Molecular communications between red blood cells and platelets during clot formation
Abstract
Hemostasis is considered a primary function of endothelial cells, platelets and soluble coagulation factors. Historically, accumulation of red blood cells (RBCs) in a clot has been attributed to passive entrapment as they flow past the site of vascular injury. Believing that erythrocytes might participate more actively in clot formation, we initiated a search for signal molecules that might mediate communication between activated platelets and adjacent RBCs. This investigation began by utilizing phorbol-12 myristate-13 acetate (PMA), an activator of protein kinase C (PKC) in RBCs. Platelet rich plasma supplemented with PMA pre-stimulated RBCs showed an increase in both platelet secretion (determined by ATP luminol assay) and platelet aggregation, indicating that the PMA “activated” RBCs enhanced clot formation as compared to control RBCs. Characterization of the signal transduction pathway responsible for this procoagulant activity in mature RBCs was pursued in order to later identify naturally occurring effectors that may enhance RBCs participation in clot formation. Fresh PMA treated RBCs were found to undergo a rapid, dose dependent influx of calcium, as demonstrated by: (i) radioactive 45Ca++ uptake, and (ii) an increase in fluorescence of the entrapped Ca2+ sensitive dye, fluo-3. At 3μM PMA, calcium influx into RBCs was minimally inhibited by nifedipine (100μM) and verapamil (100μM), both L-type calcium channel blockers, but completely inhibited by ω-agatoxin (1μM), a specific P-type calcium channel blocker. Staurosporine (3μM) as well as the specific PKC inhibitor chelerythrine chloride (10μM), also inhibited calcium uptake into PMA stimulated RBCs, indicating that the calcium influx is most likely mediated by a PKC activated channel. With the signaling role of PKC in RBC activation established, we next examined compounds released by activated platelets for a similar effect on mature RBCs. Lysophosphatidic acid (LPA), a lipid-derived second messenger generated by activated platelets that recruits cells of the circulatory system to assist in clot formation and wound healing, was found to initiate calcium influx similar to PMA stimulated RBCs. The LPA stimulated calcium influx was also similarly inhibited by ω-agatoxin and chelerythrine chloride. These data suggest that signaling pathways induced by products of platelet activation may potentiate the prothrombotic properties of RBCs.
Degree
Ph.D.
Advisors
Low, Purdue University.
Subject Area
Cellular biology|Biochemistry|Pathology
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