Covalent regulation of ULVWF string formation and elongation on endothelial cells under flow conditions

131

137

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...

Section: Discussionmentioning

confidence: 93%

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

Huang

Roth

Heuser

et al. 2009

131

137

“…Self-association has been reported to be covalent and involve unpaired cysteine residues. 12,14 Considering the cluster of potential free thiols in and around the C2 domain, 12,13 we focused on identifying the particular cysteines involved in this region and understanding how they exchange between VWF molecules during lateral self-association.…”

Section: Discussionmentioning

confidence: 99%

“…14 Mature VWF, both purified from plasma and made recombinantly, contains unpaired cysteine thiols 12,[15][16][17] that have been localized to the N-terminal D3 and C-terminal C domains. 12,13,17 Presumably, one or more of these cysteine thiols is involved in VWF self-association.…”

Section: Introductionmentioning

confidence: 99%

“…VWF secreted by sheared endothelial cells forms "strings" that are several cell diameters in length and also bind platelets. 8,12,13 The mechanism of formation of the VWF strings involves cysteine thiol/disulfide exchange, because the formation of the strings is inhibited by thiol alkylation 12 and the string structure is disrupted by the small thiol N-acetylcysteine. 14 Mature VWF, both purified from plasma and made recombinantly, contains unpaired cysteine thiols 12,15-17 that have been localized to the N-terminal D3 and C-terminal C domains.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Lateral self-association of VWF involves the Cys2431-Cys2453 disulfide/dithiol in the C2 domain

Ganderton

Wong

Schroeder

et al. 2011

VWF is a plasma protein that binds platelets to an injured vascular wall during thrombosis. When exposed to the shear forces found in flowing blood, VWF molecules undergo lateral self-association that results in a meshwork of VWF fibers. Fiber formation has been shown to involve thiol/disulfide exchange between VWF molecules. A C-terminal fragment of VWF was expressed in mammalian cells and examined for unpaired cysteine thiols using tandem mass spectrometry (MS). The VWF C2 domain Cys2431-Cys2453 disulfide bond was shown to be reduced in approximately 75% of the molecules. Fragments containing all 3 C domains or just the C2 domain formed monomers, dimers, and higher-order oligomers when expressed in mammalian cells. Mutagenesis studies showed that both the Cys2431-Cys2453 and nearby Cys2451-Cys2468 disulfide bonds were involved in oligomer formation. Our present findings imply that lateral VWF dimers form when a Cys2431 thiolate anion attacks the Cys2431 sulfur atom of the Cys2431-Cys2453 disulfide bond of another VWF molecule, whereas the Cys2451-Cys2468 disulfide/dithiol mediates formation of trimers and higher-order oligomers. These observations provide the basis for exploring defects in lateral VWF association in patients with unexplained hemorrhage or thrombosis. (Blood. 2011;118(19): 5312-5318) IntroductionVWF is a large, multimeric glycoprotein responsible for chaperoning the blood coagulation cofactor factor VIII and tethering platelets to the site of vascular endothelial injury. 1 It is synthesized by vascular endothelial cells and megakaryocytes and circulates as a series of multimers containing variable numbers of 500-kDa dimeric units. Circulating multimers range between 500 and 20 000 kDa in size, and although any size multimer is able to chaperone factor VIII, 2 it is the only the largest sizes that are able to effectively tether platelets. Each subunit contains binding sites for collagen and for platelet glycoproteins Ib and ␣IIb␤3. 1 Multimer size is regulated in the circulation. An excess of high-molecular-weight multimers can cause unwanted thrombosis and is associated with thrombotic thrombocytopenic purpura, 3 whereas a paucity of large multimers is associated with the bleeding disorder VWD. 1 Under normal conditions, the size of VWF multimers is controlled by ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type-1 motifs) proteolysis of the Tyr1605-Met1606 peptide bond in the A2 domain. 4 The tertiary and quaternary structure of VWF is influenced by the shear forces typically found in flowing blood. VWF undergoes a shear-dependent conformational change, 5 and individual molecules can self-associate under both static 6 and shear conditions. 7-9 VWF reversibly transitions from a loosely coiled ball to an elongated structure in response to shear. 5,10,11 Self-association of VWF has been reported in different systems. Soluble VWF perfused over a VWF-coated surface undergoes "homotypic" self-association that facilitates platelet adhesion. 9 In addition, VWF self-associates into ...

“…In line with the aforementioned calculations, these organelles should release strings no longer than approximately 19 m. Our data show that FVIII-expressing endothelial cells release strings that are much longer, with an average length of 340 m ( Figure 7A). This argues in favor of linkage of VWF multimers after release 44,45 or pooling of WPB content during multigranular exocytosis. 28 Moreover, tubular organization of VWF appears dispensable for the formation of UL-VWF string on the surface of endothelial cells.…”

mentioning

confidence: 98%

Factor VIII alters tubular organization and functional properties of von Willebrand factor stored in Weibel-Palade bodies

Bouwens

Mourik

Biggelaar

et al. 2011

In endothelial cells, von Willebrand factor (VWF) multimers are packaged into tubules that direct biogenesis of elongated Weibel-Palade bodies (WPBs). WPB release results in unfurling of VWF tubules and assembly into strings that serve to recruit platelets. By confocal microscopy, we have previously observed a rounded morphology of WPBs in blood outgrowth endothelial cells transduced to express factor VIII (FVIII). Using correlative lightelectron microscopy and tomography, we now demonstrate that FVIII-containing WPBs have disorganized, short VWF tubules. Whereas normal FVIII and FVIII Y1680F interfered with formation of ultralarge VWF multimers, release of the WPBs resulted in VWF strings of equal length as those from nontransduced blood outgrowth endothelial cells. After release, both WPB-derived FVIII and FVIII Y1680F remained bound to VWF strings, which however had largely lost their ability to recruit platelets. Strings from nontransduced cells, however, were capable of simultaneously recruiting exogenous FVIII and platelets. These findings suggest that the interaction of FVIII with VWF during WPB formation is independent of Y1680, is maintained after WPB release in FVIIIcovered VWF strings, and impairs recruitment of platelets. Apparently, intracellular and extracellular assembly of FVIII-VWF complex involves distinct mechanisms, which differ with regard to their implications for platelet binding to released VWF strings. (Blood. 2011; 118(22):5947-5956) IntroductionWeibel-Palade bodies (WPBs) are secretory organelles specific for vascular endothelial cells with a typical elongated shape of 100 to 200 nm in diameter and up to 5 m in length. 1 These organelles are characterized by striations running along the longitudinal axis consisting of condensed von Willebrand factor (VWF). Stimulation with agonists, such as thrombin and epinephrine, releases VWF to arrests bleeding by recruiting blood platelets to sites of vascular perturbation. 2 VWF is synthesized as a prepropolypeptide of 2813 residues that is cleaved into a propeptide (D1-D2) and mature VWF monomer (DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK) of approximately 250 kDa. 3 VWF forms multimers through C-and N-terminal disulfide linkage. [4][5][6] Multimers assemble into long, slightly twisted tubules that determine the typical cigar-shaped appearance of WPBs. [7][8][9] On fusion of WPBs with the plasma membrane, VWF tubules are exposed to physiologic pH. This results in rapid disassembly of VWF tubules, which is presumably triggered by dissociation of propeptide from DЈ-D3 regions at neutral pH. 8,10 The released VWF multimers assemble into extended string-like structures on the surface of endothelial cells onto which platelets adhere. [10][11][12] The tubular organization of VWF is thought to be essential for orderly secretion of long VWF strings without tangling. 13 Besides recruiting platelets to sites of vascular damage, VWF functions as a chaperone for factor VIII (FVIII), a cofactor for activated factor IX in the factor Xase complex. It is thus far uncl...