The effects of 22°C and 4°C storage of platelets on vascular endothelial integrity and function
Gyulnar BaimukanovaByron MiyazawaDaniel PotterStuart L. GibbSheila M. KeatingAli DaneshAshley I. BeyerYelena DayterRoberta BruhnMarcus O. MuenchP. AndrewPhilip J. NorrisPhilip C. SpinellaMitchell J. CohenShibani Pati
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BACKGROUND Although a majority of the studies conducted to date on platelet (PLT) storage have been focused on PLT hemostatic function, the effects of 4°C PLTs on regulation of endothelial barrier permeability are still not known. In this study, we compared the effects of room temperature (22°C) stored and (4°C) stored PLTs on the regulation of vascular endothelial cell (EC) permeability in vitro and in vivo. STUDY DESIGN AND METHODS Day 1, Day 5, and Day 7 leukoreduced apheresis PLTs stored at 4 or 22°C were studied in vitro and in vivo. In vitro, PLT effects on EC permeability and barrier function, adhesion, and impedance aggregometry were investigated. In vivo, using a mouse model of vascular leak, attenuation of vascular leak and circulating PLT numbers were measured. RESULTS Treatment of EC monolayers with Day 5 or Day 7 PLTs, stored at both 22°C and 4°C, resulted in similar decreases in EC permeability on average. However, analysis of individual samples revealed significant variation that was donor dependent. Additional in vitro measurements revealed a decrease in inflammatory mediators, nonspecific PLT‐endothelial aggregation and attenuated loss of aggregation over time to TRAP, ASPI, ADP, and collagen with 4°C storage. In mice, while 22°C and 4°C PLTs both demonstrated significant protection against vascular endothelial growth factor A (VEGF‐A)‐induced vascular leak 22°C PLTs exhibited increased protection compared to 4°C PLTs. Systemic circulating levels of 4°C PLTs were decreased compared to 22°C PLTs. CONCLUSIONS In vitro, 4°C‐stored PLTs exhibit a greater capacity to inhibit EC permeability than 22°C‐stored PLTs. In vivo, 22°C PLTs provide superior control of vascular leak induced by VEGF‐A. This discrepancy may be due to increased clearance of 4°C PLTs from the systemic circulation.Keywords:
Vascular permeability
Barrier function
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S ummary Measurements of platelet production in 16 patients with idiopathic thrombocytopenic purpura (ITP) demonstrate that megakaryocytopoiesis (total thrombopoiesis) and platelet turnover (effective thrombopoiesis) are increased in parallel to as much as 8 times normal. The marrow megakaryocytes show changes characteristic of stimulated thrombopoiesis.
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There is increasing evidence that an important mechanism by which platelet production is regulated depends upon a humoral substance (thrombopoietin) that affects the production of platelets by megakaryocytes. Plasma from thrombocytopenic donors increases the rate of appearance or concentrations of subsequently administered Na235SO4 or selenomethionine-75Se in platelets. Both isotopes are initially incorporated into the cytoplasm of megakaryocytes, and labeled platelets appear in the circulation after their production and release from megakaryocytes. Thrombopoiesis-stimulating activity also can be detected in the plasma of normal donors when endogenous thrombopoiesis has been suppressed in recipient assay animals by the hypertransfusion of platelets. Recent studies have indicated that certain fractions of plasma from throbocytopenic donors are also capable of stimulating thrombopoiesis in recipient animals. The nature of thrombopoietin(s) and its mechanism of action remain unknown. However, currently available data indicate that thrombopoiesis-stimulating factors may act both on diploid precursors and immature megakaryocytes and upon maturing megakaryocytes. The site of production of thrombopoietin also is unknown. Although the sensor that regulates thrombopoietin or other humoral mediators of thrombopoiesis has not been identified, it appears that platelet numbers, per se, are not the sole variable to which megakaryocytopoiesis eventually responds.
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Abstract Disruption of endothelial barrier is a crucial factor in the pathogenesis of tissue inflammation, the hallmark of inflammatory diseases such as diabetes and atherosclerosis. Increased endothelial permeability occurs because of loss of cell–cell contacts and disruption of cell–extracellular matrix (ECM) adhesions. Vascular injury is associated with activation of the coagulation cascade and release of thrombin, which increases endothelial permeability by activating endothelial cell surface thrombin receptor. This increase in endothelial permeability is typically followed by a recovery period of ≈2 hours, during which barrier integrity is restored. It has been surmised that thrombin signaling stimulates intrinsic repair mechanisms that restore barrier function. To validate our finding we determined endothelial barrier function using state of the art Electric Cell-substrate Impedance Sensing (ECIS) mechanism, a measure of trans-endothelial electrical resistance (TEER) across the endothelial monolayers using TEER electrodes. Our data show that 50mM thrombin increase endothelial permeability followed by complete recovery and reannealing of adherens junction in control endothelial cells. However, SPHK1 depleted cells showed significantly decrease in barrier disruption, and barrier failed recover completely compared to control monolayer. Upon treatment with S1P in SPHK1 depleted endothelia showed completely recovery in barrier function. In conclusion, our study for the first time shows that SPHK1-S1P-S1PR1 signaling pathway is emerging as a potential therapeutic target in improving endothelial barrier function and prevent coronary artery disease. CTRE Seed Grant to Professor Nadeem Fazal, MD, PhD
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Background : Analysis of reticulated platelets (RPs) is useful for discriminating the causes of thrombocytopenia and monitoring the thrombopoiesis. In the patients with severe thrombocytopenia, we evaluated the thrombopoiesis-discriminating ability of several indices applying forward scatter (FSC) and thiazole orange (TO) fluorescence in addition to the percentage of reticulated platelets (RPs%). Methods : Forty cases with decreased thrombopoiesis, twenty cases with increased thrombopoiesis and twenty cases with liver cirrhosis were selected. By flow cytometry with two analytic methods, dependent on or independent of the staining of CD41-PE as a platelet marker, the primary parameters including RPs% were measured and the applied parameters were calculated from them. And we compared the diagnostic efficiency of each parameter and analyzed the purity of platelet light scatter gate. Results : The purity of platelet light scatter gate was significantly lower in patients with severe thrombocytopenia than in healthy persons with normal platelet counts (P), so the use of CD41-PE for platelet gating improved the diagnostic efficiency of RPs%. Compared to the primary parameters, the applied parameters originated from RPs%, FSC and TO fluorescence improved diagnostic efficiency significantly (RPs%: 55%, RPs%s MFI: 80%) between decreased and increased thrombopoiesis groups. Conclusions : In the patients with severe thrombocytopenia, the estimate of the thrombopoiesis by a flow cytometric analysis can be more predictable by using platelet markers and by considering the fluorescence intensity of TO together with the RPs%.
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Platelets, generated from precursor megakaryocytes (MKs), are central mediators of hemostasis and thrombosis. The process of thrombopoiesis is extremely complex, regulated by multiple factors, and related to many cellular events including apoptosis. However, the role of apoptosis in thrombopoiesis has been controversial for many years. Some researchers believe that apoptosis is an ally of thrombopoiesis and platelets production is apoptosis-dependent, while others have suggested that apoptosis is dispensable for thrombopoiesis, and is even inhibited during this process. In this review, we will focus on this conflict, discuss the relationship between megakaryocytopoiesis, thrombopoiesis and apoptosis. In addition, we also consider why such a vast number of studies draw opposite conclusions of the role of apoptosis in thrombopoiesis, and try to figure out the truth behind the mystery. This review provides more comprehensive insights into the relationship between megakaryocytopoiesis, thrombopoiesis, and apoptosis and finds some clues for the possible pathological mechanisms of platelet disorders caused by abnormal apoptosis.
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Measurements of megakaryocyte number, volume, nuclear number, and cytoplasmic granulation were compared in animals with induced thrombocytosis, normal platelet counts, and induced thrombocytopenia for 4- and 10-day periods. Thrombopoiesis, as measured by megakaryocytic mass and (35)S incorporation into platelets, appeared to be regulated by the demand for circulating platelets. In addition, two mechanisms were operative in the alteration of thrombopoiesis. The first mechanism involved the regulation of endomitosis in the immature megakaryocytes which in turn determined the subsequent volume of platelet-producing cytoplasm. The second mechanism regulated the number of megakaryocytes formed from the precursor, "stem cell," compartment.
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