Von Willebrand Factor A1 Domain Research Articles

Single-Molecule Investigations of Von Willebrand Factor in Hydrodynamic Flow

Von Willebrand factor (VWF) is a multimeric blood glycoprotein that plays a pivotal role in hemostasis - it serves as a ligand for platelet adhesion and aggregation forming a hemostatic platelet plug. We are studying how hydrodynamic forces that arise in the circulation can regulate hemostasis. For example, the binding of the VWF A1 domain to the platelet protein GPIbα is correlated to conditions of strong hydrodynamic flow. We are testing the hypothesis that quaternary unfolding of VWF under hydrodynamic stress activates the VWF adhesive function by exposing A1 domains for binding to the platelet glycoprotein GPIb, and are studying the dynamics of this process.To accomplish this, we are developing powerful new techniques in single-molecule manipulation and detection to investigate how fluid forces generated in the bloodstream affect the structural conformation and function of VWF. We are creating various hydrodynamic flows to mimic a wide range of blood flow conditions, while directly visualizing the conformational dynamics of single VWF molecules in these environments. First, we have utilized a microfluidic device with a cross-slot geometry to study VWF under elongational flow. To expand these studies, we are also utilizing a microfluidic four-roll mill device that has a stagnation point that will allow prolonged observation of VWF under all flow conditions including elongational, shear, and rotational flow. In addition to the microfluidic devices, we are developing a custom “shear wheel” microscope to study fluorescently labeled VWF under shear flow. These novel techniques will enable us to understand how hydrodynamic forces in the bloodstream act on VWF to cause quaternary unfolding, and how this in turn regulates adhesive activity. These findings should also lead to a better understanding of bleeding disorders that result from abnormalities in flow conditions of the circulatory system.

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

Blockade of hyperactive von Willebrand factor (VWF) by ARC1779 blunted the platelet drop induced by desmopressin in patients with type 2B von Willebrand disease (VWD). Thus, we hypothesised that ARC1779 may increase VWF levels and correct thrombocytopenia. Three thrombocytopenic patients suffering from type 2B VWD received a loading dose of 0.23 mg/kg ARC1779 followed by 4 μg/kg/min intravenously for 72 hours in a prospective clinical trial. ARC1779 was well tolerated and safe. Plasma concentrations of ARC1779 increased to 76 μg/ml (59-130) leading to an immediate decrease of free VWF A1 domains. VWF/FVIII levels increased as early as 12 h after start of infusion, peaked near the end of infusion, and returned to baseline at follow-up. VWF ristocetin cofactor activity (VWF:RCo) showed a median 10-fold increase 8 hours after end of infusion, while the median VWF-antigen and FVIII increase was less (5-fold and 4-fold, respectively). Most importantly inhibition of hyperactive VWF rapidly increased platelet counts from 40 x 10(9)/l (38-58 x 10(9)//l) to a maximum of 146 x 10(9)//l (107-248 x 10(9)//l). In conclusion, ARC1779 markedly increases VWF/FVIII levels and most importantly improves or even corrects thrombocytopenia in VWD type 2B patients. This underscores the in vivo potency of ARC1779.

Characterization of the Binding Between the Polymeric Platelet Adhesive Proteins, Multimerin 1 and Von Willebrand Factor

Abstract 1144 Introduction:Multimerin 1 (MMRN1) is a massive variably-sized homopolymeric protein that is stored in platelet and endothelial cell secretion granules, for release with vascular injury. Recently, MMRN1 was identified to support platelet adhesion in vitro and in vivo. At high shear, MMRN1 supports platelet adhesion by a von Willebrand factor (VWF)-dependent, but integrin-independent mechanism, involving platelet glycoprotein (GP) Ibα. Direct binding of MMRN1 to GP Ibα has not been demonstrated. These data led us to postulate that VWF binds MMRN1 at site(s) distinct from the GP Ibα binding site, and test the roles of VWF A domains in MMRN1 binding. Methods:Modified enzyme linked immunosorbent assays (ELISA) and surface plasmon resonance (SPR) were used to assess binding interactions between wildtype (WT) MMRN1 and WT or domain deleted VWF constructs, and VWF polypeptides. Protein constructs tested included: multimeric VWF deletion constructs ΔA1A2A3-VWF, ΔA1A3-VWF, and ΔA1-VWF, and monomeric VWF polypeptides A1A2A3, A1A2, A1 and A3. Bovine serum albumin (BSA) coated surfaces were used as the negative control. Results:Unlike WT-VWF, VWF lacking the A domains (ΔA1A2A3-VWF) or the combination of the A1 and A3 domains (ΔA1A3-VWF) did not detectably bind to MMRN1 (p < 0.001). VWF lacking the A1 domain (ΔA1-VWF) showed MMRN1 binding comparable to WT-VWF (p = 0.39), excluding the possibility that MMRN1 binding site is located in VWF A1 domain (the region that binds GP Ibα). VWF polypeptides A1A2A3, A1A2 and A3 bound to MMRN1 (p < 0.001), unlike the VWF polypeptide A1 (p = 0.137), although the A1A2 polypeptide showed reduced binding compared to A1A2A3 (p < 0.001). SPR analyses confirmed that MMRN1 binding was supported by VWF peptides containing the A3 and/or A2 domains. Conclusions:The regions of VWF that support MMRN1 includes the A3, and possibly A2 domains, which respectively contain binding sites for collagen and ADAMTS-13. Our data suggest that the mechanism by which GP Ibα and VWF support platelet adhesion to MMRN1 at high shear include: VWF binding to GP Ibα via the A1 domain, and to MMRN1 via the A3 and possibly A2 domains. These findings have implications for the molecular mechanisms that support platelet adhesion at sites of vessel injury. Disclosures:No relevant conflicts of interest to declare.

First Case of Platelet-Type Von Willebrand Disease (PT-VWD) Associated with Type 2B Von Willebrand Disease (2B VWD)

Abstract 5313 IntroductionVWD 2B and PT-VWD are rare diseases, due to mutations inducing a gain of function respectively of von Willebrand factor (VWF) and of its platelet receptor, Glycoprotein (GP)1bα Case historyWe report here the case of a young girl, born with an extensive purpura and a severe thrombocytopenia: platelet count: 16G/L. There was no associated biological nor clinical abnormality. A high dose of 1g/kg of immunoglobulin G infused on day 1 was unsuccessful, and a HPA-1a (−) platelet concentrate infusion led to a partial and transient increase of the platelet count up to 60G/L. Thrombocytopenia then resolved spontaneously.Biological study showed no sign of materno-fetal allo- or auto-immunity, parents were not consanguineous.The diagnosis of type 2B VWD was performed when she was 5 months old: VWF:RCo < 13 IU/dl, VWF:Ag 60 IU/dl, positive ristocetin induced platelet aggregation (RIPA) at a low ristocetin concentration (0.5 mg/ml). RIPA mixing studies were unconclusive.The same biological abnormalities were found in the father, whereas the mother had normal hemostasis tests. The biological phenotype also included a study of the multimeric VWF structure, showing a marked decrease in percentage of VWF high and intermediate molecular multimers. Genetic analysis performed on VWF gene showed the heterozygous p.Pro1266Leu missense mutation in the VWF A1 domain. This mutation ( o ) is only slightly deleterious, and induces usually a mild disease, without thrombocytopenia, even in stress situations, with normal VWF multimeric distribution; therefore, it could not explain the biological phenotype severity in this family.GPIBA was then analysed, and a candidate point mutation p.Met239Ile was evidenced. This mutation had not been described yet, but p.Met255Val had already been found in diagnosed cases of PT-VWD. ConclusionThis case underlines the utmost importance to characterize precisely neonatal thrombocytopenia mechanism. Furthermore, it points out the difficulties to performing PT-VWD diagnosis, which incidence is most probably underestimated. In our case, it was the systematic and extensive biological workout performed in this case of isolated neonatal thrombocytopenia, without any obvious cause, which led to the diagnosis of a PT-VWD, inducing a severe biological phenotype, associated with type 2B VWD characterized by a mild expression. It is, to our knowledge, the first case described to date of such a morbid association. Disclosures:No relevant conflicts of interest to declare.

Inhibition of Von Willebrand Factor (VWF) by ARC1779 Normalizes Von Willebrand Factor/FVIII Levels and Corrects Thrombocytopenia in VWD Type 2b Patients,

Abstract 3377 Introduction:Infusion of the anti-VWF aptamer ARC1779 effectively elevated platelet counts in some patients with congenital thrombocytopenic purpura, and blunted the platelet drop induced by desmopressin in patients with type 2b von Willebrand disease (VWD). Thus, we hypothesized that ARC1779 may increase VWF levels and correct thrombocytopenia by blocking the hyperactive VWF A1 domain in type 2b VWD. Methods:Thrombocytopenic patients suffering from type 2b VWD received intravenous infusions of ARC1779 (4 mcg/kg/min) for 72 hours in a prospective clinical trial. Patients were monitored using previsouly published assays (Thromb Haemost. 2010;104:563–70). Data are provided as median and the range and were tested for significance by repeated measures analysis of variance. Results:All patients had moderate thrombocytopenia, low levels of VWF:RCo, VWF:CBA, FVIII:C, and collagen/ADP closure times >300s (platelet function analyzer PFA-100). ARC1779 was well tolerated and safe even in a patient who suffered from active chronic gastrointestinal bleeding due to angiodysplasias before start of infusion. Plasma concentrations of ARC1779 increased to 76 mcg/mL (59–130) which suppressed free VWF A1 domains in the REAADS assay to a minimum of <3 to 5%. Consistent with the estimated in vivo half-life of VWF, VWF/FVIII levels increased as early as 12h after start of infusion, peaked near the end of infusion, and returned to baseline at follow-up. That was best exemplified by VWF:RCo levels which increased from 32% (26–45%) 10-fold to 367% (215–418%) 8h after end of infusion, and decreased again to 33% (20–40%) during follow up. Similarly VWF:CBA levels increased 16-fold (8 to 69-fold), VWF: Ag levels 5.2 fold (3 to 27-fold), and FVIII levels 4.3 fold (3 to 6-fold). Most importantly inhibition of hyperactive VWF rapidly increased platelet counts from 40/nL (38–58/nL) to a maximum of 146/nL (107–248/nL). This was observed although patients were suffering from chronic liver disease due to hepatitis C infection or associated immune thrombocytopenia, which could have blunted the platelet response. All changes were statistically significant (p<0.007). Conclusion:This clinical trial underscores the therapeutic efficacy of the VWF A1 antagonist aptamer ARC1779. ARC1779 significantly increases VWF/FVIII levels and most importantly improves or even corrects thrombocytopenia in VWD type 2b patients. This could facilitate (i) the collection of autologous platelets for platelet transfusions pre-operatively or (ii) interferon therapy in patients whose HCV therapy could not be initiated or had to be stopped due to thrombocytopenia. As angiodysplasias may be caused by defective or deficient VWF (Blood 2011;117:1071–80), it may also be tempting to further investigate the potential of chronic anti-VWF aptamer therapy to treat angiodysplasia. Disclosures:Knoebl:Archemix Corp.: Consultancy. Pabinger:Archemix Corp.: Consultancy. Gilbert:Archemix Corp.: previous employment. Schaub:Archemix Corp.: Consultancy, previous employment. Jilma:Archemix Corp.: Consultancy, Research Funding.

Extracellular Hemoglobin Increases the Binding of the Active Form of Von Willebrand Factor to Glycoprotein Ibα and Platelet Activation Under Shear Stress: Implications for Sickle Cell Disease and Other Hemolytic Disorders

Abstract 2095Sickle cell disease (SCD) is characterized by a hypercoagulable state that accelerates vaso-occlusive events in microcirculation, leading to acute and chronic organ damage. Studies have indicated that excessive release of hemoglobin from erythrocytes into plasma may contribute to platelet activation and thrombosis. This finding is likely due to the infusion of extracellular hemoglobin (ECHb), which causes platelet aggregation and adhesion on prothrombotic surfaces, and inhibits the metalloprotease ADAMTS13, the enzyme that reduces the size of the constitutively active ultra large (UL) von Willebrand factor (VWF) multimers into a smaller non-active form of VWF. In fact, we have described that the binding of ECHb to the A2 domain of VWF prevents the cleavage of VWF by ADAMTS13. This inhibition from ECHb may raise the levels of hyperreactive VWF multimers in blood, contributing to thrombosis. Interestingly, a recent study from another research group described a direct correlation between the rate of hemolysis and the levels of circulating hyperreactive VWF multimers in plasma from SCD patients. We further analyzed the functional consequences of ECHb binding to active VWF multimers and are now describing a novel role for ECHb in VWF-mediated thrombosis. With increasing concentrations of ECHb (0 – 600 μg/ml) mixed with a constant concentration of endothelial-derived ULVWF, we observed incremental increases in the binding capacity of ULVWF for platelet GPIbα. Maximal binding capacity was obtained at 200 μg/ml of ECHb. Identical results were obtained with plasma-derived VWF using a low dose of ristocetin to activate VWF. This finding is due to the preferential binding of ECHb to the active form of VWF. The anti-A2 domain monoclonal antibody VP-1 effectively inhibited the upregulated effect of ECHb on VWF-GPIbα binding by blocking the interaction between ECHb and the A2 domain in both ULVWF and plasma VWF. We then tested the effect of ECHb on VWF-mediated platelet activation/adhesion to a surface coated with fibrin(ogen) under high shear rates. This assay required the addition of a low dose of ristocetin (0.15 mg/ml) to whole blood prior to perfusion. The effect of ristocetin on platelet activation/adhesion was effectively blocked with either EDTA or antibodies against GPIbα and αIIbβ3. ECHb (200 μg/ml) or buffer was added to whole blood prior to perfusion over the fibrin(ogen)-coated surface. At a shear rate of 1500s−1, the amount of activated/adhered platelets observed with blood containing ECHb was significantly higher (300%) than that of blood containing buffer. Comparable results were obtained when ristocetin was substituted with a gain-of-function recombinant A1A2A3 mutant (R1450E, 100nM), which exposes the A2 domain and exhibits increased GPIbα-binding activity. The number of adherent platelets from blood containing only ECHb without ristocetin was <5% of that from blood containing only ristocetin. Previously, we have demonstrated that the dissociation of A1 and A2 domains in VWF increases the binding of A1 to GPIbα. Therefore, we speculate that the interaction of ECHb with the exposed A2 domain in active VWF influences the structure of the neighboring A1 domain, provoking A1 to adopt a conformation with a higher binding affinity for GPIbα. Furthermore, these results suggest an important role for the interaction between ECHb and hyperreactive VWF in SCD pathology, and most likely other conditions presenting with microangiopathic hemolytic anemia. Disclosures:No relevant conflicts of interest to declare.

Evidence for a role of anti-ADAMTS13 autoantibodies despite normal ADAMTS13 activity in recurrent thrombotic thrombocytopenic purpura

Severe ADAMTS13 deficiency is a critical component of the pathogenesis of idiopathic thrombotic thrombocytopenic purpura but is found only in about 60% of patients clinically diagnosed with this disease. Over a period of 8 years and six episodes of thrombotic thrombocytopenic purpura we studied the evolution of the anti-ADAMTS13 antibody response in a patient using different ADAMTS13 assays and epitope mapping. Anti-ADAMTS13 autoantibodies were found in all episodes but were inhibitory only in the last two episodes. In a flow-based assay, normal ADAMTS13 activity was found only during the first disease episode, while ADAMTS13 activity was normal using a static assay in episodes 1 and 3, and severely deficient in the last two episodes. Fluorescence evolution in a modified fluorescence resonance energy transfer assay using a von Willebrand factor A2 domain peptide substrate was linear in episodes 1, 5 and 6, but increased exponentially in episodes 3 and 4. Despite the variable functional characteristics of the anti-ADAMTS13 autoantibodies, their principal epitope was the ADAMTS13 spacer domain in all episodes. The patient is unique as he displayed features of maturation or shaping of the anti-ADAMTS13 autoantibody response during the course of multiple episodes of thrombotic thrombocytopenic purpura. Anti-ADAMTS13 autoantibodies may be important in vivo despite normal ADAMTS13 activity in routine assays. Consequently, treatment decisions should not be based solely on activity assay results.

Epitope mapping of human VWF A3 recognized by monoclonal antibody SZ-123 and SZ-125 using MALDI mass spectrometry

von Willebrand factor (VWF) serves as a molecular bridge between the constituents of the subendothelium, such as collagen, and receptors of the platelet membrane, primarily GPIb. We have previously reported two monoclonal antibodies (mAbs), SZ-123 and SZ-125, which specifically bind the VWF A3 domain and block the interaction of VWF with collagen type III and ristocetin- or botrocetin-induced platelet aggregation. Here, we identified the epitopes recognized by SZ-123 and SZ-125 using matrix-assisted laser desorption ionization mass spectrometry in combination with proteolysis protection assays. Our results demonstrated that SZ-123 recognizes a discontinuous epitope, involving the residues (989)AHLLSLVDVMQR(1000) and (1017)YLTSEMHGARPGASK(1031) of VWF. SZ-125 recognizes a linear epitope, encompassing the sequence (1001)EGGPSQIGDALGFAVR(1016). Immunoassays further indicated that the synthetic peptide, NH2-EGGPSQIGDALGFAVR-COOH, is sufficient for the binding to SZ-125. These results provide insight into the mechanistic basis for the inhibition of VWF binding to collagen by SZ-123/SZ-125.

A Conformation-Sensitive Monoclonal Antibody against the A2 Domain of von Willebrand Factor Reduces Its Proteolysis by ADAMTS13

The size of von Willebrand factor (VWF), controlled by ADAMTS13-dependent proteolysis, is associated with its hemostatic activity. Many factors regulate ADAMTS13-dependent VWF proteolysis through their interaction with VWF. These include coagulation factor VIII, platelet glycoprotein 1bα, and heparin sulfate, which accelerate the cleavage of VWF. Conversely, thrombospondin-1 decreases the rate of VWF proteolysis by ADAMTS13 by competing with ADAMTS13 for the A3 domain of VWF. To investigate whether murine monoclonal antibodies (mAbs) against human VWF affect the susceptibility of VWF to proteolysis by ADAMTS13 in vitro, eight mAbs to different domains of human VWF were used to evaluate the effects on VWF cleavage by ADAMTS13 under fluid shear stress and static/denaturing conditions. Additionally, the epitope of anti-VWF mAb (SZ34) was mapped using recombinant proteins in combination with enzyme-linked immunosorbent assay and Western blot analysis. The results indicate that mAb SZ34 inhibited proteolytic cleavage of VWF by ADAMTS13 in a concentration-dependent manner under fluid shear stress, but not under static/denaturing conditions. The binding epitope of SZ34 mAb is located between A1555 and G1595 in the central A2 domain of VWF. These data show that an anti-VWF mAb against the VWF-A2 domain (A1555-G1595) reduces the proteolytic cleavage of VWF by ADAMTS13 under shear stress, suggesting the role of this region in interaction with ADAMTS13.

Biology and physics of von Willebrand factor concatamers.

Structural specialisations enable von Willebrand factor (VWF) to assemble during biosynthesis into helical tubules in Weibel-Palade bodies (WPB). Specialisations include a pH-regulated dimeric bouquet formed by the C-terminal half of VWF and helical assembly guided by the N-terminal half that templates inter-dimer disulphide bridges. Orderly assembly and storage of ultra-long concatamers in helical tubules, without crosslinking of neighboring tubules, enables unfurling during secretion without entanglement. Length regulation occurs post-secretion, by hydrodynamic force-regulated unfolding of the VWF A2 domain, and its cleavage by the plasma protease ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13). VWF is longest at its site of secretion, where its haemostatic function is most important. Moreover, elongational hydrodynamic forces on VWF are strongest just where needed, when bound to the vessel wall, or in elongational flow in the circulation at sites of vessel rupture or vasoconstriction in haemostasis. Elongational forces regulate haemostasis by activating binding of the A1 domain to platelet GPIbα, and over longer time periods, regulate VWF length by unfolding of the A2 domain for cleavage by ADAMTS13. Recent structures of A2 and single molecule measurements of A2 unfolding and cleavage by ADAMTS13 illuminate the mechanisms of VWF length regulation. Single molecule studies on the A1-GPIb receptor-ligand bond demonstrate a specialised flex-bond that enhances resistance to the strong hydrodynamic forces experienced at sites of haemorrhage.

Mechanism of von Willebrand factor scissile bond cleavage by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13)

The platelet-tethering function of von Willebrand factor (VWF) is proteolytically regulated by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which cleaves the Tyr1605-Met1606 (P1-P1') bond in the VWF A2 domain. To date, most of the functional interactions between ADAMTS13 and VWF that have been characterized involve VWF residues that are C terminal to the scissile bond. We now demonstrate that the substrate P3 position in VWF, Leu1603, is a critical determinant of VWF proteolysis. When VWF Leu1603 was substituted with Ser, Ala, Asn, or Lys in a short VWF substrate, VWF115, proteolysis was either greatly reduced or ablated (up to 400-fold reduction in k(cat)/K(m)). As Leu1603 must interact with residues proximate to the Zn(2+) ion coordinated in the active center of ADAMTS13, we sought the corresponding S3 interacting residues. Substitution of 10 candidate residues in the metalloprotease domain of ADAMTS13 identified two spatially separated clusters centered on Leu198 or Val195 (acting with Leu232 and Leu274, or with Leu151, respectively), as possible subsites interacting with VWF. These experimental findings using the short VWF115 substrate were replicated using full-length VWF. It is hypothesized that VWF Leu1603 interacts with ADAMTS13 Leu198/Leu232/Leu274 and that Val195/Leu151 may form part of a S1 subsite. The recognition of VWF Leu1603 by ADAMTS13, in conjunction with previously reported remote exosites C terminal of the cleavage site, suggests a mechanism whereby the VWF P1-P1' scissile bond is brought into position over the active site for cleavage. Together with recently characterized remote exosite interactions, these findings provide a general framework for understanding the ADAMTS family substrate interactions.

Treatment of von Willebrand disease with FVIII/VWF concentrates.

Treatment of von Willebrand disease with FVIII/VWF concentrates.

A novel calcium-binding site of von Willebrand factor A2 domain regulates its cleavage by ADAMTS13

AbstractThe proteolysis of VWF by ADAMTS13 is an essential step in the regulation of its hemostatic and thrombogenic potential. The cleavage occurs at strand β4 in the structural core of the A2 domain of VWF, so unfolding of the A2 domain is a prerequisite for cleavage. In the present study, we present the crystal structure of an engineered A2 domain that exhibits a significant difference in the α3-β4 loop compared with the previously reported structure of wild-type A2. Intriguingly, a metal ion was detected at a site formed mainly by the C-terminal region of the α3-β4 loop that was later identified as Ca2+ after various biophysical and biochemical studies. Force-probe molecular dynamic simulations of a modeled structure of the wild-type A2 featuring the discovered Ca2+-binding site revealed that an increase in force was needed to unfold strand β4 when Ca2+ was bound. Cleavage assays consistently demonstrated that Ca2+ binding stabilized the A2 domain and impeded its unfolding, and consequently protected it from cleavage by ADAMTS13. We have revealed a novel Ca2+-binding site at the A2 domain of VWF and demonstrated a relationship between Ca2+ and force in the regulation of VWF and primary hemostasis.

NBA1/MERIT40 and BRE Interaction Is Required for the Integrity of Two Distinct Deubiquitinating Enzyme BRCC36-containing Complexes

BRCC36-deubiquitinating enzyme (DUB) forms two different complexes through interactions with two different adaptor proteins Abraxas and ABRO1 in cells. Abraxas mainly localizes in the nucleus, mediating the interaction of BRCC36 with BRCA1. ABRO1 is mainly localized in the cytoplasm. Because it lacks the BRCA1-interacting motif, the ABRO1 complex does not interact with BRCA1. Both BRCC36-containing complexes contain common components including BRE and NBA1/MERIT40. Here, we found that the two complexes are assembled in a similar manner and NBA1 and BRE interaction is critical for maintaining the integrity of both of the complexes. Knockdown of NBA1 or BRE leads to decreased levels of components of the two BRCC36-containing complexes. We provided evidence that NBA1 interacts with BRE through a C-terminal conserved motif of the NBA1 protein and a C-terminal UEV domain of the BRE protein. Furthermore, the NBA1-BRE interaction is required for cellular resistance to ionizing irradiation and NBA1's role in recruiting BRCA1 to DNA damage sites. Together, these studies reveal critical interactions required for the formation and function of BRCC36-containing DUB complexes.

Diagnosis and therapeutic management in a patient with type 2B-like acquired von Willebrand syndrome

Acquired von Willebrand syndrome (AVWS) usually mimics von Willebrand disease (VWD) type 1 or 2A. However, in rare cases, the characteristics of other VWD types can predominate in AVWS that might require careful consideration of differential treatment options. The diagnosis and the treatment of a case of type 2B-like AVWS are discussed. Diagnosis of AVWS was ascertained by determining ristocetin cofactor activity, ristocetin-induced platelet aggregation, von Willebrand factor antigen, collagen binding and characterization of von Willebrand factor (VWF) multimers. Inhibitor presence was sought through mixing experiments, the Bethesda method, and calculation of the in-vivo recovery and plasma half-life of VWF after administration of factor VIII/VWF concentrate. Mutations in the A1 domain of VWF were ruled out by sequencing of exon 28 of the VWF gene. A 34-year-old male patient, putatively diagnosed with type 2B VWD, and undergoing laparoscopic cholecystectomy, did not respond adequately to perioperative hemostatic treatment with desmopressin and high doses of factor VIII/VWF concentrate, requiring the administration of recombinant activated factor VII. Further diagnostic workup revealed AVWS mimicking type 2B VWD, most likely owing to an autoantibody developed in the course of underlying monoclonal gammopathy of undetermined significance. The presence of AVWS should be considered before a diagnosis of type 2B VWD is made, especially in patients with a history atypical for inherited disease.

Mechanical Regulation of Von Willebrand Factor A1 Domain

Regulation of the bond between platelet Glycoprotein Ib (GPIb) and the von Willebrand Factor (VWF) A1 domain is critical to the balance between hemostasis and thrombosis, particularly in high shear conditions. The GPIbα-A1 interaction is known to be activated by shear stress and/or tensile mechanical force, but the structural basis of this mechanical regulation remained unknown. To address this question, we expressed a number of A1 constructs that differed in one or more residues or domains and anchored them to the surface at known concentrations and orientations. We then measured rolling velocities of platelets or GPIb-coated microspheres to determine the importance of different structural regions and the manner in which force is applied. We show that an intrinsically disordered peptide region N-terminal to the A1 domain has strong inhibitory activity. In addition, the A1-GPIb bond is much stronger when the A1 domain is anchored via the C-terminus rather than the N-terminus. This suggests that shear stress activates A1-GPIb binding in two ways. First, it applies tensile force across multimeric VWF, activating the A1 domain by dislodging this inhibitory region. Second, it applies tensile force across the A1-GPIb bond, inducing a conformational change in the C-terminal region of VWF that allosterically activates the A1 domain, perhaps in a way analogous to that seen in integrins.

Von Willebrand factor–mediated platelet adhesion is critical for deep vein thrombosis in mouse models

AbstractDeep vein thrombosis (DVT) and its complication, pulmonary embolism, are frequent causes of disability and mortality. Although blood flow disturbance is considered an important triggering factor, the mechanism of DVT initiation remains elusive. Here we show that 48-hour flow restriction in the inferior vena cava (IVC) results in the development of thrombi structurally similar to human deep vein thrombi. von Willebrand factor (VWF)–deficient mice were protected from thrombosis induced by complete (stasis) or partial (stenosis) flow restriction in the IVC. Mice with half normal VWF levels were also protected in the stenosis model. Besides promoting platelet adhesion, VWF carries Factor VIII. Repeated infusions of recombinant Factor VIII did not rescue thrombosis in VWF−/− mice, indicating that impaired coagulation was not the primary reason for the absence of DVT in VWF−/− mice. Infusion of GPG-290, a mutant glycoprotein Ibα-immunoglobulin chimera that specifically inhibits interaction of the VWF A1 domain with platelets, prevented thrombosis in wild-type mice. Intravital microscopy showed that platelet and leukocyte recruitment in the early stages of DVT was dramatically higher in wild-type than in VWF−/− IVC. Our results demonstrate a pathogenetic role for VWF-platelet interaction in flow disturbance-induced venous thrombosis.

Key role of glycoprotein Ib/V/IX and von Willebrand factor in platelet activation-dependent fibrin formation at low shear flow

AbstractA microscopic method was developed to study the role of platelets in fibrin formation. Perfusion of adhered platelets with plasma under coagulating conditions at a low shear rate (250−1) resulted in the assembly of a star-like fibrin network at the platelet surface. The focal fibrin formation on platelets was preceded by rises in cytosolic Ca2+, morphologic changes, and phosphatidylserine exposure. Fibrin formation was slightly affected by αIIbβ3 blockage, but it was greatly delayed and reduced by the following: inhibition of thrombin or platelet activation; interference in the binding of von Willebrand factor (VWF) to glycoprotein Ib/V/IX (GpIb-V-IX); plasma or blood from patients with type 1 von Willebrand disease; and plasma from mice deficient in VWF or the extracellular domain of GpIbα. In this process, the GpIb-binding A1 domain of VWF was similarly effective as full-length VWF. Prestimulation of platelets enhanced the formation of fibrin, which was abrogated by blockage of phosphatidylserine. Together, these results show that, in the presence of thrombin and low shear flow, VWF-induced activation of GpIb-V-IX triggers platelet procoagulant activity and anchorage of a star-like fibrin network. This process can be relevant in hemostasis and the manifestation of von Willebrand disease.

The in vitro effect of the new antithrombotic drug candidate ALX-0081 on blood samples of patients undergoing percutaneous coronary intervention

Compound ALX-0081 is a bivalent humanised Nanobody® that binds the A1-domain of von Willebrand factor (VWF) with high affinity. Consequently, it can block the interaction between VWF and its platelet-receptor-glycoprotein Ib, which leads inevitably to formation of arterial thrombi. It was the objective of this study to assess the in vitro effects of ALX-0081 on platelet adhesion and aggregation in coronary artery disease (CAD) patients to determine the optimal concentration of ALX-0081 and the effect of co-medication. We included nine CAD patients, who were scheduled for elective percutaneous coronary intervention (PCI), and 11 healthy volunteers. At admission all patients received aspirin, clopidogrel and heparin. Blood was drawn 24 hours (h) before and 1 h after start of the PCI procedure and was subsequently spiked with different concentrations of ALX-0081 or buffer. The efficacy of ALX-0081 was assessed by in vitro experiments: flow chamber experiments, ristocetin-induced platelet aggregation (RIPA), and the platelet function analyser (PFA-100™). VWF levels in CAD patients were significantly higher than in healthy controls. During PCI VWF levels did not rise. In all in vitro experiments, ALX-0081 led to complete inhibition of platelet adhesion and aggregation. However, the required effective concentration was higher in patients than in controls and was related to plasma VWF levels. In conclusion, ALX-0081 is able to completely inhibit in vitro platelet adhesion and aggregation in CAD patients scheduled for elective PCI. The efficacy of ALX-0081 is not influenced by PCI or co-medication. However, due to higher VWF levels in CAD patients a higher effective concentration of ALX-0081 was required than in healthy individuals.

Extracellular Hemoglobin Regulation of von Willebrand Factor Activity in Plasma of Patients with Sickle Cell Disease

Elevated levels of ultralarge (UL) von Willebrand factor (VWF) multimers in plasma play an important role in cell adhesion and vascular occlusion in sickle cell disease (SCD). Recently, we have shown that the binding of extracellular hemoglobin (ECHb) to the A2 domain of VWF significantly blocks VWF cleavage by the metalloprotease ADAMTS13in vitro. We speculated that on release from the inflamed endothelium, the VWF multimers maintain the UL structure in plasma if ECHb prevents their cleavage. We observed that a subpopulation of VWF multimers that are bound to ECHb (HbVWF), which accounted for about 14 % of the total VWF in the plasma of SCD patients. The plasma HbVWF level is parallely correlated with the ECHb and VWF-antigen levels. The HbVWF multimers are resistant to the metalloprotease ADAMTS13in vitroand are more adhesive to platelets and collagen compared with their Hb-free counterpart. Therefore, we speculate that the HbVWF, which are probably UL multimers, play an important role in tethering and stably adhering blood cells to the vascular endothelium and culminate in vascular occlusion/thrombosis/strokes in SCD patients. Thus, this article provides a new insight into the molecular pathophysiology of SCD.