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Biomedical subjects

M C Berndt

Publications and source records attributed to M C Berndt.

At least 37 records · Page 2Linked to original sources
PubMed2000

Platelet glycoprotein ibalpha is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18).

The firm adhesion and transplatelet migration of leukocytes on vascular thrombus are both dependent on the interaction of the leukocyte integrin, Mac-1, and a heretofore unknown platelet counterreceptor. Here, we identify the platelet counterreceptor as glycoprotein (GP) Ibalpha, a component of the GP Ib-IX-V complex, the platelet von Willebrand factor (vWf) receptor. THP-1 monocytic cells and transfected cells that express Mac-1 adhered to GP Ibalpha-coated wells. Inhibition studies with monoclonal antibodies or receptor ligands showed that the interaction involves the Mac-1 I domain (homologous to the vWf A1 domain), and the GP Ibalpha leucine-rich repeat and COOH-terminal flanking regions. The specificity of the interaction was confirmed by the finding that neutrophils from wild-type mice, but not from Mac-1-deficient mice, bound to purified GP Ibalpha and to adherent platelets, the latter adhesion being inhibited by pretreatment of the platelets with mocarhagin, a protease that specifically cleaves GP Ibalpha. Finally, immobilized GP Ibalpha supported the rolling and firm adhesion of THP-1 cells under conditions of flow. These observations provide a molecular target for disrupting leukocyte-platelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplasty-related restenosis.

Animals↗
PubMed2000

Phosphoinositide 3-kinase forms a complex with platelet membrane glycoprotein Ib-IX-V complex and 14-3-3zeta.

The binding of von Willebrand factor (vWF) to glycoprotein (GP) Ib-IX-V stimulates transmembrane signaling events that lead to platelet adhesion and aggregation. Recent studies have revealed that the signaling protein 14-3-3 zeta binds directly to the cytoplasmic domain of GP Ib alpha. In this study, the dynamic association of 14-3-3 zeta with GP Ib-IX, the phosphoinositide 3-kinase (PI 3-kinase), or both, was investigated in resting, thrombin, or vWF and botrocetin-stimulated platelets by analysis of discrete subcellular fractions. Results of this study demonstrate maximal coimmunoprecipitation of 14-3-3 zeta with GP Ib-IX in the nonstimulated cytosolic fraction and in the actin cytoskeletal fraction of thrombin- or vWF-stimulated human platelets. Immunoprecipitated 14-3-3 zeta or GP Ib from cytosolic fractions contained PI 3-kinase enzyme activity and an 85-kd polypeptide recognized by antibodies to the p85 subunit of PI 3-kinase. After platelet activation, the level of association between these species decreased in the cytosolic fraction. However, increased complex formation between 14-3-3 zeta and GP Ib-IX and between PI 3-kinase and GP Ib-IX was detected in actin cytoskeletal fractions derived from thrombin- or vWF-stimulated platelets. Recombinant glutathione S-transferase-14-3-3 zeta fusion protein (14-3-3 zeta-GST) inhibited affinity-captured PI 3-kinase enzyme activity up to 70% at 2 mcmol/L 14-3-3 zeta-GST. However, increasing concentrations up to 5 mcmol/L 14-3-3 zeta-GST resulted in the 3-fold enhancement of PI 3-kinase enzyme activity. We propose that the association between PI 3-kinase and 14-3-3 zeta with GP Ib-IX serves to promote the rapid translocation of these signaling proteins to the activated cytoskeleton, thereby regulating the formation of 3-position phosphoinositide-signaling molecules in this subcellular compartment. (Blood. 2000;96:577-584)

14-3-3 Proteins↗
PubMed2000

Requirement of leucine-rich repeats of glycoprotein (GP) Ibalpha for shear-dependent and static binding of von Willebrand factor to the platelet membrane GP Ib-IX-V complex.

The platelet glycoprotein (GP) Ib-IX-V complex mediates adhesion to von Willebrand factor (vWf) in (patho)physiologic thrombus formation. The vWf-binding site on GP Ib-IX-V is within the N-terminal 282 residues of GP Ibalpha, which consist of an N-terminal flanking sequence (His-1-Ile-35), 7 leucine-rich repeats (Leu-36-Ala-200), a C-terminal flank (Phe-201-Gly-268), and a sulfated tyrosine sequence (Asp-269-Glu-282). We have used mammalian cell expression of canine-human chimeras of GP Ibalpha, corresponding to precise structural boundaries, to demonstrate the first specific requirement for individual leucine-rich repeats for binding of vWf either induced by a modulator, ristocetin, or under hydrodynamic flow. Implicit in this approach was that the GP Ibalpha chimeras retained a functional conformation, a supposition confirmed by analyzing restoration of function to reversed human-canine chimeras and demonstrating that all chimeras bound vWf activated by botrocetin, a modulator that is indiscriminate between species. Leucine-rich repeats 2, 3, and 4 of GP Ibalpha were identified as being critical for vWf adhesion to GP Ib-IX-V.

Animals↗
PubMed2000

Cytoplasmic domains of GpIbalpha and GpIbbeta regulate 14-3-3zeta binding to GpIb/IX/V.

Shear stress causes the platelet glycoprotein (Gp) Ib/IX/V to bind to von Willebrand factor, resulting in platelet adhesion. GpIb/IX/V also functions to stimulate transmembranous signaling, leading to platelet activation and the expression of a ligand-receptive GpIIb-IIIa complex. The highly conserved cytoplasmic domain of GpIbalpha binds directly to a dimeric 14-3-3 adapter protein zeta isoform. To explore structural determinants of GpIb/IX/V binding to 14-3-3zeta, the authors examined 14-3-3zeta interactions with GpIbalpha and GpIbbeta in heterologous cells and platelets. Truncations of GpIbalpha at amino acid 542 or 594, or deletions of residues 542 through 590, inhibited binding of 14-3-3zeta. Deletion of GpIbalpha from Trp(570) to Ser(590) eliminated 14-3-3zeta binding, and deletion of the sequence from Arg(542)-Trp(570) enhanced binding of 14-3-3zeta to GpIbalpha. All GpIbalpha mutations that eliminated GpIbalpha binding to the GST-14-3-3zeta fusion protein also eliminated GpIbbeta binding to the fusion protein. Forskolin treatment of Chinese hamster ovary cells expressing wild-type GpIbalpha/beta/IX resulted in the phosphorylation of GpIbbeta associated with enhanced binding of GpIbbeta to GST-14-3-3zeta fusion protein and increased 14-3-3zeta coimmunoprecipitated with GpIbalpha. When intact human platelets aggregated in response to 90 dynes/cm(2) shear stress, 14-3-3zeta disassociated from GpIbalpha. Prostacyclin treatment of platelets inhibited shear stress-induced aggregation and the release of 14-3-3zeta from GpIbalpha. These data demonstrate that amino acid residues in the cytoskeletal interaction domains of GpIbalpha regulate 14-3-3zeta binding to GpIbalpha/beta/IX, and suggest that protein kinase A-dependent phosphorylation of GpIbbeta enhances 14-3-3zeta binding to the GpIb/IX/V complex in human platelets. (Blood. 2000;95:551-557)

14-3-3 Proteins↗
PubMed2000

Structure and function of the von Willebrand factor A1 domain: analysis with monoclonal antibodies reveals distinct binding sites involved in recognition of the platelet membrane glycoprotein Ib-IX-V complex and ristocetin-dependent activation.

Binding of the adhesive glycoprotein, von Willebrand factor (vWf), to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates platelet adhesion and aggregation at high shear stress in hemostasis and thrombosis. In this study, the GP Ib-IX-V binding site within the vWf A1 domain was analyzed using a panel of murine monoclonal antibodies raised against a 39/34-kd vWf fragment (Leu-480/Val-481-Gly-718) encompassing the A1 domain. One antibody, 6G1, strongly inhibited ristocetin-dependent vWf binding to platelets, but had no effect on botrocetin- or jaracetin-dependent binding, or asialo-vWf-dependent platelet aggregation. The 6G1 epitope was mapped to Glu-700-Asp-709, confirming the importance of this region for modulation of vWf by ristocetin. Like ristocetin, 6G1 activated the vWf A1 domain, because it enhanced binding of the 39/34-kd fragment to platelets. In contrast, 5D2 and CR1 completely inhibited asialo-vWf-induced platelet aggregation and ristocetin-induced vWf binding to GP Ib-IX-V. However, only 5D2 blocked botrocetin- and jaracetin-induced vWf binding to platelets and binding of vWf to botrocetin- and jaracetin-coated beads. Epitopes for 5D2 and CR1 were conformationally dependent, but not congruent. Other antibodies mapped to epitopes within the A1 domain (CR2 and CR15, Leu-494-Leu-512; CR2, Phe-536-Ala-554; CR3, Arg-578-Glu-596; CR11 and CR15, Ala-564-Ser-582) were not functional, identifying regions of the vWf A1 domain not directly involved in vWf-GP Ib-IX-V interaction. The combined results provide evidence that the proline-rich sequence Glu-700-Asp-709 constitutes a regulatory site for ristocetin, and that ristocetin and botrocetin induce, at least in part, separate receptor-recognition sites on vWf. (Blood. 2000;95:164-172)

Amino Acid Sequence↗
PubMed2000

Snake venom modulators of platelet adhesion receptors and their ligands.

In thrombosis, platelet aggregation is initiated by a specific membrane glycoprotein (GP) Ib-IX-V complex binding to its adhesive ligand, von Willebrand factor, in the matrix of ruptured atherosclerotic plaques or in plasma exposed to high hydrodynamic shear stress. This process closely resembles normal haemostasis at high shear, where GP Ib-IX-V-dependent platelet adhesion to von Willebrand factor in the injured blood vessel wall initiates platelet activation and integrin alphaIIb beta3 (GP IIb-IIIa)-dependent platelet aggregation. At low shear, other receptors such as those that bind collagen, the integrin alpha2beta1 (GP Ia-IIa) or GP VI, mediate platelet adhesion. Recently, snake venom proteins have been identified that selectively modulate platelet function, either promoting or inhibiting platelet aggregation by targeting GP Ib-IX-V, alpha2beta1, GP VI, alphaIIb beta3, or their respective ligands. Interestingly, these venom proteins typically belong to one of two major protein families, the C-type lectin family or the metalloproteinase-disintegrins. This review focuses on recent insights into structure-activity relationships of snake venom proteins that regulate platelet function, and the ways in which these novel probes have contributed in unexpected ways to our understanding of the molecular mechanisms underlying thrombosis.

Amino Acid Sequence↗
PubMed2000

Site-specific serine phosphorylation of the IL-3 receptor is required for hemopoietic cell survival.

In the hemopoietic compartment, IL-3, GM-CSF, and IL-5 receptors are major transducers of survival signals; however, the receptor-proximal events that determine this vital function have not been defined. We have found that IL-3 stimulation induces phosphorylation of Ser-585 of beta(c). This promotes the association of phospho-Ser-585 of beta(c) with 14-3-3 and the p85 subunit of PI 3-K. Mutation of Ser-585 specifically impairs the PI 3-K signaling pathway and reduces cell survival in response to IL-3. These results define a distinct IL-3 receptor-mediated survival pathway regulated by site-specific receptor serine phosphorylation and 14-3-3 binding and suggest that this novel mode of signaling may be utilized by disparate transmembrane receptors that have as a common theme the transduction of survival signals.

14-3-3 Proteins↗
PubMed2000

The cleaved peptide of PAR1 results in a redistribution of the platelet surface GPIb-IX-V complex to the surface-connected canalicular system.

The only known function of the 41 amino acid cleaved peptide (TR1-41) of the seven transmembrane domain thrombin receptor (PARI) is to activate platelets (as determined by aggregation, surface P-selectin, and fibrinogen binding to activated GPIIb-IIIa). We now demonstrate that TR1-41 results in a concentration-dependent decrease in the platelet surface expression of each component of the GPIb-IX-V complex, as determined by flow cytometry with a panel of monoclonal antibodies (including 6D1, directed against the von Willebrand factor binding site on GPIbalpha, and TM60, directed against the thrombin binding site on GPIbalpha). TR1-41 also decreased ristocetin-induced platelet agglutination. Immunoblotting after incubation of platelets with TR1-41 revealed neither a loss of platelet GPIb nor increase in supernatant GPIb fragments. As demonstrated by immunoelectron microscopy, TR1-41 resulted in a redistribution of GPIb, GPIX, and GPV from the platelet surface to the surface-connected canalicular system (SCCS). In summary, the cleaved peptide (TR1-41) of PAR1 results in a redistribution of the platelet surface GPIb-IX-V complex to the SCCS, thereby negatively regulating the GPIbalpha binding sites for von Willebrand factor and thrombin.

Animals↗
PubMed1999

Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model.

We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.

14-3-3 Proteins↗
PubMed1999

Molecular interactions between telomerase and the tumor suppressor protein p53 in vitro.

The telomere DNA polymerase (telomerase) and the tumor suppressor protein p53 are frequently associated with human cancers, and activation of telomerase and inactivation of p53 involved in cancer cell immortalization. In this report, we demonstrate a direct interaction of telomerase with p53 in the nuclear lysates of human breast cancer cells, and with recombinant human p53, by affinity chromatography and immunoprecipitation. On activity criteria, the interaction is between the carboxyl-terminal region of p53 and a region close to the amino-terminus of human telomerase-associated protein 1 (hTEP1). Incubation of recombinant p53 with nuclear telomerase extracts results in inhibition of telomerase activity, with the C-terminal region of p53 being essential for inhibition. This effect is not mediated by binding to telomerase substrate DNA, but requires the region near the N-terminus of hTEP1, in that a synthetic peptide derived from this region of hTEP1 similarly inhibits telomerase activity. Together, these in vitro interactions between telomerase and p53 suggest that the activity of telomerase may be regulated by p53, down-regulation of which in turn would favor up-regulation of telomerase activity in cancer cell development.

Amino Acid Sequence↗
PubMed1999

The glycoprotein Ib-IX-V complex is a platelet counterreceptor for P-selectin.

We have identified platelet glycoprotein (GP) Ibalpha as a counterreceptor for P-selectin. GP Ibalpha is a component of the GP Ib-IX-V complex, which mediates platelet adhesion to subendothelium at sites of injury. Cells expressing P-selectin adhered to immobilized GP Ibalpha, and GP Ibalpha-expressing cells adhered to and rolled on P-selectin and on histamine-stimulated endothelium in a P-selectin-dependent manner. In like manner, platelets rolled on activated endothelium, a phenomenon inhibited by antibodies to both P-selectin and GP Ibalpha. Unlike the P-selectin interaction with its leukocyte ligand, PSGL-1 (P-selectin glycoprotein ligand 1), the interaction with GP Ibalpha required neither calcium nor carbohydrate core-2 branching or alpha(1,3)-fucosylation. The interaction was inhibited by sulfated proteoglycans and by antibodies against GP Ibalpha, including one directed at a tyrosine-sulfated region of the polypeptide. Thus, the GP Ib-IX-V complex mediates platelet attachment to both subendothelium and activated endothelium.

Animals↗
PubMed1999

Identification of a 14-3-3 binding sequence in the common beta chain of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors that is serine-phosphorylated by GM-CSF.

The common beta chain (beta(c)) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is the major signaling subunit of these receptors coupling ligand binding to multiple biological activities. It is thought that these multiple functions arise as a consequence of the recruitment of specific signaling molecules to tyrosine-phosphorylated residues in the cytoplasmic domain of beta(c). However, the contribution of serine phosphorylation in beta(c) to the recruitment of signaling molecules is not known. We show here the identification of a phosphoserine motif in the cytoplasmic domain of beta(c) that interacts with the adaptor protein 14-3-3zeta. Coimmunoprecipitation and pull-down experiments with a glutathione S-transferase (GST):14-3-3zeta fusion protein showed that 14-3-3 directly associates with beta(c) but not the GM-CSF receptor alpha chain. C-terminal truncation mutants of beta(c) further showed that a region between amino acids 544 and 626 in beta(c) was required for its association with 14-3-3zeta. This region contains the sequence (582)HSRSLP(587), which closely resembles the RSXSXP (where S is phosphorylated) consensus 14-3-3 binding site identified in a number of signaling molecules, including Raf-1. Significantly, substitution of (582)HSRSLP(587) for EFAAAA completely abolished interaction of beta(c) with GST-14-3-3zeta. Furthermore, the interaction of beta(c) with GST-14-3-3 was greatly reduced in the presence of a peptide containing the 14-3-3 binding site, but only when (585)Ser was phosphorylated. Direct binding experiments showed that the peptide containing phosphorylated (585)Ser bound 14-3-3zeta with an affinity of 150 nmol/L. To study the regulation of (585)S phosphorylation in vivo, we raised antibodies that specifically recognized (585)Ser-phosphorylated beta(c). Using these antibodies, we showed that GM-CSF stimulation strongly upregulated (585)Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the SHC-binding site ((577)Tyr) to the 14-3-3-binding site ((582)HSRSLP(587)) and their conservation between mouse, rat, and human beta(c) but not in other cytokine receptors suggest that they form a distinct motif that may subserve specialized functions associated with the GM-CSF, IL-3, and IL-5 receptors.

14-3-3 Proteins↗
PubMed1999

PSGL-1-mediated adhesion of human hematopoietic progenitors to P-selectin results in suppression of hematopoiesis.

Cellular interactions are critical for the regulation of hematopoiesis. The sialomucin PSGL-1/CD162 mediates the attachment of mature leukocytes to P-selectin. We now show that PSGL-1 also functions as the sole receptor for P-selectin on primitive human CD34+ hematopoietic progenitor cells (HPC). More importantly, ligation of PSGL-1 by immobilized or soluble ligand or anti-PSGL-1 antibody results in a profound suppression of HPC proliferation stimulated by potent combinations of early acting hematopoietic growth factors. These data demonstrate an unanticipated but extremely marked growth-inhibitory effect of P-selectin on hematopoiesis and provide direct evidence that PSGL-1, in addition to its well-documented role as an adhesion molecule on mature leukocytes, is a potent negative regulator of human hematopoietic progenitors.

ADP-ribosyl Cyclase↗
PubMed1999

PETA-3/CD151, a member of the transmembrane 4 superfamily, is localised to the plasma membrane and endocytic system of endothelial cells, associates with multiple integrins and modulates cell function.

The Transmembrane 4 Superfamily member, PETA-3/CD151, is ubiquitously expressed by endothelial cells in vivo. In cultured human umbilical vein endothelial cells PETA-3 is present on the plasma membrane and predominantly localises to regions of cell-cell contact. Additionally, this protein is abundant within an intracellular compartment which accounts for up to 66% of the total PETA-3 expressed. Intracellular PETA-3 showed colocalisation with transferrin receptor and CD63 suggesting an endosomal/lysosomal localisation which was supported by immuno-electronmicroscopy studies. Co-immunoprecipitation experiments investigating possible interactions of PETA-3 with other molecules demonstrated associations with several integrin chains including beta1, beta3, beta4, (alpha)2, (alpha)3, (alpha)5, (alpha)6 and provide the first report of Transmembrane 4 Superfamily association with the (alpha)6beta4 integrin. Using 2-colour confocal microscopy, we demonstrated similar localisation of PETA-3 and integrin chains within cytoplasmic vesicles and endothelial cell junctions. In order to assess the functional implications of PETA-3/integrin associations, the effect of anti-PETA-3 antibodies on endothelial function was examined. Anti-PETA-3 mAb inhibited endothelial cell migration and modulated in vitro angiogenesis, but had no detectable effect on neutrophil transendothelial migration. The broad range of integrin associations and the presence of PETA-3 with integrins both on the plasma membrane and within intracellular vesicles, suggests a primary role for PETA-3 in regulating integrin trafficking and/or function.

Antigens, CD↗
PubMed1999

Identification and characterization of a novel mutation in von Willebrand factor causing type 2B von Willebrand's disease.

Type 2B von Willebrand's disease (VWD) is a variant in which the structurally abnormal von Willebrand factor (VWF) has an increased affinity for the platelet glycoprotein Ib-IX-V complex. Spontaneous binding of type 2B VWF to platelets and their subsequent clearance from the plasma appear to account for the characteristic phenotype of type 2B VWD. Several type 2B mutations have been described and shown to be grouped along the amino edge of the beta sheet of the VWF A1 domain. In this report we describe a novel missense mutation, Arg543Leu, in the VWF A1 domain in three members of a family with type 2B VWD. We have expressed and characterized the corresponding recombinant mutant VWF in transiently transfected COS-7 cells. Relative to wild-type VWF, recombinant Arg543Leu VWF showed a similar multimer composition but exhibited binding to fixed platelets both in the presence and absence of ristocetin, confirming the ability of this mutation to permit spontaneous interaction of VWF with platelets. These studies are consistent with a recently proposed model in which the VWF-A1 domain exists in either 'on' or 'off' states, with type 2B mutations switching VWF to an 'on' state to facilitate GPIb binding.

Female↗
PubMed1998

Quinine-dependent antibodies bind a restricted set of epitopes on the glycoprotein Ib-IX complex: characterization of the epitopes.

Severe immune thrombocytopenia is an idiosyncratic complication of quinine therapy. Although in most cases the responsible antibody is directed against platelet membrane glycoprotein (GP) Ib-IX, specificity for GPIIb-IIIa or both epitopes has also been reported. The objective of this study was to characterize the binding site of GPIb-IX-specific quinine-dependent antibodies. Antibody binding to Chinese hamster ovary cells or mouse L cells stably transfected with various combinations of the three genes (Ibalpha, Ibbeta, or IX) that encode this complex was detected using flow cytometry, monoclonal antibody-specific immobilization of platelet antigens assay, and differential adsorption studies. IgG in sera from 15 patients with quinine-induced thrombocytopenia binding to the cells, in the presence of quinine, showed three distinct patterns. Group 1 sera contained at least two antibody populations, one which binds to GPIbalpha and another which recognizes GPIX. Group 2 sera contained an antibody which binds drug dependently to GPIX, and Group 3 sera contained an antibody which recognizes a quinine-dependent epitope on GPIbalpha. Thus, the quinine-dependent antibodies fall into two distinct populations that bind to GPIbalpha and GPIX independently. Using proteases which cleave GPIbalpha at specific sites, we have shown that the GPIbalpha-specific antibody binds to an 11-amino acid (283 to 293) region. Peptide inhibition studies provide confirmatory evidence that this region contains the epitope for the GPIbalpha-specific quinine-dependent antibody.

Amino Acid Sequence↗