Computational protocol: HPA-1 polymorphism of αIIbβ3 modulates platelet adhesion onto immobilized fibrinogen in an in-vitro flow system

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Protocol publication

[…] Platelets adhesion rates onto fibrinogen and collagen-coated glass coverslips were measured in the rectangular flow chamber under linear shear rate of 50 s-1, 500 s-1 and 1500 s-1. A shear rate of 50 s-1 represents a venose system, 500 s-1 mimics a wall shear rate of larger arteries and 1500 s-1 represents a typical arteriolar shear rate as well as a shear rate by moderate arterial stenosis [,].A laser-scan microscope (Axiovert 100 M, Carl-Zeiss, Jena, Germany) allowed real-time visualisation of labelled platelets during perfusion through the chamber. To assess time-course of platelet adhesion a series of images (five images per series, 0,7 s pro image) were made at 15 sec, 1 and 5 minutes. Image analysis was performed using the ImageJ software (version 1.26t, NIH, USA). This program allows evaluation of platelet-surface interaction, consecutive aggregation and evaluation of thrombus generation within the defined area of each image. A single frame image corresponded to the area of 980 × 980 μm. The blood was perfused over fibrinogen-coated cover slips as described above. The number of stable, attached platelets on the surface was calculated as number of platelets, which remain their initial adhering position in the first and second image (time frame of 0.7 sec). Platelets were considered to move on the surface when exhibiting a spatial displacement greater than one platelet diameter. To estimate motion, a series of 5 images (time frame 0.7 sec) at one time point were made. Using ImageJ software images were binarised and a threshold was applied to distinguish platelet from background. The first two consecutive frames in a series were superimposed using the logical AND function and the resulting image represented only the overlapping areas of single platelet at two different times. [...] Data in text are given as mean values ± SD. The absolute fluorescence was expressed as arbitrary units (pixel units, AU) and represents sum of fluorescence of each thrombus or individual adherent platelet in one defined area. The platelet adhesion was calculated using a logic function of the applied software (ImageJ) and represented a stable platelet adhesion between the first and second image. Taking into consideration that initial platelet adhesion and platelet detachment after initial adhesion is a dynamic process, dependent from shear rate and perfusion time data were normalized. Absolute fluorescence recorded after five minute of perfusion was divided by recorded fluorescence after 1 minute or after 15 sec of perfusion and expressed as a relative adhesion. The relative adhesion represents the increase of absolute fluorescence in function of time and reflects number of stable adherent platelet. Data normalisation allow us to evaluate stability of adherent platelets and to quantify increase of stable platelet adhesion onto immobilized fibrinogen under different shear rates in function of time.Differences between experimental groups were tested using Student's t-test (two-sided). Regression analyses were based on individual measurements using Spearman's rank correlation coefficient. Statistical analyses were performed using SPSS for Windows, version 6.0.1. P-value of less than 0.05 (two-sided) was used to indicate a significant difference. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Diseases Cerebrovascular Disorders, Thrombosis, Stroke, Acute Coronary Syndrome