Low angle light scattering analysis: a novel quantitative method for functional characterization of human and murine platelet receptors

Abstract

Background: Determinations of platelet receptor functions are indispensable diagnostic indicators of cardiovascular and hemostatic diseases including hereditary and acquired receptor defects and receptor responses to drugs. However, presently available techniques for assessing platelet function have some disadvantages, such as low sensitivity and the requirement of large sample sizes and unphysiologically high agonist concentrations. Our goal was to develop and initially characterize a new technique designed to quantitatively analyze platelet receptor activation and platelet function on the basis of measuring changes in low angle light scattering. Methods: We developed a novel technique based on low angle light scattering registering changes in light scattering at a range of different angles in platelet suspensions during activation. Results: The method proved to be highly sensitive for simultaneous real time detection of changes in size and shape of platelets during activation. Unlike commonly-used methods, the

Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition

“…The K i value for inhibition by Iloprost was 0.053 6 0.008 nM and calculated as described. 20 These data indicate 2 effects of Iloprost on ADP-stimulated shape change: (1) a dose-dependent right shift of the dose-response curve for ADP stimulation, and (2) a dose-dependent increase of the hill-slope indicating effects on signal transduction pathways ( Figure 3).…”

“…20 Briefly, platelet-rich plasma was diluted in 6 mL of modified N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid (HEPES) buffer (pH 7.4, osmolarity 302 mOsm, containing 140 mM NaCl, 10 mM HEPES, 10 mM NaHCO 3 , 2 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 5.5 mM D-glucose) at a count of 1 3 10 7 platelets/mL. After 2 minutes of constant basal signal, platelets were stimulated by ADP and Iloprost at indicated concentrations, and data were recorded for another 10 minutes.…”

“…Initial functional experiments evaluating the ability of Iloprost to reverse ADP-induced platelet shape change and aggregation were performed using the recently established LaSca method, 20 which allows independent and simultaneous detection of shape change and aggregation (supplemental Figure 1). This indicated that shape change peaked at 90 nM ADP, which was completely inhibited by 1 nM Iloprost (SF 1A).…”

Determination of ATP and ADP Secretion from Human and Mouse Platelets by an HPLC Assay

“…Dense granule secretion and, in particular, the release of ATP and ADP from the platelet as secondary event during platelet aggregation have in recent years been recognized as a major step in platelet aggregate formation being crucial for the formation of stable aggregates [16][17][18]. Purinergic receptor activation and signaling can be efficiently quantified by routine tests [19][20][21] while monitoring of dense granule secretion is commonly only accomplished in a semi-quantitative manner. Current methods are either based on determination of the release of tracer from pre-loaded platelets [22], on fluorometry of derivatized serotonin [22] or on bioluminescence of an ATP-consuming reaction [23].…”

Optoacoustic spectroscopy for real-time monitoring of strongly light-absorbing solutions in applications to analytical chemistry

“…Various spectroscopic techniques have been used and most frequent are low-angle fluorescence [11], low-angle static [12] and dynamic [13] light scattering, and attenuated total reflection techniques [11,14,15]. Still, these methods have limitations (i) due to sophisticated methodology; (ii) spectral interfering effects like reabsorption; or (iii) related to the limited number of detectable substances (like luminophores or Mie-scattering entities).…”