HUMAN BETA-2-GLYCOPROTEIN-I BINDS TO ENDOTHELIAL-CELLS THROUGH A CLUSTER OF LYSINE RESIDUES THAT ARE CRITICAL FOR ANIONIC PHOSPHOLIPID-BINDING AND OFFERS EPITOPES FOR ANTI-BETA-2-GLYCOPROTEIN-I ANTIBODIES

beta 2-Glycoprotein I (beta 2GPI) is a phospholipid-binding protein re cognized by serum autoantibodies from the anti-phospholipid syndrome b oth in cardiolipin-and beta 2GPI-coated plates, We found that: 1) reco mbinant wild-type beta 2GPI bound to HUVEC and was recognized by both human monoclonal IgM and affinity-purified polyclonal IgG anti-beta 2G PI anti-phospholipid syndrome Abs; and 2) a single amino acid change f rom Lys(286) to Glu significantly reduced endothelial adhesion, Double and triple mutants (from Lys(284,287) to Glu(284,287), from Lys(286,2 87) to Glu(286,287) and from Lys(284,286,287) to Glu(284,286,287)) com pletely abolished endothelial binding. A synthetic peptide (P1) spanni ng the sequence Glu(274)-Cys(288) of the beta 2GPI fifth domain still displayed endothelial adhesion. another peptide (P8), identical with P 1 except that Cys(281) and Cys(288) were substituted with serine resid ues, did not bind to HUVEC. Anti-beta 2GPI Abs, once bound to P1 adher ed to HUVEC, induced E-selectin expression and cap-regulated IL-6 secr etion. Control experiments conducted with Irrelevant Abs as well as wi th the PS peptide did not show ang endothelial Ab binding nor E-select in and IL-6 modulation Our results suggest that: 1) beta 2GPI binds to endothelial cells through its fifth domain; 2) the major phospholipid -binding site that mediates the binding to anionic phospholipids is al so involved in endothelial binding; 3) HUVEC provide a suitable surfac e for beta 2GPI binding comparable to that displayed bg anionic phosph olipids dried on microtiter wells; and 4) the formation of the complex between beta 26PI and the specific Abs leads to endothelial activatio n in vitro.