Anticoagulation targeting membrane-bound anionic phospholipids improves outcomes of traumatic brain injury in mice.

Severe traumatic brain injury (TBI) often causes an acute systemic hypercoagulable state that rapidly develops into consumptive coagulopathy. We have recently demonstrated that TBI-induced coagulopathy (TBI-IC) is initiated and disseminated by brain-derived extracellular vesicles (BDEVs) and propagated by extracellular vesicles (EVs) from endothelial cells and platelets. Here, we present results from a study designed to test the hypothesis that anticoagulation targeting anionic phospholipid-expressing EVs prevents TBI-IC and improves the outcomes of mice subjected to severe TBI. We evaluated the effects of a fusion protein (ANV-6L15) for improving the outcomes of TBI in mouse models combined with in vitro experiments. ANV-6L15 combines the phosphatidylserine (PS)-binding annexin V (ANV) with a peptide anticoagulant modified to preferentially target extrinsic coagulation. We found that ANV-6L15 reduced intracranial hematoma by 70.2%, improved neurological function, and reduced death by 56.8% in mice subjected to fluid percussion injury at 1.9 atm. It protected the TBI mice by preventing vascular leakage, tissue edema, and the TBI-induced hypercoagulable state. We further showed that the extrinsic tenase complex was formed on the surfaces of circulating EVs, with the highest level found on BDEVs. The phospholipidomic analysis detected the highest levels of PS on BDEVs, as compared with EVs from endothelial cells and platelets (79.1, 15.2, and 3.5 nM/mg of protein, respectively). These findings demonstrate that TBI-IC results from a trauma-induced hypercoagulable state and may be treated by anticoagulation targeting on the anionic phospholipid-expressing membrane of EVs from the brain and other cells.

Novel Injury Site Targeted Fusion Protein Comprising Annexin V and Kunitz Inhibitor Domains Ameliorates Ischemia-Reperfusion Injury and Promotes Survival of Ischemic Rat Abdominal Skin Flaps.

Appropriate antithrombotic therapy is critical for successful outcomes in reconstructive microsurgical procedures involving free tissue transfer. The annexin V-6L15 (ANV-6L15) fusion protein was developed as a targeted antithrombotic reagent. Annexin V specifically binds to exposed phosphatidylserine on apoptotic or injured cells, and prevents coagulation and cell adhesion, whereas 6L15 inhibits tissue factor-VIIa pathway within the coagulation cascade. The treatment efficacy of ANV-6L15 on rat island muscle and pedicled abdominal fasciocutaneous flaps following ischemic injury and ischemia-reperfusion injury (IRI) was evaluated. MATERIALS AND METHODS: The effects of ANV-6L15 on survival of rat abdominal fasciocutaneous flaps subjected to 10 hours of critical ischemia were assessed on day 5. Near-IR imaging was applied to evaluate the distribution of ANV-6L15 and flap perfusion. The rat cremaster muscle island flap was used to evaluate the effect of ANV-6L15 on IRI-induced leukocyte-endothelial interactions via intravital microscopy. 2,3,5 triphenyl-tetrazolium chloride assay was used to determine the ratio between live-versus-dead tissue. RESULTS: ANV-6L15 significantly increased the ratio of viable tissue (68.5 ± 9.79% vs 84.8 ± 5.14%, P < 0.05), and promoted survival of rat pedicled abdominal flaps (59.3 ± 6.86 vs. 47.0 ± 8.67, P < 0.05). Intravital microscopy demonstrated a significant decrease in the number of adhesive leukocytes (1.8 ± 1.64 vs. 10.0 ± 6.32, P < 0.05), and the percentage change of functional capillaries (16.4 ± 15.1 vs. 47.3 ± 18.3, P < 0.05) in ANV-6L15-treatment group. CONCLUSIONS: ANV-6L15 promoted survival of ischemic rat cremaster muscle and abdominal fasciocutaneous flaps and ameliorated leukocyte-related IRI. Future evaluation of potential clinical application of ANV-6L15 is warranted as a flap treatment adjunct.

Investigation of a potential scintigraphic tracer for imaging apoptosis: radioiodinated annexin V-Kunitz protease inhibitor fusion protein.

Radiolabeled annexin V (ANV) has been widely used for imaging cell apoptosis. Recently, a novel ANV-Kunitz-type protease inhibitor fusion protein, ANV-6L15, was found to be a promising probe for improved apoptosis detection based on its higher affinity to phosphatidylserine (PS) compared to native ANV. The present paper investigates the feasibility of apoptosis detection using radioiodinated ANV-6L15. Native ANV and ANV-6L15 were labeled with iodine-123 and iodine-125 using Iodogen method. The binding between the radioiodinated proteins and erythrocyte ghosts or chemical-induced apoptotic cells was examined. ANV-6L15 can be radioiodinated with high yield (40%-60%) and excellent radiochemical purity (>95%). (123)I-ANV-6L15 exhibited a higher binding ratio to erythrocyte ghosts and apoptotic cells compared to (123)I-ANV. The biodistribution of (123)I-ANV-6L15 in mice was also characterized. (123)I-ANV-6L15 was rapidly cleared from the blood. High uptake in the liver and the kidneys may limit the evaluation of apoptosis in abdominal regions. Our data suggest that radiolabeled ANV-6L15 may be a better scintigraphic tracer than native ANV for apoptosis detection.

Measurement of the binding parameters of annexin derivative-erythrocyte membrane interactions.

Erythrocyte ghosts prepared from fresh blood expressed phosphatidylserine (PS) on the membrane surfaces in a rather stable fashion. The binding of fluorescein-5-isothiocyanate (FITC)-labeled annexin V (ANV) derivatives to these membranes was studied by titration with proteins and with calcium. Whereas the preaddition of ethylenediaminetetraacetic acid (EDTA) to reaction mixtures totally prevented membrane binding, Ca(2+)-dependent binding was only partially reversed by EDTA treatment, consistent with an initial Ca(2+)-dependent binding that became partially Ca(2+) independent. Data derived from saturation titration with ANV derivatives poorly fit the simple protein-membrane equilibrium binding equation and showed negative cooperativity of binding with increasing membrane occupancy. In contrast, calcium titration at low binding site occupancy resulted in excellent fit into the protein-Ca(2+)-membrane equilibrium binding equation. Calcium titrations of FITC-labeled ANV and ANV-6L15 (a novel ANV-Kunitz protease inhibitor fusion protein) yielded a Hill coefficient of approximately 4 in both cases. The apparent dissociation constant for ANV-6L15 was approximately 4-fold lower than that of ANV at 1.2-2.5mM Ca(2+). We propose that ANV-6L15 may provide improved detection of PS exposed on the membrane surfaces of pathological cells in vitro and in vivo.

Passivating Injured Endothelium with Kinexins in Thrombolytic Therapy.

Without conjunctive administration of an anticoagulant, endothelial injury-induced thrombosis is resistant to thrombolysis and prone to re-thrombosis. We hypothesized that co-delivery of recombinant tissue plasminogen activator (rtPA) with annexin V-containing anticoagulants that specifically target the injured endothelium may passivate the thrombogenic elements of the vascular injury site and enhance rtPA-induced thrombolysis. In this study, the effects of conjunctive administration of Kinexins (Kunitz inhibitor-annexin V fusion proteins) with rtPA on thrombolysis were determined in vitro and in vivo. Thromboelastometry showed that both TAP-A (tick anticoagulant peptide-annexin V fusion protein; an inhibitor of factor Xa [FXa] and prothrombinase) and A-6L15 (annexin V-6L15 fusion protein; an inhibitor of tissue factor/FVIIa) exerted concentration-dependent (10-100 nM) effects on clot formation, with TAP-A being several folds more potent than A-6L15 in whole blood. Combination of TAP-A or A-6L15 with rtPA (1 µg/mL) led to decrease in lysis index, suggesting conjunctive enhancement of thrombolysis by combined use of rtPA with TAP-A or A-6L15. In a rat cremaster muscle preparation subjected to photochemical injury, conjunctive administration of rtPA and TAP-A significantly restored tissue perfusion to 56%, which is approximately two fold of that by rtPA or TAP-A alone. Near-infrared fluorescence images demonstrated local retention of a fluorescent A-6L15-S288 at the injury site, suggesting a targeting effect of the fusion protein. Pharmacokinetic analysis showed that 123I-labelled TAP-A and A-6L15 had initial distribution half-lives (T1/2α) of approximately 6 minutes and elimination half-lives (T1/2ß) of approximately 2.3 hours. In conclusion, Kinexins were potentially useful adjunctive agents with rtPA thrombolytic therapy especially for thrombosis induced by endothelial injury.

Bolus injections of novel thrombogenic site-targeted fusion proteins comprising annexin-V and Kunitz protease inhibitors attenuate intimal hyperplasia after balloon angioplasty.

BACKGROUND: Systemic administrations of conventional antithrombotics reduce neointima formation after angioplasty in experimental animals. However, clinical translation of these results has not been successful due to high risk for bleeding. OBJECTIVES: We sought to determine whether novel annexin-V (ANV)-Kunitz protease inhibitor fusion proteins, TAP-ANV and ANV-6L15, can specifically target to vascular injury site and limit neointima formation without inducing systemic hypo-coagulation in a rat carotid artery balloon angioplasty injury model. METHODS: Near infrared imaging was carried out after balloon-injury and injection of fluorescent ANV or ANV-6L15 to examine their bio-distributions. For peri-procedure treatment, TAP-ANV or ANV-6L15 was administered as i.v. boluses 3 times: 30-minutes before balloon-injury, immediate after procedure, and 120-minutes post-balloon-injury. For extended treatment, additional i.v. bolus injection was given on day-2, day-3 and every other day thereafter. Carotid arteries were collected on day-7 and day-14 for analysis. Blood was collected for measurement of clotting parameters. RESULTS: Near infrared imaging and immunochemistry showed that fluorescent ANV and ANV-6L15 specifically localized to injured carotid artery and significant amount of ANV-6L15 remained bound to the injured artery after 24-h. Peri-procedure injections of TAP-ANV or ANV-6L15 resulted in decrease of intima/media ratio by 56%. Extended injections of both yielded similar results. Both decreased the expression of PCNA on day-7 and increased the expression calponin on day-14 in the intima post-balloon-injury. CONCLUSIONS: TAP-ANV and ANV-6L15 can specifically localize to balloon injured carotid arteries after i.v. bolus injections, resulting in substantial attenuation of intimal hyperplasia without inducing a state of systemic hypo-coagulation.

Assessment of Novel Anti-thrombotic Fusion Proteins for Inhibition of Stenosis in a Porcine Model of Arteriovenous Graft.

BACKGROUND: Hemodialysis arteriovenous synthetic grafts (AVG) provide high volumetric blood flow rates shortly after surgical placement. However, stenosis often develops at the vein-graft anastomosis contributing to thrombosis and early graft failure. Two novel fusion proteins, ANV-6L15 and TAP-ANV, inhibit the tissue factor/factor VIIa coagulation complex and the factor Xa/factor Va complex, respectively. Each inhibitor domain is fused to an annexin V domain that targets the inhibitor activity to sites of vascular injury to locally inhibit thrombosis. This study's objective was to determine if these antithrombotic proteins are safe and effective in inhibiting AVG stenosis. METHODS: A bolus of either TAP-ANV or ANV-6L15 fusion protein was administered intravenously immediately prior to surgical placement of a synthetic graft between the external jugular vein and common carotid artery in a porcine model. At surgery, the vein and artery were irrigated with the anti-thrombotic fusion protein. Control animals received intravenous heparin. At 4 weeks, MRI was performed to evaluate graft patency, the pigs were then euthanized and grafts and attached vessels were explanted for histomorphometric assessment of neointimal hyperplasia at the vein-graft anastomosis. Blood was collected at surgery, immediately after surgery and at euthanasia for serum metabolic panels and coagulation chemistries. RESULTS: No acute thrombosis occurred in the control group or in either experimental group. No abnormal serum chemistries, activated clotting times or PT, PTT values were observed after treatment in experimental or control animals. However, at the vein-graft anastomosis, there was no difference between the control and experimental groups in cross-sectional lumen areas, as measured on MRI, and no difference in hyperplasia areas as determined by histomorphometry. These results suggest that local irrigation of TAP-ANV or ANV-6L15 intra-operatively was as effective in inhibiting acute graft thrombosis as intravenous administration of heparin, but failed to inhibit hyperplasia development and stenosis in AVG.

Fusion proteins comprising annexin V and Kunitz protease inhibitors are highly potent thrombogenic site-directed anticoagulants.

The anionic phospholipid, phosphatidyl-L-serine (PS), is sequestered in the inner layer of the plasma membrane in normal cells. Upon injury, activation, and apoptosis, PS becomes exposed on the surfaces of cells and sheds microparticles, which are procoagulant. Coagulation is initiated by formation of a tissue factor/factor VIIa complex on PS-exposed membranes and propagated through the assembly of intrinsic tenase (factor VIIIa/factor IXa), prothrombinase (factor Va/factor Xa), and factor XIa complexes on PS-exposed activated platelets. We constructed a novel series of recombinant anticoagulant fusion proteins by linking annexin V (ANV), a PS-binding protein, to the Kunitz-type protease inhibitor (KPI) domain of tick anticoagulant protein, an aprotinin mutant (6L15), amyloid beta-protein precursor, or tissue factor pathway inhibitor. The resulting ANV-KPI fusion proteins were 6- to 86-fold more active than recombinant tissue factor pathway inhibitor and tick anticoagulant protein in an in vitro tissue factor-initiated clotting assay. The in vivo antithrombotic activities of the most active constructs were 3- to 10-fold higher than that of ANV in a mouse arterial thrombosis model. ANV-KPI fusion proteins represent a new class of anticoagulants that specifically target the anionic membrane-associated coagulation enzyme complexes present at sites of thrombogenesis and are potentially useful as antithrombotic agents.