A bispecific antibody targeting HLA-DQ2.5-gluten peptides potently blocks gluten-specific T cells induced by gluten ingestion in patients with celiac disease.

The gluten-free diet for celiac disease (CeD) is restrictive and often fails to induce complete symptom and/or mucosal disease remission. Central to CeD pathogenesis is the gluten-specific CD4+ T cell that is restricted by HLA-DQ2.5 in over 85% of CeD patients, making HLA-DQ2.5 an attractive target for suppressing gluten-dependent immunity. Recently, a novel anti-HLA-DQ2.5 antibody that specifically recognizes the complexes of HLA-DQ2.5 and multiple gluten epitopes was developed (DONQ52). OBJECTIVE: To assess the ability of DONQ52 to inhibit CeD patient-derived T-cell responses to the most immunogenic gluten peptides that encompass immunodominant T cell epitopes. METHODS: We employed an in vivo gluten challenge model in patients with CeD that affords a quantitative readout of disease-relevant gluten-specific T-cell responses. HLA-DQ2.5+ CeD patients consumed food containing wheat, barley, or rye for 3 days with collection of blood before (D1) and 6 days after (D6) commencing the challenge. Peripheral blood mononuclear cells were isolated and assessed in an interferon (IFN)-γ enzyme-linked immunosorbent spot assay (ELISpot) testing responses to gluten peptides encompassing a series of immunodominant T cell epitopes. The inhibitory effect of DONQ52 (4 or 40 µg/mL) was assessed and compared to pan-HLA-DQ blockade (SPVL3 antibody). RESULTS: In HLA-DQ2.5+ CeD patients, DONQ52 reduced T cell responses to all wheat gluten peptides to an equivalent or more effective degree than pan-HLA-DQ antibody blockade. It reduced T cell responses to a cocktail of the most immunodominant wheat epitopes by a median of 87% (IQR 72-92). Notably, DONQ52 also substantially reduced T-cell responses to dominant barley hordein and rye secalin derived peptides. DONQ52 had no effect on T-cell responses to non-gluten antigens. CONCLUSION: DONQ52 can significantly block HLA-DQ2.5-restricted T cell responses to the most highly immunogenic gluten peptides in CeD. Our findings support in vitro data that DONQ52 displays selectivity and broad cross-reactivity against multiple gluten peptide:HLA-DQ2.5 complexes. This work provides proof-of-concept multi-specific antibody blockade has the potential to meaningfully inhibit pathogenic gluten-specific T-cell responses in CeD and supports ongoing therapeutic development.

Review article: Follow-up of coeliac disease.

Coeliac disease is a lifelong immune-mediated enteropathy with systemic features associated with increased morbidity and modestly increased mortality. Treatment with a strict gluten-free diet improves symptoms and mucosal damage but is not curative and low-level gluten intake is common despite strict attempts at adherence. Regular follow-up after diagnosis is considered best-practice however this is executed poorly in the community with the problem compounded by the paucity of data informing optimal approaches. The aim of dietary treatment is to resolve symptoms, reduce complication risk and improve quality of life. It follows that the goals of monitoring are to assess dietary adherence, monitor disease activity, assess symptoms and screen for complications. Mucosal disease remission is regarded a key measure of treatment success as healing is associated with positive health outcomes. However, persistent villous atrophy is common, even after many years of a gluten-free diet. As the clinical significance of asymptomatic enteropathy is uncertain the role for routine follow-up biopsies remains contentious. Symptomatic non-responsive coeliac disease is common and with systematic follow-up a cause is usually found. Effective models of care involving the gastroenterologist, dietitian and primary care doctor will improve the consistency of long-term management and likely translate into better patient outcomes. Identifying suitable treatment targets linked to long-term health is an important goal.

Whole blood interleukin-2 release test to detect and characterize rare circulating gluten-specific T cell responses in coeliac disease.

Whole blood cytokine release assays (CRA) assessing cellular immunity to gluten could simplify the diagnosis and monitoring of coeliac disease (CD). We aimed to determine the effectiveness of electrochemiluminescence CRA to detect responses to immunodominant gliadin peptides. HLA-DQ2·5+ CD adults (cohort 1, n = 6; cohort 2, n = 12) and unaffected controls (cohort 3, n = 9) were enrolled. Cohort 1 had 3-day gluten challenge (GC). Blood was collected at baseline, and for cohort 1 also at 3 h, 6 h and 6 days after commencing 3-day GC. Gliadin peptide-stimulated proliferation, interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and 14- and 3-plex electrochemiluminescence CRA were performed. Poisson distribution analysis was used to estimate responding cell frequencies. In cohort 1, interleukin (IL)-2 dominated the gliadin peptide-stimulated cytokine release profile in whole blood. GC caused systemic IL-2 release acutely and increased gliadin peptide-stimulated IFN-γ ELISPOT and whole blood CRA responses. Whole blood CRA after GC was dominated by IL-2, but also included IFN-γ, C-X-C motif chemokine ligand 10/IFN-γ-induced protein 10 (CXCL10/IP-10), CXCL9/monokine induced by IFN-γ (MIG), IL-10, chemokine (C-C motif) ligand 3/macrophage inflammatory protein 1-alpha (CCL3/MIP-1α), TNF-α and IL-8/CXCL8. In cohorts 2 and 3, gliadin peptide-stimulated whole blood IL-2 release was 100% specific and 92% sensitive for CD patients on a gluten-free diet; the estimated frequency of cells in CD blood secreting IL-2 to α-gliadin peptide was 0·5 to 11 per ml. Whole blood IL-2 release successfully mapped human leucocyte antigen (HLA)-DQ2·5-restricted epitopes in an α-gliadin peptide library using CD blood before and after GC. Whole blood IL-2 release assay using electrochemiluminescence is a sensitive test for rare gliadin-specific T cells in CD, and could aid in monitoring and diagnosis. Larger studies and validation with tetramer-based assays are warranted.

Serum cytokines elevated during gluten-mediated cytokine release in coeliac disease.

Cytokines have been extensively studied in coeliac disease, but cytokine release related to exposure to gluten and associated symptoms has only recently been described. Prominent, early elevations in serum interleukin (IL)-2 after gluten support a central role for T cell activation in the clinical reactions to gluten in coeliac disease. The aim of this study was to establish a quantitative hierarchy of serum cytokines and their relation to symptoms in patients with coeliac disease during gluten-mediated cytokine release reactions. Sera were analyzed from coeliac disease patients on a gluten free-diet (n = 25) and from a parallel cohort of healthy volunteers (n = 25) who underwent an unmasked gluten challenge. Sera were collected at baseline and 2, 4 and 6 h after consuming 10 g vital wheat gluten flour; 187 cytokines were assessed. Confirmatory analyses were performed by high-sensitivity electrochemiluminescence immunoassay. Cytokine elevations were correlated with symptoms. Cytokine release following gluten challenge in coeliac disease patients included significant elevations of IL-2, chemokine (C-C motif) ligand 20 (CCL20), IL-6, chemokine (C-X-C motif) ligand (CXCL)9, CXCL8, interferon (IFN)-γ, IL-10, IL-22, IL-17A, tumour necrosis factor (TNF)-α, CCL2 and amphiregulin. IL-2 and IL-17A were earliest to rise. Peak levels of cytokines were generally at 4 h. IL-2 increased most (median 57-fold), then CCL20 (median 10-fold). Cytokine changes were strongly correlated with one another, and the most severely symptomatic patients had the highest elevations. Early elevations of IL-2, IL-17A, IL-22 and IFN-γ after gluten in patients with coeliac disease implicates rapidly activated T cells as their probable source. Cytokine release after gluten could aid in monitoring experimental treatments and support diagnosis.

Food knowledge and psychological state predict adherence to a gluten-free diet in a survey of 5310 Australians and New Zealanders with coeliac disease.

BACKGROUND: A gluten-free diet treats coeliac disease, but its efficacy depends on strict adherence. A variety of patient factors may influence adherence but have not been well described at a population level. AIM: To comprehensively assess the patient factors that influence gluten-free diet adherence in patients with coeliac disease. METHODS: Patients with coeliac disease completed an online survey comprising the validated Celiac Dietary Adherence Test in addition to data on demographics, details of diagnosis and management and assessment of diet knowledge, quality of life and psychological distress. Survey data were analysed for predictors of adherence and quality of life. RESULTS: Of 7393 responses, 5310 completed the Celiac Dietary Adherence Test and 3230 (61%) were adherent to a gluten-free diet. Multivariate regression showed older age, being male, symptoms after gluten ingestion, better food knowledge and lower risk of psychological distress were independent predictors of adherence (each P ≤ 0.008). Additionally, dietary adherence was associated with better quality of life (P < 0.001; multiple regression). Respondents who considered themselves to have poor food knowledge were more likely to incorrectly identify gluten-free foods, but could still recognise gluten-containing foods, suggesting that poor knowledge may lead to over-restriction of diet. CONCLUSIONS: Poor knowledge of a gluten-free diet and psychological wellbeing were independent modifiable risk factors for inadequate adherence to a gluten-free diet in patients with coeliac disease. Involvement of both a dietitian and mental health care professional, in the presence of psychological distress, is likely to be necessary to improve adherence and health outcomes.

Resolving incomplete single nucleotide polymorphism tagging of HLA-DQ2.2 for coeliac disease genotyping using digital droplet PCR.

A hallmark of coeliac disease (CD) is the exceptionally strong genetic association with HLA-DQ2.5, DQ8, and DQ2.2. HLA typing provides information on CD risk important to both clinicians and researchers. A method that enables simple and fast detection of all CD risk genotypes is particularly desirable for the study of large populations. Single nucleotide polymorphism (SNP)-based HLA typing can detect the CD risk genotypes by detecting a combination of six SNPs but this approach can struggle to resolve HLA-DQ2.2, seen in 4% of European CD patients, because of the low resolution of one negatively predicting SNP. We sought to optimise SNP-based HLA typing by harnessing the additional resolution of digital droplet PCR to resolve HLA-DQ2.2. Here we test this two-step approach in an unselected sample of Mexican DNA and compare its accuracy to DNA typed using traditional exon detection. The addition of digital droplet PCR for samples requiring negative prediction of HLA-DQ2.2 enabled HLA-DQ2.2 to be accurately typed. This technique is a simple addition to a SNP-based typing strategy and enables comprehensive definition of all at-risk HLA genotypes in CD in a timely and cost-effective manner.

The practice and perception of precautionary allergen labelling by the Australasian food manufacturing industry.

BACKGROUND: The precautionary allergen labelling (PAL) and Voluntary Incidental Trace Allergen Labelling (VITAL® ) tools were designed by industry to assist consumers with selecting safe foods for consumption. However, a sizeable proportion of food products bear no label, and it is unclear whether these products are free from allergens and therefore safe to consume or have simply not undergone a risk assessment and therefore remain unlabelled for that reason. OBJECTIVE: To assess the prevalence of unlabelled products that have undergone a risk assessment process and to examine the factors influencing industry's uptake of the VITAL® process. METHODS: A web-based questionnaire was distributed to Australasian food and grocery manufacturers. RESULTS: One hundred and thirty-seven Australasian manufacturers were contacted, and 59 questionnaires were returned (response rate: 43%). The respondents represented 454 different manufacturing sites. Manufacturers reported that 23% (95% CI 19-28) of products (n=102/434) that had been through the VITAL® risk assessment process had no PAL statement on the label. 34% (95% CI 30-38), (n=204/600) of products that had undergone another (non-VITAL® ) risk assessment process had no PAL statement. In examining the factors that influenced industry's uptake of the VITAL® process, 25 manufacturers reported on factors that influenced the uptake of the VITAL® process, 76% (CI 95% 55-91) reported that VITAL® was an effective tool because it was based on science; 52% (CI 95% 31-72) reported that it was too time-consuming and 36% (CI 95% 18-57) identified a concern with it not being endorsed by the government. CONCLUSION AND CLINICAL RELEVANCE: Currently, we estimate that at least 30% of products may have been through a risk assessment process and yet bear no PAL statement on the label. Permissive labelling could be incorporated onto these products if they have been assessed to be safe for consumption.

Appropriate clinical use of human leukocyte antigen typing for coeliac disease: an Australasian perspective.

The past decade has seen human leukocyte antigen (HLA) typing emerge as a remarkably popular test for the diagnostic work-up of coeliac disease with high patient acceptance. Although limited in its positive predictive value for coeliac disease, the strong disease association with specific HLA genes imparts exceptional negative predictive value to HLA typing, enabling a negative result to exclude coeliac disease confidently. In response to mounting evidence that the clinical use and interpretation of HLA typing often deviates from best practice, this article outlines an evidence-based approach to guide clinically appropriate use of HLA typing, and establishes a reporting template for pathology providers to improve communication of results.

Ex-vivo whole blood secretion of interferon (IFN)-γ and IFN-γ-inducible protein-10 measured by enzyme-linked immunosorbent assay are as sensitive as IFN-γ enzyme-linked immunospot for the detection of gluten-reactive T cells in human leucocyte antigen (HLA)-DQ2·5(+) -associated coeliac disease.

T cell cytokine release assays are used to diagnose infectious diseases, but not autoimmune or allergic disease. Coeliac disease (CD) is a common T cell-mediated disease diagnosed by the presence of gluten-dependent intestinal inflammation and serology. Many patients cannot be diagnosed with CD because they reduce dietary gluten before medical workup. Oral gluten challenge in CD patients treated with gluten-free diet (GFD) mobilizes gluten-reactive T cells measurable by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) or major histocompatibility complex (MHC) class II tetramers. Immunodominant peptides are quite consistent in the 90% of patients who possess HLA-DQ2·5. We aimed to develop whole blood assays to detect gluten-specific T cells. Blood was collected before and after gluten challenge from GFD donors confirmed to have CD (n = 27, all HLA-DQ2·5(+) ), GFD donors confirmed not to have CD (n = 6 HLA-DQ2·5(+) , 11 HLA-DQ2·5(-) ) and donors with CD not following GFD (n = 4, all HLA-DQ2·5(+) ). Plasma IFN-γ and IFN-γ inducible protein-10 (IP-10) were measured by enzyme-linked immunosorbent assay (ELISA) after whole blood incubation with peptides or gliadin, and correlated with IFN-γ ELISPOT. No T cell assay could distinguish between CD patients and controls prior to gluten challenge, but after gluten challenge the whole blood IFN-γ ELISA and the ELISPOT were both 85% sensitive and 100% specific for HLA-DQ2·5(+) CD patients; the whole blood IP-10 ELISA was 94% sensitive and 100% specific. We conclude that whole blood cytokine release assays are sensitive and specific for detection of gluten-reactive T cells in CD; further clinical studies addressing the utility of these tests in patients with an uncertain diagnosis of CD is warranted.

The risk of recurrence in breast cancer patients treated with tamoxifen: polymorphisms of CYP2D6 and ABCB1.

CYP2D6 plays a major role in the metabolism of tamoxifen, and polymorphism of P-glycoprotein has been associated with resistance of many drug therapies. This study investigates the clinical impact of genetic variants of CYP2D6 and ABCB1 in breast cancer patients treated with tamoxifen. Blood samples from 95 breast cancer patients treated with tamoxifen were collected and genotyped for CYP2D6 and ABCB1 variants using allele-specific PCR method. Recurrence risks were calculated using Kaplan-Meier analysis and compared using the log-rank test. Patients carrying CYP2D6*10/*10 and heterozygous null allele (IM) showed higher risks of developing recurrence and metastasis (OR 13.14; 95% CI 1.57-109.94; P = 0.004) than patients with CYP2D6*1/*1 and *1/*10 genotypes. Patients with homozygous CC genotypes of ABCB1 C3435T showed a shorter time to recurrence. Patients who were CYP2D6 IM and homozygous CC genotype of C3435T have statistically significant higher risks of recurrence (P = 0.002). Similarly, median time to recurrence in these patients was only 12 months (95% CI = 0.79-23.2) compared to those without this combination which was 48 months (95% CI = 14.7-81.2). Patients with CYP2D6 IM and homozygous CC genotype of ABCB1 C3435T have shorter times to recurrence. The results confirmed the findings of previous studies and support FDA recommendation to perform pre-genotyping in patients before the choice of therapy is determined in breast cancer patients.

Effect of moderate walnut consumption on lipid profile, arterial stiffness and platelet activation in humans.

BACKGROUND/OBJECTIVES: A large intake of walnuts may improve lipid profile and endothelial function. The effect of moderate walnut consumption is not known. We investigated whether a moderate intake of walnuts would affect lipid profile, arterial stiffness and platelet activation in healthy volunteers. SUBJECTS/METHODS: A total of 30 healthy males were recruited into a single-blind randomized controlled crossover trial of 4 weeks of dietary walnut supplementation (15 g/day) and 4 weeks of control (no walnuts). Arterial stiffness was assessed using pulse waveform analysis to determine the augmentation index and augmented pressure. Platelet activation was determined using flow cytometry to measure circulating platelet-monocyte aggregates. RESULTS: There were no differences in lipid profile after 4 weeks of walnut supplementation compared with control. Dietary intake of α-linolenic acid was increased during the walnut diet (2.1±0.4 g/day versus 0.7±0.4 g/day, P<0.0001). There were no differences in augmentation index or augmented pressure during walnut supplementation. Walnut supplementation did not affect platelet-monocyte aggregation. CONCLUSIONS: Dietary intervention with a moderate intake of walnuts does not affect lipid profile, arterial stiffness or platelet activation in man. Our results suggest that the potentially beneficial cardiac effects of walnuts may not be apparent at lower and more practical levels of consumption.

Dissecting the T-cell response to hordeins in coeliac disease can develop barley with reduced immunotoxicity.

BACKGROUND: Wheat, rye and barley prolamins are toxic to patients with coeliac disease. Barley is diploid with pure inbred cultivars available, and is attractive for genetic approaches to detoxification. AIM: To identify barley hordein fractions which activated the interferon-γ (IFN-γ) secreting peripheral blood T-cells from coeliac volunteers, and compare immunotoxicity of hordeins from experimental barley lines. METHODS: To reactivate a T-cell response to hordein, volunteers underwent a 3-day oral barley challenge. Peripheral blood mononuclear cells (PBMC) were isolated from twenty-one HLA DQ2(+) patients with confirmed coeliac disease. IFN-γ ELISpot assays enumerated T-cells activated by purified prolamins and positive controls. RESULTS: Hordein-specific T-cells were induced by oral barley challenge. All prolamin fractions were immunotoxic, but D- and C-hordeins were most active. Barley lines lacking B- and C-hordeins had a 5-fold reduced hordein-content, and immunotoxicity of hordein extracts were reduced 20-fold compared with wild-type barley. CONCLUSIONS: In vivo oral barley challenge offers a convenient and rapid approach to test the immunotoxicity of small amounts of purified hordeins using fresh T-cells from patients in high throughput overnight assays. Barley lines that did not accumulate B- and C-hordeins were viable, yet had substantially reduced immunotoxicity. Creation of hordein-free barley may therefore be possible.

The influence of the menstrual cycle, normal pregnancy and pre-eclampsia on platelet activation.

Platelet activation has a key role in mediating thrombotic and inflammatory events. This study aimed to determine the influence of the menstrual cycle, pregnancy and pre-eclampsia on in vivo platelet activation. Twelve healthy nulliparous, non-smoking women with regular menses were studied over a single menstrual cycle. Twenty-one healthy primigravida pregnant women were studied longitudinally at 16, 24, 32 and 37 weeks gestation and seven weeks post-partum. Sixteen primigravida women with pre-eclampsia were studied at time of diagnosis and at seven weeks post-partum. Platelet-monocyte aggregates and platelet-surface P-selectin expression were assessed by flow-cytometry. Soluble P-selectin and CD40 ligand (CD40L) were measured by ELISA. Markers of platelet activation did not vary over the menstrual cycle. Platelet-monocyte aggregates were greater in the third trimester of pregnancy compared to non-pregnant women (p=0.003). Platelet surface and plasma soluble P-selectin concentrations increased with gestation (p<0.0001) and were raised by 24 weeks of pregnancy compared to non-pregnant women (p< or =0.02 for both) and together with platelet monocyte aggregates, decreased post-partum (p< or =0.02). Soluble CD40L concentrations fell in pregnancy, reaching a nadir at mid-gestation (p=0.002). There were no differences in markers of platelet activation between normal and pre-eclamptic pregnancies. In conclusion, platelet activation is increased in pregnancy and increases with gestation but is unaffected by pre-eclampsia. This suggests that systemic platelet activation is a feature of pregnancy but this is not affected by established pre-eclampsia.

Acute endothelial tissue plasminogen activator release in pregnancy.

OBJECTIVE: Pregnancy is associated with marked changes in vascular physiology and an increased risk of thrombosis. The aim of the study was to assess the effect of pregnancy on the acute release of tissue plasminogen activator (t-PA) from the endothelium. METHODS AND RESULTS: Ten primigravida pregnant women were recruited in the third trimester of pregnancy (week 36 +/- 1) and compared with 20 age-matched non-pregnant women (day 9.8 +/- 0.3 of menstrual cycle). Blood flow and plasma fibrinolytic factors were measured in both forearms by venous occlusion plethysmography and blood sampling, respectively, during unilateral brachial artery infusions of bradykinin (100-1000 pmol min(-1)). Pregnant women had higher plasma plasminogen activator inhibitor type 1 (PAI-1) antigen concentrations (77.1 +/- 12.4 vs. 21.5 +/- 9.8 ng mL(-1); P = 0.004) that resulted in lower basal t-PA/PAI-1 ratios (0.2 +/- 0.1 vs. 0.6 +/- 0.1; P = 0.02) and plasma t-PA activity concentrations (0.17 +/- 0.02 vs. 0.58 +/- 0.06 IU mL(-1); P < 0.0004). In both groups, bradykinin caused dose-dependent increases in blood flow and local release of plasma t-PA antigen and activity (P < 0.005 for all). Both the plasma t-PA/PAI-1 ratios and the net release of active t-PA were markedly reduced in pregnant women (P < 0.05 for both). Area under the curve for net active t-PA release was reduced by 36%. CONCLUSIONS: Pregnancy is associated with major perturbations of endogenous fibrinolytic capacity with an overwhelming increase in plasma PAI-1 concentrations and an inadequate release of active t-PA. These prothrombotic effects may, in part, explain the increased risk of arterial and venous thrombosis in pregnant women.

Aspiration in the context of upper gastrointestinal endoscopy.

BACKGROUND: Pulmonary aspiration is a life-threatening complication of upper gastrointestinal endoscopy, the incidence of which has not been determined. Endoscopy-related aspiration has not been studied in procedures in which patients swallow a radiolabelled potential aspirate immediately before endoscopy and undergo nuclear scanning postprocedure. METHODS: A pilot study was conducted in which 200 MBq of nonabsorbable technetium-99m phytate in 10 mL of water was administered orally to 50 patients who were about to undergo endoscopy. Gamma camera images were obtained to ensure that there had been no aspiration before endoscopy. After endoscopy, a repeat scan was performed. Fluid aspirated through the endoscope was also collected and analyzed for radioactivity using a hand-held radiation monitor. RESULTS: No evidence of pulmonary aspiration was found in any of the patients studied. The mean estimated percentage of the initially administered radioactivity aspirated through the endoscope was 2.66% (range 0% to 10.3%). CONCLUSION: The present pilot study confirms earlier observations that clinically significant aspiration in the context of upper gastrointestinal endoscopy is uncommon. The incidence of aspiration may, however, be different in acutely bleeding patients undergoing endoscopy. For logistic reasons, this group could not be studied.

Functional interplay between platelet activation and endothelial dysfunction in patients with coronary heart disease.

Platelet-monocyte binding and surface P-selectin expression are sensitive markers of platelet activation. Endothelium-derived factors are known to inhibit platelet activation and may confer important anti-atherothrombotic effects. We assessed the relationship between platelet activation and endothelium-dependent vasomotion in patients with coronary heart disease (CHD). Twenty male patients with stable CHD were compared with 20 healthy men. Platelet-monocyte binding and platelet surface expression of P-selectin were assessed using two-colour flow cytometry on whole blood. Forearm blood flow was assessed in patients using venous occlusion plethysmography during intra-arterial infusions of substance P, acetylcholine and sodium nitroprusside. Platelet activation was higher in patients than healthy men (platelet-monocyte binding, 27 +/- 3 vs. 20 +/- 1%; P < 0.05). In patients with CHD, there was an inverse correlation between maximal substance P induced vasodilatation and both platelet-monocyte binding (P = 0.003) and P-selectin expression (P = 0.02). A similar correlation was observed between platelet-monocyte binding and the vasomotor response to acetylcholine (P = 0.08) but not with sodium nitroprusside. In patients with stable coronary heart disease, there is a strong inverse relationship between markers of platelet activation and endothelium-dependent vasomotor function. This may explain the pathophysiological mechanism linking endothelial vasomotor dysfunction and the risk of acute atherothrombotic events.

Promotion of proinflammatory interactions between platelets and monocytes by unfractionated heparin.

OBJECTIVES: To determine the in vitro effects of unfractionated heparin, fractionated heparin and direct thrombin inhibition on platelet-monocyte aggregation, and to establish the in vivo effects of unfractionated heparin and direct thrombin inhibition on platelet-monocyte aggregates in patients scheduled for percutaneous coronary intervention (PCI). DESIGN: Platelet-monocyte aggregates were assessed in whole blood from 18 healthy volunteers after the addition of unfractionated heparin (1 U/ml), enoxaparin (0.8 U/ml) or lepirudin (5.6 microg/ml), and in 28 patients scheduled for elective PCI before and after administration of 100 U/kg of unfractionated heparin or 0.75 mg/kg bivalirudin. The influence of P-selectin-mediated platelet-monocyte aggregation was assessed with specific blocking antibodies. RESULTS: Addition of unfractionated heparin in vitro was associated with a higher level of platelet-monocyte aggregates than in controls (20.1 (1.9)% v 16.2 (1.6)%, respectively, p < 0.001). However, platelet-monocyte aggregation was not affected by enoxaparin or lepirudin (16.9 (2.0)% and 17.0 (2.2)%, respectively, NS). Intravenous unfractionated heparin in vivo also resulted in an increase in platelet-monocyte aggregates (absolute Delta 7.1 (2.7)%, p < 0.01), whereas intravenous bivalirudin had no effect (absolute Delta -1.5 (2.4)%, NS). The addition of P-selectin blockade abolished any increase in platelet-monocyte aggregates associated with heparin. CONCLUSIONS: In vitro and in vivo unfractionated heparin is associated with increased platelet-monocyte aggregation through a P-selectin-dependent mechanism. These findings provide a potential explanation for the superior cardiovascular outcomes associated with fractionated heparins and direct thrombin inhibitors.