Platelets express three different splice variants of ApoER2 that are all involved in signaling.
BACKGROUND
beta2-Glycoprotein I is the most relevant antigen in antiphospholipid syndrome. We have shown that binding of dimerized beta2-GPI to platelets via ApoER2' sensitizes platelets for second activating stimuli.


OBJECTIVE
Determine the region of ApoER2 involved in the binding of dimeric beta2-GPI.


METHODS
Cultured human megakaryocytes (MK) and three different human megakaryocytic cell lines were used for mRNA isolation to clone and express recombinant soluble platelet ApoER2. Domain deletion mutants of ApoER2 were constructed to identify the binding site for dimeric beta2-GPI. The presence of ApoER2 splice variants in platelets was demonstrated by immuno-blotting.


RESULTS
Three different mRNA splice variants were isolated from all four types of megakaryocytic cells used. Sequence analysis identified the splice variants: (i) shApoER2Delta5 lacking low-density lipoprotein (LDL) binding domains 4, 5 and 6; (ii) shApoER2Delta4-5 lacking LDL binding domains 3, 4, 5, 6 and (iii) shApoER2Delta3-4-5 lacking LDL binding domains 3, 4, 5, 6 and 7. The presence of three splice variants of ApoER2 on platelets was confirmed by immuno-blotting, with ApoER2Delta4-5 being the most abundantly expressed splice variant. Upon stimulation with dimeric beta2-GPI, all three splice variants were translocated to the cytosol; however, ApoER2Delta4-5 translocation was most prominent. Dimeric beta2-GPI binds platelet ApoER2 variants via LDL-binding domain 1.


CONCLUSIONS
Three different ApoER2 mRNA splice variants were isolated from MK and platelets express all three splice variants. All splice variants were shown to be functional by translocation upon stimulation with dimeric beta2-GPI. All three splice variants express LDL-binding domain 1.
Pennings
M T T
MT
Laboratory of Thrombosis and Haemostasis, Department of Clinical Chemistry and Hematology, UMCU Utrecht, Utrecht, The Netherlands.
Derksen
R H W M
RH
Urbanus
R T
RT
Tekelenburg
W L
WL
Hemrika
W
W
de Groot
P G
PG
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001792:Blood Platelets / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D006801:Humans; D066298:In Vitro Techniques; D026502:LDL-Receptor Related Proteins; D008533:Megakaryocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D012333:RNA, Messenger / Q000097:blood / Q000235:genetics; D018110:Receptors, Lipoprotein / Q000097:blood* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D053482:beta 2-Glycoprotein I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D026502:LDL-Receptor Related Proteins; D012333:RNA, Messenger; D018110:Receptors, Lipoprotein; D011994:Recombinant Proteins; D053482:beta 2-Glycoprotein I; C098773:low density lipoprotein receptor-related protein 8