelet membrane fusion is controlled by lipid components of the membrane bilayer, by transmembrane proteins termed SNARE proteins, and by chaperone proteins that interact with SNARE proteins. This core membrane fusion machinery is controlled by activation-dependent changes in cytoskeletal organization, intracellular calcium levels, kinase activity, and intracellular protease activity. Through these mechanisms, interactions of ligands with their cognate cell-surface receptors are transmitted to the membrane fusion machinery to facilitate membrane fusion and secretion of granule contents from platelets.

Blood Platelets↗
PubMed2003

SNARE protein degradation upon platelet activation: calpain cleaves SNAP-23.

In order to better understand the molecular mechanisms of platelet granule secretion, we evaluated the effect of activation-induced degranulation on three functional platelet SNARE proteins, SNAP-23, VAMP-3, and syntaxin 4. Initial studies showed that SNAP-23 is lost upon SFLLRN-induced platelet activation. Experiments with permeabilized platelets demonstrated that proteolysis of SNAP-23 was Ca(2+)-dependent. Ca(2+)-dependent proteolysis of SNAP-23 was inhibited by the cell-permeable calpain inhibitors, calpeptin and E-64d, as well as by the naturally occurring calpain inhibitor, calpastatin. In addition, purified calpain cleaved SNAP-23 in permeabilized platelets in a dose-dependent manner. In intact platelets, calpeptin prevented SFLLRN-induced degradation of SNAP-23. In contrast, calpeptin did not prevent SFLLRN-induced degradation of VAMP-3 and syntaxin 4 did not undergo substantial proteolysis following platelet activation. Calpain-induced cleavage of SNAP-23 was a late event occurring between 2.5 and 5 min following exposure of permeabilized platelets to Ca(2+). Experiments evaluating platelet alpha-granule secretion demonstrated that incubation of permeabilized platelets with 10 microM Ca(2+) prior to exposure to ATP inhibited ATP-dependent alpha-granule secretion from permeabilized platelets. SNAP-23 was cleaved under these conditions. Incubation of permeabilized platelets with either calpeptin or calpastatin prevented Ca(2+)-mediated degradation of SNAP-23 and reversed Ca(2+)-mediated inhibition of ATP-dependent alpha-granule secretion. Thus, activation of calpain prior to secretion results in loss of SNAP-23 and inhibits alpha-granule secretion. These studies suggest a mechanism whereby calpain activation serves to localize platelet secretion to areas of thrombus formation.

Blood Platelets↗
PubMed2003

The platelet as a model for chemical genetics.

Chemical genetics is an emerging strategy in chemical biology that promises to bring the power of true genetics to mammalian systems and facilitate the transfer of biological discoveries to therapeutics. The platelet is an anucleate cell with several features that render it suitable for chemical genetic analysis. This review addresses the benefits and challenges of chemical genetics using platelets as a model system.

Animals↗
PubMed2002

Protein kinase C mediates translocation of type II phosphatidylinositol 5-phosphate 4-kinase required for platelet alpha-granule secretion.

To better understand the molecular mechanisms of platelet granule secretion, we have evaluated the role of type II phosphatidylinositol (PtdIns) 5-phosphate 4-kinase in agonist-induced platelet alpha-granule secretion. SFLLRN-stimulated alpha-granule secretion from SL-O-permeabilized platelets was inhibited by either antibodies directed at type II PtdIns 5-phosphate 4-kinase or by a kinase-impaired point mutant of type IIbeta PtdIns 5-phosphate 4-kinase. In contrast, recombinant type IIbeta PtdIns 5-phosphate 4-kinase augmented SFLLRN-stimulated alpha-granule secretion from SL-O-permeabilized platelets. SFLLRN-stimulated alpha-granule secretion was inhibited by a protein kinase C-specific inhibitor peptide or bisindolylmaleimide I. Phorbol 12-myristate 13-acetate-stimulated alpha-granule secretion was inhibited by anti-type II PtdIns 5-phosphate 4-kinase antibodies or the kinase-impaired point mutant of type IIbeta PtdIns 5-phosphate 4-kinase and augmented by recombinant type IIbeta PtdIns 5-phosphate 4-kinase. Immunoblot analysis demonstrated that type II PtdIns 5-phosphate 4-kinase remained associated with SL-O-permeabilized platelets when incubated in the presence, but not the absence, of SFLLRN. This SFLLRN-induced translocation of type II PtdIns 5-phosphate 4-kinase was blocked by either the protein kinase C-specific inhibitor peptide or bisindolylmaleimide I. In addition to stimulating alpha-granule secretion, both SFLLRN and PMA enhanced the association of a fluorescein isothiocyanate-labeled peptide derived from the PtdIns (4,5)P(2)-binding domain of gelsolin to permeabilized platelets. Agonist-induced recruitment of the PtdIns (4,5)P(2)-binding domain was inhibited by neomycin, bisindolylmaleimide I, and anti-type II PtdIns 5-phosphate 4-kinase antibody. These results suggest a mechanism whereby protein kinase C-mediated translocation of type II PtdIns 5-phosphate 4-kinase leads to the recruitment of PtdIns (4,5)P(2)-binding proteins.

Amino Acid Sequence↗
PubMed2002

Subcellular distribution of 3 functional platelet SNARE proteins: human cellubrevin, SNAP-23, and syntaxin 2.

Morphologic studies have demonstrated a process by which alpha-granule contents are released from platelets. Studies aimed at defining the molecular mechanisms of this release have demonstrated that SNARE proteins are required for alpha-granule secretion. These observations raise the possibility that morphologic features of alpha-granule secretion may be influenced by the subcellular distribution of SNARE proteins in the platelet. To evaluate this possibility, we analyzed the subcellular distribution of 3 functional platelet SNARE proteins-human cellubrevin, SNAP-23, and syntaxin 2. Exposure of streptolysin O-permeabilized platelets to antihuman cellubrevin antibody inhibited Ca(++)-induced alpha-granule secretion by approximately 50%. Inhibition of alpha-granule secretion by antihuman cellubrevin was reversed by a blocking peptide. Syntaxin 2 and SNAP-23 have previously been demonstrated to mediate platelet granule secretion. The subcellular localization of the 3 SNARE proteins was determined by ultrastructural studies, using a pre-embedding immunonanogold method, and by immunoblot analysis of subcellular fractions. Immunonanogold localization demonstrated that approximately 80% of human cellubrevin in resting platelets was localized to platelet granule membranes. In contrast, SNAP-23 localized predominantly to plasma membrane, whereas syntaxin 2 was more evenly distributed among membranes of alpha-granules, the open canalicular system, and plasma membrane. Thus, each of these SNARE proteins has a distinct subcellular distribution in platelets, and each of these membrane compartments demonstrates a unique SNARE protein composition. This distribution provides a basis for several characteristics of alpha-granule secretion that include homotypic alpha-granule fusion and the fusion of alpha-granules with the open canalicular system and plasma membrane.

Antibodies↗
PubMedDate not supplied

Interactions of platelets, blood-borne tissue factor, and fibrin during arteriolar thrombus formation in vivo.

Thrombus formation following vascular injury is an essential component of both hemostasis and pathologic vessel occlusion. This process occurs in a closed, pressurized environment in which blood flows rapidly over the injury site. Thrombus formation must occur quickly to reduce blood loss, but is carefully modulated to limit vessel occlusion. Circulating cells, plasma proteins, vessel wall components, and physical forces such as shear all influence thrombus formation. Historically, thrombus formation has been studied by isolating the separate components of blood involved in clot formation. With improved optical techniques, investigators have increasingly studied thrombus formation under conditions of flow in vitro and in live animals in vivo. Using multichannel fluorescence intravital videomicroscopy, the authors have studied the changes in the kinetics and deposition of platelets, fibrin, and tissue factor at the injury site during thrombosis in transgenic mice, bone marrow transplanted mice, and mice treated with pharmacological agents that modulate thrombosis. The differences in the kinetics of accumulation of the various components of thrombus in these mice have provided new insights about thrombus formation in arterioles. This review discusses the role of platelet intracellular signaling, P-selectin expression on platelets, and tissue factor-bearing microparticles in thrombus formation.

Animals↗