Von Willebrand Factor Release and P-Selectin Expression Is Stimulated by Thrombin and Trypsin but not IL-1 in Cultured Human Endothelial Cells

Summary

The stimulated release of von Willebrand factor (vWF) from endothelial cells by secretagogues such as thrombin is associated with the translocation of Weibel-Palade bodies to the cell membrane and the surface expression of P-selectin (also known as GMP 140, PADGEM and CD 62). P-selectin, which is stored in Weibel-Palade bodies, is a neutrophil and monocyte adhesion molecule important in the initiation of inflammation.

We have developed a simple assay for the detection of P-selectin on endothelial cells using indirect immunofluorescence and flow cytometry and have confirmed that this is temporally related to vWF release. The assay has been used to demonstrate that IL-1 does not cause Weibel-Palade body degranulation but that trypsin does. This has implications for the use of passaged endothelial cells in the study of vWF release and the assay has numerous possible applications in study of mechanisms of stimulated vWF release.

• References

• 1 Wagner DD, Olmsted JB, Marder VJ. Immunolocalisation of von Willebrand protein in Weibel-Palade bodies of human endothelial cells. J Cell Biol 1982; 95: 355-602
• 2 Zucher MB, Broekman MJ, Kaplan KL. Factor VIII-related antigen in human blood platelets. Localisation and release by thrombin and collagen. J Lab Clin Med 1979; 94: 675-682
• 3 McEver RP, Beckstead JH, Moore KL, Marshall-Carlson L, Bainton DF. GMP-140, a platelet α-granule membrane protein is synthesized by vascular endothelial cells and is localised in Weibel-Palade bodies. J Clin Invest 1989; 84: 92-99
• 4 Toothill VJ, van Mourik JA, Niewenhuis HK, Metzelaar MJ, Pearson JD. Characterization of the enhanced adhesion of neutrophil leukocytes to thrombin stimulated endothelial cells. J Immunol 1990; 145: 283-291
• 5 Geng J-G, Bevilacqua MP, Moore KL, McIntyre TM, Prescott SM, Kim JM, Bliss GA, Zimmerman GA, McEver RP. Rapid neutrophil adhesion to activated endothelium mediated by GMP 140. Nature 1990; 343: 757-760
• 6 Johnston GI, Cook RG, McEver RP. Cloning of GMP 140 a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation. Cell 1989; 56: 1033-1044
• 7 Hattori R, Hamilton KK, Fugates RD, McEver RP, Sims PJ. Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140. J Biol Chem 1989; 264: 7768-7771
• 8 Levine JD, Harlan JM, Harker LA, Joseph ML, Counts RB. Thrombin-mediated release of factor VIII antigen from human umbilical vein endothelial cells in culture. Blood 1982; 60: 531-533
• 9 Ribes JA, Francis CW, Wagner DD. Fibrin induces release of von Willebrand factor from endothelial cells. J Clin Invest 1987; 79: 117-123
• 10 Hattori R, Hamilton KK, McEver RP, Sims PJ. Complement proteins C5b-9 induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface. J Biol Chem 1989; 264: 9053-9060
• 11 Loesberg C, Gonsalves MD, Zandbergen J, Willems C, van Aken WG, Stel HV, van Mourik JA, de Groot PG. The effect of calcium on the secretion of factor VIII-related antigen by cultured human endothelial cells. Biochem Biophys Acta 1983; 763: 160-168
• 12 de Groot PG, Gonsalves MD, Loesberg C, van Buul-Wortelboer MF, van Aken WG, van Mourik JA. Thrombin-induced release of von Willebrand factor from endothelial cells is mediated by phospholipid methylation. J Biol Chem 1984; 259: 13329-13333
• 13 Sternberg PE, McEver RP, Shuman MA, Jacques YV, Bainton DF. A platelet alpha granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J Cell Biol 1985; 101: 880-886
• 14 Furie B, Bing DH, Feldman RJ, Robinson DJ, Burnier JP, Furie BC. Computer generated models of blood coagulation factor Xa factor IXa and thrombin based upon structural homology with other serine proteases. J Biol Chem 1982; 257: 3875-3882
• 15 Vu T-K, Hung D, Wheaton V, Coughlin S. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057-1068
• 16 Schorer AE, Moldow CF, Rick ME. Interleukin 1 or endotoxin increases the release of von Willebrand factor from human endothelial cells. Br J Haematol 1987; 67: 193-197
• 17 Zavoico GB, Ewenstein BM, Schafer AI, Pober JS. IL-1 and related cytokines enhance thrombin-stimulated PGI₂ production in cultured endothelial cells without affecting thrombin-stimulated von Willebrand factor secretion or platelet activating factor biosynthesis. J Immunol 1989; 142: 3993-3999
• 18 Paleolog EM, Crossman DC, McVey JH, Pearson JD. Differential regulation by cytokines of constitutive and stimulated secretion of von Willebrand factor from endothelial cells. Blood 1990; 75: 688-695
• 19 Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphological and immunological criteria. J Clin Invest 1973; 52: 2745-2756
• 20 Modderman PW. New clusters of antibodies against platelet activation antigens: CD 62 and CD 63. In: Leucocyte typing IV Knapp W, Dorker B, Gilks WR, Rieber EP, Schmidt RE, Stein H, von dem BorneAE. (eds) Oxford University Press; Oxford: 1038 1989
• 21 Coughlin SR, Vu T-K, Hung DT, Wheaton VI. Characterization of a functional thrombin receptor. J Clin Invest 1992; 89: 351-355