Morphological alterations in endothelial cells associated with the release of von Willebrand factor after thrombin generation in vivo.

Abstract

von Willebrand factor (vWF) is synthesized by endothelial cells and stored in endothelium-specific granules, the Weibel-Palade (WP) bodies. The release of vWF from endothelial cells in vitro in response to secretagogues such as thrombin is considered to result in the loss of WP bodies through the fusion of the WP bodies with the plasma membrane. Biochemical and morphological techniques, including transmission (TEM) and scanning (SEM) electron microscopy, were used to examine the plasma profile of vWF in parallel with morphological alterations in endothelial cells associated with the generation of thrombin in vivo. There was a rapid loss of high-molecular-weight multimers of the circulating vWF, with full recovery within 1 hour. Simultaneously, TEM demonstrated that the endothelial cells lost WP bodies and became V on Willebrand factor (vWF) is a large multimeric plasma protein that plays a central role in hemostasis. 12 It is synthesized in megakaryocytes 34 and endothelial cells 56 and stored in the a-granules of platelets 7 and in the endothelial cell storage organelles, the Weibel-Palade (WP) bodies. 8 P-selectin is a component of both the platelet a-granule and WP body membrane, 9 but other components of the a-granules have not been identified in WP bodies. 10 vWF is released from endothelial cells both constitutively and via the regulated pathway from WP bodies after endothelial cell stimulation." Release from WP bodies makes available the highly platelet-interactive, high-to abnormally high-molecular-weight multimeric forms of vWF. 2 vWF is released from endothelial cells in culture in response to stimulation by a variety of secretagogues, including thrombin.1112 It is generally considered that the mechanism of release involves the fusion of the limiting membrane of the WP bodies with either the luminal or abluminal membranes of the endothelial cell. This process has been inferred from observation in two settings. In vitro studies, using immunofluorescence 8 " 13 or electron mi- 10 demonstrated that the release of vWF was associated with a reduction in the numbers of WP bodies and the appearance of vWF in the supernatant. In studies in vivo, WP bodies appeared to release their contents into the circulation after fusing with the luminal membrane of endothelial cells.14 Although this observation was reported after the recognition that WP bodies stored vWF, 8 this was not appreciated by the authors, who considered the observation to indicate that WP bodies were secretory in nature but of unknown function.14 To our knowledge, there has been no in vivo study in which plasma vWF levels have been quantified in parallel with alterations in the WP bodies and vWF distribution within endothelial cells.We have developed a model for the induction of thrombin generation in vivo in various animal species. This is achieved through the infusion of two defined components of the prothrombinase complex 15 : factor Xa (activated factor X [FXa]) and a coagulant active phospholipid surface provided by phosphatidylchol...

von Willebrand factor multimerization and the polarity of secretory pathways in endothelial cells

“…This is in line with the majority of previous in vitro 10,12,14 and in vivo studies. 30 This form of secreted VWF is also composed of mainly UL-VWF multimers. This is in line with the currently accepted, yet experimentally unsupported, dogma that most highly multimerized VWF is secreted mainly toward the apical (lumen) surface of endothelial cells, which we now prove experimentally for the first time.…”

Biochemistry and Genetics of Von Willebrand Factor

“…In vivo, the plasma level of VWF is increased acutely by adrenergic stress, thrombin generation, or treatment with the vasopressin analog 1-desamino-8-Darginine vasopressin (DDAVP). These responses are presumed to reflect secretion from Weibel-Palade bodies, although this has been demonstrated directly only for thrombin generation (45). The ability of DDAVP to raise plasma VWF levels has made it a major drug for the treatment of VWD and hemophilia (46).…”

Polarity of Constitutive and Regulated von Willebrand Factor Secretion by Transfected MDCK-II Cells

“…After washing with PBS, the cells were labeled for 3 days by using the extensively, characterized Madin-Darby Caadding the following components to the basolateral compartment: 85 nine Kidney-II (MDCK-II) cell line [18][19][20]. These cells mCi 35 …”

“…Recent in vivo studies with rats, ganelles, similar to the condensation of hormones be-treated with the coagulation factor Xa and a mixture fore storage [15]. At present, however, it cannot be ex-of phosphotidylcholine and phosphoditylserine to gencluded that the divergent concepts on the structural erate thrombin, support the view that vWF secreted requirements for routing to storage organelles might via the regulated pathway is released at the apical side be due to the use of different cell types for transfection [35]. By using an electron microscopic analysis of rat experiments with vWF cDNA or variants thereof.…”