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Biomedical subjects

J P Girma

Publications and source records attributed to J P Girma.

At least 19 recordsLinked to original sources
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↗
PubMed1994

Influence of mutations and size of multimers in type II von Willebrand disease upon the function of von Willebrand factor.

We compared the properties of plasma von Willebrand factor (vWF) from normal individuals and from two patients with type IIA (Glu875Lys) and type IIB (duplication of Met 540) von Willebrand disease (vWD) with the corresponding fully multimerized recombinant proteins. We included cryosupernatant from normal human plasma and type IIA plasma (Cys509Arg). Functions of vWF were analyzed by binding assays to platelets in the presence of ristocetin or botrocetin. Parameters of binding (number of binding sites per vWF subunit, and dissociation constant Kd) were quantitatively estimated from the binding isotherms of 125I-botrocetin or glycocalicin to vWF, independently of the size of the multimers. We found that ristocetin- or botrocetin-induced binding to platelets was correlated in all cases with the size of vWF multimers. In the absence of inducer, only type IIB rvWF Met-Met540 spontaneously bound to platelets. No significant difference of binding of purified botrocetin to vWF was found between normal and patients' plasma, or between wild-type rvWF (rvWF-WT) and rvWF-Lys875. In contrast, affinity of botrocetin for type IIB rvWF Met-Met540 was decreased. Botrocetin-induced binding of glycocalicin to vWF from all plasma and cryosupernatant was similar. Compared with rvWF-WT, binding of glycocalicin to rvWF-Lys875 was normal. In contrast, the affinity for type IIB rvWF Met-Met540 was 10-fold greater. Thus, our data suggest that, in the patients tested, the abnormal IIA phenotype results from the lack of large-sized multimers and is independent of the point mutations. In contrast, the type IIB mutation is directly involved by providing a conformation to the vWF subunits that allows the high molecular weight multimers to spontaneously interact with platelet glycoprotein Ib.

Animals↗
PubMed1994

Discrepancy between IIA phenotype and IIB genotype in a patient with a variant of von Willebrand disease.

Type IIA and IIB von Willebrand disease (vWD) result from qualitative abnormalities of von Willebrand factor (vWF) characterized by an absence in plasma of high molecular weight vWF multimers and an abnormal reactivity of vWF towards platelet glycoprotein (GP) Ib, which is decreased in type IIA and increased in type IIB. In this report, we describe the case of a patient having a IIA vWD phenotype associated with an intermittent thrombocytopenia atypical in this subtype but observed in type IIB vWD. The patient plasma vWF showed an absence of high molecular weight and intermediate multimers and had a decreased binding capacity to GPIb. The affinity of botrocetin was normal for plasma vWF from the propositus. Analysis of the propositus vWF gene showed the presence of a substitution Val 551 to Phe of the mature vWF subunit. This mutation is localized within a 509-695 disulphide loop of the vWF that plays an important role in the binding to GPIb and is where most of the molecular defects described so far were associated with type-IIB vWD. We have reproduced the Val 551 Phe substitution onto the vWF cDNA, expressed it in COS-7 cells, and performed structural and functional analysis of the mutant recombinant protein (rvWFPhe 551). The rvWFPhe 551 had a normal multimeric structure and showed the capacity to spontaneously interact with GPIb. Botrocetin had a decreased affinity for rvWFPhe 551. In conclusion, the Val 551 Phe mutation modifies the affinity of vWF for platelet GPIb, as does a type IIB mutation, and may be responsible for the thrombocytopenia of the patient and the clearance of the high molecular weight and intermediate-sized multimers of vWF from the plasma. The study of the rvWFPhe 551 has confirmed the discrepancy between the IIA phenotype and the IIB genotype of the patient.

Adolescent↗
PubMed1994

Interaction of von Willebrand factor with platelets activated by thrombin or a synthetic 7-amino acid peptide derived from the cleaved thrombin receptor.

Thrombin and the 7-mer agonist peptide from its receptor (SFLLRNP) were compared for their ability to promote the binding of vWF to platelets. Identical Ca(2+)-dependence and kinetics of activation were observed. Studies of inhibition of the binding by a series of monoclonal antibodies to GPIb, GPIIb/IIIa and vWF and experiments performed using platelets from patients with Glanzmann thrombasthenia or Bernard-Soulier syndrome enabled to identify GPIIb/IIIa as the receptor of vWF. Binding isotherms of vWF in the presence of an excess of either agonist yielded a similar number of binding sites but an apparent dissociation constant slightly but consistently higher with the 7-mer peptide than with thrombin. The latter point was confirmed by studying the binding of limiting amounts of vWF to platelets as a function of the agonist concentration. The lower affinity in the presence of 7-mer peptide was not corrected by adding increasing amounts of FPR-thrombin, a derivative with irreversibly blocked active site but retaining the binding properties of the active enzyme. Conversely, the higher affinity observed with thrombin was decreased when platelets were treated with Serratia protease which selectively cleaved GPIb but did not affect the function of the thrombin receptor and GPIIb/IIa. Our data thus suggest that both the 7-mer peptide and thrombin are able to induce the assembly of functional GPIIb/IIIa.(ABSTRACT TRUNCATED AT 250 WORDS)

Bernard-Soulier Syndrome↗
PubMed1993

Functional analysis of the Arg91Gln substitution in the factor VIII binding domain of von Willebrand factor demonstrates variable phenotypic expression.

An Arg91Gln substitution in the mature von Willebrand factor (vWF) has been associated with defective binding of vWF to factor VIII (FVIII). We studied four families with members initially classified as having type I von Willebrand disease (vWD) who were either homozygous or heterozygous for the Arg91Gln change. The first family was the original case described by Nishino et al. (1) where three members were homozygous for the Gln91 allele. They had a low FVIII coagulant activity:vWF antigen (VIIIC:vWFAg) ratio, from 0.29 to 0.44, and the ability of their plasma vWF to bind FVIII was markedly decreased. All the heterozygous members had normal vWF and FVIII levels but the capacity of their plasma vWF to bind FVIII was reduced and intermediate between the homozygous members and normals. The affected individual from the second family was heterozygous for the Gln91 allele and demonstrated a VIIIC:vWFAg ratio of 0.98. The FVIII binding assay confirmed the heterozygous status indicating that the moderately low levels of vWF were due to reduced expression of both alleles. The propositus from the third family was also heterozygous and had below normal levels of vWF as well as a low VIIIC:vWFAg ratio of 0.34; however, FVIII binding to her plasma vWF was similar to that of the homozygous individuals suggesting that Gln91-vWF was the major circulating form. Her daughter who has type I vWD inherited the allele without the Gln91 mutation indicating that the expression of this allele was indeed impaired. The heterozygous patient in the fourth family had a vWF level of 24 U/dl but an VIIIC:vWFAg ratio greater than 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1992

Aurin tricarboxylic acid inhibits platelet adhesion to collagen by binding to the 509-695 disulphide loop of von Willebrand factor and competing with glycoprotein Ib.

Aurin tricarboxylic acid (ATA) is known to inhibit ristocetin-induced platelet agglutination but not arachidonic acid-, epinephrine- or ADP-induced aggregation. Its capacity to abolish human von Willebrand factor (vWF)-platelet interactions was further investigated by measurement of platelet adhesion to collagen, platelet agglutination tests and binding studies. In flowing blood using parallel-plate perfusion chambers and human collagen, ATA inhibited platelet adhesion to completion in a dose-dependent manner only at the highest shear rate tested (2,600 s-1). It was without effect at 100 and 650 s-1. ATA completely abolished vWF-dependent platelet agglutination induced by ristocetin, botrocetin and asialo-vWF, respectively. 125I-vWF binding to ristocetin- and botrocetin-treated platelets, to heparin and to sulfatides as well as 125I-botrocetin binding to vWF was competitively inhibited by ATA. By contrast, binding of 125I-vWF to collagen was not affected. To further localize the domain of vWF interacting with ATA, experiments of inhibition of binding of selected 125I-monoclonal antibodies (MoAbs) to immobilized vWF by ATA were performed. Our data led to the conclusion that: 1) the interaction of ATA with vWF involves sequences of the A1 disulphide loop of vWF (residues 509-695) and close epitopes which interact with GPIb and 2) the inhibition of platelet adhesion by ATA occurs only at a high shear rate where vWF is known to play a key role. Thus ATA, which blocks the vWF/GPIb pathway by interfering with vWF and not with platelets, is a potential tool in preventing the early stages of thrombosis.

Aurintricarboxylic Acid↗
PubMed1992

Evidence that a secondary binding and protecting site for factor VIII on von Willebrand factor is highly unlikely.

A binding domain for Factor VIII (F.VIII) has been previously identified on the N-terminal portion of human von Willebrand Factor (vWF) subunit [amino acids (AA) 1-272]. In order to characterize other possible structures of vWF involved in its capacity to bind and to protect F.VIII against human activated protein C (APC), we used a series of purified vWF fragments overlapping the whole sequence of the subunit. Among those were fragments SpIII (dimer; AA 1-1365), SpII (dimer; AA 1366-2050) and SpI (monomer; AA 911-1365) generated by Staphylococcus aureus V8 proteinase, a P34 species (monomer; AA 1-272) obtained with plasmin, a monomeric 39/34 kDa dispase fragment (AA 480-718) and a tetrameric III-T2 fragment (AA 273-511/674-728) produced from SpIII by trypsin. Three other fragments without precise extremities were located using selected monoclonal antibodies to vWF. Two C-terminal fragments of 270 and 260 kDa, overlapping SpI and SpII, were respectively generated from vWF with trypsin and protease 1 from Crotalus atrox venom. An N-terminal 120 kDa fragment, overlapping P34 and 39/34 kDa fragments, was produced by protease 1. Our results show that vWF bound to F.VIII and protected it from degradation by APC in a dose-dependent way. Among the C-terminal and central vWF fragments (SpII, tryptic 270 kDa, 260 kDa, SpI, 39/34 kDa and III-T2), none had the capacity to bind or to protect F.VIII, even at high concentrations. The three N-terminal fragments (SpIII, 120 kDa and P34) bound to F.VIII in a dose-dependent and saturable fashion. SpIII and the 120 kDa fragment had the capacity to protect F.VIII in a dose-dependent way. In contrast, the P34 species did not significantly protect F.VIII, even when using high concentrations of the fragment. In conclusion, the N-terminal end of vWF subunit (AA 1-272) plays a crucial role in binding to F.VIII, but requires additional structures of the 120 kDa fragment to protect it against APC. In addition, the presence of a secondary binding and/or protecting domain on other portions of the vWF subunit (potentially destroyed during the proteolysis of vWF) is highly unlikely.

Antibodies↗
PubMed1992

Processing and characterization of recombinant von Willebrand factor expressed in different cell types using a vaccinia virus vector.

The 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 vWAgII 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The binding domain of von Willebrand factor to sulfatides is distinct from those interacting with glycoprotein Ib, heparin, and collagen and resides between amino acid residues Leu 512 and Lys 673.

A series of proteolytic fragments of human von Willebrand Factor (vWF) was purified to characterize the functional site that supports its interaction with sulfatides. SpIII, an N-terminal homodimer generated by V-8 protease (amino acids [AA] 1 to 1365), bound to sulfatides in a dose-dependent and saturable way. SpIII also totally inhibited the binding of vWF to sulfatides and SpIII binding was completely abolished by vWF. In contrast, SpII, the complementary C-terminal homodimer (AA 1366 to 2050), did not exhibit any binding affinity for sulfatides. Four purified fragments overlapping the sequence of SpIII were also tested for their ability to interact with sulfatides. An N-terminal monomeric 34-Kd fragment (P34, AA 1 to 272) generated by plasmin, a central monomer (SpI, AA 911 to 1365) produced by digestion with V-8 protease, and a tetrameric fragment III-T2 (comprising a pair of the two sequences AA 273 to 511 and AA 674 to 728) produced by secondary digestion of SpIII with trypsin did not interact with sulfatides. In contrast, a monomeric 39/34-Kd fragment produced by dispase (AA 480 to 718) bound specifically and with a high affinity to sulfatides and totally displaced vWF or SpIII binding. Conversely, binding of the 39/34-Kd species was totally abolished by vWF or SpIII. Thus, a functional site responsible for sulfatide binding was localized between AA 480 and 718 and comparison of the binding properties of the 39/34-Kd and III-T2 fragments indicated that the sequence 512 to 673 is necessary for the binding to sulfatides. Further mapping of this new functional domain of vWF, based on experiments of competitive inhibition of binding by either heparin or monoclonal antibodies directed toward vWF, showed that the site interacting with sulfatides is distinct from those involved in binding to platelet glycoprotein Ib, collagen, or heparin. This finding was confirmed by experiments using synthetic peptides which also indicated that the sequence comprising AA 569 to 584 is part of the sulfatide-binding domain or influences its activity.

Amino Acid Sequence↗
PubMed1991

von Willebrand factor: structure and function.

Von Willebrand factor (vWF) is an adhesive, multimeric glycoprotein present in plasma, platelets, and subendothelium, which has two main functions: (1) it serves as a carrier for factor VIII and (2) it plays a crucial role in platelet adhesion to subendothelium, acting as a "bridge" between platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa and subendothelial components such as collagen and heparin. vWF is involved at high shear rates in the initial contact of platelets with the subendothelium, in their subsequent spreading, and in thrombus formation. The three pools of vWF (plasma, platelets, and subendothelium) are necessary for optimal adhesion. Specific fragments of vWF involved in binding to platelets, collagen, heparin, and factor VIII have been mapped by using a series of proteases and well-characterized monoclonal antibodies to distinct epitopes of vWF. Several groups, including ours, have identified at least eight functional domains on the 270-kd subunit that consists of 2,050 amino acids. The importance of the binding domains to GP Ib and to collagen is illustrated by the role of vWF fragment SpIII (amino acids 1 through 1,365) in promoting platelet adhesion to collagen. The role of the vWF-GP Ib axis and of the vWF-GP IIb/IIIa axis in platelet-vessel wall interactions has been demonstrated through the study of patients, monoclonal antibodies, recombinant fragments, and synthetic peptides. We have recently expressed fragments of vWF complementary DNA in Escherichia coli. One of these recombinant fragments, which spans amino acids 449 through 730, binds to platelets in the presence of ristocetin, to collagen, and to heparin and has the property of inhibiting ristocetin-induced platelet agglutination. The second fragment, which spans amino acids 914 through 1,364, binds to collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗
PubMed1990

Synthetic RGDS-containing peptides of von Willebrand factor inhibit platelet adhesion to collagen.

We compared the effect of a synthetic dodecapeptide of residues 400-411 of the gamma chain of fibrinogen (gamma Fg 400-411) and of three synthetic peptides (15 to 18 aminoacids), of human von Willebrand Factor (vWF), containing the 1744-1747 Arg-Gly-Asp-Ser (RGDS) sequence, upon platelet adhesion to collagen in flowing blood. Both types of peptides are known to inhibit the binding of adhesive proteins to platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa). Collagen was coated onto plastic cover slips and exposed in parallel-plate perfusion chambers to reconstituted human blood at various shear rates for 5 min at 37 degrees C. At a shear rate of 2,600 s-1, RGDS peptides inhibited platelet adhesion to collagen in a dose-dependent manner and appeared to be more potent inhibitors than the gamma Fg 400-411 on a molar basis. No synergetic effect between RGDS and gamma Fg 400-411 peptides was observed. These results suggest that the RGDS peptides affect adhesion by inhibiting the GPIIb/IIIa-vWF interaction and confirm the involvement of this platelet receptor in vWF-mediated platelet adhesion to collagen at high shear rate.

Amino Acid Sequence↗
PubMed1990

Ristocetin and botrocetin involve two distinct domains of von Willebrand factor for binding to platelet membrane glycoprotein Ib.

We have evidence that ristocetin and botrocetin mediate binding of von Willebrand Factor (vWF) to platelet glycoprotein Ib (GPIb) through two distinct domains on the vWF molecule. This was established by using monoclonal antibodies (MAbs) to vWF and synthetic peptides derived from the sequence of vWF. MAb 322 and MAb NMC/vW4 both recognize native vWF as well as fragments containing the GPIb-binding domain of vWF, obtained with the following enzymes: trypsin (116 kDa), V-8 protease (SpIII, 320 kDa) and V-8 protease plus subtilisin (33-28 kDa). Nevertheless, the lack of reciprocal displacement between the two MAbs in experiments of competitive inhibition for binding to vWF demonstrate that their respective epitopes are separate. Both MAbs inhibit 125I-vWF binding to platelet membrane GPIb and vWF-dependent platelet agglutination induced by ristocetin. However, only MAb NMC/vW4 inhibits these functions in the presence of botrocetin and when ristocetin-induced platelet agglutination is inhibited by MAb 322, botrocetin is still able to restore the agglutination. The involvement of two distinct domains of vWF for binding to GPIb in the presence of ristocetin or botrocetin was confirmed in experiments of binding of 125I-vWF to platelets using a competitor synthetic peptides corresponding to the GPIb binding domain of vWF (Cys 474 to Pro 488 and Ser 692 to Pro 708). At a final concentration of 2.5 mM both peptides inhibit more than 90% of the binding of vWF to ristocetin-treated platelets but are unable to modify this binding in the presence of botrocetin. In conclusion our data suggest that botrocetin and ristocetin involve distinct sites on vWF for binding to GPIb.

Antibodies, Monoclonal↗
PubMed1989

von Willebrand factor and platelet function.

vWF is an adhesive protein that binds to two distinct platelet glycoproteins, GP Ib and GP IIb-IIa complex. Its interaction with GP Ib is primarily responsible for platelet adhesion to the subendothelium. The current model is that vWF binds to collagen and/or another component of the subendothelium, after which a conformational change in the vWF molecule exposes the GP Ib binding site. This interaction may not only promote the initial attachment of platelets to the subendothelium but also play a role in thrombus formation through exposure of GP IIb-IIIa to which vWF and fibrinogen can bind. The second important function of vWF is to be a carrier for F. VIII, protecting it from degradation and playing a role in its activation by thrombin. Circulating vWF has a complex multimeric structure that ranges in Mrs from 0.5 to 20 x 10(6) Daltons. The basic subunit has a Mr of 270 kDa. Amino acid sequencing of vWF demonstrated that the basic subunit or mature vWF is made up of 2050 amino acids. Molecular cloning of the vWF cDNA revealed that the primary transcript consists of 8900 base pairs that encode for 2813 amino acids, including a 22 amino acid signal peptide and a propolypeptide of 741 amino acids, called vWF antigen II. Recent studies on the expression of recombinant vWF molecules indicate that the propolypeptide is involved in the multimerization of vWF. The domains on the vWF molecule involved in the interactions of vWF with GP Ib, GP IIb-IIIa, collagen, F. VIII and heparin have been localized to varying extents. It is anticipated that peptide analysis and recombinant DNA techniques, such as in vitro mutagenesis, will further define the structural requirements of these binding domains. vWF is synthesized in a cell-specific manner by endothelial cells and megakaryocytes. It undergoes a complex intracellular biosynthesis involving transcription of a 200 kb gene, splicing out more than 42 introns, translation of a 8900 bp mRNA, glycosylation, disulphide bond formation, sulphatation, multimerization and proteolytic cleavage. The molecule can be secreted in a constitutive or regulated manner upon perturbation of the endothelial cells with physiological and non-physiological secretagogues. The mechanisms that control the synthesis of vWF should be an exciting area of further research. vWD is probably the most common of all congenital disorders of haemostasis. It is an extremely heterogeneous syndrome involving quantitative or qualitative disorders of vWF.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1989

New variant of von Willebrand disease with defective binding to factor VIII.

A new variant of von Willebrand disease (vWD) was identified by a new analytic method which characterizes the ability of plasma von Willebrand Factor (vWF) to bind to purified factor VIII (F.VIII). vWF was isolated from small amounts of plasma by immunoadsorption with a selected monoclonal antibody to vWF previously coated onto wells of microtitration plates. Plasma F.VIII was removed from immobilized vWF by washing with 0.4 mol/L CaCl2; purified F.VIII was then added to the well. The amount of bound F.VIII was estimated directly in the wells by a chromogenic assay and immobilized vWF was estimated by an immunologic a pool of normal plasma, ten control individuals, 13 with hemophilia A and five with type I vWD. In all cases, the dose-response curves were linear and the slopes of the regression lines were essentially the same. The method was then applied to investigate the binding of vWF to F.VIII in two vWD patients (sister and brother) who demonstrated significantly lower activity of F.VIII than of vWF. The first patient, with a long history of epistaxis, bruising, and hematomas, showed a slightly prolonged bleeding time (10 minutes); 15% VIII:C and 39% of vWF:Ag and vWFRCo. Her brother, who has a bleeding syndrome but no hematomas, showed similar data (bleeding time 9 minutes, 20% VIII:C, 53% vWF:Ag and vWFRCo). Similar levels of F.VIII were observed in the two propositi by four different methods (one- and two-stage clotting and chromogenic and immunologic assays). Sodium dodecyl sulfate (SDS) 1.4% agarose gel electrophoresis showed that all multimers of vWF were present in both patients. vWF binding to F.VIII was markedly decreased in the two propositi. The abnormal binding of vWF to F.VIII was not corrected during pregnancy or after infusion of 1-deamino (8-D-arginine) vasopressin despite an increase in vWF levels. The qualitative abnormality of vWF in both patients was associated with a subtle alteration of the multimeric structure by SDS 3% agarose gel electrophoresis in which the two central subbands of the quintuplet of individual oligomers were undetectable or poorly visible. SDS-polyacrylamide gel electrophoresis under reducing conditions demonstrated a single band of 275 Kd in the plasma of both patients, and there was no evidence of a second band corresponding to pro-vWF, the precursor of the mature vWF subunit, suggesting that proteolytic processing of vWF was normal.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood Coagulation Tests↗
PubMed1988

Abnormality of the N-terminal portion of von Willebrand factor in type IIA and IIC von Willebrand disease.

We have established a new analytical method which allows the characterization of von Willebrand factor (vWF) degradation fragments in minute amounts (10 microliters) of plasma, without the need for immunopurification of vWF. Plasma vWF was hydrolysed by S aureus V-8 protease (V-8 protease) and the cleaved fragments separated by SDS-agarose gel electrophoresis followed by staining with 125I-labeled polyclonal or monoclonal antibodies against vWF and autoradiography. Quantification of the amount of each product was estimated by counting the incorporated radioactivity following excision. V-8 protease limitedly hydrolysed vWF in normal as well as type I von Willebrand disease (vWD) plasma and produced two distinct fragments with identical electrophoretic and antigenic characteristics to those produced from purified vWF, i.e. a C-terminal SpII and a series of N-terminal SpIII fragments (SpIIIa, b and c). The method was applied to further characterize the molecular abnormalities of vWF in eighteen patients with type II vWD. In seven individuals with type IIA and five patients with type IIC, SpIII appeared significantly modified as compared to normal. In type IIA, there was a marked decrease or absence of SpIIIa and an increase of SpIIIb and c. In type IIC, SpIIIb was lacking. In three patients with type IIB and in three patients with type IID, there was no significant modification of SpIII. In all cases, SpII was apparently not modified. In conclusion, the molecular abnormality of vWF in type IIA and IIC vWD appears to reside in SpIII, the N-terminal portion of the vWF-subunit (residues 1 to 1,365).

Amino Acid Sequence↗
PubMed1988

von Willebrand factor-mediated platelet adhesion to collagen involves platelet membrane glycoprotein IIb-IIIa as well as glycoprotein Ib.

Monoclonal antibodies (MAbs) directed toward distinct functional domains of human von Willebrand factor (vWF) were used to probe the involvement of platelet membrane receptors glycoprotein Ib (GPIb) and glycoprotein IIb-IIIa (GPIIb-IIIa) in vWF-mediated platelet adhesion to collagen in flowing blood. Among nine MAbs to vWF, MAb H9 inhibits binding of vWF to GPIb, MAb 9 blocks binding of vWF to GPIIb-IIIa, and MAbs B200 through B204 inhibit binding of vWF to collagen. Collagen-coated cover slips were exposed to human citrated blood at shear rates varying from 200 to 2600 sec-1 in parallel-plate perfusion chambers. Blood was reconstituted with washed radiolabeled platelets, erythrocytes, and citrated autologous plasma previously incubated with MAb IgG or F(ab')2. Platelet-collagen interactions were estimated by radioactive counting and by quantitative morphometry. Inhibition of 70% of platelet adhesion was observed at a concentration of 20 micrograms/ml MAb 9, similar to that observed with MAbs H9 or B202. The effect of each MAb was dose dependent, and their inhibitory effect on platelet adhesion was also shear rate dependent. Virtually 100% inhibition was observed at 2600 sec-1 shear rate when a mixture of the MAbs (H9 + 9 + B202) was added to reconstituted blood. Thus, blocking three functional domains of vWF virtually abolishes platelet-collagen adhesion at high shear rates, indicating that normal adhesion to collagen is mediated through binding of vWF to collagen and to both platelet membrane GPIb and GPIIb-IIIa.

Adult↗