Trial protocol: a clustered, randomised, longitudinal, type 2 translational trial of alcohol consumption and alcohol-related harm among adolescents in Australia.

BACKGROUND: This cluster randomised control trial is designed to evaluate whether the Communities That Care intervention (CTC) is effective in reducing the proportion of secondary school age adolescents who use alcohol before the Australian legal purchasing age of 18 years. Secondary outcomes are other substance use and antisocial behaviours. Long term economic benefits of reduced alcohol use by adolescents for the community will also be assessed. METHODS: Fourteen communities and 14 other non-contiguous communities will be matched on socioeconomic status (SES), location, and size. One of each pair will be randomly allocated to the intervention in three Australian states (Victoria, Queensland and Western Australia). A longitudinal survey will recruit grade 8 and 10 students (M = 15 years old, N = 3500) in 2017 and conduct follow-up surveys in 2019 and 2021 (M = 19 years old). Municipal youth populations will also be monitored for trends in alcohol-harms using hospital and police administrative data. DISCUSSION: Community-led interventions that systematically and strategically implement evidence-based programs have been shown to be effective in producing population-level behaviour change, including reduced alcohol and drug use. We expect that the study will be associated with significant effects on alcohol use amongst adolescents because interventions adopted within communities will be based on evidence-based practices and target specific problems identified from surveys conducted within each community. TRIAL REGISTRATION: The trial was retrospectively registered in September, 2017 ( ACTRN12616001276448 ), as communities were selected prior to trial registration; however, participants were recruited after registration. Findings will be disseminated in peer-review journals and community fora.

A prospective study of alcohol expectancies and self-efficacy as predictors of young adolescent alcohol misuse.

AIMS: To test the relative contribution of two key Social Learning Theory constructs, alcohol expectancies (AEs) and drinking refusal self-efficacy (DRSE), in predicting early adolescent drinking behavior and examine the possible mediational role of DRSE over AE. METHODS: High school students (N = 192, mean age 14) were administered measures assessing AE (Drinking Expectancy Questionnaire--Adolescent version; DEQ-A), DRSE (Drinking Refusal Self-Efficacy Questionnaire--Revised Adolescent version; DRSEQ-RA) and indices of alcohol consumption and problem drinking. Age, gender, peer drinking, tobacco use and positive and negative behavioral characteristics were included in the statistical models as known predictors of alcohol misuse. Subjects were followed up at 12 months, with 88.5% retention. RESULTS: Initial confirmatory factor analyses verified factor structures of the DEQ-A and DRSEQ-RA. Prospective structural models controlling for Time 1 drinking behavior, age, gender, peer alcohol use, tobacco use and behavior problems identified that DRSE but not AE was associated with problem drinking 12-month post-initial assessment. DRSE mediated AE in predicting problem drinking. CONCLUSION: Results suggest that DRSE is a more salient cognitive construct than AE in early adolescence alcohol use. In this age group, prevention and treatment strategies that build refusal self-efficacy may be more effective than strategies that challenge AEs.

Effects of tirofiban on haemostatic activation in vitro.

BACKGROUND: Thrombin plays a critical role in normal haemostasis and pathological thrombosis. Heparin has long been a mainstay choice of antithrombotic regimen in cardiac patients, but persistent thrombin generation seems to occur during heparin therapy. Because platelets are integral to primary haemostasis and clot formation, we evaluated the use of tirofiban (Aggrastat),a platelet inhibitor, as a therapy to improve heparin sensitivity and delay thrombin formation. METHODS: Blood samples were obtained from healthy subjects (n=8) and cardiac surgical patients (n=34). Thrombin formation was measured in platelet-rich plasma with a Thrombogram-Ascent fluorescent plate reader system. Platelet inhibition by tirofiban was evaluated with Plateletworks, and the interaction of tirofiban and heparin (>1.5 U ml(-1)) on clot formation was evaluated with Sonoclot Analyzer or kaolin activated clotting times (ACTs). RESULTS: Addition of tirofiban (70-280 ng ml(-1)) progressively delayed onset of thrombin generation triggered by adenosine diphosphate (ADP). Plateletworks showed platelet inhibition with tirofiban (>35 ng ml(-1)), whereas heparin per se failed to produce platelet inhibition at 7 U ml(-1). Heparin (1.5 U ml(-1)) slowed the onset and rate of fibrin formation on Sonoclot analyses, and this was further slowed after addition of tirofiban (70 ng ml(-1)) to heparin-containing blood samples. Significant increases in ACT at all heparin concentrations were observed with the addition of tirofiban (70 ng ml(-1)). The addition of antithrombin (0.2 units/ml) to heparinized blood samples further prolonged ACTs, but the difference was not statistically significant when compared with heparin alone. CONCLUSION: Tirofiban delays platelet activation-mediated thrombin generation and prolongs ACT in heparinized blood.

Helping the female partners of men abusing alcohol: a comparison of three treatments.

AIM: To evaluate the effectiveness of three approaches to assisting the female partners of male problem drinkers with the stress imposed by the male's drinking. DESIGN: Participants were assigned randomly via random number tables to one of three treatment conditions: supportive counselling, stress management or alcohol-focused couples therapy. SETTING: The intervention took place at the Behaviour Research and Therapy Centre (BRTC), The University of Queensland. This research and training centre offers outpatient psychology services to the community. PARTICIPANTS: Sixty-one married women whose husbands drank heavily. Participants reported protracted alcohol problems, severe impact of alcohol on social functioning and severe marital distress. MEASUREMENT: The women's stress, alcohol consumption by the male, and relationship functioning were assessed at pre- and post-treatment and at 6-month follow-up. INTERVENTIONS: All three treatments involved 15 1-hour sessions with the woman. In the alcohol-focused couple therapy, attempts were made to engage the man in these sessions. RESULTS: Contrary to our predictions, there were few differences between the treatments. All three treatments were associated with reductions in the women's reported stress, with trends for somewhat greater reduction in the women's stress in the stress management and alcohol-focused couples therapy conditions than for supportive counselling. None of the treatments produced clinically significant reductions in men's drinking or relationship distress. CONCLUSION: The treatments ease stresses and burden but do not improve drinking or relationships. Limited power in the design restricted the capacity to detect differential treatment effects.

Maritally distressed women with alcohol problems: the impact of a short-term alcohol-focused intervention on drinking behaviour and marital satisfaction.

AIM: To evaluate the efficacy of a short-term alcohol-focused intervention for maritally distressed women, and to explore changes in relationship functioning. DESIGN: Participants were assigned randomly to an alcohol-focused treatment or to a waiting-list control group. The waiting-list control group began the intervention at 1-month follow-up. SETTING: The intervention took place at a research and training centre offering outpatient psychology services to the community. PARTICIPANTS: A sample of 32 women with alcohol and marital problems were recruited through the media. Participants reported protracted alcohol problems, moderate to severe impact of alcohol on social and occupational functioning, and moderate to severe marital distress. MEASUREMENTS: Measures of average alcohol consumption, marital distress, relational efficacy and depression were administered at pre- and post-therapy, and at 1, 6 and 12-month follow-up. INTERVENTION: The intervention involved six 1-hour sessions, consisting of clinical assessment, motivational interviewing, cognitive-behavioural strategies and relapse prevention. RESULTS: At 1-month follow-up, the intervention was associated with statistically significant improvements in alcohol consumption, marital satisfaction, relational efficacy and depression, and these effects were sustained at 12-month follow-up. CONCLUSIONS: At 1-month follow-up the intervention was associated with decreased alcohol consumption and depression, and increased marital satisfaction and relational efficacy, with evidence of maintained effects at 12-month follow-up. However, it is unlikely that reduced problem drinking and improved confidence in resolving problems were the only factors producing low marital quality in these couples. Further research is needed to identify those individuals who might benefit from marital interventions.

Reduction in vascular lesion formation by hirudin secreted from retrovirus-transduced confluent endothelial cells on vascular grafts in baboons.

BACKGROUND: The hypothesis that thrombin mediates the formation of neointimal vascular lesions at sites of mechanical vascular injury has been tested in baboons by measurement of the effects of hirudin delivered by retrovirus-transduced hirudin-secreting vascular endothelial cells (ECs) lining surgically implanted arterial vascular grafts (AVGs). METHODS AND RESULTS: The antithrombotic efficacy of baboon ECs transduced with cDNA encoding hirudin was assessed in vitro and in vivo on thrombogenic segments in chronically exteriorized femoral arteriovenous (AV) shunts. Bilateral brachial AVGs lined with hirudin-transduced versus nonhirudin control ECs at confluent density were surgically implanted, and vascular lesion formations at distal graft-vessel anastomoses were compared after 30 days. Hirudin-transduced ECs secreted 20+/-6 ng x 10(6) cells(-1) x 24 h(-1) (range, 14 to 24 ng x 10(6) cells(-1) x 24 h(-1)) hirudin in supernatants of static cultures. Hirudin-secreting ECs on segments of collagen-coated graft interposed in chronic AV shunts decreased the accumulation of (111)In-labeled platelets to 0.52+/-0.34 x 10(9) platelets, compared with 0.82+/-0.49 x 10(9) platelets in controls (P = 0.03) and reduced platelet deposition in propagated thrombotic tails extending downstream from segments of vascular graft from 1.38+/-0.41 x 10(9) platelets in controls to 0.59+/-0.22 x 10(9) platelets (P = 0.04). ECs recovered from 30-day AVG implants generated 17+/-9 ng x 10(6) cells(-1) x 24 h(-1) (range, 9 to 25 ng x 10(6) cells(-1) x 24 h(-1)) hirudin. Hirudin-secreting ECs reduced neointimal lesion formation at distal graft-vessel anastomoses, ie, 1.02 mm(2) (range, 0.88 to 1.95 mm(2)) versus 1.82 mm(2) (range, 0.88 to 2.56 mm(2)) in contralateral AVGs bearing nonhirudin control ECs (P<0.01). CONCLUSIONS: Viral vector-directed secretion of hirudin from ECs lining implanted AVGs significantly reduces the formation of thrombus and neointimal vascular lesions.

Distinct patterns of neuropeptide gene expression in the lateral hypothalamic area and arcuate nucleus are associated with dehydration-induced anorexia.

We have investigated the hormonal and hypothalamic neuropeptidergic substrates of dehydration-associated anorexia. In situ hybridization and hormone analyses of anorexic and paired food-restricted rats revealed two distinct profiles. First, both groups had the characteristic gene expression and endocrine signatures usually associated with starvation: increased neuropeptide Y and decreased proopiomelanocortin and neurotensin mRNAs in the arcuate nucleus (ARH); increased circulating glucocorticoid but reduced leptin and insulin. Dehydrated animals are strongly anorexic despite these attributes, showing that the output of leptin- and insulin-sensitive ARH neurons that ordinarily stimulate eating must be inhibited. The second pattern occurred only in anorexic animals and had two components: (1) reduced corticotropin-releasing hormone (CRH) mRNA in the neuroendocrine paraventricular nucleus (PVH) and (2) increased CRH and neurotensin mRNAs in the lateral hypothalamic (LHA) and retrochiasmatic areas. However, neither corticosterone nor suppressed PVH CRH gene expression is required for anorexia after dehydration because PVH CRH mRNA in dehydrated adrenalectomized animals is unchanged from euhydrated adrenalectomized controls. We also showed that LHA CRH mRNA was strongly correlated with the intensity of anorexia, increased LHA CRH gene expression preceded the onset of anorexia, and dehydrated adrenalectomized animals (which also develop anorexia) had elevated LHA CRH gene expression with a distribution pattern similar to intact animals. Finally, we identified specific efferents from the CRH-containing region of the LHA to the PVH, thereby providing a neuroanatomical framework for the integration by the PVH of neuropeptidergic signals from the ARH and the LHA. Together, these observations suggest that CRH and neurotensin neurons in the LHA constitute a novel anatomical substrate for their well known anorexic effects.

The progression of thrombus in an ex-vivo shunt model evaluated by intravascular ultrasound radiofrequency analysis.

We tested the ability of ultrasound radiofrequency (RF) signal analysis to characterize thrombus accumulation in a Dacron graft incorporated into the exteriorized arteriovenous shunt in 3 baboons with constant blood flow for 60 min. Thrombus formation was quantified by sequential measurements of 111Indium-labeled platelet deposition. RF signals were acquired every 15 min at 2 sites in the graft, using a 2.9 Fr intravascular ultrasound catheter-based transducer (30 MHz) and digitized at 250 MHz in 8-bit resolution. Regions of interest were placed within a 0.5-mm perimeter adjacent to the graft wall. Integrated backscatter increased significantly (p < 0.001) with increasing platelet deposition. However, mean-to-standard deviation ratio of the RF envelope showed no significant change and the distribution pattern of the RF probability function remained constant and consistent with a Rayleigh scattering process. These results provide a basis for using RF analysis to monitor the time-course of thrombus formation.

The role of alpha and beta platelet-derived growth factor receptor in the vascular response to injury in nonhuman primates.

Restenosis remains a significant clinical problem associated with mechanical interventional procedures for arterial revascularization or repair, including coronary angioplasty and stenting. Studies with rodents have established that platelet-derived growth factor (PDGF), a potent chemotactic and mitogenic agent for vascular smooth muscle cells, is a key mediator of lesion formation after vascular injury. To further explore this hypothesis in a more clinically relevant model, neutralizing monoclonal antibodies (mAbs) were used to examine the effect of selective inhibition of alpha or beta PDGF receptor (PDGFR) on neointima formation in nonhuman primates. Carotid arteries were injured by surgical endarterectomy and femoral arteries by balloon catheter dilatation. Immunostaining revealed that both injuries induced cell proliferation and the upregulation of beta PDGFR but not alpha PDGFR. By 7 days after injury, beta PDGFR staining was limited to the luminal region of the media, the small areas of neointima, and the adventitia. Nearly all bromodeoxyuridine-positive cells were found in these regions as well. After 30 days, a concentric neointima that stained strongly for beta PDGFR had formed in the carotid and femoral arteries. Treatment of baboons with anti-beta PDGFR mAb 2A1E2 for 6 days after injury reduced the carotid artery and femoral artery lesion sizes by 37% (P<0.05) and 48% (P<0.005), respectively, when measured at 30 days. Under the same conditions, treatment with anti-alpha PDGFR mAb 2H7C5 had no effect. These findings suggest that PDGF mediates neointima formation through the beta PDGFR, and that antagonism of this pathway may be a promising therapeutic strategy for reducing clinical restenosis.

Clopidogrel inhibition of stent, graft, and vascular thrombogenesis with antithrombotic enhancement by aspirin in nonhuman primates.

BACKGROUND: A recent study showed that clopidogrel reduces thrombo-occlusive complications in patients with symptomatic atherosclerosis more effectively than aspirin. METHODS AND RESULTS: The effects of clopidogrel and aspirin have been compared, singly and in combination, for measurements of 111In-labeled platelets and 125I-labeled fibrin deposition in baboon models of arterial thrombosis and related to platelet aggregation and expression of activation epitopes induced by ADP, collagen, and thrombin receptor agonist peptide (TRAP) and to template bleeding times (BTs). Low-dose oral clopidogrel (0.2 mg. kg-1. d-1) produced cumulative (1) intermediate decreases in 111In-platelet and 125I-fibrin deposition for segments of prosthetic vascular graft, deployed endovascular metallic stents, and endarterectomized aorta (P<0.009 in all cases); (2) elimination of ADP-induced platelet aggregation (P<0.001); (3) modest inhibition of collagen-induced platelet aggregation (P<0.01); (4) no reduction in TRAP-induced platelet aggregation; and (5) minimal prolongation of BTs (P=0.03). High-dose oral clopidogrel (>/=2 mg/kg) produced the same effects within 3 hours. The effects of clopidogrel dissipated over 5 to 6 days. Aspirin 10 mg. kg-1. d-1 alone did not decrease 111In-platelet and 125I-fibrin deposition on segments of vascular graft but detectably decreased 111In-platelet and 125I-fibrin accumulation on stents (P<0.01), minimally inhibited ADP- and collagen-induced platelet aggregation (P<0.05 in both cases), and minimally prolonged BTs (P=0.004). Within 3 hours of aspirin administration, the antithrombotic effects of acute high-dose or chronic low-dose clopidogrel were substantially enhanced, and BTs were modestly prolonged without inhibiting platelet aggregation induced by TRAP (P<0.001 in all cases compared with clopidogrel alone). CONCLUSIONS: Clopidogrel produces irreversible, dose-dependent, intermediate reduction in thrombosis that is substantially enhanced by the addition of aspirin. The effects of combining aspirin and clopidogrel need to be evaluated in patients at risk of vascular thrombosis.

Treatment of thrombocytopenia in chimpanzees infected with human immunodeficiency virus by pegylated recombinant human megakaryocyte growth and development factor.

Three chimpanzees experimentally infected with human immunodeficiency virus (HIV) developed significant chronic thrombocytopenia after 5, 4, and 2 years, with peripheral platelet counts averaging 64 +/- 19 x 10(3)/microL (P = .004 compared with 228 +/- 92 x 10(3)/microL in 44 normal control animals), mean platelet volumes of 11.2 +/- 1.8 fL (P > .5 compared with 10.9 +/- 0. 7 fL in normal controls), endogenous thrombopoietin (TPO) levels of 926 +/- 364 pg/mL (P < .001 compared with 324 +/- 256 pg/mL in normal controls), uniformly elevated platelet anti-glycoprotein (GP) IIIa49-66 antibodies, and corresponding viral loads of 534, 260, and 15 x 10(3) RNA viral copies/mL. Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) was administered subcutaneously (25 microg/kg twice weekly for 3 doses) to determine the effects of stimulating platelet production on peripheral platelet concentrations in this cohort of thrombocytopenic HIV-infected chimpanzees. PEG-rHuMGDF therapy increased (1) peripheral platelet counts 10-fold (from 64 +/- 19 to 599 +/- 260 x 10(3) platelets/microL; P = .02); (2) marrow megakaryocyte numbers 30-fold (from 11.7 +/- 6.5 x 10(6)/kg to 353 +/- 255 x 10(6)/kg; P = .04); (3) marrow megakaryocyte progenitor cells fourfold (from a mean of 3.6 +/- 0.6 to 14.1 x 10(3) CFU-Meg/1, 000 CD34(+) marrow cells); and (4) serum levels of Mpl ligand from 926 +/- 364 pg/mL (endogenous TPO) to predosing trough levels of 1, 840 +/- 353 pg/mL PEG-rHuMGDF (P = .02). The peripheral neutrophil counts were also transiently increased from 5.2 +/- 2.6 x 10(3)/microL to 9.9 +/- 5.0 x 10(3)/microL (P = .01), but neither the erythrocyte counts nor the reticulocyte counts were altered significantly (P > .1). The serum levels of antiplatelet GPIIIa49-66 antibodies exhibited reciprocal reductions during periods of thrombocytosis (P < .07). PEG-rHuMGDF therapy did not increase viral loads significantly (395, 189, and 53 x 10(3) RNA viral copies/mL; P > .5 compared with baseline values). The striking increase in peripheral platelet counts produced by PEG-rHuMGDF therapy implies that thrombocytopenia in HIV-infected chimpanzees is attributable to insufficient compensatory expansion in platelet production resulting from HIV-impaired delivery of platelets despite stimulated megakaryocytopoiesis. These data suggest that PEG-rHuMGDF therapy may similarly correct peripheral platelet counts in thrombocytopenic HIV-infected patients.

The region of the pontine parabrachial nucleus is a major target of dehydration-sensitive CRH neurons in the rat lateral hypothalamic area.

Neurons in a restricted part of the lateral hypothalamic area (LHA) show increased expression of corticotropin-releasing hormone (CRH) mRNA as a consequence of cellular dehydration. In the present study, we have investigated the organization of their efferent projections by using anterograde and retrograde tracing techniques. Additionally, we have compared the distribution of CRH mRNA-containing neurons after cellular dehydration and intraventricular (i.c.v.) colchicine injections. Our results show that cellular dehydration activates a more restricted neuronal population than does i.c.v. colchicine. Iontophoretic injections of Phaseolus vulgaris leucoagglutinin (PHAL) were placed in the LHA of animals drinking hypertonic saline and their proximity to activated CRH neurons determined by in situ hybridization for CRH mRNA. Although labelled fibers from these injections were seen throughout the brain, the region of the parabrachial nucleus and nucleus of the solitary tract (NTS) were most conspicuous in also having CRH immunoreactive fibers. Injections of Fluoro-Gold placed in these two structures were used to confirm these findings in dehydrated animals. Significant numbers of neurons containing both Fluoro-Gold and CRH mRNA were seen in the lateral hypothalamus after injections in the lateral and medial parts of the parabrachial nucleus; far fewer were seen after injections in the NTS. These results strongly suggest that the CRH neurons in the LHA activated by cellular dehydration provide an input to the region of the parabrachial nucleus. The altered biochemical composition of this pathway may well be able to modify sensory and motor patterns both during and after dehydration.

Effects of Mpl ligands on platelet production and function in nonhuman primates.

Endogenous thrombopoietin (TPO) stimulates platelet production in nonhuman primates dose-dependentbyinducing megakaryocyte development from early marrow hematopoietic progenitors and subsequent proliferation and endoreduplication. Recombinant human TPO, nonpegylated or pegylated recombinant human megakaryocyte growth and development factor produce log-linear responses in peak peripheral platelet counts (or peripheral platelet mass turnover), platelet TPO receptor density, and marrow megakaryocyte volume, ploidy, number and mass. Mpl ligand therapy sustains normal peripheral platelet concentrations following myelosuppressive chemotherapy in baboons and corrects peripheral platelet counts in HIV-infected chimpanzees with severe thrombocytopenia. Whereas Mpl ligands do not directly induce platelet aggregation in vitro, they enhance aggregatory responsiveness of platelets to physiologic agonists both in vitro and transiently ex vivo following treatment with Mpl ligands. However, platelet recruitment into forming thrombus is not augmented by these agents when evaluated in quantitative rabbit or baboon models of platelet-dependent thrombus formation, except for the direct effect of platelet concentration per se. These findings indicate that appropriate dosing of these agents prevents thrombocytopenia without increasing the risk of platelet-dependent thrombo-occlusive complications.

Reduction in stent and vascular graft thrombosis and enhancement of thrombolysis by recombinant Lys-plasminogen in nonhuman primates.

BACKGROUND: To enhance thrombolytic responses without increasing hemorrhagic risks, the antithrombotic effects of recombinant Lys-plasminogen (r-LysPgn), a prothrombolytic plasminogen intermediate, were examined in baboon models of thrombus formation and dissolution. METHODS AND RESULTS: The dose-response effects of r-LysPgn, alone or in combination with subthreshold dosing of tissue plasminogen activator (TPA), were measured with respect to the accumulation of (111)In-labeled platelets and (125)I-fibrin in thrombus forming on endovascular metallic stents or thrombogenic segments of vascular graft interposed in exteriorized long-term arteriovenous (AV) femoral shunts. Thrombolytic losses have also been determined for preformed, stable, (111)In-platelet- and (125)I-fibrin-labeled graft thrombus and corresponding propagated thrombotic tails, together with changes in blood tests of thrombosis, thrombolysis, and hemostasis. Bolus intravenous r-LysPgn in escalating doses (2, 4, or 8 mg/kg) increased circulating plasminogen levels in a dose-dependent manner, was removed by log-linear clearance with a T50 of 120 minutes, and reciprocally decreased the accumulating thrombus on metallic stents and segments of vascular graft (P<.001 in all cases for 8-mg/kg doses). r-LysPgn also impaired platelet aggregatory responses to physiological agonists in vitro but not ex vivo. Prethrombosis administration of low-dose r-LysPgn (2 mg/kg) greatly enhanced the lysis of radiolabeled nonoccluding thrombus by a subthreshold dose of TPA (0.1 mg/kg) compared with TPA-only controls (P=.03). CONCLUSIONS: Elective bolus injections of r-LysPgn before stent deployment decrease the amount of thrombus formed without compromising hemostasis by facilitating endogenous TPA thrombolysis. r-LysPgn may provide effective and safe antithrombotic therapy for interventional vascular procedures.

Antithrombotic strategies targeting thrombin activities, thrombin receptors and thrombin generation.

Thrombin mediates acute vascular thrombosis and subsequent vascular lesion formation following mechanical denuding injury or spontaneous atherosclerotic plaque rupture. In the process of generating thrombin Factor VII/VIIa binds avidly with tissue factor (TF) exposed on cellular membranes, and coagulation serine proteases are sequentially cleaved via macromolecular catalytic complexes on phospholipid surfaces. Thrombin activates platelets, blood leukocytes, endothelium and vascular smooth muscle cells (SMCs) by cleaving G protein-coupled thrombin receptors (TRs), leading to SMC intimal proliferation and synthesis of extracellular matrix in the local formation of stenosing neointimal vascular lesions. Therapeutic strategies include inactivation of bound thrombin, inhibition of TR activation by thrombin, and interruption of thrombin generation. In patients having orthopedic surgery, inactivating bound thrombin with direct antithrombins markedly reduces venous thromboembolic events, compared with heparin or its derivatives, without significant impairment of hemostasis. However, acute coronary syndrome patients are not benefitted when given systemic direct antithrombins at safe levels, because interrupting TR-dependent platelet thrombosis demands systemic levels of direct antithrombins that concurrently compromise hemostatic function. Local drug delivery strategies have yet to be explored. In preclinical studies: a) enhancing the formation of endogenous activated Protein C (APC) by Protein C-selective thrombin mutants produces antithrombotic levels of APC; b) inhibiting thrombin activation of TRs abolishes platelet recruitment in arterial thrombogenesis in nonhuman primates, while sparing fibrin formation in hemostatic plugs; and c) preventing thrombin generation by inhibiting precursor serine protease function interrupts the formation of both acute thrombosis and chronic stenotic lesions after denuding vascular damage without significant hemostatic compromise. TF antagonists appear to have a highly favorable efficacy:safety therapeutic relationship for preventing the formation of thrombosis and vascular lesions.

Induction of vascular endothelial growth factor in balloon-injured baboon arteries. A novel role for reactive oxygen species in atherosclerosis.

Neovascularization is a hallmark of neointimal formation in atherosclerotic plaques and restenotic lesions. Vascular endothelial growth factor (VEGF) promotes neovascular growth, whereas oxidative stress is a potent factor in vascular cell proliferation. To investigate the mechanisms of neovascular formation, we treated human and rat vascular smooth muscle cells (VSMCs) with H2O2. Northern blot analysis demonstrated a dose- and time-dependent increase in VEGF mRNA, with a maximum of 4-fold at 3 hours (200 mumol/L). As determined by immunoblotting and enzyme-linked immunosorbent assay, VEGF protein expression and secretion were similarly increased. Human umbilical vein endothelial cells were treated with conditioned medium from VSMCs incubated with 200 mumol/L H2O2. DNA synthesis, measured by thymidine incorporation, was increased 4-fold compared with control, an effect that was blocked by a neutralizing anti-VEGF antibody. The lipid peroxidation product 4-hydroxynonenal (1 mumol/L), an endogenous reactive oxygen species present in human atherosclerotic lesions, also increased VEGF secretion in VSMCs in a similar time-dependent fashion. Immunohistochemical staining and in situ hybridization of aortic sections from balloon-injured baboons demonstrated increased VEGF expression in discrete areas of the neointima and media compared with control sections, and expression correlated with the generation of 4-hydroxynonenal. Regulators of VEGF expression, such as reactive oxygen species, may enhance neovascularization of atherosclerotic and restenotic arteries.

Anti-VLA-4 antibody reduces intimal hyperplasia in the endarterectomized carotid artery in nonhuman primates.

PURPOSE: Recently, very late antigen-4 (VLA-4) has been shown to mediate initial monocyte adhesion and migration to the injured artery. We hypothesized that blocking monocyte adhesion using a specific monoclonal antibody against VLA-4 may reduce intimal hyperplasia. METHODS: Bilateral carotid endarterectomies were performed in eight adult baboons. Among them, five animals received an intravenous bolus injection of anti-VLA-4 antibody (3 mg/kg) during surgery and again after 2 weeks. Three animals underwent bilateral carotid endarterectomies and served as untreated control subjects. Specimens were harvested at 4 weeks and subjected to morphometric analysis, cell proliferation assay, and immunostaining for macrophages. RESULTS: All of the endarterectomized arteries were patent except for one in the treated group. The number of macrophages in the intimal tissues was significantly reduced in the treated arteries compared with that in the control vessels (15.78 +/- 3.05 cells/section versus 33.50 +/- 6.13 cells/section; p < 0.001). The cell proliferation rate was significantly lower (p < 0.001) in the treated vessels (2.88% +/- 1.07%) compared with the control vessels (4.89% +/- 0.77%). The intimal area at the endarterectomized sites of carotid arteries was significantly less (p < 0.05) in the group treated with the anti-VLA-4 antibody (1.10 +/- 0.68 mm2) than in the control group (2.00 +/- 0.52 mm2). CONCLUSION: These data show that blocking monocyte adhesion by use of an anti-VLA-4 antibody significantly reduces the number of intimal macrophages, intimal cell proliferation, and intimal hyperplasia in injured carotid arteries in baboons. This study supports a central role for macrophages in the development of intimal hyperplasia and may suggest a new therapeutic strategy to prevent clinical restenosis.

Antithrombotic potency of hirudin is increased in nonhuman primates by fibrin targeting.

BACKGROUND: Inhibition of thrombin by either the indirect thrombin inhibitor heparin or by more potent direct thrombin inhibitors such as hirudin reduces thrombus formation after arterial injury. The present study was designed to determine if a fibrin-specific thrombin inhibitor could, by local thrombin inhibition, prevent thrombosis more effectively. METHODS AND RESULTS: We first studied antithrombotic potency in vitro, comparing fibrin-targeted hirudin (recombinant hirudin covalently linked to the Fab' fragment of the anti-fibrin monoclonal antibody 59D8) to recombinant hirudin in baboon plasma. Fibrin-targeted hirudin was nine times more effective than recombinant hirudin in inhibiting fibrin deposition on experimental clot surfaces in baboon plasma (P < .01). The potency of fibrin-targeted hirudin was then compared with that of recombinant hirudin in a baboon model of thrombus formation. 111In-labeled platelet deposition was measured in a synthetic graft segment of an extracorporeal arteriovenous shunt in control animals and in animals receiving either fibrin-targeted hirudin or hirudin. In these experiments, fibrin-targeted hirudin was 10-fold more potent than hirudin in inhibiting platelet deposition and thrombus formation (P < .05). CONCLUSIONS: These data indicate that targeting a thrombin inhibitors such as hirudin to an epitope present in thrombi results in increased antithrombotic potency.