Engineered Fluorescent Variants of Lactadherin C2 Domain for Phosphatidylserine Detection in Flow Cytometry

Phosphatidylserine (PS) is an essential phospholipid and an emerging biomarker involved in key biological processes. While annexin V (axV) is the most widely used tool for PS detection, its calcium-dependent binding and other limitations have spurred interest in alternative probes. The lactadherin C2 domain (lactC2) offers a promising alternative, addressing many of the drawbacks associated with axV. However, its broader adoption has been hindered by challenges in production and modification for convenient experimental use. Here, we demonstrate the successful in-house engineering of fully functional recombinant bovine lactC2-based fluorescent sensors and compare their key parameters to axV probes. We show that mNeonGreen-lactC2 fusion exhibits calcium-independent binding with a comparable dissociation constant for 20% PS liposomes. We also demonstrate the detrimental effects of primary amine modification on lactC2's PS binding efficiency, suggesting the preferential use of fluorescent protein fusion or alternative approaches. Finally, we show that unlike full-length lactadherin or axV, lactC2 inhibited thrombin generation only at high concentrations (>250 nM) in coagulation assays. These findings establish recombinant lactC2 as a versatile and promising PS sensor, with potential applications in experimental settings where axV might be unsuitable.