Abstract
Proteins secreted from Weibel-Palade bodies (WPBs) play important roles in regulating inflammatory and hemostatic responses. Inflammation is associated with the extracellular acidification of tissues and blood, conditions that can alter the behavior of secreted proteins. The effect of extracellular pH (pH(o)) on the release of von Willebrand factor (VWF), the VWF-propolypeptide (Proregion), interleukin-8, eotaxin-3, P-selectin, and CD63 from WPBs was investigated using biochemical approaches and by direct optical analysis of individual WPB fusion events in human endothelial cells expressing green or red fluorescent fusions of these different cargo proteins. Between pH(o) 7.4 and 7.0, ionomycin-evoked WPB exocytosis was characterized by the adhesion of VWF to the cell surface and the formation of long filamentous strands. The rapid dispersal of Proregion, interleukin-8, and eotaxin-3 into solution, and of P-selectin and CD63 into the plasma membrane, was unaltered over this pH(o) range. At pH(o) 6.8 or lower, Proregion remained associated with VWF, in many cases WPB failed to collapse fully and VWF failed to form filamentous strands. At pH(o) 6.5 dispersal of interleukin-8, eotaxin-3, and the membrane protein CD63 remained unaltered compared with that at pH(o) 7.4; however, P-selectin dispersal into the plasma membrane was significantly slowed. Thus, extracellular acidification to levels of pH(o) 6.8 or lower significantly alters the behavior of secreted VWF, Proregion, and P-selectin while rapid release of the small pro-inflammatory mediators IL-8 and eotaxin-3 is essentially unaltered. Together, these data suggest that WPB exocytosis during extracellular acidosis may favor the control of inflammatory processes.
Highlights
The major Weibel-Palade bodies (WPBs) core proteins are von Willebrand factor (VWF) and the VWF-propolypeptide (Proregion)
Occurs between pHo 7.0 and 6.8—To establish more precisely the level of extracellular acidification required for the retention of Proregion with VWF at the cell surface, we first determined, by immunofluorescence, the localization of Proregion, extracellular VWF (VWFo), and non-secreted VWF following stimulation at pHo buffered to 7.4, 7.2, 7.0, 6.8, and 6.5
To study the behavior of eotaxin-3 during release prediction we found that ionomycin-evoked WPB exocytosis from individual WPBs we constructed eotaxin-3-mEGFP and at pHo 6.5 resulted in a profound decrease (70 – 80%) in the expressed this in human umbilical vein endothelial cells (HUVEC) to label WPBs. appearance of soluble Proregion or VWF, and that the Consistent with immunofluorescence data, the dispersal of secreted material was readily recovered by subsequently eotaxin-3-mEGFP from WPBs during exocytosis was not affected exposing the stimulated cells to fresh stimulus-free medium by extracellular acidification (pHo 7.4; 3.0 Ϯ 3.1s, S.D. n ϭ 7 WPBs, at pHo 7.4 (Fig. 3)
Summary
The major WPB core proteins are von Willebrand factor (VWF) and the VWF-propolypeptide (Proregion). Secreted VWF adheres to the cell surface and can form long filamentous strands, under flow conditions, that are essential for the efficient capture of platelets from solution both in vitro and in vivo (e.g. Ref. 15). The aim of this study was to determine the influence of extracellular acidification in this range (pHo 7.4-6.5) on the release of a variety of soluble and membrane proteins of the WPB involved in coagulant and inflammatory responses. We define more precisely the value of pHo at which VWF fails to form filamentous strands and remains associated with Proregion at the cell surface, demonstrate the rapid reversibility of this pH-dependent association at individual sites of WPB fusion, and examine in detail the influence of extracellular acidification on the release and dispersal of fluorescent fusion proteins of the soluble mediators IL-8, eotaxin-3, and the membrane proteins P-selectin and CD63 from individual WPBs
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