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J P Girma

Publications and source records attributed to J P Girma.

At least 19 recordsLinked to original sources
PubMed2005

The von Willebrand factor self-association is modulated by a multiple domain interaction.

BACKGROUND: Platelet adhesion and aggregation at sites of vascular injury exposed to rapid blood flow require von Willebrand factor (VWF). VWF becomes immobilized by binding to subendothelial components or by a self-association at the interface of soluble and surface-bound VWF. OBJECTIVES: As this self-association has been demonstrated only under shear conditions, our first goal was to determine whether the same interaction could be observed under static conditions. Furthermore, we wanted to identify VWF domain(s) important for this self-association. RESULTS: Biotinylated VWF (b-VWF) interacted dose-dependently and specifically with immobilized VWF in an enzyme-linked immunosorbent assay (ELISA) assay, showing that shear is not necessary to induce the VWF self-association. Whereas anti-VWF monoclonal antibodies (mAbs) had no effect on the self-association, the proteolytic VWF-fragments SpII(1366-2050) and SpIII(1-1365) inhibited the b-VWF-VWF interaction by 70 and 80%, respectively. Moreover, a specific binding of b-VWF to immobilized Sp-fragments was demonstrated. Finally, both biotinylated SpII and SpIII were able to bind specifically to both immobilized SpII and SpIII. Similar results were observed under flow conditions, which confirmed the functional relevance of our ELISA system. CONCLUSION: We have developed an ELISA binding assay in which a specific VWF self-association under static conditions can be demonstrated. Our results suggest a multiple domain interaction between immobilized and soluble VWF.

Antibodies, Monoclonal↗
PubMed2002

Generation of von Willebrand factor epitope libraries expressed in E. coli.

The von Willebrand factor (VWF) subunit is composed of several domains, often coinciding with structural regions, characterized through their specific interaction with a ligand. Since several monoclonal antibodies (MoAbs) have been shown to functionally interfere with one of the specific interactions, we have created libraries of bacterial clones expressing peptidic sequences of VWF to map antibodies directed against this protein. Randomly cleaved fragments of VWF cDNA have been cloned in a plasmid designed for the expression of small peptides as part of larger fusion proteins. The NovaTope system is a useful procedure for protein analysis, allowing screening of epitopes composed of contiguous amino acid residues. To map MoAbs with conformational discontinuous epitopes displayed on small as well as large peptidic domains, this technique had to be widely modified to obtain two VWF peptide libraries expressing two ranges of peptide length (15-70 and 100-300 amino acids). Screening with six MoAbs with an epitope in a known region was performed to control both libraries. Four MoAbs were mapped through the characterization of overlapping sequences for 5-10 different positively expressed clones respectively. Two of these mapped MoAbs had no known inhibitory effect and bind reduced VWF only. The fact that the two other MoAbs mapped VWF functional interactions with ligands, platelet GPIIb/IIIa and Factor VIII, respectively, demonstrate that our libraries are valuable tools to determine conformational epitopes.

Antibodies, Monoclonal↗
PubMed2001

Specific von Willebrand factor-cleaving protease in thrombotic microangiopathies: a study of 111 cases.

Retrospective studies of patients with thrombotic microangiopathies (TMAs) have shown that a deficient activity of von Willebrand factor (vWF)-cleaving protease is involved in thrombotic thrombocytopenic purpura (TTP) but not in the hemolytic-uremic syndrome (HUS). To further analyze the relevance of this enzymatic activity in TMA diagnosis, a 20-month multicenter study of vWF-cleaving protease activity was conducted in adult patients prospectively enrolled in the acute phase of TMA. Patients with sporadic (n = 85), intermittent (n = 21), or familial recurrent (n = 5) forms of TMA (66 manifesting as TTP and 45 as HUS) were included. TMA was either idiopathic (n = 42) or secondary to an identified clinical context (n = 69). vWF-cleaving protease activity was normal in 46 cases (7 TTP and 39 HUS) and decreased in 65 cases (59 TTP and 6 HUS). A protease inhibitor was detected in 31 cases and was observed only in patients manifesting TTP with a total absence of protease activity. Among the 111 patients, mean vWF antigen levels were increased and the multimeric distribution of vWF was very heterogeneous, showing either a defect of the high-molecular-weight forms (n = 40), a normal pattern (n = 21), or the presence of unusually large multimers (n = 50). Statistical analysis showed that vWF-protease deficiency was associated with the severity of thrombocytopenia (P <.01). This study emphasizes that vWF-cleaving protease deficiency specifically concerns a subgroup of TMA corresponding to the TTP entity.

ADAM Proteins↗
PubMed2001

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

INTRODUCTION: The purpose of this study was to assess the requirement for the RGD sequence of von Willebrand factor (VWF) for its binding to the beta3 chain of integrins and the structural basis for the specificity of monoclonal antibody (MoAb) 9 which specifically binds to VWF and inhibits this interaction. MATERIAL AND METHODS: : Seven point mutations were introduced into VWF by site-directed mutagenesis. Mutated recombinant VWF were tested for their multimeric pattern and their ability to bind to purified GPIIb/IIIa, thrombin-activated platelets and MoAb 9. RESULTS: All recombinant VWF were fully multimerized. The conservative Arg 1744 to Lys substitution into the RGD sequence resulted in an 80% loss of function whereas the Arg to Ala change led to a total loss of function. The two substitutions however did not impair the binding to MoAb 9. In contrast Arg 1715 to Ala substitution had no effect on the binding to GPIIb/IIIa but the binding of the corresponding mutated recombinant VWF to MoAb 9 was strikingly decreased. CONCLUSION: Direct evidence of the role of the structure and the charge of Arg 1744 into the RGD sequence were established by changing Arg to Lys (KGD) and to Ala (AGD). Our results also demonstrate that Arg 1715 is not essential in the function but it is necessary for maintaining the conformation recognized by MoAb 9 specific for the GPIIb/IIIa-binding domain of VWF.

Amino Acid Substitution↗
PubMed2001

Ser968Thr mutation within the A3 domain of von Willebrand factor (VWF) in two related patients leads to a defective binding of VWF to collagen.

We report the identification of a new mutation of von Willebrand Factor (VWF) gene within exon 30 occurring in two related patients (mother and daughter) with a hemorrhagic syndrome. A T-->A transvertion at nucleotide 5441 was found changing the serine 968 to threonine of the mature VWF subunit (S1731T of the preproVWF). The Ser968Thr mutation is located within the VWF A3 domain which interacts with type I and III collagens. Both patients were found to be heterozygous for the mutation. The propositus (daughter) exhibited a slightly prolonged bleeding time, levels of VWF:Ag and VWF:RCo at the lower limit of normal, contrasting with normal levels of VIII:C. Her mother exhibited borderline bleeding time and moderately decreased levels of VWF and VIII:C. In both patients multimeric structure of VWF and ristocetin- as well as botrocetin-induced binding of VWF to GPIb were normal; however both patients repeatedly showed decreased binding of VWF to collagen. The Ser968Thr substitution was reproduced by site-directed mutagenesis on the full-length cDNA of VWF. The mutated recombinant VWF (rVWF), T968rVWF, and the hybrid S/T968rVWF were transiently expressed by COS-7 cells. Both rVWF exhibited normal multimeric pattern and normal ristocetin- as well as botrocetin-induced binding to GPIb. T968rVWF showed significantly decreased binding to collagen while the hybrid S/T968rVWF bound to collagen in a similar way to that of the patients' plasma VWF. Thus, our data demonstrate that the Ser968Thr mutation of the VWF A3 domain is clearly responsible for the abnormal binding of VWF to collagen observed in both patients. The Ser968Thr substitution of the VWF is the first mutation identified in two patients leading to a decreased affinity of VWF for collagen and a normal multimeric structure.

Adolescent↗
PubMed2000

Improvement of von Willebrand factor proteolysis after prostacyclin infusion in severe pulmonary arterial hypertension.

BACKGROUND: The presence of dysfunctional von Willebrand factor (vWF) in pulmonary arterial hypertension (PAH) was suggested to be related to increased proteolysis. METHODS AND RESULTS: In 10 patients with severe PAH, we studied the proteolysis of plasma vWF (vWF levels, multimeric distribution, proteolytic pattern, and cleaving protease activity) and hemodynamic variables (mean pulmonary artery pressure, cardiac index, and total pulmonary vascular resistance) at baseline and 30 days after initiation of continuous prostacyclin infusion. At baseline, vWF levels were significantly increased, vWF proteolysis was excessive, and vWF-cleaving protease activity remained normal. These biological abnormalities were reversible and paralleled the improvement of hemodynamics under vasodilator treatment with prostacyclin. CONCLUSIONS: The excessive proteolysis of vWF in PAH is likely to be related to an increased susceptibility of vWF to proteases induced by high shear rates rather than to an enhanced release of enzymes.

ADAM Proteins↗
PubMed2000

Mapping and functional studies of two alloantibodies developed in patients with type 3 von Willebrand disease.

Inhibitors against von Willebrand factor (vWF) developed in two unrelated multitransfused patients (patients 1 and 2) with severe (type 3) von Willebrand disease (vWD) were analyzed. Both inhibitors were identified as antibodies of the IgG class by ELISA using immobilized purified vWF and either serum or purified Ig from the patients. Typing, mapping and functional studies of both antibodies revealed significantly distinct properties. Patient 1 antibody contained all subclasses of IgG (1, 2, 3 and 4) whereas antibody from patient 2 was a mixture of only IgG1 and 4. By ELISA using a series of immobilized purified proteolytic fragments of vWF, patient 1 antibody mainly bound to fragment SpIII and, to a lower extent, to fragments SpII and SpI; it poorly bound to P34 and the 39/34 kDa fragment. In contrast, patient 2 antibody only bound to fragments corresponding to the N-terminal portion of vWF but failed to bind to SpII. Functional studies were performed by testing the capacity of each antibody to inhibit vWF binding to its various ligands. Both antibodies blocked vWF binding to Factor VIII (FVIII), fibrillar type III collagen, bitiscetin and the subsequent induced binding to GPIb. Patient 1 antibody also blocked vWF binding to platelet GPIb when induced by ristocetin. However it failed to block vWF binding to GPIb when induced by botrocetin as well as the binding of botrocetin itself to vWF. Our data thus suggest that this inhibitor does not recognize the GPIb-binding site on vWF but the sites of vWF involved in its interaction with ristocetin. In contrast, we observed that patient 2 antibody blocked vWF binding to platelet GPIb induced by either agonist as well as vWF binding to botrocetin. Finally, the effect of the antibodies was tested on vWF binding to GPIIb/IIIa. As expected from the mapping experiments, only IgG from patient 1 blocked the interaction while IgG from patient 2 had no effect. In conclusion, we have shown that two multitransfused patients with type 3 vWD have developed alloantibodies with similar properties to those of polyclonal antibodies but with distinct effects on the functions of vWF.

Adult↗
PubMed1999

Conformational changes in the A3 domain of von Willebrand factor modulate the interaction of the A1 domain with platelet glycoprotein Ib.

Bitiscetin has recently been shown to induce von Willebrand factor (vWF)-dependent aggregation of fixed platelets (Hamako J, et al, Biochem Biophys Res Commun 226:273, 1996). We have purified bitiscetin from Bitis arietans venom and investigated the mechanism whereby it promotes a form of vWF that is reactive with platelets. In the presence of bitiscetin, vWF binds to platelets in a dose-dependent and saturable manner. The binding of vWF to platelets involves glycoprotein (GP) Ib because it was totally blocked by monoclonal antibody (MoAb) 6D1 directed towards the vWF-binding site of GPIb. The binding also involves the GPIb-binding site of vWF located on the A1 domain because it was inhibited by MoAb to vWF whose epitopes are within this domain and that block binding of vWF to platelets induced by ristocetin or botrocetin. However, in contrast to ristocetin or botrocetin, the binding site of bitiscetin does not reside within the A1 domain but within the A3 domain of vWF. Thus, among a series of vWF fragments, 125I-bitiscetin only binds to those that overlap the A3 domain, ie, SpIII (amino acid [aa] 1-1365), SpI (aa 911-1365), and rvWF-A3 domain (aa 920-1111). It does not bind to SpII corresponding to the C-terminal part of vWF subunit (aa 1366-2050) nor to the 39/34/kD dispase species (aa 480-718) or T116 (aa 449-728) overlapping the A1 domain. In addition, bitiscetin that does not bind to DeltaA3-rvWF (deleted between aa 910-1113) has no binding site ouside the A3 domain. The localization of the binding site of bitiscetin within the A3 domain was further supported by showing that MoAb to vWF, which are specific for this domain and block the interaction between vWF and collagen, are potent inhibitors of the binding of bitiscetin to vWF and consequently of the bitiscetin-induced binding of vWF to platelets. Thus, our data support the hypothesis that an interaction between the A1 and A3 domains exists that may play a role in the function of vWF by regulating the ability of the A1 domain to bind to platelet GPIb.

Antibodies, Monoclonal↗
PubMed1999

New assay for measuring binding of platelet glycoprotein IIb/IIIa to unpurified von Willebrand factor.

Among the numerous variants of vWD, no patient with an abnormal vWF binding to GPIIb/IIIa has been described to date. To search for such potential variants, we developed a two-site assay for measuring the binding of purified GPIIb/IIIa to vWF in biological fluids and we used it to study a large series of plasmas from various types of von Willebrand disease (vWD) and recombinant vWF (rvWF). vWF in plasma or rvWF in culture medium was immobilized onto anti-vWF monoclonal antibodies (MoAb)-coated wells of microtiter plates. After incubation with either unlabeled GPIIb/IIIa and a 125I-anti-GPIIb/IIIa MoAb or 125I-GPIIb/IIIa, binding curves and binding isotherms were respectively established. Normal pool plasma and wild-type rvWF were used as reference samples. We tested plasmas from 85 normal subjects, 115 patients with different types of vWD (64 type 1, 2 type 3, 9 type 2A, 4 type 2M, 16 type 2B, 15 type 2N, 3 type IID and 2 acquired forms) and 50 patients with various bleeding disorders. Four mutated rvWF with 2A (Glu875Lys and Pro885Ser) or 2B (Dupl.Met540 and Val551Phe) substitutions and one rvWF mutated in the RGD domain of the C-terminal part of vWF-subunit (Asp1746Gly) were also studied. Among the various samples tested, only rvWF Asp1746Gly had no affinity for GPIIb/IIIa. In contrast, GPIIb/IIIa similarly bound to the other vWF, independently of the proteic environment, the factor VIII level, the degree of multimerization or the mutation of vWF. Our results indicate that subjects with an abnormal vWF binding to GPIIb/IIIa are probably rare and difficult to target for a specific screening.

Biological Assay↗
PubMed1999

Estimation of the von Willebrand factor-cleaving protease in plasma using monoclonal antibodies to vWF.

A protease present in plasma cleaves von Willebrand factor (vWF) at the peptide bond 842Tyr-843Met of the mature subunit. To quantify this vWF-cleaving protease activity in plasma we have developed a simple method based on the estimation by IRMA of the degradation of a constant amount of wild type recombinant vWF used as substrate, by serial dilutions of test plasma used as protease provider. vWFAg was estimated by two-site IRMA using as first coating antibody a monoclonal antibody (MoAb) whose epitope is localized on the C-terminal side of the cleavage site, and as second labeled antibody a pool of MoAbs specific for the N-terminal side. Because the proteolytic process leads to the progressive separation of the C- and N-terminal portions of the vWF subunit such an IRMA also shows a progressive apparent loss of vWFAg. In contrast, the levels of vWFAg estimated after proteolysis by regular IRMA remained essentially constant. Results obtained with this new method were compared with the analysis by SDS-agarose gel electrophoresis of the multimeric pattern of proteolyzed WT-rvWF and no significant difference was noted testing a series of 28 plasmas. As compared with normal pooled plasma, 14 normal individuals and 13 patients with various types of vWD had normal levels of protease activity (44-178%) by both methods. The validity of the method was confirmed by showing a lack of detectable protease activity in a patient with chronic relapsing thrombotic thrombocytopenic purpura. In conclusion our method appears as a useful tool for the quantification of the vWF-cleaving protease activity in plasma. Its sensitivity and specificity are similar to those of SDS-gel electrophoresis. However, this new IRMA has the major advantages of being much simpler and faster, and open to most research laboratories in the field.

ADAM Proteins↗
PubMed1999

[Inhibition of thrombus formation by anti-Willebrand monoclonal antibodies in the guinea pig].

To experimentally evaluate the role of von Willebrand factor in thrombosis, a monoclonal antibody-712-, directed against the binding domain of von Willebrand factor to the platelet glycoprotein Ib and a control antibody D4H1, directed against the light chain of Factor VII C, were injected into guinea pigs. A model of experimental thrombosis induced by laser in guinea pig mesenteric arteries was used. The antibody 712 induced a dose-dependent reduction of the thrombus formation without affecting the bleeding time and the platelet number. Ex vivo, the ristocetin-induced platelet aggregation was inhibited by the antibody 712 in a dose-dependent manner while the adenosine diphosphate-induced platelet aggregation was not modified. In conclusion, this study showed that antibody-712- has a significant antithrombotic activity without inducing a hemorrhagic state, suggesting that the von Willebrand factor--glycoprotein Ib axis was a promising target for the strategy of a new antithrombotic therapy.

Animals↗
PubMed1998

Platelet aggregation induced by a monoclonal antibody to the A1 domain of von Willebrand factor.

Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of alphaIIb beta3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to alphaIIb beta3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds-1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds-1, DSP was increased from 5.9% +/- 3.5% in the absence of vWF to 32.7% +/- 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to alphaIIb beta3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds-1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to alphaIIbbeta3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab')2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcgamma receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.

Adult↗
PubMed1998

Assay of factor VIII antigen (VIII:CAg) in 294 haemophilia A patients by a new commercial ELISA using monoclonal antibodies.

Monoclonal antibodies (MoAbs 833 and D4H1) directed against human factor VIII (FVIII) have been produced on a large scale to measure VIII:CAg by two-site ELISA (Asserachrom VIII:CAg, Diagnostica Stago). F(ab')2 from MoAb 833 were used for coating and bound VIII:CAg was revealed with MoAb D4H1 coupled to peroxidase. Control plasma (100 VIII:CAg U dL-1 by comparing with the International Standard) was used as reference. The assay sensitivity was 0.1 U dL-1 VIII:CAg. No apparent effect of the plasma proteins was observed provided plasma dilution was > or = 5. Thus this ELISA allowed us to estimate VIII:CAg levels of 0.5 U dL-1 in plasma. Levels of VIII:CAg were similar to those of VIII:C (correlation coefficient r = 0.87) in plasma from normal individuals (32 cases) and in patients with von Willebrand disease of various types (30 cases). Among 294 patients with haemophilia A (HA), 161 had severe HA (VIII:C < 1 U dL-1). Among those patients, 124 were cross-reacting material (CRM) negative with undetectable VIII:CAg and 37 were CRM+ (VIII:CAg 1-31 U dL-1). In 42 patients with moderate HA (VIII:C 1-5 U dL-1), 33 were CRM reduced (VIII:CAg 0.5-8 U dL-1) and nine were CRM+ with a VIII:CAg/VIII:C ratio of 6-91 (mean 34.3). In mild HA (91 cases with VIII:C > or = 6 U dL-1), 29 patients were classified as CRM+ (VIII:C 6-57 U dL-1, VIII:CAg 17-130 U dL-1 and VIII:CAg/VIII:C ratio 1.8-13.7 (mean 4.51)). In 62 CRM reduced patients there was a linear correlation between VIII:C (6-39 U dL-1) and VIII:CAg (2-36 U dL-1) levels (r = 0.88). In conclusion, this sensitive assay allows us to distinguish the quantitative CRM reduced and negative from the qualitative (CRM+) abnormalities in haemophilia A.

Antibodies, Monoclonal↗
PubMed1997

Abnormal proteolytic processing of von Willebrand factor Arg611 Cys and Arg611His.

The structural and functional properties of plasma and platelet vWF were studied in 8 patients (5 unrelated families) with vWD demonstrating a mutation at position 611 (R611C or R611H). Following reduction, electrophoresis and immunoblotting with a polyclonal anti-reduced vWF antibody, abnormal proteolysis of vWF was demonstrated in plasma and to a lesser extent in platelets from all patients, leading to the formation of a unique 209 kDa fragment undetectable in control as well as in type 2A, 2B or 2N vWF. Immunoblotting with MoAbs to reduced vWF showed that the C-terminal end of the 209 kDa fragment was located beyond residue 1744 of the subunit and that its N-terminus was between residues 523 and 1114. Multimeric analysis of patients vWF showed an abnormal pattern in both plasma and platelets, with a moderate decrease of the HMW multimers together with a significant increase of the lowest MW forms. The specific sensitivity of vWF R611C and vWF R611H to proteolysis was further evidenced using V-8 protease. In all patient's samples the enzyme produced a unique monomeric 80 kDa fragment, absent in V-8 digested normal vWF, which overlapped the N-terminal part of the subunit. The functional analysis of vWF showed a markedly decreased affinity of mutated plasma vWF for platelet GPIb in the presence of ristocetin. Infusion of DDAVP in two of these patients did not lead to significant platelet count change. It induced a limited increase of the HMW multimers in plasma together with a poor correction of the vWF binding to platelet GPIb. In conclusion, our data demonstrate that in addition to a normal proteolysis, vWF mutated at position 611 undergoes a specific cleavage in plasma and platelets. In contrast to the increased proteolysis observed in type 2A and 2B patients' plasma, this additional cleavage produced a unique 209 kDa species but maintained a HMW multimer-like structure of vWF R611C and R611H.

Arginine↗
PubMed1997

Impaired prothrombin consumption in Bernard-Soulier syndrome is corrected in vitro by human factor VIII.

The Bernard-Soulier syndrome (BSS) is characterized by thrombocytopenia with giant platelets, a prolonged bleeding time with defective platelet adhesion to the subendothelium related to a defect in platelet membrane glycoprotein Ib (GPIb) and a decreased prothrombin consumption. The mechanism of the latter abnormality remains unknown. In this study, we showed that this defect was corrected by the addition of purified human factor VIII (FVIII) to blood from four patients with BSS. The correction of prothrombin consumption was almost complete at concentrations between 1.5 and 3 IU/ml of FVIII procoagulant activity (VIII:C) and partially abolished by a monoclonal antibody which neutralizes VIII:C. This correction was specific for FVIII and was not observed after addition of purified human FIX. It was obtained, in the same magnitude range, with FVIII complexed to von Willebrand factor (vWF) but not with free vWF. These data provide a new insight into the knowledge of the physiological interaction between the platelet membrane and the vWF-FVIII complex facilitating plasma coagulation activation and may lead to helpful therapeutic advances.

Antibodies, Monoclonal↗
PubMed1996

Characterization of recombinant von Willebrand factors mutated on cysteine 509 or 695.

The interacting domain of vWF with platelet GPIb has been shown to overlap the large A1 loop formed by the intra-chain disulfide bond linking Cys 509 to Cys 695. In order to further investigate the role of the conformation of this region, we have expressed in COS-7 cells three mutated full-length recombinant vWFs (rvWFs) in which the substitutions Cys509Gly, Cys509Arg or Cys695Gly have been introduced by site-directed mutagenesis. SDS-agarose gel electrophoresis demonstrated an impaired multimerization of the mutants with undetectable high molecular weight multimers and a decrease of the relative amounts of the intermediate sized multimers. Binding analysis showed that rvWFC509G and rvWFC509R did not interact with botrocetin but spontaneously interacted with GPIb; the latter binding remained unchanged in the presence of ristocetin. This indicates that the substitution of Cys509 by Gly or Arg creates a conformation of vWF that increases its binding to GPIb. In contrast, rvWFC695G which did not react with botrocetin was also unable to interact with GPIb even in the presence of ristocetin, indicating that sequences interacting with GPIb are masked and/or disrupted. In conclusion, the substitution of each of the Cys509 and 695 results in mutant proteins which may be "locked" into active or inactive conformations in regard to the binding to platelet GPIb receptor.

Cysteine↗
PubMed1995

A monoclonal antibody (B724) to von Willebrand factor recognizing an epitope within the A1 disulphide loop (Cys509-Cys695) discriminates between type 2A and type 2B von Willebrand disease.

Monoclonal antibody (MoAb) B724 to von Willebrand factor (vWF) completely inhibits its interaction with heparin, sulphatides and botrocetin and consequently botrocetin-induced binding of vWF to platelets. MoAb B724 has no effect on the binding of vWF to collagen or to ristocetin-treated platelets nor on vWF-dependent platelet aggregation induced with ristocetin and asialo-vWF-mediated platelet aggregation. MoAb B724 preferentially recognizes a conformation of native vWF, in solution, or immobilized through a coated antibody. It exhibits a markedly lower affinity for vWF immobilized onto collagen or plastic surfaces. Using proteolytic fragments of vWF, B724 epitope was localized within the 512-673 sequence of the A1 disulphide loop of vWF, MoAb B724 was used as second antibody in a two-site ELISA to test a series of patients with type 1, 2A, 2B and 2N vWD or haemophilia A and recombinant wild type or mutated vWFs. Results were compared with those obtained by control ELISAs performed using polyclonal antibodies. Using MoAb B724, strikingly lower levels of vWFAg were observed in plasma from most patients with type 2B vWD, and in seven out of the eight rvWF mutated close to or within the A1 disulphide loop. Therefore MoAb B724, which interferes with this loop involved in the function of vWF, appears to be a useful tool for rapid screening of conformational changes in this region.

Antibodies, Monoclonal↗