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ABSTRACTPurposeTo evaluate real-world prescription digital therapeutic (PDT) use and associated clinical outcomes among patients with opioid use disorder (OUD).Patients and methodsA real-world observational evaluation of patients who filled either a 12- or 24-week (refill) prescription for the reSET-O® PDT. The PDT content consists of 67 interactive lessons unlocked in sequence during use as well as the chance to earn rewards for progress and/or negative urine screens. Engagement/retention data (ongoing engagement in weeks 9–12, or 21–24) were collected via the PDT and analyzed with descriptive statistics. Substance use was evaluated as a composite of patient self-reports and urine drug screens (UDS). Missing UDS data were assumed to be positive. A regression analyses of hospital encounters for 12- vs. 24-week prescriptions controlling for covariates was conducted.ResultsIn a cohort of 3,817 individuals with OUD who completed a 12-week PDT prescription, a cohort of 643 was prescribed a second 12-week ‘refill’ prescription, for a total treatment time of 24 weeks. Mean age of the 24-week cohort was 39 years, 56.7% female. At 24 weeks of total treatment: abstinence in the last 4 weeks of treatment was 86% in an analysis in which patients with no data are assumed to be positive for illicit opioids. Over 91% of patients were retained in treatment. An analysis of matched insurance claims showed that those treated for 24 weeks had a 27% decrease in unique hospital encounters compared to those who got the first 12-week prescription only.ConclusionsThese data present real-world evidence that a second prescription (24 weeks) of a PDT for OUD is associated with improved outcomes, high levels of retention, and fewer hospital encounters compared to a single prescription for a PDT.PLAIN LANGUAGE SUMMARYPrescription digital therapeutics (PDTs) are software-based treatments that are FDA-authorized to improve clinical outcomes for serious diseases and conditions. The reSET-O PDT consists of 67 interactive lessons unlocked in sequence during use as well as the chance to earn rewards for progress and/or negative urine screens. Multiple studies show that a single 12-week PDT prescription for opioid use disorder (OUD) helps patients engage in treatment, reduces substance use, and helps patients remain in treatment, but to date there has been no evaluation of how patients who receive a ‘refill’ second prescription engage with the therapeutic and whether the positive effects on substance use and retention are durable across a second 12 weeks (total of 24 weeks) of treatment.This real-world analysis evaluated 643 patients from 12 U.S. states who were prescribed a second PDT prescription. 93% of this cohort completed 8 or more core lesson modules in the second prescription period, 85% completed at least half of core modules, and 64% completed all 32 core modules. Patients used the PDT outside of clinic hours about 40% of the time. 94.4% of patients had 80% or greater negative reports of opioid use across the second 12 weeks of treatment. A 27% decrease in unique hospital encounters was observed in patients who completed a second prescription vs. patients who completed only one prescription.These data show that a second prescription of a PDT for OUD is associated with postive patient outcomes. Patients showed durable and high levels of engagement with the PDT, reduced substance use, and improved treatment retention through 24 weeks of treatment.

This brief report presents transmission rates from a prospective study of 15 households with pediatric index cases of severe acute respiratory coronavirus-2 in Los Angeles County from December 2020 to February 2021. Our findings support ongoing evidence that transmission from pediatric index cases to household contacts is frequent but can be mitigated with practicing well-documented control measures at home, including isolation, masking and good hand hygiene.

Purpose: To evaluate whether exposure to MyFamilyPlan—a web-based preconception health education module—changes the proportion of women discussing reproductive health with providers at well-woman visits.Design: Cluster randomized controlled trial. One hundred thirty participants per arm distributed among 34 clusters (physicians) required to detect a 20% change in the primary outcome.Setting: Urban academic medical center (California).Participants: Eligible women were 18 to 45 years old, were English speaking, were nonpregnant, were able to access the Internet, and had an upcoming well-woman visit. E-mail and phone recruitment between September 2015 and May 2016; 292 enrollees randomized.Intervention: Intervention participants completed the MyFamilyPlan module online 7 to 10 days before a scheduled well-woman visit; control participants reviewed standard online preconception health education materials.Measures: The primary outcome was self-reported discussion of reproductive health with the physician at the well-woman visit. Self-reported secondary outcomes were folic acid use, contraceptive method initiation/change, and self-efficacy score.Analysis: Multilevel multivariate logistic regression.Results: After adjusting for covariates and cluster, exposure to MyFamilyPlan was the only variable significantly associated with an increase in the proportion of women discussing reproductive health with providers (odds ratio: 1.97, 95% confidence interval: 1.22-3.19). Prespecified secondary outcomes were unaffected.Conclusion: MyFamilyPlan exposure was associated with a significant increase in the proportion of women who reported discussing reproductive health with providers and may promote preconception health awareness; more work is needed to affect associated behaviors.

Mental and behavioral health (MBH) emergencies in children and youth continue to increasingly affect not only the emergency department (ED), but the entire spectrum of emergency medical services for children, from prehospital services to the community. Inadequate community and institutional infrastructure to care for children and youth with MBH conditions makes the ED an essential part of the health care safety net for these patients. As a result, an increasing number of children and youth are referred to the ED for evaluation of a broad spectrum of MBH emergencies, from depression and suicidality to disruptive and aggressive behavior. However, challenges in providing optimal care to these patients include lack of personnel, capacity, and infrastructure, challenges with timely access to a mental health professional, the nature of a busy ED environment, and paucity of outpatient post-ED discharge resources. These factors contribute to prolonged ED stays and boarding, which negatively affects patient care and ED operations. Strategies to improve care for MBH emergencies, including systems level coordination of care, is therefore essential. The goal of this policy statement and its companion technical report is to highlight strategies, resources, and recommendations for improving emergency care delivery for pediatric MBH.

Child poverty in the United States is widespread and has serious negative effects on the health and well-being of children throughout their life course. Child health providers are considering ways to redesign their practices in order to mitigate the negative effects of poverty on children and support the efforts of families to lift themselves out of poverty. To do so, practices need to adopt effective methods to identify poverty-related social determinants of health and provide effective interventions to address them. Identification of needs can be accomplished with a variety of established screening tools. Interventions may include resource directories, best maintained in collaboration with local/regional public health, community, and/or professional organizations; programs embedded in the practice (eg, Reach Out and Read, Healthy Steps for Young Children, Medical-Legal Partnership, Health Leads); and collaboration with home visiting programs. Changes to health care financing are needed to support the delivery of these enhanced services, and active advocacy by child health providers continues to be important in effecting change. We highlight the ongoing work of the Health Care Delivery Subcommittee of the Academic Pediatric Association Task Force on Child Poverty in defining the ways in which child health care practice can be adapted to improve the approach to addressing child poverty.

The aims of this study are to describe growth trajectories in the body mass index (BMI) among the major racial and ethnic groups of US children and to identify predictors of children’s BMI trajectories. The Early Childhood Longitudinal Study-Birth Cohort (ECLS-B) was used to identify predictors of BMI growth trajectories, including child characteristics, maternal attributes, home practices related to diet and social behaviors, and family sociodemographic factors. Growth models, spanning 48 to 72 months of age, were estimated with hierarchical linear modeling via STATA/Xtmixed methods. Approximately one-third of 4-year-old females and males were overweight and/or obese. African-American and Latino children displayed higher predicted mean BMI scores and differing mean BMI trajectories, compared with White children, adjusting for time-independent and time-dependent predictors. Several factors were significantly associated with lower mean BMI trajectories, including very low birth weight, higher maternal education level, residing in a two-parent household, and breastfeeding during infancy. Greater consumption of soda and fast food was associated with higher mean BMI growth. Soda consumption was a particularly strong predictor of mean BMI growth trajectory for young Black children. Neither the child’s inactivity linked to television viewing nor fruit nor vegetable consumption was predictive of BMI growth for any racial/ethnic group. Significant racial and ethnic differences are discernible in BMI trajectories among young children. Raising parents’ and health practitioners’ awareness of how fast food and sweetened-beverage consumption contributes to early obesity and growth in BMI—especially for Blacks and Latinos—could improve the health status of young children.

The American Psychiatric Association will update its Diagnostic and Statistical Manual of Mental Disorders to its fifth edition (DSM-5). With this new edition, the classification and diagnostic criteria for the spectrum of autistic disorders will change and become more specific and potentially more restrictive. Rather than maintaining several subcategories of autism including Asperger syndrome, there will be one new category called autism spectrum disorder. This change may alter which children are diagnosed as having autism as well as modify eligibility for treatment, educational, and other support services. We review the history and rationale for the proposed changes as well as several recent studies that have attempted to gauge the impact of these changes on children and families. We also consider how the proposed changes are likely to create new challenges for parents who are attempting to organize their children’s care and for pediatricians who are providing that care and assisting with care coordination.

AbstractObjective To evaluate the impact of a public health approach to pediatric residency education on learner knowledge, skills, attitudes, beliefs, and career choice.Background Incorporating public health principles into traditional residency education can give pediatricians the population-oriented perspective to address social determinants of health.Methods The Community Health and Advocacy Training (CHAT) program is an educational intervention with a public health framework. From 2001–2007, 215 categorical pediatric residents and 37 residents in the CHAT program were evaluated by using an annual survey of community pediatrics exposure, knowledge, attitudes, and beliefs. American Board of Pediatrics (ABP) examination passage rates for both groups were also examined, as was career choice after graduation.Results While interns in both the categorical and CHAT programs scored similarly on attitudes, beliefs, skills, and knowledge of community pediatrics, the postgraduate level-3 (PL-3) year CHAT residents scored higher in attitudes (P< .001) and skills (P< .05). Exposure to both didactic (P< .05) and practical (P< .001) community pediatrics curricular experiences were higher for CHAT residents than for categorical residents. No significant differences between ABP examination scores were found for the 2 groups, although 100 of CHAT graduates passed on the first try compared to 91 of categorical graduates during this time period. A greater percentage of CHAT graduates (82) than categorical graduates (53) reported pursuing careers in primary care.Conclusion With a public health approach to residency education, residents gain the knowledge, attitudes, and skills to address child health problems from a population perspective. Participation in such a curriculum still resulted in high passage rates on the ABP examination.

The aim of this study was to examine parental reports of receiving a child developmental assessment (DA), and the child, family, and type of health care setting characteristics and well-child care processes associated with receiving this aspect of preventive developmental care.

Plasminogen activators (PAs) are used to treat life-threatening thrombosis, but not for thromboprophylaxis because of rapid clearance, risk of bleeding, and central nervous system (CNS) toxicity. We describe a novel strategy that may help to overcome these limitations by targeting a thrombin-activated PA pro-drug to circulating red blood cells (RBCs). We fused a single chain antibody (scFv Ter-119) that binds to mouse glycophorin A (GPA) with a variant human single-chain low molecular weight urokinase construct that can be activated selectively by thrombin (scFv/uPA-T). scFv/uPA-T bound specifically to mouse RBCs without altering their biocompatibility and retained its zymogenic properties until converted by thrombin into an active 2-chain molecule. As a result, RBC-bound scFv/uPA-T caused thrombin-induced fibrinolysis. One hour and 48 hours after intravenous (IV) injection in mice, approximately 70% and approximately 35% of scFv/uPA-T was retained in the blood, respectively, and approximately 95% of the circulating scFv/uPA-T remained bound to RBCs. A single IV injection of scFv/uPA-T provided effective prophylaxis against arterial and venous thrombosis for up to 24 hours. Thus, prophylactic delivery of RBC-targeted PA pro-drugs activated selectively at the site of clot formation represents a new approach to prevent thrombosis in clinical settings where the risk of clotting is high.

Plasminogen activators (PAs) are used to treat life-threatening thrombosis, but not for thromboprophylaxis because of rapid clearance, risk of bleeding, and central nervous system (CNS) toxicity. We describe a novel strategy that may help to overcome these limitations by targeting a thrombin-activated PA pro-drug to circulating red blood cells (RBCs). We fused a single chain antibody (scFv Ter-119) that binds to mouse glycophorin A (GPA) with a variant human single-chain low molecular weight urokinase construct that can be activated selectively by thrombin (scFv/uPA-T). scFv/uPA-T bound specifically to mouse RBCs without altering their biocompatibility and retained its zymogenic properties until converted by thrombin into an active 2-chain molecule. As a result, RBC-bound scFv/uPA-T caused thrombin-induced fibrinolysis. One hour and 48 hours after intravenous (IV) injection in mice, approximately 70% and approximately 35% of scFv/uPA-T was retained in the blood, respectively, and approximately 95% of the circulating scFv/uPA-T remained bound to RBCs. A single IV injection of scFv/uPA-T provided effective prophylaxis against arterial and venous thrombosis for up to 24 hours. Thus, prophylactic delivery of RBC-targeted PA pro-drugs activated selectively at the site of clot formation represents a new approach to prevent thrombosis in clinical settings where the risk of clotting is high.

To examine associations among Puerto Rican children's physical health problems and children's internalizing disorders, parental psychopathology and acculturative stress, and family factors. A population-based probability sample of 2491 Puerto Rican children, aged between 5 and 13 years, and caregivers from the South Bronx and the U.S. Commonwealth of Puerto Rico participated in this study. The parent version of the Diagnostic Interview Schedule for Children-IV was used to assess children's internalizing disorders. Children's anxiety disorders, parental psychopathology, and acculturative stress were associated with childhood asthma, abdominal pain, and headaches. Children's depressive disorders, maternal acceptance, and family functioning were associated with abdominal pain and headaches. Parents of children living in Puerto Rico were more likely to report physical health problems in their children than in the Bronx. Children's internalizing disorders, parental psychopathology, and acculturative stress may be important areas to target among Puerto Rican children with physical health problems.

Chemical coupling to carrier red blood cells (RBCs) converts tissue type plasminogen activator (tPA) from a problematic therapeutic into a safe agent for thromboprophylaxis. The goal of this study was to develop a more clinically relevant recombinant biotherapeutic by fusing a mutant tPA with a single-chain antibody fragment (scFv) with specificity for glycophorin A (GPA) on mouse RBCs. The fusion construct (anti-GPA scFv/PA) bound specifically to mouse but not human RBCs and activated plasminogen; this led to rapid and stable attachment of up to 30,000 copies of anti-GPA scFv/PA per mouse RBC that were thereby endowed with high fibrinolytic activity. Binding of anti-GPA scFv/PA neither caused RBC aggregation, hemolysis, uptake in capillary-rich lungs or in the reticuloendothelial system nor otherwise altered the circulation of RBCs. Over 40% of labeled anti-GPA scFv/PA injected in mice bound to RBC, which markedly prolonged its intravascular circulation and fibrinolytic activity compared with its nontargeted PA counterpart, anti-GPA scFv/PA, but not its nontargeted PA analog, prevented thrombotic occlusion in FeCl(3) models of vascular injury. These results provide proof-of-principle for the development of a recombinant PA variant that binds to circulating RBC and provides thromboprophylaxis by use of a clinically relevant approach.

Latinos make up the largest group of minority children in the United States. Despite great diversity among Latino children, studies have traditionally treated them as a monolithic group. Few studies have examined patterns and determinants of access to and use of health care for different subgroups of Latino children.

Urokinase-type plasminogen activator (uPA) participates in diverse (patho)physiological processes through intracellular signaling events that affect cell adhesion, migration, and proliferation, although the mechanisms by which these occur are only partially understood. Here we report that upon cell binding and internalization, single-chain uPA (scuPA) translocates to the nucleus within minutes. Nuclear translocation does not involve proteolytic activation or degradation of scuPA. Neither the urokinase receptor (uPAR) nor the low-density lipoprotein-related receptor (LRP) is required for nuclear targeting. Rather, translocation involves the binding of scuPA to the nucleocytoplasmic shuttle protein nucleolin through a region containing the kringle domain. RNA interference and mutational analysis demonstrate that nucleolin is required for the nuclear transport of scuPA. Furthermore, nucleolin is required for the induction smooth muscle α-actin (α-SMA) by scuPA. These data reveal a novel pathway by which uPA is rapidly translocated to the nucleus where it might participate in regulating gene expression.

Urokinase-type plasminogen activator (uPA) participates in diverse (patho)physiological processes through intracellular signaling events that affect cell adhesion, migration, and proliferation, although the mechanisms by which these occur are only partially understood. Here we report that upon cell binding and internalization, single-chain uPA (scuPA) translocates to the nucleus within minutes. Nuclear translocation does not involve proteolytic activation or degradation of scuPA. Neither the urokinase receptor (uPAR) nor the low-density lipoprotein-related receptor (LRP) is required for nuclear targeting. Rather, translocation involves the binding of scuPA to the nucleocytoplasmic shuttle protein nucleolin through a region containing the kringle domain. RNA interference and mutational analysis demonstrate that nucleolin is required for the nuclear transport of scuPA. Furthermore, nucleolin is required for the induction smooth muscle α-actin (α-SMA) by scuPA. These data reveal a novel pathway by which uPA is rapidly translocated to the nucleus where it might participate in regulating gene expression.

Means to prevent thrombus extension and local recurrence remain suboptimal, in part because of the limited effectiveness of existing thrombolytics. In theory, plasminogen activators could be used for this purpose if they could be anchored to the vascular lumen by targeting stably expressed, noninternalized determinants such as platelet-endothelial-cell adhesion molecule 1 (PECAM-1). We designed a recombinant molecule fusing low-molecular-weight single-chain prourokinase plasminogen activator (lmw-scuPA) with a single-chain variable fragment (scFv) of a PECAM-1 antibody to generate the prodrug scFv/lmw-scuPA. Cleavage by plasmin generated fibrinolytically active 2-chain lmw-uPA. This fusion protein (1) bound specifically to PECAM-1-expressing cells; (2) was rapidly cleared from blood after intravenous injection; (3) accumulated in the lungs of wild-type C57BL6/J, but not PECAM-1 null mice; and (4) lysed pulmonary emboli formed subsequently more effectively than lmw-scuPA, thereby providing support for the concept of thromboprophylaxis using recombinant scFv-fibrinolytic fusion proteins that target endothelium.

Means to prevent thrombus extension and local recurrence remain suboptimal, in part because of the limited effectiveness of existing thrombolytics. In theory, plasminogen activators could be used for this purpose if they could be anchored to the vascular lumen by targeting stably expressed, noninternalized determinants such as platelet-endothelial-cell adhesion molecule 1 (PECAM-1). We designed a recombinant molecule fusing low-molecular-weight single-chain prourokinase plasminogen activator (lmw-scuPA) with a single-chain variable fragment (scFv) of a PECAM-1 antibody to generate the prodrug scFv/lmw-scuPA. Cleavage by plasmin generated fibrinolytically active 2-chain lmw-uPA. This fusion protein (1) bound specifically to PECAM-1-expressing cells; (2) was rapidly cleared from blood after intravenous injection; (3) accumulated in the lungs of wild-type C57BL6/J, but not PECAM-1 null mice; and (4) lysed pulmonary emboli formed subsequently more effectively than lmw-scuPA, thereby providing support for the concept of thromboprophylaxis using recombinant scFv-fibrinolytic fusion proteins that target endothelium.

Activation of plasminogen by urokinase plasminogen activator (uPA) plays important roles in several physiologic and pathologic conditions. Cells secrete uPA as a single-chain molecule (scuPA). scuPA can be activated by proteolytic cleavage to a 2-chain enzyme (tcuPA). scuPA is also activated when it binds to its receptor (uPAR). The mechanism by which the enzymatic activity of the scuPA/suPAR complex is regulated is only partially understood. We now report that the plasminogen activator activity of the scuPA/suPAR complex is inhibited by Glu- and Lys-plasminogen, but not by mini-plasminogen. In contrast, neither Glunor Lys-plasminogen inhibits the activation of plasminogen by 2-chain uPA. Inhibition of scuPA/suPAR activity was evident at a Glu-plasminogen concentration of approximately 100 nM, and at physiologic plasma concentrations inhibition was nearly complete. A plasminogen fragment containing kringles 1-3 inhibited the enzymatic activity of scuPA/suPAR with an inhibition constant (Ki) equal to 1.9 μM, increased the Michaelis constant (Km) of scuPA/suPAR from 18 nM to 49 nM, and decreased the catalytic constant (Kcat) approximately 3-fold from 0.035 sec—1to 0.011 sec—1. Inhibition of scuPA/suPAR by plasminogen was completely abolished in the presence of fibrin clots. These studies provide insight into the regulation of uPA-mediated plasminogen activation and identify a novel mechanism for its fibrin specificity.

Activation of plasminogen by urokinase plasminogen activator (uPA) plays important roles in several physiologic and pathologic conditions. Cells secrete uPA as a single-chain molecule (scuPA). scuPA can be activated by proteolytic cleavage to a 2-chain enzyme (tcuPA). scuPA is also activated when it binds to its receptor (uPAR). The mechanism by which the enzymatic activity of the scuPA/suPAR complex is regulated is only partially understood. We now report that the plasminogen activator activity of the scuPA/suPAR complex is inhibited by Glu- and Lys-plasminogen, but not by mini-plasminogen. In contrast, neither Glunor Lys-plasminogen inhibits the activation of plasminogen by 2-chain uPA. Inhibition of scuPA/suPAR activity was evident at a Glu-plasminogen concentration of approximately 100 nM, and at physiologic plasma concentrations inhibition was nearly complete. A plasminogen fragment containing kringles 1-3 inhibited the enzymatic activity of scuPA/suPAR with an inhibition constant (Ki) equal to 1.9 μM, increased the Michaelis constant (Km) of scuPA/suPAR from 18 nM to 49 nM, and decreased the catalytic constant (Kcat) approximately 3-fold from 0.035 sec—1 to 0.011 sec—1. Inhibition of scuPA/suPAR by plasminogen was completely abolished in the presence of fibrin clots. These studies provide insight into the regulation of uPA-mediated plasminogen activation and identify a novel mechanism for its fibrin specificity.

The structural basis of the interaction between single-chain urokinase-type plasminogen activator (scuPA) and its receptor (uPAR) is incompletely defined. Several observations indicated the kringle facilitates the binding of uPA to uPAR. A scuPA variant lacking the kringle (ΔK-scuPA) bound to soluble uPAR (suPAR) with the similar “on-rate” but with a faster “off-rate” than wild-type (WT)-scuPA. Binding of ΔK-scuPA, but not WT-scuPA, to suPAR was comparably inhibited by its growth factor domain (GFD) and amino-terminal fragment (ATF). ATF and WT-scuPA, but not GFD, scuPA lacking the GFD (ΔGFD-scuPA), or ΔK-scuPA reconstituted the isolated domains of uPAR. ATF completely inhibited the enzymatic activity of WT-scuPA-suPAR unlike comparable concentrations of GFD. Variants containing mutations that alter the charge, length, or flexibility of linker sequence (residues 43-49) between the GFD and the kringle displayed a lower affinity for uPAR, were unable to reconstitute uPAR domains, and their binding to uPAR was inhibited by GFD in the same manner as ΔK-scuPA. A scuPA variant in which the charged amino acids in the heparin binding site (HBS) in the kringle domain were mutated to alanines behaved like ΔK-scuPA, indicating that that the structure of the kringle as well as its interaction with the GFD govern receptor binding. These data demonstrate an important role for the kringle in stabilizing the binding of scuPA to uPAR. (Blood. 2003;102:3600-3608)

The structural basis of the interaction between single-chain urokinase-type plasminogen activator (scuPA) and its receptor (uPAR) is incompletely defined. Several observations indicated the kringle facilitates the binding of uPA to uPAR. A scuPA variant lacking the kringle (ΔK-scuPA) bound to soluble uPAR (suPAR) with the similar “on-rate” but with a faster “off-rate” than wild-type (WT)-scuPA. Binding of ΔK-scuPA, but not WT-scuPA, to suPAR was comparably inhibited by its growth factor domain (GFD) and amino-terminal fragment (ATF). ATF and WT-scuPA, but not GFD, scuPA lacking the GFD (ΔGFD-scuPA), or ΔK-scuPA reconstituted the isolated domains of uPAR. ATF completely inhibited the enzymatic activity of WT-scuPA-suPAR unlike comparable concentrations of GFD. Variants containing mutations that alter the charge, length, or flexibility of linker sequence (residues 43-49) between the GFD and the kringle displayed a lower affinity for uPAR, were unable to reconstitute uPAR domains, and their binding to uPAR was inhibited by GFD in the same manner as ΔK-scuPA. A scuPA variant in which the charged amino acids in the heparin binding site (HBS) in the kringle domain were mutated to alanines behaved like ΔK-scuPA, indicating that that the structure of the kringle as well as its interaction with the GFD govern receptor binding. These data demonstrate an important role for the kringle in stabilizing the binding of scuPA to uPAR. (Blood. 2003;102:3600-3608)

Arterial occlusive disorders are a leading cause of human morbidity. We hypothesized that ectopic expression of fibrinolytic proteins in platelets could be used to favorably alter the hemostatic balance at sites of thrombosis. To test our hypothesis, we directed murine urokinase-type plasminogen activator transgene expression to platelets using a platelet factor 4 promoter. Urokinase was selectively expressed and stored in the platelets of these mice. These transgenic mice had altered platelet biology and a bleeding diathesis similar to that seen in patients with Quebec platelet disorder, affirming the role of ectopic urokinase expression as the etiology of this inherited disease. These mice were resistant to the development of occlusive carotid artery thrombosis in the absence of systemic fibrinolysis and displayed rapid resolution of pulmonary emboli. Moreover, transfusion of urokinase-expressing platelets into wild-type mice prevented formation of occlusive arterial thrombi. These studies show the feasibility of delivering fibrinolytic agents to sites of incipient thrombus formation through selective storage in platelets and offer a new strategy to prevent thrombosis and hemorrhage.

Arterial occlusive disorders are a leading cause of human morbidity. We hypothesized that ectopic expression of fibrinolytic proteins in platelets could be used to favorably alter the hemostatic balance at sites of thrombosis. To test our hypothesis, we directed murine urokinase-type plasminogen activator transgene expression to platelets using a platelet factor 4 promoter. Urokinase was selectively expressed and stored in the platelets of these mice. These transgenic mice had altered platelet biology and a bleeding diathesis similar to that seen in patients with Quebec platelet disorder, affirming the role of ectopic urokinase expression as the etiology of this inherited disease. These mice were resistant to the development of occlusive carotid artery thrombosis in the absence of systemic fibrinolysis and displayed rapid resolution of pulmonary emboli. Moreover, transfusion of urokinase-expressing platelets into wild-type mice prevented formation of occlusive arterial thrombi. These studies show the feasibility of delivering fibrinolytic agents to sites of incipient thrombus formation through selective storage in platelets and offer a new strategy to prevent thrombosis and hemorrhage.

Urokinase plasminogen activator (uPA) is a multifunctional protein that has been implicated in several physiological and pathological processes involving cell adhesion and migration in addition to fibrinolysis. In a previous study we found that two-chain urokinase plasminogen activator (tcuPA) stimulates phenylephrine-induced vasoconstriction of isolated rat aortic rings. In the present paper we report that uPA(-/-) mice have a significantly lower mean arterial blood pressure than do wild type mice and that aortic rings from uPA(-/-) mice show an attenuated contractile response to phenylephrine. In contrast, the blood pressure of urokinase receptor knockout (uPAR(-/-)) mice and the response of their isolated aortic rings to phenylephrine were normal, indicating that the effect of uPA on vascular contraction is independent of uPAR. Addition of mouse and human uPA almost completely reversed both the impaired vascular contractility and the lower arterial blood pressure in vivo. The in vitro and in vivo effects of infused uPA on aortic contractility and the restoration of normal blood pressure in uPA(-/-) mice were prevented by antibody to low-density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor (LRP). A modified form of uPA that lacks the kringle failed to restore the blood pressure in uPA(-/-) mice, notwithstanding having a longer half-life in the circulation. Ligands that regulate the interaction of uPA with LRP, such as PAI-1 or the PAI-1-derived peptide (EEIIMD), abolished the vasoactivity of tcuPA in vitro and in vivo. These studies identify a novel signal transducing cellular receptor pathway involved in the regulation of vascular contractility.

The influence of platelets on the cellular metabolism of atherogenic lipoproteins has not been characterized in detail. Therefore, we investigated the effect of platelet factor 4 (PF4), a cationic protein released in high concentration by activated platelets, on the uptake and degradation of low-density lipoprotein (LDL) via the LDL receptor (LDL-R). LDL-R–dependent binding, internalization, and degradation of LDL by cultured cells were inhibited 50%, 80%, and 80%, respectively, on addition of PF4. PF4 bound specifically to the ligand-binding domain of recombinant soluble LDL-R (half-maximal binding 0.5 μg/mL PF4) and partially (approximately 50%) inhibited the binding of LDL. Inhibition of internalization and degradation by PF4 required the presence of cell-associated proteoglycans, primarily those rich in chondroitin sulfate. PF4 variants with impaired heparin binding lacked the capacity to inhibit LDL. PF4, soluble LDL-R, and LDL formed ternary complexes with cell-surface proteoglycans. PF4 induced the retention of LDL/LDL-R complexes on the surface of human fibroblasts in multimolecular clusters unassociated with coated pits, as assessed by immuno-electron microscopy. These studies demonstrate that PF4 inhibits the catabolism of LDL in vitro in part by competing for binding to LDL-R, by promoting interactions with cell-associated chondroitin sulfate proteoglycans, and by disrupting the normal endocytic trafficking of LDL/LDL-R complexes. Retention of LDL on cell surfaces may facilitate proatherogenic modifications and support an expanded role for platelets in the pathogenesis of atherosclerosis.

The influence of platelets on the cellular metabolism of atherogenic lipoproteins has not been characterized in detail. Therefore, we investigated the effect of platelet factor 4 (PF4), a cationic protein released in high concentration by activated platelets, on the uptake and degradation of low-density lipoprotein (LDL) via the LDL receptor (LDL-R). LDL-R–dependent binding, internalization, and degradation of LDL by cultured cells were inhibited 50%, 80%, and 80%, respectively, on addition of PF4. PF4 bound specifically to the ligand-binding domain of recombinant soluble LDL-R (half-maximal binding 0.5 μg/mL PF4) and partially (approximately 50%) inhibited the binding of LDL. Inhibition of internalization and degradation by PF4 required the presence of cell-associated proteoglycans, primarily those rich in chondroitin sulfate. PF4 variants with impaired heparin binding lacked the capacity to inhibit LDL. PF4, soluble LDL-R, and LDL formed ternary complexes with cell-surface proteoglycans. PF4 induced the retention of LDL/LDL-R complexes on the surface of human fibroblasts in multimolecular clusters unassociated with coated pits, as assessed by immuno-electron microscopy. These studies demonstrate that PF4 inhibits the catabolism of LDL in vitro in part by competing for binding to LDL-R, by promoting interactions with cell-associated chondroitin sulfate proteoglycans, and by disrupting the normal endocytic trafficking of LDL/LDL-R complexes. Retention of LDL on cell surfaces may facilitate proatherogenic modifications and support an expanded role for platelets in the pathogenesis of atherosclerosis.

Urokinase-type plasminogen activator (uPA) binds to its receptor (uPAR) with a Kdof about 1 nm. The catalytic activity of the complex is apparent at uPA concentrations close to Kd. Other functions of the complex, such as signal transduction, are apparent at much higher concentrations (35–60 nm). In the present study, we show that uPA and recombinant soluble uPAR (suPAR), at concentrations that exceed the Kdand the theoretical saturation levels (10–80 nm), establish novel interactions that lead to a further increase in the activity of the single-chain uPA (scuPA)/suPAR and two-chain uPA (tcuPA)/suPAR complexes. Experiments performed using dynamic light scattering, gel filtration, and electron microscopy techniques indicate that suPAR forms dimers and oligomers. The three techniques provide evidence that the addition of an equimolar concentration of scuPA leads to the dissociation of these dimers and oligomers. Biacore data show that suPAR dimers and oligomers bind scuPA with decreased affinity when compared with monomers. We postulate that uPAR is present in equilibrium between oligomer/dimer/monomer forms. The binding of uPA to suPAR dimers and oligomers occurs with lower affinity than the binding to monomer. These novel interactions regulate the activity of the resultant complexes and may be involved in uPA/uPAR mediated signal transduction.

We examined the effect of urokinase (uPA) and its fragments on vascular smooth muscle cell contraction. Single‐chain uPA inhibits phenylepherine (PE) ‐induced contraction of rat aortic rings, whereas two‐chain uPA exerts the opposite effect. Two independent epitopes mediating these opposing activities were identified. Å6, a capped peptide corresponding to amino acids 136‐143 (KPSSPPEE) of uPA, increased the EC50of PE‐induced vascular contraction sevenfold by inhibiting the release of calcium from intracellular stores. Å6 activity was abolished by deleting the carboxyl‐terminal Glu or by mutating the Ser corresponding to position 138 in uPA to Glu. A single‐chain uPA variant lacking amino acids 136‐143 did not induce vasorelaxation. A second epitope within the kringle of uPA potentiated PE‐induced vasoconstriction. This epitope was exposed when single‐chain uPA was converted to a two‐chain molecule by plasmin. The isolated uPA kringle augmented vasoconstriction, whereas uPA variant lacking the kringle had no procontractile activity. These studies reveal previously undescribed vasoactive domains within urokinase and its naturally derived fragments.—Haj‐Yehia, A., Nassar, T., Sachais, B. S., Kuo, A., Bdeir, K., Al‐Mehdi, A. B., Mazar, A., Cines, D. B., Higazi, A. A. Urokinase‐derived peptides regulate vascular smooth muscle contraction in vitroand in vivo. FASEB J.14, 1411–1422 (2000)

Retention of lipoproteins within the vasculature is a central event in the pathogenesis of atherosclerosis. However, the signals that mediate this process are only partially understood. Prompted by putative links between inflammation and atherosclerosis, we previously reported that ?-defensins released by neutrophils are present in human atherosclerotic lesions and promote the binding of lipoprotein(a) [Lp(a)] to vascular cells without a concomitant increase in degradation. We have now tested the hypothesis that this accumulation results from the propensity of defensin to form stable complexes with Lp(a) that divert the lipoprotein from its normal cellular degradative pathways to the extracellular matrix (ECM). In accord with this hypothesis, defensin stimulated the binding of Lp(a) to vascular matrices approximately 40-fold and binding of the reactants to the matrix was essentially irreversible. Defensin formed stable, multivalent complexes with Lp(a) and with its components, apoprotein (a) and low-density lipoprotein (LDL), as assessed by optical biosensor analysis, gel filtration, and immunoelectron microscopy. Binding of defensin/Lp(a) complexes to matrix was inhibited (>90%) by heparin and by antibodies to fibronectin (>70%), but not by antibodies to vitronectin or thrombospondin. Defensin increased the binding of Lp(a) (10 nmol/L) to purified fibronectin more than 30-fold. Whereas defensin and Lp(a) readily traversed the endothelial cell membranes individually, defensin/Lp(a) complexes lodged on the cell surface. These studies demonstrate that ?-defensins released from activated or senescent neutrophils stimulate the binding of an atherogenic lipoprotein to the ECM of endothelial cells, a process that may contribute to lipoprotein accumulation in atherosclerotic lesions.

Single-chain urokinase plasminogen activator (scuPA), the unique form secreted by cells, expresses little intrinsic plasminogen activator activity. scuPA can be activated by proteolytic cleavage to form a two-chain enzyme (tcuPA), which is susceptible to inhibition by plasminogen activator inhibitor type I (PAI-1). scuPA is also activated when it binds to its cellular receptor (uPAR), in which case the protein remains as a single chain molecule with less susceptibility to PAIs. Fibrin clots are invested with PAI-1 derived from plasma and from activated platelets. Therefore, we compared the fibrinolytic activity of complexes between scuPA and recombinant soluble uPAR (suPAR) to that of scuPA, tcuPA, and tcuPA/suPAR complexes. scuPA/suPAR complexes mediated the lysis of plasma-derived fibrin clots 14-fold more extensively than did equimolar concentrations of scuPA and threefold more extensively than did tcuPA or tcuPA/suPAR, respectively. The enhanced catalytic activity of scuPA/suPAR required that all three domains of the receptor be present, correlated with its PAI-1 resistance, was not dependent on fibrin alone, and required a plasma cofactor that was identified as IgG. Human IgG bound specifically to suPAR and scuPA/suPAR as determined by using affinity chromatography and immunoprecipitation. Plasma depleted of IgG lost most of its capacity to promote the fibrinolytic activity of scuPA/suPAR, and the activity of the complex was restored by adding plasma concentrations of purified IgG. These studies indicate that scuPA/suPAR can function as a plasminogen activator in a physiological milieu. © 1998 by The American Society of Hematology.

Tissue factor (TF) has been implicated in several important biologic processes, including fibrin formation, atherogenesis, angiogenesis, and tumor cell migration. In that plasminogen activators have been implicated in the same processes, the potential for interactions between TF and the plasminogen activator system was examined. Plasminogen was found to bind directly to the extracellular domain of TF apoprotein (amino acids 1-219) as determined by optical biosensor interaction analysis. A fragment of plasminogen containing kringles 1 through 3 also bound to TF apoprotein, whereas isolated kringle 4 and miniplasminogen did not. Expression of TF on the surface of a stably transfected Chinese hamster ovary (CHO) cell line stimulated plasminogen binding to the cells by 70% more than to control cells. Plasminogen bound to a site on the TF apoprotein that appears to be distinct from the binding site for factors VII and VIIa as judged by a combination of biosensor and cell assays. TF enhanced two-chain urokinase (tcuPA) activation of Glu-plasminogen, but not of miniplasminogen, in a dose-dependent, saturable manner (half maximal stimulation at 59 pmol/L). TF apoprotein induced an effect similar to that of relipidated TF, but a relatively higher concentration of the apoprotein was required (half maximal stimulation at 3.8 nmol/L). The stimulatory effect of TF on plasminogen activation was confirmed when plasmin formation was examined directly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In accord with this, TF inhibited fibrinolysis by approximately 74% at a concentration of 14 nmol/L and almost totally inhibited the binding of equimolar concentrations of plasminogen to human umbilical vein endothelial cells and human trophoblasts. Further, CHO cells expressing TF inhibited uPA-mediated fibrinolysis relative to a wild-type control. TF apoprotein and plasminogen were found to colocalize in atherosclerotic plaque. These data suggest that plasminogen localization and activation may be modulated at extravascular sites through a high-affinity interaction between kringles 1 through 3 of plasminogen and the extracellular domain of TF.

Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC lead to an increase in its apparent molecular mass by approximately 40 Kd. The predominant u-PA binding protein isolated from whole cell detergent extracts migrated with a molecular mass of approximately 36 Kd using affinity chromatography. In contrast, when only cell surface proteins were radiolabeled before extraction, the predominant labeled u-PA binding protein isolated migrated with a molecular mass of approximately 46 Kd. Several pieces of evidence suggested that the difference in molecular mass between these two u-PA binding proteins resulted from glycosylation of a single receptor protein. First, a polyclonal antibody against u-PA receptor isolated from phorbol myristate acetate (PMA) stimulated U-937 cells reacted with both the 36- and 46-Kd proteins on Western blotting. Second, the size of the unmodified receptor was estimated by amplifying a full-length cDNA for u-PA receptor from an endothelial cell cDNA library using the polymerase chain reaction (PCR) and oligonucleotide primers corresponding to the ONA sequence of the receptor cloned from transformed human fibroblasts (Roldan et al, EMBO J 9:467, 1990). The size of the cDNA (~1,054 base pairs, bp) and the presence of a single 1.4-kilobase (Kb) mRNA transcript on Northern blot analysis predict an unglycosylated receptor protein of approximately 35 Kd. Third, synthesis of 35S-labeled 46-Kd cell surface receptor protein was inhibited when the cells were grown in the presence of tunicamycin, while the synthesis of the 36-Kd species was unaffected. Moreover, the apparent molecular mass of purified surface-labeled receptor (~46 Kd) was reduced by N-glycanase. These studies suggest that the u-PA receptor on the surface of HUVEC is a glycoprotein derived from a protein of approximately 35 Kd which is similar immunologically to u-PA receptors on other cell types.

To determine whether unsaturated fatty acids induce changes in the mRNA level of plasminogen activator inhibitor type-1 (PAI-1), Northern analyses were performed on human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells that were treated with two common fatty acids. Supplementation of cultured HUVEC with docosahexanoic acid (DHA) or with dihomogamma linolenic acid (DGLA), resulted in a concentration dependent, specific increase of the PAI-1 transcript levels, which was detectable within 2 h. DHA and DGLA treatment of smooth muscle cells did not result in changes in the PAI-1 mRNA levels. Homology search of the upstream regulatory region of the PAI-1 gene sequences identified a consensus nucleotide sequence for a fatty acid-responsive element. Our results indicate that unsaturated fatty acids selectively increase PAI-1 mRNA levels in endothelial cells, the primary source of circulating PAI-1 in vivo.

To determine whether unsaturated fatty acids induce changes in the mRNA level of plasminogen activator inhibitor type‐1 (PAI‐1), Northern analyses were performed on human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells that were treated with two common fatty acids. Supplementation of cultured HUVEC with docosahexanoic acid (DHA) or with dihomogamma linolenic acid (DGLA), resulted in a concentration dependent, specific increase of the PAI‐1 transcript levels, which was detectable within 2 h. DHA and DGLA treatment of smooth muscle cells did not result in changes in the PAI‐1 mRNA levels. Homology search of the upstream regulatory region of the PAI‐1 gene sequences identified a consensus nucleotide sequence for a fatty acid‐responsive element. Our results indicate that unsaturated fatty acids selectively increase PAI‐1 mRNA levels in endothelial cells, the primary source of circulating PAI‐1 in vivo.

This pilot study was a 16-week Telehealth intervention using wearable devices, automated text messaging, and trained health coaching, in primary care clinics of an academic medical center. Thirty patients were enrolled in three cohorts, ages 18-64, BMI > 27, and MVPA < 150 minutes per week. The primary outcome was weight loss per week.