High dimensional predictions of suicide risk in 4.2 million US Veterans using ensemble transfer learning.

We present an ensemble transfer learning method to predict suicide from Veterans Affairs (VA) electronic medical records (EMR). A diverse set of base models was trained to predict a binary outcome constructed from reported suicide, suicide attempt, and overdose diagnoses with varying choices of study design and prediction methodology. Each model used twenty cross-sectional and 190 longitudinal variables observed in eight time intervals covering 7.5 years prior to the time of prediction. Ensembles of seven base models were created and fine-tuned with ten variables expected to change with study design and outcome definition in order to predict suicide and combined outcome in a prospective cohort. The ensemble models achieved c-statistics of 0.73 on 2-year suicide risk and 0.83 on the combined outcome when predicting on a prospective cohort of [Formula: see text] 4.2 M veterans. The ensembles rely on nonlinear base models trained using a matched retrospective nested case-control (Rcc) study cohort and show good calibration across a diversity of subgroups, including risk strata, age, sex, race, and level of healthcare utilization. In addition, a linear Rcc base model provided a rich set of biological predictors, including indicators of suicide, substance use disorder, mental health diagnoses and treatments, hypoxia and vascular damage, and demographics.

OrganiCam: a lightweight time-resolved laser-induced luminescence imager and Raman spectrometer for planetary organic material characterization.

OrganiCam is a laser-induced luminescence imager and spectrometer designed for standoff organic and biosignature detection on planetary bodies. OrganiCam uses a diffused laser beam (12° cone) to cover a large area at several meters distance and records luminescence on half of its intensified detector. The diffuser can be removed to record Raman and fluorescence spectra from a small spot from 2 m standoff distance. OrganiCam's small size and light weight makes it ideal for surveying organics on planetary surfaces. We have designed and built a brassboard version of the OrganiCam instrument and performed initial tests of the system.

PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis.

Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in Burkholderia infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis, we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. Identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host-targeted therapies against infectious disease caused by intracellular pathogens.

The interplay between chromosome stability and cell cycle control explored through gene-gene interaction and computational simulation.

Chromosome stability models are usually qualitative models derived from molecular-genetic mechanisms for DNA repair, DNA synthesis, and cell division. While qualitative models are informative, they are also challenging to reformulate as precise quantitative models. In this report we explore how (A) laboratory experiments, (B) quantitative simulation, and (C) seriation algorithms can inform models of chromosome stability. Laboratory experiments were used to identify 19 genes that when over-expressed cause chromosome instability in the yeast Saccharomyces cerevisiae To better understand the molecular mechanisms by which these genes act, we explored their genetic interactions with 18 deletion mutations known to cause chromosome instability. Quantitative simulations based on a mathematical model of the cell cycle were used to predict the consequences of several genetic interactions. These simulations lead us to suspect that the chromosome instability genes cause cell-cycle perturbations. Cell-cycle involvement was confirmed using a seriation algorithm, which was used to analyze the genetic interaction matrix to reveal an underlying cyclical pattern. The seriation algorithm searched over 10(14) possible arrangements of rows and columns to find one optimal arrangement, which correctly reflects events during cell cycle phases. To conclude, we illustrate how the molecular mechanisms behind these cell cycle events are consistent with established molecular interaction maps.

A new protein-protein interaction sensor based on tripartite split-GFP association.

Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

RBC-coupled tPA Prevents Whereas tPA Aggravates JNK MAPK-Mediated Impairment of ATP- and Ca-Sensitive K Channel-Mediated Cerebrovasodilation After Cerebral Photothrombosis.

The sole Food and Drug Administration-approved treatment for acute stroke is tissue-type plasminogen activator (tPA), but tPA aggravates impairment of cerebrovasodilation during hypotension in a newborn pig photothrombotic model of stroke. Coupling to carrier red blood cells (RBC) enhances thrombolytic effects of tPA, while reducing its side effects. ATP- and Ca-sensitive K channels (Katp and Kca) are important regulators of cerebrovascular tone and mediate cerebrovasodilation during hypotension. Mitogen-activated protein kinase, a family of at least three kinases, ERK, p38, and c-Jun-N-terminal kinase (JNK), is upregulated after photothrombosis. This study examined the effect of photothrombosis on Katp- and Kca-induced cerebrovasodilation and the roles of tPA and JNK during/after injury. Photothrombosis blunted vasodilation induced by the Katp agonists cromakalim, calcitonin gene-related peptide, and the Kca agonist NS 1619, which was aggravated by injection of tPA. In contrast, both pre- or post-injury thrombosis injection of RBC-tPA and JNK antagonist SP 600125 prevented impairment of Katp- and Kca-induced vasodilation. Therefore, JNK activation in thrombosis impairs K channel-mediated cerebrovasodilation. Standard thrombolytic therapy of central nervous system ischemic disorders using free tPA poses the danger of further dysregulation of cerebrohemodynamics by impairing cation-mediated control of cerebrovascular tone, whereas RBC-coupled tPA both restores reperfusion and normalizes cerebral hemodynamics.

Targeted In Situ Biosynthetic Transcriptional Activation in Native Surface-Level Human Articular Chondrocytes during Lesion Stabilization.

OBJECTIVE: Safe articular cartilage lesion stabilization is an important early surgical intervention advance toward mitigating articular cartilage disease burden. While short-term chondrocyte viability and chondrosupportive matrix modification have been demonstrated within tissue contiguous to targeted removal of damaged articular cartilage, longer term tissue responses require evaluation to further clarify treatment efficacy. The purpose of this study was to examine surface chondrocyte responses within contiguous tissue after lesion stabilization. METHODS: Nonablation radiofrequency lesion stabilization of human cartilage explants obtained during knee replacement was performed for surface fibrillation. Time-dependent chondrocyte viability, nuclear morphology and cell distribution, and temporal response kinetics of matrix and chaperone gene transcription indicative of differentiated chondrocyte function were evaluated in samples at intervals to 96 hours after treatment. RESULTS: Subadjacent surface articular cartilage chondrocytes demonstrated continued viability for 96 hours after treatment, a lack of increased nuclear fragmentation or condensation, persistent nucleic acid production during incubation reflecting cellular assembly behavior, and transcriptional up-regulation of matrix and chaperone genes indicative of retained biosynthetic differentiated cell function. CONCLUSIONS: The results of this study provide further evidence of treatment efficacy and suggest the possibility to manipulate or induce cellular function, thereby recruiting local chondrocytes to aid lesion recovery. Early surgical intervention may be viewed as a tissue rescue, allowing articular cartilage to continue displaying biological responses appropriate to its function rather than converting to a tissue ultimately governed by the degenerative material property responses of matrix failure. Early intervention may positively impact the late changes and reduce disease burden of damaged articular cartilage.

The Brucella TIR-like protein TcpB interacts with the death domain of MyD88.

The pathogen Brucella melitensis secretes a Toll/interleukin-1 receptor (TIR) domain containing protein that abrogates host innate immune responses. In this study, we have characterized the biochemical interactions of Brucella TIR-like protein TcpB with host innate immune adaptor proteins. Using protein-fragment complementation assays based on Gaussia luciferase and green fluorescent protein, we find that TcpB interacts directly with MyD88 and that this interaction is significantly stronger than the interaction of TcpB with TIRAP, the only other adaptor protein that detectably interacts with TcpB. Surprisingly, the TcpB-MyD88 interaction depends on the death domain (DD) of MyD88, and TcpB does not interact with the isolated TIR domain of MyD88. TcpB disrupts MyD88(DD)-MyD88(DD), MyD88(DD)-MyD88(TIR) and MyD88(DD)-MyD88 interactions but not MyD88-MyD88 or MyD88(TIR)-MyD88(TIR) interactions. Structural models consistent with these results suggest how TcpB might inhibit TLR signaling by targeting MyD88 via a DD-TIR domain interface.

Design, synthesis, and a novel application of quorum-sensing agonists as potential drug-delivery vehicles.

Surface adhered bacterial colonies or biofilms are an important problem in medical and food industries. Bacteria use a chemical language to monitor their quorum and to express virulence factors, which eventually help them in colonization and manifestation of an infection. The LasR-LasI and RhlR-RhlI quorum-sensing (QS) systems of Pseudomonas aeruginosa control expression of virulence factors in a population density-dependent fashion. In this study we investigated the role of synthetic analogs to RhlR-RhlI system of P. aeruginosa strains (PAO-1; wild-type and mutants JP-1, PDO-100, and JP-2) responsible for production of acyl-homoserine lactones-2; butanol homoserine lactone (AHL-2; C(4)-HSL). We synthesized double (QS1207) and single (QS0108) sulfur analogs against (C(4)-HSL; AHL-2), an autoinducer of Pseudomonas QS system. Extensive biological investigation of these analogs suggested a growth promoting activity for these analogs in Pseudomonas controlling biofilm production and exo-protease secretion. We hypothesized that these thiolactone analogs could be potentially utilized as potent drug-delivery vehicles against biofilm-producing pathogens. As a proof of principle we conjugated the single sulfur analog QS0108 with the broad-spectrum antibiotic, ciprofloxacin (QS0108-Cip). The QS analog-antibiotic conjugate was significantly more effective at disrupting both the nascent and mature biofilms of P. aeruginosa than the free antibiotic.

Red blood cell-coupled tissue plasminogen activator prevents impairment of cerebral vasodilatory responses through inhibition of c-Jun-N-terminal kinase and potentiation of p38 mitogen-activated protein kinase after cerebral photothrombosis in the newborn pig.

OBJECTIVE: Pediatric ischemic stroke is a poorly understood, yet clinically important, problem. The sole approved treatment for acute stroke is tissue-type plasminogen activator. However, tissue plasminogen activator vasoactivity aggravates hypoxia/ischemia-induced impairment of cerebrovasodilation in response to hypercapnia and hypotension in newborn pigs. Mitogen-activated protein kinase (a family of 3 kinases, extracellular signal-related kinase, p38, and c-Jun-N-terminal kinase) is upregulated after hypoxia/ischemia. Coupling of tissue plasminogen activator to red blood cells prevented hypoxia/ischemia-induced impairment of dilation and suppressed extracellular signal-related kinase mitogen-activated protein kinase activation. This study investigated the differential roles of mitogen-activated protein kinase isoforms in the effects of red blood cells-tissue plasminogen activator on cerebrovasodilation in a translationally relevant injury model, photothrombosis. DESIGN: Prospective, randomized animal study. SETTING: : University laboratory. SUBJECTS: Newborn (1- to 5-day-old) pigs. INTERVENTIONS: Cerebral blood flow and pial artery diameter were determined before and after photothrombotic injury (laser 532 nm and erythrosine B) was produced in piglets equipped with a closed cranial window. Cerebral blood flow extracellular signal-related kinase, p38, and c-Jun-N-terminal kinase mitogen-activated protein kinase were determined by enzyme-linked immunosorbent assay. MEASUREMENTS AND MAIN RESULTS: Tissue plasminogen activator and red blood cells-tissue plasminogen activator alleviated reduction of cerebral blood flow after photothrombotic injury. Cerebrovasodilation was blunted by photothrombotic injury, reversed to vasoconstriction by tissue plasminogen activator, but dilation was maintained by red blood cells-tissue plasminogen activator. Cerebral blood flow c-Jun-N-terminal kinase and p38 mitogen-activated protein kinase but not extracellular signal-related kinase mitogen-activated protein kinase was elevated by photothrombotic injury, an effect potentiated by tissue plasminogen activator. Red blood cells-tissue plasminogen activator blocked c-Jun-N-terminal kinase but potentiated p38 mitogen-activated protein kinase upregulation after photothrombotic injury. A c-Jun-N-terminal kinase mitogen-activated protein kinase antagonist prevented, a p38 mitogen-activated protein kinase antagonist potentiated, whereas an extracellular signal-related kinase mitogen-activated protein kinase antagonist had no effect on dilator impairment after photothrombotic injury. CONCLUSIONS: These data indicate that in addition to restoring perfusion, red blood cells-tissue plasminogen activator prevents impairment of cerebrovasodilation after photothrombotic injury through blockade of c-Jun-N-terminal kinase and potentiation of p38 mitogen-activated protein kinase. These data suggest tissue plasminogen activator coupling to red blood cells offers a novel approach to increase the benefit/risk ratio of thrombolytic therapy to treat central nervous system ischemic disorders.

Histopomorphic evaluation of radiofrequency mediated débridement chondroplasty.

The use of radiofrequency devices has become widespread for surgical ablation procedures. When ablation devices have been deployed in treatment settings requiring tissue preservation like débridement chondroplasty, adoption has been limited due to the collateral damage caused by these devices in healthy tissue surrounding the treatment site. Ex vivo radiofrequency mediated débridement chondroplasty was performed on osteochondral specimens demonstrating surface fibrillation obtained from patients undergoing knee total joint replacement. Three radiofrequency systems designed to perform débridement chondroplasty were tested each demonstrating different energy delivery methods: monopolar ablation, bipolar ablation, and non-ablation energy. Treatment outcomes were compared with control specimens as to clinical endpoint and histopomorphic characteristics. Fibrillated cartilage was removed in all specimens; however, the residual tissue remaining at the treatment site displayed significantly different characteristics attributable to radiofrequency energy delivery method. Systems that delivered ablation-based energies caused tissue necrosis and collateral damage at the treatment site including corruption of cartilage Superficial and Transitional Zones; whereas, the non-ablation system created a smooth articular surface with Superficial Zone maintenance and without chondrocyte death or tissue necrosis. The mechanism of radiofrequency energy deposition upon tissues is particularly important in treatment settings requiring tissue preservation. Ablation-based device systems can cause a worsened state of articular cartilage from that of pre-treatment. Non-ablation energy can be successful in modifying/preconditioning tissue during débridement chondroplasty without causing collateral damage. Utilizing a non-ablation radiofrequency system provides the ability to perform successful débridement chondroplasty without causing additional articular cartilage tissue damage and may allow for other cartilage intervention success.

Signaling, delivery and age as emerging issues in the benefit/risk ratio outcome of tPA For treatment of CNS ischemic disorders.

Stroke is a leading cause of morbidity and mortality. While tissue-type plasminogen activator (tPA) remains the only FDA-approved treatment for ischemic stroke, clinical use of tPA has been constrained to roughly 3% of eligible patients because of the danger of intracranial hemorrhage and a narrow 3 h time window for safe administration. Basic science studies indicate that tPA enhances excitotoxic neuronal cell death. In this review, the beneficial and deleterious effects of tPA in ischemic brain are discussed along with emphasis on development of new approaches toward treatment of patients with acute ischemic stroke. In particular, roles of tPA-induced signaling and a novel delivery system for tPA administration based on tPA coupling to carrier red blood cells will be considered as therapeutic modalities for increasing tPA benefit/risk ratio. The concept of the neurovascular unit will be discussed in the context of dynamic relationships between tPA-induced changes in cerebral hemodynamics and histopathologic outcome of CNS ischemia. Additionally, the role of age will be considered since thrombolytic therapy is being increasingly used in the pediatric population, but there are few basic science studies of CNS injury in pediatric animals.

Native Chondrocyte Viability during Cartilage Lesion Progression: Normal to Surface Fibrillation.

OBJECTIVE: Early surgical intervention for articular cartilage disease is desirable before full-thickness lesions develop. As early intervention treatments are designed, native chondrocyte viability at the treatment site before intervention becomes an important parameter to consider. The purpose of this study is to evaluate native chondrocyte viability in a series of specimens demonstrating the progression of articular cartilage lesions to determine if the chondrocyte viability profile changes during the evolution of articular cartilage disease to the level of surface fibrillation. DESIGN: Osteochondral specimens demonstrating various degrees of articular cartilage damage were obtained from patients undergoing knee total joint replacement. Three groups were created within a patient harvest based on visual and tactile cues commonly encountered during surgical intervention: group 1, visually and tactilely intact surfaces; group 2, visually intact, tactilely soft surfaces; and group 3, surface fibrillation. Confocal laser microscopy was performed following live/dead cell viability staining. RESULTS: Groups 1 to 3 demonstrated viable chondrocytes in all specimens, even within the fibrillated portions of articular cartilage, with little to no evidence of dead chondrocytes. Chondrocyte viability profile in articular cartilage does not appear to change as disease lesion progresses from normal to surface fibrillation. CONCLUSIONS: Fibrillated partial-thickness articular cartilage lesions are a good therapeutic target for early intervention. These lesions retain a high profile of viable chondrocytes and are readily diagnosed by visual and tactile cues during surgery. Early intervention should be based on matrix failure rather than on more aggressive procedures that further corrupt the matrix and contribute to chondrocyte necrosis of contiguous untargeted cartilage.

Beryllium alters lipopolysaccharide-mediated intracellular phosphorylation and cytokine release in human peripheral blood mononuclear cells.

Beryllium exposure in susceptible individuals leads to the development of chronic beryllium disease, a lung disorder marked by release of inflammatory cytokine and granuloma formation. We have previously reported that beryllium induces an immune response even in blood mononuclear cells from healthy individuals. In this study, we investigate the effects of beryllium on lipopolysaccharide-mediated cytokine release in blood mononuclear and dendritic cells from healthy individuals. We found that in vitro treatment of beryllium sulfate inhibits the secretion of lipopolysaccharide-mediated interleukin 10, while the release of interleukin 1beta is enhanced. In addition, not all lipopolysaccharide-mediated responses are altered, as interleukin 6 release in unaffected upon beryllium treatment. Beryllium sulfate-treated cells show altered phosphotyrosine levels upon lipopolysaccharide stimulation. Significantly, beryllium inhibits the phosphorylation of signal transducer and activator of transducer 3, induced by lipopolysaccharide. Finally, inhibitors of phosphoinositide-3 kinase mimic the effects of beryllium in inhibition of interleukin 10 release, while they have no effect on interleukin 1beta secretion. This study strongly suggests that prior exposures to beryllium could alter host immune responses to bacterial infections in healthy individuals, by altering intracellular signaling.

Red blood cells-coupled tPA prevents impairment of cerebral vasodilatory responses and tissue injury in pediatric cerebral hypoxia/ischemia through inhibition of ERK MAPK activation.

Babies experience hypoxia (H) and ischemia (I) from stroke. The only approved treatment for stroke is fibrinolytic therapy with tissue-type plasminogen activator (tPA). However, tPA potentiates H/I-induced impairment of responses to cerebrovasodilators such as hypercapnia and hypotension, and blockade of tPA-mediated vasoactivity prevents this deleterious effect. Coupling of tPA to red blood cells (RBCs) reduces its central nervous system (CNS) toxicity through spatially confining the drug to the vasculature. Mitogen-activated protein kinase (MAPK), a family of at least three kinases, is upregulated after H/I. In this study we determined whether RBC-tPA given before or after cerebral H/I would preserve responses to cerebrovasodilators and prevent neuronal injury mediated through the extracellular signal-related kinase (ERK) MAPK pathway. Animals given RBC-tPA maintained responses to cerebrovasodilators at levels equivalent to pre-H/I values. cerebrospinal fluid and brain parenchymal ERK MAPK was elevated by H/I and this upregulation was potentiated by tPA, but blunted by RBC-tPA. U0126, an ERK MAPK antagonist, also maintained cerebrovasodilation post H/I. Neuronal degeneration in CA1 hippocampus after H/I was not improved by tPA, but was ameliorated by RBC-tPA and U0126. These data suggest that coupling of tPA to RBCs offers a novel approach toward increasing the benefit/risk ratio of thrombolytic therapy for CNS disorders associated with H/I.

Erythrocyte-bound tissue plasminogen activator is neuroprotective in experimental traumatic brain injury.

The purpose of this study was to test the effects of exogenous tissue plasminogen activator (tPA) in traumatic brain injury (TBI).We tested two different tPA formulations, free tPA and tPA bound to erythrocytes (RBC/tPA).Vehicle and each of the tPA treatments were injected intravenously into anesthetized rats 15 min after moderate lateral fluid percussion injury. The animals were sacrificed at 2 days for calculating microclot burden (n=13) and IgG staining area (n=13) in the brain sections as indicators of post-traumatic thrombosis and blood-brain barrier (BBB) breakdown, respectively. Another set of injured animals treated in the same way were sacrificed at 7 days to compare cortical lesion volumes (n=28) and CA3 hippocampal cell loss (n=24). All evaluations were done blinded with respect to treatment. No significant differences were found with respect to microclot burden or IgG staining volume. Injection of wild-type tPA caused significantly ( p<0.05) larger cortical injuries and greater cerebral hemorrhage. In contrast, there was significantly less cortical injury ( p<0.01) and hippocampal cell loss ( p<0.01) in the RBC=tPA group than in all other groups. These results reveal that RBC/tPA is more neuroprotective in experimental TBI than is unbound tPA.

Cerebrovascular thromboprophylaxis in mice by erythrocyte-coupled tissue-type plasminogen activator.

BACKGROUND: Cerebrovascular thrombosis is a major source of morbidity and mortality after surgery, but thromboprophylaxis in this setting is limited because of the formidable risk of perioperative bleeding. Thrombolytics (eg, tissue-type plasminogen activator [tPA]) cannot be used prophylactically in this high-risk setting because of their short duration of action and risk of causing hemorrhage and central nervous system damage. We found that coupling tPA to carrier red blood cells (RBCs) prolongs and localizes tPA activity within the bloodstream and converts it into a thromboprophylactic agent, RBC/tPA. To evaluate the utility of this new approach for preventing cerebrovascular thrombosis, we examined the effect of RBC/tPA in animal models of cerebrovascular thromboembolism and ischemia. METHODS AND RESULTS: Preformed fibrin microemboli were injected into the middle carotid artery of mice, occluding downstream perfusion and causing severe infarction and 50% mortality within 48 hours. Preinjected RBC/tPA rapidly lysed nascent cerebral thromboemboli, providing rapid, durable reperfusion and reducing morbidity and mortality. These beneficial effects were not achieved by preinjection of tPA, even at a 10-fold higher dose, which increased mortality from 50% to 90% by 10 hours after embolization. RBC/tPA injected 10 minutes after tail amputation to simulate postsurgical hemostasis did not cause bleeding from the wound, whereas soluble tPA caused profuse bleeding. RBC/tPA neither aggravated brain damage caused by focal ischemia in a filament model of middle carotid artery occlusion nor caused postthrombotic hemorrhage in hypertensive rats. CONCLUSIONS: These results suggest a potential RBC/tPA utility as thromboprophylaxis in patients who are at risk for acute cerebrovascular thromboembolism.

Prophylactic thrombolysis by thrombin-activated latent prourokinase targeted to PECAM-1 in the pulmonary vasculature.

A recombinant prodrug, single-chain urokinase-type plasminogen activator (scuPA) fused to an anti-PECAM-1 antibody single-chain variable fragment (anti-PECAM scFv/scuPA) targets endothelium and augments thrombolysis in the pulmonary vasculature.(1) To avoid premature activation and inactivation and to limit systemic toxicity, we replaced the native plasmin activation site in scFv/low-molecular-weight (lmw)-scuPA with a thrombin activation site, generating anti-PECAM scFv/uPA-T that (1) is latent and activated by thrombin instead of plasmin; (2) binds to PECAM-1; (3) does not consume plasma fibrinogen; (4) accumulates in mouse lungs after intravenous injection; and (5) resists PA inhibitor PAI-1 until activated by thrombin. In mouse models of pulmonary thrombosis caused by thromboplastin and ischemia-reperfusion (I/R), scFv/uPA-T provided more potent thromboprophylaxis and greater lung protection than plasmin-sensitive scFv/uPA. Endothelium-targeted thromboprophylaxis triggered by a prothrombotic enzyme illustrates a novel approach to time- and site-specific regulation of proteolytic reactions that can be modulated for therapeutic benefit.