Lactadherin promotes microvesicle clearance to prevent coagulopathy and improves survival of severe TBI mice

Abstract

Coagulopathy is common in patients with traumatic brain injury (TBI) and predicts poor clinical outcomes. We have shown that brain-derived extracellular microvesicles, including extracellular mitochondria, play a key role in the development of TBI-induced coagulopathy. Here, we further show in mouse models that the apoptotic cell-scavenging factor lactadherin, given at a single dose of 400 μg/kg 30 minutes before (preconditioning) or 30 minutes after cerebral fluid percussion injury, prevented coagulopathy as defined by clotting time, fibrinolysis, intravascular fibrin deposition, and microvascular bleeding of the lungs. Lactadherin also reduced cerebral edema, improved neurological function, and increased survival. It achieved these protective effects by enhancing the clearance of circulating microvesicles through phosphatidylserine-mediated phagocytosis. Together, these results identify the scavenging system for apoptotic cells as a potential therapeutic target to prevent TBI-induced coagulopathy and improve the outcome of TBI.

Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

Figure 1.

Lactadherin reduced TBI-induced coagulopathy and vasculopathy. Clotting time (A) and plasma d-dimer (B) of sham mice and FPI mice preconditioned with either PBS or lactadherin (n = 16, 1-way ANOVA). Representative images of PTAH-stained lungs from a sham mouse (C) and FPI mice receiving PBS (D, red arrow indicates extensive blue PTAH stain for intravascular fibrin deposition) or lactadherin (E, arrow indicates significantly reduced intravascular PTAH stain, bar in C-E = 10 μm). Representative images of H&E-stained lungs from a sham mouse (F) and FPI mice receiving PBS (G) or lactadherin (H, bar in F-H = 20 μm). Perivascular space (denoted with an asterisk) is enlarged with extravascular accumulation of erythrocytes (hemorrhage) in the lungs of TBI mice receiving PBS. The C-H images are representatives of the 26 mice examined.

Figure 2.

Lactadherin reduced cerebral edema and improved outcomes of FPI. (A) Representative topical and cross-sectional views of brains from a sham mouse (left) and from FPI mice preconditioned with PBS (middle) or lactadherin (right). (B-D) Representative fluorescence images of brain cryosections from a sham mouse and mice receiving lactadherin or PBS. (E) Levels (OD unite) of Evans blue in the supernatants of tissue homogenates from FPI mice receiving different treatments (n = 9, 1-way ANOVA). (F) Platelet counts at the baseline (white bars) and 3 hours after FPI (black bars) of sham mice and TBI mice preconditioned with PBS or lactadherin (n = 16, 1-way ANOVA). A Kaplan-Meier survival analysis (G, n = 18; P < .005 vs mice received PBS) and modified Neurological Severity Score (H, n = 18, 1-way ANOVA) of sham and FPI mice receiving PBS or lactadherin. (I) A Kaplan-Meier survival analysis of BDMV-infused and control mice (P < .005 vs mice that received PBS).

Figure 3.

Lactadherin reduced MPs and its deficiency increased TBI-induced BDMV release and coagulopathy. (A) A schematic illustration of lactadherin-mediated MV clearance. (B) Levels of circulating NSE+ (scale on the left) and mtMVs (MitoTracker Green+, scale on the right) measured 3 hours after injury of sham mice and FPI mice preconditioned with PBS or lactadherin (n = 15, 1-way ANOVA). (C) Levels of circulating NSE+ MVs (scale on the left) and mtMVs (scale on the right) in noninjured mice infused with 1.5 × 107/mouse of BDMVs followed by lactadherin or PBS (n = 12, 1-way ANOVA). Plasma samples from lactadherin−/− mice and their wild-type littermates were examined for dynamic changes in plasma levels of (D) NSE+ MVs, (E) annexin V-binding MVs, (F) clotting time, and (G) plasma levels of d-dimer (n = 24, repeated-measures ANOVA; *P < .001).

Figure 4.

Lactadherin promoted BDMV clearance through phagocytosis. (A) Plasma levels of biotinylated BDMVs infused into noninjured mice that also received lactadherin or PBS (n = 12, paired t test; *P < .01). (B) Representative images of liver sections from mice infused with (1) PBS, (2) biotinylated BDMVs, and (3) biotinylated BDMVs with lactadherin were stained with horseradish peroxidase–conjugated streptavidin (bar = 20 μm, biotinylated BDMVs are stained in brown color); (4) integrated optical densities from scans from multiple mice (n = 9, 1-way ANOVA). (C) Bright field (left) and fluorescence images (right) of CD45+ monocytes and MitoTracker Green+ mtMVs (right overlay) detected by imaging flow cytometry. (D) mtMV binding to CD45+ monocytes in the absence and presence of PS, CL, and phosphatidylcholine (PC; all 200 nM, n = 12, 1-way ANOVA). (E) Blocking the binding of fluorescein isothiocyanate-lactadherin to BDMVs by 100-fold excess of either unlabeled lactadherin or annexin V (n = 24, 1-way ANOVA).

Figure 5.

BDMV clearance required the integrin-binding domain of lactadherin. (A) The transmigration of PKH26-labeled BDMVs through activated endothelial cells in the presence and absence of lactadherin and monocytes (n = 15, 1-way ANOVA). C57BL/6J mice (n = 16) were preconditioned with an equal molar concentration of either lactadherin or its C1C2 domain (PBS as control) before being subjected to FPI. They were then examined for plasma levels of (B) NSE+ MVs, (C) mtMVs, (D) clotting time, and (E) plasma levels of d-dimer. Data presented in C-F were analyzed with repeated measures ANOVA, *P < .01 and **P < .001 vs PBS-injected mice.

Figure 6.

Plasma lactadherin and its therapeutic potential for TBI-induced coagulopathy. (A) Plasma levels of lactadherin in sham mice and in FPI mice preconditioned with lactadherin or PBS (n = 15, repeated measures ANOVA; *P < .001 vs sham). Plasma levels of (B) NSE+ MVs and (C) mtMVs in C57Bl/6J mice subjected to FPI and that received 400 μg/kg lactadherin or PBS 30 minutes after injury (sham mice as control, repeated measures ANOVA, *P < .01, #P < .05). (D) A comparison in plasma NSE+ (top) and mtMVs (bottom) between mice preconditioned with lactadherin (a) and those receiving lactadherin after the injury (b; n = 32, 1-way ANOVA). (E) Clotting time and (F) plasma levels of d-dimer in mice receiving lactadherin 30 minutes after TBI (n = 21, repeated measures ANOVA, *P < .01 vs PBS-infused mice).