Lactadherin, a glycoprotein of the milk-fat globule membrane, contains tandem C domains with homology to discoidin-type lectins and to membrane-binding domains of blood-clotting factors V and VIII. We asked whether the structural homology confers the capacity to compete for the membrane-binding sites of factor VIII and factor V and to function as an anticoagulant. Our results indicate that lactadherin competes efficiently with factor VIII and factor V for binding sites on synthetic phosphatidylserine-containing membranes with half-maximal displacement at lactadherin concentrations of 1 to 4 nM. Binding competition correlated to functional inhibition of factor VIIIa-factor IXa (factor Xase) enzyme complex. In contrast to annexin V, lactadherin was an efficient inhibitor of the prothrombinase and the factor Xase complexes regardless of the degree of membrane curvature and the phosphatidylserine content. Lactadherin also inhibited the factor VIIa-tissue factor complex efficiently whereas annexin V was less effective. Because the inhibitory concentration of lactadherin was proportional to the phospholipid concentration, and because lactadherin was not an efficient inhibitor in the absence of phospholipid, the major inhibitory effect of lactadherin relates to blocking phospholipid sites rather than forming inhibitory protein-protein complexes. Lactadherin was also an effective inhibitor of a modified whole blood prothrombin time assay in which clotting was initiated by dilute tissue factor; 60 nM lactadherin prolonged the prothrombin time 150%
IntroductionLactadherin is a 47 000-Da molecular weight (MW) glycoprotein of milk-fat globules. It has also been known as PAS-6/7, indicating the 2 glycosylation variants, 1 bovine-associated mucoprotein, BA-46, P47, and MFG-E8. 2 Lactadherin has a domain structure of EGF1-EGF2-C1-C2 in which EGF indicates epidermal growth factor homology domains, and the C domains share homology with the discoidin family, including the lipid-binding C domains of blood coagulation factor VIII and factor V. 2 The second EGF domain displays an Arg-Gly-Asp motif, 3 which binds to the ␣ v  3 and ␣ v  5 integrins. 1,[4][5][6] The second C domain binds to phospholipids. 6 In milk-fat globules, lactadherin lines the surface of the phospholipid bilayer that surrounds the central triglyceride droplet, apparently stabilizing the bilayer. 7 Lactadherin decreases the symptoms of rotavirus infection in infants, possibly by binding to rotavirus particles via carbohydrate moieties. 8 In tissue sections, lactadherin is found localized on the apical portion of secretory epithelium in the breast. 7 Abundant expression by breast carcinoma tissue makes lactadherin a potential target for antigen-guided radiation therapy. 9 Lactadherin is also found on the apical surface of epithelia in the biliary tree, the pancreas, and sweat glands 7 and is synthesized by aortic medial smooth muscle cells. 10 Function in these tissues remains unknown. Lactadherin has been identified as a zona pellucida-binding protein on the a...