The 33-kDa Platelet α-granule Membrane Protein (GMP-33) Is an N-terminal Proteolytic Fragment of Thrombospondin

2001 ◽  
Vol 86 (09) ◽  
pp. 887-893 ◽  
Author(s):  
Conchi Damas ◽  
Tom Vink ◽  
Karel Nieuwenhuis ◽  
Jan Sixma
Keyword(s):  
Plasma Membrane ◽  
Alternative Splicing ◽  
Membrane Protein ◽  
Immunoaffinity Chromatography ◽  
Terminal Sequence ◽  
Terminal Part ◽  
Proteolytic Fragment ◽  
Activated Platelets ◽  
N Terminus ◽  
Granule Membrane

SummaryGMP-33 is a platelet membrane associated protein that is recognised by RUU-SP 1.77, an antibody raised against activated platelets. GMP-33 is predominantly associated with the membrane of platelet α-granules and it is translocated to the plasma membrane upon platelet activation (Metzelaar et al. Blood 1992; 79: 372-9). In this study we have isolated the protein by immunoaffinity chromatography. The N-terminus was sequenced and was identical to the N-terminal sequence of human thrombospondin. The protein was N-glycosylated and bound to heparin as would be expected of the N-terminal part of thrombospondin. RUU-SP 1.77 reacted only with reduced thrombospondin. Plasmin and trypsin digestion of thrombospondin yielded fragments of approximately the same size as GMP 33 that reacted with RUU-SP 1.77 after reduction. No evidence for alternative splicing was found. We postulate that GMP 33 is an N-terminal proteolytic fragment of thrombospondin that is membrane associated.

scholarly journals Platelet dense granule membranes contain both granulophysin and P- selectin (GMP-140)

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 143-152 ◽  
Author(s):  
SJ Israels ◽  
JM Gerrard ◽  
YV Jacques ◽  
A McNicol ◽  
B Cham ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Serotonin Release ◽  
Dense Granule ◽  
Double Labeling ◽  
Platelet Surface ◽  
Thin Sections ◽  
Dense Granules ◽  
Granule Membrane ◽  
Dense Granule Secretion

We recently reported the characterization of a platelet granule membrane protein of molecular weight (mol wt) 40,000 called granulophysin (Gerrard et al: Blood 77:101, 1991), identified by a monoclonal antibody (MoAb D545) raised to purified dense granule membranes. Using immunoelectron-microscopic techniques on frozen thin sections, this protein was localized in resting and thrombin-stimulated platelets. In resting platelets, labeled with antigranulophysin antibodies and immunogold probes, label was localized to the membranes of one or two clear granules per platelet thin section. D545 also labeled dense granules in permeabilized whole platelets and isolated dense granule preparations examined by whole-mount techniques. Expression of granulophysin on the platelet surface paralleled dense granule secretion as measured by 14C-serotonin release under conditions in which lysosomal granule release, as measured by beta-glucuronidase secretion, was less than 5%. After thrombin stimulation, both the surface-connected canalicular system and the plasma membrane were labeled, demonstrating redistribution of granulophysin associated with degranulation. Double labeling experiments with D545 and antibodies to the alpha-granule membrane protein, P-selectin, demonstrated labeling of both P-selectin and granulophysin on dense granule membranes. Distribution of both proteins on the plasma membrane after platelet stimulation was similar. The results demonstrate that granulophysin is localized to the dense granules of platelets and is redistributed to the plasma membrane after platelet activation.

Recycling of a secretory granule membrane protein after stimulated secretion

1993 ◽  
Vol 106 (2) ◽  
pp. 649-655 ◽  
Author(s):  
S.M. Hurtley
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Secretory Granule ◽  
Chromaffin Cells ◽  
Secretory Granules ◽  
Primary Cultures ◽  
Dopamine Beta Hydroxylase ◽  
Granule Membrane ◽  
Antibody Complex ◽  
Adrenal Chromaffin

Recycling of a secretory granule membrane protein, dopamine-beta-hydroxylase, was examined in primary cultures of bovine adrenal chromaffin cells. Cells were stimulated to secrete in the presence of antibodies directed against the luminal domain of dopamine-beta-hydroxylase. The location of the antibodies after various times of reincubation and after a second secretory stimulus was assessed using immunofluorescence microscopy. Stimulation led to the exposure of dopamine-beta-hydroxylase at the plasma membrane, which could be detected by a polyclonal antibody in living and fixed cells. The plasma membrane dopamine-beta-hydroxylase, either alone or complexed with antibody, was rapidly internalized after removal of the secretagogue. Internalized protein-antibody complex remained stable for at least 24 hours of reculture. Twenty four hours after stimulation the cells with internalized antibody could respond to further stimulation and some of the antibody was re-exposed at the plasma membrane. These findings were confirmed using FACS analysis. This suggests that the antibody-protein complex had returned to secretory granules that could respond to further secretagogue stimulation.

scholarly journals Identification of a 33-Kd protein associated with the alpha-granule membrane (GMP-33) that is expressed on the surface of activated platelets

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 372-379
Author(s):  
MJ Metzelaar ◽  
HF Heijnen ◽  
JJ Sixma ◽  
HK Nieuwenhuis
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Binding Sites ◽  
Major Protein ◽  
Activated Platelets ◽  
Granule Membrane ◽  
Microscopic Studies ◽  
Number Of Binding Sites ◽  
Thrombin Activation ◽  
A Minor

To identify antigens on the platelet plasma membrane that are exposed after activation, we developed a monoclonal antibody (MoAb) designated RUU-SP 1.77. The RUU-SP 1.77 antigen is present on the membrane of resting platelets at a basal level and is strongly expressed on the plasma membrane after thrombin activation. Freshly fixed platelets bound 4,150 +/- 1,935 (mean +/- SD) RUU-SP 1.77 molecules per platelet; on fixed thrombin-stimulated platelets the number of binding sites was upregulated to 19,050 +/- 5,120 (kd 4.5 +/- 0.8 nmol/L). MoAb RUU-SP 1.77 recognized a major protein of 33 Kd and a minor 28-Kd protein, both under nonreduced and reduced conditions. Immunoelectron microscopic studies showed the presence of the protein associated with the membrane of alpha-granules. Due to the localization associated with the alpha-granule membrane, we have designated it GMP-33 (granule membrane protein with a molecular weight of 33 Kd). Based on structural properties, we conclude that GMP-33 is a protein associated with the alpha-granule membrane that has not been described before.

scholarly journals Platelet plasma membrane glycoproteins and .ALPHA.-Granule membrane protein of long-term liquid-preserved platelets in a double-bag system.

1993 ◽  
Vol 39 (6) ◽  
pp. 967-973
Author(s):  
Eiji Kino ◽  
Tsuneo A. Takahashi ◽  
Chiaki Yakushiji ◽  
Takami Maekawa ◽  
Seizo Fujikawa ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Membrane Glycoproteins ◽  
Granule Membrane

scholarly journals A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation.

1985 ◽  
Vol 101 (3) ◽  
pp. 880-886 ◽  
Author(s):  
P E Stenberg ◽  
R P McEver ◽  
M A Shuman ◽  
Y V Jacques ◽  
D F Bainton
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Plasma Membranes ◽  
Polyclonal Antibodies ◽  
Fold Increase ◽  
Immunogold Label ◽  
Thin Sections ◽  
Granule Membrane ◽  
Immunocytochemical Techniques ◽  
Intracellular Organelles

We have previously characterized a monoclonal antibody, S12, that binds only to activated platelets (McEver, R.P., and M.N. Martin, 1984, J. Biol. Chem., 259:9799-9804). It identifies a platelet membrane protein of Mr 140,000, which we have designated as GMP-140. Using immunocytochemical techniques we have now localized this protein in unstimulated and thrombin-stimulated platelets. Polyclonal antibodies to purified GMP-140 were used to enhance the sensitivity of detection. Nonpermeabilized, unstimulated platelets, incubated with anti-GMP-140 antibodies, and then with IgG-gold probes, showed very little label for GMP-140 along their plasma membranes. In contrast, thrombin-stimulated platelets exhibited at least a 50-fold increase in the amount of label along the plasma membrane. On frozen thin sections of unstimulated platelets we observed immunogold label along the alpha-granule membranes. We also employed the more sensitive technique of permeabilizing with saponin unstimulated platelets in suspension, and then incubating the cells with polyclonal anti-GMP-140 antibodies and Fab-peroxidase conjugate. Alpha-granule membranes showed heavy reaction product, but no other intracellular organelles were specifically labeled. These results demonstrate that GMP-140 is an alpha-granule membrane protein that is expressed on the platelet plasma membrane during degranulation.

Expression of Thrombospondin 1 on the Surface of Activated Platelets Mediates their Interaction with the Heavy Chains of Human Kininogens through Lys 244-Pro 254

1998 ◽  
Vol 79 (01) ◽  
pp. 186-194 ◽  
Author(s):  
Satya Kunapuli ◽  
Daniel Walz ◽  
Robert Colman ◽  
Raul DeLa Cadena
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Binding Site ◽  
Cyclic Form ◽  
Inhibitory Effects ◽  
Cell Binding ◽  
Terminal Sequence ◽  
Proteolytic Fragment ◽  
Heavy Chains ◽  
Activated Platelets

SummaryPlatelet thrombospondin (TSP1) forms a complex with high (HK) and low (LK) molecular weight kininogens. We isolated a proteolytic fragment from HK and LK heavy chains (12 kDa) recognized by TSP1 with a N-terminal sequence, K244ICVGCPRDIP254. Lys244-Pro254 oxidized to cyclic form prevented binding of 125I-LK to TSP1. This effect was abolished by reduction and alkylation. Oxidized peptide KICVGCPRDIP (100 μM) reversed the known inhibitory effects of LK or HK (1 μM), on thrombin-induced platelet activation, suggesting this peptide forms part of the cell binding site on HK and LK for activated platelets. KICVGCPRDIP completely inhibited the binding of 125I-LK to activated platelets. However, the peptide only partially inhibited binding of 125I-HK to platelets, suggesting an additional binding site on the HK light chain. Fluorescein-labeled KICVGCPRDIP bound directly and specifically to activated platelets. A monoclonal antibody directed to TSP1 partially inhibited the binding of 125I-HK to activated but not inactivated platelets. We conclude residues Lys244-Pro254 on kininogen heavy chain is responsible for binding to thrombospondin on the surface of activated platelets.

scholarly journals Identification of a 33-Kd protein associated with the alpha-granule membrane (GMP-33) that is expressed on the surface of activated platelets

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 372-379 ◽  
Author(s):  
MJ Metzelaar ◽  
HF Heijnen ◽  
JJ Sixma ◽  
HK Nieuwenhuis
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Binding Sites ◽  
Major Protein ◽  
Activated Platelets ◽  
Granule Membrane ◽  
Microscopic Studies ◽  
Number Of Binding Sites ◽  
Thrombin Activation ◽  
A Minor

Abstract To identify antigens on the platelet plasma membrane that are exposed after activation, we developed a monoclonal antibody (MoAb) designated RUU-SP 1.77. The RUU-SP 1.77 antigen is present on the membrane of resting platelets at a basal level and is strongly expressed on the plasma membrane after thrombin activation. Freshly fixed platelets bound 4,150 +/- 1,935 (mean +/- SD) RUU-SP 1.77 molecules per platelet; on fixed thrombin-stimulated platelets the number of binding sites was upregulated to 19,050 +/- 5,120 (kd 4.5 +/- 0.8 nmol/L). MoAb RUU-SP 1.77 recognized a major protein of 33 Kd and a minor 28-Kd protein, both under nonreduced and reduced conditions. Immunoelectron microscopic studies showed the presence of the protein associated with the membrane of alpha-granules. Due to the localization associated with the alpha-granule membrane, we have designated it GMP-33 (granule membrane protein with a molecular weight of 33 Kd). Based on structural properties, we conclude that GMP-33 is a protein associated with the alpha-granule membrane that has not been described before.

scholarly journals Platelet dense granule membranes contain both granulophysin and P- selectin (GMP-140)

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 143-152 ◽  
Author(s):  
SJ Israels ◽  
JM Gerrard ◽  
YV Jacques ◽  
A McNicol ◽  
B Cham ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Serotonin Release ◽  
Dense Granule ◽  
Double Labeling ◽  
Platelet Surface ◽  
Thin Sections ◽  
Dense Granules ◽  
Granule Membrane ◽  
Dense Granule Secretion

Abstract We recently reported the characterization of a platelet granule membrane protein of molecular weight (mol wt) 40,000 called granulophysin (Gerrard et al: Blood 77:101, 1991), identified by a monoclonal antibody (MoAb D545) raised to purified dense granule membranes. Using immunoelectron-microscopic techniques on frozen thin sections, this protein was localized in resting and thrombin-stimulated platelets. In resting platelets, labeled with antigranulophysin antibodies and immunogold probes, label was localized to the membranes of one or two clear granules per platelet thin section. D545 also labeled dense granules in permeabilized whole platelets and isolated dense granule preparations examined by whole-mount techniques. Expression of granulophysin on the platelet surface paralleled dense granule secretion as measured by 14C-serotonin release under conditions in which lysosomal granule release, as measured by beta-glucuronidase secretion, was less than 5%. After thrombin stimulation, both the surface-connected canalicular system and the plasma membrane were labeled, demonstrating redistribution of granulophysin associated with degranulation. Double labeling experiments with D545 and antibodies to the alpha-granule membrane protein, P-selectin, demonstrated labeling of both P-selectin and granulophysin on dense granule membranes. Distribution of both proteins on the plasma membrane after platelet stimulation was similar. The results demonstrate that granulophysin is localized to the dense granules of platelets and is redistributed to the plasma membrane after platelet activation.

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