Ethanol-induced increased surface-localized fibrinolytic activity in cultured human endothelial cells: kinetic analysis.

Background: Moderate alcohol consumption is associated with reduced risk for coronary heart disease and this cardioprotection may be due, in part, to increased fibrinolysis. We have previously demonstrated that low concentrations of ethanol (0.1%, v/v) induce the short-term (<1 hr) and sustained, long-term (24 hr) increase in surface-localized fibrinolytic activity; it up-regulates t-PA, u-PA, and the candidate plasminogen receptor (PmgR), annexin II, and gene transcription in cultured human umbilical vein endothelial cells (HUVECs). These studies describe the short- and long-term effects of low concentrations of ethanol on the kinetics of cell-bound 125 I-Iabeled Glu-plasminogen (Glu-Pmg) activation by receptor (R)-bound t-PA, resulting in increased fibrinolytic activity in cultured HUVECs. Methods: Live cultured HUVECs were incubated with varying concentrations of Glu-Pmg (0.25-2 μM) and ethanol (0.025-0.1%, v/v) (in the presence of Aprotinin and α 2 -antiplasmin) and the direct activation of cell-bound 125 I-labeled Glu-Pmg quantitated by measurement of 125 I-labeled M r 20 kDa plasmin light-chain, after reduction/SDS-PAGE. The effects of ethanol on 125 I-labeled Glu-Pmg and t-PA ligand binding were determined by Scatchard analysis (B max , sites/cell). Results: Cell-bound t-PA (endogenous/exogenous) activation of cultured HUVEC-bound 125 I-labeled Glu-Pmg (short- and long-term) obeyed Michaelis-Menten kinetics, both in the absence/presence of low ethanol, as shown by Lineweaver-Burke plot analysis. In the short-term, ethanol (at 0.1%) increased the V max (2.5-fold), k cat (2-fold) and the apparent k cat /K m (4-fold), commensurate with a significant decrease in the apparent K m (6-fold) and increase in 125 I-labeled Glu-Pmg ligand binding, B max (2-fold). In the long-term, ethanol increased the V max (2- to 3-fold), k cat (2.5-fold), apparent k cat /K m (5-fold), and Bmax (2-fold) for 125 I-labeled Glu-Pmg ligand binding, without significantly affecting the apparent K m . Conclusions: Low concentrations of ethanol induce the short- versus long-term increase in surface-localized fibrinolytic activity in cultured HUVECs via different mechanisms. Short-term effects may be mediated by ethanol-induced membrane conformational changes that simultaneously facilitate increased surface-localized HUVEC PmgR availability and fibrinolytic protein/receptor interactions, resulting in the increased affinity of t-PA for Glu-Pmg and the accelerated activation of Glu-Pmg (increased B max , decreased apparent K m ). The long-term effects may be attributed primarily to the ethanol-induced increased availability of both newly synthesized t-PA and PmgR and, hence, the accelerated activation of Glu-Pmg (increased B max ).