A unique function for LRP‐1: a component of a two‐receptor system mediating specific endocytosis of plasma‐derived factor V by megakaryocytes

Abstract

Summary. Background: Factor V is endocytosed by megakaryocytes from plasma via a specific, receptor-mediated, clathrin-dependent mechanism to form the unique plateletderived 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. Results and conclusion: 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 a-granules and not destined for lysosomal degradation.

Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor–related protein 1 (LRP1) on megakaryocytes

“…Alternatively, binding of PF4 to LRP1 may prevent interaction with another ligand that would then promote proliferation. Our shRNA and anti-LRP1 blocking antibody in vitro megakaryocyte colonyformation studies data suggest that the former mechanism is more likely given that directly blocking LRP1 results in an increase in megakaryocyte colony formation.While our studies were under way, Bouchard et al reported the presence of LRP1 on human megakaryocytes and its role in the endocytosis of factor V. 18 Our studies confirm their observation that LRP1 is present on the surface of megakaryocytes. In addition, we extended these findings to show that this receptor is only transiently expressed during megakaryopoiesis.…”

“…18 Briefly, 4 ϫ 10 5 purified human CD34 ϩ cells were cultured in media consisting of IMDM with 10 mg/mL BSA (Sigma-Aldrich), 740 g/mL partially saturated transferrin (Sigma-Aldrich), 10 g/mL human insulin (Sigma-Aldrich), 40 g/mL low-density lipoprotein (Calbiochem), 2 mM L-glutamine (Fisher), and 100 ng/mL recombinant human TPO (R&D Systems). Cells were incubated at 37°C and medium was replaced on days 4, 7, 11, and 14 of culture.…”

Residual platelet factor V ensures thrombin generation in patients with severe congenital factor V deficiency and mild bleeding symptoms

“…5,6 Although megakaryocytes are capable of FV synthesis, 7,8 platelet FV originates from the plasma pool. [9][10][11] Bone marrow megakaryocytes internalize plasma FV via a specific receptormediated process 12 and store it in secretory ␣-granules. 13 After endocytosis, FV gains several unique modifications that make platelet FV structurally and functionally different from plasma FV.…”

Novel Role for Galectin-8 Protein as Mediator of Coagulation Factor V Endocytosis by Megakaryocytes

“…This suggests that Gal8 may also require a counter receptor to mediate the uptake of FV. Interestingly, Bouchard et al (22) previously proposed that FV uptake by megakaryocytes involves a two-step mechanism in which FV first binds an unidentified cell surface element, after which it is transferred to LRP-1 for receptor-mediated endocytosis. If so, our data suggest that the initial binding event may involve Gal8.…”

“…The actual mechanism behind the endocytic uptake of FV, however, is still unclear. It has been proposed that FV first binds to the cell surface via an unknown cellular component, after which it is internalized via low density lipoprotein receptor-related protein 1 (LRP-1) (22).…”