VWF Cleavage Research Articles

Abstract 17158: Effects of Human Monoclonal Antibodies on ADAMTS13 in Immune Thrombotic Thrombocytopenic Purpura

Introduction: Immune thrombotic thrombocytopenic purpura (iTTP) is caused by antibody-mediated deficiency of the metalloprotease ADAMTS13, which cleaves VWF. Myocardial infarction and ischemic strokes are common and often fatal sequelae of iTTP. Inhibitory anti-ADAMTS13 antibodies appear to affect catalytic turnover more than apparent substrate binding affinity (PMID 37023370). Exploring how antibodies affect VWF cleavage by ADAMTS13 in physiologic conditions is needed to better understand the pathophysiology of iTTP. Hypothesis: Anti-ADAMTS13 antibodies may influence ADAMTS13 function by affecting catalytic turnover and/or apparent substrate binding affinity differently in physiologic conditions than in standard assay conditions. Aims: Characterize how single chain fragments of the variable loop (scFv’s) previously identified from a phage display library derived from several iTTP patients’ antibodies affect ADAMTS13-mediated VWF cleavage in conditions that more closely represent plasma and see if/how they differ from scFv-mediated effects in standard conditions. Methods: ADAMTS13 in normal human plasma was titrated with increasing concentrations of the substrate FRETS-VWF73 to derive maximum velocity ( V max ) and apparent Michaelis constant ( K M,app ) in standard assay conditions (pH 6.0) and conditions closer to physiologic (pH 7.0). The same parameters were derived in the presence of saturating concentrations of scFv’s. Results: Compared to VWF cleavage by ADAMTS13 in the absence of scFv, the inhibitory scFv4-20 decreased V max at both pH 6.0 and pH 7.0; K M,app decreased at pH 6.0 and increased at pH 7.0. The stimulatory scFv3-3 and scFv4-41 increased V max at each pH, while K M,app in the presence of each scFv decreased only at pH 7.0. Conclusions: Anti-ADAMTS13 antibody-mediated effects on VWF cleavage differ when comparing standard and more physiologic pH conditions, guiding future investigative directions into iTTP pathophysiology.

A Mutation in Complement Factor H (W1183R) Enhances VWF Proteolysis By ADAMTS13 Under Shear

Introduction: Complement factor H (CFH) is the primary negative regulator of complement activation in the alternative pathway in both fluidic phase and on cell surface. Loss of function of CFH such as W1183R is associated with atypical hemolytic uremic syndrome (aHUS). Homozygous mutation W1206R in the murine CFH, corresponding to W1183R in human CFH, results in acute aHUS. Interestingly, in these mice, while plasma VWF antigen levels are significantly increased, plasma VWF multimer size is markedly reduced in ADAMTS13-depedent manner. However, the molecular mechanism underlying such a dramatic effect of the mutant CFH (W1206R) on VWF multimer distribution is not known. The goal of this study is to determine how wild type CFH and mutant CFH W1183R affects VWF proteolysis under shear, and the mechanism of action. Methods: Recombinant WT CFH, CFH W1183R, ADAMTS13, and VWF fragments were expressed in mammalian cells and purified to homogeneity. The effects of WT CFH or CFH W1183R on proteolytic cleavage of VWF73 peptide and full-length multimeric VWF were determined. Additionally, the binding and unfolding events were determined by micro scale thermophoresis (MST) and optical tweezer, respectively. Results : WT CFH showed no effect on the cleavage of peptide VWF73 substrate, but CFH W1183R enhanced the proteolytic cleavage of VWF73 by ADAMTS13 in a dose-dependent manner ( Fig. 1). Counterintuitively, WT CFH appeared to inhibit the proteolytic cleavage of multimeric VWF by ADAMTS13 under arterial shear, but CFH W1183R exhibited an opposite effect, modestly enhancing the proteolytic cleavage of multimeric VWF by ADAMTS13 under the same conditions. The MST binding assay demonstrated that both WT CFH and CFH W1183R bind to a short A1, a long AIM-A1, and A1-A2-A3 fragments with a dissociation constant ( K D) of 6.4 x 10 -7, 3.0 x 10 -9 M, and 3.3 x10 -9 mol/L, respectively. These results suggest that CFH may bind to the AIM, A1-A2 junction, and A2 domain. To further assess the impact of CFH binding to A2, we performed optical tweezer experiments. The results showed that both WT CFH (Lc: from 27.4 nm at 12 nM to 40 nm at 1.0 mM) and CFH W1183R (Lc: from 26 nm at 12 nM to 31.8 nm at 1.0 mM) resulted in significantly extended contour length (Lc) of VWF-A2 domain in a concentration-dependent manner ( Fig. 2), suggesting a dramatic conformational change in the A2 domain upon binding by WT CFH or CFH W1183R. Conclusions : Our results demonstrate that WT CFH at physiological concentrations may exhibit a modest inhibitory activity towards cleavage of VWF by ADAMTS13 under shear, but such an inhibitory activity disappears as the result of a point mutation in the C-terminal end of CFH (e.g., W1183R). Instead, the mutant CFH W1183R appears to enhance the VWF cleavage by ADAMTS13, likely through direct binding to the AIM, A1-A2 junction, and A2 domain of VWF, resulting in conformational changes in the central A2 domain.

OC 26.5 Regulation of VWF Cleavage is Mediated by Coupling ADAMTS13 Dand C- Domain Binding Sites

OC 26.5 Regulation of VWF Cleavage is Mediated by Coupling ADAMTS13 Dand C- Domain Binding Sites

Von Willebrand factor processing in patients with advanced chronic liver disease and its relation to portal hypertension and clinical outcome

Background and aimsEndothelial dysfunction and portal hypertension (PH) are reflected by increased von Willebrand factor antigen (VWF-Ag) levels in advanced chronic liver disease (ACLD). This study investigated VWF release and cleavage and their association with PH and clinical outcomes.MethodsLevels of VWF-Ag, VWF-N (VWF-propeptide), and VWF-A (VWF processed by the main VWF-cleaving protease ADAMTS13) were assessed in 229 patients with clinically stable ACLD (hepatic venous pressure gradient [HVPG] ≥ 6 mmHg; absence of bacterial infections or acute decompensation) undergoing HVPG-measurement. Liver-healthy individuals served as controls (n = 24).ResultsVWF-Ag and VWF-N were similarly accurate for the identification of clinically significant PH (CSPH; HVPG ≥ 10 mmHg) in compensated ACLD (AUROC: VWF-Ag 0.748; VWF-N 0.728). ADAMTS13 activity was similar between patients with ACLD and controls and did not correlate with PH and disease severity, whereas VWF cleavage decreased in patients with CSPH (i.e., VWF-Ag/-A-ratio increased). In vitro VWF activity strongly reflected VWF-Ag levels (Spearman’s r = 0.874, p < 0.001), but decreased (vs. controls) in patients with CSPH when normalized to VWF-Ag levels (VWF-activity/-Ag-ratio). VWF-Act/-Ag ratio correlated negatively with ADAMTS13 activity (r =– 0.256, p < 0.001). ADAMTS13 activity was independently predictive for (i) portal vein thrombosis (PVT) and (ii) hepatic decompensation or liver-related death.ConclusionsVWF-Ag levels and its propeptide are similarly suitable surrogates of PH in patients with compensated ACLD. ADAMTS13-Act was not linked to disease and PH severity, however, when normalized to VWF-Ag, both VWF cleavage and VWF activity were decreased in patients with CSPH, as compared to liver-healthy individuals. Low ADAMTS13-Act was associated with presumably more procoagulant VWF and adverse outcomes.Clinical trial numberNCT03267615

Dynamic and functional linkage between von Willebrand factor and ADAMTS-13 with aging: an Atherosclerosis Risk in Community study

Backgroundvon Willebrand factor (VWF) is a multimeric glycoprotein critically involved in hemostasis, thrombosis, and inflammation. VWF function is regulated by its antigen levels, multimeric structures, and the state of enzymatic cleavage. Population studies in the past have focused almost exclusively on VWF antigen levels in cross-sectional study designs. ObjectiveTo identify subjects in the Atherosclerosis Risk in Community study who had persistently low and high VWF antigen over 10 years and to quantify longitudinal changes in the biological activities and cleavage of VWF in these subjects. MethodsWe measured VWF antigen, propeptide, adhesive activities, and cleavage by ADAMTS-13 quantified using a mass spectrometry method that detected the cleaved VWF peptide EQAPNLVY, as well as coagulation factor VIII activity. ResultsWe determined the mean subject-specific increase in VWF to be 22.0 International Units (IU)/dL over 10 years, with 95% between -0.3 and 59.7 IU/dL. This aging-related increase was also detected in VWF propeptide levels, ristocetin cofactor activity, and VWF binding to collagen. We identified 4.1% and 25.0% of subjects as having persistently low (<50 IU/dL) and high (>200 IU/dL) VWF antigen, respectively. Subjects with persistently low VWF had enhanced ristocetin cofactor activity, whereas those with persistently high VWF had elevated levels of ADAMTS-13, resulting in a comparable rate of VWF cleavage between the 2 groups. ConclusionsThese results provide new information about the effects of aging on VWF antigens and adhesive activity and identify a functional coordination between VWF and the rate of its cleavage by ADAMTS-13.

Abstract 220: Post-gwas Functional Genomics Analysis Of Abo Locus To Identify Novel Pathogenic Mechanisms For Chronic Thromboembolic Pulmonary Hypertension

Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is a rare and mysterious complication of pulmonary embolism, characterized by chronic pulmonary arterial thrombosis, occlusions and vasculopathy. Emerging evidence has suggested genetic relevance of CTEPH, particularly to ABO blood type. Genome-wide association study (GWAS) has identified ABO gene as the most significant genetic locus associated with CTEPH. This locus, however, consists only intronic SNPs and there is no mechanistic explanation on how these non-coding SNPs control the risk of CTEPH. Methods: GWAS-identified CTEPH-associated ABO locus was analyzed, and haplotype SNPs were identified based on linkage disequilibrium (LD). Electrophoretic mobility shift assay (EMSA) was applied to screen functional SNPs (fSNPs) based on allele-imbalanced nuclear protein binding. SNP-binding nuclear transcription factors (TFs) were identified by proteomics. Hi-C chromatin-interaction analysis was utilized to determine potential target genes affected by fSNPs. Knockdown of fSNP-associated TFs in human pulmonary artery endothelial cells (hPAECs) was applied to validate the regulation of TFs on target genes. Results: Based on GWAS, the minor allele of tag SNP rs2519093 is associated with higher risk of CTEPH (Odds ratio 2.22). It defines 11 haplotype SNPs in a LD with R 2 &gt; 0.8. Screened by EMSA with hPAEC nuclear proteins, intronic SNPs rs579459 and rs550057 were identified functional given their allele-specific protein binding. Proteomics analysis identified 3 candidate TFs that allele-specifically bind to fSNP rs579459: PBX1, CTCF and SAFB2. Candidate target gene ADAMTS13 is chosen based on its prominent interaction with fSNP revealed by Hi-C analysis, and the potential role in thromboembolism through vWF cleavage. TF knockdown by siRNA downregulates ADAMTS13 in hPAECs, suggesting their regulatory function as activators. Conclusion: Leveraging our post-GWAS functional genomics approach, we identified fSNPs in ABO locus that define a genetic architecture controlling the susceptibility of CTEPH. This genetic architecture, along with the newly identified fSNP-associated TFs and target gene ADAMTS13, provides a biological explanation for the pathogenesis of CTEPH.

Transcription factor AP-2 gamma/Krüppel-like factor 10 axis is involved in miR-3656-related dysfunction of endothelial cells in hypertension.

Dysfunction of endothelial cells links to microvascular rarefaction, reflecting the pathogenesis of hypertension. Our previous studies found that miR-3656 reduces nitric oxide generation and von Willebrand factor (vWF) cleavage, thereby retarding blood flow and potentially increasing blood pressure. In this paper, we investigated mechanism of transcription regulation contributing to miR-3656-damaged endothelial cells in hypertension. The effects of miR-3656 on function of endothelial cells were analyzed on the basis of proliferation, migration, tube formation, and apoptosis. The mRNA level and protein level of genes were examined using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Dual-luciferase reporter assay was performed to confirm the binding between miR-3656 and 3' untranslated region (UTR) of transcription factor AP-2 gamma ( TFAP2C ). The binding between TFAP2C and the promoter region of Krüppel-like factor 10 ( KLF10 ) was confirmed by chromatin immunoprecipitation-qPCR assay. miR-3656 impaired the cell proliferation, migration, tube formation, and apoptosis of endothelial cells. miR-3656 inhibited the expression of TFAP2C by directly targeting 3'UTR of TFAP2C ; moreover, miR-3656-induced injury of endothelial cells was rescued by TFAP2C overexpression. Furthermore, downregulated TFAP2C decreased KLF10 expression by binding to KLF10 promoter region, and upregulated KLF10 reversed the effects of silencing TFAP2C on endothelial cells. These inhibitory processes led to interference of miR-3656 to KLF10-promoted function of endothelial cells. TFAP2C/KLF10 axis is involved in miR-3656-related dysfunction of endothelial cells in hypertension. The 3'UTR of TFAP2C and KLF10 promoter region are the hubs of the TFAP2C/KLF10 axis.

A case of acquired thrombotic thrombocytopenic purpura (TTP) mimicking transient ischemic attack (TIA)

TTP is a rare multisystem disease among children due to dysfunction of ADAMTS 13 enzyme in plasma causing defective vWF cleavage, thus giving rise to platelet aggregation, micro thrombi formation, platelet consumption, capillary blockage and finally organ dysfunction leading to a severe life-threatening condition with mortality around 90% if untreated. It can be congenital (5%) or acquired (95%) and Plasmapheresis works wonder for both the subtypes.: Here we have described a case of acquired TTP masquerading TIA in an 8-year-old girl child who had attacks of sudden onset aphasia and upper limb weakness lasting for few hours with spontaneous recovery. On detailed examination we found pallor and few petechial spots on face and upper limbs with a peripheral blood picture suggestive of thrombocytopenia with microangiopathic hemolytic anemia but a normal renal function. We sent blood for ADAMTS 13 activity which reported a critically low level of ADAMTS 13 enzymatic activity with presence of inhibitors and absent genetic mutation. Immediate Plasmapheresis was started along with oral prednisolone therapy for a span of one month which resulted both clinical and hematological recovery. Platelet count acted as a good indicator of clinical remission. A high degree of clinical suspicion is required to make a prompt diagnosis of this life threatening disease (TTP) since this carries a very high mortality rate and can mimic a more common disease HUS.

Complement promotes endothelial von Willebrand factor and angiopoietin-2 release in obstructive sleep apnea.

Obstructive sleep apnea (OSA) is highly prevalent and triples vascular thromboembolic risk. Intermittent hypoxia (IH) during transient cessation of breathing in OSA impairs endothelial protection against complement. Complement activation stimulates the endothelial release of a pro-thrombotic von Willebrand factor (vWF). We investigated whether increased complement activity in OSA promotes the endothelial release of vWF and pro-inflammatory angiopoietin-2. We further investigated whether improving complement protection with statins reverses these changes. Using endothelial cells (ECs) and blood collected from OSA patients (n = 109) and controls (n = 67), we assessed whether altered cellular localization of complement inhibitor CD59 in OSA modulates exocytosis of Weibel-Palade bodies (WPB), secretory granules that store vWF and angiopoietin-2. These interactions were also assessed in vitro in ECs exposed to normoxia or IH with or without recombinant complement C9 and with or without atorvastatin. Circulating levels of angiopoietin-2 were greater in OSA than controls and levels of vWF cleavage products correlated with OSA severity. In cultured ECs, IH enhanced complement-stimulated angiopoietin-2 and vWF release by reducing EC surface and increasing intracellular expression of complement inhibitor CD59. Intracellular CD59 co-localized with WPB in OSA. IH increased binding of intracellular CD59 to syntaxin-3, which dissociated syntaxin-3 from voltage-sensitive calcium channel Cav1.2, and activated WPB exocytosis in a calcium-dependent manner. Atorvastatin reversed IH-enhanced endothelial release of vWF and angiopoietin-2. IH promotes the complement-mediated release of vWF and angiopoietin-2, which may contribute to pro-thrombotic and pro-inflammatory conditions in OSA. Statin reversed these effects, suggesting a potential approach to reduce cardiovascular risk in OSA.

Temporal map of the pig polytrauma plasma proteome with fluid resuscitation and intravenous vitamin C treatment

BackgroundFluid resuscitation plays a prominent role in stabilizing trauma patients with hemorrhagic shock yet there remains uncertainty with regard to optimal administration time, volume, and fluid composition (e.g., whole blood, component, colloids) leading to complications such as trauma‐induced coagulopathies (TIC), acidosis, and poor oxygen transport. Synthetic fluids in combination with antioxidants (e.g., vitamin C) may resolve some of these problems. ObjectivesWe applied quantitative mass spectrometry‐based proteomics [liquid chromatography‐mass spectrometry (LC‐MS/MS)] to map the effects of fluid resuscitation and intravenous vitamin C (VitC) in a pig model of polytrauma (hemorrhagic shock, tissue injury, liver reperfusion, hypothermia, and comminuted bone fracture). The goal was to determine the effects of VitC on plasma protein expression, with respect to changes associated with coagulation and trauma‐induced coagulopathy (TIC). MethodsLongitudinal blood samples were drawn from nine male Sinclair pigs at baseline, 2 h post trauma, and 0.25, 2, and 4 h post fluid resuscitation with 500 mL hydroxyethyl starch. Pigs were treated intravenously (N = 3/treatment group) with saline, 50 mg VitC/kg (Lo‐VitC), or 200 mg VitC/kg (Hi‐VitC) during fluid resuscitation. ResultsA total of 436 plasma proteins were quantified of which 136 changed following trauma and resuscitation; 34 were associated with coagulation, complement cascade, and glycolysis. Unexpectedly, Lo‐VitC and Hi‐VitC treatments stabilized ADAMTS13 levels by ~4‐fold (P = .056) relative to saline and enhanced ADAMTS13/von Willebrand factor (VWF) cleavage efficiency based on LC‐MS/MS evidence for the semitryptic VWF cleavage product (VWF1275‐1286). ConclusionsThis study provides the first comprehensive map of trauma‐induced changes to the plasma proteome, especially with respect to proteins driving the development of TIC.

Blood group antigen A on von Willebrand factor is more protective against ADAMTS13 cleavage than antigens B and H

BackgroundADAMTS13 specifically cleaves the peptide bond between Y1605 and M1606 within the VWF‐A2 domain. ObjectiveThe VWF contains ABO(H) blood group antigens, which may influence the susceptibility of VWF to ADAMTS13. MethodsUsing a unique monoclonal antibody recognizing the Y1605 residue, we have developed a sandwich ELISA to analyze the generation of a VWF‐DP by ADAMTS13 quantitatively. ResultsProduction of VWF‐DP after exposure to four different degrees of high shear stress was validated in comparison to the reduction in high‐molecular‐weight multimers using VWF multimer analysis. In analysis of plasma from 259 healthy individuals, plasma levels of VWF antigen (VWF:Ag) were significantly lower in blood group O than in the other groups and were significantly correlated with plasma VWF‐DP levels. The ratio between VWF‐DP and VWF:Ag was significantly higher in blood group O than in blood groups A and AB. The ratio in blood group B was also significantly higher than those in A and AB, but did not differ from blood group O. Finally, to examine whether ABO(H) blood group antigens contributed to VWF cleavage, 82 plasma samples were exposed to high shear stress using a cone‐plate shear stress inducer. The difference in the VWF‐DP/VWF:Ag ratio before and after high shear stress in blood group O was significantly greater than those in groups A and AB. ConclusionThese results indicate that blood group antigen A on VWF was more protective against ADAMTS13 cleavage than antigens B and H.

Von Willebrand factor enhances microvesicle-induced vascular leakage and coagulopathy in mice with traumatic brain injury

Abstractvon Willebrand factor (VWF) is an adhesive ligand, and its activity is proteolytically regulated by the metalloprotease ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat 13). An elevated level of plasma VWF has been widely considered a marker for endothelial cell activation in trauma and inflammation, but its causal role in these pathological conditions remains poorly defined. Using a fluid percussion injury mouse model, we demonstrated that VWF released during acute traumatic brain injury (TBI) was activated and became microvesicle-bound. The VWF-bound microvesicles promoted vascular leakage and systemic coagulation. Recombinant ADAMTS-13 given either before or after TBI reduced the VWF reactivity with minimal influence on VWF secretion. rADAMTS-13 protected the integrity of endothelial cell barriers and prevented TBI-induced coagulopathy by enhancing VWF cleavage without impairing basal hemostasis. Promoting microvesicle clearance by lactadherin had efficacy similar to that of rADAMTS-13. This study uncovers a novel synergistic action between VWF and cellular microvesicles in TBI-induced vascular leakage and coagulopathy and demonstrates protective effects of rADAMTS-13.

Targeting von Willebrand Factor in Ischaemic Stroke: Focus on Clinical Evidence.

Despite great efforts in stroke research, disability and recurrence rates in ischaemic stroke remain unacceptably high. To address this issue, one potential target for novel therapeutics is the glycoprotein von Willebrand factor (vWF), which increases in thrombogenicity especially under high shear rates as it bridges between vascular sub-endothelial collagen and platelets. The rationale for vWF as a potential target in stroke comes from four bodies of evidence. (1) Animal models which recapitulate the pathogenesis of stroke and validate the concept of targeting vWF for stroke prevention and the use of the vWF cleavage enzyme ADAMTS13 in acute stroke treatment. (2) Extensive epidemiologic data establishing the prognostic role of vWF in the clinical setting showing that high vWF levels are associated with an increased risk of first stroke, stroke recurrence or stroke-associated mortality. As such, vWF levels may be a suitable marker for further risk stratification to potentially fine-tune current risk prediction models which are mainly based on clinical and imaging data. (3) Genetic studies showing an association between vWF levels and stroke risk on genomic levels. Finally, (4) studies of patients with primary disorders of excess or deficiency of function in the vWF axis (e.g. thrombotic thrombocytopenic purpura and von Willebrand disease, respectively) which demonstrate the crucial role of vWF in atherothrombosis. Therapeutic inhibition of VWF by novel agents appears particularly promising for secondary prevention of stroke recurrence in specific sub-groups of patients such as those suffering from large artery atherosclerosis, as designated according to the TOAST classification.

Role of calcium in regulating the intra- and extracellular cleavage of von Willebrand factor by the protease ADAMTS13

Abstractvon Willebrand factor (VWF) and the metalloprotease a disintegrin and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13) are present both within endothelial cells (ECs) and in peripheral blood. Calcium concentrations are lower in intracellular compartments (80-400 μM) compared with the extracellular milieu (∼1.25 mM). Because low calcium favors VWF A2-domain proteolysis by ADAMTS13, the dependence of proteolysis rates on calcium was assayed both within ECs and in blood. Confocal microscopy studies demonstrate partial perinuclear colocalization of VWF with ADAMTS13 in human umbilical vein ECs (HUVECs). Consequently, low levels (5%-10%) of VWF cleavage products were detected in HUVEC lysates and also culture-supernatant following EC stimulation. This proteolysis occurred before disulfide bond formation. Compared with wild-type VWF A2-domain, calcium-binding mutants including the common von Willebrand disease (VWD) type 2A R1597W mutant were expressed in an open conformation in ECs and were highly susceptible to intracellular proteolysis. Fluorescence resonance energy transfer measurements demonstrate strong calcium-dependent VWF-A2 conformation changes at concentrations ≮500 μM, with unfolding rates being fourfold higher for monomeric VWF A2-domain compared with multimeric, full-length VWF. Under shear, physiological levels of ADAMTS13 did not cleave VWF strings on HUVECs, unless platelets were attached to stretch these strings under flow. Further, VWF-platelet string cleavage under shear proceeded with equal efficiency in the absence and presence of calcium at shear stress ≥1 dyn/cm2. Overall, low calcium levels may promote intracellular VWF proteolysis particularly during VWD type 2A disease. Calcium has a negligible effect on VWF-platelet string proteolysis under physiologically relevant fluid shear.

Effect of Vitamin D Status on Von Willebrand Factor and ADAMTS13 in Diabetic Patients on Chronic Hemodialysis.

Von Willebrand factor (vWF) is a glycoprotein with a crucial role in the formation of platelet thrombi, and ADAMTS13 is the main enzyme responsible for vWF cleavage. Both are important in the relationship between diabetic nephropathy, hypercoagulability, and cardiovascular disease. This study evaluated a potential relationship between vitamin D (vitD) levels, vWF, ADAMTS13 activity, and inflammation in diabetic patients on chronic hemodialysis (HD). Blood samples from 52 diabetic patients on chronic HD were obtained to determine vitD levels, vWF, and ADAMTS13 activity, and inflammatory markers. HD patients were grouped according to 25-hydroxyvitamin D [25(OH) VitD]<25 nmol/L (n=16) or >25 nmol/L (n=36). vWF antigen and vWF activity were elevated in both groups, with an average of 214.3±82.6% and 175.8±72.6%, respectively. Average ADAMTS13 activity was within the normal range in both groups. Blood samples from the vitD <25 nmol/L group showed a positive correlation between c-reactive protein (CRP) and vWF levels (P=0.023; r=0.564; 95% confidence interval=0.095-0.828), with a negative correlation between HbA1c and 25(OH) VitD (P=0.015; r=-0.337; 95% confidence interval=-0.337-0.19). Diabetic patients on chronic HD had elevated vWF levels and activity with no significant change in ADAMTS13 activity. The correlation between CRP and vWF levels in the 25(OH) VitD<25 nmol/L group suggests inflammatory-related endothelial dysfunction in these patients.

VWF Cleavage Products Inhibit Shear-Induced Self-Association

BackgroundVon Willebrand factor (VWF) self-association is key to hemostasis in high-shear regions of the vasculature. Further, in pathologic small vessel thrombosis, VWF can also self-associate, even on an intact endothelium in the absence of vessel injury (Dong et al. Blood, 2002, 100:40339). VWF self-association preferentially occurs between large multimers, which are more sensitive to shear-induced unfolding. This process is regulated by ADAMTS13 proteolysis, which converts circulating ultra-large VWF multimers to smaller, less-adhesive multimers(Sadler. Proc Natl Acad Sci USA, 2002, 99(18):11552-4). ADAMTS13 not only cleaves large multimers, in the process it also generates small VWF cleavage fragments. This process is sometimes excessive, such as in von Willebrand disease (VWD) type 2A, in which large amounts of cleavage products may be present. However, the role of these small VWF fragments in regulating VWF function has not been studied. Here, we propose that small VWF fragments inhibit VWF self-association by competitively inhibiting self-association sites without providing functional substrates to propagate VWF strand formation.MethodsDevice: We developed a technique based on a device recently used by the Diamond laboratory to visualize VWF self-association in vitro (Zhu et al. Biorheology, 2015, 52:303; Herbig and Diamond. J. Thromb. Haem. 2015, 13:1699). The device consists of a PDMS microfluidic channel of width 60 µm with a 30 µm square pillar in the center. Flow through the channel produces high shear gradients next to the block. The inlet shear rate was set at 5000 s-1.VWF fragment preparation: Purified plasma VWF was incubated with purified recombinant ADAMTS13 (with a biotin tag) at a ratio of 5:1 at 37°C in 1.5 M urea for 16 hr. Recombinant ADAMTS13 was then removed with streptavidin-coated beads, and urea was removed with a PD10 column. This produced fragments corresponding to N- and C-termini VWF dimers.Assay: VWF fragments were mixed with purified plasma VWF (5 µg/mL) at a molar ratio (fragment to VWF monomer) of 1:1 and 5:1 and the mixture was immediately perfused into the channel. Results were compared to those from samples that contained either only fragments or only purified VWF (30 µg/mL). A total sample volume of 200 µL was perfused over 10 minutes, and VWF strand formation was monitored in real-time with differential interference contrast and immunofluorescence microscopy.Patient Samples: Samples from patients with type 2A VWD and thrombotic thrombocytopenic purpura (TTP) were obtained from consented adults under a protocol approved by the University of Washington Institutional Review Board. A total of 200 µL of sample was perfused in the channel over 10 minutes.ResultsWe were able to visualize VWF strand formation induced by shear stress alone when plasma was perfused through the channel. The strands formed from the initial adhesion of VWF to the pillar and subsequent self-association of VWF with the adherent VWF. Strands formed at physiologic VWF concentration and shear stress. The addition of small VWF fragments at 1:1 and 5:1 ratios delayed the formation and elongation of VWF strands and reduced the final extent of strand formation by approximately 20% and 60%, respectively. Compared to normal, strand formation was more pronounced in the absence of small VWF fragments (TTP) and less pronounced in the presence of larger amounts of small VWF fragments (VWD 2A).SummaryWe present a microfluidic assay to visualize in real-time VWF strand formation by generating elongational flow (shear gradients). VWF strands thus formed were reduced in the presence of small VWF fragments generated by ADAMTS13 both in a purified system and in patient samples. This result suggests that the hemostatic defect associated with type 2 VWD is a consequence not only of the deficiency of large multimers, but also of the presence of excessive quantities of proteolytic fragments, and that the profound thrombotic phenotype of TTP results not only from excess ULVWF, but also from the deficiency of smaller fragments. DisclosuresNo relevant conflicts of interest to declare.

Pharmacodynamic Profile of a Recombinant ADAMTS13 (BAX930) in Hereditary Thrombotic Thrombocytopenic Purpura (Upshaw-Schulman Syndrome (USS))

Pharmacodynamic Profile of a Recombinant ADAMTS13 (BAX930) in Hereditary Thrombotic Thrombocytopenic Purpura (Upshaw-Schulman Syndrome (USS))

Tissue factor: A potent stimulator of Von Willebrand factor synthesis by human umbilical vein endothelial cells.

Inflammation and dysfunction of endothelial cells are thought to be triggers for the secretion of Von Willebrand factor. The aim of this study was to examine the effects of the inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-α) and the coagulation factors, tissue factor and thrombin on the release and cleavage potential of ultra-large von Willebrand factor (ULVWF) and its cleavage protease by cultured human umbilical vein endothelial cells (HUVEC). HUVEC were treated with IL-6, IL-8, and TNF-α, tissue factor (TF) and thrombin, and combinations thereof for 24 hours under static conditions. The cells were then exposed to shear stress after which the VWF-propeptide levels and the VWF cleavage protease, ADAMTS13 content were measured. All treatments and their combinations, excluding IL-6, significantly stimulated the secretion of VWF from HUVEC. The VWF secretion from the HUVEC was stimulated most by the combination of TF with TNF-α. Slightly lower levels of ADAMTS13 secretion were found with all treatments. This may explain the thrombogenicity of patients with inflammation where extremely high VWF levels and slightly lower ADAMTS13 levels are present.

Acquired von Willebrand factor deficiency caused by LVAD is ADAMTS-13 and platelet dependent

IntroductionThe high shear rates induced by left ventricular assist devices cause acquired von Willebrand disease (aVWD). We hypothesised that an ex vivo model could be established to study whether mechanical shear stress alone causes aVWD or whether this process depends also on the VWF cleavage protein ADAMTS-13 and on platelets. Materials and methodsHealthy volunteers and two patients with congenital ADAMTS-13 deficiency donated blood. In vitro closed extracorporeal circuits were established using medically approved left ventricular assist devices (LVAD). VWF multimers were quantified by gel electrophoresis; VWF antigen, ristocetin cofactor activity (VWF:RCo), ADAMTS-13 levels and platelet function were assessed. ResultsThe high shear stress in the extracorporeal circulation rapidly decreased VWF:RCo and thereby the VWF:RCo/VWF:Ag ratio by 47% (p<0.01) to pathologically low values. Concomitantly, high molecular weight multimers (HMWM) decreased: up to 14–15 mers were visible on the gels at baseline, which were reduced by a maximum of 6–7 mers, corresponding to an average 68% lower densitometry signal of HMWM (p<0.001). This was accompanied by marked reduction of aggregation by various agonists (p<0.005). In contrast, the two patients with congenital thrombocytopenic purpura with virtually complete deficiency of ADAMTS-13 activity had only a minimal or no decrease in multimers (p<0.005 vs. healthy controls). Similarly, no or minimal depletion of large multimers occurred, when normal plasma circulated without platelets. ConclusionAn in vitro model for LVAD associated aVWD demonstrated that ADAMTS-13 and platelets contribute to the depletion of HMWM of VWF.

Patients With End Stage Congestive Heart Failure Have Decreased High Molecular Weight Von Willebrand Factor Multimers and Higher Plasma Levels Of Total TGF-β1: Implantation Of a Left Ventricular Assist Devices (LVAD) Exacerbates Both Abnormalities

Left ventricular assist devices (LVADs) have improved survival of patients with end stage congestive heart failure (HF), but patients are at high risk of both hemorrhage and thrombosis. Acquired von Willebrand (vW) disease is observed in most patients after continuous flow LVAD insertion, presumably due to shear-dependent vW factor (vWf) cleavage, which may contribute to hemorrhage, but there are no validated thrombosis biomarkers. Since shear can also activate platelets, and platelets contain high concentration of transforming growth factor β1 (TGF-β1), we analyzed plasma TGF-β1 levels in patients before and after LVAD implantation and correlated the data with the loss of high molecular weight (HMW) vWf multimers.Blood samples were collected from 14 HF patients enrolled either as a destination therapy (n=2) or as a bridge to transplantation (n=12) at the Columbia University before and after LVAD implantation. Plasma was prepared by centrifuging blood at 12,000 rpm for 5 min at 4°C within 5 min of blood drawing. Total TGF-β1 levels were measured after acidification of samples using a 2-antibody ELISA (R&D Systems). The platelet α-granule proteins thrombospondin 1 (TSP-1) and platelet factor 4 (PF4) were detected by immunoblotting. vWf multimers were analyzed by electrophoresis of plasma after running in 1.2% discontinuous agarose gel followed by in-gel immunoreactivity band quantification using the LI-COR imaging system. HMW-vWf multimers were defined as the percentage of total vWf antigen contributed by multimers above the 11th identifiable band, starting from the cathodal position.Plasma total TGF-ß1 levels were higher in HF patients before LVAD implantation than in 16 healthy controls (3.76 ± 1.55 vs. 1.0 ± 0.60 ng/ml; p <0.001). After LVAD insertion, the levels in HF patients increased further to 5.20 ± 2.30 ng/ml (p=0.014). Plasma TGF-ß1 levels in HF patients before and after LVAD implantation were positively correlated with TSP-1 levels (r=0.86; p<0.0001), but PF4 was not detectable, suggesting in vivo release of TGF-β1 from platelets. A reduced percentage of HMW-vWf multimers was observed in HF patients before LVAD insertion compared to healthy controls (18.0 ± 10.0 vs. 29.5 ± 2.5 %; p=0.018), and the percentage was further reduced after LVAD implantation (11.0 ± 9.0 %; p=0.027). There was a weak negative correlation between the percentage of HMW-vWf multimers and TGF-ß1 after LVAD implantation (r= - 0.40; p=0.05).We conclude that HF itself, even before LVAD insertion, is associated with both loss of HMW-vWf multimers and elevation of plasma TGF-ß1 levels and both abnormalities are exacerbated by LVAD implantation. Disclosures:No relevant conflicts of interest to declare.