Transmembrane Calcium Influx Associated with von Willebrand Factor Binding to GP Ib in the Initiation of Shear-Induced Platelet Aggregation

1993 ◽  
Vol 69 (05) ◽  
pp. 496-502 ◽  
Author(s):  
Yasuo Ikeda ◽  
Makoto Handa ◽  
Tetsuji Kamata ◽  
Koichi Kawano ◽  
Yohko Kawai ◽  
...  
Keyword(s):  
Shear Force ◽  
Calcium Influx ◽  
Fold Increase ◽  
Intracellular Camp ◽  
Recombinant Fragment ◽  
Transmembrane Flux ◽  
Irreversible Aggregation ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Camp Levels

SummaryWe found that the binding of multimeric vWF to GP Ib under a shear force of 108 dynes/cm2 resulted in the transmembrane flux of Ca2+ ions with a two-to three-fold increase in their intracellular concentration ([Ca2+]i). The blockage of this event, obtained by inhibiting the vWF-GP Ib interaction, suppressed aggregation. In contrast, the blockage of vWF binding to GP IIb-IIIa, as well as the prevention of activation caused by increased intracellular cAMP levels, inhibited aggregation but had no significant effect on [Ca2+]i increase. A monomeric recombinant fragment of vWF containing the GP Ib-binding domain of the molecule (residues 445-733) prevented all effects mediated by multimeric vWF but, by itself, failed to support the increase in [Ca2+]i and aggregation. These results suggest that the binding of multimeric vWF to GP Ib initiates platelets aggregation induced by high shear stress by mediating a transmembrane flux of Ca2+ ions, perhaps through a receptor-dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of GP IIb-IIIa; the latter receptor then binds vWF and mediates irreversible aggregation.

Porcine von Willebrand Factor Binding to Human Platelet GPIb Induces Transmembrane Calcium Influx

1996 ◽  
Vol 75 (04) ◽  
pp. 655-660 ◽  
Author(s):  
Mario Mazzucato ◽  
Luigi De Marco ◽  
Paola Pradella ◽  
Adriana Masotti ◽  
Francesco I Pareti
Keyword(s):  
Monoclonal Antibody ◽  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Human Platelet ◽  
Platelet Glycoprotein ◽  
Dependent Manner ◽  
Transmembrane Flux ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryPorcine von Willebrand factor (P-vWF) binds to human platelet glycoprotein (GP) lb and, upon stirring (1500 rpm/min) at 37° C, induces, in a dose-dependent manner, a transmembrane flux of Ca2+ ions and platelet aggregation with an increase in their intracellular concentration. The inhibition of P-vWF binding to GP lb, obtained with anti GP lb monoclonal antibody (LJ-Ib1), inhibits the increase of intracellular Ca2+ concentration ([Ca2+]i) and platelet aggregation. This effect is not observed with LJ-Ib10, an anti GP lb monoclonal antibody which does not inhibit the vWF binding to GP lb. An anti GP Ilb-IIIa monoclonal antibody (LJ-CP8) shown to inhibit the binding of both vWF and fibrinogen to the GP IIb-IIIa complex, had only a slight effect on the [Ca2+]i rise elicited by the addition of P-vWF. No inhibition was also observed with a different anti GP IIb-IIIa monoclonal antibody (LJ-P5), shown to block the binding of vWF and not that of fibrinogen to the GP IIb-IIIa complex. PGE1, apyrase and indomethacin show a minimal effect on [Ca2+]i rise, while EGTA completely blocks it. The GP lb occupancy by recombinant vWF fragment rvWF445-733 completely inhibits the increase of [Ca2+]i and large aggregates formation. Our results suggest that, in analogy to what is seen with human vWF under high shear stress, the binding of P-vWF to platelet GP lb, at low shear stress and through the formation of aggregates of an appropriate size, induces a transmembrane flux of Ca2+, independently from platelet cyclooxy-genase metabolism, perhaps through a receptor dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of the GP IIb-IIIa complex.

scholarly journals Structure-Function Studies of Factor VIII/Von Willebrand Protein(s)

1977 ◽  
Author(s):  
Patrick A. McKee
Keyword(s):  
Factor Viii ◽  
Single Molecule ◽  
Gel Filtration ◽  
Protein S ◽  
Fold Increase ◽  
Procoagulant Activity ◽  
Void Volume ◽  
Pas Staining ◽  
Von Willebrand ◽  
Willebrand Factor

Factor VIII (FVIII) procoagulant activity was initially thought to be a glycoprotein with a molecular weight (MW) >1 million and composed of disulfide-1inked ~200,000 MW subunits. A protein with similar properties, except lacking procoagulant activity, is in hemophilic plasma; it was identical to normal FVIII by SDS-gel analyses, isoelectric focusing, and PAS staining. Subsequently it was shown that the FVIII glycoprotein also has von Willebrand factor (vWF) activity, suggesting that both FVIII and vWF activities might be properties of the same molecule. When the FVIII/vWF protein(s) is rechromatographed on 4% agarose and 0.25 M CaCl2, virtually all the protein and vWF activity elute in the void volume, but most of the FVIII procoagulant activity elutes much later. The extent of separation of the two activities depends on the amount of protein applied to the column. Also, exposure of the FVIII/vWF to thrombin before gel filtration strikingly accentuates separation of the two activities. The reduced SDS-gel pattern of the void volume protein peak showed the 200,000 MW subunit while that of the procoagulant peak contained several subunit bands which ranged from ~30,000–100,000 MW. Removal of sialic acid from FVIII/vWF is associated with reduced ristocetin induced platelet aggregation and causes a 50-fold increase in the rate of clearance of protein from the circulation by the hepatocyte. Currently, our data suggest that FVIII procoagulant and vWF activities are properties of a single molecule composed of disulfide-bound identical subunits. Cleavage by thrombin then results in FVIII procoagulant activity. Additional cleavages, to which the molecule appears very sensitive, results in FVIII inactivation. The vWF activity is very stable—even to proteolysis—and it appears to be a function of the carbohydrate side chains of the molecule.

Unique Antiplatelet Effects of a Novel S-Nitrosoderivative of a Recombinant Fragment of von Willebrand Factor, AR545C: In Vitro and Ex Vivo Inhibition of Platelet Function

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1693-1700
Author(s):  
Aida Inbal ◽  
Osnat Gurevitz ◽  
Ilia Tamarin ◽  
Regina Eskaraev ◽  
Angela Chetrit ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Platelet Aggregation ◽  
Platelet Function ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Aggregate Formation ◽  
Recombinant Fragment ◽  
Von Willebrand ◽  
Willebrand Factor

The recombinant fragment of von Willebrand factor (vWF) spanning Ala444 to Asp730 and containing an Arg545Cys mutation (denoted AR545C) has antithrombotic properties that are principally a consequence of its ability to inhibit platelet adhesion to subendothelial matrix. Endothelial-derived nitric oxide (NO) can also inhibit platelet function, both as a consequence of inhibiting adhesion as well as activation and aggregation. Nitric oxide can react with thiol functional groups in the presence of oxygen to form S-nitrosothiols, which are naturally occurring NO derivatives that prolong the biological actions of NO. Because AR545C has a single free cysteine (Cys545), we attempted to synthesize the S-nitroso-derivative of AR545C and to characterize its antiplatelet effects. We successfully synthesized S-nitroso-AR545C and found that it contained 0.96 mol S-NO per mole peptide. S-nitroso-AR545C was approximately 5-fold more potent at inhibiting platelet agglutination than was the unmodified peptide (IC50 = 0.02 ± 0.006 μmol/L v 0.1 ± 0.03 μmol/L, P = .001). In addition and by contrast, S-nitroso-AR545C was a powerful inhibitor of adenosine diphosphate–induced platelet aggregation (IC50 = 0.018 ± 0.002 μmol/L), while AR545C had no effect on aggregation. These effects were confirmed in studies of adhesion to and aggregation on extracellular matrix under conditions of shear stress in a cone-plate viscometer, where 1.5 μmol/L S-nitroso-AR545C inhibited platelet adhesion by 83% and essentially completely inhibited aggregate formation, while the same concentration of AR545C inhibited platelet adhesion by 74% and had significantly lesser effect on aggregate formation on matrix (P ≤ .004 for each parameter by ANOVA). In an ex vivo rabbit model, we also found that S-nitroso-AR545C had a more marked and more durable inhibitory effect on botrocetin-induced platelet aggregation than did AR545C, and these differences were also reflected in the extent and duration of effect on the prolongation of the bleeding time in these animals. These data show that S-nitroso-AR545C has significant and unique antiplatelet effects, inhibiting both adhesion and aggregation, by blocking platelet GPIb receptor through the AR545C moiety and elevating platelet cyclic 3′,5′-guanosine monophosphate through the -SNO moiety. These observations suggest that this NO-modified fragment of vWF may have potential therapeutic benefits as a unique antithrombotic agent.

A Reliable and Reproducible ELISA Method to Measure Ristocetin Cofactor Activity of von Willebrand Factor

2000 ◽  
Vol 83 (01) ◽  
pp. 107-113 ◽  
Author(s):  
Stephan Vauterin ◽  
Agota Schlammadinger ◽  
Claudine Mazurier ◽  
Hans Deckmyn ◽  
Karen Vanhoorelbeke ◽  
...  
Keyword(s):  
Recombinant Fragment ◽  
Agglutination Assay ◽  
Different Types ◽  
Single Determination ◽  
A1 Domain ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

SummaryThe ristocetin induced binding of vWF to GPIb, which is routinely tested in a platelet agglutination assay, can be reproducibly studied in an ELISA where plasma vWF binds to a captured rGPIbα-fragment (His1-Val289) in the presence of ristocetin. This binding is specific since the vWF-GPIb interaction could (i) be blocked by inhibitory antiGPIb or anti-(vWF A1 domain) monoclonal antibodies (mAbs) and (ii) be enhanced by an anti-vWF mAb that also facilitates ristocetin induced platelet agglutination. Further studies were undertaken to determine whether the test could be used to differentiate vWF from patients with different types of von Willebrand’s disease. The median vWF:RiCof activity in controls (n = 24) was 0.75 U/ml, in type 1 vWD patients (n = 17) 0.28 U/ml, in type 2A (n = 18) 0.055 U/ml, in type 2B (n = 4) 0.094 U/ml and in type 3 (n = 3) <0.0005 U/ml. Moreover, the values correlated well with those obtained from the vWF:RiCof-agglutination assay (r = 0.873). The vWF:RiCof-ELISA has several advantages: the use of a recombinant fragment instead of donor platelets results in a more reproducible test with a low inter-and intra-assay variability (<14% CV), the test can further be readily automated and for a single determination, only minimal amounts of patient plasma are required (8 µl).

scholarly journals Sialylation on O-linked glycans protects von Willebrand factor from macrophage galactose lectin mediated clearance

Haematologica ◽  
2021 ◽  
Author(s):  
Soracha E. Ward ◽  
Jamie M. O’Sullivan ◽  
Alan B. Moran ◽  
Daniel I. R. Spencer ◽  
Richard A. Gardner ◽  
...  
Keyword(s):  
Residence Time ◽  
Von Willebrand Factor ◽  
Mean Residence Time ◽  
Half Life ◽  
Fold Increase ◽  
Von Willebrand ◽  
Willebrand Factor

Terminal sialylation determines plasma VWF half-life. A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we show that MGL influences physiological plasma VWF clearance. MGL inhibition was associated with a significantly extended mean residence time and 3-fold increase in endogenous plasma VWF:Ag levels (p

Response of Factor VIII/Von Willebrand Factor to Intranasal DDAVP in Healthy Subjects and Mild Haemophiliacs (with Observations in Patients with Combined Deficiency of Factors V and VIII)

1982 ◽  
Vol 48 (01) ◽  
pp. 091-093 ◽  
Author(s):  
V Vicente Garcia ◽  
I Alberca Silva ◽  
A Lopez Borrasca
Keyword(s):  
Healthy Subjects ◽  
Standard Dose ◽  
Fold Increase ◽  
The Body ◽  
High Dose ◽  
Factor V ◽  
Single Standard ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Combined Deficiency

SummaryThe effect of a single standard dose of intranasal DDAVP (260 μg) was investigated in healthy subjects and in patients with mild deficiencies of VIII:C. Changes in FVIII/VWF activities were measured from baseline after 30, 60, 120 and 360 min of administration of the drug. Intranasal DDAVP was followed by a two-fold increase of VIII:C in both groups studied. VIIIR : AG and VIIIR:RCo increased to a lesser extent. Even though FVIII/VWF activities reached their maximum after 60–120 min, a significant increase over baseline was still observed after 360 min. The rise of VIII:C was unrelated to the body weight of the patients and was proportional to the baseline levels of this factor. In two sisters with combined deficiencies of FV/FVIII, the responses in all activities of FVIII/VWF were similar to those seen in mild hemophiliacs. Factor V did not undergo any variation. No alteration in serum osmolarity and no consistent variation in blood pressure or pulse rate were noted. It is concluded that the i.n. administration of a single high dose of DDAVP might be adopted to provide an emergency aid in bleeding patients with mild to moderate haemophilia A and to yield higher VIII:C levels in blood donors.

The anti-von Willebrand factor aptamer ARC1779 increases von Willebrand factor levels and platelet counts in patients with type 2B von Willebrand disease

2012 ◽  
Vol 108 (08) ◽  
pp. 284-290 ◽  
Author(s):  
Petra Jilma-Stohlawetz ◽  
Paul Knöbl ◽  
James C. Gilbert ◽  
Bernd Jilma
Keyword(s):  
Von Willebrand Factor ◽  
Plasma Concentrations ◽  
Fold Increase ◽  
Von Willebrand Disease ◽  
Platelet Counts ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

SummaryBlockade of hyperactive von Willebrand factor (VWF) by ARC1779 blunted the platelet drop induced by desmopressin in patients with type 2B von Willebrand disease (VWD). Thus, we hypothesised that ARC1779 may increase VWF levels and correct thrombocytopenia. Three thrombocytopenic patients suffering from type 2B VWD received a loading dose of 0.23 mg/kg ARC1779 followed by 4 μg/kg/min intravenously for 72 hours in a prospective clinical trial. ARC1779 was well tolerated and safe. Plasma concentrations of ARC1779 increased to 76 μg/ml (59–130) leading to an immediate decrease of free VWF A1 domains. VWF/FVIII levels increased as early as 12 h after start of infusion, peaked near the end of infusion, and returned to baseline at follow-up. VWF ristocetin cofactor activity (VWF:RCo) showed a median 10-fold increase 8 hours after end of infusion, while the median VWF-antigen and FVIII increase was less (5-fold and 4-fold, respectively). Most importantly inhibition of hyperactive VWF rapidly increased platelet counts from 40x109/l (38–58 x109/l) to a maximum of 146 x109/l (107–248 x109/l). In conclusion, ARC1779 markedly increases VWF/FVIII levels and most importantly improves or even corrects thrombocytopenia in VWD type 2B patients. This underscores the in vivo potency of ARC1779.

scholarly journals Adhesion of Blood Platelets Is Inhibited by VCL, a Recombinant Fragment (Leucine 504 to Lysine 728 ) of von Willebrand Factor

1996 ◽  
Vol 16 (1) ◽  
pp. 64-71 ◽  
Author(s):  
Jan J. Sixma ◽  
Martin J.W. IJsseldijk ◽  
George Hindriks ◽  
G. Henrita van Zanten ◽  
Marian Gorecki ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Blood Platelets ◽  
Recombinant Fragment ◽  
Von Willebrand ◽  
Willebrand Factor
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