Dermot Kenny

Conformation-Specific Blockade of the Integrin GPIIb/IIIa: A Novel Antiplatelet Strategy That Selectively Targets Activated Platelets

Platelet activation causes conformational changes of integrin GPIIb/IIIa (&agr;IIb&bgr;3), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to design agents that specifically block activated platelets only. We used phage display of single-chain antibody (scFv) libraries in combination with several rounds of depletion/selection to obtain human scFvs that bind specifically to the activated conformation of GPIIb/IIIa. Functional evaluation of these scFv clones revealed that fibrinogen binding to human platelets and platelet aggregation can be effectively inhibited by activation-specific scFvs. In contrast to clinically used GPIIb/IIIa blockers, which are all conformation unspecific, activation-specific GPIIb/IIIa blockers do not induce conformational changes in GPIIb/IIIa or outside-in signaling, as evaluated by ligand-induced binding-site (LIBS) exposure in flow cytometry or P-selectin expression in immunofluorescence microscopy, respectively. In contrast to the conformation-unspecific blocker abciximab, activation-specific scFvs permit cell adhesion and spreading on immobilized fibrinogen, which is mediated by nonactivated GPIIb/IIIa. Mutagenesis studies and computer modeling indicate that exclusive binding of activation-specific scFv is mediated by RXD motifs in the heavy-chain complementary-determining region (CDR) 3 of the antibodies, which in comparison with other antibodies forms an exceptionally extended loop. In vivo experiments in a ferric-chloride thrombosis model of the mouse carotid artery demonstrate similar antithrombotic potency of activation-specific scFv, when compared with the conformation-unspecific blockers tirofiban and eptifibatide. However, in contrast to tirofiban and eptifibatide, bleeding times are not prolonged with the activation-specific scFvs, suggesting lower bleeding risks. In conclusion, activation-specific GPIIb/IIIa blockade via human single-chain antibodies represents a promising novel strategy for antiplatelet therapy.

Screening for lung cancer using low dose CT scanning

Background: Lung cancer is the most common cause of cancer related death in Ireland. The majority of lung cancers are inoperable at the time of diagnosis and consequently the overall 5 year survival is less than 10%. The objective of the ProActive Lung Cancer Detection (PALCAD) study was to evaluate whether low dose chest computed tomographic scanning (LDCCT) can detect early stage asymptomatic lung cancer in a high risk urban population. Methods: Four hundred and forty nine subjects of median age 55 years (range 50–74) with a median pack year smoking history of 45 years (range 10–160), with no previous cancer history and medically fit to undergo thoracic surgery were recruited. After informed consent, LDCCT was performed on all subjects. Non-calcified nodules (NCNs) of ⩾10 mm in diameter were referred for biopsy. Follow up with interval LDCCT at 6, 12 and 24 months to exclude growth was recommended for NCNs <10 mm in diameter. Results: Six (1.3%) NCNs of ⩾10 mm were detected of which one (0.23%) had non-small cell lung cancer stage 1; 145 NCNs of <10 mm were detected in 87 (19.4%) subjects. Mediastinal masses were detected in three subjects (0.7%)—one small cell lung cancer and two benign duplication cysts. Incidental pathology was noted in 276 patients (61.5%), most commonly emphysema and coronary artery calcification. Conclusion: The prevalence of resectable lung cancer detected by LDCCT at baseline screening was low at 0.23%, but there was a high rate of significant incidental pathology.

Screening for lung cancer using low dose CT scanning: results of 2 year follow up

Background: Screening with low dose chest computed tomographic scanning (LDCCT) may improve survival by identifying early asymptomatic lung cancer. Methods: Four hundred and forty nine high risk subjects were screened with serial LDCCT scanning over 2 years. Fine needle aspiration biopsy was recommended for non-calcified nodules (NCNs) of >10 mm diameter or demonstrating interval growth. Results: NCNs were identified in 111 subjects (24.7%), three of which were lung cancer. The overall prevalence (0.4%) and incidence (1.3%) rates of lung cancer detection were low. Three of the six lung cancers detected in the study were stage 1 non-small cell lung cancer; the remainder were unresectable central tumours. By contrast, eight subjects developed extrathoracic malignancy during the study period and other incidental pathology was noted in 221 subjects (49.2%). Smoking cessation rates at 19% were higher than in the general population, but 60.8% of subjects continued to smoke. Conclusion: LDCCT scanning is useful in detecting early peripheral non-small cell lung cancers but its usefulness as a screening tool is limited by low specificity and by poor sensitivity for central tumours.

Platelet Adhesion and Degranulation Induce Pro-Survival and Pro-Angiogenic Signalling in Ovarian Cancer Cells

Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.

Diagnosis of suspected inherited platelet function disorders: results of a worldwide survey

Diagnosis of inherited platelet function disorders (IPFDs) is important for appropriate management and to improve epidemiologic and clinical knowledge. However, there remains a lack of consensus on the diagnostic approach.

Electrophysiologic findings in Fabry's disease with a short PR interval

Fabry’s disease is an X-linked lysosomal storage disease caused by a deficiency of a-galactosidase-A that results in an accumulation of intracellular glycolipid in the heart and other organs. l Several electrocardiographic changes have been described in affected patients, including varying degrees of atrioventricular block, ST and T-wave changes, and a short PR interval.24 These changes are thought to be due to glycosphingolipid deposition involving the myocardial fibers and conduction system.24 However, the etiology of a short PR interval in Fabry’s disease is not completely understood. We report a patient with heterozygous Fabry’s disease who presented with palpitations and a short PR interval, and underwent detailed electrophysiologic testing. A 43-year-old white woman with a well-documented history of a carrier state for Fabry’s disease (based on decreased levels of a-galactosidase)presented with palpitations and dyspnea. On examination, her pulse was irregular, with a rate of 150 beats1 min. An initial electrocardiogram demonstrated atrialBbrillation, with an average ventricular response of approximately 150 beatslmin and shortest RR interval of 350 ms. A total of 0.75 mg of digoxin was administered in sequential doses, followed by conversion to normal sinus rhythm. Further digoxin dosing was held. An electrocardiogram recorded during sinus rhythm (Figure 1) showed a short PR interval of 110 ms, with a normal QRS duration and no evidence of preexcitation. The echocardiogram was normal. To assess the etiology of the short PR interval, an electrophysiologic

Platelet function and HIV: a case-control study.

Objective:Cardiovascular disease and myocardial infarction are of increasing concern in HIV-infected populations. Although platelets mediate arterial thrombosis, central to myocardial infarction, data on platelet function in HIV infection are lacking. We hypothesized that HIV-infected patients would have altered platelet reactivity. Design:A case–control study of platelet reactivity in 20 HIV-infected (HIVpos) and 20 age and sex-matched HIV-negative (HIVneg) individuals. Methods:Time-dependent platelet aggregation was measured in response to increasing concentrations of platelet agonists: epinephrine, collagen, thrombin receptor-activating peptide and ADP using light absorbance. Results:In both groups, mean age was 34 years, and 65% were men. Sixteen out of 20 (80%) of the HIVpos patients were on antiretroviral therapy with 12 out of 20 (60%) patients having HIV RNA less than 50 copies/ml. There were significant between-group differences in platelet reactivity across all four agonists. Platelets from HIVpos patients were more reactive to epinephrine [mean (SD) log concentration required to induce 50% maximal aggregation, 1.9 (1.2) versus 3.0 (1.7) μmol/l in HIVneg individuals, P = 0.028], whereas less platelet aggregation was observed in response to submaximal concentrations of the other agonists [thrombin receptor-activating peptide 72.5 (14.5)% versus 82.2 (7.6)% at 10 μmol/l, P = 0.011; ADP 67.3 (12.1)% versus 75.2 (8.8)% at 10 μmol/l, P = 0.035; collagen 16.6 (25.1)% versus 35.4 (31.5)% at 71.25 μg/ml, P = 0.007]. Conclusion:Between-group differences in platelet responses to all agonists suggest multiple underlying defects in platelet function in HIV infection. Further research is required to determine the contribution of antiretroviral therapy and relationships between platelet function and the increased cardiovascular disease observed in HIV-infected populations.

Increased Platelet Reactivity in HIV-1–Infected Patients Receiving Abacavir-Containing Antiretroviral Therapy

BACKGROUND Current or recent use of abacavir for treating human immunodeficiency virus type 1 (HIV-1) infection has been associated with increased rates of myocardial infarction (MI). Given the role of platelet aggregation in thrombus formation in MI and the reversible nature of the abacavir association, we hypothesized that patients treated with abacavir would have increased platelet reactivity. METHODS In a prospective study in adult HIV-infected patients, we determined associations between antiretrovirals (ARVs), and in particular the nucleoside reverse transcriptase inhibitor abacavir, and platelet reactivity by measuring time-dependent platelet aggregation in response to agonists: adenosine diphosphate (ADP), thrombin receptor-activating peptide (TRAP), collagen, and epinephrine. RESULTS Of 120 subjects, 40 were ARV-naive and 80 ARV-treated, 40 of whom were receiving abacavir. No consistent differences in platelet reactivity were observed between the ARV-naive and ARV-treated groups. In contrast, within the ARV-treated group, abacavir-treated subjects had consistently higher percentages of platelet aggregation upon exposure to ADP, collagen, and epinephrine (P = .037, P = .022, and P = .032, respectively) and had platelets that were more sensitive to aggregation upon exposure to TRAP (P = .025). CONCLUSIONS The consistent increases in platelet reactivity observed in response to a range of agonists provides a plausible underlying mechanism to explain the reversible increased rates of MI observed in abacavir-treated patients.

The role of Nox1 and Nox2 in GPVI-dependent platelet activation and thrombus formation

Background Activation of the platelet-specific collagen receptor, glycoprotein (GP) VI, induces intracellular reactive oxygen species (ROS) production; however the relevance of ROS to GPVI-mediated platelet responses remains unclear. Objective The objective of this study was to explore the role of the ROS-producing NADPH oxidase (Nox)1 and 2 complexes in GPVI-dependent platelet activation and collagen-induced thrombus formation. Methods and results ROS production was measured by quantitating changes in the oxidation-sensitive dye, H2DCF-DA, following platelet activation with the GPVI-specific agonist, collagen related peptide (CRP). Using a pharmacological inhibitor specific for Nox1, 2-acetylphenothiazine (ML171), and Nox2 deficient mice, we show that Nox1 is the key Nox homolog regulating GPVI-dependent ROS production. Nox1, but not Nox2, was essential for CRP-dependent thromboxane (Tx)A2 production, which was mediated in part through p38 MAPK signaling; while neither Nox1 nor Nox2 was significantly involved in regulating CRP-induced platelet aggregation/integrin αIIbβ3 activation, platelet spreading, or dense granule and α-granule release (ATP release and P-selectin surface expression, respectively). Ex-vivo perfusion analysis of mouse whole blood revealed that both Nox1 and Nox2 were involved in collagen-mediated thrombus formation at arterial shear. Conclusion Together these results demonstrate a novel role for Nox1 in regulating GPVI-induced ROS production, which is essential for optimal p38 activation and subsequent TxA2 production, providing an explanation for reduced thrombus formation following Nox1 inhibition.

Human IgG antibody profiles differentiate between symptomatic patients with and without colorectal cancer

Objective: Patients with cancer have antibodies against tumour antigens. Characterising the antibody repertoire may provide insights into aberrant cellular mechanisms in cancer development, ultimately leading to novel diagnostic or therapeutic targets. The aim of this study was to characterise the antibody profiles in patients whose symptoms warranted colonoscopy, to see if there was a difference in patients with and without colorectal cancer. Methods: Patients were recruited from a colonoscopy clinic. Individual serum samples from 43 patients with colorectal cancer and 40 patients with no cancer on colonoscopy were profiled on a 37 830 clone recombinant human protein array. Antigen expression was evaluated by quantitative reverse transcription-PCR and by immunohistochemistry on tissue microarrays. Results: Using a sex- and age-matched training set, 18 antigens associated with cancer and 4 associated with the absence of cancer (p<0.05) were identified and confirmed. To investigate the mechanisms triggering antibody responses to these antigens, antigen expression was examined in normal colorectal mucosa and colorectal carcinoma of the same patients. The identified antigens showed cellular accumulation (p53), aberrant cellular expression (high mobility group B1 (HMGB1)) and overexpression (tripartite motif-containing 28 (TRIM28), p53, HMGB1, transcription factor 3 (TCF3), longevity assurance gene homologue 5 (LASS5) and zinc finger protein 346 (ZNF346)) in colorectal cancer tissue compared with normal colorectal mucosa. Conclusions: It is demonstrated for the first time that screening high-density protein arrays identifies unique antibody profiles that discriminate between symptomatic patients with and without colorectal cancer. The differential expression of identified antigens suggests their involvement in aberrant cellular mechanisms in cancer.