Littérature scientifique sur le sujet « High on-treatment platelet reactivity »

We describe, to the best of our knowledge, the first incidence of stent thrombosis in a patient treated with ticagrelor, who exhibited high on-treatment platelet reactivity (HTPR) according to platelet reactivity testing. He was on clopidrogel and tested for platelet reactivity using the VerifyNow P2Y12 assay. The test showed a PRU of 249 and only 12% platelet inhibition. The patient was then switched to ticagrelor, with a loading dose of 180 mg given. The patient had stent thrombosis three weeks later with an acute myocardial infarction (MI). The patient had good platelet inhibition when started on Ticagrelor treatment (PRU=33), but had HTPR when the stent thrombosis occurred three weeks later (PRU=339).

SummaryIn the last decade, several studies revealed inter-patient response variability to antiplatelet agents: patients who display negligible or no responses to these drugs are considered poor responders, or “resistant” to treatment. In order to identify poor responders to an antiplatelet drug, laboratory tests of platelet function that specifically explore the platelet activation pathway that is targeted by the drug should be utilised. In addition, they should be performed both at baseline and during treatment: however, most studies explored platelet function during antiplatelet treatment, in order to identify those patients with “high on-treatment platelet reactivity” (HPR), which exposes them to increased risk of major adverse cardiovascular events (MACE). Many tests of platelet function have been used, most of which are able to identify patients at risk of MACE. Unfortunately, universal cut-off values for HPR have not been clearly established yet. In addition, the concordance among different tests in the identification of patients at risk is very poor and the most effective and safe treatment for patients at risk is still unknown.

AbstractPlatelet multidrug resistance protein 4 (MRP4) plays a modulating role on platelet activation. Platelet function and thrombus formation are impaired in MRP4 knockout mice models, and, among aspirin-treated patients, high on-aspirin residual platelet reactivity (HARPR) positively correlates with MRP4 levels. To better understand the effects of MRP4 on platelet function, the aim of this investigation was to assess the impact of cilostazol-induced inhibition of MRP4-mediated transport and assess aspirin-induced antiplatelet effects and rates of HARPR in human subjects.Cilostazol-dependent inhibition of MRP4-mediated transport was assessed with the release of the fluorescent adduct bimane-glutathione and aspirin entrapment. Effect of Cilostazol on cAMP inhibition was evaluated by vasodilator-stimulated phosphoprotein (VASP). Platelet function was studied by collagen and TRAP-6-induced platelet aggregation and secretion.Cilostazol reduced the release of bimane-glutathione and enhanced aspirin entrapment demonstrating an inhibitory effect on MRP4 in platelets. VASP phosphorylation was absent until 10 seconds after addition of cilostazol, and becomes evident after 30 seconds. An inhibitory effect on platelet aggregation and secretion was found in activated platelets, with threshold concentration of agonists, 10 seconds after addition of cilostazol, supporting a role of MRP4 on platelet function that is cAMP independent. Cilostazol effects were also shown in aspirin-treated platelets. A reduction of platelet aggregation and secretion were observed in aspirin-treated patients with HARPR.This study supports the role of MRP4 on modulating platelet function which occurs through cAMP-independent mechanisms. Moreover, inhibition of MRP4 induced by cilostazol enhances aspirin-induced antiplatelet effects and reduces HARPR.

BACKGROUND Dual antiplatelet therapy is recommended in patients undergoing primary percutaneous coronary intervention (p-PCI) for ST-segment elevation myocardial infarction (STEMI). In the past few decades, oral antiplatelet agents have proved to significantly reduce the incidence of ischemic events in patients with atherothrombotic diseases. Nevertheless, recurrent ischemic events often occur in patients undergoing stent implantation. High platelets reactivity has been associated with a higher risk for major cardiovascular events in patients with acute coronary syndromes (ACS). Several pre-analytical variables may influence platelet function analysis. The aim of our study was to assess the on-treatment platelet reactivity in peripheral and coronary blood, in a group of patients receiving dual antiplatelet therapy undergoing primary percutaneous coronary intervention (p-PCI) for STEMI. METHODS Eligible patients for the study were considered as consecutively admitted patients to the emergency department of University-Hospital of Padua with a diagnosis of ACS with ST-segment elevation scheduled for an urgent procedure of coronary angioplasty. One hundred nine patients who consecutively underwent p-PCI (males: 72%, females: 28%; mean age: 64±13 years) were enrolled. Before the coronary angioplasty intervention, the patients were treated with dual antiplatelet therapy (aspirin 250mg I.V in association with one another oral thienopyridines; Clopidogrel 300/600mg, Prasugrel 60 mg or Ticagrelor 180 mg) and with anticoagulant therapy (unfractionated heparin 70U/Kg I.V). During the coronary angioplasty intervention two different samples were obtained, one from peripheral artery and the other from coronary blood. The platelet aggregation was studied using the impedance aggregometry Multiplate®, according to manufacturer’s indications. For each patient the values of “Area Under the Curve” (AUC) in ADP-test and ASPI-test were considered, both in the peripheral and in coronary blood. “Low responders of antiplatelet therapy” were considered when an AUC value of ASPI-test or ADP-test greater than or equal to a pre-established cut-off. RESULTS The Multiplate® analysis of ADP-test revealed that mean values were slightly higher in peripheral blood compared to coronary blood (peripheral blood: 41±28 U; coronary blood: 39±28 U), However these values with no statistically significant difference (p=0.68). Likewise, for the ASPI-test; no statistically significant difference between the mean values in the peripheral blood compared to the coronary blood (peripheral blood: 23±4 U; coronary blood: 17±2 U; p=0.06). The percentage of low-responders to ADP-receptor inhibitors was significantly greater than the percentage of low-responders to acetylsalicylic acid at time of primary PCI both in the peripheral and in the coronary blood samples (peripheral ADP-test: 38%; peripheral ASPI-test: 14%; p<0.01, Coronary ADP-test: 36%; coronary ASPI-test: 11%; p<0.01). In peripheral blood, the prevalence of “low Clopidogrel responders” was higher (45%) than that observed for Prasugrel (36%) and Ticagrelor (33%). Similar results were observed in coronary blood, the prevalence of “low Clopidogrel responders” was higher (40%) than that observed for Prasugrel (36%) and Ticagrelor (29%) however these results were with no significant statistical difference (p >0.05). Finally, a positive and statistically significant linear correlation was observed for both ASPI-test and ADP-test in peripheral and coronary blood (r2 0.23, p <0.001 and r2 0.12, p <0.001; respectively). That means; those who are resistant to acetylsalicylic acid tend to be resistant to ADP receptor inhibitors, and vice versa; those who are sensitive to acetylsalicylic acid therapy tend to be sensitive to ADP inhibitor therapy also. Our observed data did not show a correlation between platelet function and clinical outcome both for in-hospital and 1-year clinical outcomes. CONCLUSIONS In this study we observed that the overall platelet reactivity in coronary blood is lower than in peripheral blood, though not statistically significant. This more likely appears to be due to high antiplatelet drugs effect at plaque ulceration/thrombus site, where the hemostatic process is highly active at onset of STEMI. Larger studies are needed for better evaluation of these mechanisms in term of pharmacodynamic, pharmacokinetic and receptor kinetic properties of antiplatelet agents. The other interesting result emerging from data processing is the high incidence (about 30%) of low response to thienopyridine type antiplatelet drugs at the time of primary angioplasty. This result, moreover known for Clopidogrel in addition our results include patients treated with Prasugrel and Ticagrelor also. An explanation of this phenomenon which also involves potent recent drugs, requires careful analysis and further studies. The significant direct correlation between platelet reactivity in peripheral and in coronary blood is still a matter of debate. Larger studies are needed for in-depth assessment of any correlation between on–treatment platelet reactivity measured in coronary blood and clinical outcome.
INTRODUZIONE La doppia terapia antiaggregante (DAPT) è raccomandata in pazienti sottoposti ad intervento di angioplastica coronarica primaria (p-PCI) per infarto miocardico acuto con sopraslivellamento del tratto ST (STEMI). Infatti, il trattamento con farmaci antipiastrinici orali ha dimostrato di ridurre significativamente l'incidenza di eventi ischemici nei pazienti con malattie aterotrombotiche sia in fase acuta che in fase cronica. Tuttavia, spesso si verificano eventi ischemici ricorrenti nei pazienti sottoposti ad angioplastica ed impianto di stent. È stato dimostrato che una delle cause di recidiva ischemica sia l’elevata reattività delle piastrine. Pertanto, lo studio della funzione piastrinica diventa un elemento sempre più importante per valutare questo tipo di pazienti. Diverse variabili pre-analitiche possono influenzare l'analisi della funzione piastrinica. Lo scopo del nostro studio è stato quello di valutare la reattività piastrinica del sangue periferico e coronarico in un gruppo di pazienti trattati con DAPT e sottoposti p-PCI per STEMI. METODI Abbiamo considerato eleggibili allo studio tutti i pazienti consecutivamente giunti in urgenza al Pronto Soccorso dell’Azienda Ospedaliera di Padova con diagnosi di sindrome coronarica acuta con sopraslivellamento del tratto ST per i quali fosse indicata l’esecuzione in urgenza di una procedura di angioplastica coronarica. Sono stati arruolati 109 pazienti (maschi: 72%, femmine: 28%; età media: 64±13 anni). I pazienti arruolati nello studio sono stati trattati, prima di essere sottoposti alla procedura di angioplastica primaria, con doppia terapia antiaggregante (aspirina 250 mg e.v in associazione con uno dei seguenti tre farmaci: Clopidogrel 300/600 mg per os, Prasugrel 60 mg per os o Ticagrelor 180 mg per os) e con terapia anticoagulante (eparina non frazionata 70 U/Kg e.v). Durante la procedura di angioplastica primaria sono stati eseguiti due tipi di prelievo, uno dal sangue arterioso periferico ed uno dal sangue arterioso coronarico. L’aggregazione piastrinica è stata studiata con l’aggregometro Multiplate® secondo le indicazioni fornite dal costruttore. Per ogni paziente sono stati valutati i valori di “Area Under the Curve” (AUC) nell’ADP-test e nell’ASPI-test, ottenuti sul sangue periferico e sul sangue coronarico. “Low responders alla terapia antiaggregante” sono stati definiti quei pazienti con valori di “Area Under Curve” (AUC) all’ASPI test o all’ADP test sono maggiore o uguale a un range prestabilito. RISULTATI Non abbiamo osservato differenza statisticamente significativa tra i valori medi di ADP-test calcolati su sangue periferico e su sangue coronarico. I valori medi delle AUC sono risultati lievemente superiori nel sangue periferico che nel sangue coronarico (sangue periferico: 41±28 U; sangue coronarico: 39±28 U; p=0.68). Allo steso modo, non è stata riscontrata differenza statisticamente significativa tra i valori medi di ASPI-test calcolati su sangue periferico e su sangue coronarico. Anche in questo caso abbiamo osservato valori medi di AUC lievemente superiori nel sangue periferico che nel sangue coronarico (sangue periferico: 23±4 U; sangue coronarico: 17±2 U; p=0.06). Sia nel sangue periferico che nel sangue coronarico la percentuale di pazienti “low responders” al trattamento con inibitori del recettore per l’ADP è risultata essere statisticamente superiore alla percentuale di pazienti “low responders” alla terapia con acido acetilsalicilico al momento dell’angioplastica primaria (ADP-test periferico: 38%; ASPI-test periferico: 14%; p<0.01. ADP-test coronarico: 38%; ASPI-test coronarico: 11%; p<0.01). Nel sangue periferico la prevalenza di "low responders” al Clopidogrel era superiore (45%) a quella osservata rispettivamente per Prasugrel (36%) e Ticagrelor (33%). Risultati simili sono stati osservati nel sangue coronarico. In particolare, la prevalenza di "low responders” al Clopidogrel è stata superiore (40%) rispetto a quella osservata per Prasugrel (36%) e Ticagrelor (29%). Non è stata osservata alcuna differenza significativa (p> 0,05) nella prevalenza dei pazienti con valori di ADP-test superiori al cut-off prestabilito, considerando separatamente le tre diverse tienopiridine. Infine è stata individuata una correlazione lineare statisticamente significativa tra “low responders” all’acido acetilsalicilico e “low responders” agli inibitori del recettore dell’ADP. Questa osservazione indica come i pazienti resistenti al trattamento con acido acetilsalicilico tendono ad essere resistenti anche al trattamento con inibitori del recettore per l’ADP e, viceversa, pazienti “sensibili” alla terapia con acido acetilsalicilico tendono ad essere “sensibili” anche al trattamento con inibitori del recettore per l’ADP. Questi resultati sono stati osservati sia su sangue periferico (r2 0.23, p<0.001) che su sangue coronarico (r2 0.12, p<0.001). I dati che abbiamo osservato non mostrano un’associazione tra funzione piastrinica e outcome clinico nè per quanto riguarda gli “in-hospital outcome” né per quanto riguarda gli outcome a distanza di 1 anno. CONCLUSIONI I dati analizzati ci hanno permesso di dimostrare che la reattività piastrinica nel sangue coronarico era inferiore rispetto a quella osservata nel sangue periferico. Sembrerebbe quindi che, la risposta alla terapia farmacologica con doppia antiaggregante prima della procedura sia maggiore proprio laddove il processo emostatico è più attivo, ossia a livello della placca aterosclerotica sede della formazione del trombo responsabile dell’insorgenza della STEMI. Questo meccanismo necessità di conferma in termini di farmacodinamica, farmacocinetica e di cinetica recettoriale. L’altro dato estremamente interessante emerso dall’elaborazione dei dati è l’elevata incidenza (circa 30%) dei pazienti “low responders” al trattamento con farmaci antiaggreganti di tipo tienopiridinico al momento della angioplastica primaria. Questo risultato, peraltro noto per il Clopidogrel, comprende anche pazienti trattati con Prasugrel e Ticagrelor. Una possibile spiegazione di questo fenomeno, che coinvolge anche i farmaci di “seconda generazione”, necessita di un’attenta analisi. Abbiamo infine osservato una significativa correlazione tra reattività piastrinica nel sangue periferico e nel coronario. I nostri risultati, che alla luce dei limiti del nostro lavoro devono considerarsi come preliminari, necessitano di essere confermati su casistiche più numerose soprattutto per quanto riguarda la correlazione tra “on-treatment platelet reactivity” misurata nel sangue coronarico e outcomes clinici.

Doutoramento em Bioquímica
Os polissacarídeos são os componentes maioritários dos grãos de café verde e torrado e da bebida de café. Os mais abundantes são as galactomananas, seguindo-se as arabinogalactanas. Durante o processo de torra, as galactomananas e arabinogalactanas sofrem modificações estruturais, as quais estão longe de estar completamente elucidadas devido à sua diversidade e à complexidade estrutural dos compostos formados. Durante o processo de torra, as galactomananas e arabinogalactanas reagem com proteínas, ácidos clorogénicos e sacarose, originando compostos castanhos de alto peso molecular contendo nitrogénio, designados de melanoidinas. As melanoidinas do café apresentam diversas atividades biológicas e efeitos benéficos para a saúde. No entanto, a sua estrutura exata e os mecanismos envolvidos na sua formação permanecem desconhecidos, bem como a relação estrutura-atividade biológica. A utilização de sistemas modelo e a análise por espectrometria de massa permitem obter uma visão global e, simultaneamente, detalhada das modificações estruturais nos polissacarídeos do café promovidas pela torra, contribuindo para a elucidação das estruturas e mecanismos de formação das melanoidinas. Com base nesta tese, oligossacarídeos estruturalmente relacionados com a cadeia principal das galactomananas, (β1→4)-Dmanotriose (Man3), e as cadeias laterais das arabinogalactanas, (α1→5)-Larabinotriose (Ara3), isoladamente ou em misturas com ácido 5-Ocafeoilquínico (5-CQA), o ácido clorogénico mais abundante nos grãos de café verde, e péptidos compostos por tirosina e leucina, usados como modelos das proteínas, foram sujeitos a tratamento térmico a seco, mimetizando o processo de torra. A oxidação induzida por radicais hidroxilo (HO•) foi também estudada, uma vez que estes radicais parecem estar envolvidos na modificação dos polissacarídeos durante a torra. A identificação das modificações estruturais induzidas por tratamento térmico e oxidativo dos compostos modelo foi feita por estratégias analíticas baseadas principalmente em espectrometria de massa, mas também em cromatografia líquida. A cromatografia de gás foi usada na análise de açúcares neutros e ligações glicosídicas. Para validar as conclusões obtidas com os compostos modelo, foram também analisadas amostras de polissacarídeos do café obtidas a partir de resíduo de café e café instantâneo. Os resultados obtidos a partir dos oligossacarídeos modelo quando submetidos a tratamento térmico (seco), assim como à oxidação induzida por HO• (em solução), indicam a ocorrência de despolimerização, o que está de acordo com estudos anteriores que reportam a despolimerização das galactomananas e arabinogalactanas do café durante a torra. Foram ainda identificados outros compostos resultantes da quebra do anel de açúcares formados durante o tratamento térmico e oxidativo da Ara3. Por outro lado, o tratamento térmico a seco dos oligossacarídeos modelo (individualmente ou quando misturados) promoveu a formação de oligossacarídeos com um maior grau de polimerização, e também polissacarídeos com novos tipos de ligações glicosídicas, evidenciando a ocorrência de polimerização através reações de transglicosilação não enzimática induzidas por tratamento térmico a seco. As reações de transglicosilação induzidas por tratamento térmico a seco podem ocorrer entre resíduos de açúcares provenientes da mesma origem, mas também de origens diferentes com formação de estruturas híbridas, contendo arabinose e manose como observado nos casos dos compostos modelo usados. Os resultados obtidos a partir de amostras do resíduo de café e de café instantâneo sugerem a presença de polissacarídeos híbridos nestas amostras de café processado, corroborando a ocorrência de transglicosilação durante o processo de torra. Além disso, o estudo de misturas contendo diferentes proporções de cada oligossacarídeo modelo, mimetizando regiões do grão de café com composição distinta em polissacarídeos, sujeitos a diferentes períodos de tratamento térmico, permitiu inferir que diferentes estruturas híbridas e não híbridas podem ser formadas a partir das arabinogalactanas e galactomananas, dependendo da sua distribuição nas paredes celulares do grão e das condições de torra. Estes resultados podem explicar a heterogeneidade de estruturas de melanoidinas formadas durante a torra do café. Os resultados obtidos a partir de misturas modelo contendo um oligossacarídeo (Ara3 ou Man3) e 5-CQA sujeitas a tratamento térmico a seco, assim como de amostras provenientes do resíduo de café, mostraram a formação de compostos híbridos compostos por moléculas de CQA ligadas covalentemente a um número variável de resíduos de açúcar. Além disso, os resultados obtidos a partir da mistura contendo Man3 e 5-CQA mostraram que o CQA atua como catalisador das reações de transglicosilação. Por outro lado, nas misturas modelo contendo um péptido, mesmo contendo também 5-CQA e sujeitas ao mesmo tratamento, observou-se uma diminuição na extensão das reações transglicosilação. Este resultado pode explicar a baixa extensão das reações de transglicosilação não enzimáticas durante a torra nas regiões do grão de café mais ricas em proteínas, apesar dos polissacarídeos serem os componentes maioritários dos grãos de café. A diminuição das reações de transglicosilação na presença de péptidos/proteínas pode dever-se ao facto de os resíduos de açúcares redutores reagirem preferencialmente com os grupos amina de péptidos/proteínas por reação de Maillard, diminuindo o número de resíduos de açúcares redutores disponíveis para as reações de transglicosilação. Além dos compostos já descritos, uma diversidade de outros compostos foram formados a partir dos sistemas modelo, nomeadamente derivados de desidratação formados durante o tratamento térmico a seco. Em conclusão, a tipificação das modificações estruturais promovidas pela torra nos polissacarídeos do café abre o caminho para a compreensão dos mecanismos de formação das melanoidinas e da relação estrutura-atividade destes compostos.
Polysaccharides are the major components of green and roasted coffee beans, and coffee brew. The most abundant ones are galactomannans, followed by arabinogalactans. During the roasting process, galactomannans and arabinogalactans undergo structural modifications that are far to be completely elucidated due to their diversity and complexity of the compounds formed. During the roasting process, galactomannans and arabinogalactans react with proteins, chlorogenic acids, and sucrose, originating high molecular weight brown compounds containing nitrogen, known as melanoidins. Several biological activities and beneficial health effects have been attributed to coffee melanoidins. However, their exact structures and the mechanisms involved in their formation remain unknown, as well as the structure-biological activity relationship. The use of model systems and mass spectrometry analysis allow to obtain an overall view and, simultaneously, detailed, of the structural modifications in coffee polysaccharides promoted by roasting, contributing to the elucidation of the structures and formation mechanisms of melanoidins. Based on this thesis, oligosaccharides structurally related to the backbone of galactomannans, (β1→4)-D-mannotriose, and the side chains of arabinogalactans, (α1→5)-Larabinotriose, alone or in mixtures with 5-O-caffeoylquinic acid, the most abundant chlorogenic acid in green coffee beans, and dipeptides composed by tyrosine and leucine, used as models of proteins, were submitted to dry thermal treatments, mimicking the coffee roasting process. The oxidation induced by hydroxyl radicals (HO•) was also studied, since these radicals seem to be involved in the modification of the polysaccharides during roasting. The identification of the structural modifications induced by thermal and oxidative treatment of the model compounds was performed mostly by mass spectrometry-based analytical strategies, but also using liquid chromatography. Gas chromatography was used in the analysis of neutral sugars and glycosidic linkages. To validate the conclusions achieved with the model compounds, coffee polysaccharide samples obtained from spent coffee grounds and instant coffee were also analysed. The results obtained from the model oligosaccharides when submitted to thermal treatment (dry) or oxidation induced by HO• (in solution) indicate the occurrence of depolymerization, which is in line with previous studies reporting the depolymerization of coffee galactomannans and arabinogalactans during roasting. Compounds resulting from sugar ring cleavage were also formed during thermal treatment and oxidative treatment of Ara3. On the other hand, the dry thermal treatment of the model oligosaccharides (alone or when mixed) promoted the formation of oligosaccharides with a higher degree of polymerization, and also polysaccharides with new type of glycosidic linkages, evidencing the occurrence of polymerization via non-enzymatic transglycosylation reactions induced by dry thermal treatment. The transglycosylation reactions induced by dry thermal treatment can occur between sugar residues from the same origin, but also of different origins, with formation of hybrid structures, containing arabinose and mannose in the case of the model compounds used. The results obtained from spent coffee grounds and instant coffee samples suggest the presence of hybrid polysaccharides in these processed coffee samples, corroborating the occurrence of transglycosylation during the roasting process. Furthermore, the study of mixtures containing different proportions of each model oligosaccharide, mimicking coffee bean regions with distinct polysaccharide composition, subjected to different periods of thermal treatment, allowed to infer that different hybrid and non-hybrid structures may be formed from arabinogalactans and galactomannans, depending on their distribution in the bean cell walls and on roasting conditions. These results may explain the heterogeneity of melanoidins structures formed during coffee roasting. The results obtained from model mixtures containing an oligosaccharide (Ara3 or Man3) and 5-CQA and subjected to dry thermal treatment, as well as samples derived from spent coffee grounds, showed the formation of hybrid compounds composed by CQA molecules covalently linked to a variable number of sugar residues. Moreover, the results obtained from the mixture containing Man3 and 5-CQA showed that CQA acts as catalyst of transglycosylation reactions. On the other hand, in the model mixtures containing a peptide, even if containing 5-CQA and subjected to the same treatment, it was observed a decrease in the extent of transglycosylation reactions. This outcome can explain the low extent of non-enzymatic transglycosylation reactions during roasting in coffee bean regions enriched in proteins, although polysaccharides are the major components of the coffee beans. The decrease of transglycosylation reactions in the presence of peptides/proteins can be related with the preferential reactivity of reducing residues with the amino groups of peptides/proteins by Maillard reaction, decreasing the number of reducing residues available to be directly involved in the transglycosylation reactions. In addition to the compounds already described, a diversity of other compounds were formed from model systems, namely dehydrated derivatives formed during dry thermal treatment. In conclusion, the identification of the structural modifications in coffee polysaccharides promoted by roasting pave the way to the understanding of the mechanisms of formation of melanoidins and structure-activity relationship of these compounds.

Background and Aim: Lots of studies demonstrated that a different entity of on-treatment platelet function inhibition is associated with different clinical outcomes in patients with acute coronary syndromes. In particular, high on-treatment platelet reactivity (HPR) has been associated with an increased risk of ischemic complications (especially stent thrombosis), and there is a growing body of evidence that, on the contrary, low on-treatment platelet reactivity (LPR) could be associated with bleeding risk. Few data are available in the literature on the association between a different entity of platelet inhibition on-antiplatelet treatment and clinical outcomes in patients with peripheral artery disease (PAD). Aim of this study was to evaluate, in patients with PAD undergoing percutaneous revascularization, the degree of on-treatment platelet reactivity, and its association with ischemic and hemorrhagic adverse events at follow-up. Methods: In this observational, prospective, single center study, consecutive patients with PAD undergoing percutaneous transluminal angioplasty (PTA) with or without stenting, were enrolled. All patients were treated with dual antiplatelet therapy with aspirin and a P2Y12 inhibitor. Platelet function was assessed by Light Transmission Aggregometry (LTA) using arachidonic acid (AA) and adenosine diphosphate (ADP) as agonists of platelet aggregation, on blood samples obtained within 24 hours from PTA. HPR was defined by LTA ≥20% if induced by AA, and LTA ≥70% if induced by ADP. Follow-up was performed in order to record the occurrence of ischemic and bleeding events. Results: The study enrolled 177 patients [118 males, median age 75 (IQR 68-81) years]. HPR by AA was found in 52% of patients, and showed a non significant association with older age and a higher prevalence of renal failure, whereas HPR by ADP was found in 32% of patients, and was significantly associated with older age. During follow-up [median duration 23 (IQR 13-27) months] 23 deaths (13%) were recorded; 27 patients (17.5%) underwent target limb revascularization, 2 (1.3%) amputation, and 6 (3.9%) myocardial revascularization. Twenty-four patients (15.6%) experienced a minor bleeding complication. At multivariate analysis HPR by AA and HPR by ADP were independent predictors of death [HR 3.75 (1.20-11.66), P=0.023 and HR 4.78 (1.57-14.52), P=0.006, respectively]. Moreover, patients with dual HPR both by AA and by ADP showed a significantly higher risk of death than those without (P<0.001). The median value of LTA by ADP was significantly lower in patients with bleeding complications than in those without [26.5 (22-39.2)% vs 62 (44.5-74)%, P<0.001). At ROC curve analysis the cut-off of platelet aggregation induced by ADP with the best sensitivity and specificity for increased risk of bleeding was 41%. LTA by ADP lower than 41% was independently associated with bleeding [HR 14.59 (2.55-24.01), P=0.001] at multivariate analysis. Conclusions: In PAD patients undergoing PTA, HPR by ADP and AA were predictors of death, whereas LPR by ADP was predictor of bleeding complications. These results suggest the potential utility of assessing platelet function, even in the setting of PAD, in order to ensure the patient the best tailored antiplatelet therapy.

The purpose of the monograph, which contains a modern view of the problem of adaptation of children with extremely low body weight, is to provide a wide range of doctors with basic information about the clinical picture, functional activity of innate and adaptive immunity, prognostic criteria of postnatal pathology, based on their own research. The specific features of the immunological reactivity of premature infants of various gestational ages who have developed bronchopulmonary dysplasia (BPD) and retinopathy of newborns (RN) from the moment of birth and after reaching postconceptional age (37-40 weeks) are described separately. The mechanisms of their implementation with the participation of factors of innate and adaptive immunity are considered in detail. Methods for early prediction of BPD and RN with the determination of an integral indicator and an algorithm for the management of premature infants with a high risk of postnatal complications at the stage of early rehabilitation are proposed. The information provided makes it possible to personify the treatment, preventive and rehabilitation measures in premature babies. The monograph is intended for obstetricians-gynecologists, neonatologists, pediatricians, allergists-immunologists, doctors of other specialties, residents, students of the system of continuing medical education. This work was done with financial support from the Ministry of Education and Science, grant of the President of the Russian Federation No. MK-1140.2020.7.

The purpose of the monograph, which contains a modern view of the problem of adaptation of children with extremely low body weight, is to provide a wide range of doctors with basic information about the clinical picture, functional activity of innate and adaptive immunity, prognostic criteria of postnatal pathology, based on their own research. The specific features of the immunological reactivity of premature infants of various gestational ages who have developed bronchopulmonary dysplasia (BPD) and retinopathy of newborns (RN) from the moment of birth and after reaching postconceptional age (37-40 weeks) are described separately. The mechanisms of their implementation with the participation of factors of innate and adaptive immunity are considered in detail. Methods for early prediction of BPD and RN with the determination of an integral indicator and an algorithm for the management of premature infants with a high risk of postnatal complications at the stage of early rehabilitation are proposed. The information provided makes it possible to personify the treatment, preventive and rehabilitation measures in premature babies. The monograph is intended for obstetricians-gynecologists, neonatologists, pediatricians, allergists-immunologists, doctors of other specialties, residents, students of the system of continuing medical education. This work was done with financial support from the Ministry of Education and Science, grant of the President of the Russian Federation No. MK-1140.2020.7.

The purpose of the monograph, which contains a modern view of the problem of adaptation of children with extremely low body weight, is to provide a wide range of doctors with basic information about the clinical picture, functional activity of innate and adaptive immunity, prognostic criteria of postnatal pathology, based on their own research. The specific features of the immunological reactivity of premature infants of various gestational ages who have developed bronchopulmonary dysplasia (BPD) and retinopathy of newborns (RN) from the moment of birth and after reaching postconceptional age (37-40 weeks) are described separately. The mechanisms of their implementation with the participation of factors of innate and adaptive immunity are considered in detail. Methods for early prediction of BPD and RN with the determination of an integral indicator and an algorithm for the management of premature infants with a high risk of postnatal complications at the stage of early rehabilitation are proposed. The information provided makes it possible to personify the treatment, preventive and rehabilitation measures in premature babies. The monograph is intended for obstetricians-gynecologists, neonatologists, pediatricians, allergists-immunologists, doctors of other specialties, residents, students of the system of continuing medical education. This work was done with financial support from the Ministry of Education and Science, grant of the President of the Russian Federation No. MK-1140.2020.7.

The purpose of the monograph, which contains a modern view of the problem of adaptation of children with extremely low body weight, is to provide a wide range of doctors with basic information about the clinical picture, functional activity of innate and adaptive immunity, prognostic criteria of postnatal pathology, based on their own research. The specific features of the immunological reactivity of premature infants of various gestational ages who have developed bronchopulmonary dysplasia (BPD) and retinopathy of newborns (RN) from the moment of birth and after reaching postconceptional age (37-40 weeks) are described separately. The mechanisms of their implementation with the participation of factors of innate and adaptive immunity are considered in detail. Methods for early prediction of BPD and RN with the determination of an integral indicator and an algorithm for the management of premature infants with a high risk of postnatal complications at the stage of early rehabilitation are proposed. The information provided makes it possible to personify the treatment, preventive and rehabilitation measures in premature babies. The monograph is intended for obstetricians-gynecologists, neonatologists, pediatricians, allergists-immunologists, doctors of other specialties, residents, students of the system of continuing medical education. This work was done with financial support from the Ministry of Education and Science, grant of the President of the Russian Federation No. MK-1140.2020.7.

The purpose of the monograph, which contains a modern view of the problem of adaptation of children with extremely low body weight, is to provide a wide range of doctors with basic information about the clinical picture, functional activity of innate and adaptive immunity, prognostic criteria of postnatal pathology, based on their own research. The specific features of the immunological reactivity of premature infants of various gestational ages who have developed bronchopulmonary dysplasia (BPD) and retinopathy of newborns (RN) from the moment of birth and after reaching postconceptional age (37-40 weeks) are described separately. The mechanisms of their implementation with the participation of factors of innate and adaptive immunity are considered in detail. Methods for early prediction of BPD and RN with the determination of an integral indicator and an algorithm for the management of premature infants with a high risk of postnatal complications at the stage of early rehabilitation are proposed. The information provided makes it possible to personify the treatment, preventive and rehabilitation measures in premature babies. The monograph is intended for obstetricians-gynecologists, neonatologists, pediatricians, allergists-immunologists, doctors of other specialties, residents, students of the system of continuing medical education. This work was done with financial support from the Ministry of Education and Science, grant of the President of the Russian Federation No. MK-1140.2020.7.

The purpose of the monograph, which contains a modern view of the problem of adaptation of children with extremely low body weight, is to provide a wide range of doctors with basic information about the clinical picture, functional activity of innate and adaptive immunity, prognostic criteria of postnatal pathology, based on their own research. The specific features of the immunological reactivity of premature infants of various gestational ages who have developed bronchopulmonary dysplasia (BPD) and retinopathy of newborns (RN) from the moment of birth and after reaching postconceptional age (37-40 weeks) are described separately. The mechanisms of their implementation with the participation of factors of innate and adaptive immunity are considered in detail. Methods for early prediction of BPD and RN with the determination of an integral indicator and an algorithm for the management of premature infants with a high risk of postnatal complications at the stage of early rehabilitation are proposed. The information provided makes it possible to personify the treatment, preventive and rehabilitation measures in premature babies. The monograph is intended for obstetricians-gynecologists, neonatologists, pediatricians, allergists-immunologists, doctors of other specialties, residents, students of the system of continuing medical education. This work was done with financial support from the Ministry of Education and Science, grant of the President of the Russian Federation No. MK-1140.2020.7.

Water pollution by metalloids is a global environmental concern. Owing to their propensity for bioaccumulation, water solubility, and interaction with environment, they are threatening both human and ecosystem health. Inherent limitations like low efficiency, sensitive operating conditions, and high capital and operating costs are associated with conventional removal methods which restricts adoption of these technologies on large scale. While adsorption is commonly recognized as both an effective and affordable remediation technology, many common adsorbents often have inherited limitations including non-renewability and high operating costs. Thus, limitations in conventional remediation technologies have headed to the rapid progression of new avenues for advanced treatment technologies for metalloid pollutant removal such as green nanotechnology. In contrast to many of the currently available adsorbents, nanoparticles often have unique properties such as tiny size, more active sites and big surface area, easy separation, and high reactivity that enhance removal efficiencies.

The interest in platelet functions in HIV/AIDS is due to the high incidence of microvascular thrombosis in these individuals. A lot of laboratory data have been generated regarding platelet functions in this population. The tests demonstrate platelet hyperactivity but decreased aggregation, though results are inconsistent depending on the study design. Antiretroviral treatments currently in use display complex interactions. Many studies on platelet functions in these patients have been for research purposes, but none have found utility in guiding drug treatment of thrombosis.

Water pollution by metalloids is a global environmental concern. Owing to their propensity for bioaccumulation, water solubility, and interaction with environment, they are threatening both human and ecosystem health. Inherent limitations like low efficiency, sensitive operating conditions, and high capital and operating costs are associated with conventional removal methods which restricts adoption of these technologies on large scale. While adsorption is commonly recognized as both an effective and affordable remediation technology, many common adsorbents often have inherited limitations including non-renewability and high operating costs. Thus, limitations in conventional remediation technologies have headed to the rapid progression of new avenues for advanced treatment technologies for metalloid pollutant removal such as green nanotechnology. In contrast to many of the currently available adsorbents, nanoparticles often have unique properties such as tiny size, more active sites and big surface area, easy separation, and high reactivity that enhance removal efficiencies.

Thrombus formation involves activation of aggregating platelets as well as the formation of fibrin after activation of the coagulation cascade. Fibrin eventually binds to glycoprotein IIB/IIIA receptors on platelets thereby forming a very solid occlusive clot. There are several molecules interfering with platelet activation: (1) aspirin, which blocks the formation of thromboxane A₂; (2) clopidogrel, prasugrel, and ticagrelor which block P₂Y₁₂ receptors and their activation by ADP; (3) glycoprotein inhibitors that interfere with glycoprotein IIB/IIIA on platelets, the final common pathway of platelet activation, and finally (4) thrombin inhibitors blocking PAR-1 receptors on platelets. Commonly, after an acute coronary syndrome (ACS) or after percutaneous coronary interventions (PCI) or even bypass surgery, dual antiplatelet therapy (DAPT) is recommended consisting of aspirin plus a P₂Y₁₂ inhibitor. The duration of treatment after the acute event is commonly 12 months for those with ACS and 6 months for those with a high bleeding risk. In patients with high ischaemic risk, prolonged DAPT treatment can be considered, but often such patients also have a high bleeding risk. Intravenous platelet inhibitors, such as Reopro, tirofiban, and others, are used mainly during PCI, particularly in patients with ACS. Inhibitors of coagulation cascade, such as factor Xa or factor II inhibitors or vitamin K antagonists are mainly used in patients with ACS and concomitant atrial fibrillation. In these patients, mainly aspirin or in some the P₂Y₁₂ inhibitor is skipped for some weeks or months.

CPA are considered as a possible cause of blood flow impairment and as a risk factor of sudden death in patients with CHD. The relevance of a low PCR to the presence of CPA is still debated. Indeed in a critical and well designed evaluation of this index, Sanibaldi et al.(1986) have shown that PCR obtained by counting free platelets in diluted whole blood was not lowered in patients with “thrombotic tendency”. They suggested that low CPA reported in other clinical studies may result from an artifact of blood sampling or from ex-vivo procedures.In order to reevalute the meaning of a low PCR, selected patients with CHD have been submitted to an exercice resulting in platelet stimulation. Free platelets were counted, whole blood and plasma β-tg were simultaneously determined by RIA after careful blood sampling performed before the exercice, at its end and 30 min later. Patients were previously treated by Placebo (P)or by T 500 mg per day given in a cross-over double blind manner.It was shown that : 1) after P treatment, PCR was lowered and β-tg was increased, both significantly, at the end of exercice; 30 min later, PCR was normalized whereas β-tg remained high presumably because β-tg is not cleared from plasma within 30 min. 2) T treatment significantly inhibited both platelet responses.In conclusion, when platelets are submitted to systemic and sustained stimuli occurring during exercice stress, lowered PCR reflects the presence of circulating platelet aggregates which can be dispersed ex-vivo by EDTA. In these conditions, the meaning of this index is strongly confirmed by the simultaneous release of βtg. T treatment significantly reduces platelet reactivity to exer cice stress and may reduce the effects of platelet aggregation and release on compromised microcirculation.

Heparin-induced thrombocytopenia (HIT), an adverse effect of heparin therapy, may be associated with serious thrombosis resulting in severe disability or death. An IgG heparin-dependent antibody may be demonstrated in HIT by platelet aggregation studies with patient serum/plasma and standard (s) heparin. A recent study showed high cross-reactivity of the antibody with low molecular weight (LMW) heparins as most of the 22 patient sera tested gave a positive reaction with various LMW heparins including CY222, CY216, PK10169 and Kabi 2165 (Messmore HL et al, Blood 64(5), 1984 suppl). However, cross-reactivity rate with Org 10172, a LMW heparinoid which has strong anti-Xa but negligible antithrombin activity, is unknown. We tested the plasma of 17 patients with HIT for cross-reactivity with Org 10172. Although all 17 patient plasmas reacted positively with s.heparin (0.2 1.0 IU/ml), only 3 patient plasmas gave a positive but weaker reaction with Org 10172 (0.2-1.0 IU/ml).Further studies were performed to investigate the effect of adding Org 10172 (0.2 to 2.0 anti-Xa U/ml) to a reaction mixture of normal platelet-rich plasma, patient plasma and s.heparin (0.2 IU/ml). With 7 patient plasmas which showed no cross-reactivity with Org 10172, the antibody-induced platelet aggregation was inhibited when the concentration of Org 10172 exceeded 0.5 to 1.0 anti-Xa U/ml. When the study was repeated with other s.heparin concentrations (0.05, 0.1, 0.4 IU/ml), this inhibitory effect was again present provided the ratio of Org 10172 concentration (anti-Xa U/ml) to heparin concentration (IU/ml) exceeds 2.5 to 5. However, this inhibitory effect was not observed with the 3 patient plasmas which showed cross-reactivity with the heparinoid whqp. the same concentrations of Org 10172 were added. This inhibitory effect appeared to be specific for platelet aggregation induced by the heparin-dependent antibody as Org 10172 (<10 anti-Xa U/ml) did not affect platelet aggregation induced by collagen (2 ug/ml) and ADP (2.5 uM). These findings together with our experience of 3 cases of HIT successfully treated with Org 10172 suggest that this LMW heparinoid may be a useful drug for the treatment of HIT.

Patients with familial hyperlipoproteinaemia (HLP) are at high risk for the premature development of atherosclerosis. This study was designed to investigate the effect of long-term treatment (8 months) with the HMG-CoA-reductase inhibitor SYN (20-40 mg/day) on platelet reactivity and PGIo receptors in 12 HLP patients (B) as compared with 11 untreated HLP patients (A) and 11 healthy subjects (C). Treatment with SYN reduced the plasma cholesterol to 234 ± 12 mg/dl as compared to 307 ± 21 (A) and 195 ± 14 mg/dl (C), respectively. Collagen (0.6 pg/ml) induced platelet thromboxane (TXB2) formation was 50±6 ng/ml in group A and significantly reduced to 32 ± 3 (B) which was not different from the 28±3 ng/ml in group C. Similar results were obtained by measuring pTatelet ATP secretion and aggregation. SYN treatment significantly improved the reduced number of PGI2 receptors (determined according to Schillinger & Prior, 1980) and normalized the iloprost (ILO, 30 nM) induced cAMP stimulation in platelet-rich plasma while the kg remained unchanged:These data demonstrate that SYN-treatment of HLP patients at doses that reduce plasma cholesterol by about 25% results in sig-ficant improvement of platelet function. This includes both normalization of platelet hyperreactivity against proaggregatory agents as well as platelet hyporeactivity aganist PGI2. The last effect might involve an increase of the (reduced) number of PGI2 receptors in HLP type IIa.

The platelet response to angioplasty or spontaneous plaque rupture leads to acute thrombotic occlusion under certain conditions. We analyzed the role of local shear rate (flow and vessel cross-section related), the nature of the exposed matrix and the effect of thrombin inhibition in platelet acute response to injury. Collagen type I (exposed in plaque rupture) and de-endothelialized pig aorta (mild injury) were exposed to pig blood in a tubular perfusion chamber with well characterized flow conditions, placed within an extracorporeal circuit in swine (N=20). Platelet deposition was measured by labeling autologous platelets with Indium and optical morphometry of epoxy embedded specimens. Selected specimens were analyzed by electron microscopy. Unanticoagulated blood and blood from animals treated with 300u/Kg of heparin were perfused over the substrate for 3 and 10 min at local shear rates typical of unobstructed arteries (212s™1 - 424s™1) and of stenotic vessel (824s™1 - 1690s™1). Platelet deposition (Platelets × 106/cm2 ± SE) for 3 min perfusions were:Platelet deposition is dependent on the reactivity of the vascular matrix exposed to blood and on the local shear rate. The greatest rate of thrombus growth is observed with collagen and high shear rate conditions which may precipitate acute thrombotic occlusion in stenotic regions, mainly when the coagulation pathway is not inhibited (255×l06 platelets/ cm2 in 3 minutes. The relative contribution of rheology and the isolated components of the atherosclerotic plaque matrix exposed to blood in the onset of acute coronary syndromes will be differentiated with this experimental model.

Lymphocyte subsets, platelet counts, immune globulin levels and antibody to HIV (Elisa, WB) were determined in 87 multitransfused asymptomatic haemophiliacs in 1982/83. Between 1982 and 1987 6 patients developed AIDS and 5 ARC (3 immune thrombocytopenia and 2 lymphadenopathy). AIDS or ARC developed in seropositive patients only (11/49). Patients who subsequently developed AIDS or ARC showed significantly lower numbers of T helper lymphocytes (378/mm3 versus 605/mm3; p 0.01), lower platelet counts (157x109 versus 194x109; p 0.05) and higher levels of IgG (2528 mg/dl versus 1992 mg/dl; p 0.01). AIDS or ARC occured in 4 of 7 patients(57.1%) with a low HIV antibody level ( 2000), but only in 7 of 42 (16.6%) with a high level of antibody to HIV ( 2000). A weak gag p 24 in WB was found in 4 of 11 patients (36.3%) who subsequently acquired AIDS or ARC , while none of the patients whq remained asymptomatic displayed this reactivity pattern in WB. 9 patients showed a weak gag p 18 in WB. 8 of them (88.8%) have platelet counts below 120x109 /1, 3 developed imiruine thrombocytopenia with platelet counts of less than 50xl09. Oily 6 of 40 patients (15%) without this reactivity pattern in WB have platelet counts lower than 120x109 and none below 50xl09.We conclude that a weak gag p 24 in WB has a strong positive predictive power for the development of AIDS or ARC in seropositive haemophiliacs. A weak gag p 18 in WB could possibly be associated with the occurence of immune thrombocytopenia in these patients.

The biochemical structure of arterial microfibrils (MFS) is unknown. Presently, the most probable hypothesis is that elastin associated MFS contain several antigenic determinants with MW varying between 31 and 200 KD.From our previous studies we know that MFS extracted by 6 M GuCl contain a major glycoprotein with a 128 KD MV (GP128). GP 128 is essential for the reactivity of MFS towards blood platelets but due tc the high insolubility of the extracted material it was not possible to isolate and study this GP 128. We have used immunoblotting to determine if MFS contain determinants recognized by antibodies against connective tissue glycoproteins such as fibronectin, type VI collagen or anti-platelet thrombospondin (TSP). 'The results showed that MFS do not contain fibronectin or type VI collagen but that anti-TSP IgG reacted with GP 128. Furthermore, the Fab fragments from anti-TSP IgG inhibited platelet aggregation induced by MFS but not by collagen or ADP . In a second step,to raise antibodies against GP 128, we prepared blots from entire MFS, the nitrocellulose band corresponding to GP 128 was cut, dissolved in DMSO, and wrs injected to rabbits. Such obtained antibodies recognized only GP 128 in arterial MFS and also TSP in a platelet lysate confirming that GP 128 and TSP have a common antigenic structure. IgG from anti-GP 128 inhibit platelet aggregation induced by MFS but not by collagen or ADP. Previously reported observations showed that tissue TSP and endothelial cells derived GP 128 have a similar affinity for chromatography supports and have the same effect on platelet-MFS interactions. All these results led us to propose that TSP, GP 128, and MFS recognize a common determinant on platelet membrane. This assumption would be strenghened if GP 128 indeed is derived from tissue TSP.

Earlier studies indicate an increased platelet reactivity in patients with hypercholesterolaemia. Physical and mental stress have also been reported to cause increased reactivity. The present study was undertaken to evaluate the effect of gemfibrozil (G) a new lipid lowering drug on platelet reactivity during physical stress. Ten otherwise healthy male subjects with serum cholesterol levels above 7 mmol/l were involved in a double-blind study. It consisted of two treatment periods of 8 weeks during which the patients were given either G (600 mg b.i.d.) or placebo (PI) and an 8 weeks wash-out period before the cross-over. At the end of the treatment periods an exercise test was carried out and platelet reactivity tested. Adrenaline, ADP and collagen were used to induce aggregation and 5-HT and T×B2 release measured. Plasma beta-TG and fibrinogen were also determined.The treshold concentration of adrenaline necessary to evoke secondary aggregation was increased in 8/10 patients during exercise after G treatment and in 2/10 after PI. When the lowest ADP concentration to cause secondary aggregation (2-4 uM) was used there was a significant decrease in the 5-HT (− 44%) and T×B2 (− 48%) secretion and a significant decrease in the area under the aggregation curve (− 28%). A decrease in 5-HT secretion was also seen after G treatment when a fixed ADP concentration of 10 uM was used. During collagen stimulation no changes were seen between the two groups. Beta-TG remained unchanged irrespective of treatment and fibrinogen showed a modest increase during exercise in both treatment groups. These results indicate a new anti-platelet effect of gemfibrozil which might be of importance in prevention of acute thrombotic events in hypercho1estero1aemic patients.

In unstable coronary artery disease (UCAD), i.e. unstable angina pectoris (UAP) or non-Q-myocardial infarction (NMI), treatment with heparin or ASA have given encouraging results. The present study attempts to verify the effects of i.v. heparin (5 days) and to evaluate the utility of ASA 75 mg/day (one year). Patients, admitted because of chest pain, who either develops NMI or signs of ischemia in resting or exercise ECG.s are included. Within 72 hours patients are randomized to obtain Heparin+ASA, Heparin+Placebo, Placebo + ASA or Placebo+Placebo . Platelet reactivity is studied in vitro in platelet rich plasma (PRP) in a subgroup of patients.The aggregation response is studied after addition of collagen and ADP and after preincubation with prostacyclin before aggregation with ADP. The figures present results from 85 randomized patients tested before, 5 days, one month and one year after start of therapy.Conclusion: In patients with unstable CAD long term treatment with ASA 75 mg/day inhibits collagen induced platelet aggregation and hampers the ADP response. I.v. heparin tends to raise platelet reactivity and reduce the inhibitory effect of prostacyclin. Heparin induced platelet activation is reduced by simultaneous ASA therapy.

Dipyridamole (DIP) is a pirydo-pirymidine drug widely used as an antiplatelet agent.DIP has also been demonstrated to have effects on various cultured cells. We studied platelet reactivity of extracellular matrices (ECMs) produced by untreated (ECM-c) and DIP-treated (ECM-DIP) endothelial cells (ECs).Confluent monolayers of EC were incubated in buffer containing 0 or 10 pM. DIP for 48 h. The ECMs were prepared by removing the EC with 2% EGTA (1 h at 37°C). Platelet deposition onto the ECMs was measured under flow conditions using a flat chamber perfusion model ECM-c and ECM-DIP were exposed for 5 min to whole blood at shear rates of 300 or 1300 sec~1.Then, the perfused ECMs were processed for cross sectional morphometric evaluation using a computer system. The platelet, deposition is expressed as total surface covered with platelets (mean±SEM ) and shown in the table.Platelet aggregate formation on ECM-DIP at 1300 sec-1 was also reduced(by 25%)as measured morphometrically. We conclude that DIP-treatment of EC results in ECM modifications,which in turn decrease ECM-platelet interactions.These data also suggest that pharmacological treatment of the endothelium influences basement membrane reactivity.

We previously demonstrated that dexamethasone increases fibronectin (FN) expression by human umbilical vein endothelial cell (HUVEC) as well as its incorporation in the extracellular matrix (EM). FN is primarily involved in platelet/vessel wall interactions as well as in cell attachment to substrates. The in vivo anti-haemorragic effect of glucocorticoids could therefore be related either to a better spreading and attachment of endothelial monolayer to the basement membrane or to an increase of vessel wall reactivity to platelets. To further investigate this, we studied the effect of dexamethasone treatment on EM reactivity to platelets in an in vitro perfusion system. Second passage HUVEC were seeded on glass coverslips at a density of 105/cm2 and treated with dexamethasone 1.0 μM for 48 hours. EM were prepared by exposure to 0.1 M NH4OH for 30’. Coverslips carrying the EM were perfused with citrated whole blood in a flat perfusion chamber(*) at wall shear rates of 300 and 900 sec™1 for 5’. Platelet adhesion to EM was evaluated by a morphometric method. The number of platelet adhering to EM at both shear rates was significantly increased by dexamethasone treatment, (+51.9% at 300 sec™1 (p<0.001) and +24.3% at 900 sec™1 (p<0.01)). Our results demonstrate that dexamethasone increases endothelial EM reactivity to platelets possibly through an increase of FN deposition. This, together with FN effect on cell spreading and anchorage to EM, might be responsible for the antl-haemorrhagic action of glucocorticoids.(*) K. Sakanassen et al . , J . Lab. Cl in . Med. 102:522, 1983

The overall objectives of the project were: (a) To measure and study in situ the effect of irrigation with reclaimed sewage effluents on redox processes and related chemical dynamics in soil profiles of agricultural fields. (b) To study under controlled conditions the kinetics and equilibrium states of selected processes that affect redox conditions in field soils or that are effected by them. Specifically, these include the effects on heavy metals sorption and desorption, and the effect on pesticide degradation. On the basis of the initial results from the field study, increased effort was devoted to clarifying and quantifying the effects of plants and water regime on the soil's redox potential while the study of heavy metals sorption was limited. The use of reclaimed sewage effluents as agricultural irrigation water is increasing at a significant rate. The relatively high levels of suspended and, especially, dissolved organic matter and nitrogen in effluents may affect the redox regime in field soils irrigated with them. In turn, the changes in redox regime may affect, among other parameters, the organic matter and nitrogen dynamics of the root zone and trace organic decomposition processes. Detailed data of the redox potential regime in field plots is lacking, and the detailed mechanisms of its control are obscure and not quantified. The study established the feasibility of long-term, non-disturbing monitoring of redox potential regime in field soils. This may enable to manage soil redox under conditions of continued inputs of wastewater. The importance of controlling the degree of wastewater treatment, particularly of adding ultrafiltration steps and/or tertiary treatment, may be assessed based on these and similar results. Low redox potential was measured in a field site (Site A, KibutzGivat Brenner), that has been irrigated with effluents for 30 years and was used for 15 years for continuous commercial sod production. A permanently reduced horizon (Time weighted averaged pe= 0.33±3.0) was found in this site at the 15 cm depth throughout the measurement period of 10 months. A drastic cultivation intervention, involving prolonged drying and deep plowing operations may be required to reclaim such soils. Site B, characterized by a loamy texture, irrigated with tap water for about 20 years was oxidized (Time weighted average pe=8.1±1.0) throughout the measurement period. Iron in the solid phases of the Givat Brenner soils is chemically-reduced by irrigation. Reduced Fe in these soils causes a change in reactivity toward the pesticide oxamyl, which has been determined to be both cytotoxic and genotoxic to mammalian cells. Reaction of oxamyl with reduced-Fe clay minerals dramatically decreases its cytotoxicity and genotoxicity to mammalian cells. Some other pesticides are affected in the same manner, whereas others are affected in the opposite direction (become more cyto- and genotoxic). Iron-reducing bacteria (FeRB) are abundant in the Givat Brenner soils. FeRB are capable of coupling the oxidation of small molecular weight carbon compounds (fermentation products) to the respiration of iron under anoxic conditions, such as those that occur under flooded soil conditions. FeRB from these soils utilize a variety of Fe forms, including Fe-containing clay minerals, as the sole electron acceptor. Daily cycles of the soil redox potential were discovered and documented in controlled-conditions lysimeter experiments. In the oxic range (pe=12-8) soil redox potential cycling is attributed to the effect of the daily temperature cycle on the equilibrium constant of the oxygenation reaction of H⁺ to form H₂O, and is observed under both effluent and freshwater irrigation. The presence of plants affects considerably the redox potential regime of soils. Redox potential cycling coupled to the irrigation cycles is observed when the soil becomes anoxic and the redox potential is controlled by the Fe(III)/Fe(II) redox couple. This is particularly seen when plants are grown. Re-oxidation of the soil after soil drying at the end of an irrigation cycle is affected to some degree by the water quality. Surprisingly, the results suggest that under certain conditions recovery is less pronounced in the freshwater irrigated soils.