HYDROPHOBIC AMINO-ACID-RESIDUES OF HUMAN ANTICOAGULATION PROTEIN-C THAT CONTRIBUTE TO ITS FUNCTIONAL BINDING TO PHOSPHOLIPID-VESICLES

The contributions to functional phospholipid (PL) binding of the cluster of amino acid side chains of human protein C (PC) comprising F-4, L(5), and L(8) have been assessed by construction of mutants of PC and activated protein C (APC) designed wherein a hydrophilic side chain replaced the wild-type hydrophobic groups at these positions. The PL-dependent plasma-based anticoagulant activities of [F(4)Q]r-APC and [L(8)Q]r-APC were severely reduced to 5% and <2%, respectively, of wild-type r-APC. Activity losses of the mutants toward inactivation of coagulation factor VIII, measured in the complete in vitro tenase system, have also been observed. As evidenced through Ca2+-induced intrinsic fluorescence changes, both [F(4)Q]r-PC and [L(8)Q]r-PC were able to adopt Ca2+-dependent conformations that appeared similar to that of wtr-PC, ruling out shortcomings associated with such Ca2+-induced transitions as the basis for their anticoagulant activity losses. However, despite this, [L(8)Q]r-PC showed greatly defective macroscopic binding properties to PL vesicles, as did to a lesser extent [F(4)Q]r-PC. These findings were similar to those reported previously for [L(5)Q]r-PC/APC [Zhang, L., and Castellino, F. J. (1994) J. Biol. Chern. 269, 3590-3595]. We thus propose that the PL-dependent activity losses of these mutants are related to their suboptimal binding to PL or to their misorientation on the PL surface leading to poor alignment of the active sites of the r-APC mutants with the complementary cleavage sites on fVIII/fVIIIa and fV/fVa. These investigations confirm the importance of hydrophobic components of functional PL binding by PC and APC and implicate L(8), along with L(5), as the principal amino acid residue involved in these interactions. F-4 has a lesser direct involvement in this regard but may contribute to proper alignment of PC and APC on the PL surface.