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Redistribution of membrane glycoproteins in platelets activated under flow conditions
Abstract:
A reduction in the ability of GPIb to bind specific MoAbs or ligands (vWF) has been reported in platelets exposed to thrombin in suspension. We have analyzed modifications in the presence of glycoproteins (GPs) on platelets activated under flow conditions in a system which allows limited thrombin and fibrin generation. Normal blood anticoagulated with low molecular weight heparin (LMWH, Dalteparin 20 IU/ml) was recirculated for up to 10 min at 800 s-1 through annular chambers containing denuded arterial segments. Aliquots of blood were removed from the reservoir at 0, 1, 5 and 10 min and immediately mixed with paraformaldehyde. Membrane glycoproteins: GPIb (CD42b), GPIIb-IIIa (CD41a), GPIV (CD36); and activation dependent antigens: P-selectin (CD62P) and lysosomal glycoprortein (CD63), were detected in whole blood by dual color flow cytometry. Circulation of through the perfusion system resulted in platelet activated as demonstrated by the increased percentage of platelets positive for antigens CD62P and CD63. A gradual increase in the binding of MoAbs directed against GPIb, GPIIb-IIIa, and GPIV epitopes was noted during the entire perfusion period. Observed differences in mean fluorescence intensities at all the observation times were statistically significant (P < 0.001). Our results obtained on platelets in an experimental thrombosis system indicate that GPIb, GPIIb-IIIa and GPIV remain on the surface of activated platelets and actually increase their expression. Alterations detected at the level of GPIb in platelets activated by thrombin in suspension may not take place under in vivo situations.
Insights
Platelet glycoproteins (GPs) like GPIb, GPIIb-IIIa, and GPIV expression increases on activated platelets under flow conditions. This suggests in vivo thrombosis may differ from in vitro thrombin activation studies.
Area of Science:
- Hematology
- Biomedical Engineering
- Thrombosis Research
Background:
- Previous studies indicated reduced GPIb binding on thrombin-activated platelets in suspension.
- The behavior of platelet glycoproteins under physiological flow conditions remained unclear.
Purpose of the Study:
- To investigate changes in platelet glycoprotein expression during activation under simulated flow conditions.
- To compare platelet activation in a flow system with findings from suspension studies.
Main Methods:
- Normal blood anticoagulated with low molecular weight heparin was recirculated through an artificial arterial system.
- Platelet activation markers (CD62P, CD63) and glycoprotein expression (GPIb, GPIIb-IIIa, GPIV) were analyzed using dual-color flow cytometry at various time points.
- Platelets were exposed to shear rates of 800 s-1 for up to 10 minutes.
Main Results:
- Platelet activation was confirmed by increased expression of CD62P and CD63.
- Binding of monoclonal antibodies against GPIb, GPIIb-IIIa, and GPIV epitopes gradually increased during perfusion.
- Observed increases in mean fluorescence intensities were statistically significant (P < 0.001).
Conclusions:
- Platelet glycoproteins GPIb, GPIIb-IIIa, and GPIV remain expressed on the surface of activated platelets under flow.
- Their expression actually increases during activation in this experimental thrombosis model.
- Findings suggest that GPIb alterations observed in suspension may not reflect in vivo conditions.