A picogram BA-ELISA quantification assay for rLj-RGD3, a platelet fibrinogen receptor antagonist, in the rat plasma and its application to a pharmacokinetic study.

rLj-RGD3, a new member of the RGD (Arginine-Glycine-Aspartate)-motif toxin protein family obtained from Lampetra japonica by means of recombinant DNA techniques, has been demonstrated to be a platelet fibrinogen receptor antagonist and holds potential as a drug candidate for a specific indication. The present article reports an innovative validated highly sensitive and specific biotin-avidin enzyme linked immunosorbent assay (BA-ELISA) to provide a bio-analytical method for pharmacokinetic (PK) studies of rLj-RGD3. The concentration of picogram level rLj-RGD3 in rat plasma was measured using the developed double sandwich BA-ELISA assay, which used two mouse anti-rLj-RGD3 monoclonal antibodies that recognize different epitopes for capture and detection. This method was verified to be highly specific (blank plasma did not interfere with detection), precise (RSD <15%), and accurate (86%-113%). Absolute recovery was in the 94%-119% range. The calibration curve showed good linearity within the 50 to 1600 pg/mL range. The LOQ was as low as 50 pg/mL. The above validated assay was successfully employed to assess PK of rLj-RGD3 in rats. After i.v. and s.c. dosing with 30 µg/kg, the rLj-RGD3 plasma concentration declined bi-exponentially with time. This decay was best fitted to a two-compartment model. In conclusion, the BA-ELISA method described here meets all requirements for PK studies of rLj-RGD3 with an effective pharmacological dose in the µg/kg BW range.

Glanzmann Thrombasthenia in Pakistani Patients: Identification of 7 Novel Pathogenic Variants in the Fibrinogen Receptor αIIbß3.

Glanzmann thrombasthenia (GT) is a rare autosomal recessive inherited platelet disorder occurring frequently in populations with high incidence of consanguineous marriages. GT is characterized by quantitative and/or qualitative defect of the platelet αIIbß3 (GPIIb/IIIa) receptor caused by pathogenic variants of the encoding genes: ITGA2B and ITGB3. Patients present with a moderate to severe bleeding tendency with normal platelet count. Platelets show reduced/absent aggregation for all agonists except ristocetin in light transmission aggregometry and reduced/absent αIIbß3 expression in flow cytometry (FC). In this study, we investigated a cohort of 20 Pakistani patients and 2 families collected from the National Institute of Blood Disease, Karachi and Chughtai's Lab, Lahore. Platelet aggregation studies, FC (platelet CD41, CD61, CD42a, CD42b) and direct sequencing of the candidate genes were performed. All patients showed altered platelet aggregation, but normal agglutination after stimulation with ristocetin. Absent/reduced αIIbß3 receptor expression was present in the platelets of 16 patients, in 4 patients expression was borderline/normal. Candidate gene sequencing identified pathogenic/likely pathogenic variants in 15 patients. Seven variants are novel. One patient with absent receptor expression remained without genetic finding. 13 (86.7%) of 15 patients stated consanguinity reflected by homozygosity finding in 14 (93.3%) patients.

Platelets Contribution to Thrombin Generation in Philadelphia-Negative Myeloproliferative Neoplasms: The "Circulating Wound" Model.

Current cytoreductive and antithrombotic strategies in MPNs are mostly based on cell counts and on patient's demographic and clinical history. Despite the numerous studies conducted on platelet function and on the role of plasma factors, an accurate and reliable method to dynamically quantify the hypercoagulability states of these conditions is not yet part of clinical practice. Starting from our experience, and after having sifted through the literature, we propose an in-depth narrative report on the contribution of the clonal platelets of MPNs-rich in tissue factor (TF)-in promoting a perpetual procoagulant mechanism. The whole process results in an unbalanced generation of thrombin and is self-maintained by Protease Activated Receptors (PARs). We chose to define this model as a "circulating wound", as it indisputably links the coagulation, inflammation, and fibrotic progression of the disease, in analogy with what happens in some solid tumours. The platelet contribution to thrombin generation results in triggering a vicious circle supported by the PARs/TGF-beta axis. PAR antagonists could therefore be a good option for target therapy, both to contain the risk of vascular events and to slow the progression of the disease towards end-stage forms. Both the new and old strategies, however, will require tools capable of measuring procoagulant or prohaemorrhagic states in a more extensive and dynamic way to favour a less empirical management of MPNs and their potential clinical complications.

Influence of long-term proteasome inhibition on platelet responsiveness mediated by bortezomib.

INTRODUCTION: Although platelets contain a full proteasome system, its role in platelet function is not completely understood yet. Since the proteasome system may be involved in time-delayed processes, platelet responsiveness was investigated after long-term, bortezomib-mediated proteasome inhibition. MATERIALS AND METHODS: Citrate-anticoagulated whole blood was stored with 5 nM and 1 µM bortezomib for 24 h. Consecutively, aggregation was measured by light transmission in platelet-rich-plasma (PRP). Flow cytometry was performed to determine phosphorylation levels of the vasodilator-stimulated phosphoprotein (VASP), fibrinogen binding, PAC1-antibody binding and purinergic receptor expression in PRP, P2Y12 activity or glycoprotein (GP) Ib and IIb expression in whole blood. P2Y1 and P2X1 activities were assessed by calcium flux-induced fluorescence in washed platelets. Using PRP, adherent platelets on fibrinogen-, collagen- and ristocetin-coated surfaces were visualized and quantified by immunostaining. RESULTS: Under bortezomib, VASP phosphorylation was less inducible and nitric oxide-induced inhibition of fibrinogen binding was slightly reduced. Proteasome inhibition did not tamper adenosine diphosphate-mediated aggregation or purinergic receptor expression and activity. Induced expression of activated fibrinogen receptors and fibrinogen binding were not significantly influenced by incubation with bortezomib for 24 h. Aggregation values with threshold agonist concentrations were increased under bortezomib. Despite unchanged GPIb expression, bortezomib-treated platelets showed enhanced adhesion on coated surfaces. CONCLUSIONS: In platelets incubated for 24 h, bortezomib mediates a slight attenuation of inhibitory signaling, associated with facilitated platelet aggregation using threshold agonist concentrations and enhanced adhesion on agonist-coated surfaces.

Female platelets have distinct functional activity compared with male platelets: Implications in transfusion practice and treatment of trauma-induced coagulopathy.

BACKGROUND: Females are hypercoagulable and have survival benefit in trauma-induced coagulopathy (TIC). The mechanism for this sex-specific hypercoagulability is unknown. Platelets and platelet function are central in providing hemostatic potential and are the largest contributor to clot strength. Ligands (adenosine diphosphate [ADP] and platelet-activating factor [PAF]) bind distinct platelet receptors to potentiate activation and aggregation. We hypothesize that female platelets have a differential response to ADP and PAF, resulting in greater aggregation and activation compared to males, and that estradiol pretreatment of male or female platelets enhances this activity. METHODS: Platelets were collected from healthy volunteers: premenopausal/postmenopausal females (≤54 years, >54 years) and similarly aged males. Platelet aggregometry and flow cytometry (fibrinogen binding capacity) were examined. After treatment with ADP or PAF, platelet aggregation was assessed with Chronolog and activation assessed by CD41 receptor surface expression using flow cytometry. Aggregation and activation were again assessed after platelet pretreatment with estradiol. RESULTS: Healthy volunteers included 12 premenopausal and 13 postmenopausal females and 18 similarly aged males. Female platelets (combined premenopausal and postmenopausal) had increased aggregation with ADP stimulation, as compared to male platelets. Male and female platelets had differential fibrinogen receptor expression, with female platelets (combined premenopausal and postmenopausal) demonstrating robust activation with ADP versus male platelets with PAF. In the presence of estradiol incubation, male platelets' activation with PAF approximated that of females (combined premenopausal and postmenopausal) and activation with PAF was enhanced in both male and female platelets. CONCLUSION: Male and female platelets have differential response to stimuli, suggesting sex-dependent signaling and cellular activation. Female platelets have both increased aggregation and activation potential, and estradiol pretreatment feminizes male platelets to approximate female platelet activation with PAF. These findings offer potential explanation for sex-based differences in hemostatic potential in TIC and question whether donor sex of transfused platelets should be considered in resuscitation. Estradiol may also serve as a novel therapeutic adjunct in TIC.

Association of Fibrinogen Receptor (Integrin αIIbß3) Polymorphism in Sudanese Ischemic Stroke Patients.

BACKGROUND AND OBJECTIVE: The fibrinogen receptor the human platelet antigen (HPA1 and HPA3) have an essential role in Atherothrombosis. This study aimed to detect the association of αIIbß3 polymorphism with ischemic stroke in Sudanese patients and its association with the common risk factors. METHODOLOGY: This is a case-control study. Fifty atherosclerotic with ischemic stroke Sudanese patients were included in present study and were compared to apparently 50 healthy Sudanese subjects at the same ages. The ages of both groups were >18 years. About 5 mL of venous blood sample was taken from each patient and control. The laboratory analyses were done for HbA1c, lipid profile and DNA genotyping by polymerase chain reaction (PCR) followed by FokI and ScrFI digestion. RESULTS: The result showed that, the risk factors (TRI.G, HDL, HbA1C, and body mass index were associated with the increased risk of ischemic stroke). None of the cholesterol levels and LDL increased the risk of stroke. The risk of ischemic stroke was higher with B/B genotype in HPA3 (p-value 0.009) and A/B genotype in HPA1 (p-value 0.041) and HPA1 (p-value 0.041). CONCLUSION: The αIIbß3 polymorphism were with ischemic stroke in Sudanese patients.

The 95RGD97 sequence on the Aα chain of fibrinogen is essential for binding to its erythrocyte receptor.

BACKGROUND: Erythrocyte aggregation, a cardiovascular risk factor, is increased by high plasma fibrinogen levels. Here, the effect of different fibrinogen mutations on binding to its human erythrocyte receptor was assessed in order to identify the interaction sites. METHODS: Three fibrinogen variants were tested, specifically mutated in their putative integrin recognition sites on the Aα chain (mutants D97E, D574E and D97E/D574E) and compared with wild-type fibrinogen. RESULTS: Atomic force microscopy-based force spectroscopy measurements showed a significant decrease both on the fibrinogen-erythrocyte binding force and on its frequency for fibrinogen with the D97E mutation, indicating that the corresponding arginine-glycine-aspartate sequence (residues 95-97) is involved in this interaction, and supporting that the fibrinogen receptor on erythrocytes has a ß3 subunit. Changes in the fibrin clot network structure obtained with the D97E mutant were observed by scanning electron microscopy. CONCLUSION: These findings may lead to innovative perspectives on the development of new therapeutic approaches to overcome the risks of fibrinogen-driven erythrocyte hyperaggregation.

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor αIIbß3 and activated platelets.

Thromboembolic events (TEE) underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs) functionalized with bitistatin (Bit), a disintegrin that specifically binds to the αIIbß3 integrin, platelet fibrinogen receptor (PFR) on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP-Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, αIIbß3 receptor antagonist, specifically blocked F-NDP-Bit-PFR complex formation. Moreover, F-NDP-Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP). Our results suggest that engineered F-NDP-Bit particles could serve as noninvasive, "real-time" optical diagnostics for clots present in blood vessels.

Role of P2Y12 Receptor in Thrombosis.

P2Y12 receptor is a 342 amino acid Gi-coupled receptor predominantly expressed on platelets. P2Y12 receptor is physiologically activated by ADP and inhibits adenyl cyclase (AC) to decrease cyclic AMP (cAMP) level, resulting in platelet aggregation. It also activates PI3 kinase (PI3K) pathway leading to fibrinogen receptor activation, and may protect platelets from apoptosis. Abnormalities of P2Y12 receptor include congenital deficiencies or high activity in diseases like diabetes mellitus (DM) and chronic kidney disease (CKD), exposing such patients to a prothrombotic condition. A series of clinical antiplatelet drugs, such as clopidogrel and ticagrelor, are designed as indirect or direct antagonists of P2Y12 receptor to reduce incidence of thrombosis mainly for patients of acute coronary syndrome (ACS) who are at high risk of thrombotic events. Studies on novel dual-/multi-target antiplatelet agents consider P2Y12 receptor as a promising part in combined targets. However, the clinical practical phenomena, such as "clopidogrel resistance" due to gene variations of cytochrome P450 or P2Y12 receptor constitutive activation, call for better antiplatelet agents. Researches also showed inverse agonist of P2Y12 receptor could play a better role over neutral antagonists. Personalized antiplatelet therapy is the most ideal destination for antiplatelet therapy in ACS patients with or without other underlying diseases like DM or CKD, however, there is still a long way to go.

Structural and Physico-Chemical Interpretation (SPCI) of QSAR Models and Its Comparison with Matched Molecular Pair Analysis.

This paper describes the Structural and Physico-Chemical Interpretation (SPCI) approach, which is an extension of a recently reported method for interpretation of quantitative structure-activity relationship (QSAR) models. This approach can efficiently be used to reveal structural motifs and the major physicochemical factors affecting the investigated properties. Its efficacy was demonstrated both on the classical Free-Wilson data set and on several data sets with different end points (permeability of the blood-brain barrier, fibrinogen receptor antagonists, acute oral toxicity). Structure-activity patterns extracted from QSAR models with SPCI were in good correspondence with experimentally observed relationships and molecular docking, regardless of the machine learning method used. Comparison of SPCI with the matched molecular pair (MMP) method clearly shows an advantage of our approach over MMP, especially for small or structurally diverse data sets. The developed approach has been implemented in the SPCI software tool with a graphical user interface, which is publicly available at http://qsar4u.com/pages/sirms_qsar.php .

Biomarcadores, índices y algoritmos para el diagnóstico de fibrosis hepática en pacientes con hepatitis crónica por virus C / Biomarkers, indexes and algorithms for the detection of liver fibrosis in chronic hepatitis C patients

Determinar el estadio de fibrosis hepática es esencial en el manejo de la enfermedad hepática en pacientes con hepatitis crónica por virus C. La biopsia hepática ha sido considerada el gold standard para diagnosticar la enfermedad, la actividad necroinflamatoria y el estadio de fibrosis, pero tiene algunas limitaciones técnicas y riesgos. Teniendo en cuenta estas limitaciones, existe cierta exigencia en introducir biomarcadores séricos no invasivos para el diagnóstico de fibrosis que podrían ser capaces de reemplazar parcialmente a la biopsia hepática. El biomarcador sérico ideal debería ser específico para el hígado y fácil de determinar. Los marcadores séricos se dividen en directos e indirectos. Los directos reflejan el recambio de la matriz extracelular, mientras que los indirectos reflejan alteraciones en la función hepática. Aunque todavía está limitado el grado de implementación de los test no invasivos de fibrosis hepática, esta revisión es una visión de conjunto de los biomarcadores, índices y algoritmos diagnósticos de fibrosis hepática estudiados en hepatitis crónica C pero con un interés potencial en otras hepatopatías crónicas (AU)

Precise staging of liver fibrosis is essential in the management of liver disease activity in chronic hepatitis C patients. Liver biopsy has been considered the reference method for diagnosing disease, grading necroinflammatory activity, and staging fibrosis, but it has some technical limitations and risks. Taking into account these limitations, there is a need to introduce non-invasive serum markers for fibrosis that would be able to partially replace liver biopsy. Ideal serum markers should be specific for the liver and easy to perform. Serum markers of hepatic fibrosis are divided into direct and indirect. Direct markers reflect extracellular matrix turnover, whereas indirect markers reflect alterations in hepatic function. The degree of implementation of non-invasive diagnostic tests for liver fibrosis still remains limited. This review provides a current overview of biomarkers, indexes and algorithms for hepatic fibrosis diagnosis in chronic hepatitis C patients (AU)

Asymmetric alcohol C-H allylation and syn-crotylation: C9-C20 of tetrafibricin.

The C9-C20 segment of the fibrinogen receptor inhibitor tetrafibricin was prepared in 10 steps (longest linear sequence). Ruthenium catalyzed enantioselective syn-crotylation is used to construct C9-C13. Iridium catalyzed asymmetric alcohol C-H allylation of a commercial malic acid derived alcohol is used to construct C14-C20. Recovery and recycling of the iridium catalyst is described.

Genetic variation of platelet function and pharmacology: an update of current knowledge.

Platelets are critically involved in atherosclerosis and acute thrombosis. The platelet phenotype shows a wide variability documented by the inherited difference of platelet reactivity, platelet volume and count and function of platelet surface receptors. Several candidate genes have been put into focus and investigated for their functional and prognostic role in healthy individuals and patients with cardiovascular (CV) disease treated with antiplatelet agents. In addition to genetic variation, other clinical, disease-related and demographic factors are important so-called non-genetic factors. Due to the small effect sizes of single nucleotide polymorphisms (SNP) in candidate genes and due to the low allele frequencies of functional relevant candidate SNPs, the identification of genetic risk factors with high predictive values generally depends on the sample size of study cohorts. In the post-genome era new array and bioinformatic technologies facilitate high throughput genome-wide association studies (GWAS) for the identification of novel candidate genes in large cardiovascular cohorts. One of the crucial aspects of platelet genomic studies is the precise definition of a specific clinical phenotype (e.g. stent thrombosis) as this will impact importantly the findings of genomic studies like GWAS. Here, we provide an update on genetic variation of platelet receptors and drug metabolising enzymes under specific consideration of data derived by GWAS. The potential impact of this information and the role in personalised therapeutic concepts will be discussed.

N-octanoyl-dopamine is a potent inhibitor of platelet function.

Dopamine (DA) is a co-agonist for platelet activation; yet, donor DA treatment is associated with improved transplantation outcome in renal and heart recipients. Recently, N-octanoyl-dopamine (NOD) was developed which displays superior effects compared to DA in terms of graft protecting properties. Whereas DA is a known platelet co-agonist, the effect of NOD on platelet function is unknown. This is a hypothesis generating study with the aim to assess the effects and molecular mechanisms of NOD and NOD-like compounds on platelet function. The influence of DA, NOD, and NOD-like compounds on platelet responses to classical agonists (adenosine 5'-diphosphate (ADP), U46619) was investigated in six healthy donors by applying whole blood aggregometry (Multiplate®) and flow cytometry for Pac-1, CD62P, and CD63 expression. Changes in platelet cAMP concentrations were assessed by ELISA. While DA showed synergy in platelet activation by ADP and U46619, NOD caused significant inhibition of platelet function both in whole blood aggregometry and flow cytometry. The inhibitory effect of NOD was not mediated via cAMP levels. The nonredox-active NOD-analog N-octanoyl-tyramine had no effects on platelet function. Acetylated NOD conferred to NOD by intracellular esterases showed similar inhibitory effects as NOD. In contrast to DA, NOD is a potent inhibitor of platelet function most likely through intracellular redox-active processes. This adds to the overall protective effect of NOD on pre-transplantation injury and makes NOD an attractive candidate compound for donor or organ conditioning prior to transplantation.

Atomic force microscopy-based force spectroscopy--biological and biomedical applications.

The use of atomic force microscopy (AFM) applied to biological systems to generate high resolution images is gaining a wider acceptance. However, the most remarkable advances are being achieved on the use of the AFM to measure inter- and intramolecular interaction forces with piconewton resolution, not only to demonstrate this ability but also actually to solve biological and biomedical relevant questions. Single-molecule force spectroscopy recognition studies enable the detection of specific interaction forces, based on the AFM sensitivity and the possibility of manipulating individual molecules. In this review, we describe the basic principles of this methodology and some of the practical aspects involved. The ability to measure interactions at the single-molecule level is illustrated by some relevant examples. A special focus is given to the study of the fibrinogen-erythrocyte binding and its relevance as a cardiovascular risk factor. An approach to the latter problem by single-molecule force spectroscopy allowed the molecular recognition, characterization, and partial identification of a previously unknown receptor for fibrinogen on human erythrocytes.

Random phage-epitope library based identification of a peptide antagonist of Mac-1 ß2 integrin ligand binding.

The leukocyte ß2 integrin Mac-1 (CD11b/CD18) plays a pivotal role in inflammation and host defense. To develop peptide antagonists selectively inhibiting the function of Mac-1, we used a random constrained 6-mer (cys-6aa-cys) peptide library to map the structural features of CD11b, by determining the epitope of neutralizing monoclonal antibody mAb 44a (anti-CD11b). We have used a stringent phage display strategy, which resulted in the identification of one disulfide C-RLKEKH-C constrained peptide by direct biopanning of library on decreasing amounts of purified mAb 44a. The selected peptide mimics a discontinuous epitope, a peculiar shape on the CD11b-I-domain surface. Competitive ELISA experiments with different Mac-1 ligands showed that C-RLKEKH-C is able to bind to fibrinogen, iC3b, and C1q. Furthermore, the monomeric circular peptide C-RLKEKH-C, was effective in blocking the interaction between (125)I-fibrinogen and Mac-1 (IC(50)=3.35±0.1×10(-6)M), and inhibited the adhesion of human neutrophils to fibrinogen and iC3b. These data provide information about the relative location of amino acids on the I-domain surface using mAb 44a imprint of the CD11b protein. The derived mimotope may help in the design of future anti-inflammatory therapeutic agents that can act as specific therapeutic agents targeting PMNs mediated inflammation.

RGD mimetics containing phthalimidine fragment as novel ligands of fibrinogen receptor.

The novel RGD mimetics with phthalimidine central fragment were synthesized with the use of 4-piperidine-4-yl-butyric, 4-piperidine-4-yl-benzoic, 4-piperazine-4-yl-benzoic and 1,2,3,4-tetrahydroisoquinoline-7-carboxylic acids as surrogates of Arg motif. The synthesized compounds potently inhibited platelet aggregation in vitro and blocked FITC-Fg binding to α(IIb)ß(3) integrin in a suspension of washed human platelets. The key α(IIb)ß(3) protein-ligand interactions were determined in docking experiments.

Lytic resistance of fibrin containing red blood cells.

OBJECTIVE: Arterial thrombi contain variable amounts of red blood cells (RBCs), which interact with fibrinogen through an eptifibatide-sensitive receptor and modify the structure of fibrin. In this study, we evaluated the modulator role of RBCs in the lytic susceptibility of fibrin. METHODS AND RESULTS: If fibrin is formed at increasing RBC counts, scanning electron microscopy evidenced a decrease in fiber diameter from 150 to 96 nm at 40% (v/v) RBCs, an effect susceptible to eptifibatide inhibition (restoring 140 nm diameter). RBCs prolonged the lysis time in a homogeneous-phase fibrinolytic assay with tissue plasminogen activator (tPA) by up to 22.7±1.6%, but not in the presence of eptifibatide. Confocal laser microscopy using green fluorescent protein-labeled tPA and orange fluorescent fibrin showed that 20% to 40% (v/v) RBCs significantly slowed down the dissolution of the clots. The fluorescent tPA variant did not accumulate on the surface of fibrin containing RBCs at any cell count above 10%. The presence of RBCs in the clot suppressed the tPA-induced plasminogen activation, resulting in 45% less plasmin generated after 30 minutes of activation at 40% (v/v) RBCs. CONCLUSIONS: RBCs confer lytic resistance to fibrin resulting from modified fibrin structure and impaired plasminogen activation through a mechanism that involves eptifibatide-sensitive fibrinogen-RBC interactions.

In vitro inhibition of human and rat platelets by NO donors, nitrosoglutathione, sodium nitroprusside and SIN-1, through activation of cGMP-independent pathways.

Three different NO donors, S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP) and 3-morpholino-sydnonimine hydrochloride (SIN-1) were used in order to investigate mechanisms of platelet inhibition through cGMP-dependent and -independent pathways both in human and rat. To this purpose, we also evaluated to what extent cGMP-independent pathways were related with the entity of NO release from each drug. SNP, GSNO and SIN-1 (100 µM) effects on platelet aggregation, in the presence or absence of a soluble guanylate cyclase inhibitor (ODQ), on fibrinogen receptor (α(IIb)ß(3)) binding to specific antibody (PAC-1), and on the entity of NO release from NO donors in human and rat platelet rich plasma (PRP) were measured. Inhibition of platelet aggregation (induced by ADP) resulted to be greater in human than in rat. GSNO was the most powerful inhibitor (IC(50) values, µM): (a) in human, GSNO=0.52±0.09, SNP=2.83 ± 0.53, SIN-1=2.98 ± 1.06; (b) in rat, GSNO = 28.4 ± 6.9, SNP = 265 ± 73, SIN-1=108 ± 85. GSNO action in both species was mediated by cGMP-independent mechanisms and characterized by the highest NO release in PRP. SIN-1 and SNP displayed mixed mechanisms of inhibition of platelet aggregation (cGMP-dependent and independent), except for SIN-1 in rat (cGMP-dependent), and respectively lower or nearly absent NO delivery. Conversely, all NO-donors prevalently inhibited PAC-1 binding to α(IIb)ß(3) through cGMP-dependent pathways. A modest relationship between NO release from NO donors and cGMP-independent responses was found. Interestingly, the species difference in NO release from GSNO and inhibition by cGMP-independent mechanism was respectively attributed to S-nitrosylation of non-essential and essential protein SH groups.