Fuelling conditions at staging sites can mitigate Arctic warming effects in a migratory bird

Under climate warming, migratory birds should align reproduction dates with advancing plant and arthropod phenology. To arrive on the breeding grounds earlier, migrants may speed up spring migration by curtailing the time spent en route, possibly at the cost of decreased survival rates. Based on a decades-long series of observations along an entire flyway, we show that when refuelling time is limited, variation in food abundance in the spring staging area affects fitness. Bar-tailed godwits migrating from West Africa to the Siberian Arctic reduce refuelling time at their European staging site and thus maintain a close match between breeding and tundra phenology. Annual survival probability decreases with shorter refuelling times, but correlates positively with refuelling rate, which in turn is correlated with food abundance in the staging area. This chain of effects implies that conditions in the temperate zone determine the ability of godwits to cope with climate-related changes in the Arctic

Critical role for the mitochondrial permeability transition pore and cyclophilin D in platelet activation and thrombosis

Many of the cellular responses that occur in activated platelets resemble events that take place following activation of cell-death pathways in nucleated cells. We tested the hypothesis that formation of the mitochondrial permeability transition pore (MPTP), a key signaling event during cell death, also plays a critical role in platelet activation. Stimulation of murine platelets with thrombin plus the glycoprotein VI agonist convulxin resulted in a rapid loss of mitochondrial transmembrane potential (Δψm) in a subpopulation of activated platelets. In the absence of cyclophilin D (CypD), an essential regulator of MPTP formation, murine platelet activation responses were altered. CypD-deficient platelets exhibited defects in phosphatidylserine externalization, high-level surface fibrinogen retention, membrane vesiculation, and procoagulant activity. Also, in CypD-deficient platelet-rich plasma, clot retraction was altered. Stimulation with thrombin plus H2O2, a known activator of MPTP formation, also increased high-level surface fibrinogen retention, phosphatidylserine externalization, and platelet procoagulant activity in a CypD-dependent manner. In a model of carotid artery photochemical injury, thrombosis was markedly accelerated in CypD-deficient mice. These results implicate CypD and the MPTP as critical regulators of platelet activation and suggest a novel CypD-dependent negative-feedback mechanism regulating arterial thrombosis

Gradual increase in thrombogenicity of juvenile platelets formed upon offset of prasugrel medication Gradual increase in thrombogenicity of juvenile platelets formed upon offset of prasugrel medication

Abstract In patients with acute coronary syndrome, dual antiplatelet therapy with aspirin and a P2Y 12 inhibitor like prasugrel is prescribed for one year. Here, we investigated how the hemostatic function of platelets recovers after discontinuation of prasugrel treatment. Therefore, sixteen patients who suffered from ST-elevation myocardial infarction were investigated. Patients were treated with aspirin (100 mg/day, long-term) and stopped taking prasugrel (10 mg/day) after one year. Blood was collected at the last day of prasugrel intake and at 1, 2, 5, 12 and orange and the novel 5'Cy5-oligo-dT probe revealed that this subpopulation consisted of juvenile platelets, which progressively contributed to platelet aggregation and thrombus formation under flow. During offset, juvenile platelets were overall more reactive than older platelets. Interestingly, the responsiveness of both juvenile and older platelets increased in time, pointing towards a residual inhibitory effect of prasugrel on the megakaryocyte level. In conclusion, the gradual increase in thrombogenicity after cessation of prasugrel treatment is due the increased activity of juvenile platelets.

Gradual increase in thrombogenicity of juvenile platelets formed upon offset of prasugrel medication

In patients with acute coronary syndrome, dual antiplatelet therapy with aspirin and a P2Y(12) inhibitor like prasugrel is prescribed for one year. Here, we investigated how the hemostatic function of platelets recovers after discontinuation of prasugrel treatment. Therefore, 16 patients who suffered from ST-elevation myocardial infarction were investigated. Patients were treated with aspirin (100 mg/day, long-term) and stopped taking prasugrel (10 mg/day) after one year. Blood was collected at the last day of prasugrel intake and at 1, 2, 5, 12 and 30 days later. Platelet function in response to ADP was normalized between five and 30 days after treatment cessation and in vitro addition of the reversible P2Y(12) receptor antagonist ticagrelor fully suppressed the regained activation response. Discontinuation of prasugrel resulted in the formation of an emerging subpopulation of ADP-responsive platelets, exhibiting high expression of active integrin α(IIb)β(3). Two different mRNA probes, thiazole orange and the novel 5′Cy5-oligo-dT probe revealed that this subpopulation consisted of juvenile platelets, which progressively contributed to platelet aggregation and thrombus formation under flow. During offset, juvenile platelets were overall more reactive than older platelets. Interestingly, the responsiveness of both juvenile and older platelets increased in time, pointing towards a residual inhibitory effect of prasugrel on the megakaryocyte level. In conclusion, the gradual increase in thrombogenicity after cessation of prasugrel treatment is due to the increased activity of juvenile platelets

2009

ABSTRACT © F e r r a t a S t o r t i F o u n d a t i o n function of platelets recovers after discontinuation of prasugrel treatment. We hypothesized an immediate recovery of newly formed, juvenile platelets to the level of untreated platelets. Our data provide evidence for a critical role of the juvenile platelets in the regained aggregation of platelets and thrombus formation, and also show that these platelets gradually increase in responsiveness. Methods Patients and control subjects This study was approved by the local medical ethics committee . All patients and healthy volunteers gave written informed consent to participate in the study according to the Declaration of Helsinki. Sixteen patients were studied who were treated with prasugrel (10 mg/day) for one year and long-term aspirin (80-100 mg/day) due to a myocardial infarction with ST elevation. After one year of prasugrel treatment, blood was collected on the last treatment day, and at 1, 2, 5 and 30 days later. From 2 patients, blood samples were also collected after 12 days to better understand the delayed regain of platelet function. Patients with a malignancy, active infection or a known platelet disorder were not included. Blood was obtained by venipuncture into Vacuette tubes, containing K2-EDTA, for measurement of hemostatic variables and immature platelet fraction (IPF) using a Sysmex XN-9000 analyzer (Sysmex, Chuo-ku Kobe, Japan); 3.2% (w/v) trisodium citrate for platelet function measurements, or hirudin for whole blood platelet aggregation. Control experiments were performed with blood drawn from healthy volunteers collected in trisodium citrate or acidic citrate dextrose. 28 Preparation of platelet-rich plasma, platelets and red cells Platelet-rich plasma (PRP), platelet-free plasma and washed platelets were prepared as described. 30 Irreversible P2Y 12 inhibition in vitro PRP from healthy donors was treated with lysine aspirin. Platelet aggregation Aggregation of platelets in PRP was measured using a Chronolog aggregometer (Stago, Asnières sur Seine Cedex, France). 32 Ticagrelor (1 mM), being more potent than prasugrel, Flow cytometric analysis of platelet subpopulations Flow cytometry was performed on an Accuri C6 flow cytometer with CFlow Plus software (Becton-Dickinson Bioscience). To check for integrin α IIb β 3 activation, platelets were activated with 2MeS-ADP in the presence of FITC-conjugated PAC-1 antibody against the activated α IIb β 3 integrin. Activated platelets were identified as before. 31 Ticagrelor (1 mM) was added, where indicated. Juvenile platelets were identified using two different methods of mRNA staining, i.e. with thiazole orange The average percentage of juvenile platelets as analyzed by the thiazole orange staining and the oligo-dT staining was 6.7% (±1.9%) and 21.5% (±5.8%), respectively. The discrepancy in the percentage of detected juvenile platelets can be explained by the higher sensitivity of the oligo-dT staining to detect mRNA in comparison to thiazole orange. In order to use a uniform definition of juvenile platelets, the threshold for juvenile platelets was based on the IPF as determined by the Sysmex XN9000 analyzer, which is an internationally validated method in the clinic. An alternative analysis of juvenile platelets, based on the negative controls of both stainings, is presented in Online Supplementary Thrombus formation in whole blood Whole blood thrombus formation on microspots in a parallelplate flow chamber was measured, basically as described before. 35 Patient blood samples were perfused through the chamber for 4 min at a wall-shear rate of 1600 s -1 , while 2MeS-ADP (0.1 mM, f.c.) was co-perfused with a second pump. Ticagrelor was added where indicated. Thrombi were stained with AF647-labeled fi-brinogen and, when indicated, with DiOC 6 . 35 For measurement of thrombus formation of reconstituted blood samples from healthy controls, mixtures of CAM-and vehicletreated platelets were added to washed red cells and plasma. In these experiments, the CAM-treated and vehicle-treated platelets were pre-labeled with the membrane probes CellVue Maroon and PKH26, respectively. Microscopic DIC and confocal fluorescent images were taken using a Zeiss LSM7 microscope (Oberkochen, Germany). 35 Statistical analysis Statistical analysis was performed using the SPSS Statistics 22 package (Armonk, NY, USA). Statistical analysis was performed using a one-way-repeated-measures-ANOVA or with a Friedman test with a post hoc Wilcoxon signed rank test. Bonferroni correction was applied when comparing multiple groups. For a detailed description of the methods, please see the Online Supplementary Appendix. Results P2Y 12 -inhibited platelets participate less in thrombus formation In order to determine how platelets with non-responsive P2Y 12 receptors interact with responsive platelets in aggregation and thrombus formation, platelets from control subjects were treated with the clopidogrel active metabolite (CAM) and mixed in various proportions with untreated platelets. All platelets were also treated with aspirin, in order to mimic conditions as in patients. Flow cytometric C.C.F.M.J. Baaten et al. 1132 haematologica | 2015; 100(9) © F e r r a t a S t o r t i F o u n d a t i o n analysis indicated that, upon stimulation with ADP, these platelet mixtures formed two distinct populations in terms of activation of integrin α IIb β 3 and binding of OG488-fibrinogen. The population of fibrinogen-binding platelets decreased with increasing fractions of CAM-treated platelets ( To investigate this further, we assessed how the CAMtreated platelets participated in thrombus formation on immobilized collagen under high-shear flow conditions. Therefore, the P2Y 12 -inhibited platelets were labeled with the red-exciting membrane label CellVue Maroon, whereas the untreated platelets were labeled with the green-exciting membrane label PKH26. This labeling did not affect platelet activation responses (data not shown). Mixtures with 0%, 50% or 100% of CAM-treated platelets were added to red blood cells and plasma from the same donor to obtain reconstituted blood with different proportions of P2Y 12 -inhibited platelets. In comparison to reconstituted blood with solely uninhibited platelets, increasing proportions of CAM-treated platelets had limited impact on platelet adhesion to the collagen surface, but markedly suppressed the formation of large platelet aggregates (Online Supplementary Gradual restoration of platelet aggregation in patients upon prasugrel offset The offset phase of prasugrel medication was studied in 16 patients. The patients had a mean age of 59±9 years (mean±SD); 3 patients were diagnosed with type II diabetes mellitus (Online Measurements of whole blood aggregation (Multiplate assay) showed a gradual increase in ADP-induced aggregation upon offset from days 2 to 30 ( t o r t i F o u n d a t i o n that the increase in aggregation during prasugrel offset was fully antagonized, confirming that the regained platelet reactivity was fully due to increased P2Y 12 receptor function ( Formation of a highly reactive population of juvenile platelets upon prasugrel offset Based on earlier experiments with rats, 18 we expected during the offset phase of prasugrel medication the rapid formation of a population of newly formed, fully P2Y 12 -responsive platelets. A pertinent question was how the regained response in P2Y 12 receptor activity was linked to the appearance of this new platelet population. To investigate this, flow cytometry was used to analyze platelets stimulated with the stable (nucleotidase-resistant) ADP analog, 2MeS-ADP, for binding of FITC-labeled PAC-1 antibody, indicative of integrin α IIb β 3 activation. At day 0, a limited fraction of 26±9% of the platelets showed activated α IIb β 3 , and this fraction (recognized as a separate peak in the histograms) gradually increased to 56±5% at day 5 and 72±7% at day 30 ( Several assays were performed to determine whether the accumulating platelets with activated α IIb β 3 indeed consisted of newly formed, juvenile platelets. Therefore, platelet mRNA was quantified using two different mRNA probes: thiazole orange as an established, but weak fluorescent mRNA dye; 22 and 5'Cy5-labeled oligo-dT, binding to the platelet mRNA poly-A tails, 21 which was added to pre-activated and permeabilized platelets. Online Supplementary 1134 haematologica | 2015; 100(9) Gradual increase in thrombus size upon prasugrel offset To investigate whether the increased reactivity of juvenile platelets after prasugrel cessation translates into enhanced thrombus formation, whole blood was perfused over microspots containing vWF/fibrinogen or type I collagen. 35 Given the major role of P2Y 12 signaling in thrombus buildup, Staining of thrombi with AF647-labeled fibrinogen allowed assessment of integrin α IIb β 3 activation. On both the vWF/fibrinogen and collagen microspots, a marked increase in fibrinogen binding to the aggregated platelets was detected for the later blood samples (Online Supplementary A B C © F e r r a t a S t o r t i F o u n d a t i o n Discussion In this paper, we confirm earlier findings 9 However, we also found that other platelet function tests, such as ADP-induced whole blood aggregation and integrin α IIb β 3 activation, were incompletely recovered at day 5 in comparison to day 30. In vitro addition of ticagrelor completely antagonized the time-dependent increase in platelet responses, thereby proving that the recuperation was due to regained P2Y 12 signaling. Detailed flow cytometric analysis indicated that the functional recovery during prasugrel offset was caused by the appearance of a population of juvenile platelets that was increasingly responsive towards ADP. Separation of newly formed and older platelets with two mRNA probes, thiazole orange and a new Cy5-conjugated oligo-dT probe, revealed increased responsiveness to ADP of the positively stained platelet population in terms of integrin α IIb β 3 activation and fibrinogen binding. However, both probes also gave unexpected results. First, we observed a marked increase in ADP responsiveness of the juvenile platelet population after only two days of offset, and for the older platelet population after five days. This suggested that the majority of juvenile platelets formed during the first days still had inhibited P2Y 12 receptors, taking into account the presence of the prasugrel active metabolite in the circulation for 7-8 h post prasugrel administration. To determine how the increased reactivity of juvenile platelets translates into hemostasis, we studied thrombus formation under flow on two different adhesive surfaces using whole blood. Platelet deposition and aggregate formation on the vWF/fibrinogen and the collagen surfaces restored during the offset and was only maximal at day C.C.F.M.J. Baaten et al. 1136 haematologica | 2015; 100(9) © F e r r a t a S t o r t i F o u n d a t i o n 30. The regained P2Y 12 activity was most apparent from thrombus size, with larger thrombi towards the end phase of the offset. This is in agreement with earlier work showing that signaling via P2Y 12 is crucial for thrombus formation and stabilization. The present study has potential limitations, as we have investigated a relatively small number of patients. Further, in our initial ex vivo studies we used the active metabolite of clopidogrel. Although prasugrel is a more potent P2Y 12 antagonist in comparison to clopidogrel, 5 we added the active metabolite of clopidogrel at concentrations high enough for maximal inhibition. Patients on dual antiplatelet therapy who require surgery are recommended to stop prasugrel intake seven days beforehand. 9 Our findings that prasugrel can still affect the reactivity of juvenile platelets during several days after treatment cessation does not plea for a shortening of this period. The compromised reactivity of juvenile platelets during the initial days of offset can contribute to a risk of bleeding upon surgery. When urgent surgery is required, or when bleeding has to be controlled, platelet transfusions have shown to be effective in restoring hemostasis at 6 h after a loading dose of prasugrel. Acknowledgments The authors thank S. Lelieveld and the central diagnostic laboratory for technical support and R. Dennert for assisting in patient recruitment. Authorship and Disclosures Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org. Thrombogenicity of platelets upon prasugrel offset haematologica | 2015; 100 © F e r r a t a S t o r t i F o u n d a t i o