scholarly journals Sialic acid prevents loss of large von Willebrand factor multimers by protecting against amino-terminal proteolytic cleavage

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1790-1796 ◽  
Author(s):  
SD Berkowitz ◽  
AB Federici
Keyword(s):  
Sialic Acid ◽  
Von Willebrand Factor ◽  
Proteinase Inhibitors ◽  
Terminal Region ◽  
Cleavage Sites ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Beta Galactosidase

Removal of sialic acid from the von Willebrand factor (vWF) subunit exposes additional cleavage sites in the amino-terminal region that are associated with loss of large multimers. The extent of large multimer loss was evaluated by examining the sites of subunit cleavage of native and carbohydrate-modified vWF after treatment with trypsin, chymotrypsin, or plasmin. In the presence of proteinase inhibitors, purified vWF was treated with neuraminidase alone to remove 90% to 95% of the sialic acid or with neuraminidase and beta-galactosidase to remove the sialic acid and 45% to 50% of the D-galactose, with little or no loss of large multimers observed. Digestion of native vWF with trypsin produced the greatest loss of large multimers, while chymotrypsin produced less and plasmin produced the least. Large multimer loss was more extensive with each enzyme after carbohydrate modification of vWF. The extent and approximate location of subunit cleavage was determined by immunoblotting and monoclonal antibody epitope mapping. Trypsin, chymotrypsin, and plasmin were shown to produce both amino- and carboxyl-terminal fragments. The number, location, and relative quantities of carboxyl-terminal fragments produced were unchanged after carbohydrate modification. However, digestion of the amino-terminal region was considerably more extensive after carbohydrate modification as judged by a marked decrease or absence of the larger fragments seen when native vWF was digested, and by the appearance of new smaller molecular mass species. Therefore, the greater loss of large multimers that occurs after carbohydrate modification is likely to be the result of cleavages in the amino- terminal region of the molecule. By protecting the vWF subunit against amino-terminal cleavage, sialic acid inhibits the loss of large multimers.

scholarly journals Sialic acid prevents loss of large von Willebrand factor multimers by protecting against amino-terminal proteolytic cleavage

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1790-1796 ◽  
Author(s):  
SD Berkowitz ◽  
AB Federici
Keyword(s):  
Sialic Acid ◽  
Von Willebrand Factor ◽  
Proteinase Inhibitors ◽  
Terminal Region ◽  
Cleavage Sites ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Beta Galactosidase

Abstract Removal of sialic acid from the von Willebrand factor (vWF) subunit exposes additional cleavage sites in the amino-terminal region that are associated with loss of large multimers. The extent of large multimer loss was evaluated by examining the sites of subunit cleavage of native and carbohydrate-modified vWF after treatment with trypsin, chymotrypsin, or plasmin. In the presence of proteinase inhibitors, purified vWF was treated with neuraminidase alone to remove 90% to 95% of the sialic acid or with neuraminidase and beta-galactosidase to remove the sialic acid and 45% to 50% of the D-galactose, with little or no loss of large multimers observed. Digestion of native vWF with trypsin produced the greatest loss of large multimers, while chymotrypsin produced less and plasmin produced the least. Large multimer loss was more extensive with each enzyme after carbohydrate modification of vWF. The extent and approximate location of subunit cleavage was determined by immunoblotting and monoclonal antibody epitope mapping. Trypsin, chymotrypsin, and plasmin were shown to produce both amino- and carboxyl-terminal fragments. The number, location, and relative quantities of carboxyl-terminal fragments produced were unchanged after carbohydrate modification. However, digestion of the amino-terminal region was considerably more extensive after carbohydrate modification as judged by a marked decrease or absence of the larger fragments seen when native vWF was digested, and by the appearance of new smaller molecular mass species. Therefore, the greater loss of large multimers that occurs after carbohydrate modification is likely to be the result of cleavages in the amino- terminal region of the molecule. By protecting the vWF subunit against amino-terminal cleavage, sialic acid inhibits the loss of large multimers.

CARBOHYDRATE PREVENTS LOSS OF LARGE VON WILLEBRAND FACTOR MULTIMERS BY PROTECTING AGAINST AMINO TERMINAL PROTEOLYTIC CLEAVAGE

1987 ◽  
Author(s):  
A B Federici ◽  
S D Berkowitz
Keyword(s):  
Von Willebrand Factor ◽  
Proteinase Inhibitors ◽  
Enzymatic Digestion ◽  
Terminal Region ◽  
Cleavage Sites ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

We have previously shown that carbohydrate (CHO) protects von Willebrand factor (vWF) from proteolytic degradation. We have now shown that removal of CHO from the vWF subunit exposes additional cleavage sites in the amino terminal region and that cleavages in this region are associated with loss of large multimers. We examined and compared the extent of large multimer loss with sites of subunit cleavage of native and GHO-modified vWF after treatment with plasmin, chymotrypsin, and trypsin. Highly purified vWF was treated with neuraminidase and β-galactosidase in the presence of proteinase inhibitors to remove 90-95% of the sialic acid and 45-50% of the D-galactose without loss of large multimers or diminution of the ristocetin cofactor activity. The extent and approximate location of subunit cleavage was determined by immunoblotting and monoclonal antibody epitope mapping. Multimeric analysis revealed an increasingly greater loss of large multimers when native vWF was digested with plasmin, chymotrypsin, and trypsin, respectively. Large multimer loss was more extensive with each enzyme after CHO-modification of vWF. On subunit analysis, plasmin, chymotrypsin, and trypsin were shown to produce both amino and carboxy terminal fragments. The number, location, and relative quantities of carboxy terminal fragments produced by these enzymes were unchanged after CHO modification. However, digestion of the amino terminal region was considerably more extensive as judged by a marked decrease or absence of the larger fragments seen when native vWF was digested, and by the appearance of new smaller molecular weight species. Thus, enzymatic digestion of vWF after removal of carbohydrate produced new cleavages in the amino terminal region but did not alter the location or extent of carboxy terminal cleavages. Therefore, the greater loss of large multimers that occurs after CHO modification is likely to be the result of cleavages in the amino terminal region of the molecule. It appears that by protecting the vWF subunit against amino terminal cleavage, carbohydrate inhibits the loss of large multimers.

Altered Von Willebrand Factor Subunit Proteolysis and Multimer Processing Associated with the Cys1599(2362)Phe Mutation in the B2 Domain.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 179-179 ◽  
Author(s):  
Luigi De Marco ◽  
Alessandra Casonato ◽  
Lisa Gallinaro ◽  
Marita Sztukowska ◽  
Mario Mazzucato ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Terminal Region ◽  
Von Willebrand Disease ◽  
Proteolytic Processing ◽  
Large Species ◽  
Main Band ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Cysteine residues clustered in carboxyl- and amino-terminal domains mediate the assembly of von Willebrand factor (VWF) subunits into dimers and multimers, respectively. We have identified a patient with bleeding symptoms and very low plasma VWF antigen and ristocetin cofactor activity, compatible with clinically severe von Willebrand disease (VWD), whose abnormal plasma VWF multimers show a distinctly altered proteolytic processing. Analysis of plasma VWF multimers in low resolution agarose gels demonstrated a smearing extending to the origin of the separating gel in both the proband and her parents, suggestive of the presence of unusually large species with low electrophoretic mobility. In high resolution gels, these large multimers were stacked at the origin of the gel, clearly seen in a sample of proband’s plasma obtained after DDAVP infusion even though the treatment caused only a modest increase in VWF:Ag and VWF:RCo (from 4.7 to 7.5 U/dL and from 5.2 to 8 U/dL, respectively). Individual VWF multimers in the proband’s plasma comprised only one clearly visible band with different mobility as compared to the main band of normal multimers, and lacked the satellite bands that form the normal triplet structure. Analysis of the proband’s plasma VWF subunit composition using anti M7 antibodies (which probe the aminoterminal region of VWF) revealed the apparent 225 kDa mature form and a single 205 kDa fragment, but notably absent was the 140 kDa aminoterminal fragment generated by ADAMTS-13 cleavage. Subunit analysis using anti-M31 antibodies (which probe the carboxyl-terminal region of VWF) revealed the 225 kDa intact subunit and the 205 kDa fragment, but notably absent were the fragments generated by ADAMTS-13 cleavage. Analysis with anti-22K antibodies, which react with one or more epitope(s) located in the extreme carboxyl terminal region of VWF downstream of Val1927, revealed the presence of only the intact subunit of 225kDa. Concentration of ADAMTS-13 antigen and VWF-cleaving activity in the patient plasma were within normal limits.The patient carried a homozygous Cys to Phe mutation at position 1599 in the mature VWF subunit B2 domain (Cys2362 of pre-pro-VWF). In her heterozygous parents, the 205 kDa fragment was distinctly visible along with the normal VWF cleavage products. Our finding indicate that a proper conformation of the B2 domain, dependent on critical Cys residues, may be required for normal proteolytic processing of VWF multimers. The association of these structural and functional abnormalities with a missense mutation in the B2 domain provides insights into the mechanism that may regulate the post-secretion processing of VWF multimers, and, thus, their prothrombotic potential.

Visualization of von Willebrand Factor Multimers by Enzyme-Conjugated Secondary Antibodies

1986 ◽  
Vol 55 (02) ◽  
pp. 276-278 ◽  
Author(s):  
F Brosstad ◽  
Inge Kjønniksen ◽  
B Rønning ◽  
H Stormorken
Keyword(s):  
Von Willebrand Factor ◽  
Chromogenic Substrate ◽  
Agarose Electrophoresis ◽  
Secondary Antibodies ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Beta Galactosidase

SummaryA method for visualization of the multimeric forms of von Willebrand Factor (vWF) in plasma and platelets is described. The method is based upon: 1) Separation of the vWF multimers by SDS-agarose electrophoresis, 2) Subsequent blotting of the vWF multimers onto nitrocellulose, 3) Immunolocalization and visualization of the vWF pattern by the sequential incubation of the blot with a) primary vWF antiserum, b) peroxidase- or beta-galactosidase-conjugated secondary antibodies and a relevant chromogenic substrate.

Abnormal Structure of von Willebrand Factor in Myeloproliferative Syndrome Is Associated to Either Thrombotic or Bleeding Diathesis

1987 ◽  
Vol 58 (02) ◽  
pp. 753-757 ◽  
Author(s):  
M F López-Fernández ◽  
C López-Berges ◽  
R Martín ◽  
A Pardo ◽  
F J Ramos ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Proteinase Inhibitors ◽  
Relative Proportion ◽  
Variable Degree ◽  
Bleeding Diathesis ◽  
Myeloproliferative Syndrome ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe multimeric and subunit patterns of plasma von Willebrand factor (vWF) were analyzed in eight patients with myeloproliferative syndrome (MS) in order to investigate the possible existence of heterogeneity in the “in vivo” proteolytic cleavage of the protein, previously observed in this entity. Six patients lacked large vWF multimers, five of them having normal bleeding times (BT) and clinically documented episodes of thrombotic origin, whereas one patient had long BT and bleeding symptoms. Seven patients showed a relative increase in the 176 kDa subunit fragment while the 189 kDa polypeptide was increased in only one. In addition, another patient (and prior to any therapy) showed the presence of a new fragment of approximately 95 kDa which disappeared after Busulfan therapy. The collection of blood from these patients with proteinase inhibitors did not correct the abnormalities.The infusion of DDAVP to two patients with abnormal vWF was accompanied by: the appearance of larger vWF multimers which disappeared rapidly from plasma; an increase in the relative proportion of the satellite bands of each multimer and a further increase of the 176 kDa fragment. These data point to some heterogeneity in the vWF abnormality present in MS which may be related in part to a variable degree of proteolysis of vWF occurring “in vivo” rather than “in vitro”, and which may be associated to either a thrombotic or a bleeding diathesis. They also suggest that despite the presence of abnormal, already proteolyzed vWF, DDAVP-enhanced proteolysis occurs in MS to a similar extent to what is described in normal individuals.

A Factor VIII Neutralizing Monoclonal Antibody and a Human Inhibitor Alloantibody Recognizing Epitopes in the C2 Domain Inhibit Factor VIII Binding to von Willebrand Factor and to Phosphatidylserine

1993 ◽  
Vol 69 (03) ◽  
pp. 240-246 ◽  
Author(s):  
Midori Shima ◽  
Dorothea Scandella ◽  
Akira Yoshioka ◽  
Hiroaki Nakai ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Neutralizing Monoclonal Antibody ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.

scholarly journals Epitope mapping by cDNA expression of a monoclonal antibody which inhibits the binding of von Willebrand factor to platelet glycoprotein IIb/IIIa

1992 ◽  
Vol 284 (3) ◽  
pp. 711-715 ◽  
Author(s):  
G Piétu ◽  
A S Ribba ◽  
G Chérel ◽  
D Meyer
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Fusion Proteins ◽  
Solid Phase ◽  
Platelet Glycoprotein ◽  
Rgd Sequence ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Beta Galactosidase

In order to study the structure-function relationship of von Willebrand Factor (vWF), we have located the epitope of a well-characterized monoclonal antibody (MAb) to vWF (MAb 9). This MAb reacts with the C-terminal portion of the vWF subunit, SPII fragment [amino acids (aa) 1366-2050], which includes an Arg-Gly-Asp (RGD) sequence at positions 1744-1746, and totally inhibits vWF and SPII binding to platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa). A recombinant DNA library was constructed by cloning small (250-500 nucleotides) vWF cDNA fragments into the lambda gt11 vector and these inserts were expressed as fusion proteins with beta-galactosidase. Immunological screening of the library with 125I-MAb 9 identified three immunoreactive clones. vWF inserts were amplified by the PCR and their sequences demonstrated overlapping nucleotides from positions 7630 to 7855 of vWF cDNA, coding for aa residues 1698-1773 of the mature subunit, indicating that this is the epitope of MAb 9. vWF-beta-galactosidase fusion protein reacted with 125I-MAb 9 by Western blotting. In a solid-phase radioimmunoassay, the purified fusion proteins decreased the binding of vWF to 125I-MAb 9 by 50%, and this inhibition was dose-dependent between 3.5 and 120 nM. Therefore the epitope of MAb 9 is located within aa 1698-1773 of the vWF subunit, which includes the RGD sequence implicated in the binding of adhesive proteins of GPIIb/IIIa.

Modulation by Heparin of the Interaction of the A1 Domain of von Willebrand Factor With Glycoprotein Ib

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4186-4194 ◽  
Author(s):  
Christelle Perrault ◽  
Nadine Ajzenberg ◽  
Paulette Legendre ◽  
Ghassem Rastegar-Lari ◽  
Dominique Meyer ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Cell Aggregation ◽  
Cell Aggregates ◽  
Critical Determinant ◽  
Amino Terminal ◽  
A Cell ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Amino Terminal Fragment

Abstract The conformation of the A1 domain of von Willebrand factor (vWF) is a critical determinant of its interaction with the glycoprotein (GP) Ib/V/IX complex. To better define the regulatory mechanisms of vWF A1 domain binding to the GPIb/V/IX complex, we studied vWF-dependent aggregation properties of a cell line overexpressing the GPIb, GPIbβ, and GPIX subunits (CHO-GPIbβ/IX cells). We found that CHO-GPIbβ/IX cell aggregation required the presence of both soluble vWF and ristocetin. Ristocetin-induced CHO-GPIbβ/IX cell aggregation was completely inhibited by the recombinant VCL fragment of vWF that contains the A1 domain. Surprisingly, the substitution of heparin for ristocetin resulted in the formation of CHO-GPIbβ/IX cell aggregates. Using monoclonal antibodies blocking vWF interaction with GPIb/V/IX or mocarhagin, a venom metalloproteinase that removes the amino-terminal fragment of GPIb extending from aa 1 to 282, we demonstrated that both ristocetin- and heparin-induced aggregations involved an interaction between the A1 domain of vWF and the GPIb subunit of the GPIb/V/IX complex. The involvement of heparin in cell aggregation was also demonstrated after treatment of heparin with heparinase that abolished CHO-GPIbβ/IX cell aggregation. These results indicated that heparin was able to induce vWF-dependent CHO-GPIbβ/IX cell aggregation. In conclusion, we demonstrated that heparin is capable of positively modulating the vWF interaction with the GPIb/V/IX complex.

scholarly journals Partial characterization of a binding site for von Willebrand factor on glycocalicin

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 19-26 ◽  
Author(s):  
AD Michelson ◽  
J Loscalzo ◽  
B Melnick ◽  
BS Coller ◽  
RI Handin
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Binding Activity ◽  
Proteolytic Fragment ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Beta Galactosidase

The binding of von Willebrand factor (vWF) to platelet membrane glycoprotein Ib (GpIb) facilitates platelet adhesion to vascular subendothelium. In this study, we provide evidence that the vWF binding site is on glycocalicin (GC), a proteolytic fragment of GpIb, and we examine the role of the carbohydrate portion of GC on that binding. The binding to platelets of 6D1, a monoclonal antibody that recognizes an epitope on GpIb and blocks ristocetin-induced vWF binding to platelets, was inhibited by purified GC. In addition, purified GC inhibited ristocetin-dependent binding of 125I-labeled vWF to platelets. Since GC contains 60% carbohydrate by weight, we assessed the role of carbohydrate sequences on its interaction with antibody 6D1 and vWF. Based on the known sequence of the major oligosaccharide chain of GC--N- acetyl neuraminic acid, galactose, N-acetyl glucosamine, N-acetyl galactosamine--we treated GC sequentially with neuraminidase, beta- galactosidase, and beta-N-acetylglucosaminidase. Removal of sialic acid and galactose residues did not affect GC binding. Removal of N-acetyl glucosamine residues did not affect GC binding to 6D1 but did decrease the ability of GC to inhibit vWF binding to platelets, increasing the concentration needed to inhibit binding by 50% (IC50) 40-fold. This suggests that a portion of the oligosaccharide chains on GC contributes to the vWF binding activity of this molecule.

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