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Published online before print February 27, 2008

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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.1207843

Received for publication December 19, 2007.
Revised February 1, 2008.
Accepted for publication February 2, 2008.

Article

Membrane environment rather than tissue factor expression determines thrombin formation triggered by monocytic cells undergoing apoptosis

Jan Julius Stampfuss ^*, Petra Censarek ^*, Daniela Bein ^*, Karsten Schrör ^*, Maria Grandoch , Christoph Naber , and Artur-Aron Weber ^@

*Institut für Pharmakologie und Klinische Pharmakologie, Heinrich-Heine-Univesität Düsseldorf, Germany; and Institut für Pharmakologie Universität Duisburg-Essen, and Klinik für Kardiologie, Universitätsklinikum Essen, Germany

^@ To whom correspondence should be addressed. E-mail: artur.weber{at}uk-essen.de.

	Abstract

Monocyte apoptosis is an important determinant of atherothrombosis. Two major mechanisms for apoptosis-associated thrombogenicity have been described: exposure of negatively charged membrane phospholipids and up-regulation of tissue factor (TF). However, the relative importance of these mechanisms is unclear. Thus, procoagulant functions (thrombin generation) of apoptotic (staurosporine, 2 µM, 24 h) U937 cells versus cell-derived microparticles (MPs) were studied. In apoptotic U937 cells, a significant increase in TF mRNA (real-time PCR), surface expression of TF (flow cytometry), and total cellular amount of TF (Western blotting) was observed. Control cells only minimally triggered thrombin generation (endogenous thrombin potential), and apoptotic cells were highly procoagulant. However, addition of negatively charged membranes completely restored the thrombin generation capacity of control U937 cells to the levels of apoptotic cells. MPs (defined as CD45⁺ particles of subcellular size), derived from apoptotic U937 cells, were highly procoagulant but did not exhibit an increased TF expression or annexin V binding. Taken together, our data support the concept that the membrane environment, independent of TF expression, determines the extent of thrombin formation triggered by apoptosis of monocytic cells. Externalization of negatively charged phospholipids represents the most important mechanisms for whole cells. Additional yet unknown mechanisms appear to be involved in the procoagulant actions of MPs derived from apoptotic monocytes.

Key Words: U937 cells • monocytes • staurosporine