Cleavage of human von Willebrand factor by platelet calcium-activated protease

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 352-356
Author(s):  
TJ Kunicki ◽  
RR Montgomery ◽  
J Schullek
Keyword(s):  
Von Willebrand Factor ◽  
Sodium Dodecylsulfate ◽  
Apparent Molecular Weight ◽  
Aminocaproic Acid ◽  
Crossed Immunoelectrophoresis ◽  
Nonionic Detergent ◽  
Epsilon Aminocaproic Acid ◽  
Triton X ◽  
Von Willebrand ◽  
Willebrand Factor

In human platelet lysates prepared by addition of nonionic detergent (Triton X-100) or by sonication, the multimer composition and electrophoretic mobility of platelet von Willebrand factor (vWF) were consistently modified under conditions that would favor activation of the endogenous calcium-activated, sulfhydryl-dependent neutral protease (CAP). By sodium dodecylsulfate-agarose gel electrophoresis, native platelet vWF contained some multimers that were larger than those characteristic of plasma vWF. Modified platelet vWF contained a multimer population equivalent to or smaller than that of plasma vWF plus an additional fast-migrating band. In crossed immunoelectrophoresis (CIE), modified platelet vWF was characterized by a more anodic distribution and the appearance of a distinct, cross- reactive, anodic component previously designated VIIIR:Ag fragment. In the presence of calcium, radiolabeled purified plasma vWF was also degraded by the protease in question, with a decrease in the apparent molecular weight of the reduced monomer from 230,000 to 205,000. The VIIR:Ag fragment isolated from the same degraded plasma vWF by preparative CIE was shown to be composed of an identical mol wt 205,000 subunit. Because cleavage of plasma or platelet vWF was inhibited by prior addition of leupeptin, EDTA, ethylene glycol bis (beta-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA), or N-ethylmaleimide (agents known to inhibit platelet CAP) but was unaffected by numerous other protease inhibitors, including soybean trypsin inhibitor, benzamidine, hirudin, phenylmethylsulfonyl fluoride, aprotonin, or epsilon-aminocaproic acid (none of which inhibits platelet CAP), we conclude that proteolysis of vWF observed in this study is a direct effect of CAP and is not mediated by way of secondary proteases.

Cleavage of human von Willebrand factor by platelet calcium-activated protease

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 352-356 ◽  
Author(s):  
TJ Kunicki ◽  
RR Montgomery ◽  
J Schullek
Keyword(s):  
Von Willebrand Factor ◽  
Sodium Dodecylsulfate ◽  
Apparent Molecular Weight ◽  
Aminocaproic Acid ◽  
Crossed Immunoelectrophoresis ◽  
Nonionic Detergent ◽  
Epsilon Aminocaproic Acid ◽  
Triton X ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In human platelet lysates prepared by addition of nonionic detergent (Triton X-100) or by sonication, the multimer composition and electrophoretic mobility of platelet von Willebrand factor (vWF) were consistently modified under conditions that would favor activation of the endogenous calcium-activated, sulfhydryl-dependent neutral protease (CAP). By sodium dodecylsulfate-agarose gel electrophoresis, native platelet vWF contained some multimers that were larger than those characteristic of plasma vWF. Modified platelet vWF contained a multimer population equivalent to or smaller than that of plasma vWF plus an additional fast-migrating band. In crossed immunoelectrophoresis (CIE), modified platelet vWF was characterized by a more anodic distribution and the appearance of a distinct, cross- reactive, anodic component previously designated VIIIR:Ag fragment. In the presence of calcium, radiolabeled purified plasma vWF was also degraded by the protease in question, with a decrease in the apparent molecular weight of the reduced monomer from 230,000 to 205,000. The VIIR:Ag fragment isolated from the same degraded plasma vWF by preparative CIE was shown to be composed of an identical mol wt 205,000 subunit. Because cleavage of plasma or platelet vWF was inhibited by prior addition of leupeptin, EDTA, ethylene glycol bis (beta-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA), or N-ethylmaleimide (agents known to inhibit platelet CAP) but was unaffected by numerous other protease inhibitors, including soybean trypsin inhibitor, benzamidine, hirudin, phenylmethylsulfonyl fluoride, aprotonin, or epsilon-aminocaproic acid (none of which inhibits platelet CAP), we conclude that proteolysis of vWF observed in this study is a direct effect of CAP and is not mediated by way of secondary proteases.

scholarly journals Direct radioimmune detection in human plasma of the association between factor VIII procoagulant protein and von Willebrand factor, and the interaction of von Willebrand factor-bound procoagulant VIII with platelets

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1163-1173 ◽  
Author(s):  
JL Moake ◽  
MJ Weinstein ◽  
JH Troll ◽  
LE Chute ◽  
NM Colannino
Keyword(s):  
Factor Viii ◽  
Human Plasma ◽  
Von Willebrand Factor ◽  
Sodium Dodecylsulfate ◽  
Coagulation Factors ◽  
Cysteine Protease Inhibitors ◽  
Proteolytic Activation ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The predominant procoagulant factor VIII (VIII:C) form in normal human plasma containing various combinations of anticoagulants and serine/cysteine protease inhibitors is a protein with mol wt 2.6 +/- 0.2 X 10(5). This protein can be detected by 125I-anti-VIII:C Fab binding and gel electrophoresis in the presence and absence of sodium dodecylsulfate (SDS) and is distinct from the subunit of factor VIII/von Willebrand factor (VIII:vWF) multimers. No larger VIII:C form is present in plasma from patients with severe congenital deficiencies of each of the coagulation factors, other than VIII:C. The mol wt approximately 2.6 X 10(5) VIII:C form is, therefore, likely to be the in vivo procoagulant form of VIII:C, rather than a partially proteolyzed, partially activated derivative of a larger precursor. About 60% of this procoagulant mol wt approximately 2.6 X 10(5) VIII:C form in plasma is present in noncovalent complexes with larger VIII:vWF multimers, which attach reversibly to platelet surfaces in the presence of ristocetin. This VIII:vWF-bound protein of mol wt approximately 2.6 X 10(5) may be the plasma procoagulant form of VIII:C which, after proteolytic activation, accelerates the IXa-mediated cleavage and activation of X postulated to occur on platelet surfaces.

scholarly journals Plasminogen interactions with platelets in plasma

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1530-1535 ◽  
Author(s):  
B Adelman ◽  
A Rizk ◽  
E Hanners
Keyword(s):  
Monoclonal Antibody ◽  
Binding Site ◽  
Platelet Rich Plasma ◽  
Aminocaproic Acid ◽  
Binding Domains ◽  
Fibrinogen Binding ◽  
Platelet Binding ◽  
Epsilon Aminocaproic Acid ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In this report we used a fluorescent flow cytometry-based assay to examine plasminogen binding to platelets in plasma. Our data indicate that platelets activated in platelet-rich plasma (PRP) by adenosine-5′- diphosphate (ADP) or thrombin bind plasminogen to their surface. Fab fragments of the monoclonal antibody LJ-CP8 that are directed against the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex inhibit both plasminogen and fibrinogen binding to ADP-stimulated platelets as does 5 mmol/L EDTA. Platelet aggregation and plasminogen and fibrinogen binding are also concurrently inhibited by the Gly-Arg- Asp (RGD) analogue Gly-Arg-Gly-Asp-Ser (GRGDS) when it is added to PRP before ADP stimulation. The scrambled peptide analogue SDGRG has no effect. The monoclonal antibody 6D1, directed against the von Willebrand factor binding site on GPIb, has no effect on plasminogen- platelet binding, nor does antithrombospondin antibody. epsilon- Aminocaproic acid (EACA), however, inhibits plasminogen binding to ADP- activated platelets. These data indicate that plasminogen binds to platelets activated in plasma, that binding occurs on platelet GPIIb/IIIa, and that binding may be mediated via plasminogen association with fibrinogen via lysine binding domains. Finally, we found both plasminogen and fibrinogen on resting platelets in PRP and demonstrated that they are equally displaced by EDTA, LJ-CP8, and 10E5 (an additional anti-GPIIb/IIIa monoclonal antibody). Plasminogen is also equally displaced by EACA. These data suggest that plasminogen is also bound to GPIIb/IIIa on resting platelets, possibly also via interaction with fibrinogen.

scholarly journals Plasminogen interactions with platelets in plasma

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1530-1535
Author(s):  
B Adelman ◽  
A Rizk ◽  
E Hanners
Keyword(s):  
Monoclonal Antibody ◽  
Binding Site ◽  
Platelet Rich Plasma ◽  
Aminocaproic Acid ◽  
Binding Domains ◽  
Fibrinogen Binding ◽  
Platelet Binding ◽  
Epsilon Aminocaproic Acid ◽  
Von Willebrand ◽  
Willebrand Factor

In this report we used a fluorescent flow cytometry-based assay to examine plasminogen binding to platelets in plasma. Our data indicate that platelets activated in platelet-rich plasma (PRP) by adenosine-5′- diphosphate (ADP) or thrombin bind plasminogen to their surface. Fab fragments of the monoclonal antibody LJ-CP8 that are directed against the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex inhibit both plasminogen and fibrinogen binding to ADP-stimulated platelets as does 5 mmol/L EDTA. Platelet aggregation and plasminogen and fibrinogen binding are also concurrently inhibited by the Gly-Arg- Asp (RGD) analogue Gly-Arg-Gly-Asp-Ser (GRGDS) when it is added to PRP before ADP stimulation. The scrambled peptide analogue SDGRG has no effect. The monoclonal antibody 6D1, directed against the von Willebrand factor binding site on GPIb, has no effect on plasminogen- platelet binding, nor does antithrombospondin antibody. epsilon- Aminocaproic acid (EACA), however, inhibits plasminogen binding to ADP- activated platelets. These data indicate that plasminogen binds to platelets activated in plasma, that binding occurs on platelet GPIIb/IIIa, and that binding may be mediated via plasminogen association with fibrinogen via lysine binding domains. Finally, we found both plasminogen and fibrinogen on resting platelets in PRP and demonstrated that they are equally displaced by EDTA, LJ-CP8, and 10E5 (an additional anti-GPIIb/IIIa monoclonal antibody). Plasminogen is also equally displaced by EACA. These data suggest that plasminogen is also bound to GPIIb/IIIa on resting platelets, possibly also via interaction with fibrinogen.

scholarly journals Human plasma alpha 2-macroglobulin and von Willebrand factor possess covalently linked ABO(H) blood group antigens in subjects with corresponding ABO phenotype

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 663-668
Author(s):  
T Matsui ◽  
Y Fujimura ◽  
S Nishida ◽  
K Titani
Keyword(s):  
Blood Group ◽  
Von Willebrand Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Enzyme Linked Immunosorbent Assay ◽  
Blood Group Antigens ◽  
Ulex Europaeus ◽  
Von Willebrand ◽  
Willebrand Factor

We recently identified ABO(H) blood group structures in Asn-linked sugar chains of human von Willebrand factor (vWF) purified from factor VIII concentrates (J Biol Chem 267:8723, 1992). We surveyed plasma glycoproteins carrying ABO(H) blood group antigens by Western blotting analysis and sandwich enzyme-linked immunosorbent assay using blood group-specific monoclonal antibodies (MoAbs) and a lectin. Two major plasma proteins showing apparent molecular weight of about 180 Kd and 270 Kd by sodium dodecyl sulfate polyacrylamide gel electrophoresis reacted with blood group-specific MoAbs and Ulex europaeus lectin I in accordance with donor blood group. Direct sequence analysis of the protein bands showed their identity with the N-terminal sequences of alpha 2-macroglobulin (alpha 2M) and vWF, respectively. The two bands also reacted with anti-alpha 2M and anti-vWF antibodies. The alpha 2M and vWF prepared from plasma by immunoprecipitation showed the appropriate blood group antigenicity. After incubation with endoglycosidase F, both alpha 2M and vWF lost almost all reactivity with anti-blood group reagents. About 90% of plasma vWF, but only approximately 10% of alpha 2M, was immunoprecipitated with anti-blood group antibody. These results indicate that at least two plasma glycoproteins, vWF and alpha 2M, possess Asn-linked ABO(H) blood group antigens in normal individuals with corresponding ABO phenotype. Therefore, ABO(H) blood group antigens in plasma glycoproteins should be considered during preparation of plasma materials for therapeutic use.

Identification of Platelet Antigens by Monoclonal Antibodies Using Crossed Immunoelectrophoresis with Immunoblotting of the Monoclonal Antibody

1987 ◽  
Vol 57 (02) ◽  
pp. 212-216 ◽  
Author(s):  
L I Thorsen ◽  
G Gaudernack ◽  
F Brosstad ◽  
T M Pedersen ◽  
N O Solum
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Passive Transfer ◽  
Cellular Proteins ◽  
Nitrocellulose Membrane ◽  
Crossed Immunoelectrophoresis ◽  
Antigen Complex ◽  
Triton X ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA method is described for the identification of antigens by monoclonal antibodies. This is applicable whenever precipitating antibodies to the same antigens from a different species are available. The method is based upon: 1) Separation and immunoprécipitation of cellular proteins with a polyspecific antiserum in crossed immunoelectrophoresis in the presence of the non-denaturing detergent Triton X-100 and the monoclonal antibody. 2) Coprecipitation of the monoclonal antibody with its antigen. 3) Subsequent passive transfer of the monoclonal antibody in the antibody-antigen complex onto a nitrocellulose membrane. 4) Visualization of the blotted antibody using an enzyme-linked secondary antibody and a chromogenic substrate. 5) Identification of the corresponding antigen by comparisons to the immunoprecipitate pattern of the original immunoplate. To test this method we have analyzed the detection of the antigens recognized by six previously described monoclonal antibodies against platelet membrane proteins and von Willebrand factor. Specific immunoblots were obtained in each case using small amounts of monoclonal antibodies. Thus, the technique provides an alternative when epitopes are denatured by SDS, and avoids the use of radioactively labelled monoclonal antibodies.

CHARACTERIZATION OF A 40 kDa FRAGMENT OF VON WILLEBRAND FACTOR THAT CONTAINS THE GLYCOPROTEIN IIb/IIIa-BINDING DOMAIN

1987 ◽  
Author(s):  
K Niiya ◽  
P Kostel ◽  
T S Zimmerman ◽  
Z M Ruggeri
Keyword(s):  
Molecular Weight ◽  
Von Willebrand Factor ◽  
Apparent Molecular Weight ◽  
Binding Domain ◽  
Size Exclusion ◽  
Amino Terminus ◽  
Single Chain ◽  
Amino Terminal ◽  
Von Willebrand ◽  
Willebrand Factor

We have isolated a 40 kDa fragment of von Willebrand factor (vWF) that contains the glycoprotein (GP) Ilb/IIIa-binding domain. The Staphylococcus aureus V8 protease-generated fragment II was digested with trypsin (1:50 enzyme:substrate ratio on a weight-to-weight basis). After addition of a 100fold molar excess of (p-amidinophenyl)methanesulfonyl fluoride in order to inhibit any residual trypsin activity, the whole digest was subjected to ion-exchange and size-exclusion high pressure liquid chromatography. Two major fragments were separated. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) demonstrated that one of the two purified polypeptides had an apparent molecular weight of 40 kDa under both reducing and nonreducing conditions, suggesting that it was a single chain polypeptide. The other fragment had an apparent molecular weight of 22 kDa after reduction and 44 kDa unreduced, suggesting that it was a homodimer. Amino terminal sequence analysis of both fragments was performed by classical Edman degradation following electroelution from reduced SDS-polyacrylamide gels. The amino terminus of the 40 kDa fragment corresponded to residue Glu (1366) (as did the fragment II from which it was derived), while the amino terminus of the 22 kDa fragment corresponded to residue Val (1927) of the constituent 2050 residue subunit. The effect of both fragments on vWF binding to the platelet membrane GP IIb/IIIa complex was evaluated by measuring the residual binding of 125I-labeled vWF to thrombin-stimulated platelets in the presence of varying amounts of the unreduced fragments. The 40 kDa polypeptide inhibited 64 percent of vWF binding when tested at a concentration of 20 μK, whereas the 22kDa dimer was without effect. This study establishes that the GP IIb/IIIa-binding domain of vWF resides in a discrete, single-chain 40 kDa fragment derived from the 220 kDa, homodimeric fragment II generated by V8 protease. Moreover, we found evidence for the existence of inter-chain disulfide bonds within 22 kDa from the carboxyl terminus of the constituent subunit.

Stabilization of the Antihemophilic Factor (VIII :AHF) by the Von Willebrand Factor (VIII :VWF): A Possible Model for Von Willebrand’s Disease

1977 ◽  
Author(s):  
H. J. Weiss ◽  
I. I. Sussman ◽  
L. W. Hoyer
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Crossed Immunoelectrophoresis ◽  
Factor Viii Antigen ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Antihemophilic Factor

When compared with VIII:AHF in normal citrated plasmas, VIII:AHF activity showed increased lability at 37°C in the ‘late’ post-transfusion plasmas (VIII:AHF≫VIII:VWF) of a patient with von Willebrand’s disease, but not in the ‘early’ post-transfusion plasmas in which VIII:AHF~VIII:VWF. VIII:AHF was also labile in the baseline plasmas of 3 patients with von Willebrand’s disease in whom VIII:AHF≫VIII:VWF. In two of these patients the mobility of Factor VIII antigen (on crossed Immunoelectrophoresis) was increased. (VIII:AHF was not excessively labile in 4 other patients in whom VIII :AHF~VIII:VWF). In all of the above cases, VIII:AHF was stabilized by the addition of either purified von Willebrand factor or plasmas of patients with hemophilia, but not by plasmas of patients with severe von Willebrand’s disease. Thus, VIII:VWF may serve to stabilize VIII:AHF and this might explain the post-transfusion findings in von Willebrand’s disease.

scholarly journals Proteolysis of platelet glycoprotein Ib by plasmin is facilitated by plasmin lysine-binding regions

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1280-1284 ◽  
Author(s):  
B Adelman ◽  
AD Michelson ◽  
J Greenberg ◽  
RI Handin
Keyword(s):  
Binding Sites ◽  
Direct Interaction ◽  
Aminocaproic Acid ◽  
Platelet Glycoprotein ◽  
Binding Region ◽  
Glycoprotein Ib ◽  
Platelet Membrane Receptor ◽  
Epsilon Aminocaproic Acid ◽  
Von Willebrand ◽  
Willebrand Factor

We have characterized the effects of plasmin on glycoprotein Ib (GpIb), a platelet membrane receptor for von Willebrand factor (vWF), and on glycocalicin, a fragment of the alpha chain of GpIb that contains the vWF-binding region. The addition of 4.5 X 10(-7) mol/L plasmin to washed platelets caused a time-dependent decrease in ristocetin- induced, vWF-dependent platelet agglutination. epsilon-Aminocaproic acid (EACA) inhibited plasmin release of glycocalicin-related antigen from washed platelets and preserved vWF-dependent platelet agglutination, thus indicating that the lysine-binding sites on plasmin facilitated its degradation of GpIb. To demonstrate a direct interaction between plasmin and the vWF-binding region of GpIb we incubated purified glycocalicin with plasmin. Plasmin degraded the glycocalicin into two small carbohydrate-poor peptides and into a larger carbohydrate-rich fragment. EACA was able to inhibit plasmin- mediated degra dation of glycocalicin in a concentration-dependent fashion. These studies indicated that plasmin degradation of GpIb was due to a direct interaction between plasmin and GpIb and that this effect was mediated by the lysine-binding region of the plasmin molecule.

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