Autor:innen:
K. von Bargen (Bad Oeynhausen, DE)
M. Kraus (Koblenz, DE)
M. Dittrich (Würzburg, DE)
C. Brandenburger (Bad Oeynhausen, DE)
M. Wolny (Bad Oeynhausen, DE)
T. Flieder (Bad Oeynhausen, DE)
J. Rheinlaender (Tübingen, DE)
T. Schäffer (Tübingen, DE)
G. Kappert (Duisburg, DE)
S. Halimeh (Duisburg, DE)
C. Knabbe (Bad Oeynhausen, DE)
E. Strasser (München, DE)
H. Neeb (Koblenz, DE)
I. Birschmann (Bad Oeynhausen, DE)
Introduction
After vascular injury, platelet adhesion to the extracellular matrix leads to platelet activation, which in turn induces platelet shape change. Thereby the reorganization of the cytoskeleton is mediated by a variety of signaling pathways. Among other things, the fibrinogen receptor (GPIIb/IIIa complex) is also involved. This complex plays a crucial role in primary hemostasis by mediating platelet aggregation. Therefore, the study aimed to investigate the shape change (spreading) in dependence of the GPIIb/IIIa complex. For this purpose, platelets from Glanzmann thrombasthenia patients (GT-patients) were studied. This is a platelet dysfunction with a quantitative or qualitative defect of the GPIIb/IIIa complex.
Methods
In the present study, platelet shape change was investigated in a total of five healthy donors and six GT patients. For this purpose, platelets were allowed to spread on fibrinogen under different conditions (without activator, with ADP, or with TRAP), the actin cytoskeleton was stained with phalloidin, and then 40 immunofluorescence images were acquired per condition and time point. The immunofluorescence images were then evaluated using an algorithm (automated quantitative morphometry analysis) by determining various parameters such as area, fractal dimension, number of pseudopodia, etc. In addition, the morphometry of the platelets was examined by electron microscopy.
Results
Analysis of the immunofluorescence images shows that the GT platelets have a spreading defect, which is particularly characterized by the absence of lamellipodia formation. Whereas the healthy platelets have mostly a fully spread shape after 45 min, the GT platelets persist in the early phase of spreading, which is characterized by a large number of long pseudopodia. Overall, the results of the algorithm show that the individual parameters (such as number of pseudopodia, FD, circularity, and area) describing the morphometry differ significantly between the studied collectives (healthy and GT). Here, the differences are particularly distinct at the late spreading time points. Thus, the GT platelets are much smaller due to the large number of pseudopodia, and the FD is increased. Electron microscopy also shows the altered morphometry of the GT platelets.
Conclusion
Spreading analysis show that platelet shape change is impaired in GT platelets. Both immunofluorescence microscopy and electron microscopy could show an absence of lamellipodia formation. Quantitative morphometry analysis was used to better describe cytoskeletal reorganization and to show the differences between the two collectives. Overall, quantitative morphometry analysis is a useful tool to better describe the different stages of platelet shape change in patients with thrombocytopathy.