PI3K/Akt in platelet integrin signaling and implications in thrombosis

Blood platelets are anucleated circulating cells that play a critical role in hemostasis and are also implicated in arterial thrombosis, a major cause of death worldwide. The biological function of platelets strongly relies in their reactiveness to a variety of extracellular agonists that regulate their adhesion to extracellular matrix at the site of vascular injury and their ability to form rapidly growing cell aggregates. Among the membrane receptors expressed on the cell surface, integrins are crucial for both platelet activation, adhesion and aggregation. Integrin affinity for specific ligands is regulated by intracellular signaling pathways activated in stimulated platelets, and, once engaged, integrins themselves generate and propagate signals inside the cells to reinforce and consolidate platelet response and thrombus formation. Phosphatidylinositol 3-Kinases (PI3Ks) have emerged as crucial players in platelet activation, and they are directly implicated in the regulation of integrin function. This review will discuss the contribution of PI3Ks in platelet integrin signaling, focusing on the role of specific members of class I PI3Ks and their downstream effector Akt on both integrin inside-out and outside-in signaling. The contribution of the PI3K/Akt pathways stimulated by integrin engagement and platelet activation in thrombus formation and stabilization will also be discussed in order to highlight the possibility to target these enzymes in effective anti-thrombotic therapeutic strategies

Mismatch in early life traits between settlers and recruits in a Mediterranean fish: Clue of the relevance of the settlement tail?

Background. Life stage transitions (e.g., settlement and recruitment), characterized by high mortality rates, act as selective bottlenecks for fishes with a bipartite life cycle. Mortality at these stages is usually selective and potentially affected by larval history. This process is reflected in an inconsistency in larval traits’ distribution between subsequent life stages (e.g., settlers and recruits) originating from the same reproductive season. Despite the importance of this issue only very scarce information is available about this aspect of Mediterranean fishes life histories. Materials and methods. Here we described settlement and investigated the match/mismatch of larval traits between settlers and recruits coming from the same reproductive season, using the white seabream, Diplodus sargus sargus (Linnaeus, 1758), as a model species along ~ 200 km of the Apulian Adriatic coast (south-western Adriatic Sea, Italy). Both microstructure and chemistry analyses were carried out on otoliths of settlers (n = 140) and recruits (n = 113). Results. We highlighted a mismatch in two life traits, i.e., PLD (pelagic larval duration) and natal origin, between settlers and recruits. Recruits showed PLD longer than the maximum recorded for settlers, and a higher number of natal sources compared to settlers. Mismatch in PLD could suggest selective juvenile mortality related to PLD, and recruits with higher PLD potentially originated from the settlement tail (i.e., settled after the settlement peak). Conclusion. Our findings can support hypotheses suggesting that 1) a fraction of juveniles are selectively eliminated; 2) settlement tail could play a relevant role in replenishing local populations of white seabream

The small GTPase Rap1b regulates the cross talk between platelet integrin α2β1 and integrin αIIbβ3

The involvement of the small GTPase Rap1b in platelet integrin α2β1-dependent outside-in signaling was investigated. Platelet adhesion to 4 different specific ligands for integrin α2β1, monomeric collagen, decorin, and collagen-derived peptides CB8(II) and CB11(II), induced a robust and rapid activation of Rap1b. This process did not require secreted ADP or thromboxane A2 production but was critically regulated by phospholipase C (PLC)–derived second messengers. Both Ca2+ and protein kinase C were found to organize independent but additive pathways for Rap1b activation downstream of integrin-α2β1, which were completely blocked by inhibition of PLC with U73122. Moreover, integrin α2β1 engagement failed to trigger Rap1b activation in murine platelets lacking CalDAG-GEFI, a guanine nucleotide exchange factor regulated by Ca2+ and diacylglycerol, despite normal phosphorylation and activation of PLCγ2. In addition, CalDAG-GEFI–deficient platelets showed defective integrin α2β1-dependent adhesion and spreading. We found that outside-in signaling through integrin α2β1 triggered inside-out activation of integrin αIIbβ3 and promoted fibrinogen binding. Similarly to Rap1b stimulation, this process occurred downstream of PLC activation and was dramatically impaired in murine platelets lacking the Rap1 exchange factor CalDAG-GEFI. These results demonstrate that Rap1b is an important element in integrin-dependent outside-in signaling during platelet adhesion and regulates the cross talk between adhesive receptors

Early reduction of serum TARC levels may predict for success of ABVD as frontline treatment in patients with Hodgkin Lymphoma

Background Many efforts have been made to predict prognosis of newly diagnosed Hodgkin Lymphoma (HL) patients. Objective of this study was to investigate the association between early reduction of Thymus and Activation-Regulated Chemokine after the first ABVD cycle (TARC-1) and prognosis of HL patients. Methods Serum samples of 116 HL patients were collected at baseline, after every ABVD cycle and during follow-up. The 99th centile of TARC distribution in a group of 156 independent healthy subjects (800 pg/ml) was considered as cut-off for discriminating between abnormal and normal TARC values. Findings 101 patients out of 116 had baseline TARC above 800 pg/ml (median value 27515 pg/ml (IQR, 11001\ue2\u80\u9068139)) and were the object of this analysis. TARC-1 significantly decreased to a median value of 556 pg/ml (IQR, 378\ue2\u80\u93977 pg/ml). TARC-1 values below 800 pg/ml were associated with success of therapy (p = 0.0003) and PET-2 negativity (p = 0.001). TARC-1 \ue2\u89\ua4 800 pg/ml identified a population with a significantly higher 5-years PFS in the whole cohort (90.1% vs 55.6%; p < 0.0001) and in both subgroups of advanced (p = 0.003) and early stage patients (p = 0.021). At multivariable analysis, TARC-1 was significant independent predictor of PFS (p = 0.0035). Interpretation Early reduction of TARC serum levels can predict success of treatment, being associated with achievement of interim PET-2 negative and favorable long-term outcome in HL patients receiving ABVD as front-line therapy

Autosomal dominant thrombocytopenias with reduced expression of glycoprotein Ia

We have recently studied a case series of 46 unrelated patients with inherited thrombocytopenias and identified 18 cases that did not fit any known platelet disorder. In two unrelated families, a mild thrombocytopenia with normal platelet size was transmitted in an autosomal dominant fashion. Bleeding time was prolonged in 5 investigated patients. In all of them, flow cytometry and SDS-PAGE of platelet glycoproteins (GP) showed a reduced content of GPIa, a subunit of the GPIa-IIa complex (also known as integrin alpha 2 beta(1)) that is a major collagen receptor on platelets. All other membrane GPs were within the normal range. GPIa deficiency was associated with severely reduced in vitro platelet adhesion to molecules known to interact selectively with GPIa. In vitro platelet aggregation was normal in all subjects, except for a suboptimal platelet response to fibrillar collagen in two patients. A mild defect of alpha-granules was observed in all affected subjects. No mutation was identified in the genes encoding for GPIa or GPIIa. Since no other similar cases have been reported in the literature, we suggest that an autosomal dominant thrombocytopenia associated with GPIa deficiency and alpha-granule defect represents a new form of inherited thrombocytopenia

The Impact of Platelet Isolation Protocol on the Release of Extracellular Vesicles

Background: Platelet-derived extracellular vesicles (PEVs) are small vesicles released by activated platelets that arc gaining growing interest in the field of vascular biology. The mode of platelet activation is a critical determinant of PEVs release, phenotype and function. However, only very limited information is available concerning the impact of the platelet purification procedure on PEVs release. Methods: Washed or isolated platelets were separated by differential centrifugations. For washed platelets, the platelet pellet was washed by resuspension in PIPES buffer and finally resuspended in HEPES buffer. Isolated platelets were obtained by directly resuspending the platelet pellet in HEPES, skipping the washing steps in PIPES buffer. PEVs release was induced in washed or isolated platelets by stimulation with different agonist and analysed by Nanoparticle Tracking Analysis. Results: Isolated platelets showed a higher release of PEVs upon adenosine diphosphate (ADP) stimulation compared to washed platelets, whereas PEVs released upon stimulation with strong agonists (thrombin, collagen, A23187, U46619) were similar in the two groups. This different responsiveness to ADP was also observed as a higher a-granules release and protein kinase C activation in isolated platelets compared to washed ones. Residual plasma contamination appeared to be essential for the ability of platelets to release PEVs in response to ADP. Conclusions: In conclusion, our study strongly suggests that procedure adopted for platelets preparation is a critical determinant of PEVs release upon ADP stimulation

Absorbed dose and biologically effective dose in patients with high-risk non-Hodgkin\u2019s lymphoma treated with high-activity myeloablative 90Y-ibritumomab tiuxetan (Zevalin\uae)

Purpose: The aim of this study was to carry out two different dose estimation approaches in patients with non-Hodgkin's lymphoma (NHL) treated with a myeloablative amount of 90Y-labelled ibritumomab tiuxetan (Zevalin\uae) in an open-label dose escalation study. Methods: Twenty-seven patients with relapsed/refractory or de novo high-risk NHL receiving one myeloablative dose of 90Y-ibritumomab tiuxetan followed by tandem stem cell reinfusion were evaluated for dose estimate. The injected activity was 30 MBq/kg in 12 patients and 45 MBq/kg in 15 patients. Dose estimation was performed 1 week prior to 90Y-ibritumomab tiuxetan by injection of 111In-ibritumomab tiuxetan (median activity: 200 MBq). The absorbed dose (D) and the biologically effective dose (BED) were calculated. Results: The absorbed doses per unit activity (Gy/GBq) were [median (range)]: heart wall 4.6 (2.5-9.7), kidneys 5.1 (2.8-10.5), liver 6.1 (3.9-10.4), lungs 2.9 (1.5-6.8), red marrow 1.0 (0.5-1.7), spleen 7.0 (1.5-14.4) and testes 4.9 (2.9-16.7). The absorbed dose (Gy) for the 15 patients treated with 45 MBq/kg were: heart wall 17.0 (8.7-25.4), kidneys 17.1 (7.9-22.4), liver 20.8 (15.4-28.3), lungs 8.1 (5.4-11.4), red marrow 3.1 (2.0-4.0), spleen 26.2 (17.0-35.6) and testes 17.3 (9.0-28.4). At the highest activities the acute haematological toxicity was mild or moderate and of very short duration, and it was independent of the red marrow absorbed dose. No secondary malignancy or treatment-related myelodysplastic syndrome was observed. No non-haematological toxicity (liver, kidney, lung) was observed during a follow-up period of 24-48 months. Conclusion: The use of 45 MBq/kg of 90Y-ibritumomab tiuxetan in association with stem cell autografting resulted in patients being free of toxicity in non-haematological organs. These clinical findings were in complete agreement with our dose estimations, considering both organ doses and BED values