Complete downmodulation of P-selectin glycoprotein ligand in monocytes undergoing apoptosis.

OBJECTIVE: Apoptotic monocytes release membrane microparticles which may play a major role in thrombogenicity through a P-selectin glycoprotein ligand (PGSL-1)-mediated mechanism. We have studied systematically the regulation of PSGL-1 expression and function in apoptotic monocytic cells. METHODS AND RESULTS: PSGL-1 expression (flow cytometry, immunofluorescence microscopy, immunoblot) was virtually abolished in apoptotic monocytes by proteolytic shedding. This was accompanied by a complete loss of PSGL-1-mediated platelet-leukocyte (flow cytometry) and leukocyte-endothelial cell (parallel plate flow chamber) interactions. Systematic screening of protease inhibitors combined with knock-out and siRNA experiments characterized the PSGL-1-cleaving enzyme as an N-ethylmaleimide-inhibitable metalloproteinase of the ADAM family. CONCLUSIONS: Downmodulation of PGSL-1 in apoptotic monocytes may prevent ectopic cell clearance in the peripheral vasculature to reduce local inflammatory and proliferative responses. Depletion of PSGL-1 expression on apoptotic microparticles may also act as a molecular switch to modulate their thrombogenic activity.

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

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 muM, 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.

Rapid release of active tissue factor from human arterial smooth muscle cells under flow conditions.

Circulating tissue factor (TF) is an important determinant of coronary thrombosis. Among other cell types, such as monocytes, vascular smooth muscle cells (SMCs) are capable of releasing TF. When studied under static conditions, SMCs do release TF, but this process is slow and, thus, cannot explain the elevated levels of circulating TF, as observed in patients with acute coronary syndromes. The present study demonstrates that cultured human mammary artery SMCs very rapidly (minutes) release active, microparticle-bound TF when exposed to flow conditions. There was a clear log-linear correlation between the shear rate (range 10 s(-1) to 1500 s(-1)) and the procoagulant activity of SMC perfusates. Flow-dependent release of TF was transient (10 minutes) and did not measurably reduce cell surface TF content. Interestingly, a time-dependent (t(1/2) 30 minutes) re-exposure of releasable TF was detected after a no-flow period. These data demonstrate that SMCs may become a pathophysiologically relevant source of TF that can be rapidly released into the circulation in situations in which endothelial damage occurs and SMCs come into a close contact with the flowing blood.