Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 44
Filter
Add more filters










Publication year range
1.
J Thromb Haemost ; 13(10): 1888-99, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26256688

ABSTRACT

BACKGROUND: Megakaryocytes express and store platelet factor 4 (PF4) in alpha granules. In vivo, PF4 is a clinically relevant, negative regulator of megakaryopoiesis and hematopoietic stem cell replication. These findings would suggest a regulated source of free intramedullary PF4. OBJECTIVES: Define the source of free intramedullary PF4 and its intramedullary life cycle. METHODS: We interrogated both murine and human bone marrow-derived cells during megakaryopoiesis in vitro by using confocal microscopy and enzyme-linked immunosorbent assay. With immunohistochemistry, we examined in vivo free PF4 in murine bone marrow before and after radiation injury and in the setting of megakaryocytopenia and thrombocytopenia. RESULTS: Exogenously added human PF4 is internalized by murine megakaryocytes. Human megakaryocytes similarly take up murine PF4 but not the related chemokine, platelet basic protein. Confocal microscopy shows that internalized PF4 colocalizes with endogenous PF4 in alpha granules and is available for release on thrombin stimulation. Immunohistochemistry shows free PF4 in the marrow, but not another alphagranule protein, von Willebrand factor. Free PF4 increases with radiation injury and decreases with megakaryocytopenia. Consistent with the known role of low-density lipoprotein receptor-related protein 1 in the negative paracrine effect of PF4 on megakaryopoiesis, PF4 internalization is at least partially low-density lipoprotein receptor-related protein 1 dependent. CONCLUSIONS: PF4 has a complex intramedullary life cycle with important implications in megakaryopoiesis and hematopoietic stem cell replication not seen with other tested alpha granule proteins.


Subject(s)
Cytoplasmic Granules/metabolism , Megakaryocytes/metabolism , Platelet Factor 4/metabolism , Thrombocytopenia/metabolism , Thrombopoiesis , Animals , Biological Transport , Cells, Cultured , Cytoplasmic Granules/radiation effects , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunohistochemistry , Low Density Lipoprotein Receptor-Related Protein-1 , Megakaryocytes/radiation effects , Mice, Knockout , Microscopy, Confocal , Platelet Factor 4/deficiency , Platelet Factor 4/genetics , RNA Interference , Receptors, LDL/genetics , Receptors, LDL/metabolism , Thrombocytopenia/blood , Thrombocytopenia/genetics , Time Factors , Transfection , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism
2.
Blood ; 98(10): 3143-9, 2001 Nov 15.
Article in English | MEDLINE | ID: mdl-11698303

ABSTRACT

Because human CD34+ and murine Sca-1+ hematopoietic stem-progenitor cells (HSPCs) express platelet-binding sialomucin P-selectin (CD162) and integrin Mac-1 (CD11b-CD18) antigen, it was inferred that these cells might interact with platelets. As a result of this interaction, microparticles derived from platelets (PMPs) may transfer many platelet antigens (CD41, CD61, CD62, CXCR4, PAR-1) to the surfaces of HSPCs. To determine the biologic significance of the presence of PMPs on human CD34+ and murine Sca-1+ cells, their expressions on mobilized peripheral blood (mPB) and on nonmobilized PB- and bone marrow (BM)-derived CD34+ cells were compared. In addition, the effects of PMPs on the proliferation of CD34+ and Sca-1+ cells and on adhesion of HSPCs to endothelium and immobilized SDF-1 were studied. Finally, the hematopoietic reconstitution of lethally irradiated mice receiving transplanted BM mononuclear cells covered or not covered with PMPs was examined. It was found that PMPs are more numerous on mPB than on BM CD34+ cells, do not affect the clonogenicity of human and murine HSPCs, and increase adhesion of these cells to endothelium and immobilized SDF-1. Moreover, murine BM cells covered with PMPs engrafted lethally irradiated mice significantly faster than those not covered, indicating that PMPs play an important role in the homing of HSPCs. This could explain why in a clinical setting human mPB HSPCs (densely covered with PMPs) engraft more rapidly than BM HSPCs (covered with fewer PMPs). These findings indicate a new role for PMPs in stem cell transplantation and may have clinical implications for the optimization of transplantations.


Subject(s)
Blood Platelets/metabolism , Graft Survival , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Animals , Antigens, CD34/analysis , Antigens, Human Platelet/metabolism , Antigens, Ly/analysis , Blood Platelets/ultrastructure , Bone Marrow Cells/metabolism , Cell Adhesion , Cell Division , Cell Membrane/metabolism , Cells, Cultured , Chemokine CXCL12 , Chemokines, CXC/metabolism , Colony-Forming Units Assay , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Female , HL-60 Cells , Hematopoietic Stem Cell Mobilization , Humans , Membrane Glycoproteins/analysis , Membrane Proteins/analysis , Mice , Mice, Inbred C57BL , Platelet Activation , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Radiation Chimera , Time Factors , Umbilical Veins
3.
Blood ; 98(3): 610-7, 2001 Aug 01.
Article in English | MEDLINE | ID: mdl-11468158

ABSTRACT

The genes for the related human (h) chemokines, PBP (platelet basic protein) and PF4 (platelet factor 4), are within 5.3 kilobases (kb) of each other and form a megakaryocyte-specific gene locus. The hypothesis was considered that the PBP and PF4 genes share a common distal regulatory region(s) that leads to their high-level megakaryocyte-specific expression in vivo. This study examined PBP and PF4 expression in transgenic mice using 4 distinct human PBP/PF4 gene locus constructs. These studies showed that within the region studied there was sufficient information to regulate tissue-specific expression of both hPBP and hPF4. Indeed this region contained sufficient DNA information to lead to expression levels of PBP and PF4 comparable to the homologous mouse genes in a position-independent, copy number-dependent fashion. These studies also indicated that the DNA domains that led to this expression were distinct for the 2 genes; hPBP expression is regulated by a region that is 1.5 to 4.4 kb upstream of that gene. Expression of hPF4 is regulated by a region that is either intergenic between the 2 genes or immediately downstream of the hPF4 gene. Comparison of the available human and mouse sequences shows conserved flanking region domains containing potential megakaryocyte-related transcriptional factor DNA-binding sites. Further analysis of these regulatory regions may identify enhancer domains involved in megakaryopoiesis that may be useful in the selective expression of other genes in megakaryocytes and platelets as a strategy for regulating hemostasis, thrombosis, and inflammation. (Blood. 2001;98:610-617)


Subject(s)
Chemokines/genetics , Genes, Regulator/genetics , Megakaryocytes/metabolism , Platelet Factor 4/genetics , Animals , Base Sequence , Binding Sites , Chemokines/chemistry , Conserved Sequence , Humans , Immunohistochemistry , Mice , Mice, Transgenic , Molecular Sequence Data , Platelet Factor 4/metabolism , Protein Structure, Tertiary , Spleen/chemistry , Spleen/cytology , Transcription Factors , beta-Thromboglobulin
4.
Blood ; 97(10): 3075-85, 2001 May 15.
Article in English | MEDLINE | ID: mdl-11342433

ABSTRACT

The aim of this study was to explore further the hypothesis that early stages of normal human hematopoiesis might be coregulated by autocrine/paracrine regulatory loops and by cross-talk among early hematopoietic cells. Highly purified normal human CD34(+) cells and ex vivo expanded early colony-forming unit-granulocyte-macrophage (CFU-GM)-derived, burst forming unit-erythroid (BFU-E)-derived, and CFU-megakaryocyte (CFU-Meg)-derived cells were phenotyped for messenger RNA expression and protein secretion of various growth factors, cytokines, and chemokines to determine the biological significance of this secretion. Transcripts were found for numerous growth factors (kit ligand [KL], FLT3 ligand, fibroblast growth factor-2 [FGF-2], vascular endothelial growth factor [VEGF], hepatocyte growth factor [HGF], insulinlike growth factor-1 [IGF-1], and thrombopoietin [TPO]); cytokines (tumor necrosis factor-alpha, Fas ligand, interferon alpha, interleukin 1 [IL-1], and IL-16); and chemokines (macrophage inflammatory protein-1alpha [MIP-1alpha], MIP-1beta, regulated upon activation, normal T cell expressed and secreted [RANTES], monocyte chemotactic protein-3 [MCP-3], MCP-4, IL-8, interferon-inducible protein-10, macrophage-derived chemokine [MDC], and platelet factor-4 [PF-4]) to be expressed by CD34(+) cells. More importantly, the regulatory proteins VEGF, HGF, FGF-2, KL, FLT3 ligand, TPO, IL-16, IGF-1, transforming growth factor-beta1 (TGF-beta1), TGF-beta2, RANTES, MIP-1alpha, MIP-1beta, IL-8, and PF-4 were identified in media conditioned by these cells. Moreover, media conditioned by CD34(+) cells were found to inhibit apoptosis and slightly stimulate the proliferation of other freshly isolated CD34(+) cells; chemo-attract CFU-GM- and CFU-Meg-derived cells as well as other CD34(+) cells; and, finally, stimulate the proliferation of human endothelial cells. It was also demonstrated that these various hematopoietic growth factors, cytokines, and chemokines are expressed and secreted by CFU-GM-, CFU-Meg-, and BFU-E-derived cells. It is concluded that normal human CD34(+) cells and hematopoietic precursors secrete numerous regulatory molecules that form the basis of intercellular cross-talk networks and regulate in an autocrine and/or a paracrine manner the various stages of normal human hematopoiesis.


Subject(s)
Chemokines/metabolism , Cytokines/metabolism , Growth Substances/metabolism , Hematopoiesis , Hematopoietic Stem Cells/physiology , Homeostasis , Antigens, CD34/analysis , Cell Division , Cell Separation , Cell Survival , Cells, Cultured , Chemokines/genetics , Chemotaxis , Culture Media, Conditioned , Cytokines/genetics , Erythroblasts/physiology , Flow Cytometry , Gene Expression , Granulocytes/physiology , Growth Substances/genetics , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Humans , Megakaryocytes/physiology , RNA, Messenger/analysis , Rh-Hr Blood-Group System/physiology
5.
J Biol Chem ; 276(17): 13975-81, 2001 Apr 27.
Article in English | MEDLINE | ID: mdl-11278919

ABSTRACT

To determine the molecular basis for the insensitivity of rat alpha(IIb)beta(3) to inhibition by RGD-containing peptides, hybrids of human and rat alpha(IIb)beta(3) and chimeras of alpha(IIb)beta(3) in which alpha(IIb) was composed of portions of human and rat alpha(IIb) were expressed in Chinese hamster ovary cells and B lymphocytes, and the ability of the tetrapeptide RGDS to inhibit fibrinogen binding to the various forms of alpha(IIb)beta(3) was measured. These measurements indicated that sequences regulating the sensitivity of alpha(IIb)beta(3) to RGDS are located in the seven amino-terminal repeats of alpha(IIb). Moreover, replacing the first three or four (but not the first two) repeats of rat alpha(IIb) with the corresponding human sequences enhanced sensitivity to RGDS, whereas replacing the first two or three repeats of human alpha(IIb) with the corresponding rat sequences had little or no effect. Nevertheless, RGDS bound to Chinese hamster ovary cells expressing alpha(IIb)beta(3) regardless whether the alpha(IIb) in the heterodimers was human, rat, or a rat-human chimera. These results indicate that the sequences determining the sensitivity of alpha(IIb)beta(3) to RGD-containing peptides are located in the third and fourth amino-terminal repeats of alpha(IIb). Because RGDS binds to both human and rat alpha(IIb)beta(3), the results suggest that differences in RGDS sensitivity result from differences in the allosteric changes induced in these repeats following RGDS binding.


Subject(s)
Blood Platelets/metabolism , Fibrinogen/chemistry , Fibrinogen/metabolism , Oligopeptides/chemistry , Platelet Glycoprotein GPIIb-IIIa Complex/chemistry , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Adenosine Diphosphate/metabolism , Allosteric Site , Animals , B-Lymphocytes/metabolism , CHO Cells , Carcinogens , Cell Adhesion , Cricetinae , DNA, Complementary/metabolism , Dithiothreitol/pharmacology , Dose-Response Relationship, Drug , Humans , Inhibitory Concentration 50 , Ligands , Platelet Aggregation , Protein Binding , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/chemistry , Tetradecanoylphorbol Acetate , Transfection
6.
Blood ; 96(13): 4142-51, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11110685

ABSTRACT

The role of the chemokine binding stromal-derived factor 1 (SDF-1) in normal human megakaryopoiesis at the cellular and molecular levels and its comparison with that of thrombopoietin (TPO) have not been determined. In this study it was found that SDF-1, unlike TPO, does not stimulate alpha(IIb)beta(3)(+) cell proliferation or differentiation or have an antiapoptotic effect. However, it does induce chemotaxis, trans-Matrigel migration, and secretion of matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) by these cells, and both SDF-1 and TPO increase the adhesion of alpha(IIb)beta(3)(+) cells to fibrinogen and vitronectin. Investigating the intracellular signaling pathways induced by SDF-1 and TPO revealed some overlapping patterns of protein phosphorylation/activation (mitogen-activated protein kinase [MAPK] p42/44, MAPK p38, and AKT [protein kinase B]) and some that were distinct for TPO (eg, JAK-STAT) and for SDF-1 (eg, NF-kappa B). It was also found that though inhibition of phosphatidyl-inositol 3-kinase (PI-3K) by LY294002 in alpha(IIb)beta(3)(+) cells induced apoptosis and inhibited chemotaxis adhesion and the secretion of MMP-9 and VEGF, the inhibition of MAPK p42/44 (by the MEK inhibitor U0126) had no effect on the survival, proliferation, and migration of these cells. Hence, it is suggested that the proliferative effect of TPO is more related to activation of the JAK-STAT pathway (unique to TPO), and the PI-3K-AKT axis is differentially involved in TPO- and SDF-1-dependent signaling. Accordingly, PI-3K is involved in TPO-mediated inhibition of apoptosis, TPO- and SDF-1-regulated adhesion to fibrinogen and vitronectin, and SDF-1-mediated migration. This study expands the understanding of the role of SDF-1 and TPO in normal human megakaryopoiesis and indicates the molecular basis of the observed differences in cellular responses. (Blood. 2000;96:4142-4151)


Subject(s)
Chemokines, CXC/physiology , Megakaryocytes/cytology , Thrombopoietin/physiology , Adult , Apoptosis/drug effects , Blood Platelets/drug effects , Blood Platelets/metabolism , Calcium/metabolism , Calcium Signaling/drug effects , Caspase 3 , Caspases/metabolism , Cell Adhesion/drug effects , Cell Cycle/drug effects , Chemokine CXCL12 , Chemokines, CXC/pharmacology , Chemotaxis/drug effects , Chromones/pharmacology , Collagen , Drug Combinations , Endothelial Growth Factors/biosynthesis , Endothelial Growth Factors/genetics , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Fibrinogen/metabolism , Gene Expression Regulation/drug effects , Humans , Ion Transport/drug effects , Laminin , Lymphokines/biosynthesis , Lymphokines/genetics , MAP Kinase Signaling System/drug effects , Megakaryocytes/drug effects , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/genetics , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Platelet Glycoprotein GPIIb-IIIa Complex/physiology , Poly(ADP-ribose) Polymerases/metabolism , Protein Kinases/metabolism , Protein Processing, Post-Translational/drug effects , Proteoglycans , Recombinant Proteins/pharmacology , Thrombopoietin/pharmacology , Transcription Factors/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Vitronectin/metabolism
7.
Eur J Haematol ; 64(3): 164-72, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10997882

ABSTRACT

The aim of this study was to identify pathways which are involved in signal transduction from the CXCR4 receptor stimulated by stromal derived factor-1alpha (SDF-1alpha) in human malignant hematopoietic cells and normal megakaryoblasts. First, we found that activation of CXCR4 in human T cell lines (Jurkat and ATL-2) rapidly induced phosphorylation of mitogen-activated protein kinases (MAPK) (p44 ERK-1 and p42 ERK-2). Next, we became interested in CXCR4-mediated signaling in normal hematopoietic cells, and employed human megakaryoblasts, which highly express CXCR4 as a model. We found that stimulation of these cells with SDF-1alpha led to the phosphorylation of MAPK and serine/threonine kinase AKT as well. Activation of MAPK further led to the phosphorylation of the nuclear transcription factor ELK-1. Phosphorylation of ELK-1 in megakaryoblasts implies that phosphorylated MAPK translocate from cytoplasm into the nucleus where they may phosphorylate some nuclear proteins. Note that neither MAPK nor AKT was phosphorylated in normal human platelets after stimulation by SDF-1. We conclude that both MAPK and AKT are involved in signal transduction pathways from the CXCR4 receptor in malignant and normal human hematopoietic cells. The biological consequences of MAPK, ELK-1 and AKT phosphorylation in megakaryoblasts after stimulation with SDF-1alpha require further studies.


Subject(s)
Blood Platelets/metabolism , Chemokines, CXC/metabolism , DNA-Binding Proteins , Hematopoietic Stem Cells/metabolism , Megakaryocytes/metabolism , Phosphoproteins/metabolism , Receptors, CXCR4/metabolism , Calcium/metabolism , Chemokine CXCL12 , Chemokines, CXC/pharmacology , Chemotaxis, Leukocyte , Humans , Jurkat Cells , Leukemia-Lymphoma, Adult T-Cell , Mitogen-Activated Protein Kinase 1/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Signal Transduction , Transcription Factors/metabolism , Tumor Cells, Cultured , ets-Domain Protein Elk-1
8.
Proc Natl Acad Sci U S A ; 97(18): 9984-9, 2000 Aug 29.
Article in English | MEDLINE | ID: mdl-10954748

ABSTRACT

Heterotrimeric G proteins mediate the earliest step in cell responses to external events by linking cell surface receptors to intracellular signaling pathways. G(z) is a member of the G(i) family of G proteins that is prominently expressed in platelets and brain. Here, we show that deletion of the alpha subunit of G(z) in mice: (i) impairs platelet aggregation by preventing the inhibition of cAMP formation normally seen at physiologic concentrations of epinephrine, and (ii) causes the mice to be more resistant to fatal thromboembolism. Loss of G(zalpha) also results in greatly exaggerated responses to cocaine, reduces the analgesic effects of morphine, and abolishes the effects of widely used antidepressant drugs that act as catecholamine reuptake inhibitors. These changes occur despite the presence of other G(ialpha) family members in the same cells and are not accompanied by detectable compensatory changes in the level of expression of other G protein subunits. Therefore, these results provide insights into receptor selectivity among G proteins and a model for understanding platelet function and the effects of psychoactive drugs.


Subject(s)
Blood Platelets/physiology , Brain/physiology , GTP-Binding Protein alpha Subunits , Heterotrimeric GTP-Binding Proteins/metabolism , Platelet Activation/physiology , Psychotropic Drugs/pharmacology , Animals , Blood Platelets/drug effects , Brain/drug effects , Cocaine/pharmacology , Collagen/pharmacology , Cyclic AMP/blood , Epinephrine/pharmacology , Heterotrimeric GTP-Binding Proteins/deficiency , Heterotrimeric GTP-Binding Proteins/genetics , Humans , Lung/drug effects , Lung/physiopathology , Male , Mice , Mice, Knockout , Morphine/pharmacology , Motor Activity , Platelet Aggregation , Signal Transduction , Thromboembolism/genetics , Thromboembolism/physiopathology , Thromboembolism/prevention & control
9.
Blood ; 96(1): 50-7, 2000 Jul 01.
Article in English | MEDLINE | ID: mdl-10891429

ABSTRACT

Platelets play roles in both thrombosis and inflammation, and chemokines that are released at sites of inflammation could potentially activate platelets. Among the chemokine receptors expressed on platelets, the CXCR4 is the receptor for chemokine stromal cell-derived factor-1 (SDF-1), and the CCR4 is the receptor for macrophage-derived chemokine (MDC). Of the chemokines tested, SDF-1 and MDC were the only 2 that activated platelets. Both are weak agonists, but they enhanced response to low-dose adenosine 5'-diphosphate (ADP), epinephrine, or serotonin. When SDF-1 and MDC were added together, full and brisk platelet aggregation occurred. Platelet activation by these 2 chemokines appears to involve distinct pathways: SDF-1 inhibited an increase in cyclic adenosine monophosphate (cAMP) following prostaglandin (PG) I(2), while MDC had no effect. In contrast, MDC, but not SDF-1, lead to Ca(++) mobilization by platelets. Further, second-wave aggregation induced by MDC in platelet-rich plasma was inhibited by aspirin, ADP scavenger creatine phosphate/creative phosphokinase (CP/CPK), and ARL-66096, an antagonist of the ADP P2T(AC) receptor involved in adenylyl cyclase inhibition. But the aggregation was not affected by A3P5PS, an inhibitor of the ADP P2Y receptor. SDF-1-induced aggregation was inhibited by aspirin, but it was only slightly affected by CP/CPK, ARL-66096, or A3P5PS. Finally, the presence of chemokines in platelets was determined. Reverse transcriptase-polymerase chain reaction studies with platelet RNA did not detect the presence of SDF-1 or MDC. In summary, SDF-1 and MDC are platelet agonists that activate distinct intracellular pathways. Their importance in the development of thrombosis at sites of inflammation needs to be further evaluated.


Subject(s)
Blood Platelets/physiology , Chemokines, CXC/pharmacology , Chemokines/pharmacology , Platelet Activation/drug effects , Actins/blood , Actins/genetics , Adenosine Diphosphate/pharmacology , Blood Platelets/drug effects , Calcium/blood , Chemokine CXCL12 , Chemokine CXCL2 , Chemokines/blood , Chemokines/genetics , Cyclic AMP/blood , Cytosol/metabolism , Drug Synergism , Epinephrine/pharmacology , Humans , In Vitro Techniques , RNA, Messenger/genetics , Receptors, CCR4 , Receptors, Chemokine/drug effects , Receptors, Chemokine/genetics , Serotonin/pharmacology , Stromal Cells/physiology , Thromboxane B2/blood , Transcription, Genetic
10.
Stem Cells ; 18(2): 128-38, 2000.
Article in English | MEDLINE | ID: mdl-10742385

ABSTRACT

In order to better define the role of HIV-related chemokines in human erythropoiesis we studied: A) the expression of chemokine receptors, both on human CD34(+) cells which include erythroid progenitors and on more mature erythroid cells; B) the functionality of these receptors by calcium flux, chemotaxis assay and phosphorylation of mitogen-activated protein kinases (MAPK) p42/44 (ERK1/ERK2) and AKT, and finally C) the influence of chemokines on BFU-E formation. We found that HIV-related chemokine receptor CXCR4, but not CCR5, is detectable on human CD34(+) BFU-E cells. CXCR4 surface expression decreased during erythroid maturation, although CXCR4 mRNA was still present in cells isolated from differentiated erythroid colonies. SDF-1, a CXCR4 ligand, induced calcium flux and phosphorylation of MAPK (p42/44) and AKT in CD34(+)KIT(+) bone marrow mononuclear cells which contain BFU-E, as well as chemotactic activity of both human CD34(+) BFU-E progenitors and erythroid cells isolated from day 2-6 BFU-E colonies. Responsiveness to SDF-1 decreased when the cells differentiated to the point of surface expression of the erythroid-specific marker Glycophorin-A. In contrast, the CCR5 ligands (macrophage inflammatory protein-1alpha [MIP-1alpha], MIP-1beta, and RANTES) did not activate calcium flux, MAPK and AKT phosphorylation or chemotaxis of CD34(+)KIT(+) cells or cells isolated from the BFU-E colonies. Interestingly, none of the chemokines tested in this study had any effect on BFU-E colony formation. In conclusion, only CXCR4 is functional, and its specific ligand SDF-1 may therefore play an important role in the homing and/or retention of early erythroid precursors in the bone marrow environment.


Subject(s)
Chemokines/physiology , Erythropoiesis/physiology , HIV/metabolism , Receptors, Chemokine/physiology , Antigens, CD34 , Calcium/metabolism , Cell Division , Cells, Cultured , Chemotaxis , Culture Media, Serum-Free , Erythroid Precursor Cells/cytology , Erythroid Precursor Cells/metabolism , Gene Expression , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Ligands , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Receptors, Chemokine/genetics
11.
Blood ; 93(8): 2605-16, 1999 Apr 15.
Article in English | MEDLINE | ID: mdl-10194440

ABSTRACT

The expression and function of a glycoprotein Ib (GPIb) complex on human umbilical vein endothelial cells (HUVECs) is still a matter of controversy. We characterized HUVEC GPIb using viper venom proteins: alboaggregins A and B, echicetin, botrocetin, and echistatin. Echicetin is an antagonist, and alboaggregins act as agonists of the platelet GPIb complex. Botrocetin is a venom protein that alters von Willebrand factor (vWF) conformation and increases its binding affinity for the GPIb complex. Echistatin is a disintegrin that blocks alphavbeta3. Echistatin, but not echicetin, inhibited the adhesion to vWF of Chinese hamster ovary (CHO) cells transfected with alphavbeta3. We found the following: (1) Binding of monoclonal antibodies against GPIbalpha to HUVECs was moderately increased after stimulation with cytokines and phorbol ester. Echicetin demonstrated an inhibitory effect. (2) Both echicetin and echistatin, an alphavbeta3 antagonist, inhibited the adhesion of HUVECs to immobilized vWF in a dose-dependent manner. The inhibitory effect was additive when both proteins were used together. (3) Botrocetin potentiated the adhesion of HUVECs to vWF, and this effect was completely abolished by echicetin, but not by echistatin. (4) CHO cells expressing GPIbalphabeta/IX adhered to vWF (in the presence of botrocetin) and to alboaggregins; GPIbalpha was required for this reaction. Echicetin, but not echistatin, inhibited the adhesion of cells transfected with GPIbalphabeta/IX to immobilized vWF. (5) HUVECs adhered strongly to immobilized vWF and alboaggregins with extensive spreading, which was inhibited by LJ1b1, a monoclonal antibody against GPIb. The purified alphavbeta3 receptor did not interact with the alboaggregins, thereby excluding the contribution of alphavbeta3 in inducing HUVEC spreading on alboaggregins. In conclusion, our data confirm the presence of a functional GPIb complex expressed on HUVECs in low density. This complex may mediate HUVEC adhesion and spreading on immobilized vWF and alboaggregins.


Subject(s)
Cell Adhesion/drug effects , Endothelium, Vascular/physiology , Platelet Glycoprotein GPIb-IX Complex/physiology , Viper Venoms/pharmacology , Animals , CHO Cells , Carrier Proteins , Cell Adhesion/physiology , Cells, Cultured , Cricetinae , Crotalid Venoms/pharmacology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Humans , Intercellular Signaling Peptides and Proteins , Models, Cardiovascular , Peptides/pharmacology , Platelet Glycoprotein GPIb-IX Complex/drug effects , Proteins/pharmacology , Umbilical Veins , von Willebrand Factor/antagonists & inhibitors , von Willebrand Factor/physiology
12.
Br J Haematol ; 104(2): 220-9, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10050701

ABSTRACT

Thrombocytopenia is a late complication of human immunodeficiency virus (HIV) infection. The chemokine receptor CXCR4 has been shown to be a co-receptor for lymphocyte-tropic HIV-1 strains. CXCR4 is also a natural receptor for the chemokine SDF-1. We have previously shown that CXCR1 and CXCR2 are present on megakaryocytes and platelets. Although interleukin-8 (IL-8) and other chemokines that bind to these two receptors do not activate platelets, they are able to inhibit megakaryocytopoiesis, presumably through these receptors. We therefore examined whether CXCR4 is present on developing and mature megakaryocytes and on platelets. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated the presence of CXCR4 message. Immature and mature alphaIIbbeta3+ megakaryocytes, and platelets were also positive for CXCR4 by flow cytometric studies using a CXCR4-specific antibody. We then tested whether SDF-1 can affect the biology of these cells. CD34+ cells and immature alphaIIbbeta3+ cells responded to SDF-1 as indicated by Ca2+ mobilization and chemotaxis. However, mature megakaryocytes failed to demonstrate either of these responses, in spite of their continued ability to bind 125I-SDF-1. Further, SDF-1 failed to inhibit megakaryocyte colony growth. Platelets bound 125I-SDF-1 with a K(D) similar to the affinity seen for CXCR4 on other cells, yet SDF-1 did not aggregate washed platelets nor augment aggregation by low-dose ADP or thrombin. SDF-1 also failed to stimulate Ca2+ mobilization, granular release or expression of P-selectin in platelets. Accordingly, although our studies demonstrate that CD34+ precursors, megakaryocytes and platelets all express CXCR4 and bind SDF-1, biological effects were only demonstrable of SDF-1 on CD34+ precursors. The potential biological implications of CXCR4 expression on maturing megakaryocytes and platelets in normal individuals and following HIV infection are discussed.


Subject(s)
Anti-HIV Agents/pharmacology , Blood Platelets/metabolism , Chemokines, CXC/pharmacology , Megakaryocytes/metabolism , Receptors, CXCR4/metabolism , Acquired Immunodeficiency Syndrome/metabolism , Anti-HIV Agents/metabolism , Calcium/metabolism , Chemokine CXCL12 , Chemokines, CXC/metabolism , Flow Cytometry , Hematopoietic Stem Cells/metabolism , Humans , Neutrophils/metabolism , Platelet Activation , Reverse Transcriptase Polymerase Chain Reaction/methods
13.
Thromb Haemost ; 80(6): 982-8, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9869171

ABSTRACT

Platelet microparticles (PMP) were isolated from outdated platelets by a combination of differential centrifugation and gel filtration, and the concentration of PMP was expressed in the equivalent of GPIIb/IIIa complex measured by captured ELISA. PMP bound to isolated neutrophils and macrophages in a dose-dependent manner, but they did not bind to lymphocytes. Incubation of PMP with neutrophils did not activate these cells as measured by up-regulation of Mac-1, release of human granulocyte elastase, and calcium mobilization. Incubation of PMP with macrophages did not enhance IL-8 production and the oxygen burst but slightly and significantly increased production of MCP-1. After 10 min incubation of PMP with macrophages, an increase of GPIIb/IIIa antigen was observed suggesting that PMP may be endocytosed by macrophages. In conclusion, PMP bind to leukocytes, but, in contrast to activated platelets, do not play a significant role in leukocyte activation.


Subject(s)
Blood Platelets/cytology , Blood Preservation , Leukocytes/cytology , Biomarkers , Blood Platelets/metabolism , Cell Separation , Centrifugation , Chemokine CCL2/analysis , Chromatography, Gel , Endocytosis , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Interleukin-8/analysis , Leukocytes/metabolism , Macrophage Activation , Macrophages/cytology , Macrophages/physiology , Neutrophils/cytology , Neutrophils/metabolism , P-Selectin/analysis , Time Factors
14.
Thromb Haemost ; 79(3): 609-13, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9531050

ABSTRACT

Viper venoms contain a variety of platelet binding proteins including those which bind to platelet GPIb/GPIX. Most of these proteins inhibit von Willebrand factor mediated platelet agglutination. Here we report the primary structures of unique members of this family, alboaggregins A and B, isolated from Trimeresurus albolabris, which have the ability to stimulate platelet agglutination and aggregation. Four chains of alboaggregin A and two chains of alboaggregin B share a high degree of homology and all cysteines in both alboaggregins are conserved. Both alboaggregins caused similar agglutination of fixed platelets. Alboaggregin A induced platelet aggregation and release reaction with EC50 = 10 and 30 nM, respectively, which is 20-fold lower than those for alboaggregin B. These observations suggest that the dimeric structure of alboaggregin B is sufficient to mediate its binding to GPIb and induce agglutination of platelets whereas aggregation and release reaction are significantly enhanced by tetrameric structure of alboaggregin A.


Subject(s)
Blood Platelets/drug effects , Crotalid Venoms/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , Amino Acid Sequence , Blood Platelets/pathology , Humans , Molecular Sequence Data , Sequence Alignment , Structure-Activity Relationship
15.
Proc Natl Acad Sci U S A ; 94(16): 8750-5, 1997 Aug 05.
Article in English | MEDLINE | ID: mdl-9238049

ABSTRACT

G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca2+ mobilization. Present studies demonstrated that platelet phospholipase A2 (cytosolic and membrane) activity in the patient was normal. Receptor-mediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca2+ release induced by guanosine 5'-[gamma-thio]triphosphate (GTP[gammaS]) was diminished in the patient's platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of alpha-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A2 analog U46619. Binding of 35S-labeled GTP[gammaS] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I2 analog) -induced rise in cAMP (mediated by Galphas) and its inhibition (mediated by Galphai) by thrombin in the patient's platelet membranes were normal. Immunoblot analysis of Galpha subunits in the patient's platelet membranes showed a decrease in Galphaq (<50%) but not Galphai, Galphaz, Galpha12, and Galpha13. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein alpha-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of Galphaq in thrombin-induced responses.


Subject(s)
Blood Platelets/metabolism , GTP-Binding Proteins/metabolism , Hemorrhage/blood , Platelet Aggregation , Signal Transduction , Blood Platelets/pathology , Female , Flow Cytometry , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/genetics , Humans , Middle Aged
16.
FEBS Lett ; 409(2): 183-7, 1997 Jun 09.
Article in English | MEDLINE | ID: mdl-9202142

ABSTRACT

The design of smaller functional mimics of large proteins has long been an important challenge. In this study we use the natural leucine zipper as a structural template to design a 31-residue peptide analog that mimics the function of the larger platelet factor 4 (PF4) protein. The heparin binding activity of PF4 has been introduced into an unrelated leucine zipper sequence only by virtue of incorporating four lysines of PF4. Circular dichroism and binding experiments have shown that the designed leucine zipper peptide adopts a stable helical conformation and shows significant PF4-like heparin binding activity. These results strongly suggest that the lysine residues play an important role in the binding of PF4 to heparin. The de novo generation of the PF4 function in a designed leucine zipper peptide demonstrates that the leucine zipper motif is a useful scaffold for the design of functional peptides and proteins.


Subject(s)
DNA-Binding Proteins , Fungal Proteins/physiology , Leucine Zippers , Peptides/physiology , Platelet Factor 4/chemistry , Platelet Factor 4/physiology , Protein Engineering , Protein Kinases/physiology , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Antigens, CD/physiology , Fungal Proteins/chemical synthesis , Heparin/metabolism , Interleukin-8/metabolism , Molecular Sequence Data , Neutrophil Activation/drug effects , Peptides/chemical synthesis , Peptides/pharmacology , Protein Kinases/chemical synthesis , Receptors, Interleukin/physiology , Receptors, Interleukin-8A , Structure-Activity Relationship
17.
Thromb Res ; 85(2): 115-26, 1997 Jan 15.
Article in English | MEDLINE | ID: mdl-9058485

ABSTRACT

beta 3 integrin-derived peptides 214-218 and 217-231 have been shown previously to inhibit platelet aggregation and fibrinogen binding to platelets and to purified receptor. In this paper we study the activity of both peptides in inhibition of binding of biotinylated fibrinogen to activated platelets and to immobilized alpha IIb beta 3 receptor. We found that the mechanism of this inhibition by both peptides is different 125I-labeled 214-218 peptide binds to alpha IIb beta 3 but in contrast, 125I-labeled 217-231 peptide binds to the A alpha-chain of native and gamma' fibrinogen, as judged by the cross-linking study. In solid phase assay both purified alpha IIb beta 3 and 217-231 peptide bound extensively to native and recombinant fibrinogen, and to fibrinogen with either D574E or D97E mutations in the A alpha-chain. Binding of purified alpha IIb beta 3 to gamma' fibrinogen was markedly impaired whereas binding of 217-231 was only slightly impaired in comparison with native fibrinogen. Binding of 217-231 to fibrinogen fragment X was also reduced suggesting that sequences other than RGDS and RGDF may represent binding sites for this peptide. We hypothesize that the close vicinity of fibrinogen binding site (217-231) and of the site participating in conformational changes of the alpha IIb beta 3 receptor (214-218) may facilitate fibrinogen interaction with its receptor.


Subject(s)
Antigens, CD/metabolism , Fibrinogen/metabolism , Integrins/metabolism , Peptide Fragments/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Platelet Membrane Glycoproteins/metabolism , Adenosine Diphosphate/metabolism , Blood Platelets/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Integrin beta3
18.
Protein Expr Purif ; 8(1): 68-74, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8812836

ABSTRACT

Vitronectin receptor (alphavbeta3 integrin) is present on the surface of many types of cells. We describe a simple, fast, and reliable method of purification of recombinant human alphavbeta3 from Chinese hamster ovary (CHO) cells transfected with alphavbeta3 (VNRC3 cells). The method consists of two steps: lysis of the cells and affinity chromatography of the lysate on a GRGDSPK-Sepharose column. The yield of the procedure was about 79%. The purified receptor migrated as two bands on a silver stained SDS-polyacrylamide gel, corresponding to the alphav and beta3 subunits, and was recognized by monoclonal antibodies directed against alphav and the alphavbeta3 complex, but not by monoclonal antibody specific for the alphaIIbbeta3 complex. This receptor also bound to immobilized vitronectin, von Willebrand factor, and echistatin. However, binding to immobilized fibrinogen was not observed. Purified recombinant alphavbeta3 demonstrated greater immunoreactivity with LM 609, an alphavbeta3 complex-specific monoclonal antibody, than alphavbeta3 purified from placenta. As visualized by SDS-polyacrylamide gel electrophoresis, preparations of placenta-derived alphavbeta3 contained several contaminating proteins that were not present in preparations of recombinant alphavbeta3 purified from the transfected CHO cells.


Subject(s)
Receptors, Vitronectin/isolation & purification , Recombinant Proteins/isolation & purification , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , CHO Cells , Chromatography, Affinity , Cricetinae , Disintegrins/metabolism , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Extracellular Matrix/chemistry , Extracellular Matrix/metabolism , Humans , Integrins/isolation & purification , Integrins/metabolism , Transfection/genetics , Vitronectin/metabolism , von Willebrand Factor/metabolism
20.
Thromb Haemost ; 74(3): 954-7, 1995 Sep.
Article in English | MEDLINE | ID: mdl-8571328

ABSTRACT

Echicetin, a protein isolated from Echis carinatus snake venom, inhibited platelet aggregation and secretion induced by low concentrations of thrombin ( < 0.2 U/ml), by binding to platelet glycoprotein Ib (GPIb). The inhibition was not observed when the platelets were stimulated with higher concentrations of thrombin ( > 0.2 U/ml). Echicetin competed with thrombin for binding to the high affinity site on GPIb. Thrombin also inhibited 50% of the binding of 125I-echicetin to the platelets.


Subject(s)
Platelet Aggregation Inhibitors/pharmacology , Platelet Glycoprotein GPIb-IX Complex/metabolism , Proteins/pharmacology , Receptors, Thrombin/metabolism , Viper Venoms/pharmacology , Amino Acid Sequence , Blood Platelets/drug effects , Blood Platelets/metabolism , Carrier Proteins , Epitopes , Humans , Molecular Sequence Data , Peptides/blood , Platelet Membrane Glycoproteins/metabolism , Proteins/immunology , Proteins/metabolism , Receptors, Cell Surface/metabolism , Secretory Rate/drug effects , Thrombin/pharmacology , Viper Venoms/blood
SELECTION OF CITATIONS
SEARCH DETAIL
...