Abstract
In the present study, the interaction between the endocytic receptor low density lipoprotein receptor-related protein (LRP) and coagulation factor VIII (FVIII) was investigated. Using purified components, FVIII was found to bind to LRP in a reversible and dose-dependent manner (K(d) approximately 60 nM). The interaction appeared to be specific because the LRP antagonist receptor-associated protein readily inhibited binding of FVIII to LRP (IC(50) approximately 1 nM). In addition, a 12-fold molar excess of the physiological carrier of FVIII, i.e. von Willebrand factor (vWF), reduced the binding of FVIII to LRP by over 90%. Cellular degradation of (125)I-labeled FVIII by LRP-expressing cells ( approximately 8 fmol/10(5) cells after a 4.5-h incubation) was reduced by approximately 70% in the presence of receptor-associated protein. LRP-directed antibodies inhibited degradation to a similar extent, indicating that LRP indeed contributes to binding and transport of FVIII to the intracellular degradation pathway. Degradation of FVIII was completely inhibited by vWF. Because vWF binding by FVIII involves its light chain, LRP binding to this subunit was studied. In ligand blotting experiments, binding of FVIII light chain to LRP could be visualized. More detailed analysis revealed that FVIII light chain interacts with LRP with moderate affinity (k(on) approximately 5 x 10(4) M(-1) s(-1); k(off) approximately 2.5 x 10(-3) s(-1); K(d) approximately 50 nM). Furthermore, experiments using recombinant FVIII C2 domain showed that this domain contributes to the interaction with LRP. In contrast, no association of FVIII heavy chain to LRP could be detected under the same experimental conditions. Collectively, our data demonstrate that in vitro LRP is able to bind FVIII at the cell surface and to mediate its transport to the intracellular degradation pathway. FVIII-LRP interaction involves the FVIII light chain, and FVIII-vWF complex formation plays a regulatory role in LRP binding. Our findings may explain the beneficial effect of vWF on the in vivo survival of FVIII.
Highlights
In the present study, the interaction between the endocytic receptor low density lipoprotein receptor-related protein (LRP) and coagulation factor VIII (FVIII) was investigated
Activated FVIII consists of the A2 domain, which is noncovalently associated with the metal-ion linked A1/A3-C1-C2 dimer, whereas modified Eagle’s medium:F12; FVIII, factor VIII; GST, glutathione S-transferase; RAP, receptor-associated protein; vWF, von Willebrand factor; SPR, surface plasmon resonance
We investigated the possibility that FVIII is recognized by the multifunctional receptor LRP
Summary
Materials—Glutathione-Sepharose 4B and protein A-Sepharose CL-4B were from Amersham Pharmacia Biotech. 125I-labeled FVIII (20 nM) was added in a volume of 200 l in DMEM:F12 medium containing 1% (w/v) bovine albumin and 5 mM CaCl2. 125I-labeled FVIII was added in the presence of vWF (500 nM), GST-RAP (1 M), or protein A-Sepharose purified polyclonal antibodies directed against LRP (0.9 mg/ml). Incubation was allowed to proceed for another 4.5 h at 37 °C in a volume of 200 l of DMEM:F12 medium containing 1% (w/v) bovine albumin and 5 mM CaCl2, in the presence of freshly added competitor where appropriate. The blots were subsequently incubated with FVIII light chain (50 nM) in the absence or presence of GST-RAP (125 nM) in the same buffer containing 5 mM CaCl2 for 16 h at room temperature. Binding was visualized using 3,3-diaminobenzidine tablets (Ken-En-Tec, Copenhagen, Denmark)
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