Abstract 561: The ex vivo RAS-Fingerprint - A Novel Approach for the Biochemical Chatracterization of the Renin-Angiotensin-System in Clinical Samples

Angiotensin concentrations are affected by multiple molecular components including receptors and enzymes which might be either dissolved in plasma or attached to blood cells or endothelial surfaces throughout the body, giving rise to a concentration determining local enzymatic environment. This environment substantially changes during blood collection leading to a rapid and fundamental shift in angiotensin peptide levels. Therefore, a clearly defined and properly controlled sample stabilization procedure is essential for the accurate measurement of in vivo angiotensin peptide levels. Surprisingly, standard samples collected by anti-coagulation with heparin can be used for analyzing the human RAS under well-defined steady-state conditions, allowing RAS-Fingerprint based conclusions about the activities of circulating enzymes involved in angiotensin metabolism. The mass spectrometry based measurements of in vivo RAS-Fingerprints (immediate sample stabilization) or heparin plasma derived ex vivo RAS-Fingerprints in plasma or whole blood provide unique insights into the physiology of the human RAS. RAS-Fingerprinting provides an integrated view about the activity of the enzymes involved in angiotensin metabolism in a plasma sample and therefore represents a powerful tool for characterization of the patient specific “Biochemical Hardware”, which determines angiotensin peptide levels in vivo. The assay is compatible with undiluted plasma and whole blood and can be further applied to long-term stored frozen plasma samples. The utilization of RAS-Fingerprinting in clinical studies will substantially enhance our understanding of the human RAS and could lead to the development of personalized approaches for the treatment and prevention of cardiovascular diseases in the near future.

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Abstract 291: RAS Fingerprinting: Patient-Specific Evaluation of the Renin-Angiotensin-System in Plasma by LC-MS/MS-Based Multiplex Quantification of Angiotensin Peptides

Circulation Research ◽

10.1161/res.111.suppl_1.a291 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Marko Poglitsch ◽

Cornelia Schwager ◽

Manfred Schuster ◽

Hans Loibner

Keyword(s):

Whole Blood ◽

Ex Vivo ◽

Renin Angiotensin System ◽

Patient Specific ◽

Angiotensin System ◽

Renin Angiotensin ◽

Associated Diseases ◽

Angiotensin Peptides ◽

Specific Evaluation

The Renin-Angiotensin-System (RAS) is critically involved in the regulation of important physiological functions including blood pressure and fluid balance. It is constituted by multiple enzymes giving rise to a meshwork of effector peptides, which mediate their functions through binding to specific receptor molecules. Therefore, the modification of angiotensin peptide levels represents a common strategy for the treatment of RAS-associated diseases and is frequently achieved by the pharmacologic regulation of enzymes taking part in angiotensin metabolism. We developed a highly sensitive method, which allows the simultaneous absolute quantification of up to 10 different angiotensin peptides in human plasma and whole blood (RAS-Fingerprinting). Either the measurement of in vivo RAS-Fingerprints (immediate sample stabilization) or heparin derived ex vivo RAS-Fingerprints in plasma or whole blood, provide unique insights into the biochemical constitution of the human RAS and therefore represent powerful tools for the patient specific evaluation of this physiologically important peptide hormone system. During the development and validation of a new LC-MS/MS based method for angiotensin quantification, comprehensive datasets for various applications of RAS-Fingerprinting have been generated. RAS-Fingerprints were measured in blood of healthy volunteers during the treatment with different RAS-blockers, which provided deep insights in the physiology and regulation of the human RAS and revealed surprising differences between individual patients. With the help of RAS-Fingerprinting, we were able to re-draw the picture of the human RAS at a previously unachieved level of detail and accuracy. Due to the availability of new selection criteria for pharmacologic screens, RAS-Fingerprinting could contribute to the development of innovative therapeutic approaches affecting the RAS. The extensive utilization of RAS-Fingerprinting in clinical studies will substantially enhance our understanding of the human RAS and could lead to the development of personalized treatment schemes in the management of RAS-associated diseases in the near future.

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A Critical Investigation into the Existence of Circulating Platelet Aggregates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661600 ◽

1986 ◽

Vol 56 (01) ◽

pp. 045-049 ◽

Cited By ~ 10

Author(s):

A R Saniabadi ◽

G D O Lowe ◽

R Madhok ◽

K Spowart ◽

B Shaw ◽

...

Keyword(s):

Whole Blood ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Venous Blood ◽

White Blood Cells ◽

Blood Platelet ◽

Blood Collection ◽

Significant Difference ◽

Platelet Aggregates

SummaryBy a method of counting single platelets in diluted whole blood, platelet aggregates were quantified ex-vivo. Four groups: 20 thrombotic patients, 10 non-thrombotic patients, 10 healthy old controls and 10 healthy young controls were included in the study. Using a 19 gauge needle, with and without tubing, venous blood was taken into buffered EDTA, as a disaggregating agent and buffered EDTA-formalin, as the fixative. The amount of platelet aggregates quantified was affected by the quality of venepuncture or the rate of blood flow through the needle, but was unaffected by the presence of the tubing. There was no statistically significant difference between the four groups, in terms of the platelet aggregates quantified, but scanning electron microscopy revealed the presence of irreversible aggregates, composed of platelet red and white blood cells, in the blood of a greater number of thrombotic patients than non-thrombotic or healthy controls. Platelet aggregates were also quantified in aliquots of platelet rich plasma, and were found to be significantly greater than the corresponding values in whole blood. The difference appeared to be due to increased viscosity of the plasma, induced by the fixative which reduces platelet mobility during centrifugation. It is concluded that the platelet aggregates which disaggregate in bufffered EDTA may represent an artifact of blood collection; the irreversible aggregates are suspected to represent the in vivo circulating aggregates.

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The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000116 ◽

2012 ◽

Vol 82 (3) ◽

pp. 228-232 ◽

Cited By ~ 7

Author(s):

Mauro Serafini ◽

Giuseppa Morabito

Keyword(s):

Antioxidant Capacity ◽

Dietary Intervention ◽

Ex Vivo ◽

The Body ◽

Dietary Polyphenols ◽

Enzymatic Antioxidant ◽

Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

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Adenosine Diphosphate (ADP)-Induced ⍺-Granules Release from Platelets of Native Whole Blood Is Reduced by Ticlopidine but Not by Aspirin or Dipyridamole

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661629 ◽

1986 ◽

Vol 56 (02) ◽

pp. 147-150 ◽

Cited By ~ 18

Author(s):

V Pengo ◽

M Boschello ◽

A Marzari ◽

M Baca ◽

L Schivazappa ◽

...

Keyword(s):

Whole Blood ◽

Light Transmission ◽

Ex Vivo ◽

Early Stage ◽

Shape Change ◽

Adenosine Diphosphate ◽

Dose Dependent ◽

Plateletderived Growth Factor ◽

Platelet Shape

SummaryA brief contact between native whole blood and ADP promotes a dose-dependent release of platelet a-granules without a fall in the platelet number. We assessed the “ex vivo” effect of three widely used antiplatelet drugs, aspirin dipyridamole and ticlopidine, on this system. Aspirin (a single 800 mg dose) and dipyridamole (300 mg/die for four days) had no effect, while ticlopidine (500 mg/die for four days) significantly reduced the a-granules release for an ADP stimulation of 0.4 (p <0.02), 1.2 (p <0.01) and 2 pM (p <0.01). No drug, however, completeley inhibits this early stage of platelet activation. The platelet release of α-granules may be related to platelet shape change of the light transmission aggregometer and may be important “in vivo” by enhancing platelet adhesiveness and by liberating the plateletderived growth factor.

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Effect of inhibition of 5-lipoxygenase metabolism of arachidonic acid on response to endotoxemia in sheep

Journal of Applied Physiology ◽

10.1152/jappl.1988.65.3.1351 ◽

1988 ◽

Vol 65 (3) ◽

pp. 1351-1359 ◽

Cited By ~ 28

Author(s):

J. W. Coggeshall ◽

B. W. Christman ◽

P. L. Lefferts ◽

W. E. Serafin ◽

I. A. Blair ◽

...

Keyword(s):

Arachidonic Acid ◽

Whole Blood ◽

Ex Vivo ◽

Calcium Ionophore ◽

Lung Mechanics ◽

Gas Chromatography Mass Spectrometry ◽

Lipoxygenase Inhibitor ◽

Pulmonary Hemodynamics ◽

Lung Lymph

We studied the effects of a 5-lipoxygenase inhibitor, L-651,192, on the pulmonary dysfunction caused by endotoxemia in chronically instrumented unanesthetized sheep. The efficacy and selectivity of L-651,392 were tested by measuring in vivo production of leukotriene B4 (LTB4) and cyclooxygenase products of arachidonic acid after endotoxemia before and after pretreatment with L-651,392 and ex vivo from granulocytes and whole blood stimulated with calcium ionophore from sheep before and 24 h after pretreatment with L-651,392. A novel assay for LTB4 by high-performance liquid chromatography/gas chromatography/mass spectrometry techniques was developed as a measure of 5-lipoxygenase metabolism of arachidonic acid. L-651,392 proved to be an effective in vivo 5-lipoxygenase inhibitor in sheep. L-651,392 blocked the increase in LTB4 observed in lung lymph after endotoxemia in vivo in sheep as well as inhibited by 80% the ex vivo production of LTB4 by granulocytes removed from sheep treated 24 h earlier with L-651,392. Although L-651,392 blocked the increase in cyclooxygenase products of arachidonic acid observed in lung lymph after endotoxemia in vivo in sheep, the drug probably did not function directly as a cyclooxygenase inhibitor. L-651,392 did not attenuate the ex vivo production of thromboxane B2 by whole blood from sheep treated 24 h earlier with the drug. L-651,392 attenuated the alterations in pulmonary hemodynamics, lung mechanics, oxygenation, and lung fluid and solute exchange observed after endotoxemia in sheep. We speculate that 5-lipoxygenase products are a major stimulus for cyclooxygenase metabolism of arachidonic acid after endotoxemia in sheep.

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4081 Quantifying pH buffering capacity and kinetics of tumor and healthy tissue to understand and exploit differences in biology

Journal of Clinical and Translational Science ◽

10.1017/cts.2020.89 ◽

2020 ◽

Vol 4 (s1) ◽

pp. 15-15

Author(s):

A. Colleen Crouch ◽

Emily A. Thompson ◽

Mark D. Pagel ◽

Erik N.K. Cressman

Keyword(s):

Tumor Tissue ◽

Ex Vivo ◽

Buffering Capacity ◽

The Body ◽

Healthy Tissue ◽

Buffer System ◽

Bicarbonate Buffer ◽

Ph Buffering ◽

Acidocest Mri

OBJECTIVES/GOALS: The purpose of this work is to investigate natural buffering capacity of liver tissue and tumors, to understand and exploit differences for therapy. Using this work, we will determine the concentrations of reagents (acids or bases) used in ablation treatment to optimize treatment by increasing tumor toxicity and minimizing healthy tissue toxicity. METHODS/STUDY POPULATION: For this preliminary study, two methods will be used: benchtop pH experiments ex vivo and non-invasive imaging using acidoCEST MRI in vivo. For ex vivo, two types of tissues will be tested: non-cancerous liver and tumor tissue from HepG2 inoculated mice (n = 10). After mice are euthanized, pH will be measured in tissue homogenates at baseline and then the homogenates will be placed in either acidic (acetic acid) or basic (sodium hydroxide) solutions with varied concentrations (0.5–10M) and time recorded until pH returns to baseline. For in vivo imaging, Mia PaCA-2 flank model mice (n = 10) will be imaged with acidoCEST MRI to quantify pH at baseline. Mice will then be injected intratumorally with (up to 100 μL of) acid or base at increasing concentrations and imaged to quantify pH changes in the tumor. RESULTS/ANTICIPATED RESULTS: For this study, buffering capacity is defined as the concentration threshold for which tissue can buffer pH back to within normal range. Non-cancerous tissue is likely to buffer a wider range of concentrations compared to tumor tissue. From the benchtop experiment, comparison of time-to-buffer will be made for each concentration of acid/base for the two tissue types. AcidoCEST MRI will provide in vivo buffering capacity and potentially demonstrate tumor heterogeneity of buffering capacity. For both experiments, a pH vs. concentration curve for the two tissue types will allow for comparison of ex vivo to in vivo experiments, which will differentiate contributions of local tissue buffering capacity from the full body’s natural bicarbonate buffer system that depends on respiration and blood flow. DISCUSSION/SIGNIFICANCE OF IMPACT: The pH of the body must be maintained within a narrow range. With cancer, impairment in regulation of tumor metabolism causes acidosis, lowering extracellular pH in tumors. It remains unclear if pH plays a role in local recurrence or tumor toxicity. This work will determine if acidoCEST MRI can measure deliberate alteration of pH and how this change affects biology.

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Lipolysis generates platelet dysfunction after in vivo heparin administration

Clinical Science ◽

10.1042/cs1030433 ◽

2002 ◽

Vol 103 (4) ◽

pp. 433-440 ◽

Cited By ~ 9

Author(s):

Elijah W. MURIITHI ◽

Philip R. BELCHER ◽

Stephen P. DAY ◽

Mubarak A. CHAUDHRY ◽

Muriel J. CASLAKE ◽

...

Keyword(s):

Superoxide Dismutase ◽

Cardiopulmonary Bypass ◽

Lipoprotein Lipase ◽

Whole Blood ◽

Ex Vivo ◽

Hepatic Lipase ◽

Platelet Factor 4 ◽

Platelet Factor

Heparin, when administered to patients undergoing operations using cardiopulmonary bypass, induces plasma changes that gradually impair platelet macroaggregation, but heparinization of whole blood in vitro does not have this effect. The plasma changes induced by heparin in vivo continue to progress in whole blood ex vivo. Heparin releases several endothelial proteins, including lipoprotein lipase, hepatic lipase, platelet factor-4 and superoxide dismutase. These enzymes, which remain active in plasma ex vivo, may impair platelet macroaggregation after in vivo heparinization and during cardiopulmonary bypass. In the present study, proteins were added in vitro to hirudin (200units·ml-1)-anticoagulated blood from healthy volunteers, and the platelet macroaggregatory responses to ex vivo stimulation with collagen (0.6μg·ml-1) were assessed by whole-blood impedance aggregometry. Over a 4h period, human lipoprotein lipase and human hepatic lipase reduced the platelet macroaggregatory response from 17.0±2.3 to 1.5±1.3 and 1.2±0.6Ω respectively (means±S.D.) (both P<0.01; n = 6). Other lipoprotein lipases also impaired platelet macroaggregation, but platelet factor-4 and superoxide dismutase did not. Platelet macroaggregation showed an inverse linear correlation with plasma concentrations of non-esterified fatty acids (r2 = 0.69; two-sided P<0.0001; n = 8), suggesting that heparin-induced lipolysis inhibits platelet macroaggregation. Lipoprotein degradation products may cause this inhibition by interfering with eicosanoids and other lipid mediators of metabolism.

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Highly Potent GalNAc-Conjugated Tiny LNA Anti-miRNA-122 Antisense Oligonucleotides

Pharmaceutics ◽

10.3390/pharmaceutics13060817 ◽

2021 ◽

Vol 13 (6) ◽

pp. 817

Author(s):

Tsuyoshi Yamamoto ◽

Yahiro Mukai ◽

Fumito Wada ◽

Chisato Terada ◽

Yukina Kayaba ◽

...

Keyword(s):

Antisense Oligonucleotides ◽

Ex Vivo ◽

The Body ◽

Locked Nucleic Acid ◽

Imaging Studies ◽

Phosphodiester Bond ◽

Chemically Modified ◽

Ex Vivo Imaging ◽

Mirna Targeting

The development of clinically relevant anti-microRNA antisense oligonucleotides (anti-miRNA ASOs) remains a major challenge. One promising configuration of anti-miRNA ASOs called “tiny LNA (tiny Locked Nucleic Acid)” is an unusually small (~8-mer), highly chemically modified anti-miRNA ASO with high activity and specificity. Within this platform, we achieved a great enhancement of the in vivo activity of miRNA-122-targeting tiny LNA by developing a series of N-acetylgalactosamine (GalNAc)-conjugated tiny LNAs. Specifically, the median effective dose (ED50) of the most potent construct, tL-5G3, was estimated to be ~12 nmol/kg, which is ~300–500 times more potent than the original unconjugated tiny LNA. Through in vivo/ex vivo imaging studies, we have confirmed that the major advantage of GalNAc over tiny LNAs can be ascribed to the improvement of their originally poor pharmacokinetics. We also showed that the GalNAc ligand should be introduced into its 5′ terminus rather than its 3′ end via a biolabile phosphodiester bond. This result suggests that tiny LNA can unexpectedly be recognized by endogenous nucleases and is required to be digested to liberate the parent tiny LNA at an appropriate time in the body. We believe that our strategy will pave the way for the clinical application of miRNA-targeting small ASO therapy.

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In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation

Blood ◽

10.1182/blood-2011-11-393900 ◽

2012 ◽

Vol 119 (17) ◽

pp. 4066-4072 ◽

Cited By ~ 55

Author(s):

Bethan Psaila ◽

James B. Bussel ◽

Matthew D. Linden ◽

Bracken Babula ◽

Youfu Li ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Function ◽

Whole Blood ◽

Immune Thrombocytopenia ◽

Ex Vivo ◽

Platelet Reactivity ◽

Adenosine Diphosphate ◽

High Dose ◽

Platelet Surface

Abstract The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased.

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