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Introduction: von Willebrand factor (VWF) is rich in cysteine; next to important structural disulfide bonds, free thiol groups are present. Free thiols on the surface of plasmatic VWF have been shown to play a role in VWF self-association and in platelet binding under pathologically high levels of shear stress. The present study explores the role of VWF free thiol groups under physiological levels of shear stress and in interactions with collagen and platelet-GPIbα receptor., Materials and Methods: Free and accessible thiol groups were blocked with N-ethylmaleimide (NEM) and the derivatized molecule was evaluated in functional assays. Reduced cysteine residues were identified using biotin-linked maleimide (MPB) followed by analysis of multimer and domain incorporation and by analysis of derivatized tryptic peptides by mass spectrometry., Results: Blockade of free thiol groups significantly reduced VWF-mediated platelet recruitment to collagen under physiological flow conditions. This resulted from inhibition of VWF binding to both collagen and the platelet GPIb receptor. Evaluation of derivatization sites revealed a high level of derivatization in the cysteine-rich N- and C-termini of VWF. 19 MPB-derivatized peptides, 13 of which are described here for the first time, were identified by mass spectrometry., Conclusions: This study shows a significant contribution of free thiol groups in VWF to the mediation of platelet adhesion under physiological shear stress conditions. The free thiol groups are shown to be involved in VWF binding to both collagen III and platelet GP1b receptor., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: The multimeric form of von Willebrand factor (VWF), is the largest soluble protein in mammals and exhibits a multidomain structure resulting in multiple functions. Upon agonist stimulation endothelial cells secrete VWF multimers from Weibel-Palade bodies into the blood stream where VWF plays an essential role in platelet-dependent primary hemostasis. Elongation of VWF strings on the cells' surface leads to accessibility of VWF binding sites for proteins, such as platelet membrane glycoprotein Ib. The prothrombotic strings are size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage., Material and Methods: VWF string formation was induced by histamine stimulation of HUVEC cells under unidirectional shear flow and VWF strings were detected employing the VWF binding peptide of platelet glycoprotein Ib coupled to latex beads. VWF strings were then used as substrate for kinetic studies of recombinant and plasma ADAMTS13., Results: To investigate specific aspects of the shear-dependent functions of VWF and ADAMTS13, we developed a shear flow assay that allows observation of VWF string formation and their degradation by ADAMTS13 without the need for isolated platelets. Our assay specifically detects VWF strings, can be coupled with fluorescent applications and allows semi-automated, quantitative assessment of recombinant and plasma ADAMTS13 activity., Conclusions: Our assay may serve as a valuable research tool to investigate the biochemical characteristics of VWF and ADAMTS13 under shear flow and could complement diagnostics of von Willebrand Disease and Thrombotic Thrombocytopenic Purpura as it allows detection of shear flow-dependent dysfunction of VWD-associated VWF mutants as well as TTP-associated ADAMTS13 mutants., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy (TMA), related to a severe functional deficiency of ADAMTS13 activity (<10% of normal). ADAMTS13 activity is thus crucial to confirm the clinical suspicion of TTP, to distinguish it from other TMAs, and to perform the follow-up of TTP patients., Material and Methods: We compared the performance of the commercial chromogenic assay Technozym ADAMTS13 Activity ELISA (chromogenic VWF73 substrate, Chr-VWF73, Technoclone, Vienna, Austria), to that of our in-house FRETS-VWF73 used as reference method. A large group of 247 subjects (30 healthy volunteers and 217 patients with miscellaneaous TMAs) was studied., Results: The lower limit of detection of the Chr-VWF73 was 3%, which is well adapted to the clinically relevant threshold for TTP diagnosis (10%). Our results showed a reasonable agreement between FRETS-VWF73 and Chr-VWF73 assays to distinguish samples with an ADAMTS13 activity <10% from those with an ADAMTS13 activity >10%. However, Chr-VWF73 assay provided false negative results in ~12% of acute TTP patients. Inversely, the Chr-VWF73 assay globally underestimated ADAMTS13 activity in detectable values ranging from 11 to 100% (with a great variability compared to FRETS-VWF73), which may be a concern for the follow-up of TTP patients in remission., Conclusion: In-house assays developed and performed by expert laboratories remain the reference methods that should be used without limitation to control values provided by commercial assays when needed. Also, the development of an international reference preparation will be crucial to improve standardization., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) is a metalloprotease enzyme that regulates the size and activity of the von Willebrand factor (VWF). ADAMTS13, like many other ADAMTSs, has a WXXW motif in its thrombospondin type 1 repeat domain (TSR1). However, the function of the WXXW motif in ADAMTSs is unclear., Materials and Methods: The constructs of wild-type (WT) and WXXW mutant (W387A) ADAMTS13 was generated by PCR site-directed mutagenesis. The secretion of the protein was quantified with western blotting methods. The binding affinity of the WT or W387A mutant ADAMTS13 with Plasma-derived human VWF (pVWF) was investigated by using enzyme linked immunosorbent assay. The Cleaving activity of the WT or W387A mutant ADAMTS13 against full length pVWF was measured under denatured conditions or shear stress. The proteolytic activity was also validated with the FRETS-VWF73 assay., Results: The W387A mutant was secreted less efficiently and had a reduced binding affinity for pre-denatured pVWF in comparison to WT ADAMTS13. However, both the WT and mutant ADAMTS13 interacted equally with native pVWF. The W387A mutant showed less cleaving activity against VWF under denaturing conditions, and the same result was observed when the fluorescence resonance energy transfer substrate VWF73 (FRETS-VWF73) was used as the substrate. However, under high shear stress conditions the mutant and WT ADAMTS13 were equally able to cleave VWF., Conclusion: The WXXW motif is important for the secretion of ADAMTS13 and that it modulates the proteolytic cleavage of VWF by ADAMTS13 under denaturing conditions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Introduction: Hemophilia A is routinely treated by administration of exogenous coagulation factor VIII (FVIII). As safety and efficacy of FVIII products have improved over the years, development of FVIII-neutralizing antibodies (FVIII inhibitors) has emerged as the most serious complication. The new human cell line-derived recombinant human FVIII (human-cl rhFVIII) is the first recombinant FVIII product produced in a human cell line without additive animal proteins, with a goal of minimizing the risk of inhibitor development., Materials and Methods: Biochemical analyzes of purity, molecular and functional attributes of the novel human-cl rhFVIII were undertaken for product characterization., Results and Conclusions: Human-cl rhFVIII was shown to be highly pure, with host-cell protein and DNA traces comparable to, or lower than, currently marketed recombinant FVIII (rFVIII) products. Human-cl rhFVIII was shown to have high specific FVIII activity and characteristics similar to full-length rFVIII products. Furthermore, no significant discrepancy between one-stage and chromogenic assay results were observed for human-cl rhFVIII, indicating potency ratios of these assays comparable to the full-length rFVIII products. In functional tests, human-cl rhFVIII exhibited physiological thrombin generation and a normal rate of inactivation by activated protein C. Importantly, human-cl rhFVIII displayed higher binding capacity with von Willebrand factor than comparator products, thus minimizing circulating unbound FVIII and further reducing the potential risk of inhibitor development., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Phenotypic diagnosis of VWD, in particular type 2, is challenging. Molecular diagnosis may fail to provide clarity since mutations within a short stretch of the same domain may cause various phenotypes, and since even experts will ascribe different subtypes to similar mutations. We assessed diagnostic difficulty in VWD by investigating five cases where phenotypic data was unclear. We identified 3 novel mutations within the A1 domain of the VWF gene: L1460F (2 related patients), Y1363C (1 patient), E1389K (2 related patients). These were not found in 100 normal individuals or documented in the VWF mutation database. Detailed functional analysis of recombinant mutants included VWF multimers, VWF:Ag, VWF:RCo, VWF:CB, and Platelet-VWF binding studies, and results assessed against recombinant WT and plasma derived (pd) VWF. Multimer analysis showed clear loss of HMW VWF with E1389K only, consistent with coincident low relative CB/Ag ratio. VWF-platelet binding studies using two independent approaches showed enhanced activity for L1460F, but reduced activity for E1389K and Y1363C. A novel finding was that WT rVWF showed enhanced platelet binding in RIPA analysis compared to pdVWF with this being dependent on the dilution material used. Through these extensive studies, we assigned L1460F to type 2B, E1389K to 2A, and Y1363C to 2M VWD. Thus, although molecular analysis is not required to classify VWD patient subtypes, a thorough and combined phenotypic, genotypic and functional analysis will assist assignment of the VWD subtype., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Platelets play a central role in maintaining hemostasis mainly by binding to subendothelial collagen exposed upon vascular injury, thereby initiating thrombus formation. Platelets can bind directly to the exposed collagen through two major receptors i.e. the integrin a2b1 and glycoprotein (GP) VI. However, under high shear conditions the GPIb-V-IX receptor complex and its main ligand von Willebrand Factor are additionally needed for firm platelet adhesion to the vessel wall. In this review, we summarize the current knowledge on the individual roles and structure-function relationships of these main platelet adhesion receptors., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Introduction: ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 repeat motif. 13) is the major metalloprotease for VWF degradation. ADAMTS13 deficiency causes the accumulation of uncleaved VWF and might lead to a lethal thrombotic thrombocytopenic purpura (TTP). Thrombospondin-1 (TSP1) is considered as a reductase of VWF (von Willebrand factor) which can mildly downregulate the size of VWF by targeting on disulfide bond between VWF dimers. It was reported that TSP1 might protected VWF from cleaving by ADAMTS13, yet the underlying mechanism of this VWF protection has remained unknown., Materials and Methods: Full-length ADAMTS13 and different domains (A1,A2,A3) of human VWF were constructed and expressed respectively. The binding ability of TSP1 or ADAMTS13 with each VWF domain or full-length VWF was investigated by using enzyme linked immunosorbent assay. The inhibition of ADAMTS13 activities by the different concentrations of TSP1 were observed by western blot and residual-collagen binding assay (R-CBA) under the denaturing condition., Results: We found that ADAMTS13 interacted with the rVWF A1, A2, A3 domains and full-length VWF, while TSP1 also bound to three A domains, especially to A2 and A3 domains. We observed that TSP1 partially blocked ADAMTS13 binding to A2 domain, A3 domain and full length VWF. The results of our assays showed that TSP1 could restrain ADAMTS13 activity up to 70%., Conclusions: Our study suggested that TSP1 played competitively inhibitory role in ADAMTS13 binding and cleaving of VWF, and the potential competition might happen within A2 and A3 domains., (Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.)

Introduction: Upon stimulation, endothelial cells release von Willebrand factor (VWF) in the unusually large (UL) and hyperactive forms that are rapidly cleaved by ADAMTS-13. Mutations in the ADAMTS13 gene result in ULVWF-mediated thrombosis found in patients with familial thrombotic thrombocytopenia purpura (TTP). ADAMTS-13 fits in the consensus of the ADAMTS family metalloproteases, but also contains two unique C- terminal CUB domains. Studying mutations in CUB domains could provide insights into the functional role of these domains., Methods: Three naturally occurring mutations (C1213Y, W1245del and K1256FS) in the CUB-1 domain found in patients with TTP were expressed in Hela cells. The secretion, stability and VWF-cleaving activity of the mutants under static and flow conditions were examined., Results: The mutations impaired secretion of ADAMTS-13 to apical surface, but not to extracellular matrix of transfected Hela cells. C1213Y and K1256FS also accelerated, whereas W1245del delayed, extracellular degradation of the mutants. The mutations also resulted in a moderate decrease in cleaving plasma VWF under static conditions. However, the mutated ADAMTS-13 bound to VWF substrate similarly as the wild-type metalloprotease and remained active in cleaving (UL)VWF under flow conditions., Conclusions: The CUB-1 domain is critical for ADAMTS-13 secretion and stability upon secretion. ADAMTS-13 deficiency found in TTP patients could be resulted from reduced ADAMTS-13 secretion and, in the case of C1213Y and K1256FS accelerated degradation. W1245del is highly resistant to degradation and active in cleaving VWF.

We evaluated vWF profiles in children and adolescents with SCD and sleep hypoxemia. Mean vWF:Ag levels were significantly elevated in the SCD-hypoxemia group when compared with SCD-normoxia and control groups (p=0.007); and correlated inversely with pulse oximetry (r=-0.54, p=0.01). Densitographic analyses of vWF multimer distribution also showed an inverse correlation between %HMW-multimers and oxygen saturation (r=-0.62, p=0.03). The previously reported association between nocturnal desaturation and SCD vascular complications, including stroke, may be influenced by hypoxemic modulation of vWF as noted in this study.

At sites of vascular injury, free-flowing platelets attach to the vessel wall to initiate thrombus formation in areas of high haemodynamic shear stress, a process that is critical for both haemostasis and thrombosis. This reaction is mediated by the binding of the platelet glycoprotein (GP) Iba to the A1 domain of immobilized von Willebrand factor (VWF), resulting in a complex series of events that includes platelet adhesion, activation and aggregation. Under elevated rates of shear stress these events support the formation of platelet-derived tethers and microparticles (MPs), which contain adhesive receptors and glycoproteins, and can bind to immobilized and soluble VWF, thereby facilitating thrombus formation. Understanding of the unique functional attributes of VWF has helped elucidate the interaction between VWF and platelets and its subsequent impact on thrombus formation. This interaction is multifaceted and modulated by different environmental conditions. A novel mechanism for initiating thrombus formation under high haemodynamic forces has recently been demonstrated. Results from direct real-time visualization of blood flow studies demonstrate activation-independent platelet aggregation, which occurs exclusively through binding of soluble VWF to platelet GPIb at very high shear stress, and may thus contribute to acute thrombotic occlusion. This article reviews the attributes of VWF and their impact on thrombus formation.

In this study, cDNA of von Willebrand factor domain A1 (VWF-A1) was cloned from human umbilical vein endothelial cells, and expressed in E. coli. The expressed VWF-A1 was used as antigen to immunize Balb/c mice. Reverse transcribed-polymerase chain reaction (RT-PCR) was used to amplify VH and VL gene segment from the RNA isolated from the immunized mouse spleens. Single chain Fv (ScFv) genes were generated by splicing overlap extension and subcloned into a phagemid vector pHEN-1. Phage display library of repertoire single chain antibodies of anti-VWF-A1 was then constructed and screened for ScFvs that interact with VWF-A1. The ScFv gene of phage clone 28 that has best binding activity to VWF-A1 was cloned into pET20b vector for higher expression in E. coli strain BL-21(DE3) pLysS. The recombinant ScFv specifically reacted with VWF, rVWF-A1 and rVWF-A1/A3, but not with rVWF-A3, P-selectin and BSA. It inhibited ristocetin-induced platelet aggregation with an IC50 of 0.75 micromol/l, and its maximal extent of inhibition is 62.5%. The ScFv had no effect on thrombin-induced platelet aggregation. These data show that the ScFv is specific against VWF-A1 and could have a potential to be used as an antithrombotic agent.

Introduction: Recent studies demonstrate that deficiency of ADAMTS13, the metalloprotease that cleaves von Willebrand factor (VWF) in a shear-dependent manner, causes thrombotic thrombocytopenic purpura (TTP). Previously, ultralarge multimers of VWF were detected in the fetuses, the umbilical cords and the newborns. However, the significance of this finding is unknown., Materials and Methods: The activity of ADAMTS13 and the multimer pattern of VWF in the cord blood, as well as the VWF antigenic and ristocetin cofactor levels, were analyzed using previously published methods. The presence of ultralarge multimers was determined by densitometric analysis., Results: On the average, the level of VWF antigen and ristocetin cofactor activity are slightly increased in the newborns (mean+/-standard deviation, 1.66+/-0.76 and 1.45+/-0.64 U/ml, respectively). Ultralarge VWF multimers are detected in 11 of 17 umbilical cord plasma samples. However, the ADAMTS13 is normal (0.99+/-0.15 U/ml) in all but one sample in which it is mildly decreased (0.71 U/ml)., Conclusions: Since ADAMTS13 activity is normal, we speculate that the presence of ultralarge multimers in the umbilical cord blood may be due to the low levels of shear stress in the umbilical circulation. These results may have important implications for understanding the manifestation of ADAMTS13 deficiency during the neonatal period.

The results described in this report demonstrate the feasibility of using AFM in combination with immuno-gold labeling to investigate the accessibility of various binding sites on vWF and to localize the binding site within a vWF multimer. With the aid of monoclonal antibodies [5, 11 and 23] it should be possible to use this approach to perform a quantitative assessment of the differential accessibility of various binding sites on vWF. This should allow localization and quantification of binding sites within the observed tertiary structure of the vWF, providing a measure of the accessibility, a point of reference with which the tertiary structure of vWF could be correlated to the primary sequence, and a means to determine the structural features of the antibody binding regions under physiologic buffer conditions. There are a number of obvious questions that are not addressed here: The role of different biologic and artificial surfaces; time-dependent effects; vWF orientation with respect to different thrombogenic surfaces; and the location of critical binding sites, such as for platelet GPIalpha and GPIIb-IIIa binding regions in the hydrated tertiary structure of vWF. Nevertheless, the work described in this report provides essential groundwork that should provide a novel basis on which to explore the molecular steps, both structural and functional, of vWF in thrombus development. In a wider sense, this experimental approach is applicable to structure-function studies on a wide variety of proteins in physiologic environments.

A highly sensitive and rapid clinical method for the visualization of the multimeric structure of von Willebrand Factor in plasma and platelets is described. The method utilizes submerged horizontal agarose gel electrophoresis, followed by transfer of the von Willebrand Factor onto a polyvinylidine fluoride membrane, and immunolocalization and luminographic visualization of the von Willebrand Factor multimeric pattern. This method distinguishes type 1 from types 2A and 2B von Willebrand disease, allowing timely evaluation and classification of von Willebrand Factor in patient plasma. It also allows visualization of the unusually high molecular weight multimers present in platelets. There are several major advantages to this method including rapid processing, simplicity of gel preparation, high sensitivity to low concentrations of von Willebrand Factor, and elimination of radioactivity.

Activation of the von Willebrand Factor (vWF) A1 domain is a critical factor in regulating the interaction of vWF with its platelet membrane receptor, the glycoprotein (GP) Ib-IX-V complex. This activation controls vWF-dependent platelet adhesion at high shear. The vWF-GP Ib-IX-V interaction is induced in vivo by exposure of platelet-rich plasma to high shear force, or by association of vWF with one or more unidentified components of the subendothelial matrix. In vitro, soluble vWF is activated to bind to platelets by nonphysiological modulators, such as the bacterial glycopeptide, ristocetin, or the snake venom protein, botrocetin, or by removal of negatively-charged sialic acid residues. Analysis of vWF modulators and the very marked charge asymmetry of amino acid sequences within the A1 domain has led to an electrostatic model for vWF modulation. Endothelial membrane/matrix and detergent-soluble fractions of human placenta were screened for the ability to bind vWF by electrophoresis of extracts on SDS-polyacrylamide gels, electrotransferring to nitrocellulose and probing with fluid-phase 125I-labeled vWF or a 39/34-kDa vWF fragment (Leu-480-Gly-718) that encompasses the A1 domain. In the course of these studies, it was found that both vWF and the 39/34-kDa vWF fragment bound strongly to histone. Purified soluble histone also bound vWF since, like ristocetin, it induced vWF flocculation. Histone binding to vWF did not activate or inhibit vWF binding to platelets. While the vWF-histone interaction has no conceivable physiological role, it suggests that binding to the A1 domain of vWF alone is insufficient to modulate vWF adhesive activity. This implies that specific interactions of the vWF A1 domain with either ristocetin or botrocetin are required for GP Ib-IX-V recognition to occur.

The smallest circulating von Willebrand factor (vWF) molecule is a dimer composed of two identical subunits containing binding sites for heparin, collagen, platelet glycoproteins and coagulation factor VIII (FVIII). Interdimeric disulfide linking leads to multimers composed of up to 40 dimers. vWF serves as a carrier of FVIII and is required for normal interactions of platelets with the subendothelium of the injured vessel wall. Von Willebrand factor was purified from human plasma cryoprecipitate and fermentation supernatant of recombinant CHO cells by anion exchange chromatography. Heparin affinity chromatography was used to isolate vWF polymers of different degree of multimerization. Analysis of collagen binding and platelet aggregation revealed that these activities increase with increasing degree of multimerization of vWF. Binding of FVIII to vWF was studied by real-time biospecific interaction analysis and surface plasmon technology. The binding data showed that the binding of FVIII is independent of vWF multimerization. Using recombinant FVIII and recombinant vWF, real-time biospecific interaction analysis resulted in a potential stoichiometry of 2 to 2.5 vWF-subunits per bound FVIII molecule. The kinetic analysis of the vWF-FVIII interaction resulted in a binding rate constant of about 3 x 10(6) M-1 s-1 and an equilibrium dissociation constant of about 0.4 x 10(-9) M.

Antithrombotic activity of two recombinant GPIb-binding fragments of vWF, RG12986 (residues 445-733), and VCL (residues 504-728), were assessed in an ex vivo capillary perfusion chamber exposing human type III collagen to native nonanticoagulated guinea pig blood. Platelet adhesion and thrombus formation were evaluated by computer assisted morphometry for two shear rates (650 and 1800 s-1) and for two perfusion times (1.5 and 4 min). At 1800 s-1 and 4 min of perfusion, platelet adhesion decreased from 63 +/- 7% for control, to 46 +/- 4% for 20 mg/kg RG12986, and to 29 +/- 5% for 4 mg/kg VCL, and the mean thrombus height dropped from 40 +/- 8 microns to 24 +/- 3 microns and 7.5 +/- 1 microns, respectively. The two doses did not change bleeding time values. Our results suggest that guinea pig blood and the circular perfusion chamber represent a good model for the evaluation of limited amount of GPIb/IX-vWF axis inhibitors.

A monoclonal antibody purified factor VIII concentrate containing FVIII/vWF complex has been assayed by one-stage clotting (CA) and chromogenic substrate (CSA) methods. The influences of potassium iodide (KI) and albumin in combination with predilution buffers, standards and storage of samples have been examined. These components are compared for their effect on FVIII potencies in final product and in-process controls. FVIII:C purified by immunoaffinity chromatography can not be measured reliably by CA or CSA, because of KI which interfere on the assay. Overall yield of FVIII, efficiency of IAC step and purity of FVIII can be determined by assaying the desalted samples.

To detect von Willebrand factor multimers in plasma samples and factor VIII concentrates, a vertical discontinuous SDS electrophoresis was developed. A vacuum blotting system allowed to improve the transfer to the nitrocellulose membrane. The visualization of the separated multimers was sensitized by applying an alkaline phosphatase anti-alkaline phosphatase staining technique. The reported method clearly shows structural abnormalities of von Willebrand factor and deficiency of high multimers, the vacuum transfer is efficient and the sensitivity of the staining system is very high.

Plasma von Willebrand factor (vWf) consists of a series of multimers of different molecular sizes. When analyzed for subunit composition, plasma vWf contained a major polypeptide of approximately 225 kD, and at least three smaller proteolytic fragments. In the present study, the subunit composition of each of these multimers was analyzed using a two dimensional electrophoresis technique. Although nearly all the multimers, as separated by SDS 1.5% agarose gel electrophoresis, contained both the major polypeptide and the smaller fragments, the relative amount of the fragments varied among the multimers. The smaller multimers contained relatively more proteolytic fragments. After the larger multimers were depleted by incubation of plasma with formaldehyde-fixed platelets and ristocetin, the intact subunit decreased, while the amount of the proteolytic fragments remained relatively unchanged. The findings of this study are consistent with the scheme that plasma multimers undergo proteolytic cleavage after multimer assembly. Furthermore, an apparent increase in the amount of these proteolytic fragments may result from mechanisms unrelated to proteolysis, such as selective adsorption of the larger multimers onto platelets.

We report the development and optimisation of an agarose gel electrophoretic method for the separation and detection of von Willebrand Factor (vWF) multimers. The method has been specifically developed for use in the clinical evaluation and classification of patients with von Willebrand's Disease (vWD) and clearly shows structural multimer abnormalities associated with the bleeding diathesis of this inherited bleeding disorder.

This study compares selected properties of purified tryptic fragments of human von Willebrand (vW) protein in order to better understand how the M(r) 116,000 fragment retains activity in ristocetin-induced platelet agglutination. Wheat germ agglutinin (WGA) aggregation of platelet-rich plasma was potentiated by preincubation of WGA with intact vW protein and with the M(r) 116,000 moiety, but not by the smaller (M(r) 45,000) or larger (M(r) 220-250,000) tryptic fragments. Since all of the fragments bound to WGA, the unique potentiating effect of the M(r) 116,000 fragment on platelet aggregation could not be explained by differential ability to form a complex with this lectin. No clearcut difference in binding of tryptic fragments to platelets in the presence of ristocetin was found, and all of the tryptic fragments had approximately equal content of carbohydrate, as reflected by periodic acid-Schiff staining and binding to the lectin Ricinus communis type II. The M(r) 116,000 fragment contains a subunit polypeptide chain of M(r) 73,000 that is lacking from both larger and smaller fragments in the tryptic digest. This chain may possess critical features that mimic the optimal structure of vW protein that forms intercellular bridges and promotes agonist stimulation by WGA via membrane receptors.