Gravity sedimentation reveals functionally and morphologically different platelets in human blood.

In contrast to red blood cells, platelets float rather than sediment when a column of blood is placed in the gravitational field. By the analogy of erythrocyte sedimentation (ESR), it can be expressed with the platelet antisedimentation rate (PAR), which quantitates the difference in platelet count between the upper and lower halves of the blood column after 1 h of 1 g sedimentation. Venous blood samples from 21 healthy subjects were analyzed for PAR. After a 1-h sedimentation, the upper and lower fractions of blood samples were analyzed for platelet count, mean platelet volume (MPV), immature platelet fraction (IPF), and high-fluorescence IPF (H-IPF). The mechanisms behind platelet flotation were explored by further partitioning of the blood column, time-dependent measurements of platelet count and comparison with ESR. The structure and function of the platelets were assessed by electron microscopy (EM) and atomic force microscopy (AFM), and platelet aggregometry, respectively. Platelet antisedimentation is driven by density differences and facilitated by a size-exclusion mechanism caused by progressive erythrocyte sedimentation. The area under the curve (AUC) of the whole blood adenosine diphosphate (ADP) aggregation curves showed significant differences between the upper and lower samples (p < .005). AUC in the upper samples of 38% of healthy subjects exceeded the top of the normal range (53-122) suggesting that ascending platelets show an intensified ADP-induced aggregability ex vivo. H-IPF was significantly higher in the upper samples (p < .05). EM and AFM revealed that platelets in the upper samples were larger in volume and contained 1.6 times more alpha granules compared to platelets in the lower samples. Our results indicate that antisedimentation is able to differentiate platelet populations based on their structural and functional properties. Therefore, PAR may be a suitable laboratory parameter in various thromboinflammatory disorders.

It is less known that platelets do not sediment in response to gravitational force but float on the top of the blood column. This phenomenon is called antisedimentation, the rate of which, however, can be different, yet this feature has not been widely studied and used in clinical practice or diagnosis. We tested the idea that antisedimentation of platelets from venous blood samples can be a potential biomarker. We have found that platelet antisedimentation is driven by density differences and facilitated by a size-exclusion mechanism caused by progressive erythrocyte sedimentation and after 1-h upper and lower fractions develop. Interestingly, the aggregation curves showed significant differences between the upper and lower samples, suggesting that the ascending platelets show ex vivo hyperaggregability. Electron and atomic force microscopy revealed that platelets in the upper samples were larger in volume and contained more alpha granules than platelets in the lower samples. Subsequently, antisedimentation can be used to differentiate platelet populations based on their structural and functional properties; thus, it may be a promising biomarker for various thromboinflammatory disorders.

Morphological study of the postnatal hippocampal development in the TRPV1 knockout mice.

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel with polymodal sensory function. TRPV1 links to fever, while, according to previous studies on TRPV1 knock-out (KO) mice, the role of the channel in the generation of febrile seizure is debated. In the hippocampal formation, functional TRPV1 channels are expressed by Cajal-Retzius cells, which have a role in guidance of migrating neurons during development. Despite the developmental aspects of febrile seizure as well as of Cajal-Retzius cells, no information is available about the hippocampal development in TRPV1 KO mouse. Therefore, in the present work postnatal development of the hippocampal formation was studied in TRPV1 KO mice. Several morphological characteristics including neuronal positioning and maturation, synaptogenesis and myelination were examined with light microscopy following immunohistochemical detection of protein markers of various neurons, synapses, and myelination. Regarding the cytoarchitectonics, neuronal migration, morphological, and neurochemical maturation, no substantial difference could be detected between TRPV1 KO and wild-type control mice. Our data indicate that synapse formation and myelination occur similarly in TRPV1 KO and in control animals. We have found slightly, but not significantly larger numbers of persisting Cajal-Retzius cells in the KO mice than in controls. Our result strengthens previous suggestion concerning the role of TRPV1 channel in the postnatal apoptotic cell death of Cajal-Retzius cells. However, the fact that the hippocampus of KO mice lacks major developmental abnormalities supports the use of TRPV1 KO in various animal models of diseases and pathological conditions.

Development of parvalbumin-immunoreactive neurons in the postnatal human hippocampal formation.

Introduction: Parvalbumin (PV) is a calcium-binding protein present in fast-spiking GABAergic neurons, such as basket and axo-axonic cells. Previous studies in non-human primates reported prenatal expression of PV in the temporal archicortex including entorhinal cortex and hippocampal formation. In contrast, PV-immunoreactivity was observed only postnatally in the human entorhinal cortex. Regarding PV expression in the human hippocampal formation, no information is available. Methods: In this study, the neurochemical maturation of PV-immunoreactive interneurons was studied in the postnatal developing human hippocampal formation. Results: Before birth, no PV-immunoreactive neurons could be detected in the human hippocampus. At birth, only a few PV-immunoreactive neurons were visible in Ammon's horn. The first PV-immunoreactive cells in the hilus of the dentate gyrus appeared at the age of 1 month. Even at the age of 5 months, only a few PV-immunopositive cells were present in the dentate hilus. The number of cells and their dendritic and axonal arborization in Ammon's horn and in the dentate gyrus gradually increased with age. Even at the age of 2 years, dendritic tree and axons of PV-immunoreactive neurons were less complex than can be seen in 8 and 11 years old children. Discussion: Our results showed that long-lasting maturation of PV-immunoreactive interneurons follows the developmental sequence of the subfields of the human hippocampal formation and provides further morphological evidence for the long-lasting functional maturation of the human cortex.

Circulating Microvesicles in Convalescent Ischemic Stroke Patients: A Contributor to High-On-Treatment Residual Platelet Reactivity?

INTRODUCTION: Exploration of novel and effective antiplatelet strategies for the secondary prevention of ischemic stroke is utmost. Some platelet derived microparticles (PMVs) in convalescent stroke subjects were found to be predictive for the next vascular event. Patients with high-on-treatment platelet reactivity (HTPR) had a significantly higher risk for ischemic stroke. Here, we aimed to explore associations among circulating microparticles and responsivness to antiplatelet (clopidogrel) therapy. METHODS: A total of 18 patients on clopidogrel therapy due to secondary stroke prevention were rospectively recruited into this study. Twenty age-matched healthy subjects served as controls. Flow cytometric measurements of microparicles (MVs) and data analysis were performed on Beckman-Coulter FC-500 cytometer with CXP software. Besides, platelet aggregometry data were revealed. Both measurements were performed in whole blood and from the lower and upper blood fractions separated after 1-hour gravity sedimentation by the analogy with erythrocyte sedimentation rate. RESULTS: The total number of circulating MVs, and particularly the platelet derived CD42+ and PAC-1+ were significantly higher in post-stroke patients (p < 0.001). The platelet aggregation in the whole blood (area under the curve, AUC) showed a significant negative correlation with the total number of MPs in the lower blood sample after 1-hour gravity sedimentation (r = -0.650, p = 0.005). Next, we analyzed associations among MPs and aggregometry data obtained from clopidogrel responders and non-responders. Both, area under the curve (AUC) and velocity in the whole blood showed opposite correlation with the total number of MVs in the lower blood sample after 1-hour gravity sedimentation. Importantly, a significant negative correlation was observed for the velocity (r = -0.801, p = 0.005), but not for the AUC in responders. Platelet derived CD42+ and PAC-1+ MVs showed positive correlations with neutrophils in the lower blood sample (p = 0.008 and p = 0.006 respectively). CONCLUSIONS: Circulating MVs may allow to monitor the response to antiplatelet therapy in post-stroke patients. In addition, the link between platelet derived MVs and neutrophil granulocytes might become therapeutic targets in the future.