Molecular Weight Dependency in the Uptake of Fractionated [³H]Heparin in Isolated Rat Kupffer Cells

Abstract

The uptake of low molecular weight fractionated [³H]heparin (LMWFH, 10000 Da) was compared with that of high molecular weight fractionated [³H]heparin (HMWFH, 23000 Da) in isolated rat Kupffer cells. Several heparin analogs, including HMWFH and ligands of scavenger receptors, inhibited both the surface binding and internalization of LMWFH, suggesting the involvement of scavenger receptors in the uptake of LMWFH in isolated rat Kupffer cells as well as HMWFH, in spite of a large difference in molecular weight. Metabolic inhibitors(2, 4-dinitrophenol and rotenone), receptor-mediated and adsorptive endocytosis of polypeptides (phenylarsine oxide) and phagocytosis inhibitors (cytochalasine B and colchicine) did not inhibit the internalization of LMWFH. These results suggest that the scavenger receptor-mediated uptake of LMWFH is ATP-independent and different from receptor-mediated and adsorptive endocytosis of polypeptides and phagocytosis, in agreement with our previous results for HMWFH. The equilibrium binding of LMWFH to Kupffer cells was concentration-dependent with the dissociation constant (K_d) of 50 nM and maximum binding capacity (B_max) of 2.3 pmol/10⁶ cells. The dissociation constant of LMWFH was an order of magnitude larger than that of HMWFH (5.7 nM), suggesting a decrease in binding affinity to scavenger receptors with a decrease in the molecular weight of fractionated heparin. It was also shown that LMWFH is internalized by scavenger receptors according to first-order kinetics with an apparent internalization rate constant (k_{int, app}) of 0.0053 min^-1, which is about half that for HMWFH (0.0118 min^-1). Molecular weight thus appears to be one of dominant factors determining the uptake of fractionated heparin by scavenger receptors in Kupffer cells, and may partly explain the reported lower hepatic uptake of low molecular weight heparin than that of unfractionated heparin.