Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an “eat-me” signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface. Interestingly, tumor cell-derived extracellular vesicles (EVs) externalize PS. Recent studies have proposed PS-exposing EVs as a potential biomarker for the early detection of cancer and other diseases. However, there are confounding results regarding subtypes of PS-positive EVs, and knowledge of PS exposure on the EV surface requires further elucidation. In this study, we enriched small EVs (sEVs) and medium/large EVs (m/lEVs) from conditioned media of breast cancer cells (MDA-MB-231, MDA-MB-468) and non-cancerous cells (keratinocytes, fibroblasts). Since several PS-binding molecules are available to date, we compared recombinant proteins of annexin A5 and the carboxylated glutamic acid domain of Protein S (GlaS), also specific for PS, to detect PS-exposing EVs. Firstly, PS externalization in each EV fraction was analyzed using a bead-based EV assay, which combines EV capture using microbeads and analysis of PS-exposing EVs by flow cytometry. The bulk EV assay showed higher PS externalization in m/lEVs derived from MDA-MB-468 cells but not from MDA-MB-231 cells, while higher binding of GlaS was also observed in m/lEVs from fibroblasts. Second, using single EV flow cytometry, PS externalization was also analyzed on individual sEVs and m/lEVs. Significantly higher PS externalization was detected in m/lEVs (annexin A1⁺) derived from cancer cells compared to m/lEVs (annexin A1⁺) from non-cancerous cells. These results emphasize the significance of PS-exposing m/lEVs (annexin A1⁺) as an undervalued EV subtype for early cancer detection and provide a better understanding of PS externalization in disease-associated EV subtypes.

Activated protein C (APC) is a pleiotropic coagulation protease with anticoagulant, anti-inflammatory, and cytoprotective activities. Selective modulation of these APC activities contributes to our understanding of the regulation of these physiological mechanisms and permits the development of therapeutics for the pathologies associated with these pathways. An antibody library targeting the nonactive site of APC was generated using llama antibodies (nanobodies). Twenty-one nanobodies were identified that selectively recognize APC compared with the protein C zymogen. Overall, 3 clusters of nanobodies were identified based on the competition for APC in biolayer interferometry studies. APC functional assays for anticoagulant activity, histone H3 cleavage, and protease-activated receptor 1 (PAR1) cleavage were used to understand their diversity. These functional assays revealed 13 novel nanobody-induced APC activity profiles via the selective modulation of APC pleiotropic activities, with the potential to regulate specific mechanisms for therapeutic purposes. Within these, 3 nanobodies (LP2, LP8, and LP17) inhibited all 3 APC functions. Four nanobodies (LP1, LP5, LP16, and LP20) inhibited only 2 of the 3 functions. Monofunction inhibition specific to APC anticoagulation activity was observed only by 2 nanobodies (LP9 and LP11). LP11 was also found to shift the ratio of APC cleavage of PAR1 at R46 relative to R41, which results in APC-mediated biased PAR1 signaling and APC cytoprotective effects. Thus, LP11 has an activity profile that could potentially promote hemostasis and cytoprotection in bleedings associated with hemophilia or coagulopathy by selectively modulating APC anticoagulation and PAR1 cleavage profile.

Phosphatidylserine (PS) is a negatively charged phospholipid normally localized to the inner leaflet of the plasma membrane of cells but is externalized onto the cell surface during apoptosis as well as in malignant and infected cells. Consequently, PS may comprise an important molecular target in diagnostics, imaging, and targeted delivery of therapeutic agents. While an array of PS‐binding molecules exist, their utility has been limited by their inability to internalize diagnostic or therapeutic payloads. We describe the generation, isolation, characterization, and utility of a PS‐binding motif comprised of a carboxylated glutamic acid (GLA) residue domain that both recognizes and binds cell surface‐exposed PS, and then unlike other PS‐binding molecules is internalized into these cells. Internalization is independent of the traditional endosomal–lysosomal pathway, directly entering the cytosol of the target cell rapidly. We demonstrate that this PS recognition extends to stem cells and that GLA‐domain‐conjugated probes can be detected upon intravenous administration in animal models of infectious disease and cancer. GLA domain binding and internalization offer new opportunities for specifically targeting cells with surface‐exposed PS for imaging and delivery of therapeutics.

Phosphatidylserine (PS) is a negatively charged phospholipid normally localized to the inner leaflet of the plasma membrane of cells but is externalized onto the cell surface during apoptosis as well as in malignant and infected cells. Consequently, PS may comprise an important molecular target in diagnostics, imaging and targeted delivery of therapeutic agents. While an array of PS binding-molecules exist, their utility has been limited by their inability to recognize PS and internalize diagnostic or therapeutic payloads. We describe the generation, isolation, characterization, and utility of a PS binding motif comprised of a carboxylated glutamic acid (GLA) residue domain, that both recognizes cell surface-exposed PS and is internalized into these cells after binding. Internalization is independent of the traditional endosomal-lysosomal pathway, directly entering the cytosol of the target cell in a rapid and energy-independent fashion. We demonstrate that this PS recognition extends to extracellular vesicles and stem cells and that GLA-domain conjugated probes can be detected upon intravenous administration in animal models of infectious disease and cancer. GLA domain binding and internalization offers new opportunities for targeting specific cells for imaging and delivery of therapeutics.

Activated protein C (APC) is a pleiotropic plasma protease with diverse functions derived from its anticoagulant, anti-inflammatory, and cytoprotective activities. The selective uncoupling and/or modulation of these APC activities by antibodies may have therapeutic benefit in diseases such as traumatic bleeding, hemophilia, sepsis, and ischemia. TPP-26870 is an antibody that targets a nonactive site of APC for the selective modulation of APC activities. To optimize the potency of TPP-26870, variants with single amino acid mutation in the complementarity-determining regions (CDRs) were screened, and 21 variants with improved affinity constant were identified. Interestingly, the affinity maturation of TPP-26870 did not merely generate a panel of variants with higher potency in functional assays. Functional data demonstrated that the pleiotropic functions of APC were very sensitive to epitope-CDR interactions. Single amino acid mutations within the CDRs of TPP-26870 were sufficient to elicit divergent antagonistic and agonistic effects on the various APC functional activities. These include prolonged in vitro APC plasma half-life, increased inhibition of anticoagulant activity, and agonistic enhancement of histone H3 cleavage, while having less impact on protease-activated receptor 1 cleavage, compared with TPP-26870. This study illustrates that APC is highly sensitive to non–active site targeting that can lead to unpredictable changes in its activity profile of this pleiotropic enzyme. Furthermore, this study demonstrates the ability to modify APC functions to advance the potential development of APC-targeted antibodies as therapeutics for the treatment of diseases including trauma bleeding, hemophilia, ischemia, and sepsis.