Early Platelet Activation by Low Density Lipoprotein via p38MAP Kinase

Abstract

SummaryLow Density Lipoprotein (LDL) is known to sensitize platelets for physiological agonists. To clarify the basis of this sensitization, we investigated the involvement of p38MAP Kinase (p38MAPK). As dual phosphorylation on Thr180 and Tyr182 of p38MAPK is the trigger for activation of the kinase, p38MAPK-activity was measured with an antibody that recognizes the dual-phosphorylated sequence. LDL induced a rapid and dose dependent activation of p38MAPK. The activation was not inhibited by a wide variety of inhibitors of platelet signalling, including TxA2-formation, Phospholipase C-activation, Ca2+-mobilization and ERK 1/2-activation. Only a slight reduction in p38MAPK-activation was observed when protein kinase C was inhibited. Activation of p38MAPK was strongly inhibited by a rise in cAMP. Thus, p38MAPK-activation was upstream of most signalling pathways and close to the LDL-receptor. A number of platelet receptors was screened with the use of antibodies. Integrins αIIbβ3 and α2β1, as well as the FcγRII-receptor, CD36 (platelet glycoprotein IV), CD68 (gp110) and Low Density Lipoprotein-receptor related protein (LRP) were not implicated in LDL-induced p38MAPK-activation. Inhibition of LDL binding by modification of apo B100 lysines reduced p38MAPK-activation by 80 %. Activation of p38MAPK resulted in an increase in release of arachidonic acid, the precursor for thromboxane A2 synthesis. In conclusion, activation of p38MAPK might be the first step in platelet sensitization by LDL, leading to formation of arachidonate metabolites and increased aggregation and secretion responses to physiological agonists.

Intracellular events in platelet activation induced by antiphospholipid antibodies in the presence of low doses of thrombin

“…Blood was collected in acidcitrate-dextrose anticoagulant (9/1 volume/volume). Plasmarich platelets (PRPs) were obtained by centrifugation for 20 minutes at 120g at room temperature (21).…”

“…The phosphorylated form of cPLA 2 is detected as a slower-moving second band. Thrombin-induced anti-cPLA 2 was detectable after 2 minutes of stimulation (approximately half of the protein was in the phosphorylated form) (21).…”

“…The emission peaks of calcium green and Fura Red signal intensities were determined at 530/30 nm and 640/40 nm, respectively (21). Elevations in [Ca 2ϩ ] result in increased fluorescence intensity of calcium green and decreased fluorescence intensity of Fura Red, at 485/20 nm excitation wavelength.…”

“…Elevations in [Ca 2ϩ ] result in increased fluorescence intensity of calcium green and decreased fluorescence intensity of Fura Red, at 485/20 nm excitation wavelength. Changes in [Ca 2ϩ ] were evaluated on the basis of a ratio of signal intensity at the G-channel (calcium green, sensitive peak at 530 nm) relative to the R-channel (red, sensitivity peak at 640 nm) (21).…”

Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) Inhibits Low Density Lipoprotein-induced Signaling in Platelets

“…Part of this sensitization is mediated via activation of p38 MAPK and cytosolic phospholipase A 2 (cPLA 2 ), which, together with a second stimulus, leads to more liberation of arachidonic acid and the formation of thromboxane A 2 (1,6). A second mechanism involved in sensitization is the activation of p125 focal adhesion kinase (FAK) with its still poorly characterized effects on the formation of focal adhesions and cytoskeletal rearrangements (7,8).…”