Platelet aggregation studies: autologous platelet-poor plasma inhibits platelet aggregation when added to platelet-rich plasma to normalize platelet count

Adjusting platelet count (PC) in platelet-rich plasma (PRP) using platelet-poor plasma (PPP) is recommended for platelet aggregation (PA) studies, but it could also affect PA independently of the decrease in PC. Analysis of aggregation tracings from healthy controls showed that PC correlated with PA in 47 diluted-PRPs, but not in 104 undiluted-PRPs. Dilution of 9 PRPs with PPP progressively decreased PA, while dilution of washed platelets with buffer hardly affected PA. Apyrase partially prevented the inhibitory effect of PPP. Therefore, the practice of diluting PRP with PPP to adjust platelet count should be avoided because it artefactually inhibits PA

Short communication:Intra- and inter-individual milk microbiota variability in healthy and infected water buffalo udder quarters

The concept that ruminant mammary gland quarters are anatomically and physiologically unrelated has been recently challenged by immunological evidence. How this interdependence reflects on individual quarter milk microbiota is unknown. The aim of the present study was to cover this gap by investigating the interdependence of quarters among the same mammary gland at the milk microbiota level using next-generation sequencing of the V4\u201316S rRNA gene. A total of 52 samples were included in this study and classified as healthy or affected by subclinical mastitis. Extraction of DNA, amplification of the V4\u201316S rRNA gene, and sequencing using Ion Torrent Personal Genome Machine (Thermo Fisher Scientific, Waltham, MA) were carried out. We found that the intra-individual variability was lower than the inter-individual one. The present findings further support at milk microbiota level the hypothesis of the interdependence of quarters, as previously demonstrated following immunological studies, suggesting that individual factors (e.g., immunity, genetics) may have a role in modulating milk microbiota

Characterization of skin surface and dermal microbiota in dogs with mast cell tumor

The skin microbiota interacts with the host immune response to maintain the homeostasis. Changes in the skin microbiota are linked to the onset and the progression of several diseases, including tumors. We characterized the skin surface and dermal microbiota of 11 dogs affected by spontaneous mast cell tumor (MCT), using skin contralateral sites as intra-animal healthy controls. The microbial profile differed between healthy and tumor skin surfaces and dermis, demonstrating that the change in microbiota composition is related to the presence of MCT. The number of observed taxa between MCT and healthy skin surfaces was detected, showing a decrease in number and heterogeneity of taxa over the skin surface of MCT, at both inter- and intra-individual level. Preliminary data on bacterial population of MCT dermis, obtained only on three dogs, demonstrated an intra-individual reduction of taxa number when compared to the skin surface. Taxonomy reveals an increase of Firmicutes phylum and Corynebacteriaceae family in MCT skin surface when compared to the healthy contralateral. In conclusion, we demonstrate that microbial population of skin surface and dermis is related to mast cell tumor. Our study provides the basis for future investigations aiming to better define the interaction between mast cell tumors, microbiota and host immune response

Antiaggregatory activity in human platelets of potent antagonists of the P2Y1 receptor

Activation of the P2Y1 nucleotide receptor in platelets by ADP causes changes in shape and aggregation, mediated by activation of phospholipase C (PLC). Recently, MRS2500 (2-iodo-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate) was introduced as a highly potent and selective antagonist for this receptor. We have studied the actions of MRS2500 in human platelets and compared these effects with the effects of two acyclic nucleotide analogues, a bisphosphate MRS2298 and a bisphosphonate derivative MRS2496, which act as P2Y1 receptor antagonists, although less potently than MRS2500. Improved synthetic methods for MRS2500 and MRS2496 were devised. The bisphosphonate is predicted to be more stable in general in biological systems than phosphate antagonists due to the non-hydrolyzable C–P bond. MRS2500 inhibited the ADP-induced aggregation of human platelets with an IC50 value of 0.95 nM. MRS2298 and MRS2496 also both inhibited the ADP-induced aggregation of human platelets with IC50 values of 62.8 nM and 1.5 μM, respectively. A similar order of potency was observed for the three antagonists in binding to the recombinant human P2Y1 receptor and in inhibition of ADP-induced shape change and ADP-induced rise in intracellular Ca2+. No substantial antagonism of the pathway linked to the inhibition of cyclic AMP was observed for the nucleotide derivatives, indicating no interaction of these three P2Y1 receptor antagonists with the proaggregatory P2Y12 receptor, which is also activated by ADP. Thus, all three of the bisphosphate derivatives are highly selective antagonists of the platelet P2Y1 receptor, and MRS2500 is the most potent such antagonist yet reported

Acquired platelet dysfunction and overproduction of platelet cyclic AMP in two patients with myeloid malignancies

The pathophysiology of impaired platelet function in acquired disorders is often poorly understood. We report two unrelated patients with hematologic malignancies associated with acquired severe bleeding diathesis, and complex platelet function abnormalities, including overproduction of the physiological inhibitor cyclic-AMP (cAMP). Patient 1, with mild macrocytic anemia and thrombocytopenia (100 x 109/L), was diagnosed with chronic myelomonocytic leukemia a few months after the onset of her bleeding diathesis and our analysis of platelet function. Patient 2, with bleeding diathesis of recent onset, was studied when his myelodysplastic syndrome with excess blasts had already progressed to acute myeloid leukemia. In both patients, platelet aggregation/ATP secretion, serum thromboxane B2, intraplatelet content of ADP, ATP, serotonin, and fibrinogen were severely impaired. Baseline platelet cAMP levels were mildly elevated and markedly increased after stimulation by prostaglandin E1. In conclusion, these are the first patients with myeloid malignancies associated with acquired severe platelet dysfunction and overproduction of cAMP

Standard internazionali per raccolta, manipolazione, caratterizzazione, conservazione, selezione e distribuzione del sangue cordonale. Seconda edizione (2001) degli standard elaborati e pubblicati congiuntamente da NETCORD e FAHCT

Consiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7 , Rome / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Novel variant in HPS3 gene in a patient with Hermansky Pudlak syndrome (HPS) type 3

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder caused by defects in 10 human HPS genes, characterized by oculocutaneous albinism (OCA) and bleeding diathesis associated to platelet δ-storage pool defect (SPD). We report a case of 4-year-old boy from non-consanguineous parents with OCA and negative personal and familiar hemorrhagic history, referred to us for severe bleeding after mild trauma. His platelet function, studied by lumi-aggregometry, showed normal first wave of aggregation in response to exogenous agonists and impaired second wave with defective ATP release. This, in combination with impaired platelet δ-granules content (serotonin, ATP, ADP) and the OCA phenotype suggested the HPS diagnosis. HPS3 sequencing revealed a novel pathogenic homozygous variant (NM_032383.4:c.7>T, p.Gln3*) resulting in a premature stop codon at the amino acid 3. Moreover, our report highlights the importance of evaluating platelet function in children with OCA without bleeding diathesis to identify HPS early and prevent bleeding complications