Cross Linking of Human Platelet Membrane Aminophospholipids:Changes Induced by Thrombin

The proximity of the aminophospholipids, phosphatidylethanolamine (PE) and phosphatidyl-serine (PS), in platelet membranes was probed with difluorodinitrobenzene (D) which reacts with and cross-links aminophospholipids when they are less than 5 A° apart. D can penetrate intact cells and labels lipids on either side of the membrane bilayer. The incubation of mixtures of pure PE and PS can result in the formation of the following derivatives: PE-D-PE, PE-D-PS, PE-D, PS-D-PS, PS-D. Following incubation of washed platelets with D, lipids and derivatives were extracted, separated by thin-layer chromatography, and quantitated by lipid phosphorus. The incubation of platelets with D (125μM) for 2 hours resulted in derivatization of 49% PE and 34.6%PS. When platelets were treated with thrombin (0.1U/ml) for 5 minutes prior to incubation with D, 71.4% PE and 53.8% PS reacted. Results are shown in Table; %PE(PS)=% total platelet PE(PS).

In controls significant amounts of only PE-D-PE and PE-D-PS were detected and thus are intimately associated within platelet membranes. In thrombin-treated platelets significant amounts of PS-D-PS were formed which did not occur in controls. However, there was no increase in PE-D-PE and PE-D-PS. Also, in thrombin-treated platelets additional PE and PS reacted but were not close enough to be cross-linked. The study indicates that incubation of platelets with thrombin results in the unmasking or rearrangement of specific compartments of PE and PS within platelet membranes which may be critical for platelet hemostatic activities.

 

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