P-selectin glycoprotein ligand-1-independent transfer of tissue factor to platelets

Bein, Daniela1, Prof. Dr. Weber, Artur-Aron2, Prof. Dr. Schrör, Karsten1, Dr. Censarek, Petra 1. 1Uni-Klinikum Düsseldorf Institut für Pharmakologie und Klinische Pharmakologie, Universitätsstrasse 1, 40225 Düsseldorf, Germany ; 2Universität Duisburg-Essen/Universitätsklinikum Essen Institut für Pharmakologie, Hufelandstrasse 55, 45147 Essen, Germany.

Background and purpose: Microparticle (MP)-bound circulating tissue factor (TF) is critical for thrombin generation and fibrin clot formation. Several cell types including smooth muscle cells (SMC) and monocytes release TF-bearing MP that interact with platelets. The present study elucidates the transfer of TF from SMC and apoptotic monocytes to platelets.

Experimental approach: TF was tagged with a FLAG-tag sequence and overexpressed in SMC and the monocytic cell line U937. TF-FLAG functional expression was confirmed with an α-FLAG antibody and measurement of the endogenous thrombin potential (ETP). Apoptosis was induced by staurosporine. MP were prepared and incubated with platelets. TF-FLAG transfer to platelets was determined in washed platelets by Western blotting. Thrombogenic activity was expressed in terms of lagtime and time-to-peak thrombin formation.

Key results: TF-FLAG transfer from MP led to a significant 1.70 ± 0.03 fold (p<0.05, n=3) increase in thrombogenicity as compared to platelets incubated with MP from mock-transfected SMC. Incubation of platelets with MP from TF-FLAG overexpressing U937-cells doubled the thrombogenic potential. Even with MP prepared from apoptotic TF-FLAG expressing U937-cells the increase still significantly amounted to 1.80 ± 0.11 fold (p<0.001, n=3), in both the presence and absence of platelets.

Conclusions and implications: MP from SMC and apoptotic U937-cells transfer TF to platelets, thus delivering TF into a highly thrombogenic environment. TF-transfer to platelets contributes to thrombotic events in certain clinical conditions, e.g. coronary angioplasty. The TF transfer from monocytes to platelets is normally mediated by a P-selectin glycoprotein ligand-1 (PSGL-1)/P-selectin interaction. However neither SMC nor apoptotic monocytes express PSGL-1 on their surface, thus the observed TF transfer must involve a hitherto undescribed transfer mechanism independent of PSGL-1.