Mature Von Willebrand Factor Research Articles

A novel type 2A (Group II) von Willebrand disease mutation (L1503Q) associated with loss of the highest molecular weight von Willebrand factor multimers

Type 2A von Willebrand disease (VWD) is characterized by decreased platelet-dependent function of von Willebrand factor (VWF) associated with an absence of high-molecular-weight multimers. In this study, sequence analysis of the VWF gene from a Type 2A VWD patient showed a novel, heterozygous T-->A transversion at nucleotide 4510, resulting in the non-conservative substitution of L1503Q in the mature VWF subunit. This substitution, which was not found in 55 unrelated normal individuals, was reproduced by in vitro site directed mutagenesis of a full-length VWF cDNA and was subsequently expressed in COS-7 cells. The corresponding recombinant mutant VWF protein was partially retained in COS-7 cells yet the full spectrum of multimers was observed, suggesting that the absence of the highest molecular weight multimers results from increased proteolysis. The recombinant mutant VWF protein was digested with the ADAMTS13 protease from VWF-depleted plasma and the aberrant VWF multimer pattern was observed. These results suggest that the L1503Q substitution induces a conformational change in the VWF protein, which increases the protein's susceptibility to proteolysis. A three-dimensional model of the A2 domain demonstrates that the L1503Q mutation and the physiological proteolytic cleavage site for ADAMTS13 (Y(1605)-M(1606)) are localized close together in two adjacent parallel beta-sheets. The mutation L1503Q does not significantly disrupt the conformation of the protein; thus the subtle loss of multimers in this patient may be due to altered interactions with the ADAMTS13 protease.

How to roll an endothelial cigar: the biogenesis of Weibel-Palade bodies.

Weibel-Palade bodies (WPB) are the regulated secretory organelles of endothelial cells. These cigar-shaped membrane-bound structures function in both hemostasis and inflammation but their biogenesis is poorly understood. Here, we review what is currently known about their formation. The content of WPBs is dominated by the hemostatic factor von Willebrand factor (VWF), whose complex biogenesis ends in the formation of high molecular weight multimers. VWF is also organized into proteinaceous tubules which underlie the striated interior of WPBs as seen in the EM. VWF expression is necessary for formation of WPBs, and its heterologous expression can even lead to the specific recruitment of WPB membrane proteins, including the leukocyte receptor P-selectin, the tetraspanin CD63, and Rab27a. Unusually, the VWF propeptide is implicated in the biogenesis of WPBs, being essential for formation of the storage compartment. The elongation of the cigars and the formation of the tubules are determined by non-covalent interactions between pro- and mature VWF proteins. Surprisingly, high molecular weight multimers seem neither necessary nor sufficient to trigger formation of a storage compartment, and do not seem to have any role in WPB biogenesis. Von Willebrand's disease, usually caused by mutations within VWF, has provided many of the insights into the way in which VWF drives the formation of these organelles.

Karl landsteiner and his major contributions to haematology

Karl landsteiner and his major contributions to haematology

Critical independent regions in the VWF propeptide and mature VWF that enable normal VWF storage

Von Willebrand factor (VWF) is synthesized in endothelial cells, where it is stored in Weibel-Palade bodies. Administration of 1-desamino-8-D-arginine-vasopressin (DDAVP) to patients with type 1 von Willebrand disease and to healthy individuals causes a rapid increase in plasma VWF levels. This increase is the result of stimulated release of VWF from Weibel-Palade bodies in certain beds of endothelial cells. The VWF propeptide (VWFpp) targets VWF to storage granules through a noncovalent association. The nature of the VWFpp/VWF interaction was investigated by using cross-species differences in VWF storage. While canine VWFpp traffics to storage granules and facilitates the multimerization of human VWF, it does not direct human VWF to storage granules. Since storage takes place after furin cleavage, this defect appears to be due to the defective interaction of canine VWFpp and human VWF. To determine the regions within VWFpp and VWF important for this VWFpp/VWF association and costorage, a series of human-canine chimeric VWFpp and propeptide-deleted VWF (Deltapro) constructs were produced and expressed in AtT-20 cells. The intracellular localization of coexpressed proteins was examined by confocal microscopy. Two amino acids, 416 in VWFpp and 869 in the mature VWF molecule, were identified as being critical for the association and granular storage of VWF.

Two novel mutations, Q1053H and C1060R, located in the D3 domain of von Willebrand factor, are responsible for decreased FVIII‐binding capacity

In type 2N von Willebrand disease (VWD), von Willebrand factor (VWF) is characterized by a markedly decreased affinity for Factor VIII (FVIII), and the mutations responsible are essentially located in the D' domain of VWF. We report the identification, in seven unrelated French families, of two novel type 2N VWD mutations, Q1053H and C1060R (Gln290His and Cys297Arg in mature VWF sequence), in exon 24 of the VWF gene. These missense mutations have been identified in the heterozygous, homozygous or hemizygous states. Using site-directed mutagenesis and transient expression in COS-7 cells, we showed that both mutations, although located in the D3 domain of VWF, outside the tryptic fragment containing the FVIII domain, dramatically decrease the binding of VWF to FVIII. In contrast, the R924Q substitution, which was identified in a patient who was heterozygous for C1060R, was shown to be a polymorphism.

The role of the D1 domain of the von Willebrand factor propeptide in multimerization of VWF

While studying patient plasma containing an unusual pattern of von Willebrand factor (VWF) multimers, we discovered a previously unreported phenomenon: heavy predominance of dimeric VWF. Genomic analysis revealed a new congenital mutation (Tyr87Ser) that altered the final stages of VWF biosynthesis. This mutation in the propeptide (VWFpp) resulted in synthesis of dimeric VWF with an almost complete loss of N-terminal multimerization. The multimer pattern in patient plasma appears to result from separate alleles' synthesizing wild-type or mutant (dimeric) VWF, with homodimers composing the predominant protomeric species. We have expressed VWF protein containing the Tyr87Ser mutation and analyzed the intracellular processing and resulting VWF biological functions. The expressed dimeric VWF displayed a loss of several specific functions: collagen binding, factor VIII binding, and ristocetin-induced platelet binding. However, granular storage of dimeric VWF was normal, demonstrating that the lack of multimerization does not preclude granular storage. Although the tertiary structure of the VWFpp remains unknown, the mutant amino acid is located in a region that is highly conserved across several species and may play a major role in the multimerization of VWF. Our data suggest that one function of the highly cysteine-rich VWFpp is to align the adjacent subunits of VWF into the correct configuration, serving as an intramolecular chaperone. The integrity of the VWFpp is essential to maintain the proper spacing and alignment of the multiple cysteines in the VWFpp and N-terminus of the mature VWF.

The von Willebrand factor propeptide (VWFpp) traffics an unrelated protein to storage.

The von Willebrand factor (VWF) propeptide (VWFpp) is critical for the targeting of VWF multimers to storage granules. VWFpp alone efficiently navigates the storage pathway in AtT-20 and endothelial cells and chaperones mature VWF multimers to storage granules when the two proteins are expressed in cis or in trans. To further define the role of VWFpp in granular sorting, we examined its ability to sort an unrelated protein, C3alpha into the regulated secretory pathway. Chimeric constructs of VWFpp and the alpha-chain of C3 were developed. The C3alpha protein expressed alone did not sort to granules in AtT-20 cells. The trans expression of C3alpha and VWFpp resulted in granular storage of VWFpp but no corresponding storage of C3alpha. When C3alpha is expressed as a single chain molecule with VWFpp that was rendered uncleavable by furin, C3alpha is re-routed to storage and is colocalized with VWFpp. The uncleavable protein was expressed in bovine aortic endothelial cells where it sorted to Weibel-Palade bodies, colocalized with bovine VWF, and was released when agonist stimulated. We now demonstrate that VWFpp re-routes a constitutively secreted protein to the regulated storage pathway. Furthermore, our studies suggest that the VWFpp storage signal is contained within amino acids 201 to 741.

Biosynthesis, processing and secretion of von Willebrand factor: biological implications

von Willebrand factor is a multimeric plasma glycoprotein that is required for normal haemostasis. von Willebrand factor is synthesized by endothelial cells and megakaryocytes, and originates from its precursor pro-von Willebrand factor. The endoproteolytic processing of pro-von Willebrand factor results in mature von Willebrand factor and von Willebrand factor propeptide (also known as von Willebrand Ag II). In endothelial cells, the propeptide controls the polymerization and subsequent targeting of von Willebrand factor to the storage vesicles, the so-called Weibel–Palade bodies. Upon stimulation of the endothelial cells, the Weibel–Palade bodies are translocated to the plasma membrane of the cell, and mature von Willebrand factor and its propeptide are co-secreted. After release, these polypeptides have divergent fates and serve different biological functions. Mature von Willebrand factor both controls platelet adhesion and aggregation at sites of vascular injury and acts as a chaperone protein for coagulation factor VIII. The von Willebrand factor propeptide may serve a role in modulating inflammatory processes. This still growing body of information indicates that the biological function of the von Willebrand factor gene product is more diverse than was previously thought.

Type 2N von Willebrand disease: clinical manifestations, pathophysiology, laboratory diagnosis and molecular biology

Type 2N von Willebrand disease encompasses all patients with factor VIII deficiency caused by a markedly decreased affinity of von Willebrand factor for factor VIII. It is recessively inherited and clinically similar to mild haemophilia. The differential biological diagnosis is of major importance for providing the optimal treatment and relevant genetic counselling. This accurate diagnosis is based on an evaluation of the factor VIII-binding capacity of plasma von Willebrand factor. Furthermore, molecular biology techniques allow the identification of missense mutations in the von Willebrand factor gene. All of these induce the substitution of amino acid residues located in the N terminal part of the mature von Willebrand factor molecule, which contains the factor VIII binding site. Most of them induce a classical type 2N von Willebrand disease phenotype with factor VIII deficiency but a normal level and multimeric pattern of von Willebrand factor.

A Standard Nomenclature for von Willebrand Factor Gene Mutations and Polymorphisms

Examination of the entire von Willebrand factor (VWF) gene for mutations, particularly in types 1 and 3 von Willebrand disease (VWD) is becoming more widely practised. The sequence of the entire VWF gene will soon be compiled as a single sequence. For these reasons, a clearly defined nomenclature to use for numbering the VWF nucleotide and amino acid sequence is required. The following recommendations are made for VWF numbering. VWF cDNA nucleotide sequence should be numbered from the A of the initiator ATG as the +1 position. Genomic DNA should be prefixed with a "g" and also numbered from this position. Amino acid (aa) numbering should be from the initiator methionine as the +1 position with sequential numbering of aa throughout VWF. To avoid confusion with previously used numbering schemes for mature VWF, which started from serine 764 of pre-pro VWF, the use of the single letter amino acid code is recommended.

Type 2M von Willebrand disease variant characterized by abnormal von Willebrand factor multimerization

We describe a von Willebrand disease (VWD) variant characterized by low plasma and platelet von Willebrand factor (VWF), impaired ristocetin-induced VWF binding to platelet glycoprotein Ib (GPIb), and abnormal VWF multimer pattern not associated with the absence of large forms. A C-to-T transition at nucleotide 4120 in exon 28 of the VWF gene was found; this mutation introduces a cysteine at the codon for Arg 611 of mature VWF. In addition to the decreased factor VIII (FVIII) and VWF levels, ristocetin-induced platelet aggregation (RIPA) was almost absent, and VWF ristocetin cofactor activity (VWF:RCo) was significantly more decreased than VWF antigen. The patients (mother and son) also showed a defect in VWF collagen-binding activity. Plasma VWF multimers were decreased, with no limit in the size of large forms, and the normal discontinuous multimer organization was replaced by a diffuse smear, especially detectable in the large forms. This picture was emphasized by 1-deamino-8-D -arginine vasopressin (DDAVP) infusion, so that the abnormal VWF multimers appeared to have a molecular weight higher than those present in, or released by, human umbilical vein endothelial cells. DDAVP also increased FVIII and VWF levels but did not normalize the GPIb-dependent VWF functions expressed as RIPA and VWF:RCo. We include this variant in type 2M VWD, focusing on the abnormality in GPIb-dependent VWF function. We advance that this defect depends on the mutation in the GPIb binding domain of VWF rather than the abnormal VWF multimer pattern. (J Lab Clin Med 2001;137:70-6)

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.

Von Willebrand antigen II (vWAgII) distinguishes acquired von Willebrand Syndrome (AvWS) due to decreased synthesis from that due to increased clearance of vWF. † 795

Acquired von Willebrand syndrome (AvWS) is bleeding disorder that develops de novo in an individual with a negative past personal and family history of bleeding in association with laboratory studies indicative of von Willebrand disease. This diagnosis can be difficult to distinguish from hereditary von Willebrand syndrome, especially in children. Potential mechanisms causing AvWS include decreased protein synthesis, increased proteolysis of mature von Willebrand factor or accelerated clearance of mature vWF(on an immunologic or adsorptive basis). VWF is synthesized in endothelial cells and megakaryocytes as a pre-propolypeptide that undergoes proteolytic cleavage forming the propolypeptide (vWAgII) and mature vWF. These two proteins are immunologically distinct. We hypothesized that since vWAgII has different functional and immunological characteristics from mature vWF, levels of vWAgII should be normal or increased in states of increased vWF clearance or proteolysis. In contrast, levels of vW AgII should parallel those of mature vWF in states of decreased protein synthesis. We studied 24 individuals, 3-79 years of age, referred for evaluation of AvWS. All 24 had reduced levels of vWF antigen, mean 17±3 u/dL. There was a striking difference in vWAgII levels for the 5 individuals with hypothyroidism when compared to all other individuals, mean 55±4 for hypothyroid subjects v. 120±13 u/dL for all other diagnoses (p=.0001). We had previously shown that the ratio of vWF antigen to vWAgII in normal plasma and in plasma from individuals with type 1 von Willebrand disease was unity. This ratio was markedly different between the groups with AvWS, mean vWF/vWAgII for hypothyroid subjects was 0.53±.11 v. 0.14±.04 for other diagnoses (p=.02). In support of the hypothesis that decreased vWF and vWAgII levels in hypothyroidism are secondary to decreased protein synthesis, 4 patients demonstrated normalization of both vWF and vWAgII following thyroid replacement. Although no subject studied had an inhibitor of vWF ristocetin cofactor activity, 3 euthyroid subjects who underwent DDAVP trials demonstrated accelerated clearance of vWF antigen. vWAgII successfully differentiates AvWS caused by decreased synthesis from AvWS due to increased clearance of vWF.

Leu 697-->Val mutation in mature von Willebrand factor is responsible for type IIB von Willebrand disease

Type IIB von Willebrand disease is characterized by the selective loss of high molecular weight von Willebrand factor (vWF) multimers from plasma and enhanced platelet agglutination of platelet-rich-plasma in the presence of low concentrations of ristocetin. We identified, in two related patients, a C-->G transversion resulting in the substitution of Valine for Leucine at position 697 of the mature subunit of vWF. We reproduced this mutation in vWF cDNA and expressed the recombinant protein in Cos-7 cells. The subunit composition and multimeric structure of mutated protein (rvWFLeu697Val) were similar to the wild- type recombinant (WTrvWF). Ristocetin-induced binding of rvWFLeu697Val to platelets was markedly increased in the presence of low doses of ristocetin and slightly increased with botrocetin as compared with that for WTrvWF, whereas collagen binding was not affected by the mutation. These data show that the Leu 697-->Val substitution is not a rare polymorphism but is responsible for the subtype IIB characteristic abnormalities identified in the two affected patients; however, it is not located in the area of vWF (amino acid 540 to amino acid 578) where most of the other type IIB mutations have already been reported.

Functional Analysis of the Arg91GIn 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)

Substitution of cysteine for phenylalanine 751 in mature von Willebrand factor is a novel candidate mutation in a family with type IIA von Willebrand disease

Type IIA is a variant form of von Willebrand disease (vWD) characterized by the absence of von Willebrand factor (vWF) high molecular weight multimers in plasma. Most of the candidate missense mutations potentially responsible for type IIA vWD have been found clustered within a short segment of vWF, lying between Gly742 and Glu875 of the mature subunit. The present work reports a single heterozygous T-->G transversion in eight patients from a large type IIA vWD family, resulting in the substitution Phe751-->Cys. The absence of this mutation in 100 normal vWF genes as well as the lack, in these patients, of any other abnormality within the whole exon 28 encoding amino acids 463-921 of mature vWF, provide a strong support that this non-conservative mutation may be at the origin of the disease in this family. The presence of an additional cysteine at position 751 may induce a conformational change of the vWF subunit affecting either its 'in vivo' sensitivity to proteolytic cleavage or, more likely, its intracellular transport as suggested by the abnormal multimeric pattern of platelet vWF observed in these patients.

Defects in type IIA von Willebrand disease: a cysteine 509 to arginine substitution in the mature von Willebrand factor disrupts a disulphide loop involved in the interaction with platelet glycoprotein Ib‐IX

Type IIA von Willebrand disease (vWD) is characterized by the loss of high and intermediate weight multimers of von Willebrand factor (vWF) from plasma. The 3' end of exon 28 in the vWF gene from four type IIA vWD patients was amplified by the polymerase chain reaction, cloned and sequenced. Sequencing identified two potential missense mutations resulting in the amino acid substitutions Arg 834-->Gln and Glu 875-->Lys in the mature vWF subunit within an area of vWF where mutations in type IIA vWD have been reported. Neither of these amino acid substitutions was found in over 100 normal alleles tested by allele specific oligonucleotide hybridization. A polymorphism (Val 802-->Leu) was identified in another patient. Other areas of exon 28 were analysed by denaturing gradient gel electrophoresis (DGGE) and DNA from one patient demonstrated an irregular DGGE pattern on the 5' end of the exon. Sequencing demonstrated an amino acid substitution of an arginine for cysteine at position 509 adjacent to an area of vWF where defects associated with type IIB vWD have been found. This substitution was not found in 100 normal chromosomes tested by restriction enzyme digestion. The Cys 509-->Arg substitution eliminates an intramolecular disulphide bridge formed by Cys 509 and Cys 695 which is important to maintain the configuration of vWF functional domains that interact with platelet glycoprotein Ib-IX.

The pro-polypeptide of von Willebrand factor is required for the formation of a functional factor VIII-binding site on mature von Willebrand factor

We have established that a recombinant von Willebrand Factor (vWF) mutant (vWFdelpro) that lacks the propolypeptide, in contrast with mature wild-type vWF, with which it is identical in terms of primary amino acid sequence, is not able to form a complex with Factor VIII. Wild-type vWF (flvWF) and vWFdelpro were expressed in AtT-20 cells. Under the culture conditions employed, completely processed multimerized flvWF and dimeric vWFdelpro were secreted into the medium. FlvWF and vWFdelpro were compared for their Factor VIII-binding properties in two distinct assay systems. In a direct binding assay, purified human Factor VIII was shown to bind to flvWF that had been immobilized on the surface of microtitre wells by using an anti-vWF monoclonal antibody. In contrast, Factor VIII did not bind to immobilized vWFdelpro. In a competition assay, fluid-phase flvWF appeared to inhibit efficiently the binding of Factor VIII to immobilized vWF isolated from plasma, whereas vWFdelpro did not influence Factor VIII binding. From these observations, it is argued that the pro-polypeptide serves an essential role in the post-translational processes that lead to the expression of a functional Factor VIII-binding site on the mature vWF subunit.

A major factor VIII binding domain resides within the amino-terminal 272 amino acid residues of von Willebrand factor.

We have identified a Factor VIII (FVIII) binding domain residing within the amino-terminal 272 amino acid residues of the mature von Willebrand Factor (vWF) subunit. Two-dimensional crossed immunoelectrophoresis showed direct binding of purified human FVIII to purified human vWF. After proteolytic digestion of vWF with Staphylococcus aureus V8 protease (SP), FVIII binding was seen only with the amino-terminal SP fragment III and not with the carboxyl-terminal SP fragment II. A monoclonal anti-vWF antibody (C3) partially blocked FVIII binding to vWF and SP fragment III. FVIII also bound to vWF which had been adsorbed to polystyrene beads. This binding was inhibited in a dose-dependent manner by whole vWF, SP fragment III, and by monoclonal antibody C3. Binding could not be inhibited by SP fragment I, which contains the middle portion of the vWF molecule, or by reduced and alkylated whole vWF. SP fragment II caused only minimal inhibition. Trypsin cleavage of SP fragment III produced a monomeric 35-kDa fragment containing the amino-terminal 272 amino acid residues of vWF. This fragment reacted with monoclonal antibody C3 and inhibited the binding of FVIII to vWF in a dose-dependent manner. These studies demonstrate that a major FVIII binding site resides within the amino-terminal 272 amino acid residues of vWF.

Divergent fates of von Willebrand factor and its propolypeptide (von Willebrand antigen II) after secretion from endothelial cells.

The intracellular site of cleavage of pro-von Willebrand factor subunit and the subsequent fate of the propolypeptide (von Willebrand antigen II) and of the mature von Willebrand factor (vWf) were investigated. Both the propolypeptide, which was found to be a homodimer of noncovalently linked subunits, and mature vWf were released from Weibel-Palade bodies of endothelial cells following stimulation with secretagogues. The stoichiometry of the two released proteins was essentially equimolar. This indicates that vWf and the propolypeptide were packaged into the Weibel-Palade bodies as one unit, pro-vWf, and that the proteolytic cleavage of pro-vWf is likely to be a post-Golgi event. The association of prosequences into dimers supports their hypothetical role in the multimerization process. After secretion, the two proteins were distributed differently, as based on the following observations. The propolypeptide did not associate with vWf in the culture medium, did not codistribute with vWf in the extracellular "patches of release" on stimulated endothelial cells, and was not detected in the endothelial cell extracellular matrix, which did contain vWf. Additionally, in contrast to vWf, the propolypeptide did not bind to the matrix of human foreskin fibroblasts. Since the propolypeptide does not associate with vWf and does not interact with extracellular matrices in vitro, it is highly unlikely that it would promote platelet adhesion to subendothelium in vivo.