scholarly journals Identification of platelet proteins that bind alloantibodies and autoantibodies

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1240-1245 ◽  
Author(s):  
DV Devine ◽  
WF Rosse
Keyword(s):  
Membrane Proteins ◽  
Western Blot ◽  
Hla Antigens ◽  
Cell Types ◽  
Igg Antibody ◽  
Hla Antigen ◽  
Molecular Weights ◽  
Plasma Factors ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We have used the techniques of radioimmunoprecipitation (RIP) and Western blot to identify the membrane proteins that bind certain alloantibodies. Anti-PlA1 sera precipitated two bands, corresponding to platelet glycoproteins IIb and III, whether or not calcium was present during the procedure. By Western blot, this antibody bound only glycoprotein III. Anti-PlA1 serum does not precipitate proteins from the platelets of a patient with Glanzmann's thrombasthenia. Two monoclonal antibodies reacting with lymphocyte HLA antigens, as well as sera from highly allosensitized patients, precipitated bands of 38,500 and 13,500 daltons. These bands correspond to the molecular weights of the two subunits of the HLA antigen, as it has been described for other cell types. The patients' sera also precipitated a protein of 72,000 daltons from some platelets. The sera of two patients with quinidine- induced thrombocytopenia precipitated a 138,000-dalton band (glycoprotein Ib-alpha) in the presence of quinidine. The purified IgG antibody from one patient did not require other plasma factors to bind to platelets in the presence of quinidine, while purified antibody from a second patient required plasma factors other than, or in addition to von Willebrand factor. Although several sera from patients with idiopathic thrombocytopenic purpura (ITP) were tested, only one precipitated membrane proteins by the RIP method; this serum identified binding proteins corresponding to glycoproteins IIb and III.

scholarly journals Identification of platelet proteins that bind alloantibodies and autoantibodies

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1240-1245
Author(s):  
DV Devine ◽  
WF Rosse
Keyword(s):  
Membrane Proteins ◽  
Western Blot ◽  
Hla Antigens ◽  
Cell Types ◽  
Igg Antibody ◽  
Hla Antigen ◽  
Molecular Weights ◽  
Plasma Factors ◽  
Von Willebrand ◽  
Willebrand Factor

We have used the techniques of radioimmunoprecipitation (RIP) and Western blot to identify the membrane proteins that bind certain alloantibodies. Anti-PlA1 sera precipitated two bands, corresponding to platelet glycoproteins IIb and III, whether or not calcium was present during the procedure. By Western blot, this antibody bound only glycoprotein III. Anti-PlA1 serum does not precipitate proteins from the platelets of a patient with Glanzmann's thrombasthenia. Two monoclonal antibodies reacting with lymphocyte HLA antigens, as well as sera from highly allosensitized patients, precipitated bands of 38,500 and 13,500 daltons. These bands correspond to the molecular weights of the two subunits of the HLA antigen, as it has been described for other cell types. The patients' sera also precipitated a protein of 72,000 daltons from some platelets. The sera of two patients with quinidine- induced thrombocytopenia precipitated a 138,000-dalton band (glycoprotein Ib-alpha) in the presence of quinidine. The purified IgG antibody from one patient did not require other plasma factors to bind to platelets in the presence of quinidine, while purified antibody from a second patient required plasma factors other than, or in addition to von Willebrand factor. Although several sera from patients with idiopathic thrombocytopenic purpura (ITP) were tested, only one precipitated membrane proteins by the RIP method; this serum identified binding proteins corresponding to glycoproteins IIb and III.

Processing and Characterization of Recombinant von Willebrand Factor Expressed in Different Cell Types Using a Vaccinia Virus Vector

1992 ◽  
Vol 67 (01) ◽  
pp. 154-160 ◽  
Author(s):  
P Meulien ◽  
M Nishino ◽  
C Mazurier ◽  
K Dott ◽  
G Piétu ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Von Willebrand Factor ◽  
Human Fibroblasts ◽  
Active Form ◽  
Cell Types ◽  
Biologically Active ◽  
L Cells ◽  
Von Willebrand ◽  
Different Cell Types ◽  
Willebrand Factor

SummaryThe cloning of the cDNA encoding von Willebrand factor (vWF) has revealed that it is synthesized as a large precursor (pre-pro-vWF) molecule and it is now clear that the prosequence or vWAgll is responsible for the intracellular multimerization of vWF. We have cloned the complete vWF cDNA and expressed it using a recombinant vaccinia virus as vector. We have characterized the structure and function of the recombinant vWF (rvWF) secreted from five different cell types: baby hamster kidney (BHK), Chinese hamster ovary (CHO), human fibroblasts (143B), mouse fibroblasts (L) and primary embryonic chicken cells. Forty-eight hours after infection, the quantity of vWF antigen found in the cell supernatant varied from 3 to 12 U/dl depending on the cell type. By SDS-agarose gel electrophoresis, the percentage of high molecular weight forms of vWF varied from 39 to 49% relative to normal plasma for BHK, CHO, 143B and chicken cells but was less than 10% for L cells. In all cell types, the two anodic subbands of each multimer were missing. The two cathodic subbands were easily detected only in BHK and L cells. By SDS-PAGE of reduced samples, pro-vWF was present in similar quantity to the fully processed vWF subunit in L cells, present in moderate amounts in BHK and CHO and in very low amounts in 143B and chicken cells. rvWF from all cells bound to collagen and to platelets in the presence of ristocetin, the latter showing a high correlation between binding efficiency and degree of multimerization. rvWF from all cells was also shown to bind to purified FVIII and in this case binding appeared to be independent of the degree of multimerization. We conclude that whereas vWF is naturally synthesized only by endothelial cells and megakaryocytes, it can be expressed in a biologically active form from various other cell types.

scholarly journals von Willebrand Factor Activity in Plasmin Digests of Factor VIII

1977 ◽  
Author(s):  
J. A. Guisasola ◽  
C. Cockburn ◽  
R. M. Hardisty
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Human Factor ◽  
Factor Activity ◽  
Group Iii ◽  
Molecular Weights ◽  
Human Factor Viii ◽  
Group Ii ◽  
Von Willebrand ◽  
Willebrand Factor

Purified human factor VIII was incubated for up to 24 hours with plasmin, and the activity of the breakdown products studied at intervals. Factor VIII coagulant activity was lost within the first hour, but von Willebrand factor activity (FVIIIR:WF) was retained for two hours, and then declined slowly during the subsequent incubation. Analysis of the 24-hour breakdown products by immuno-electrophoresis, sepharose 4B chromatography and SDS Polyacrylamide electrophoresis revealed three main groups of fragments recognised by rabbit anti-human factor VIII anti-serum, and having molecular weights in the following ranges: Group 1 300,000=500,000; Group II, 150–200,000; Group III, 100,000. FVIIIR:WF activity, which was found only in Group II, appeared to be associated with glycopeptide(s) of up to 155,000 daltons.

Molecular weights of antihaemophilic factor and von Willebrand factor proteins in human plasma

Nature ◽  
1976 ◽  
Vol 263 (5578) ◽  
pp. 612-613 ◽  
Author(s):  
JACK NEWMAN ◽  
ROBERT B. HARRIS ◽  
ALAN J. JOHNSON
Keyword(s):  
Human Plasma ◽  
Von Willebrand Factor ◽  
Molecular Weights ◽  
Von Willebrand ◽  
Willebrand Factor

Elevated Procoagulant Microparticles Expressing Endothelial and Platelet Markers in Essential Thrombocythemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2793-2793
Author(s):  
Marijke Trappenburg ◽  
Muriel van Schilfgaarde ◽  
Marina Marchetti ◽  
Henri Spronk ◽  
Hugo ten Cate ◽  
...  
Keyword(s):  
Essential Thrombocythemia ◽  
Thrombin Generation ◽  
Cell Types ◽  
Endothelial Activation ◽  
Peak Height ◽  
Cellular Origin ◽  
Thrombin Generation Assay ◽  
Von Willebrand ◽  
Design And Methods ◽  
Willebrand Factor

Abstract Background: Most cell types, including blood - and vascular cells, produce microparticles (MPs) upon activation. Since cellular MPs are known to be elevated in thromboembolic diseases, we hypothesized a role for MPs in the pathogenesis of thrombosis in Essential Thrombocythemia (ET). Design and methods: In plasma samples from 21 ET patients and 10 healthy subjects, the levels and the cellular origin of MPs were determined by flowcytometric analysis, while the MP-associated procoagulant activity was measured by the thrombin generation assay. Results: ET patients had significantly higher numbers of circulating AnnexinV-positive MPs than controls (median 4500 vs 2500×106 events/L; p=0.039), including significantly higher number of MPs positive for the platelet marker CD61 (median 4000 vs 2400×106/L; p=0.043) the endothelial marker CD62E (median 875 vs 14×106/L; p=0.009), and for Tissue Factor (median 1.8 vs 0.9×106/L; p=0.036). CD62E was co-expressed with the platelet marker CD41 on MPs, suggesting a bilineal origin of such MPs, that were observed only in patients with risk factors for thrombosis. ET patients had higher plasma mature von Willebrand factor (vWF) levels (median 50 vs 35 nM, p=0.045) but similar propeptide levels (median 7 vs 5 nM, p=0,07) compared to controls, indicating chronic endothelial activation. In thrombin generation analyses, MP rich plasma from ET patients had a shorter lag time (9.7 min, 95%CI: 8.7–10.7 versus 15.9 min, 95%CI: 10.9–20.9, p=0.001) and higher peak height (215 nM, 95%CI: 189–241 versus 142 nM, 95%CI: 87–189, p=0.038) than from controls. Peak height correlated significantly with the total number of MPs (R=0.634, p<0.001). Conclusions: ET patients showed higher number of circulating MPs with platelet and endothelial markers, suggesting ongoing platelet and endothelial activation. This is confirmed by an increased mature vWF level and an abnormal mature vWF/propeptide ratio, and a hypercoagulable state reflected in thrombin generation. These findings suggest a role for MPs in thrombosis in ET.

scholarly journals Cleavage of human von Willebrand factor by porcine pancreatic elastase

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 673-681
Author(s):  
MA Howard ◽  
T Greco ◽  
M Coghlan
Keyword(s):  
Von Willebrand Factor ◽  
Structural Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Pancreatic Elastase ◽  
Molecular Weights ◽  
Sds Page ◽  
Porcine Pancreatic Elastase ◽  
Cofactor Activity ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (vWF) was purified from pooled normal plasma, radiolabeled with iodine then cleaved by porcine pancreatic elastase. Cleavage was monitored by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), SDS-agarose electrophoresis, crossed immuno- electrophoresis, ristocetin and botrocetin cofactor activities, and ristocetin and botrocetin induced binding to fixed washed platelets. Cleavage of vWF by porcine elastase was dependent on both the concentration of porcine elastase and period of incubation. Incubation of vWF with concentrations of porcine elastase greater than 20 U/mL for 30 minutes resulted in loss of the 240 Kd vWF subunit and a corresponding loss of the high molecular weight multimers and formation of fragments with molecular weights on reduced SDS-PAGE of 150, 125, 115 Kd and minor bands at 44, 28, and 24 Kd and a band at 20 Kd, which increased in intensity as either the concentration of porcine elastase or the period of incubation increased. More than 50% of the ristocetin cofactor activity was lost during a five-minute incubation of vWF with 0.56 U/mL porcine elastase when no detectable structural changes in the vWF molecule had occurred. Botrocetin cofactor activity was more resistant. Similarly botrocetin induced vWF binding to platelets was retained by a fraction produced by digestion of vWF with porcine elastase, which contained predominantly a 20 Kd fragment of vWF. This fragment retained insignificant amounts of ristocetin related functions and therefore represents a useful piece of the vWF molecule for further exploration of the site involved in botrocetin induced binding to platelets.

The N-Glycans Modulate the Binding of ADAMTS13 with von Willebrand Factor.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2710-2710
Author(s):  
Wenhua Zhou ◽  
Han-Mou Tsai
Keyword(s):  
Von Willebrand Factor ◽  
Chinese Hamster ◽  
Complex Type ◽  
Molecular Weights ◽  
Igg Binding ◽  
Severe Deficiency ◽  
Pngase F ◽  
High Mannose ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract ADAMTS13, a circulating multidomain protease consisting a metalloprotease (MP), a disintegrin, a thrombospondin type 1 repeat [TSR], a cysteine rich region, a spacer, 7 additional TSR and 2 CUB domains, is critical for maintaining the homeostasis of von Willebrand factor (VWF). Severe deficiency of the enzyme can lead to VWF-platelet thrombosis in the microcirculation characteristic of thrombotic thrombocytopenic purpura (TTP). Sequence analysis predicts that ADAMTS13 contains 2 and 6 likely N-glycosylation sites respectively at or near the MP and spacer domains, and 2 possible sites in the CUB domains. To investigate the N-glycosylation status and the role of N-glycans in the function of ADAMTS13, we digested plasma derived ADAMTS13 and recombinant ADAMTS13 or its truncated variants with peptide-N-glycosidase F (PNGase F) or endoglycosidase H. SDS PAGE analysis showed that digestion with PNGase F, but not with endo H, decreased the molecular weights of plasma derived ADAMTS13 as well as recombinant ADAMTS13 proteins, including AD7 (residues 1-1427, full-length), AD5 (residues 1-745, MP-TSR#1), AD2 (residues 1-448, MP-TSR#1), AD8 (residues 556-748, spacer-TSR#2) and AD13 (residues 1016-1427, TSR#8-CUB#2), indicating that the MP, spacer and CUB domains were all N-glycosylated as predicted, and contained complex type carbohydrate chains. Enzyme linked immunoassay showed that removal of the N-glycans did not affect the binding of proteases to immobilized VWF multimers or VWF73 peptide. Removal of the N-glycans modestly affected the protease-TTP IgG binding, increasing the Kd by 190% for AD7 and by 50% for AD5. Expression experiments in Lec1 mutant Chinese hamster ovary (CHO) cells with defective GlcNAc-TI activity due to mutations in the Mgat1 gene showed that the resulting high-mannose type AD7 and AD5 variant proteases were secreted normally, showed no difference in VWF cleaving and TTP IgG binding activities, but exhibited increased binding to VWF multimers or VWF73 peptide. In summary, ADAMTS13 are N-glycosylated with complex type oligosaccharides at the MP, spacer and CUB domains. Intracellular conversion from high mannose to complex type glycans modulates the binding of the protease to VWF, suggesting that this process may help prevent depletion of the protease under conditions of high VWF levels in the circulation.

scholarly journals Selective and Signal-dependent Recruitment of Membrane Proteins to Secretory Granules Formed by Heterologously Expressed von Willebrand Factor

2002 ◽  
Vol 13 (5) ◽  
pp. 1582-1593 ◽  
Author(s):  
Anastasia D. Blagoveshchenskaya ◽  
Matthew J. Hannah ◽  
Simon Allen ◽  
Daniel F. Cutler
Keyword(s):  
Endothelial Cells ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Von Willebrand Factor ◽  
De Novo ◽  
Secretory Granules ◽  
Synaptotagmin I ◽  
Organelle Membrane ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (vWF) is a large, multimeric protein secreted by endothelial cells and involved in hemostasis. When expressed in AtT-20 cells, vWF leads to the de novo formation of cigar-shaped organelles similar in appearance to the Weibel-Palade bodies of endothelial cells in which vWF is normally stored before regulated secretion. The membranes of this vWF-induced organelle, termed the pseudogranule, are uncharacterized. We have examined the ability of these pseudogranules, which we show are secretagogue responsive, to recruit membrane proteins. Coexpression experiments show that the Weibel-Palade body proteins P-selectin and CD63, as well as the secretory organelle membrane proteins vesicle-associated membrane protein-2 and synaptotagmin I are diverted away from the endogenous adrenocorticotropic hormone-containing secretory granules to the vWF-containing pseudogranules. However, transferrin receptor, lysosomal-associated membrane protein 1, and sialyl transferase are not recruited. The recruitment of P-selectin is dependent on a tyrosine-based motif within its cytoplasmic domain. Our data show that vWF pseudogranules specifically recruit a subset of membrane proteins, and that in a process explicitly driven by the pseudogranule content (i.e., vWF), the active recruitment of at least one component of the pseudogranule membrane (i.e., P-selectin) is dependent on residues of P-selectin that are cytosolic and therefore unable to directly interact with vWF.

scholarly journals The role of the 5′-flanking region in the cell-specific transcription of the human von Willebrand factor gene

1993 ◽  
Vol 293 (3) ◽  
pp. 641-648 ◽  
Author(s):  
V Ferreira ◽  
Z Assouline ◽  
J L Schwachtgen ◽  
B R Bahnak ◽  
D Meyer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Thymidine Kinase ◽  
Von Willebrand Factor ◽  
Transcriptional Activity ◽  
Cell Types ◽  
Flanking Region ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Cat Gene ◽  
Cpae Cells

Transcriptional regulation of the human von Willebrand factor (vWF) gene was investigated in calf pulmonary artery endothelial (CPAE), HeLa, COS 7 and Hep G2 cells. Various lengths of flanking sequences extending up to 2123 bp 5′ of the transcription initiation site and containing 19 bp of the first exon, were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and these constructs were assayed in transient transfection assays. Sequences up to 89 bp upstream of the cap site showed transcriptional activity in all cell types. Sequences between -147 and -419 bp markedly reduced CAT activity in CPAE cells and abolished it in other cell lines. A domain from -592 to -810 bp generated low levels of expression only in CPAE cells. This transcriptional activity was repressed with constructs containing 1041 to 1240 bp upstream of the cap site. Endothelial cell-specific transcription was restored by a construct that contained 1286 bp upstream of the cap site. The additional 46 bp upstream of the negative regulatory domain were within the 5′ end of an inverse human Alu-family DNA repeat. RNAase-protection assays confirmed the correct transcriptional initiation. The sequence between -89 and -420 contained at least one negative regulatory element able to repress the CAT gene expression controlled by the heterologous thymidine kinase promoter in all cell types. A construct that included the sequence from -89 to -1286 bp increased the transcriptional activity directed by the thymidine kinase promoter only in CPAE cells. These results indicate that negative and positive elements in the 5′-flanking region interact to regulate vWF gene expression.

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