A unique function for LRP-1: a component of a two-receptor system mediating specific endocytosis of plasma-derived factor V by megakaryocytes.
BACKGROUND
Factor V is endocytosed by megakaryocytes from plasma via a specific, receptor-mediated, clathrin-dependent mechanism to form the unique platelet-derived FV pool.


OBJECTIVE
The role of low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1), or a related family member, in FV endocytosis by megakaryocytes was examined because of its known interactions with other proteins involved in hemostasis.


METHODS
LRP-1 expression by megakaryocytes and its functional role in FV endocytosis was confirmed using reverse transcription polymerase chain reaction (RT-PCR) and specific antibodies. FV binding to megakaryocytes was performed under Ca(2+)-free conditions to quantify binding in the absence of endocytosis.


CONCLUSIONS
Cell surface expression of LRP-1 by CD34+ ex vivo-derived megakaryocytes and the megakaryocyte-like cell line CMK was confirmed using anti-LRP-1 antibodies and was consistent with the detection of LRP-1 message in these cells. All cells capable of endocytosing FV expressed LRP-1. Anti-LRP-1 antibodies and receptor-associated protein (RAP), a known antagonist of LDL receptor family members, displaced only 50% of the [(125)I]FV bound to megakaryocytes. FV binding to megakaryocytes showed positive cooperativity (Hill coefficient = 1.92 +/- 0.18) that was substantially reduced in the presence of RAP (1.47 +/- 0.26). As FV endocytosis is specific to this cofactor, a model is hypothesized where FV binding to a specific receptor facilitates binding and endocytosis of a second FV molecule by LRP-1, or a related family member. These combined observations describe a unique role for LRP-1 in endocytosis of a coagulation protein trafficked to alpha-granules and not destined for lysosomal degradation.
Bouchard
B A
BA
Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT, USA. beth.bouchard@uvm.edu
Meisler
N T
NT
Nesheim
M E
ME
Liu
C-X
CX
Strickland
D K
DK
Tracy
P B
PB
D002118:Calcium / Q000494:pharmacology; D002460:Cell Line / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D004705:Endocytosis / Q000502:physiology*; D005165:Factor V / Q000378:metabolism* / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006801:Humans; D006834:Hydrazines / Q000493:pharmacokinetics; D026563:LDL-Receptor Related Protein-Associated Protein / Q000494:pharmacology; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D008533:Megakaryocytes / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
C417561:Alexa 488 hydrazide; D005456:Fluorescent Dyes; D006834:Hydrazines; D026563:LDL-Receptor Related Protein-Associated Protein; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D012333:RNA, Messenger; C067297:8-aminohexylamino cAMP; D005165:Factor V; D000242:Cyclic AMP; D002118:Calcium