Serum Beta-Defensin-2 is a biomarker for psoriasis but not subclinical atherosclerosis: Role of IL17a, PI-3 kinase and Rac1.

Background: Beta-defensins (BDs) are antimicrobial peptides secreted upon epithelial injury. Both chemotactic and antimicrobial properties of BDs function as initial steps in host defense and prime the adaptive immune system in the body. Psoriasis, a chronic immune-mediated inflammatory disease, has both visible cutaneous manifestations as well as known associations with higher incidence of cardiometabolic complications and vascular inflammation. Objectives: We aimed to investigate the circulating expression of beta-defensin-2 (BD2) in psoriasis at baseline compared to control subjects, along with changes in BD2 levels following biologic treatment at one-year. The contribution of BD2 to subclinical atherosclerosis is also assessed. In addition, we have sought to unravel signaling mechanisms linking inflammation with BD2 expression. Methods: Multimodality imaging as well inflammatory biomarker assays were performed in biologic naïve psoriasis (n=71) and non-psoriasis (n=53) subjects. A subset of psoriasis patients were followed for one-year after biological intervention (anti-Tumor Necrosis Factor-α (TNFα), n=30; anti-Interleukin17A (IL17A), n=21). Measurements of circulating BD2 were completed by Enzyme-Linked Immunosorbent Assay (ELISA). Using HaCaT transformed keratinocytes, expression of BD2 upon cytokine treatment was assessed by quantitative polymerase chain reaction (qPCR) and ELISA. Results: Herein, we confirm that human circulating BD2 levels associate with psoriasis, which attenuate upon biologic interventions (anti-TNFα, anti-IL-17A). A link between circulating BD2 and sub-clinical atherosclerosis markers was not observed. Furthermore, we demonstrate that IL-17A-driven BD2 expression occurs in a Phosphatidylinositol 3-kinase (PI3-kinase) and Rac1 GTPase-dependent manner. Conclusions: Our findings expand on the potential role of BD2 as a tractable biomarker in psoriasis patients and describes the role of an IL-17A-PI3-kinase/Rac signaling axis in regulating BD2 levels in keratinocytes.

The psoriasis area and severity index is an independent risk factor for cardiovascular events: A prospective register study.

BACKGROUND: Patients treated with systemics for moderate-to-severe psoriasis have increased risk of cardiovascular disease (CVD). However, to the best of our knowledge, there are no data on the association between clinical disease activity and future cardiovascular events in this population. Such data could help identify patients at increased CVD risk and inform the potential for CVD prevention with effective psoriasis treatment. OBJECTIVE: To estimate the association between the Psoriasis Area and Severity Index (PASI) and cardiovascular events defined as hospitalization for CVD and cardiovascular death. METHODS: We linked prospectively collected data on PASI and CVD risk factors to population-based administrative data on hospitalizations and causes of death. We estimated the association between PASI and cardiovascular events using Cox proportional hazard models with PASI and Framingham 10-year cardiovascular risk as time-varying covariates. RESULTS: A total of 767 patients with 6264 PASI scores were included. After adjusting for 10-year cardiovascular risk and prior CVD, a one-point increase in PASI was associated with a hazard ratio of 1.04 (95% CI: 1.01-1.07) for cardiovascular events. The findings were robust in sensitivity analyses. CONCLUSION: PASI is an independent marker for future cardiovascular events in patients with moderate-to-severe psoriasis.

Spatial transcriptomics landscape of lesions from non-communicable inflammatory skin diseases.

Abundant heterogeneous immune cells infiltrate lesions in chronic inflammatory diseases and characterization of these cells is needed to distinguish disease-promoting from bystander immune cells. Here, we investigate the landscape of non-communicable inflammatory skin diseases (ncISD) by spatial transcriptomics resulting in a large repository of 62,000 spatially defined human cutaneous transcriptomes from 31 patients. Despite the expected immune cell infiltration, we observe rather low numbers of pathogenic disease promoting cytokine transcripts (IFNG, IL13 and IL17A), i.e. >125 times less compared to the mean expression of all other genes over lesional skin sections. Nevertheless, cytokine expression is limited to lesional skin and presented in a disease-specific pattern. Leveraging a density-based spatial clustering method, we identify specific responder gene signatures in direct proximity of cytokines, and confirm that detected cytokine transcripts initiate amplification cascades of up to thousands of specific responder transcripts forming localized epidermal clusters. Thus, within the abundant and heterogeneous infiltrates of ncISD, only a low number of cytokine transcripts and their translated proteins promote disease by initiating an inflammatory amplification cascade in their local microenvironment.

Inflammatory bowel disease and psoriasis: modernizing the multidisciplinary approach.

Psoriasis and inflammatory bowel disease (IBD) are immune-mediated diseases occurring in barrier organs whose main task is to protect the organism from attack. These disorders are highly prevalent especially in northern Europe where psoriasis has a prevalence of around 3-4% and IBD around 0.3%. The prevalence of IBD in North America has been estimated at around 0.4%. The total incidence rates in northern Europe have been estimated at around 6 for Crohn's disease and 11 for ulcerative colitis per 100 000 person-years, compared with an incidence rate of around 280 per 100 000 person-years for psoriasis. Both diseases are less common in countries with a lower index of development. The rise in IBD appears to occur as populations adopt a westernized lifestyle, whereas psoriasis seems more stable and prevalence differences may derive more from variation in genetic susceptibility. The gut microbiota is clearly an important driver of IBD pathogenesis; in psoriasis, changes in gut and skin microbiota have been reported, but it is less clear whether and how these changes contribute to the pathogenesis. Large studies show that most identified genes are involved in the immune system. However, psoriasis and IBD are highly heterogeneous diseases and there is a need for more precise and deeper phenotyping to identify specific subgroups and their genetic, epigenetic and molecular signatures. Epigenetic modifications of DNA such as histone modifications, noncoding RNA effects on transcription and translation and DNA methylation are increasingly recognized as the mechanism underpinning much of the gene-environment interaction in the pathogenesis of both IBD and psoriasis. Our understanding of underlying pathogenetic mechanisms has deepened fundamentally over the past decades developing hand in hand with novel therapies targeting pathways and proinflammatory cytokines incriminated in disease. There is not only substantial overlap between psoriasis and IBD, but also there are differences with implication for therapy. In psoriasis, drugs targeting interleukin-23 and interleukin-17 have shown superior efficacy compared with anti-TNFs, whilst in IBD, drugs targeting interleukin-17 may be less beneficial. The therapeutic toolbox for psoriasis is impressive and is enlarging also for IBD. Still, there are unmet needs reflecting the heterogeneity of both diseases and there is a need for closer molecular diagnostics to allow for the development of precise therapeutics.

Characteristics and outcomes of patients treated with apremilast in the real world: results from the APPRECIATE study.

BACKGROUND: APPRECIATE is a multinational, observational, retrospective, cross-sectional study in patients treated for psoriasis with apremilast, an oral phosphodiesterase 4 inhibitor. OBJECTIVES: To describe the characteristics of patients with psoriasis treated with apremilast in the clinical setting, to evaluate real-world outcomes of psoriasis treatment with apremilast and to better understand the perspectives of patients and physicians on treatment outcomes. METHODS: In six European countries, patients with chronic plaque psoriasis treated in clinical practice who could be contacted 6 (±1) months after apremilast initiation were enrolled. Patient characteristics, Dermatology Life Quality Index (DLQI) and Psoriasis Area and Severity Index (PASI) were obtained from medical records when available. Outcomes were evaluated using patient/physician questionnaires. RESULTS: In 480 patients at treatment initiation, mean [median; 95% confidence interval (CI)] PASI and DLQI scores were 12.5 (10.7; 11.6-13.4) and 13.4 (13.0; 11.4-14.2), respectively. At 6 (±1) months, 72.3% of patients (n = 347) continued apremilast treatment [discontinuations: lack of efficacy (13.5%), safety (11.7%), other (2.5%)]. In patients continuing treatment, 48.6% achieved a ≥75% reduction in PASI score; mean (95% CI) DLQI score was 5.7 (4.5-6.9), and mean (SD) Patient Benefit Index score was 2.8 (1.2). Physicians perceived clinical improvement in 75.6% of patients. Physicians' perspective on overall success of apremilast in meeting expectations correlated with patients' perception of treatment benefit (r = 0.691). Most commonly reported adverse events (>5% of patients) were diarrhoea, nausea and headache. CONCLUSIONS: Patients in APPRECIATE reported high disease burden despite more moderate skin involvement than those who enrolled in clinical trials of apremilast. Findings from APPRECIATE demonstrate the real-world value of apremilast for psoriasis treatment, as 7 of 10 patients continued therapy and showed notable improvement in disease severity and quality of life 6 (±1) months after apremilast initiation.

Circulating microRNAs in extracellular vesicles as potential biomarkers for psoriatic arthritis in patients with psoriasis.

BACKGROUND: Psoriatic arthritis (PsA) develops in ~30% of patients with psoriasis. The diagnosis of PsA is challenging, and there are no reliable molecular markers in clinical use. MicroRNAs are short non-coding regulatory RNAs, which can be actively packaged into extracellular vesicles (EVs) and secreted to the circulation. OBJECTIVES: To explore whether plasma-derived EV microRNAs may serve as biomarkers for PsA in patients with psoriasis. METHODS: Plasma samples were obtained from patients with cutaneous-only psoriasis (PsC) and patients with psoriasis and PsA. Plasma EVs were isolated using miRCURY™ Exosome Isolation Kit. RNA sequencing was used to identify differentially expressed EV miRNAs in the discovery phase (PsC, n = 15; PsA, n = 14). In the validation phase (PsC, n = 29; PsA, n = 28), 41 selected miRNAs were analysed in plasma EVs by qPCR. The association of the identified miRNAs with PsA was assessed by logistic regression analysis. RESULTS: RNA sequencing identified 19 plasma EV miRNAs with significantly different levels between PsA and PsC in the discovery cohort. Significantly lower levels of plasma EV let-7b-5p and miR-30e-5p in PsA vs. PsC were confirmed in the validation cohort, and their decreased levels were found to be associated with the presence of PsA. ROC analysis revealed an AUC of 0.68 (95% CI 0.53-0.83) for let-7b-5p and 0.69 (95% CI 0.55-0.84) for miR-30e-5p. CONCLUSIONS: Circulating EV microRNA levels are altered in patients with PsA as compared with PsC. Findings of this exploratory study suggest that circulating EV microRNAs may serve as biomarkers for arthritis in psoriasis patients.

Real-world comparative effectiveness of adalimumab, etanercept and methotrexate: a Swedish register analysis.

BACKGROUND: The advent of biosimilars may render etanercept (ETN) and adalimumab (ADA) viable alternatives to methotrexate (MTX) as first-line systemics in psoriasis. However, real-world relative effectiveness data comparing ADA and ETN to MTX are limited. OBJECTIVE: To estimate the relative effectiveness of ADA and ETN compared to MTX. METHODS: We analysed data from DermaReg, a regional register in Stockholm, Sweden, to estimate drug survival and mean Psoriasis Area and Severity Index (PASI) and Dermatology Life Quality Index (DLQI) during maintenance treatment. RESULTS: A total of 524 patients initiated 727 treatment episodes with ADA, ETN or MTX. After adjusting for confounders, patients treated with ADA had better drug survival (HR: 0.67; P = 0.003), lower mean PASI (-2.0; P < 0.001) and lower mean DLQI (-0.9; P < 0.001) during maintenance treatment compared to patients treated with MTX. The results for ETN compared to MTX were mixed. After adjusting for confounding, there was no significant difference in drug survival (HR 1.23; P = 0.082), but patients on ETN had lower mean PASI (-0.7; P = 0.006) during maintenance treatment. CONCLUSION: Adalimumab is superior to MTX in clinical practice whereas the relative effectiveness between ETN and MTX is less clear. This study also highlights the importance of appropriate confounding control in effectiveness analysis.

Secukinumab treatment in new-onset psoriasis: aiming to understand the potential for disease modification - rationale and design of the randomized, multicenter STEPIn study.

BACKGROUND: To date, biological treatments have been assessed in subjects with a long-term history of psoriasis and previous failures to systemic and topical therapies. In rheumatoid arthritis and other immune-mediated inflammatory diseases, early intensive systemic treatment prolongs treatment-free remission. We hypothesize that, by treating patients with psoriasis early with an effective systemic therapy, we may be able to alter the clinical outcome and the natural course of the disease. The STEPIn study (NCT03020199) investigates early intervention with secukinumab versus narrow-band ultraviolet B (nb-UVB) phototherapy in subjects with new-onset psoriasis. OBJECTIVE: To determine whether early intervention with either nb-UVB treatment or secukinumab in subjects with new-onset plaque psoriasis might modify the natural course of the disease. METHODS: One hundred and sixty subjects aged 18-50 years with new-onset (≤12 months) moderate-to-severe plaque psoriasis and naïve to systemic treatment and phototherapy will be randomized to secukinumab 300 mg or nb-UVB. The Main Study has two treatment arms: Arm A1, subcutaneous secukinumab 300 mg at baseline, Weeks 1, 2, 3 and 4, and every 4 weeks thereafter until and including Week 52; Arm B1, one/two cycles of nb-UVB for 12 weeks each (maximum 28-week break between cycles). After treatment discontinuation, patients will be followed up and monitored for disease activity up to Week 208. A Mechanistic Sub-study will assess immunological changes and pathogenic tissue-resident memory T cells in skin biopsies. CONCLUSIONS: STEPIn is the first study to investigate whether early intensive treatment in new-onset psoriasis can modify the long-term natural course of the disease and thus become a novel treatment strategy for patients with psoriasis.

Sebum lipids influence macrophage polarization and activation.

BACKGROUND: As lipids are known to regulate macrophage functions, it is reasonable to suppose that a sebocyte-macrophage axis mediated by sebum lipids may exist. OBJECTIVES: To investigate if sebocytes could contribute to the differentiation, polarization and function of macrophages with their secreted lipids. METHODS: Oil Red O lipid staining and Raman spectroscopy were used to assess the dermal lipid content and penetration. Immunohistochemistry was used to analyse the macrophage subsets. Human peripheral blood monocytes were differentiated in the presence of either supernatant from human SZ95 sebocytes or major sebum lipid components and activated with Propionibacterium acnes. Macrophage surface markers and their capacity to uptake fluorescein isothiocyanate-conjugated P. acnes were detected by fluorescence-activated cell sorting measurements. Cytokine protein levels were evaluated by enzyme-linked immunosorbent assay and Western blot analysis. RESULTS: Sebaceous gland-rich skin had an increased dermal lipid content vs. sebaceous gland-poor skin to which all the tested sebum component lipids could contribute by penetrating the dermoepidermal barrier. Of the lipids, oleic acid and linoleic acid promoted monocyte differentiation into alternatively activated macrophages. Moreover, linoleic acid also had an anti-inflammatory effect in P. acnes-activated macrophages, inhibiting the secretion of interleukin (IL)-1ß, IL-6 and tumour necrosis factor (TNF)-α. Squalene, palmitic acid, stearic acid and oleic acid augmented the secretion of IL-1ß, even in the absence of P. acnes, whereas oleic acid had a selective effect of inducing IL-1ß but downregulating IL-6 and TNF-α secretion. CONCLUSIONS: Our results suggest a role for sebaceous glands in modulating innate immune responses via their secreted lipids that are of possible pathological and therapeutic relevance.

Disease activity in and quality of life of patients with psoriatic arthritis mutilans: the Nordic PAM Study.

OBJECTIVE: To describe the social status and health-related quality of life of patients with psoriatic arthritis mutilans (PAM) in the Nordic countries. METHOD: Patients with at least one mutilated joint confirmed by radiology were studied. Disease activity involving joints and skin, physician-assessed disease activity, and patient's education and work status were recorded. Data from the 36-item Short Form Health Survey, Health Assessment Questionnaire and Dermatology Life Quality Index questionnaire were gathered and correlated with disease duration, pain, and general well-being (visual analogue scale). The controls were 58 Swedish patients with long-standing psoriatic arthritis sine PAM. RESULTS: Sixty-seven patients were included. Patients with PAM had a protracted disease history (33 ± 14 years) and disease onset at a relatively early age (30 ± 12 years). Overall inflammatory activity at inclusion was mild to moderate. The mean number of mutilated joints was 8.2 and gross deformity was found in 16% of patients. Forty per cent were treated with biological and 32% with conventional synthetic disease-modifying anti-rheumatic drugs. Forty-two per cent had retired early or were on sick leave. Impaired functional capacity with little or no ability to perform self-care or everyday tasks was reported by 21% of the patients. Patients between 45 and 60 years of age reported the most impaired quality of life in comparison to the control group. CONCLUSION: PAM seriously affects social functioning. Whether early recognition of PAM and new forms of therapy can improve disease outcome and quality of life remains to be studied.

Meta-analysis of the TNFAIP3 region in psoriasis reveals a risk haplotype that is distinct from other autoimmune diseases.

Tumor necrosis factor alpha-inducible protein 3 (TNFAIP3) encodes a ubiquitin-modifying protein, A20, that is a critical regulator of inflammatory responses. TNFAIP3 polymorphisms are associated with the susceptibility to multiple autoimmune diseases (AIDs) including psoriasis, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis and celiac disease. In order to refine the TNFAIP3 association signal in psoriasis and identify candidate causal variants, we performed imputation and meta-analysis of the TNFAIP3 region in five European ancestry cohorts totaling 4704 psoriasis cases and 7805 controls. We identified 49 variants whose significance exceeded a corrected Bonferroni threshold, with the top variant being rs582757 (P = 6.07 × 10(-12), odds ratio (OR) = 1.23). Conditional analysis revealed a suggestive independent association at rs6918329 (P(cond) = 7.22 × 10(-5), OR = 1.15). Functional annotation of the top variants identified several with a strong evidence of regulatory potential and several within long noncoding RNAs. Analysis of TNFAIP3 haplotypes revealed that the psoriasis risk haplotype is distinct from other AIDs. Overall, our findings identify novel candidate causal variants of TNFAIP3 in psoriasis and highlight the complex genetic architecture of this locus in autoimmune susceptibility.

Early intervention in psoriasis and immune-mediated inflammatory diseases: A hypothesis paper.

Psoriasis is an immune-mediated inflammatory disease (IMID) which may have a major impact on a patient's life, especially when the disease is moderate to severe. There is evidence that treatment of psoriasis during the first years is conservative and frequently based on topical agents which rarely clear lesions. Treatment with systemic agents including biologics is often undertaken only when topical agents have proved unsuitable, even in patients with moderate to severe disease. However, there is evidence that in other IMIDs (rheumatoid arthritis and Crohn's disease), targeted systemic treatment given early in the treatment pathway may improve long-term patient outcomes. We hypothesize that a patient-centered therapeutic approach, undertaken early in the psoriasis treatment pathway ("early intervention") with the goal of complete clearance, may improve control of cutaneous symptoms and may also modify disease course and burden. Critical points to address when designing an early intervention study would include: the definition of psoriasis disease activity; patient selection; intervention selection; and dosing strategies.

Treatment patterns with topicals, traditional systemics and biologics in psoriasis - a Swedish database analysis.

BACKGROUND: Little data exist on real-world treatment patterns in psoriasis, especially from European settings. OBJECTIVE: To estimate, for topicals, systemics and biologics, the time to non-persistency, switching, augmentation and insufficient treatment result (only for biologics), as well as to estimate the time to restart, in patients treated with each treatment class in Sweden based on registry data. METHODS: This database analysis utilized data from patients with psoriasis from several Swedish administrative registers. Patients were identified through combinations of diagnoses from two regional registers and filled prescriptions for relevant treatments from the Swedish Prescribed Drug Register. Kaplan-Meier time-to-event ('survival') functions were estimated with relevant treatment events as failure and the proportions of patients having experienced an event at specific time-points were derived from the failure rates. RESULTS: For topicals, systemics and biologics the number of indexed treatment episodes were 25,396, 2963, and 628 respectively. One year after treatment initiation, the proportion of patients who were classed as non-persistent with topicals, systemics and biologics were estimated at 88.3%, 47.9% and 43.2% respectively. Among patients who remained persistent, within 1 year of treatment start the proportions of treatment episodes in which patients were augmented were estimated at 56.0% for topicals, 45.3% for systemics and 58.9% for biologics. In addition, within 1 year of non-persistence, 49.0% of topicals, 60.8% of systemics and 80.2% of biologics treatment episodes were re-initiated, with 35.4-52.5% re-initiated on the non-persistent treatment depending on treatment class. In addition, among patients on biologics, 29.2% of treatment episodes had an insufficient treatment result within 1 year of treatment start. CONCLUSION: Persistency to psoriasis treatments may be sub-optimal and patients who remain persistent relatively frequently receive augmentation therapy or switch to another therapy. Therefore, current treatment options in psoriasis may be insufficient.

Demography, baseline disease characteristics and treatment history of patients with psoriasis enrolled in a multicentre, prospective, disease-based registry (PSOLAR).

BACKGROUND: Psoriasis is associated with several comorbidities and behavioural risk factors. OBJECTIVES: To evaluate demographic and disease characteristics in patients enrolled in the Psoriasis Longitudinal Assessment and Registry (PSOLAR). METHODS: PSOLAR is a global, prospective, longitudinal, disease-based registry that includes a postmarketing commitment to evaluate safety in patients with psoriasis. Enrolled patients had to be receiving, or be eligible to receive, conventional systemic or biological agents. Demographic/disease characteristics, medical histories, lifestyle risk factors and previous treatments are collected at enrolment. Efficacy and safety data are collected every 6 months for 8 years, and data are extracted annually. Selected parameters are evaluated by age quartile using post hoc analyses. RESULTS: As of 23 August 2012, 11 900 patients were enrolled at 301 sites in North America, Europe and Latin America. Over half of the PSOLAR population (54·7%) is male, with a mean age of 48·6 years and mean body mass index of 30·9 kg m(-2) at enrolment. Mean duration of disease at enrolment was 17·5 years, and mean Physician's Global Assessment score was 2·0. Psoriatic arthritis (35·5%) and cardiovascular diseases (38·2%) were highly prevalent. Diabetes mellitus type II was reported in 11·4% of patients. Depression and anxiety were noted in 14·7% and 11·1% of patients, respectively; 79·0% reported any alcohol use and 56·7% reported smoking or a history of smoking. The occurrence of most comorbidities, including cardiovascular disease and risk factors, increased with age. CONCLUSIONS: In the PSOLAR population, multiple and age-appropriate comorbidities are associated with psoriasis and may affect the selection of psoriasis treatments.

Psoriasis beyond the skin: an expert group consensus on the management of psoriatic arthritis and common co-morbidities in patients with moderate-to-severe psoriasis.

BACKGROUND: Psoriatic arthritis (PsA) and co-morbidities of psoriasis represent a significant clinical and economic burden for patients with moderate-to-severe psoriasis. Often these co-morbidities may go unrecognized or undertreated. While published data are available on the incidence and impact of some of them, practical guidance for dermatologists on detection and management of these co-morbidities is lacking. OBJECTIVE: To prepare expert recommendations to improve the detection and management of common co-morbidities in patients with moderate-to-severe psoriasis. METHODS: A systematic literature review was conducted on some common co-morbidities of psoriasis-cardiovascular (CV) diseases (including obesity, hypertension, hyperglycaemia and dyslipidaemia), psychological co-morbidities (including depression, alcohol abuse and smoking) and PsA-to establish the incidence and impact of each. Data gaps were identified and a Delphi survey was carried out to obtain consensus on the detection and management of each co-morbidity. The expert panel members for the Delphi survey comprised 10 dermatologists with substantial clinical expertise in managing moderate-to-severe psoriasis patients, as well as a cardiologist and a psychologist (see appendix) with an interest in dermatology. Agreement was defined using a Likert scale of 1-7. Consensus regarding agreement for each statement was defined as ≥75% of respondents scoring either 1 (strongly agree) or 2 (agree). RESULTS: The expert panel members addressed several topics including screening, intervention, monitoring frequency, and the effects of anti-psoriatic treatment on each co-morbidity. Consensus was achieved on 12 statements out of 22 (3 relating to PsA, 4 relating to psychological factors, 5 relating to CV factors). The panel members felt that dermatologists have an important role in screening their psoriasis patients for PsA and in assessing them for psychological and CV co-morbidities. In most cases, however, patients should be referred for specialist management if other co-morbidities are detected. CONCLUSION: This article provides useful and practical guidance for the detection and management of common co-morbidities in patients with moderate-to-severe psoriasis.

Mechanisms of hemolysis-associated platelet activation.

BACKGROUND: Intravascular hemolysis occurs after blood transfusion, in hemolytic anemias, and in other conditions, and is associated with hypercoagulable states. Hemolysis has been shown to potently activate platelets in vitro and in vivo, and several mechanisms have been suggested to account for this, including: (i) direct activation by hemoglobin (Hb); (ii) increase in reactive oxygen species (ROS); (iii) scavenging of nitric oxide (NO) by released Hb; and (iv) release of intraerythrocytic ADP. OBJECTIVE: To elucidate the mechanism of hemolysis-mediated platelet activation. METHODS: We used flow cytometry to detect PAC-1 binding to activated platelets for in vitro experiments, and a Siemens' Advia 120 hematology system to assess platelet aggregation by using platelet counts from in vivo experiments in a rodent model. RESULTS: We found that Hb did not directly activate platelets. However, ADP bound to Hb could cause platelet activation. Furthermore, platelet activation caused by shearing of red blood cells (RBCs) was reduced in the presence of apyrase, which metabolizes ADP to AMP. The use of ROS scavengers did not affect platelet activation. We also found that cell-free Hb enhanced platelet activation by abrogating the inhibitory effect of NO on platelet activation. In vivo infusions of ADP and purified (ADP-free) Hb, as well as hemolysate, resulted in platelet aggregation, as shown by decreased platelet counts. CONCLUSION: Two primary mechanisms account for RBC hemolysis-associated platelet activation: ADP release, which activates platelets; and cell-free Hb release, which enhances platelet activation by lowering NO bioavailability.