scholarly journals Structure of Plasmodium falciparum TRAP (thrombospondin-related anonymous protein) A domain highlights distinct features in apicomplexan von Willebrand factor A homologues

2013 ◽  
Vol 450 (3) ◽  
pp. 469-476 ◽  
Author(s):  
Tero Pihlajamaa ◽  
Tommi Kajander ◽  
Juho Knuuti ◽  
Kaisa Horkka ◽  
Amit Sharma ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Von Willebrand Factor ◽  
Metal Ion ◽  
Cell Attachment ◽  
Protein A ◽  
Site Directed Mutagenesis ◽  
Mammalian Host ◽  
Functional Roles ◽  
Von Willebrand ◽  
Willebrand Factor

TRAP (thrombospondin-related anonymous protein), localized in the micronemes and on the surface of sporozoites of the notorious malaria parasite Plasmodium, is a key molecule upon infection of mammalian host hepatocytes and invasion of mosquito salivary glands. TRAP contains two adhesive domains responsible for host cell recognition and invasion, and is known to be essential for infectivity. In the present paper, we report high-resolution crystal structures of the A domain of Plasmodium falciparum TRAP with and without bound Mg2+. The structure reveals a vWA (von Willebrand factor A)-like fold and a functional MIDAS (metal-ion-dependent adhesion site), as well as a potential heparan sulfate-binding site. Site-directed mutagenesis and cell-attachment assays were used to investigate the functional roles of the surface epitopes discovered. The reported structures are the first determined for a complete vWA domain of parasitic origin, highlighting unique features among homologous domains from other proteins characterized hitherto. Some of these are conserved among Plasmodiae exclusively, whereas others may be common to apicomplexan organisms in general.

Glycoprotein Ib Can Mediate Endothelial Cell Attachment to a von Willebrand Factor Substratum

1995 ◽  
Vol 73 (02) ◽  
pp. 309-317 ◽  
Author(s):  
Dorothy A Beacham ◽  
Miguel A Cruz ◽  
Robert I Handin
Keyword(s):  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Cell Attachment ◽  
Umbilical Vein ◽  
Single Amino Acid ◽  
Cell Surface Expression ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryIntroduction of single amino acid substitutions into the C-terminal Arg-Gly-Asp-Ser (RGDS) site of von Willebrand Factor, referred to as RGD mutant vWF, selectively abrogated vWF binding to platelet glycoprotein IIb/IIIa (GpIIb/IIIa, αIIbβ3 and abolished human umbilical vein endothelial cell (HUVEC) spreading, but not attachment, to RGD mutant vWF (Beacham, D. A., Wise, R. J., Turci, S. M. and Handin, R. I. 1992. J. Biol. Chem. 167, 3409-3415). These results suggested that in addition to the vitronectin receptor (VNR, αvβ3), a second endothelial membrane glycoprotein can mediate HUVEC adhesion to vWF. HUVEC attachment to wild-type (WT) and RGD-mutant vWF was reduced by two proteins known to block the vWF-platelet glycoprotein Ib/IX (GpIb/IX) interaction, the monoclonal antibody AS-7 and the recombinant polypeptide, vWF-A1. The addition of cytochalasin B or DNase I to disrupt potential GPIbα-cytoskeletal interactions enhanced the immunoprecipitation of endothelial GPIbα, caused HUVEC to round up, and increased HUVEC adhesion to RGD mutant vWF. These results indicate that while the VNR is the primary adhesion receptor for vWF, endothelial GPIbα can mediate HUVEC attachment to vWF. GpIb-dependent attachment could contribute to HUVEC adhesion under conditions when cell surface expression of the VNR is downregulated, and VNR-dependent adhesion is reduced.

scholarly journals Adhesive properties of the beta 3 integrins: comparison of GP IIb-IIIa and the vitronectin receptor individually expressed in human melanoma cells.

1991 ◽  
Vol 113 (2) ◽  
pp. 451-461 ◽  
Author(s):  
N Kieffer ◽  
L A Fitzgerald ◽  
D Wolf ◽  
D A Cheresh ◽  
D R Phillips
Keyword(s):  
Von Willebrand Factor ◽  
Cell Attachment ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Adhesive Properties ◽  
Vitronectin Receptor ◽  
Hel Cells ◽  
Alpha V Beta 3 ◽  
Von Willebrand ◽  
Willebrand Factor

Glycoprotein IIb-IIIa (alpha IIb beta 3) and the vitronectin receptor (alpha v beta 3), two integrins that share the common beta 3 subunit, have been reported to function as promiscuous receptors for the RGD-containing adhesive proteins fibrinogen, vitronectin, fibronectin, von Willebrand factor, and thrombospondin. The present study was designed to establish a cell system for the expression of either GP IIb-IIIa or the vitronectin receptor in an otherwise identical cellular environment and to compare the adhesive properties of these two integrins with those of native GP IIb-IIIa and the vitronectin receptor constitutively expressed in HEL cells or platelets. M21 human melanoma cells lack GP IIb-IIIa and use the vitronectin receptor to attach to vitronectin, fibrinogen, fibronectin, and von Willebrand factor. To study the functional properties of GP IIb-IIIa in these cells, we transfected GP IIb into M21-L cells, a variant of M21 cells (Cheresh, D.A., and R.C. Spiro. 1987. J. Biol. Chem. 262:17703-17711), which lack the expression of functional alpha v and are therefore unable to attach to vitronectin, fibrinogen, and von Willebrand factor. Transfectants expressing GP IIb were isolated by immunomagnetic beads and surface expression of the GP IIb-IIIa complex was documented by FACS analysis and immunoprecipitation experiments performed with 125I-labeled M21-L/GP IIb cells. Comparative functional studies demonstrated that GP IIb-IIIa expressed in M21-L/GPIIb cells as well as native GP IIb-IIIa constitutively expressed in HEL-5J20 cells (an HEL variant lacking alpha v beta 3) mediated cell attachment to immobilized fibrinogen, but not to vitronectin or von Willebrand factor, whereas the vitronectin receptor expressed in M21 cells and HEL-AD1 cells (an HEL variant expressing alpha v beta 3) mediated cell attachment to fibrinogen, vitronectin, and von Willebrand factor. Similarly, PGl2-treated resting platelets attached to immobilized fibrinogen but not to vitronectin or von Willebrand factor, and this attachment could be inhibited by mAb A2A9 (directed against a functional site on the GP IIb-IIIa complex). However, in contrast to platelets, which adhered to vitronectin and von Willebrand factor after stimulation by thrombin or PMA, activation of the protein kinase C pathway in M21-L/GP IIb or HEL cells did not induce cell adhesion to vitronectin or von Willebrand factor.(ABSTRACT TRUNCATED AT 400 WORDS)

PRODUCTION OF ANTIBODIES TO HUMAN VON WILLEBRAND FACTOR IN LAYING HENS. ISOLATION OF IMMUNOGLOBULINS AND APPLICATIONS TO THE DETECTION OF MOLECULAR DEFECTS OF VON WILLEBRAND FACTOR

1987 ◽  
Author(s):  
F Toti ◽  
A Stierlé ◽  
M L Wiesel ◽  
A Schwartz ◽  
J M Freyssinet ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Laying Hens ◽  
Protein A ◽  
Von Willebrand Disease ◽  
Primary Hemostasis ◽  
Normal Platelet ◽  
Electrophoretic Patterns ◽  
Egg Yolks ◽  
Von Willebrand ◽  
Willebrand Factor

Von Willebrand disease (vWD) is an inherited disorder of primary hemostasis caused by deficiency or structural abnormalities of von Willebrand factor (vWF). VWF circulates in plasma and is also present in platelets. Plasma vWF, the carrier protein for factor VIII, is a large multimeric glycoprotein composed of identical subunits linked by disulfide bridges. Plasma and platelet vWF display distinct multimeric electrophoretic patterns. The different vWD subtypes can be classified either by the determination of vWFantigen (vWFíAg) and/or by multimer distribution. Antibodies to human vWF were raised in laying hens by intramuscular injections of purified human vWF. Immunoglobulins were isolated from egg yolks by selective polyethylene glycol and ammonium sulfate precipitations. These antibodies appeared to be monospecific, as they did not react with the plasma proteins of a patient with severe vWD. The pullets received weekly 50 μg vWF for 4 weeks and then had monthly injections. The antibodies occurred as early as the third injection, the yield being 300 to 500 mg of immunoglobulin per week (6-7 eggs). The titre could be constant over periods greater than 1 year. These immunoglobulins to vWF were tested in vWFíAg electroimmunoassays and for the multimer analysis of plasma and platelet vWF by electrophoresis and immunoblotting techniques. In no case could a difference be detected between assays performed with rabbit monospecific antiserum or with yolk immunoglobulins to human vWF. Ten to 12 multimers could be revealed for normal plasma vWF and up to 12 to 14 bands for normal platelet vWF (1.7% agarose). In the case of vWD, the electrophoresis patterns were identical with both antibodies. Thus, antibodies to vWF raised in laying hens are a suitable tool to detect and to characterize vWD. Although they do not interact with protein A, yolk antibodies are certainly advantageous to produce, as they do not contain IgM or IgA. Immunoglobulin fractions can contain up to 10 % of specific antibodies. Since they are available in larger quantities and are easy to isolate, larger homogeneous batches of antibodies can be obtained. This method may easily be applied to develop antibodies to a variety of antigens.

von Willebrand Factor Protects Ca2+-Dependent Structure of the Factor VIII Light Chain.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1008-1008 ◽  
Author(s):  
Masahiro Takeyama ◽  
Midori Shima ◽  
Keiji Nogami ◽  
Masahiro Okuda ◽  
Yoshihiko Sakurai ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Divalent Cation ◽  
Light Chain ◽  
Metal Ion ◽  
Exchange Resin ◽  
Cation Exchange Resin ◽  
The Other ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Factor VIII (FVIII) cofactor function is dependent on the presence of divalent cation, since its active form is metal-linked heterotrimer as well as factor V (FV). The activity of FVIII (FVIII:C) and FV in normal plasma were completely and selectively inactivated by the treatment with divalent cation exchange resin, imidinoacetate. However, FVIII antigen (FVIII:Ag) was preserved, suggesting that deprivation of FVIII:C by the resin was not due to absorption of FVIII but due to inactivation. Both FVIII:Ag and FVIII:C of recombinant FVIII were decreased by treatment with the resin. However, FVIII:Ag but not FVIII:C, was preserved in FVIII and von Willebrand factor complex (FVIII/VWF). Sandwich ELISA using anti-A3 and anti-A2 monoclonal antibodies revealed that association with heavy and the light chains were impaired in the resin-treated FVIII. These results suggested that FVIII was inactivated by deprivation of the metal ion by the resin and that VWF protected cation-dependent FVIII structure. Therefore, we tested if the resin deprives the metal ion such as Ca2+ from FVIII or FVIII/VWF. [Ca2+] in FVIII preparation was decreased from 1.30 to 0.07 mM by the resin. Furthermore, [Ca2+] was decreased from 0.39 to 0.01 mM in FVIII/VWF preparation. [Ca2+] was recovered completely by elution with 1 N HCl from the resins used in both preparations. FVIII:C of the resin-treated FVIII/VWF was partially recovered by addition of Ca2+, whilst FVIII:C of the resin-treated FVIII was not recovered. When FVIII was treated with the resin after addition of [Ca2+], the inactivation of FVIII was dose-dependently inhibited by ~20 % (at [Ca2+]: ~75 mM). On the other hand, the inactivation of FVIII was inhibited by ~60 % (at [Ca2+]: ~25 mM) in FVIII/VWF preparation. Present results demonstrated that FVIII was selectively inactivated by the cation exchange resin due to deprivation of Ca2+. Kinetic experiments by surface plasmon resonance using BIAcore demonstrated that the resin-treated FVIII as well as treated C2 didn’t interact with phospholipid and VWF. Furthermore, immunoblot analysis using the resin-treated FVIII revealed that anti-A2 monoclonal antibody reacted with the heavy chain, whilst anti-A3 and anti-C2 antibodies failed to react with the light chain. On the other hand, these antibodies reacted with the light chain in the experiment using FVIII/VWF, indicating that VWF protects antigenic conformation of the FVIII light chain. Present findings suggest another protection mechanism of VWF on FVIII through stabilization of Ca2+-dependent structure of the FVIII light chain.

Aberrant dimerization of von Willebrand factor as the result of mutations in the carboxy-terminal region: identification of 3 mutations in members of 3 different families with type 2A (phenotype IID) von Willebrand disease

Blood ◽  
2001 ◽  
Vol 98 (3) ◽  
pp. 674-680 ◽  
Author(s):  
M. Said Enayat ◽  
Andrea M. Guilliatt ◽  
Gurcharan K. Surdhar ◽  
P. Vincent Jenkins ◽  
K. John Pasi ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Site Directed Mutagenesis ◽  
Direct Dna Sequencing ◽  
Mismatch Detection ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The 3′ end of the VWF gene was screened in the affected members of 3 different families with type 2A (phenotype IID) von Willebrand disease (vWD). Exons 49 to 52 of the VWF gene were amplified and screened for mutations by chemical cleavage mismatch detection. Mismatched bands were detected in exon 52 of 2 patients and in exon 51 of a third patient. Using direct DNA sequencing, a heterozygous G8562A transition leading to a Cys2008Tyr substitution was found in all the patients in family 1, and a T8561A transversion leading to a Cys2008Ser substitution was found in both patients from family 2. In a patient from a third family, an 8-base deletion from nucleotide 8437 to 8444 was identified in exon 51. The 2 mutations in exon 52 were reproduced by in vitro site-directed mutagenesis of full-length von Willebrand factor (vWF) cDNA and transiently expressed in COS-7 cells. The corresponding recombinant VWFs for these 2 mutations exhibited the typical aberrant vWF:Ag multimer pattern seen in the plasma of the patients. These 3 mutations demonstrate the importance of other carboxy-terminal cysteines in addition to the reported Cys2010 residue, in the normal dimerization of vWF, and their essential role in the assembly of normal multimeric vWF.

scholarly journals The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of  2  subunits is key to trafficking voltage-gated Ca2+ channels

2005 ◽  
Vol 102 (32) ◽  
pp. 11230-11235 ◽  
Author(s):  
C. Canti ◽  
M. Nieto-Rostro ◽  
I. Foucault ◽  
F. Heblich ◽  
J. Wratten ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Metal Ion ◽  
Voltage Gated ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ca2 Channels ◽  
Adhesion Site

scholarly journals Role of the glycoprotein Ib-binding A1 repeat and the RGD sequence in platelet adhesion to human recombinant von Willebrand factor

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 1035-1042 ◽  
Author(s):  
H Lankhof ◽  
YP Wu ◽  
T Vink ◽  
ME Schiphorst ◽  
HG Zerwes ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Static Condition ◽  
Site Directed Mutagenesis ◽  
Flow Conditions ◽  
Rgd Sequence ◽  
Absolute Requirement ◽  
Platelet Spreading ◽  
Von Willebrand ◽  
Willebrand Factor

To assess the relative importance of the glycoprotein (GP) Ib binding domain and the RGDS binding site in platelet adhesion to isolated von Willebrand factor (vWF) and to collagen preincubated with vWF, we deleted the A1 domain yielding delta A1-vWF and introduced an aspartate- to-glycine substitution in the RGDS sequence by site-directed mutagenesis (RGGS-vWF). Recombinant delta A1-vWF and RGGS-vWF, purified from transfected baby hamster kidney cells, were compared with recombinant wild-type vWF (WT-vWF) in platelet adhesion under static and flow conditions. Purified mutants were coated on glass or on a collagen type III surface and exposed to circulating blood in a perfusion system. Platelet adhesion under static condition, under flow conditions, and in vWF-dependent adhesion to collagen has an absolute requirement for GPIb-vWF interaction. The GPIIb/IIIa-vWF interaction is required for adhesion to coated vWF under flow conditions. Under static condition and vWF-dependent adhesion to collagen, platelet adhesion to RGGS-vWF is similar as to WT-vWF, but platelet spreading and aggregation are abolished.

scholarly journals Retention of glycoprotein Ib/IX receptors on external surfaces of thrombin-activated platelets in suspension

Blood ◽  
1995 ◽  
Vol 86 (9) ◽  
pp. 3468-3478 ◽  
Author(s):  
JG White ◽  
MD Krumwiede ◽  
D Cocking-Johnson ◽  
GH Rao ◽  
G Escolar
Keyword(s):  
Von Willebrand Factor ◽  
Protein A ◽  
Glycoprotein Ib ◽  
Platelet Surface ◽  
Thin Sections ◽  
Activated Platelets ◽  
Minute Period ◽  
Study Results ◽  
Von Willebrand ◽  
Willebrand Factor

The present study has evaluated the hypothesis stating that glycoprotein (GP) Ib/IX, the receptor for von Willebrand factor (vWF), is downregulated and cleared from exposed surfaces to channels of the open canalicular system (OCS) on platelets activated by thrombin in suspension. Cryosections of resting and thrombin-activated platelets fixed at intervals of 1 to 30 minutes after stimulation by thrombin and stained with antiglycocalicin antibody and protein A gold showed no decrease in the density of GPIb/IX receptors on the platelet surface or increase on linings of the OCS at any interval after stimulation by thrombin. Thin sections of platelets exposed to thrombin in suspension followed by settling onto a plastic chamber for intervals of 1 to 30 minutes revealed retention of GPIb/IX receptors on exposed surfaces detected by vWF, anti-vWF, and protein A gold throughout the 30-minute period of study. Results of this investigation indicate that GPIb/IX receptors remain on the surface of platelets activated by thrombin in suspension, are not cleared to the OCS, and retain the ability to bind vWF for at least 30 minutes.

Selective inactivation of Von Willebrand factor binding to glycoprotein IIb/IIIa and to inhibitor monoclonal antibody 9 by site-directed mutagenesis

2001 ◽  
Vol 2 (3) ◽  
pp. 180-187 ◽  
Author(s):  
Anne-Lise Jumilly ◽  
Agnès Veyradier ◽  
Anne-Sophie Ribba ◽  
Dominique Meyer ◽  
Jean-Pierre Girma
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Factor Binding ◽  
Von Willebrand ◽  
Willebrand Factor
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