Integrin αvβ3 on human endothelial cells binds von Willebrand factor strings under fluid shear stress

Abstract

Acutely secreted von Willebrand factor (VWF) multimers adhere to endothelial cells, support platelet adhesion, and may induce microvascular thrombosis. Immunofluorescence microscopy of live human umbilical vein endothelial cells showed that VWF multimers rapidly formed strings several hundred micrometers long on the cell surface after stimulation with histamine. Unexpectedly, only a subset of VWF strings supported platelet binding, which depended on platelet glycoprotein Ib. Electron microscopy showed that VWF strings often consisted of bundles and networks of VWF multimers, and each string was tethered to the cell surface by a limited number of sites. Several approaches implicated P-selectin and integrin ␣ v ␤ 3 in anchoring VWF strings. An RGDS peptide or a function-blocking antibody to integrin ␣ v ␤ 3 reduced the number of VWF strings formed. In addition, integrin ␣ v decorated the VWF strings by immunofluorescence microscopy. Furthermore, lentiviral transduction of shRNA against the ␣ v subunit reduced the expression of cellsurface integrin ␣ v ␤ 3 and impaired the ability of endothelial cells to retain VWF strings. Soluble P-selectin reduced the number of platelet-decorated VWF strings in the absence of Ca 2؉ and Mg 2؉ but had no effect in the presence of these cations. These results indicate that VWF strings bind specifically to integrin ␣ v ␤ 3 on human endothelial cells. Introductionvon Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays an important role in hemostasis and thrombosis, primarily by interacting with platelet adhesion receptors. 1 VWF is synthesized by vascular endothelial cells and megakaryocytes, and so-called ultralarge (UL) VWF multimers are stored in endothelial Weibel-Palade bodies and platelet ␣-granules for later secretion. 2,3 After secretion, some ULVWF remains on the cell surface as very long strings that become decorated with platelets. Eventually, ULVWF multimers are converted into smaller, less thrombogenic fragments by the metalloprotease ADAMTS13, which cleaves the Tyr1605-Met1606 bond in the central A2 domain of VWF. 4,5 Unlike VWF in solution, which interacts weakly with platelets, surface-immobilized VWF strings spontaneously mediate platelet adhesion under fluid shear stress in vitro or in vivo. For example, platelets bind to VWF on cultured human umbilical vein endothelial cells (HUVECs) to form "beads-on-a-string" structures under laminar flow, and these structures are attached to the cell surface at relatively few discrete sites and are disrupted by plasma or recombinant ADAMTS13. [5][6][7] Studies using intravital microscopy in mice also found that platelets adhere to VWF strings on the endothelium of mesenteric venules within seconds after endothelial stimulation, and ADAMTS13 deficiency prolongs these VWFmediated platelet-endothelial cell interactions. 8,9 The molecules responsible for the attachment of VWF strings to endothelial cells have not been identified conclusively and may differ between species. During VWF biosynthesis, the DЈD3 region...

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

“…Previous findings from our laboratory (see supplemental Figure 4C in reference 29) and others 31,33,34 have shown that unstimulated cells only rarely form VWF strings under flow. This is consistent with our proposed role for basal VWF, again suggesting that, although being of similar high multimeric states (except for the dimer band), basal and regulated VWF play different functional roles.…”

In vitro microvessels for the study of angiogenesis and thrombosis

“…In healthy scenarios, the endothelium provides a nonadhesive and antithrombotic interface for the transport of blood through the vessel whereas during inflammation, the endothelium promotes the formation of the thrombi via local expression and secretion of adhesive proteins (33). The interaction of blood with endothelium has been studied extensively in vitro in planar formats that lack appropriate vessel geometries or a 3D matrix (34,35). We examined the interaction of whole blood and the endothelium in μVNs in a quiescent state (after 7 to 14 d in our standard microfluidic culture; see Methods and SI Methods) and a stimulated state induced by brief exposure to phorbol-12-myristate-13-acetate (PMA).…”

“…S7 D and E). Such structures have not been reported in planar cultures in which the flow is uniaxial and parallel to the endothelium (35), or in mouse models, where details of the progression of thrombosis have been difficult to resolve (40).…”

High-density lipoprotein modulates thrombosis by preventing von Willebrand factor self-association and subsequent platelet adhesion

“…6 Self-association also allows VWF to form strands long enough to reach above the endothelial boundary layer under flow to bind platelets, which may explain the findings of a study showing that platelets bound only a subset of endothelial VWF strings identified with a VWF antibody. 14 On the basis of our observation that highly purified VWF was lost from solution by first binding to the wall of the container and then by There is an Inside Blood Commentary on this article in this issue.…”