Apolipoprotein(a) inhibits the conversion of Glu-plasminogen to Lys-plasminogen on the surface of vascular endothelial and smooth muscle cells.

Lipoprotein(a) [Lp(a)] is an enigmatic lipoprotein which has been identified as a causal risk factor for coronary heart disease and calcific aortic valve disease. Lp(a) consists of a low-density lipoprotein (LDL) moiety covalently linked to the unique glycoprotein apolipoprotein(a) [apo(a)]. Apo(a) is homologous to the fibrinolytic zymogen plasminogen and thus may interfere with plasminogen activation. Conversion of native Glu-plasminogen by plasmin to the more readily activatable Lys-plasminogen greatly accelerates plasminogen activation and is necessary for optimal stimulation of plasminogen activation on endothelial cells. Lp(a)/apo(a) has been previously shown to inhibit pericellular plasminogen activation on vascular cells, but the mechanism underling these observations is unknown. We therefore explored whether apo(a) can inhibit pericellular Glu- to Lys-plasminogen conversion on cell surfaces. A physiologically relevant recombinant version of apo(a) (17K) significantly inhibits plasmin-mediated Glu- to Lys-plasminogen conversion on human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs). All isoforms of apo(a) that were analyzed, ranging in size from 3 to 21 kringle IV type 2 repeats, were able to inhibit conversion to a similar extent. Removal of the kringle V and protease domain of apo(a) strongly reduces the ability of apo(a) to inhibit conversion on HUVECs and SMCs. Removing the strong lysine binding site in KIV10 of apo(a) abolishes its ability to inhibit conversion on HUVECs and, to a lesser extent, on SMCs. These results indicate a novel mechanism in which apo(a) inhibits the positive feedback mechanism that accelerates plasmin formation on vascular cells.

P-glycoprotein trafficking at the blood-brain barrier altered by peripheral inflammatory hyperalgesia.

P-glycoprotein (ABCB1/MDR1, EC 3.6.3.44), the major efflux transporter at the blood-brain barrier (BBB), is a formidable obstacle to CNS pharmacotherapy. Understanding the mechanism(s) for increased P-glycoprotein activity at the BBB during peripheral inflammatory pain is critical in the development of novel strategies to overcome the significant decreases in CNS analgesic drug delivery. In this study, we employed the λ-carrageenan pain model (using female Sprague-Dawley rats), combined with confocal microscopy and subcellular fractionation of cerebral microvessels, to determine if increased P-glycoprotein function, following the onset of peripheral inflammatory pain, is associated with a change in P-glycoprotein trafficking which leads to pain-induced effects on analgesic drug delivery. Injection of λ-carrageenan into the rat hind paw induced a localized, inflammatory pain (hyperalgesia) and simultaneously, at the BBB, a rapid change in colocalization of P-glycoprotein with caveolin-1, a key scaffolding/trafficking protein. Subcellular fractionation of isolated cerebral microvessels revealed that the bulk of P-glycoprotein constitutively traffics to membrane domains containing high molecular weight, disulfide-bonded P-glycoprotein-containing structures that cofractionate with membrane domains enriched with monomeric and high molecular weight, disulfide-bonded, caveolin-1-containing structures. Peripheral inflammatory pain promoted a dynamic redistribution between membrane domains of P-glycoprotein and caveolin-1. Disassembly of high molecular weight P-glycoprotein-containing structures within microvascular endothelial luminal membrane domains was accompanied by an increase in ATPase activity, suggesting a potential for functionally active P-glycoprotein. These results are the first observation that peripheral inflammatory pain leads to specific structural changes in P-glycoprotein responsible for controlling analgesic drug delivery to the CNS.