LDL Receptor Mediates Effective Endocytosis of Activated Factor V
| Autor: | Koen Mertens, Carmen van der Zwaan, Sigrid D. Roosendaal, Kees W. Rodenburg, Alexander B. Meijer, Vincent Limburg |
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| Rok vydání: | 2007 |
| Předmět: |
Yellow fluorescent protein
biology Endosome Chemistry media_common.quotation_subject Immunology Endocytic cycle food and beverages Lipoprotein receptor-related protein Cell Biology Hematology Endocytosis Biochemistry Molecular biology LDL receptor biology.protein lipids (amino acids peptides and proteins) Receptor Internalization media_common |
| Zdroj: | Blood. 110:1766-1766 |
| ISSN: | 1528-0020 0006-4971 |
| DOI: | 10.1182/blood.v110.11.1766.1766 |
| Popis: | The platelet α-granules contain a unique pool of partially activated factor V, which has been suggested to originate form megakaryocyte endocytosis of factor V (FV) from plasma. The presence of activated FV (FVa) in plasma itself should be tightly controlled as dysfunction therein may predispose to thrombotic disorders. Previously, we have reported that FVa but not FV can bind the low-density lipoprotein receptor related protein (LRP). This multifunctional receptor can bind a multitude of ligands including FV’s homologue factor VIII (FVIII). We now investigated whether FV or FVa can, like FVIII, also bind the more restricted LDL receptor (LDLR). To this end, the endocytosis of FV and FVa by CHO cells expressing LDLR (CHO-LDLR+ cells) was assessed utilizing confocal microscopy. In the experimental setup, FV and FVa were visualized employing immuno-fluorescence staining techniques. The results showed that within 10 minutes after addition of FVa, fluorescent spots appeared inside the CHO-LDLR+ cells. In contrast, no fluorescent spots were observed after 10 minutes of incubation with FV. These observations suggest that FVa but not FV effectively interacts with the LDLR expressing CHO cells. We then assessed whether FVa can compete with FVIII for endocytosis by CHO-LDLR+ cells. In the presence of an equimolar amount of FVa and a FVIII derivative (FVIIIYFP) containing yellow fluorescent protein, both proteins were detected within the same vesicles inside the CHO-LDLR+ cells. Employing co-localization studies, we established that these vesicles represented early endosomes. In the presence of an access of FVa, however, the yellow fluorescence of FVIIIYFP was no longer observed. These results demonstrate that FVa can block the FVIII endocytosis by CHO-LDLR+ cells. The observations together suggest that LDLR can not only bind FVIII but also FVa. In line with this notion, we established that CHO-ldlA cells, which lack functional expression of LDLR, were unable to internalize neither FVa nor FVIIIYFP. In addition, the ligand-binding clusters II and cluster IV of LRP effectively inhibited the endocytic uptake of FVa by CHO-LDLR+ cells. This implies that structural elements of FVa involved in LRP binding may overlap with those required for LDLR dependent internalization. Our results suggest a so far unidentified role for members of the LDL receptor family in the regulation of FVa in plasma. |
| Databáze: | OpenAIRE |
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