Lactadherin C2 Domain Exhibits Ptd-L-Ser Specificity and Anticoagulant Properties Distinct From Homologous Factor VIII C2 Domain and Full-Length Lactadherin
Authors: Novakovic Valerie A , Gilbert Eugene R , Shi Jialan , Gilbert Gary E. 
Published: October 2019

Abstract
Abstract 1105

Background:
Lactadherin (aka mfg-e8) is a milk-fat globule membrane protein with a domain structure of EGF1-EGF2-C1-C2, where the lectin-like C1 and C2 domains are homologous to the membrane binding domains of factor VIII and factor V. Like factor VIII and factor V, lactadherin exhibits calcium-independent membrane binding that is selective for phosphatidyl-L-serine (Ptd-L-Ser). Lactadherin also binds preferentially to convex membranes, competes efficiently for binding sites of factor VIII and factor V, and can function as an anticoagulant via competition for these binding sites. On stressed endothelial cells lactadherin binds to filopodia and the cell margins, identifying sites that have exposed Ptd-L-Ser and support assembly of the prothrombinase complex.
The crystallographic structure of the lactadherin C2 domain (Lact-C2) and structure-function studies have shown that membrane binding is mediated by a longer β-hairpin turn, with different residues than fVIII-C2 and fV-C2. Further, we have shown that Lact-C2 maintains specificity for phosphatidylserine, in contrast to fVIII-C2, and that fluorescent fusion proteins containing Lact-C2 can be used as intracellular phosphatidylserine probes (Yeung et al. Science 2008;319:210). However, the extent to which Lact-C2 retains the membrane binding and anticoagulant properties of full-length lactadherin, has not been studied.

Methods:
Lact-C2 was produced in E. coliand purified by metal ion chromatography followed by gel filtration. Competition experiments were performed by flow cytometry using phospholipid bilayers supported by glass microspheres to determine Lact-C2's ability to block binding sites of FITC-labeled lactadherin, or fluorescein-labeled factor VIII and factor V. Lact-C2 was also labeled with FITC to measure its binding to sonicated or 100 nm diameter, extruded vesicles with varying PS content in order to assess Ptd-L-Ser selectivity and membrane curvature sensitivity. Two-step amidolytic factor Xase and prothrombinase assays were used to assess the ability of Lact-C2 to block activity. Fluorescence microscopy experiments were used to compare the binding of Alexa 647-labeled Lact-C2 vs. FITC-labeled lactadherin on staurosphorine-treated HeLa cells.

Results:
Lact-C2 showed stereospecific binding to Ptd-L-Ser vs. Ptd-D-Ser in vesicles of 4% and 10% PS. Lact-C2 was sensitive to vesicle curvature, detecting as little as 1% Ptd-L-Ser on sonicated vesicles but requiring 4% Ptd-L-Ser on extruded vesicles. Lact-C2 competed for 89% of lactadherin binding sites and 84% of factor VIII binding sites. Inhibition of factor Xase activity plateaued at 89% reduction vs. >99% reduction for lactadherin. Lact-C2 also competed for 61% of factor V binding sites corresponding to an 82% reduction in prothrombinase activity. We are currently comparing the distribution of binding sites for Lact-C2 vs. lactadherin on stressed HeLa cells, with preliminary data showing distinct, but overlapping, binding site distribution.

Discussion:
Lact-C2 exhibits stereospecific Ptd-L-Ser binding and convex curvature preference similar to full-length lactadherin. Lact-C2 contrasts with fVIII-C2 in Ptd-L-Ser specificity and capacity to compete with factor VIII and inhibit factor Xase activity and prothrombinase activity.
These results provide a framework for interpreting experiments in which Lact-C2 is used as an anticoagulant or as a calcium-independent probe for exposed membrane Ptd-L-Ser. Lact-C2 is able to bind to only a subset of lactadherin binding sites, highlighting the importance of the lactadherin C1 domain for high affinity binding and underscoring the largely unappreciated complexity of phospholipid membrane binding sites.

Disclosures:
Shi: Brigham and Women's Hospital: Use of Lactadherin to detect phosphataidylserine, Use of Lactadherin to detect phosphataidylserine Patents & Royalties.