Antibodies to platelets in patients with anti-phospholipid antibodies

Abstract Binding of anti-phospholipid antibodies to circulating platelets and its consequences on platelet activation and aggregation was investigated in 11 patients with anti-phospholipid antibodies. Seven patients had mild thrombocytopenia. Nine healthy donors served as controls. Binding to platelets was investigated by performing enzyme- linked immunosorbent assays (ELISAs) with phospholipids as antigen on platelet eluates. Platelet activation was measured by flow cytofluorometry using monoclonal antibodies to an activation-specific lysosomal membrane protein. Findings in ELISA were compared with results of a conventional immunofluorescence method to detect platelet autoantibodies. In seven patients antibodies to negatively charged phospholipids were present in platelet eluates. In all thrombocytopenic patients and controls the platelets were not activated and aggregation was not impaired. There was a positive concordance of 50% between the results of immunofluorescence and ELISA. No apparent relation was found between the results of ELISA or immunofluorescence and platelet counts. It is concluded that anti-phospholipid antibodies can bind to circulating platelets. This binding is not associated with measurable aggregation abnormalities nor with platelet activation characterized by exposure of lysosomal membrane proteins. More studies are necessary to determine the exact role of anti-phospholipid antibodies in the pathogenesis of thrombocytopenia and thrombosis.

Download Full-text

Antibodies to platelets in patients with anti-phospholipid antibodies

Blood ◽

10.1182/blood.v77.12.2655.bloodjournal77122655 ◽

1991 ◽

Vol 77 (12) ◽

pp. 2655-2659 ◽

Cited By ~ 1

Author(s):

HJ Out ◽

PG de Groot ◽

M van Vliet ◽

GC de Gast ◽

HK Nieuwenhuis ◽

...

Keyword(s):

Platelet Activation ◽

Lysosomal Membrane ◽

Immunofluorescence Method ◽

Exact Role ◽

Flow Cytofluorometry ◽

Healthy Donors ◽

Lysosomal Membrane Proteins ◽

Lysosomal Membrane Protein ◽

Platelet Autoantibodies

Binding of anti-phospholipid antibodies to circulating platelets and its consequences on platelet activation and aggregation was investigated in 11 patients with anti-phospholipid antibodies. Seven patients had mild thrombocytopenia. Nine healthy donors served as controls. Binding to platelets was investigated by performing enzyme- linked immunosorbent assays (ELISAs) with phospholipids as antigen on platelet eluates. Platelet activation was measured by flow cytofluorometry using monoclonal antibodies to an activation-specific lysosomal membrane protein. Findings in ELISA were compared with results of a conventional immunofluorescence method to detect platelet autoantibodies. In seven patients antibodies to negatively charged phospholipids were present in platelet eluates. In all thrombocytopenic patients and controls the platelets were not activated and aggregation was not impaired. There was a positive concordance of 50% between the results of immunofluorescence and ELISA. No apparent relation was found between the results of ELISA or immunofluorescence and platelet counts. It is concluded that anti-phospholipid antibodies can bind to circulating platelets. This binding is not associated with measurable aggregation abnormalities nor with platelet activation characterized by exposure of lysosomal membrane proteins. More studies are necessary to determine the exact role of anti-phospholipid antibodies in the pathogenesis of thrombocytopenia and thrombosis.

Download Full-text

A dileucine signal situated in the C-terminal tail of the lysosomal membrane protein p40 is responsible for its targeting to lysosomes

Biochemical Journal ◽

10.1042/bj20071626 ◽

2008 ◽

Vol 414 (3) ◽

pp. 431-440 ◽

Cited By ~ 7

Author(s):

Marielle Boonen ◽

Roberta Rezende de Castro ◽

Gaëlle Cuvelier ◽

Isabelle Hamer ◽

Michel Jadot

Keyword(s):

Subcellular Fractionation ◽

Lysosomal Membrane ◽

Consensus Sequences ◽

Cell Surface Biotinylation ◽

Dileucine Motif ◽

Lysosomal Sorting ◽

Critical Residues ◽

Lysosomal Membrane Proteins ◽

Golgi Network

Transport of newly synthesized lysosomal membrane proteins from the TGN (trans-Golgi network) to the lysosomes is due to the presence of specific signals in their cytoplasmic domains that are recognized by cytosolic adaptors. p40, a hypothetical transporter of 372 amino acids localized in the lysosomal membrane, contains four putative lysosomal sorting motifs in its sequence: three of the YXXϕ-type (Y6QLF, Y106VAL, Y333NGL) and one of the [D/E]XXXL[L/I]-type (EQERL360L361). To test the role of these motifs in the biosynthetic transport of p40, we replaced the most critical residues of these consensus sequences, the tyrosine residue or the leucine–leucine pair, by alanine or alanine–valine respectively. We analysed the subcellular localization of the mutated p40 proteins in transfected HeLa cells by confocal microscopy and by biochemical approaches (subcellular fractionation on self-forming Percoll density gradients and cell surface biotinylation). The results of the present study show that p40 is mistargeted to the plasma membrane when its dileucine motif is disrupted. No role of the tyrosine motifs could be put forward. Taken together, our results provide evidence that the sorting of p40 from the TGN to the lysosomes is directed by the dileucine EQERL360L361 motif situated in its C-terminal tail.

Download Full-text

Role of the Lysosomal Membrane Protein, CLN3, in the Regulation of Cathepsin D Activity

Journal of Cellular Biochemistry ◽

10.1002/jcb.26039 ◽

2017 ◽

Vol 118 (11) ◽

pp. 3883-3890 ◽

Cited By ~ 10

Author(s):

Jaime Cárcel‐Trullols ◽

Attila D. Kovács ◽

David A. Pearce

Keyword(s):

Membrane Protein ◽

Cathepsin D ◽

Lysosomal Membrane ◽

Lysosomal Membrane Protein ◽

Cathepsin D Activity

Download Full-text

Studies with a monoclonal antibody against activated platelets: evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation

Blood ◽

10.1182/blood.v70.3.838.838 ◽

1987 ◽

Vol 70 (3) ◽

pp. 838-845 ◽

Cited By ~ 192

Author(s):

HK Nieuwenhuis ◽

JJ van Oosterhout ◽

E Rozemuller ◽

F van Iwaarden ◽

JJ Sixma

Keyword(s):

Cardiopulmonary Bypass ◽

Platelet Activation ◽

Binding Sites ◽

Double Labeling ◽

Flow Cytofluorometry ◽

Activated Platelets ◽

Normal Individuals

Abstract To define the role of activated platelets we have attempted to prepare monoclonal antibodies specific for activated platelets. The IgG2b antibody of one of the clones, designated 2.28, was studied in more detail. Native platelets from normal individuals bound 650 125I-2.28 molecules/platelet, whereas thrombin-activated platelets bound 12,600 molecules/platelet with high affinity (4.6 nmol/L). Immunoelectrophoretic analysis revealed that 2.28 reacted with a 53,000- mol wt protein. Immunocytochemistry showed that the antigen is located in a special subclass of platelet granules in unstimulated platelets and is exposed on the surface of thrombin-activated platelets. Double- labeling studies with immunogold labels disclosed simultaneous localization of 2.28 binding sites and cathepsin D in the same granules both in megakaryocytes and endothelial cells, thereby indicating that the antigen may be localized in lysosomes. By using flow cytofluorometry, in vivo platelet activation was studied in patients undergoing cardiac surgery with cardiopulmonary bypass. Increased numbers of platelets that expressed the 2.28 antigen on their surface were observed after extracorporeal perfusion. The percentage of 2.28- positive platelets in the circulation was 3.9% +/- 2.7% (SD) in controls (n = 20), 5.5% +/- 3.0% in patients (n = 10) before cardiopulmonary bypass surgery, 24.6% +/- 13.5% after the bypass, and 8.5% in two patients with acute deep venous thrombosis. These data indicate that 2.28 may serve as a useful probe of in vitro and in vivo platelet activation.

Download Full-text

Lysosomal membrane proteins: life between acid and neutral conditions

Biochemical Society Transactions ◽

10.1042/bst0381420 ◽

2010 ◽

Vol 38 (6) ◽

pp. 1420-1423 ◽

Cited By ~ 48

Author(s):

Paul Saftig ◽

Bernd Schröder ◽

Judith Blanz

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Hydrolytic Degradation ◽

Cell Types ◽

Transport Processes ◽

Lysosomal Membrane ◽

Cell Physiology ◽

Depth Analysis ◽

Lysosomal Membrane Proteins

Whereas we have a profound understanding about the function and biogenesis of the protein constituents in the lumen of the lysosomal compartment, much less is known about the functions of proteins of the lysosomal membrane. Proteomic analyses of the lysosomal membrane suggest that, apart from the well-known lysosomal membrane proteins, additional and less abundant membrane proteins are present. The identification of disease-causing genes and the in-depth analysis of knockout mice leading to mutated or absent membrane proteins of the lysosomal membrane have demonstrated the essential role of these proteins in lysosomal acidification, transport of metabolites resulting from hydrolytic degradation and interaction and fusion with other cellular membrane systems. In addition, trafficking pathways of lysosomal membrane proteins are closely linked to the biogenesis of this compartment. This is exemplified by the recent finding that LIMP-2 (lysosomal integral membrane protein type-2) is responsible for the mannose 6-phosphate receptor-independent delivery of newly synthesized β-glucocerebrosidase to the lysosome. Similar to LIMP-2, which could also be linked to vesicular transport processes in certain polarized cell types, the major constituents of the lysosomal membrane, the glycoproteins LAMP (lysosome-associated membrane protein)-1 and LAMP-2 are essential for regulation of lysosomal motility and participating in control of membrane fusion events between autophagosomes or phagosomes with late endosomes/lysosomes. Our recent investigations into the role of these proteins have not only increased our understanding of the endolysosomal system, but also supported their major role in cell physiology and the development of different diseases.

Download Full-text

Lamp-1 does not acquire the large polylactosaminoglycans characteristic of F9 cells

Biochemical Journal ◽

10.1042/bj2960253 ◽

1993 ◽

Vol 296 (1) ◽

pp. 253-257 ◽

Cited By ~ 1

Author(s):

P A Romero ◽

T Way ◽

A Herscovics

Keyword(s):

Monoclonal Antibodies ◽

Retinoic Acid ◽

Membrane Protein ◽

Acid Treatment ◽

Datura Stramonium ◽

Lysosomal Membrane ◽

Retinoic Acid Treatment ◽

F9 Cells ◽

Lysosomal Membrane Proteins ◽

Lysosomal Membrane Protein

Although F9 cells labelled with [3H]glucosamine synthesize many glycoproteins that bind to Datura stramonium agglutinin-agarose, only a small proportion of these were immunoprecipitated with monoclonal antibodies to lamp-1 and lamp-2 (lamp = lysosomal membrane protein). Differentiation of F9 cells by retinoic acid increased labelling of all Datura stramonium-bound glycoproteins, including lamp-1 and lamp-2. Although the large polylactosaminoglycans excluded from Bio-Gel P-6 that are characteristic of F9 cells were obtained from total glycoproteins, little of these large polylactosaminoglycans was found on lamp-1 and lamp-2. There was no increase in large polylactosaminoglycans of lamp-1 and lamp-2 after retinoic acid treatment, but an increase in the size of small polylactosaminoglycans (included on Bio-Gel P-6) and tri- and tetra-antennary complex oligosaccharides. Therefore, other factors besides the expression of specific glycosyltransferases determine the extent of elongation of polylactosaminoglycans on lysosomal membrane proteins.

Download Full-text

Studies with a monoclonal antibody against activated platelets: evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation

Blood ◽

10.1182/blood.v70.3.838.bloodjournal703838 ◽

1987 ◽

Vol 70 (3) ◽

pp. 838-845 ◽

Cited By ~ 2

Author(s):

HK Nieuwenhuis ◽

JJ van Oosterhout ◽

E Rozemuller ◽

F van Iwaarden ◽

JJ Sixma

Keyword(s):

Cardiopulmonary Bypass ◽

Platelet Activation ◽

Binding Sites ◽

Double Labeling ◽

Flow Cytofluorometry ◽

Activated Platelets ◽

Normal Individuals

To define the role of activated platelets we have attempted to prepare monoclonal antibodies specific for activated platelets. The IgG2b antibody of one of the clones, designated 2.28, was studied in more detail. Native platelets from normal individuals bound 650 125I-2.28 molecules/platelet, whereas thrombin-activated platelets bound 12,600 molecules/platelet with high affinity (4.6 nmol/L). Immunoelectrophoretic analysis revealed that 2.28 reacted with a 53,000- mol wt protein. Immunocytochemistry showed that the antigen is located in a special subclass of platelet granules in unstimulated platelets and is exposed on the surface of thrombin-activated platelets. Double- labeling studies with immunogold labels disclosed simultaneous localization of 2.28 binding sites and cathepsin D in the same granules both in megakaryocytes and endothelial cells, thereby indicating that the antigen may be localized in lysosomes. By using flow cytofluorometry, in vivo platelet activation was studied in patients undergoing cardiac surgery with cardiopulmonary bypass. Increased numbers of platelets that expressed the 2.28 antigen on their surface were observed after extracorporeal perfusion. The percentage of 2.28- positive platelets in the circulation was 3.9% +/- 2.7% (SD) in controls (n = 20), 5.5% +/- 3.0% in patients (n = 10) before cardiopulmonary bypass surgery, 24.6% +/- 13.5% after the bypass, and 8.5% in two patients with acute deep venous thrombosis. These data indicate that 2.28 may serve as a useful probe of in vitro and in vivo platelet activation.

Download Full-text

Characterising the role of the lysosomal membrane proteins MFSD1 and TMEM106b in osteoclasts

Bone Abstracts ◽

10.1530/boneabs.5.p185 ◽

2016 ◽

Author(s):

David Massa Lopez ◽

Markus Damme ◽

Paul Saftig

Keyword(s):

Membrane Proteins ◽

Lysosomal Membrane ◽

Lysosomal Membrane Proteins

Download Full-text

Expansion of Myeloid Derived Suppressor Cells Contributes to Platelet Activation by L-Arginine Deprivation during SARS-CoV-2 Infection

Cells ◽

10.3390/cells10082111 ◽

2021 ◽

Vol 10 (8) ◽

pp. 2111

Author(s):

Alessandra Sacchi ◽

Germana Grassi ◽

Stefania Notari ◽

Simona Gili ◽

Veronica Bordoni ◽

...

Keyword(s):

Platelet Activation ◽

Ex Vivo ◽

Critical Role ◽

Suppressor Cells ◽

Myeloid Derived Suppressor Cells ◽

Direct Role ◽

Healthy Donors ◽

Arginine Deprivation

Massive platelet activation and thrombotic events characterize severe COVID-19, highlighting their critical role in SARS-CoV-2-induced immunopathology. Since there is a well-described expansion of myeloid-derived suppressor cells (MDSC) in severe COVID-19, we evaluated their possible role in platelet activation during SARS-CoV-2 infection. During COVID-19, a lower plasmatic L-arginine level was observed compared to healthy donors, which correlated with MDSC frequency. Additionally, activated GPIIb/IIIa complex (PAC-1) expression was higher on platelets from severe COVID-19 patients compared to healthy controls and inversely correlated with L-arginine plasmatic concentration. Notably, MDSC were able to induce PAC-1 expression in vitro by reducing L-arginine concentration, indicating a direct role of PMN-MDSC in platelet activation. Accordingly, we found a positive correlation between ex vivo platelet PAC-1 expression and PMN-MDSC frequency. Overall, our data demonstrate the involvement of PMN-MDSC in triggering platelet activation during COVID-19, highlighting a novel role of MDSC in driving COVID-19 pathogenesis.

Download Full-text