Circulating Human Platelets Express LRP-1 and uPAR mRNAs and Synthesize the Proteins: The Complex LRP-1, uPAR, PAI-1 and uPA Play a Role in Modulating Fibrinolysis in Platelet-Rich Thrombi

to study the presence, origin, association and functional role of components of the fibrinolytic system in human platelets. Also, we aim to getting insight into the dynamic balance and modulation of the fibrinolytic process by the interplay of pro- and anti-fibrinolytic platelet factors.

in washed, leukocyte-free human platelets we detected expression of LRP-1, uPAR, PAI-1 mRNAs, and synthesis of these proteins (metabolic radiolabeling). Neither uPA mRNA nor synthesis of uPA was evidenced. All of these proteins, including uPA were detected in membrane or cytosol fractions by western blotting (WB). LRP-1 and uPAR were present in the outer leaflet of platelet membranes, with increased uPAR labeling after platelet activation (confocal microscopy-immunofluorescence). Non-stimulated whole platelets exhibit a low basal uPA activity (specific chromogenic substrate) selectively inhibited by amiloride. uPA activity falls slightly immediately after VWF-Ristocetin (VWF-R) and TRAP stimulation, but recovers to basal levels after 15min. Biotinylated washed platelets were immunoprecipitated (IP) with α -uPAR MoAb at different times before and after activation with either TRAP or VWF-Ristocetin. Co-precipitations with LRP-1, PAI-1 and uPA were detected in WB only after platelet activation with TRAP for 5 min, denoting the formation of a tetrameric complex, likely involved in endocytosis and receptor recycling. Interestingly, 5min after TRAP stimulation, uPA was sharply reduced, disappearing at 15 min, either in membrane or cytosol fractions, suggesting degradation of the protein. Similar pattern of co-precipitations were observed when IP was done with α -LRP-1 MoAb. Co-precipitations were more prominent in purified platelet membrane than in cytosolic fractions.

human platelets express LRP-1, uPAR and PAI-1 mRNAs, and synthesize these proteins. uPA activity is present in whole, purified, washed platelets, and the protein is likely bound to the external platelet membrane. Co-precipitation of all these fibrinolytic components presumably denotes the formation of a tetrameric complex with endocytic and recycling capacities, as demonstrated in other cell lineages. Sequential IP′s after platelet activation disclose the disappearance of uPA, but not of PAI-1, from the complex, probably explained by a degradation process. Taken together, these results suggest that platelets play a predominantly antifibrinolytic role during early stages of formation of platelet-rich clots.

No relevant conflicts of interest to declare.