Proteolysis Of Von Willebrand Factor Research Articles

Improving our understanding on the clinical role of plasmin-mediated von Willebrand factor degradation.

Von Willebrand factor (VWF) plays a pivotal role in primary hemostasis. A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13 (ADAMTS13) is primarily responsible for cleaving ultra-large VWF multimers into smaller, less adhesive forms. However, plasmin has also been shown to cleave VWF multimers. This proteolytic cleavage of VWF results in a decreased multimer size and, hence, a lower VWF activity. This review aims to present a comprehensive overview of the involvement of plasmin-mediated VWF proteolysis in (micro)thrombosis. Plasmin-mediated VWF proteolysis has been suggested to play a role in various pathologies involving microthrombosis in combination with an imbalance in VWF antigen levels and ADAMTS13 activity, as well as activation of the fibrinolytic system, but quantitative assays to demonstrate this were lacking. Recently, a V H H-based bioassay was developed designed specifically to quantify plasmin-cleaved VWF (cVWF). The novel ELISA assay holds significant promise for gaining further insights into the clinical relevance of plasmin-mediated VWF proteolysis in several pathologies. Furthermore, local plasmin activation at the site of microthrombosis has been shown to be a promising treatment strategy by degrading VWF-rich microthrombi. Plasmin-mediated proteolysis of VWF is observed during microthrombosis; however, it remains unclear whether it impacts disease severity. A novel ELISA method to detect cVWF will improve our understanding of the clinical role of plasmin-mediated VWF degradation.

Coagulation factor VIII regulates von Willebrand factor homeostasis invivo

BackgroundCoagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex, but each has its distinct functions. Binding of FVIII to VWF results in a prolongation of FVIII’s half-life in circulation and modulates FVIII’s immunogenicity during hemophilia therapy. However, the biological effect of FVIII and VWF interaction on VWF homeostasis is not fully understood. ObjectivesTo determine the effect of FVIII in VWF proteolysis and homeostasis in vivo. MethodsMouse models, recombinant FVIII infusion, and patients with hemophilia A on a high dose FVIII for immune tolerance induction therapy or emicizumab for bleeding symptoms were included to address this question. ResultsAn intravenous infusion of a recombinant B-domain less FVIII (BDD-FVIII) (40 and 160 μg/kg) into wild-type mice significantly reduced plasma VWF multimer sizes and its antigen levels; an infusion of a high but not low dose of BDD-FVIII into Adamts13+/- and Adamts13-/- mice also significantly reduced the size of VWF multimers. However, plasma levels of VWF antigen remained unchanged following administration of any dose BDD-FVIII into Adamts13-/- mice, suggesting partial ADAMTS-13 dependency in FVIII-augmented VWF degradation. Moreover, persistent expression of BDD-FVIII at ∼50 to 250 U/dL via AAV8 vector in hemophilia A mice also resulted in a significant reduction of plasma VWF multimer sizes and antigen levels. Finally, the sizes of plasma VWF multimers were significantly reduced in patients with hemophilia A who received a dose of recombinant or plasma-derived FVIII for immune tolerance induction therapy. ConclusionOur results demonstrate the pivotal role of FVIII as a cofactor regulating VWF proteolysis and homeostasis under various (patho)physiological conditions.

ADAMTS13 inhibition to treat acquired von Willebrand syndrome during mechanical circulatory support device implantation

BackgroundAcquired von Willebrand syndrome (aVWS) is common in patients with mechanical circulatory support (MCS) devices. In these patients, the high shear stress in the device leads to increased shear‐induced proteolysis of von Willebrand factor (VWF) by A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats, number 13 (ADAMTS13). As a result, the high molecular weight (HMW) VWF multimers are lost, leading to a decreased VWF function and impaired hemostasis that could explain the bleeding complications that are frequently observed in these patients. To counteract this abnormal VWF degradation by ADAMTS13, we developed a novel targeted therapy, using an anti‐ADAMTS13 monoclonal antibody (mAb) that inhibits the shear‐induced proteolysis of VWF by ADAMTS13. MethodsHuman or bovine blood was circulated through in vitro MCS device systems with either inhibitory anti‐ADAMTS13 mAb 3H9 or 17C7 (20 μg/ml) or control anti‐ADAMTS13 mAb 5C11 or phosphate buffered saline (PBS). VWF multimers and function (collagen binding activity) were determined at different time points. Next, Impella pumps were implanted in calves and the calves were injected with PBS and subsequently treated with mAb 17C7. VWF, ADAMTS13, and blood parameters were determined. ResultsWe demonstrated that blocking ADAMTS13 could prevent the loss of HMW VWF multimers in in vitro MCS device systems. Importantly, our antibody could reverse aVWS in a preclinical Impella‐induced aVWS calf model. ConclusionHence, inhibition of ADAMTS13 could become a novel therapeutic strategy to manage aVWS in MCS device patients.

Coagulation Factor VIII Enhances the Cleavage of Von Willebrand Factor By ADAMTS13 In Vivo

Introduction: Coagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex. This interaction helps increase the half-life of circulating FVIII and modulates FVIII immunogenicity. Our previous studies demonstrate that FVIII accelerates VWF proteolysis by ADAMTS13 in vitro under fluidic shear or under certain pathophysiological conditions. However, the physiological relevance of the FVIII-enhanced VWF proteolysis remains questionable. Here, we sought to determine if addition of recombinant FVIII at various concentrations to mice with or without circulating ADAMTS13 or to patients with hereditary thrombotic thrombocytopenic purpura (TTP) affects plasma VWF multimer distribution and its antigen levels. Methods: Recombinant human B-domain deleted FVIII (rFVIII) was intravenously administered into wild-type (WT),Adamts13+/-(A13+/-) and Adamts13-/- (A13-/-) mice. Blood samples were collected prior to and 24 hours following rFVIII infusion. In addition, FVIII was stably expressed in fviii-/-mice via administration of AAV8-BDDFVIII. Moreover, plasma was collected from patients with hereditary TTP who were treated with plasma derived FVIII concentrates. Plasma VWF multimers and antigen levels were determined by SDS-agarose gel electrophoresis followed by Western blotting and enzyme-linked immunoassay (ELISA), respectively. Paired T and Mann Whitney tests were performed to determine the statistical significance of the difference between two groups. Results: Our results demonstrated that an infusion of rFVIII at a low dose (40 ug/kg) and a high dose (160 ug/kg) into WT mice resulted in a dramatic reduction in plasma VWF multimer sizes (Fig. 1) and VWF antigen levels (not shown). FVIII at high dose but not at low dose could also significantly reduce the sizes of VWF multimers in Adamts13+/- and Adamts13-/- mice (Fig. 1). The enhanced degradation of VWF multimers in Adamts13-/- mice suggests that other VWF-cleaving enzymes may participate in the degradation of VWF-FVIII complex. In either Adamts13+/- or Adamts13-/- mice, administration of rFVIII at both low and high doses didn't result in significant changes in VWF antigen levels (not shown). Furthermore, a simultaneous infusion of both rFVIII and recombinant murine ADAMTS13 (rADAMTS13) into Adamts13-/- mice resulted in similar changes in plasma VWF multimer distribution (Fig. 1) and antigen levels (not shown). Importantly, all mice tolerated well to the high dose of rFVIII infusion without major thrombotic events during weeks of follow-up. The restoration of plasma FVIII to 100-200% of normal levels in the fviii-/- mice with AAV8-mediated gene therapy also led to a profound degradation of plasma VWF multimers and significantly reduced plasma levels of VWF antigen (not shown). Finally, a similar pattern of dramatically reduced plasma VWF multimers was observed in 3 out of 4 patients with hereditary TTP following treatment with plasma-derived FVIII concentrates (not shown). Conclusions: Our results demonstrate that supplementation of FVIII at physiological or super-physiological concentrations dramatically accelerates the degradation of VWF multimers in mice and human. These findings further support our hypothesis that FVIII is a physiological cofactor of ADAMTS13 in regulating VWF homeostasis. Our findings may also offer a scientific explanation how FVIII concentrates or perhaps rFVIII might work for the treatment of hereditary TTP and other arterial thrombosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Regulation of VWF Cleavage Is Mediated By Coupling ADAMTS13 D- and C- Domain Binding Sites

Introduction: The metalloprotease ADAMTS13 consists of metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains (proximal domains), followed by 7 T and 2 CUB domains (distal domains). ADAMTS13 cleaves the cryptic Tyr1605-Met1606 bond in the A2 domain of von Willebrand factor (VWF) to prevent ultra-large multimeric VWF-induced platelet aggregation. We and others previously described the allosteric regulation of ADAMTS13 by which the distal domains fold back and inhibit the proximal domains. VWF D4-CK domains bind the ADAMTS13 T8-CUB region to disrupt this allostery and accelerate VWF proteolysis. We recently discovered that ADAMTS13 and VWF have not evolved to be optimal enzyme-substrate pairs (Muia, Blood, 2019). For example, c-VWF71 (C-71), a wild-type bovine VWF domain A2-derived peptide substrate, is cleaved extremely well by many vertebrate ADAMTS13. Conversely, similar substrates prepared from wild-type human h-VWF71 (H-71) and other VWF domain A2 sequences mostly tolerated cleavage by their ADAMTS13 or additional few species-specific ADAMTS13. Although unfolded VWF A2 domain engages ADAMTS13 M, D, C, and S exosites for substrate specificity and efficient cleavage, sequence differences among the various vertebrates VWF71 substrates may hold clues about specificity determinants of ADAMTS13 substrate recognition and cleavage. Methods: Multiplex FRET ADAMTS13 assays were performed using 10 novel fluorogenic substrates. Substrates P3-P10 are chimeric substrates between C-71 and H-71; species-specific amino acid residues were swapped sequentially (Fig 1A). Plasmas from various vertebrates have been characterized and reported before. Recombinant ADAMTS13 variants were transiently expressed in HEK293 cells. Assays were performed as previously described and mean results are reported. Results: The chimeric substrates (P3-P10) were designed to substitute wild-type, species-specific amino acid residues at sites known to interact with ADAMTS13 D and C domain exosites. However, amino acid substitutions also occurred at the sites that do not interact directly with any ADAMTS13 exosite. Compared to C-71 (P2), only P3 and P6 exhibited reduced rates of cleavage by all vertebrate plasma containing ADAMTS13 (Fig 1B). The P3 substrate substituted the D binding residues from human VWF71 into C-71, whereas P6 substituted the C binding residues from human VWF71 into C-71. For P3, all ADAMTS13 assayed showed ~30-100% reduction in the rate of cleavage except for armadillo and dog plasma ADAMTS13. Likewise, P6 was cleaved ~0% to 60% slowly relative to P2 by all ADAMTS13. Substituting the D, nb-1(non-binding region 1), and nb-2 sequences from human into C-71, named P8, did not further reduce rates of cleavage compared to P3. These results suggest the nb-1 and nb-2 regions of VWF71 do not participate in protease engagement, consistent with previous studies. We wondered if swapping human VWF D- and C- binding sites to C-71 as well as the bovine's to H-71 would result in ADAMTS13 cleavage rates similar to wild-type sequences. Interestingly, there was no major change in cleavage rates when D- and C- VWF binding sites were of the same species, i.e., P9 ~ P1 and P10 ~ P2, irrespective of the peptide backbone. We also sought to know if allosteric regulation of ADAMTS13 attenuates the binding of these chimeric substrates to ADAMTS13. Pigeon (potently activated by low pH) and human plasma ADAMTS13 were activated ~15.0-28.0 and ~4.8-8.0-fold, respectively when assayed with all 10 peptides. Whether ADAMTS13 was activated or not, the ratio of cleavage for individual substrates to the parental P2 remained relatively constant (~ 0.4-1.3), implying the substrate-dependent activity is independent of allosteric activation of ADAMTS13. Conclusions: The fact that D- and C- VWF binding sites from human and bovine VWF71 cannot be combined without negatively affecting the Tyr-Met bond cleavage may suggest coupling of ADAMTS13 D and C domains to regulate its activity. It is plausible to claim that ADAMTS13 VWF D- and C- binding sites and/or ADAMTS13 D and C domains may have evolved to provide a fail-safe point for allosterically activated ADAMTS13. This may prevent substrate-activated ADAMTS13, which precedes the unraveling of the VWF domain A2, from engaging and cleaving off-targets. We conclude that VWF as a substrate serves both as an ADAMTS13 activator and activity regulator. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Acquired von Willebrand syndrome in patients with monoclonal gammopathy of undetermined significance investigated using a mechanistic approach.

Acquired von Willebrand syndrome (AVWS) has been reported to occur in association with monoclonal gammopathy, usually of undetermined significance (MGUS). It may present as a type 1 or type 2 von Willebrand factor (VWF) defect depending on the patient's representation of large VWF multimers. The mathematical model by Galvanin et al., already employed for studying inherited von Willebrand disease (VWD), was used to explore the pathogenic mechanisms behind MGUS-associated AVWS. The patients studied showed significantly reduced VWF levels and function; an increased VWF propeptide to VWF antigen ratio; and all VWF multimers present but in reduced quantities, with the low-molecular-weight VWF forms being significantly more represented than those of higher molecular weight. Our mathematical model revealed a significantly increased VWF elimination rate constant, with values similar to those of type Vicenza VWD. An even more increased VWF proteolysis rate constant was observed, with values one order of magnitude higher than in type 2A VWD but, in contrast, no loss of large multimers. The model predicted the same elimination rate for high- and low-molecular-weight VWF multimers, but proteolysis of the high-molecular-weight forms also contributes to the pool of low-molecular-weight oligomers, which explains why they were relatively over-represented. In MGUS-associated AVWS the increase of both clearance and proteolysis contributes to the circulating levels and multimer pattern of VWF, with a phenotype that appears to be a combination of type Vicenza and type 2A VWD. Hence, the mechanisms behind the onset of AVWS seem to differ from those of inherited VWD.

Apolipoprotein B-100 Binds Von Willebrand Factor and Accelerates Its Proteolysis By ADAMTS13 Under Fluidic Shear

Background : ADAMTS13 cleaves von Willebrand factor (VWF) on the surface of endothelial cells, at the site of thrombus formation, and in circulating blood under shear stress. Previous studies demonstrate that binding of VWF by coagulation factor VIII, platelet glycoprotein 1b, and ristocetin accelerates the proteolysis of VWF by ADAMTS13 under fluidic shear stress. However, it remains to be determined if other cellular and protein factors in circulation might interact with VWF and/or ADAMTS13 to regulate VWF proteolysis.Methods : To address this question, we developed a DNA aptamer-based VWF purification method to purify VWF from human plasma to homogeneity. We then determined its potential binding partners, and their role in regulation of VWF proteolysis by ADAMTS13 using a vortex-induced flow assay. Enzyme-linked immunosorbent assay (ELISA) or agarose gel electrophoresis determined the binding interactions between the novel binding partners and VWF (or ADAMTS13).Results : Purified plasma VWF exhibited a size of 250-kDa in a SDS-polyacrylamide gel under denatured and reduced conditions. Several other bands were visible on the gel and were sent for protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). One of such binding partners was apolipoprotein B-100 (ApoB-100), a primary apolipoprotein of chylomicrons, VLDL, IDL, and LDL particles, which are collectively called the “bad” cholesterol. When incubated with recombinant VWF in the presence of recombinant ADAMTS13, a purified LDL/ApoB-100 accelerated the proteolytic cleavage of multimeric VWF by ADAMTS13 under arterial shear stress (~20 dyne/cm2) in a concentration-dependent manner (see figure). Such a rate enhancing effect was abolished by the addition of EDTA (10 mM), but not by other serine protease inhibitors. VWF bound to LDL/ApoB-100 immobilized on a microtiter plate and in solution. SDS- and urea-stable VWF/ApoB complexes (500-600 kDa) were detected in normal human plasma by Western blotting analysis after electrophoresis on a SDS-PAGE gel and an agarose gel.Conclusions : Our results demonstrate a novel physiological function of ApoB-100 (or LDL) in accelerating VWF proteolysis by ADAMTS13 under fluidic shear stress. These findings may shed new light on how abnormalities in lipid metabolism may affect normal hemostasis and thrombosis under various pathological conditions. [Display omitted] DisclosuresZheng:Alexion: Speakers Bureau; Ablynx: Consultancy.

Von Willebrand disease type Vicenza: In search of a classification for the archetype of reduced von Willebrand factor survival.

Type Vicenza von Willebrand disease (VWD) features a von Willebrand factor (VWF) with a very short half‐life, and is classified as a form of type 1 VWD. To test the appropriateness of type Vicenza VWD classification, the main features of 17 patients from eight unrelated families were analysed. They had low VWF antigen levels and function (always below 20 U/dl); ristocetin‐induced platelet aggregation sometimes normal, sometimes reduced/absent (even in the same patient); normal platelet VWF levels; an increased VWF propeptide to VWF antigen ratio (8.74 ± 1.65 vs. normal 1.04 ± 0.28) and a reduced VWF half‐life. Plasma VWF multimer levels were homogeneously reduced, and unusually large VWF multimers were sometimes present. Recombinant p.R1205H VWF showed a normal synthesis, release, function, and multimer pattern, with no ultra‐large VWF multimers. The mathematical model by Galvanin et al. was used to explore the kinetic changes in VWF after DDAVP. It showed that the release, but especially the proteolysis (k proteol 1.0−3 ± 2.5−3 vs. normal 4.5−4 ± 6.4−4) and elimination (k el 1.0−2 ± 5.2−3 vs. normal 1.1−3 ± 6.8−4) of type Vicenza VWF were significantly higher than normal. The increased elimination is consistent with the short half‐life, while the increased proteolysis was unexpected. As a shorter survival of VWF is wholly responsible for the type Vicenza VWD phenotype (VWF synthesis, structure and function are normal), it might be better to classify it as a type 2 VWD (rather than type 1) to emphasise the greater interaction with clearance receptors as a new VWF functional defect.

Binding of Gpibα to VWF A2 Domain Alters Mechanical Unraveling of the A2 Domain

Background: Von Willebrand factor (VWF), is a large, multimeric plasma glycoprotein that plays a critical role in hemostasis. VWF is synthesized and secreted as ultra large (UL) multimers that contain up to 100 protomers. If not processed by ADAMTS13, a plasma metalloprotease, ULVWF may initiate the spontaneous formation of life-threatening thrombi, as seen in thrombotic thrombocytopenic purpura (TTP). The cleavage site is buried under the central β-sheet within the VWF-A2 domain and tensile force is required to expose the cleavage site for ADAMTS13. Our prior studies demonstrate that several VWF-binding proteins, including coagulation factor VIII, apoB100/LDL, as well as the ectodomains of platelet glycoprotein Iba (GPIba), appear to function as cofactors that facilitate the proteolytic cleavage of VWF by ADAMTS13 under shear. However, the mechanism underlying GPIba enhancing effect on ADAMTS13-mediated VWF proteolysis is yet to be determined. Methods: Recombinant human GPIbα was purchased from Sino Biological. The recombinant VWF-A2 domain fragment with a SpyTag on the N-terminus and an AviTag-HisTag on the C-terminus was expressed in the HEK 293T cells and affinity-purified by the Ni-NTA affinity chromatography. Biotinylation was performed in vitro using a biotin-labeling kit. The binding between VWF-A2 and GPIbα was studied by a custom-built atomic force microscope (AFM) using our established single-molecule binding study protocol. MicroScale Thermophoresis was conducted by a Monolith device to detect the binding affinity between the VWF-A2 and GPIbα. A dual-beam mini-tweezers instrument was utilized to characterize the force-induced conformational changes of the A2 domain in the absence and presence of GPIbα with a pulling speed of 200 nm/s. Results: AFM results indicated that specific binding interactions occurred between GPIbα and VWF-A2. Monolith MST assay revealed a strong binding affinity (Kd of ~20 nM) between GPIbα and the VWF-A2 fragment. In the optical tweezer study, pulling on a single VWF-A2 resulted in an unfolding event at 10-30 pN with an extension ranging from 30 to 40 nm (Fig. 1A). Gaussian fits of the unfolding extension distributions revealed a most probable force-induced extension of 34.87 ± 2.2 nm (mean ± SEM) (Fig. 1B). Addition of 100 nM of GPIbα led to a noticeable decrease in both unfolding force and extension of VWF-A2 (Fig. 1A). The most probable unfolding extension reduced to 16.05 ± 0.2 nm in the presence of 100 nM of GPIbα (Fig. 1B), indicating the binding of GPIbα may influence mechanical unfolding of VWF-A2. Further, the unfolding results were analyzed by a worm-like chain model fit, which yielded a contour length for the initially folded structure of VWF-A2 at 58.83 ± 2.0 nm (mean ± SEM) and 24.53 ± 0.2 nm in the absence and presence of GPIbα, respectively (Fig. 1C), indicating that the specific interactions between GPIbα and A2 domain may partially unfold the A2 domain. Conclusions: These results demonstrate for the first time that binding of GPIbα to VWF-A2 may alter the force-induced conformational changes in the A2 domain. Under physiological conditions, the glycocalicin (or soluble GP1bα) may bind the VWF-A2 and cause A2 partial unfolding, which may result in excessive cleavage of VWF by ADAMTS13, thus regulating hemostasis. Figure legend: Fig.1. GPIbα influences the mechanical unfolding of the A2 domain of VWF. (A) Typical optical tweezer pulling traces of A2 domain in the absence (red) and presence (blue) of GPIbα (100 nM). The arrows point to the unfolding events. The pulling speed is 200 nm/s. (B) The histograms of the unfolding extension of pulling VWF-A2 in the absence (red) or presence (blue) of 100 nM of GPIbα at 200 nm/s. Solid lines are Gaussian fits to the distributions. (C) The relationship between unfolding force (pN) and unfolding extension (nm) of pulling the VWF-A2 in the absence (red) or presence (blue) of 100 nM of GPIbα. The data are fitted to the worm-like chain model (solid lines). Horizontal and vertical error bars are one standard deviation for force and half width of the half bin width for extension, respectively. Figure Disclosures Cao: Ivygen: Consultancy; Bayer: Research Funding. Zheng:Sanofi: Consultancy, Speakers Bureau; Clotsolution: Other: Co-Founder; Alexion: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau.

Inhibition of adamts13: a novel therapy to treat mechanical circulatory support-induced acquired von willebrand syndrome

Abstract Introduction Bleeding is the most frequent adverse event in patients with continuous flow mechanical circulatory support (CF-MCS) and has been linked to the occurrence of acquired von Willebrand syndrome (aVWS). MCS devices cause an increased shear-induced proteolysis of von Willebrand factor (VWF) by ADAMTS13, leading to aVWS. Hence, specifically blocking ADAMTS13 might be an efficient way to rescue the loss of HMW VWF multimers in CF-MCS patients. Purpose To investigate if blocking ADAMTS13, using an in-house developed inhibitory anti-ADAMTS13 monoclonal antibody (mAb), prevents the loss of high molecular weight (HMW) VWF multimers in in vitro CF-MCS systems and to determine the efficacy of this therapy in a CF-MCS calf model. Methods Human blood was perfused through in vitro CF-MCS systems (Heartmate II and Impella CP, axial flow heart pumps) in the presence of the inhibitory or control mAb (20 μg/mL). Bovine blood was perfused through an in vitro Impella 5.5 system with the inhibitory mAb (20 μg/mL) or PBS. Next, Impella 5.5 pumps were implanted in calves. One dose of the inhibitory mAb (600 μg/kg) or PBS was injected eight days after Impella implantation. Plasma samples were analysed for VWF multimers, VWF antigen (VWF:Ag) and VWF collagen binding activity (VWF:CB). Results A time-dependent decrease in HMW VWF multimers was observed in both in vitro CF-MCS systems in the presence of the control mAb, leading to a 70% reduction of HMW VWF multimers, 180 minutes (min) after blood perfusion (p=0.01 for HM II and p=0.0003 for Impella). This was also reflected by a severely decreased VWF:CB/VWF:Ag ratio (0.59±0.11 and 0.52±0.10 at 180 min versus 1.00±0.06 and 1.07±0.09 before perfusion, for the HM II (p=0.03) and Impella (p=0.001) respectively). Interestingly, blocking ADAMTS13 using the inhibitory mAb prevented the loss of HMW VWF multimers in both systems (p=0.50 for the HM II and p=0.06 for the Impella, 180 min after the start of perfusion). The preservation of HMW VWF multimers was also reflected by normal VWF:CB/VWF:Ag ratios (0.92±0.16 and 0.97±0.11 at 180 min versus 0.93±0.09 and 1.19±0.12 before perfusion for the HM II (p=0.75) and Impella (p=0.06) respectively). Blocking bovine ADAMTS13 using the inhibitory mAb could prevent the loss of HMW VWF multimers in the in vitro Impella 5.5 system, showing that the calf is a good preclinical animal model to study the in vivo effect of this novel therapy. Impella implantation in the calves led to a decrease in HMW VWF multimers (Figure 1A and B). Hence, this animal model represents the VWF laboratory features of MCS-induced aVWS. Moreover, the loss of HMW VWF multimers after pump implantation could be rescued after injection of the inhibitory mAb (Figure 1A and B). Conclusion Blocking ADAMTS13 rescues MCS-induced VWF proteolysis in calves. Hence, inhibiting ADAMTS13 function could become a promising therapeutic strategy to rescue aVWS-induced bleeding in MCS patients. Figure 1. Impella calf model Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Fund for Scientific Research Flanders

Factor VIII binding affects the mechanical unraveling of the A2 domain of von Willebrand factor

BackgroundProteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 is crucial for normal hemostasis. Our previous studies demonstrate that binding of coagulation factor VIII (or FVIII) to VWF enhances the proteolytic cleavage of VWF by ADAMTS13 under shear. ObjectivesPresent study aims to determine the mechanism underlying FVIII‐mediated enhancing effect on VWF proteolysis by ADAMTS13 under force. MethodsSingle molecular force spectroscopy, atomic force microscopy, and surface plasmon resonance are all used. ResultsUsing single molecule force spectroscopy, we show that an addition of FVIII (~5 nmol/L) to D′D3 or D′D3A1 does not significantly alter force‐induced unfolding of these fragments; however, an addition of FVIII at the same concentration to D′D3A1A2 eliminates its long unfolding event at ~40 nm, suggesting that binding of FVIII to D′D3 and/or A2 may result in force‐induced conformational changes in A2 domain. Atomic force spectroscopy further demonstrates the direct binding between FVIII and D′D3 (or A2) with an intrinsic 2‐dimensional off‐rate (k0) of 0.02 ± 0.01/s (or 0.3 ± 0.1/s). The direct binding interaction between FVIII and A2 is further confirmed with the surface plasmon resonance assay, with a dissociation constant of ~0.2 μmol/L; no binding is detected between FVIII and A1 under the same conditions. ConclusionsOur results suggest that binding of FVIII to D′D3 and/or A2 may alter the mechanical property in the central A2 domain. The findings provide novel insight into the molecular mechanism underlying FVIII‐dependent regulation of VWF proteolysis by ADAMTS13 under mechanical force.

Binding of Factor VIII to D'D3 and A2 Domains of Von Willebrand Factor Facilitates Mechanical Unraveling of the A2 Domain

Background: Proteolytic cleavage of von Willebrand factor (VWF) by a plasma metalloprotease ADAMTS13 is critical for normal hemostasis. Our previous studies demonstrate that binding of coagulation factor VIII (FVIII) through its light chain to VWF significantly accelerates the proteolytic cleavage of VWF by ADAMTS13 under fluidic shear. However, the mechanism underlying the FVIII-cofactor activity enhancing VWF proteolysis by ADAMTS13 under shear is not fully understood. Present study aims to test a hypothesis that FVIII binding to the D'D3 and/or other adjacent domains may facilitate a mechanic-induced conformational change in the central A2 domain of VWF, resulting in an increased accessibility and the cleavage of Tyr1605-Met1606 bond by ADAMTS13. Methods: Recombinant B-domainless FVIII and various VWF fragments (D'D3, D'D3A1, D'D3A1A2, and A2) with an Avi-His-tag on their N-termini and a Spy-tag on their C-termini were expressed and affinity-purified to homogeneity. Laser optical tweezers analysis determined the force-induced conformational changes of VWF fragments in the absence or presence of FVIII. Atomic force microscopy (AFM) and surface plasmon resonance (SPR) determined the binding events and kinetics between FVIII and various VWF domains. Results: Laser optical tweezers analysis demonstrated a Gaussian distribution of unfolding extension events upon pulling the single D'D3A1 fragment with the most probable extension distance of 22.8 ± 1.9 nm (mean ± SEM). Addition of 5 nM FVIII to the D'D3A1 or D'D3 fragment resulted in only a small decrease (~25%) or no change in the pulling extension profile. However, two peaks of Gaussian distribution were detected when pulling the D'D3A1A2 with one peak at ~18 nm and the other at ~40 nm. Addition of 5 nM FVIII to the D'D3A1A2 fragment, only one unfolding event was detected with the most probable unfolding extension distance of 24.5 ± 0.4 nm. Moreover, a single Gaussian distribution of unfolding extension profile was detected when pulling an isolated A2 fragment with the most probable unfolding extension of 31 ± 1 nm. Interestingly, addition of 5 nM FVIII resulted in a significant reduction of the most probable unfolding extension to 16.0 ± 0.4 nm, suggesting that FVIII may directly interact with A2 domain. AFM and SPR demonstrated the binding kinetics between FVIII and various VWF fragments. As shown, the intrinsic off-rate (k0) between FVIII and D'D3 was 0.19 ± 0.9 s-1 and the barrier width (Δx‡) between the naive state and the transition state was 1.5 ± 0.2 Å; the k0 between FVIII and D'D3A1A2 was 2.8 ± 1.0 s-1 and the barrier width (Δx‡) between the naive state and the transitional state was 1.7 ± 0.2 Å. Surprisingly, there was a detectable, but relatively weak binding between FVIII and A2 domain using AFM. SPR experiments confirmed the binding of FVIII to D'D3, D'D3A1A2, and A2 with the dissociation constant (KD) of ~2.9 nM, ~5.2 nM and ~1.6 nM, respectively. These results demonstrate that the specific interaction between FVIII and D'D3 or A2 domain. Conclusions: We have demonstrated for the first time using single-molecule force spectroscopy that binding of FVIII to the D'D3 and/or A2 domain of VWF may facilitate the force-induced conformational changes of the A2 domain, resulting in the enhanced proteolysis by ADAMTS13 under force. Our findings shed new light on how FVIII may regulate VWF proteolysis and function under (patho) physiological conditions. Disclosures Zheng: Shire/Takeda: Research Funding; Alexion: Speakers Bureau; Clotsolution: Other: Co-Founder; Ablynx/Sanofi: Consultancy, Speakers Bureau.

Apolipoprotein B100/Low-Density Lipoprotein Regulates Proteolysis and Functions of von Willebrand Factor under Arterial Shear.

Proteolytic cleavage of von Willebrand factor (VWF) by a plasma a disintegrin and metalloproteinase with a thrombospondin type 1 motifs, member 13 (ADAMTS13) is regulated by shear stress and binding of coagulation factor VIII, platelets or platelet glycoprotein 1b, and ristocetin to VWF. Current study aims to identify novel VWF binding partners that may modulate VWF functions under physiological conditions. A deoxyribonucleic acid aptamer-based affinity purification of VWF, followed by tandem mass spectrometry, functional, and binding assays was employed. Apolipoprotein B100/low-density lipoprotein (apoB100/LDL) was identified as a novel VWF-binding partner. Purified apoB100/LDL was able to accelerate the proteolytic cleavage of VWF by ADAMTS13 under shear in a concentration-dependent manner. This rate-enhancing activity was dramatically reduced when apoB100/LDL was oxidized. More interestingly, the oxidized apoB100/LDL appeared to compete with native apoB100/LDL for its enhancing activity on VWF proteolysis under shear. As a control, a purified apoA1/high-density lipoprotein (apoA1/HDL) or apoB48 exhibited a minimal or no activity enhancing VWF proteolysis by ADAMTS13 under the same conditions. Both VWF and ADAMTS13 were able to bind native or oxidized apoB100/LDL with high affinities. No binding interaction was detected between VWF (or ADAMTS13) and apoA1/HDL (or apoB48). Moreover, apoB100/LDL but not its oxidized products inhibited the adhesion of platelets to ultra large VWF released from endothelial cells under flow. Finally, significantly reduced ratios of high to low molecular weight of VWF multimers with increased levels of plasma VWF antigen were detected in LDLR-/- mice fed with high cholesterol diet. These results indicate that apoB100/LDL may be a novel physiological regulator for ADAMTS13-VWF functions.

Assessment of ADAMTS-13 Level in Hospitalized Children with Serious Bacterial Infections as a Possible Prognostic Marker.

Background and objectives: In children, acute infection is the most common cause of visits in the primary care or emergency department. In 2002, criteria for diagnostics of pediatric sepsis were published, and then revised in 2016 as “life-threatening organ dysfunction due to a dysregulated host response to infection”. In the pathophysiology of sepsis endothelial dysfunction plays a very important role. Deficient proteolysis of von Willebrand factor, due to reduced ADAMTS-13 activity, results in disseminated platelet-rich thrombi in the microcirculation. ADAMTS-13 deficiency has been detected in systemic inflammation. The clinical relevance of ADAMTS-13 during sepsis is still unclear. We aimed to investigate the possible use of ADAMTS-13 as a prognostic marker in children with serious bacterial infection (SBI). Materials and Methods: Inclusion criteria were hospitalized children with SBI, aged from 1 month to 17 years. SBI was defined based on available clinical, imaging, and later also on microbiological data. Sepsis was diagnosed using criteria by The International Consensus Conference. In all the patients, the levels of ADAMTS-13 were measured at the time of inclusion. Results: Data from 71 patients were analyzed. A total of 47.9% (34) had sepsis, 21.1% (15) were admitted to the ICU, 8.5% (6) had mechanical ventilator support, and 4.2% (3) patients had a positive blood culture. The median level of ADAMTS-13 in this study population was 689.43 ng/mL. Patients with sepsis, patients admitted to the Intensive Care Unit, and patients in need of mechanical ventilator support had significantly lower levels of ADAMTS-13. None of the patients had ADAMTS-13 deficiency. In patients with SBI, the area under the curve (AUC) to predict sepsis was 0.67. A cut-off ADAMTS-13 level of ≤730.49 had 82% sensitivity and 60% specificity for sepsis in patients with SBI. Conclusions: ADATMS-13 levels were lower in patients with SBI and sepsis, but AUC and sensitivity were too low to accept it as a prognostic marker.

Involvement of the ADAMTS13-VWF axis in acute Kawasaki disease and effects of intravenous immunoglobulin

IntroductionADAMTS13 modulates shear-dependent platelet thrombus formation (PTF) by limited proteolysis of von Willebrand factor (VWF). A high-plasma-ratio of VWF antigen to ADAMTS13 activity (VWF:Ag/ADAMTS13:AC) promotes PTF and aggravates shear-induced inflammation mediated by VWF. A role of ADAMTS13 in Kawasaki disease (KD) remains unknown, however. We investigated the involvement of ADAMTS13-VWF axis in the acute-phase of KD (acute-KD). MethodsVWF:Ag and ADAMTS13:AC in 77 KD infants were measured at three time-points; immediately before (Pre), one-week (1 W) and one-month (1 M) after intravenous-immunoglobulin (IVIG) treatment. VWF multimer (VWFM) distribution and ADAMTS13-isoelectrofocusing (IEF) patterns were compared between the responders and non-responders to IVIG. ResultsA high VWF:Ag (195.7 ± 85.6%, p < 0.05), low ADAMTS13:AC (60.3 ± 23.8%, p < 0.05) and high VWF:Ag/ADAMTS13:AC ratio (3.70 ± 2.12, p < 0.05) at Pre were seen compared to control plasmas. These parameters returned to normal levels time-dependently after IVIG treatment. Non-responders to IVIG demonstrated high VWF:Ag and low ADAMTS13:AC at Pre, and high VWF:Ag/ADAMTS13:AC ratio at 1 W compared to responders, but there were no significant differences in VWFM distribution between both groups. IEF analyses revealed the decreased free form of ADAMTS13 and increased complex form with ADAMTS13 and high-molecular-weight-VWFM at Pre in non-responders. A high VWF:Ag/ADAMTS13:AC ratio was associated with increased white blood cell counts, together with decreased serum albumin and sodium at Pre and 1 W. ConclusionsA high VWF:Ag/ADAMTS13:AC ratio in acute-KD persisted after primary treatment in non-responders, and unbalanced substrate-to-enzyme ratio appeared to associate with vascular endothelial damage. Analysis of existing mode of ADAMTS13 may help to clarify pathogenesis of IVIG resistance in acute-KD.

Dissecting the pathophysiology of immune thrombotic thrombocytopenic purpura: interplay between genes and environmental triggers.

Although outstanding progress has been made in understanding the pathophysiology of thrombotic thrombocytopenic purpura (TTP), knowledge of the immunopathogenesis of the disease is only at an early stage. Anti-ADAMTS13 auto-antibodies were shown to block proteolysis of von Willebrand factor and/or induce ADAMTS13 clearance from the circulation. However, it still remains to identify which immune cells are involved in the production of anti-ADAMTS13 autoantibodies, and therefore account for the remarkable efficacy of the B-cell depleting agents in this disease. The mechanisms leading to the loss of tolerance of the immune system towards ADAMTS13 involve the predisposing genetic factors of the human leukocyte antigen class II locus DRB1*11 and DQB1*03 alleles as well as the protective allele DRB1*04, and modifying factors such as ethnicity, sex and obesity. Future studies have to identify why these identified genetic risk factors are also frequently to be found in the healthy population although the incidence of immune-mediated thrombotic thrombocytopenic purpura (iTTP) is extremely low. Moreover, the development of recombinant ADAMTS13 opens a new therapeutic era in the field. Interactions of recombinant ADAMTS13 with the immune system of iTTP patients will require intensive investigation, especially for its potential immunogenicity. Better understanding of iTTP immunopathogenesis should, therefore, provide a basis for the development of novel therapeutic approaches to restore immune tolerance towards ADAMTS13 and thereby better prevent refractoriness and relapses in patients with iTTP. In this review, we address these issues and the related challenges in this field.

A Mechanistic Model to Quantify von Willebrand Factor Release, Survival and Proteolysis in Patients with von Willebrand Disease.

A reduced von Willebrand factor (VWF) synthesis or survival, or its increased proteolysis, alone or in combination, contributes to the development of von Willebrand disease (VWD).We describe a new, simple mechanistic model for exploring how VWF behaves in well-defined forms of VWD after its 1-desamino-8-D-arginine vasopressin (DDAVP)-induced release from endothelial cells. We aimed to ascertain whether the model can consistently predict VWF kinetic changes. The study involved 9 patients with VWD types Vicenza (a paradigmatic form with a reduced VWF survival), 8 type 2B, 2 type 2A-I, 1 type 2A-II (associated with an increased VWF proteolysis), and 42 normal controls, whose VWF levels were measured after a 24-hour-long DDAVP test. The rate constants considered were: k0, associated with the VWF release phase; k1, illustrating the phase of conversion from high- to low-molecular-weight VWF multimers; and ke, associated with the VWF elimination phase. The amount of VWF released (D) was also measured. ke and D were significantly higher in O than in non-O blood group controls; k1 was also higher, but less markedly so. All the parameters were accelerated in type Vicenza, especially ke (p < 0.0001), which explains the significant reduction in VWF half-life. In types 2B and 2A-II, k1 was one order of magnitude higher than in controls, which explains their loss of large VWF multimers. All parameters except ke were lower in type 2A-I.The proposed mechanistic model clearly describes the altered biochemical pathways in well-characterized VWD, prompting us to suggest that it might help clarify elusive forms of VWD too.

Amplified endogenous plasmin activity resolves acute thrombotic thrombocytopenic purpura in mice

Background Thrombotic thrombocytopenic purpura (TTP) is an acute life-threatening pathology, caused by occlusive von Willebrand factor (VWF)-rich microthrombi that accumulate in the absence of ADAMTS-13. We previously demonstrated that plasmin can cleave VWF and that plasmin is generated in patients during acute TTP. However, the exact role of plasmin in TTP remains unclear. Objectives Investigate if endogenous plasmin-mediated proteolysis of VWF can influence acute TTP episodes. Results In mice with an acquired ADAMTS-13 deficiency, plasmin is generated during TTP as reflected by increased plasmin-α2-antiplasmin (PAP)-complex levels. However, mice still developed TTP, suggesting that this increase is not sufficient to control the pathology. As mice with TTP also had increased plasminogen activator inhibitor 1 (PAI-1) levels, we investigated whether blocking the plasmin(ogen) inhibitors would result in the generation of sufficient plasmin to influence TTP outcome in mice. Interestingly, when amplified plasmin activity was allowed (α2-antiplasmin-/- mice with inhibited PAI-1) in mice with an acquired ADAMTS-13 deficiency, a resolution of TTP signs was observed as a result of an increased proteolysis of VWF. In line with this, in patients with acute TTP, increased PAP-complex and PAI-1 levels were also observed. However, neither PAP-complex levels nor PAI-1 levels were related to TTP signs and outcome. Conclusions In conclusion, endogenous plasmin levels are increased during acute TTP, although limited via suppression through α2-antiplasmin and PAI-1. Only when amplified plasmin activity is allowed, plasmin can function as a back-up for ADAMTS-13 in mice and resolve TTP signs as a result of an increased proteolysis of VWF.

Von Willebrand factor disruption and continuous-flow circulatory devices

Bleeding events remain a significant and frequent complication of continuous-flow left ventricular assist devices (VADs). von Willebrand factor (VWF) is critical to hemostasis by acting as a bridging molecule at sites of vascular injury for normal platelet adhesion as well as promoting platelet aggregation under conditions of high shear. Clinical and experimental data support a role for acquired von Willebrand disease in VAD bleeding episodes caused by shear-induced qualitative defects in VWF. Pathologic shear induces VWF unfolding and proteolysis of large multimers into smaller less hemostatic multimers via ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). This review outlines the pathobiology of VWF disruption in the context of VADs as well as current diagnostic and management strategies of the associated acquired von Willebrand disease.

Potent Thrombolytic Effect of N-Acetylcysteine on Arterial Thrombi.

Platelet cross-linking during arterial thrombosis involves von Willebrand Factor (VWF) multimers. Therefore, proteolysis of VWF appears promising to disaggregate platelet-rich thrombi and restore vessel patency in acute thrombotic disorders such as ischemic stroke, acute coronary syndrome, or acute limb ischemia. N-Acetylcysteine (NAC, a clinically approved mucolytic drug) can reduce intrachain disulfide bonds in large polymeric proteins. In the present study, we postulated that NAC might cleave the VWF multimers inside occlusive thrombi, thereby leading to their dissolution and arterial recanalization. Experimental models of thrombotic stroke induced by either intra-arterial thrombin injection or ferric chloride application followed by measurement of cerebral blood flow using a combination of laser Doppler flowmetry and MRI were performed to uncover the effects of NAC on arterial thrombi. To investigate the effect of NAC on larger vessels, we also performed ferric chloride-induced carotid artery thrombosis. In vitro experiments were performed to study the molecular bases of NAC thrombolytic effect, including platelet aggregometry, platelet-rich thrombi lysis assays, thromboelastography (ROTEM), and high-shear VWF string formation using microfluidic devices. We also investigated the putative prohemorrhagic effect of NAC in a mouse model of intracranial hemorrhage induced by in situ collagenase type VII injection. We demonstrated that intravenous NAC administration promotes lysis of arterial thrombi that are resistant to conventional approaches such as recombinant tissue-type plasminogen activator, direct thrombin inhibitors, and antiplatelet treatments. Through in vitro and in vivo experiments, we provide evidence that the molecular target underlying the thrombolytic effects of NAC is principally the VWF that cross-link platelets in arterial thrombi. Coadministration of NAC and a nonpeptidic GpIIb/IIIa inhibitor further improved its thrombolytic efficacy, essentially by accelerating thrombus dissolution and preventing rethrombosis. Thus, in a new large-vessel thromboembolic stroke model in mice, this cotreatment significantly improved ischemic lesion size and neurological outcome. It is important to note that NAC did not worsen hemorrhagic stroke outcome, suggesting that it exerts thrombolytic effects without significantly impairing normal hemostasis. We provide evidence that NAC is an effective and safe alternative to currently available antithrombotic agents to restore vessel patency after arterial occlusion.