The factor V light chain mediates the binding and endocytosis of plasma-derived factor V by megakaryocytes.

Plasma- and platelet-derived factor (F) Va are essential for thrombin generation catalyzed by Prothrombinase assembled on the activated platelet surface [1]. While both the plasma- and platelet-derived molecules can form a functional complex, several studies demonstrate that the two cofactor pools are physically and functionally distinct [2–7]. Despite these differences, the entire platelet-derived pool of the procofactor, FV, is endocytosed from the plasma by megakaryocytes [4,8] via a two-receptor system consisting of a specific, unknown FV receptor and low density lipoprotein (LDL) receptor-related protein-1 (LRP-1) [9], an endocytic receptor belonging to a super-family of proteins related to the LDL receptor [10]. Flow cytometric analyses and fluorescence microscopy indicate that all megakaryocytes express LRP-1 [9,11]. However, cells that endocytose FV must express both LRP-1 and an unidentified, specific FV receptor. FV binds to the specific FV receptor in a Ca2+-independent manner [9]. This binding interaction facilitates binding of another FV molecule to LRP-1, which mediates endocytosis through Ca2+- [9] and clathrin-dependent [8] mechanisms. Endocytosed FV is modified intracellularly to form the unique platelet-derived cofactor molecule [2–7] and trafficked to α-granules. The platelet-derived cofactor molecule plays the predominant hemostatic role at sites of vascular injury [12]; therefore, elucidation of the mechanisms regulating its endocytosis, modification and storage in α-granules will provide insight into the events leading to formation of a fibrin clot. The goal of these studies was to identify the specific region(s) of FV involved in its interactions with the membrane receptors that mediate its endocytosis. Plasma FV is converted to FVa via cleavage by thrombin at Arg709, Arg1018 and Arg1545, to release the large central B domain and form the active cofactor [1]. FVa consists of a heavy chain (residues 1–709) and light chain (residues 1546– 2196) non-covalently linked with Ca2+ (Fig. 1A). Endocytosis assays were performed using iodine125-labeled FV (125I-FV), which was prepared and characterized as described previously [9]. Pre-incubation of megakaryocyte-like CMK cells with 50-fold molar excesses of unlabeled, human and bovine FV and FVa inhibited endocytosis of 125I-FV nearly 100% (Fig. 1B). Similarly, a recombinant human FV single chain construct missing most of the B-domain (recombinant FVdel811-1491; rFV-DT) [13] (see Fig. 1A) inhibited endocytosis by 86% ± 0%, suggesting that the B domain is not involved. Pre-incubation of cells with the isolated light chain [14] inhibited endocytosis of 125I-FV by 84% ± 2%. In contrast, the isolated heavy chain [14] had a small effect on endocytosis (19% ± 15% inhibition). Inhibition of 125I- FV endocytosis by various anti-FV antibodies provided and characterized by Dr Kenneth Mann (University of Vermont, Burlington, VT, USA) was determined in parallel. Antibodies directed against the FV light chain (E9, #2 and #9) or FV heavy chain (#17) [15] were pre-incubated with 125I-FV prior to the addition of CMK cells. Consistent with the results described above, anti-FV light chain antibodies, E9 and anti-FV#2 inhibited FV endocytosis by megakaryocytes by greater than 80% (Fig. 1C). Anti-FV#9 as well as anti-FV#17 had little or no effect on FV endocytosis (≤ 20% inhibition). Fig. 1 The FV light chain mediates its binding to and endocytosis by megakaryocytes. (A) The domain structures of FV, rFV-DT and FVa. (B) CMK cells (1 & times;106 mL−1) were pre-incubated (1 h, 37 °C) with unlabeled, human and bovine ... One of the anti-FV light chain antibodies, E9, inhibited 125I-FV endocytosis to the same extent as unlabeled FV. As the two-receptor model of endocytosis dictates that FV binding to the specific FV receptor is necessary for and precedes FV binding to LRP-1 [9], additional experiments were performed in an attempt to define which of the receptor-FV interactions E9 inhibits. Previously, it was demonstrated that 125I-FV binding was inhibited ∼50% by receptor-associated protein, a known ligand of LRP-1 [9]. In contrast, unlabeled FV inhibited binding by ∼90%. These experiments were performed in the absence of Ca2+, which is essential for FV endocytosis but not binding [9]. Similar experiments were performed using E9. Inhibition of FV binding by ∼50% would suggest that E9, like RAP, is inhibiting FV binding to LRP-1 alone. One of two scenarios is possible if complete inhibition is observed: E9 is inhibiting binding to the specific FV receptor alone and, therefore, binding to LRP-1 cannot occur; or, similar to the observations made using FV [9], E9 is directly inhibiting binding of 125I-FV to both receptors. As the E9 concentration increased, steady state binding of 125I-FV decreased with inhibition, nearing 100% at a 3.3-fold molar excess of E9 (Fig. 1D). These overall data suggest that amino acid residues in the FV light chain mediate the binding to and endocytosis of FV by megakaryocytes and are likely to encompass all or part of the E9 epitope. Experiments are currently being performed to identify this epitope. As the FV heavy chain and B domain appear to have no role in the endocytic process, our combined data suggest that the FV light chain mediates FV's interactions with both receptors at the megakaryocyte cell surface.