Von Willebrand Factor Binding Research Articles

R1205H (Vicenza) causes conformational changes in the VWF-D’D3 domains and enhances VWF binding to clearance receptors LRP1 and SR-A1.

Von Willebrand factor (VWF)-R1205H variant (Vicenza) results in markedly enhanced VWF clearance in humans that has been shown to be largely macrophage-mediated. However, the biological mechanisms underlying this enhanced clearance remain poorly understood. This study aimed to investigate the roles of (i) specific VWF domains and (ii) different macrophage receptors in regulating enhanced VWF-R1205H clearance. In vivo clearance of full-length and truncated wild-type (WT)-VWF and VWF with R1205 substitutions was investigated in VWF-/- mice. Plate-binding assays were employed to characterize VWF binding to purified scavenger receptor class A member 1 (SR-A1), low-density lipoprotein receptor-related protein-1 (LRP1) cluster II or cluster IV receptors, and macrophage galactose-type lectin (MGL). In full-length VWF missing the A1 domain (VWF-ΔA1), introduction of R1205H led to significantly enhanced clearance in VWF-/- mice compared to WT-VWF-ΔA1. Importantly, R1205H in a truncated VWF-D'D3 fragment also triggered increased clearance compared to WT-VWF-D'D3. Additional in vivo studies demonstrated that VWF-R1205K (which preserves the positive charge at 1205) exhibited normal clearance, whereas VWF-R1205E (which results in loss of the positive charge) caused significantly enhanced clearance, pinpointing the importance of the positive charge at VWF-R1205. In vitro plate-binding studies confirmed increased VWF-R1205H interaction with SR-A1 compared to WT-VWF. Furthermore, significantly enhanced VWF-R1205H binding to LRP1 cluster IV (p<0.001) and less marked enhanced binding to LRP1 cluster II (p=0.034) was observed. In contrast, VWF-R1205H and WT-VWF demonstrated no difference in binding affinity to MGL. Disruption of the positive charge at amino acid 1205 causes conformational changes in the VWF-D'D3 domains, and triggers enhanced LRP1 and SR-A1 mediated clearance.

Essential role of glycoprotein Ibα in platelet activation

AbstractGlycoprotein Ibα (GPIbα), the ligand-binding subunit of platelet GPIb-IX complex, interacts with von Willebrand factor (VWF) exposed at the injured vessel wall, initiating platelet adhesion, activation, hemostasis, and thrombus formation. The cytoplasmic tail of GPIbα interacts with 14-3-3ζ, regulating the VWF-GPIbα–elicited signal transduction and VWF binding function of GPIbα. However, we unexpectedly found that the GPIbα–14-3-3ζ association, beyond VWF-dependent function, is essential for general platelet activation. We found that the myristoylated peptide of GPIbα C-terminus MPαC, a potential GPIbα inhibitor, by itself induced platelet aggregation, integrin αIIbβ3 activation, granule secretion, and phosphatidylserine (PS) exposure. Conversely, the deletion of the cytoplasmic tail of GPIbα in mouse platelets (10aa−/−) decreased platelet aggregation, integrin αIIbβ3 activation, granule secretion, and PS exposure induced by various physiological agonists. Phosphoproteome-based kinase activity profiling revealed significantly upregulated protein kinase C (PKC) activity in MPαC-treated platelets. MPαC-induced platelet activation was abolished by the pan-PKC inhibitor and PKCα deletion. Decreased PKC activity was observed in both resting and agonist-stimulated 10aa−/− platelets. GPIbα regulates PKCα activity by sequestering 14-3-3ζ from PKCα. In vivo, the deletion of the GPIbα cytoplasmic tail impaired mouse hemostasis and thrombus formation and protected against platelet-dependent pulmonary thromboembolism. Therefore, our findings demonstrate an essential role for the GPIbα cytoplasmic tail in regulating platelet general activation and thrombus formation beyond the VWF–GPIbα axis.

Impact of rise and fall phases of shear on platelet activation and aggregation using microfluidics.

Blood flow disorders are often the result of the non-physiological narrowing of blood arteries caused by atherosclerosis and thrombus. The blood then proceeds through rising-peak-decreasing phases as it passes through the narrow area. Although abnormally high shear is known to activate platelets, the shear process that platelets undergo in small arteries is complex. Thus, understanding how each shear phase affects platelet activation can be used to improve antiplatelet therapy and decrease the risk of side effects like bleeding. Blood samples were sheared (68.8 ms,5200s-1) in vitro by the microfluidic technique, and platelet activation levels (P-selectin and integrin αIIbβ3) and von Willebrand factor (vWF) binding to platelets were analyzed by flow cytometry. Post-stenosis platelet aggregation was dynamically detected using microfluidic technology. We studied TXA2, P2Y12-ADP, and integrin αIIbβ3-fibrinogen receptor pathways by adding antiplatelet drugs, such as acetylsalicylic acid (ASA, an active ingredient of aspirin that inhibits platelet metabolism), ticagrelor (hinders platelet activation), and tirofiban (blocks integrin αIIbβ3 receptor) in vitro, respectively, to determine platelet activation function mediated by transient non-physiological high shear rates. We demonstrated that platelets can be activated under transient pathological high shear rates. The shear rise and fall phases influenced shear-induced platelet activation by regulating the binding of vWF to platelets. The degree of platelet activation and aggregation increased with multiple shear rise and fall phases. ASA did not inhibit shear-mediated platelet activation, but ticagrelor and tirofiban effectively inhibited shear-mediated platelet activation. Our data demonstrated that the shear rise and fall phases play an important role in shear-mediated platelet activation and promote platelet activation and aggregation in a vWF-dependent manner. Blocking integrin αIIbβ3 receptor and hindering P2Y12-ADP were beneficial to reducing shear-mediated platelet activation.

Cost-Effectiveness of Caplacizumab in the Warranty Program in Immune Thrombotic Thrombocytopenic Purpura in the USA

Background Immune thrombotic thrombocytopenic purpura (iTTP) is a rare, life-threatening thrombotic microangiopathy. Caplacizumab is a humanized nanobody that inhibits formation of microthrombi in iTTP by disrupting binding of von Willebrand factor to platelets. It is the only treatment approved in combination with plasma exchange therapy (PEX) and immunosuppression (IS) for the treatment of iTTP. The United Kingdom National Institute for Health and Care Excellence (NICE) committee reviewed cost-effectiveness of caplacizumab. The committee concluded that addition of caplacizumab to PEX+IS is cost-effective and recommended that the drug be reimbursed in the UK. The Sanofi Promise Warranty Program ® (SPWP) was introduced in January 2023 in the US. It refunds the full cost of caplacizumab to the hospital when treatment is discontinued due to non-response defined as refractoriness (patient with platelet counts &amp;lt;50 × 10 9/L after 4 days of combined treatment with caplacizumab and PEX+IS; up to 6 doses are refunded), or exacerbation (new drop in platelet count after initial platelet count normalization [&amp;gt;150 × 10 9/L], necessitating re-initiation of PEX after ruling out any causes for the drop in platelet count other than iTTP pathophysiology, up to 12 pre-exacerbation doses are refunded). Objectives To evaluate the cost-effectiveness of adding caplacizumab to PEX+IS from a US perspective within the context of SPWP. Methods The NICE model was adapted to the US setting by replacing UK costs with US costs and discount rates. All clinical assumptions and structure remain unchanged. Clinical inputs were derived from the HERCULES trial (NCT02553317) intent-to-treat population (mean age: 46 years; females: 69%, exacerbation rates: 4.17% during double blind phase and 12.50% in total; refractoriness: 0%). The model consisted of a 3-month decision tree, which captured patient treatment and outcomes over the acute iTTP episode, followed by a long-term Markov model (time horizon: up to 55 years; 3-monthly cycles; Figure 1). An annual discount rate of 3% was applied for both costs and quality-adjusted life-years (QALYs). Under the SPWP, the cost of caplacizumab was assumed to be $0 for refractory patients and those who exacerbate early, no discount was applied for patients who experience exacerbations after drug discontinuation, however, clinical outcomes were captured. Results Prior to the SPWP, caplacizumab addition to PEX+IS delivered a gain of 1.75 QALYs (2.96 life-years [LYs]) as compared with PEX+IS alone, at an increased lifetime cost of $256,000. The incremental cost effectiveness ratio (ICER) was $146,350 per QALY and $86,420 per LY gained ( Table 1). An estimated 4.17% of treated patients qualified for the SPWP. With this outcomes-based program, the ICER improved to $138,480 per QALY and $81,780 per LY gained, respectively ( Table 1). Conclusion Based on these analyses using the UK NICE model adapted with US costs, caplacizumab is cost-effective considering WTP threshold of $150,000 to $200,000 and has improved cost-effectiveness when the warranty program is taken into account. Outcomes based programs can improve the cost effectiveness of a treatment in addition to mitigating concerns of perceived uncertainty of efficacy in treating patients with iTTP in the US.

Studies of Protein Disulfide Isomerase (PDI) Binding to Platelets and Production of a Murine Monoclonal Antibody (mAb) to Integrin αIIbβ3 That Inhibits PDI Binding and Platelet Aggregation

L.W. and J.W. contributed equally Introduction: The platelet integrin αIIbβ3 plays a pivotal role in both hemostasis and thrombosis, and is an established target for antiplatelet therapy. Upon activation, αIIbβ3 undergoes a conformational change from a low-affinity, bent-closed conformation to a high-affinity, extended-open conformation, resulting in the binding of fibrinogen and von Willebrand factor and platelet aggregation. αIIbβ3 activation is regulated by protein disulfide isomerase (PDI), an enzyme that catalyzes disulfide bond formation, reduction, and isomerization. One of us (LW) previously showed that PDI binds to thrombin-activated platelets, but the αIIb or β3 conformation(s) and domain(s) to which PDI binds is unknown. Methods: PDI was produced in E. coli and purified and labeled with Alexa-488. Human platelets and Alexa-488-PDI were stimulated with a variety of agonists either in the absence or presence of unlabeled PDI (background control) and then analyzed by flow cytometry. For monoclonal antibody (mAb) production, BALB/cAnN mice were immunized with human platelets and their spleen cells isolated and fused with a non-secretory myeloma cell line. Hybridomas were isolated by colony picking in semi-solid media and culture supernatants were assayed for their ability to inhibit PDI binding to platelets activated with the PAR1 thrombin receptor activating peptide SFLLRN (T6). One mAb that inhibited PDI binding (21D10) was assessed for its effects on platelet aggregation. The epitope of 21D10 on αIIbβ3 was explored by comparing the binding properties of 21D10 to that of previously characterized anti-αIIbβ3 mAbs. Using cryo-electron microscopy (cryo-EM) single particle analysis, we determined the structure of the Fab fragment of 21D10 in complex with full-length αIIbβ3 purified from human platelets. Results: T6, the PAR4 thrombin receptor activating peptide AYPGKF, and collagen-related peptide induced PDI binding to washed platelets [mean fluorescence intensity (MFI) of 547 ± 21 compared with 229 ± 12 for background control, p &amp;lt; 0.001; 576 ± 111 vs 215 ± 27 for background control, p &amp;lt; 0.05; and 934 ± 99 vs 167 ± 14 for background control, p &amp;lt; 0.001, respectively]. The reducing agent dithiothreitol (DTT) also enhanced PDI binding (MFI of 595 ± 147 vs 178 ± 24 for background control, p &amp;lt; 0.05). mAb 21D10 and its Fab fragment (40 μg/ml) inhibited PDI binding to platelets [MFI of 357 ± 25 vs 567 ± 60 for buffer control, p &amp;lt; 0.01 (figure); and 286 ± 38 vs 507 ± 86 for buffer control; p &amp;lt; 0.05)], fibrinogen binding (6,493 ± 815 vs 15,058 ± 1,935 for buffer control, p &amp;lt; 0.001; 1,714 ± 276 vs 14,917 ± 2,129 for buffer control, p &amp;lt; 0.0001), and maximum platelet aggregation (mean ± SD of 32 ± 8 vs 67 ± 2% for buffer control, p &amp;lt; 0.01; and 30 ± 8 vs 69 ± 3% for buffer control; p &amp;lt; 0.01). For comparison, the PDI inhibitor rutin at 45 μM produced similar inhibition. 21D10 did not inhibit the binding of mAbs 10E5 or 7E3, both of which inhibit fibrinogen binding to αIIbβ3, to platelets, and vice versa. EDTA treatment of platelets at either 22°C, pH 7.4, or 37°C, pH 8.0, had minimal effect on 21D10 binding, while both conditions inhibited 7E3 binding and the latter inhibited 10E5 binding. 21D10 reacted with the nonreduced but not reduced β3 subunit by immunoblotting. With chymotrypsin digested platelets, both 21D10 and the mAb 7H2, which binds to the PSI domain of β3, bound to bands extending down to 66 kDa, but 21D10 also bound to the bands below 66 kDa, whereas 7H2 did not. The cryo-EM structure of the 21D10 Fab-αIIbβ3 complex revealed that 21D10 binds to two different αIIbβ3 conformations that differ in the orientation of the leg domains (in one the legs are nearly bent and in the other the legs are partially extended). In both conformations, 21D10 binds to the PSI, I-EGF-1 and I-EGF-2 domains of β3 (figure). Conclusion: We have shown that in addition to thrombin, T6, a PAR4 activating peptide, collagen-related peptide, and DTT can induce PDI binding to platelets. We produced a mAb to human platelets (21D10) that can inhibit T6-induced PDI binding to platelets. Immunoblotting and cryo-EM analysis shows that 21D10 binds to the PSI, I-EGF1, and I-EGF2 regions of β3. If 21D10 prevents PDI binding by steric hindrance, it is likely that PDI binds to β3 at a site distant from the RGD ligand binding pocket. The functional effects of 21D10 may reflect its inhibition of PDI to platelets, but it may also reflect inhibition of other related proteins, in particular, ERp57.

Phase II Study of Danazol with Plasma Exchange and Corticosteroids for the Treatment of Thrombotic Thrombocytopenic Purpura

Background: Ahn and colleagues reported a decrease in plasma exchange (PE)/plasma infusion requirement with danazol in seven cases of thrombotic thrombocytopenic purpura (TTP) (Ahn Blood 2006;108(11):1057a). We reported an increase in platelet count (PLT) within 24 hours with danazol in two of three cases of human immunodeficiency virus-associated TTP resistant to PE (Torri Transfusion 2010;50:86a). In 2009, we initiated a phase II study of danazol with PE and corticosteroids (CS) for the treatment of TTP. This trial was approved by the institutional review board and was registered at ClinicalTrials.gov (ID# NCT00953771). We report the final results. Aims: To determine if danazol reduces number of PE's, time to remission (TR), and length of stay (LOS) when administered with standard therapy for the initial treatment of TTP. Methods: Experimental Group (EG): Patients with TTP (PLT &amp;lt; 100,000/µL and microangiopathy) receive daily PE, prednisone 1 mg/kg, and danazol 600 mg. Following remission (PLT &amp;gt; 150,000/µL for three consecutive days), PE is tapered to every other day for one week then stopped. Then prednisone is tapered weekly over four weeks then stopped. After one week danazol is tapered by 200 mg weekly then stopped. Historical controls (HC): Patients treated for TTP at St. Luke's-Roosevelt Hospital Center with PE with or without CS from 2000 to 2007 (n=20). Relapse definition: PLT &amp;lt; 100,000/µL with microangiopathy &amp;gt; 30 days after remission. Total number of PE's, TR, LOS, and continuous response rates are compared between the 2 groups. Accrual target: 16 patients to detect a 40% decrease in the number of PE's with 80% power and a level of significance of 0.05.The study was terminated in 2018 due to low accrual. Results: Nine patients were enrolled. Mean number of PE's (days) was 14.8 (EG) and 16.1 (HC), mean TR (days) was 11.4 (EG) and 11 (HC), and mean LOS (days) was 27.3 (EG) and 24.9 (HC), p &amp;gt; 0.05. Four patients in HC had a total of 6 relapses at our institution at a median of 28.25 months, range 2.75 to 97 months after the initial event. One patient in EG relapsed 43 months after study enrollment. One patient in EG died from an unrelated cause. None of the additional seven patients in EG have relapsed during a median follow-up of 49 months, range 12 to 125 months. One episode of pulmonary edema and one episode of hypotension occurred in EG, for which treatment was not discontinued. Discussion: Our study shows that danazol does not reduce PE requirement, TR, or LOS during treatment of TTP, but may prevent relapse. This pattern of response is similar to that which has been observed with rituximab. In a phase 2 study of rituximab in conjunction with PE and CS for the treatment of TTP (Scully Blood 2011), in which results were compared with historical controls who did not receive rituximab, median number of PE's (days) and median LOS (days) were 16.5 and 16.5 in the rituximab group versus 18.0 and 20 in the historical control group, p=ns. Ten percent of patients relapsed at a median of 27 months in the rituximab group whereas 57% of patients relapsed at a median of 18 months in the historical control group. Of interest, the cost of rituximab 375 mg/m 2 weekly for 4 weeks as given would have been approximately $ 25,366.50. The cost of danazol in our protocol would have been approximately $ 2196.00. Caplacizumab is a humanized monoclonal antibody fragment that inhibits von Willebrand factor binding to platelet glycoprotein lb-IX-V. In a phase 3 study (Scully N Engl J Med 2019), caplacizumab in conjunction with PE and CS for the treatment of TTP led to reduced PE's, time to normalization of platelet counts, and LOS, but increased relapses in comparison with placebo. In 2020, the International Society on Thrombosis and Haemostasis strongly recommended adding CS to PE for the treatment of TTP and conditionally recommended adding rituximab and caplacizumab to PE and CS (Zheng J Thromb Haemost 2020). Conclusion: Danazol in conjunction with PE and CS may be a low-cost option to prevent relapses of TTP.

Angiopoietin-2 binds to multiple interactive sites within von Willebrand factor

BackgroundBiosynthesis of von Willebrand factor (VWF) in endothelial cells drives the formation of storage-organelles known as Weibel-Palade bodies (WPBs). WPBs also contain several other proteins, including angiopoietin-2 (Ang-2). ObjectivesAt present, the molecular basis of the VWF–Ang-2 interaction is poorly understood. Here, we used immunosorbent-binding assays and specific recombinant VWF fragments to analyze VWF–Ang-2 interactions. ResultsWe found that VWF bound to immobilized Ang-2 most efficiently (half-maximal binding at 0.5 ± 0.1 μg/mL) under conditions of high CaCl2 (10 mM) and slightly acidic pH (6.4-7.0). Interestingly, several isolated recombinant VWF domains (A1/Fc, A2/Fc, D4/Fc, and D’D3-HPC4) displayed dose-dependent binding to immobilized Ang-2. Binding appeared specific, as antibodies against D’D3, A1, and A2 significantly reduced the binding of these domains to Ang-2. Complexes between VWF and Ang-2 in plasma could be detected by immunoprecipitation- and immunosorbent assays. Unexpectedly, control experiments also revealed complexes between VWF and angiopoietin-1 (Ang-1), a protein structurally homologous to Ang-2. Furthermore, direct binding studies showed dose-dependent binding of VWF to immobilized Ang-1 (half-maximal binding at 1.8 ± 1.0 μg/mL). Interestingly, rather than competing for Ang-1 binding, Ang-2 enhanced the binding of VWF to Ang-1 about 3-fold. Competition experiments further revealed that binding to VWF does not prevent Ang-1 and Ang-2 from binding to Tie-2. ConclusionOur data show that both Ang-1 and Ang-2 bind to VWF, seemingly using different interactive sites. Ang-2 modulates the binding of VWF to Ang-1, the (patho)-physiological consequences of which remain to be investigated.

Effect of far-infrared radiation therapy on von Willebrand factor in patients with chronic kidney disease.

Hemostatic abnormality has contributed to vascular access thrombosis in patients with chronic kidney disease (CKD). Previous studies have demonstrated that far-infrared radiation (FIR) therapy can maintain the patency and maturity of arteriovenous fistulas of patients undergoing hemodialysis (HD). However, prolonged access bleeding is observed once FIR is conducted at the end of dialysis. FIR can block the binding of platelet and von Willebrand factor (vWF), a predictor of hemostatic abnormality and vascular access thrombosis. However, clinical studies exploring FIR and vWF are sparse. We recruited 20 HD patients, 21 CKD patients, and 20 controls to examine the alteration of vWF and a disintegrin and metalloproteinase with thrombospondin type 1 repeats 13 (ADAMTS13) following a single 40-min session of FIR therapy. In addition, the alteration of these factors in the HD group was examined following a 40-min FIR session thrice a week for 3 months. A decreasing trend in the vWF activity-antigen ratio of participants in all groups following a single FIR session was observed. In addition, the ratio in the HD group was significantly lower following 3 months of FIR therapy. The subgroup analysis revealed a consistent trend and multiple regression analysis showed that participants not taking hydroxymethylglutaryl-coenzyme A reductase inhibitor, diabetes mellitus, and higher hemoglobin levels were the significant factors. The alteration of the vWF activity-antigen ratio correlated moderately to that of ADAMTS13 antigen and activity. FIR may alter the ratio of ultra-large vWF multimers through ADAMTS13, contributing to inhibiting platelet-endothelium interactions of CKD patients.

Increased Binding of von Willebrand Factor to Sub-Endothelial Collagen May Facilitate Thrombotic Events Complicating Bothrops lanceolatus Envenomation in Humans.

Consumption coagulopathy and hemorrhagic syndrome exacerbated by blood anticoagulability remain the most important causes of lethality associated with Bothrops snake envenomation. Bothrops venom also engages platelet aggregation on the injured endothelium via von Willebrand factor (vWF) interactions. Besides platelet aggregation, some Bothrops venom toxins may induce qualitative thrombopathy, which has been in part related to the inhibition of vWF activation. We tested whether B. lanceolatus venom impaired vWF to collagen(s) binding (vWF:CB) activity. Experiments were performed with B. lanceolatus crude venom, in the presence or absence of Bothrofav, a monospecific B. lanceolatus antivenom. Venom of B. lanceolatus fully inhibited vWF to collagen type I and III binding, suggesting venom interactions with the vWF A3 domain. In contrast, B. lanceolatus venom increased vWF to collagen type VI binding, suggesting the enhancement of vWF binding to collagen at the vWF A1 domain. Hence, B. lanceolatus venom exhibited contrasting in vitro effects in terms of the adhesive properties of vWF to collagen. On the other hand, the antivenom Bothrofav reversed the inhibitory effects of B. lanceolatus venom on vWF collagen binding activity. In light of the respective distribution of collagen type III and collagen type VI in perivascular connective tissue and the sub-endothelium, a putative association between an increase in vWF:CB activity for collagen type VI and the onset of thrombotic events in human B. lanceolatus envenomation might be considered.

Mechanisms of inhibition of human monoclonal antibodies in immune thrombotic thrombocytopenic purpura

AbstractAntibody binding to a plasma metalloprotease, a disintegrin and metalloproteinase with thrombospondin type 1 repeats 13 (ADAMTS13), is necessary for the development of immune thrombotic thrombocytopenic purpura (iTTP). Inhibition of ADAMTS13-mediated von Willebrand factor (VWF) cleavage by such antibodies clearly plays a role in the pathophysiology of the disease, although the mechanisms by which they inhibit ADAMTS13 enzymatic function are not fully understood. At least some immunoglobulin G–type antibodies appear to affect the conformational accessibility of ADAMTS13 domains involved in both substrate recognition and inhibitory antibody binding. We used single-chain fragments of the variable region previously identified via phage display from patients with iTTP to explore the mechanisms of action of inhibitory human monoclonal antibodies. Using recombinant full-length ADAMTS13, truncated ADAMTS13 variants, and native ADAMTS13 in normal human plasma, we found that, regardless of the conditions tested, all 3 inhibitory monoclonal antibodies tested affected enzyme turnover rate much more than substrate recognition of VWF. Hydrogen-to-deuterium exchange plus mass spectrometry experiments with each of these inhibitory antibodies demonstrated that residues in the active site of the catalytic domain of ADAMTS13 are differentially exposed to solvent in the presence and absence of monoclonal antibody binding. These results support the hypothesis that inhibition of ADAMTS13 in iTTP may not necessarily occur because the antibodies directly prevent VWF binding, but instead because of allosteric effects that impair VWF cleavage, likely by affecting the conformation of the catalytic center in the protease domain of ADAMTS13. Our findings provide novel insight into the mechanism of autoantibody-mediated inhibition of ADAMTS13 and pathogenesis of iTTP.

A comparative study in patients with type 2 von Willebrand disease using 4 different platelet-dependent von Willebrand factor assays

BackgroundSeveral assays are now available to evaluate platelet-dependent von Willebrand factor (VWF) activity. ObjectiveTo report the results obtained using 4 different assays in patients with von Willebrand disease (VWD) carrying variants mainly in the A1 domain, which is critical for VWF binding to glycoprotein Ib (GPIb) and ristocetin. MethodsWe evaluated 4 different assays, 2 gain-of-function mutant GPIb binding (VWF:GPIbM) and 2 ristocetin cofactor (VWF:RCo) assays, in 76 patients with type 2 VWD. Patients and healthy controls were tested using VWF:GPIbM enzyme-linked immunosorbent assay (ELISA), VWF:GPIbM automated, VWF:RCo aggregometric, and VWF:RCo automated assays. ResultsThere was a good correlation (Pearson’s r>0.82) and agreement (Bland-Altman plots assessment) between the 4 assays, although several outliers existed among the type 2B without high-molecular-weight multimers (HMWM). The VWF activity/VWF:antigen ratios, calculated for each assay, were used to establish the percentage of a correct diagnosis of type 2 (ratio<0.60) in these patients: VWF:RCo aggregometric, 2A(100%), 2M(78%), 2M/2A(100%), 2B(68%); VWF:RCo automated, 2A(88%), 2M(89%), 2M/2A(100%), 2B(63%); VWF:GPIbM ELISA, 2A(96%), 2M(67%), 2M/2A(67%), 2B(0%); VWF:GPIbM automated, 2A(73%), 2M(44%), 2M/2A(75%), 2B(84%). In type 2B patients with HMWM, all assays gave a ratio ≥0.60. ConclusionThe VWF:GPIbM-automated assay is the most effective to diagnose as type 2 the 2B variants, whereas the VWF:RCo assays are the most effective in detecting 2M and 2M/2A variants. The VWF:GPIbM ELISA greatly overestimates the activity of the type 2B patients lacking HMWM. In this study, the use of a VWF activity/VWF:antigen ratio cut-off of 0.70 halved the number of misdiagnosed patients.

Abstract 94: Randomized, Double-blind, Placebo-controlled Phase 1 Study Of Vwf-binding Aptamer, Bb-031: Safety, Tolerability, Pharmacokinetic And Pharmacodynamic Activity In Healthy Volunteers

Introduction: Von Willebrand Factor (VWF) is a ubiquitous component of thrombi extracted by endovascular thrombectomy from patients with large vessel occlusion (LVO) stroke. BB-031 is a modified RNA aptamer that targets von Willebrand Factor (VWF) that lyses occlusive thrombi and improves outcomes in pre-clinical models of acute ischemic stroke (AIS). Hypothesis: BB-031 is safe, tolerable, and inhibits both VWF binding and activity in a dose-dependent manner. Methods: Healthy participants were randomized to receive BB-031 or placebo (6:2) by intravenous bolus injection at single ascending doses (SAD) of 0.1, 0.3, 1.0, 2.0 and 4.0 mg/kg (total n=40). Serial Clinical assessment, and blood sample collection for pharmacokinetic (PK) and pharmacodynamic (PD) analysis were performed. Non-compartmental PK analysis was conducted using Phoenix WinNonlin. PD evaluation included measurement of VWF inhibition and whole blood thrombosis (Platelet Function Analyzer [PFA-200]). Results: BB-031 was safe and well-tolerated for 28 days following single IV doses up to 4.0 mg/kg. There were no significant adverse events (SAEs), or treatment-emergent adverse events (TEAEs) leading to dose discontinuation. Minor events included bleeding at an IV site in 1 participant, and minor bleeding of an ulcer on the tongue in 1 participant. None of the minor TEAEs were dose-dependent. BB-031 demonstrated nonlinear, dose-dependent plasma PK across the dose range tested, with an apparent mean terminal half-life (t 1/2 ) of 18 min at 0.1 mg/kg to 67 min at 4.0 mg/kg. Dose-dependent changes in VWF binding were observed; &gt;95% maximal mean change from baseline was reached following a single IV dose of 4.0 mg/kg. Finally, PFA-200 results showed complete inhibition of clot formation (closing time ≥300 seconds) at all doses tested with a dose-dependent duration of inhibition and return to normal range. Conclusion: This first-in-human SAD study of BB-031 demonstrated safety following a single IV dose of 0.1 to 4 mg/kg, and dose-dependent patterns of VWF binding and platelet function changes. These results lay the foundation for future studies in patients suffering from thrombotic conditions including AIS.

Von Willebrand factor: from figurant to main character in the scene of inflammation

von Willebrand factor: from figurant to main character in the scene of inflammation

Comprehensive analysis of platelet glycoprotein Ibα ectodomain glycosylation

BackgroundPlatelet glycoprotein (GP) Ibα is the major ligand-binding subunit of the GPIb-IX-V complex that binds von Willebrand factor. GPIbα is heavily glycosylated, and its glycans have been proposed to play key roles in platelet clearance, von Willebrand factor binding, and as target antigens in immune thrombocytopenia syndromes. Despite its importance in platelet biology, the glycosylation profile of GPIbα is not well characterized. ObjectivesThe aim of this study was to comprehensively analyze GPIbα amino acid sites of glycosylation (glycosites) and glycan structures. MethodsGPIbα ectodomain that was recombinantly expressed or that was purified from human platelets was analyzed by Western blot, mass spectrometry glycomics, and mass spectrometry glycopeptide analysis to define glycosites and the structures of the attached glycans. ResultsWe identified a diverse repertoire of N- and O-glycans, including sialoglycans, Tn antigen, T antigen, and ABO(H) blood group antigens. In the analysis of the recombinant protein, we identified 62 unique O-glycosites. In the analysis of the endogenous protein purified from platelets, we identified 48 unique O-glycosites and 1 N-glycosite. The GPIbα mucin domain is densely O-glycosylated. Glycosites are also located within the macroglycopeptide domain and mechanosensory domain. ConclusionsThis comprehensive analysis of GPIbα glycosylation lays the foundation for further studies to determine the functional and structural roles of GPIbα glycans.

Serum miR-377 Can Be Used as a Diagnostic Marker for Acute Coronary Syndrome and Can Regulate Proinflammatory Factors and Endothelial Injury Markers.

The diagnostic value of microRNA-377 (miR-377) in patients with acute coronary syndrome (ACS) and explored miR-377's potential mechanisms. We performed an qRT-PCR to assess serum miR-377 levels in ACS patients and coronary artery ligation rat models. The diagnostic value of miR-377 was evaluated by determining the ROC curve. An ELISA assay was conducted to detect the model rat endothelial damage markers von Willebrand factor (vWF) and heart-type fatty acid binding protein (H-FABP), and proinflammatory cytokines TNF-α, IL-6, and IL-1β. The serum miR-377 level was elevated in the ACS patients and significantly increased in the ACS rats. MiR-377 has a high diagnostic value in ACS patients, with a 0.844 ROC, 76.47% specificity, and 87.10% sensitivity. MiR-377 was positively correlated with the expressions of vWF, H-FABP, cTnI, TNF-α, IL-6, and IL-1β. In ACS rats, reducing the expression of miR-377 significantly inhibited the increases in vWF, H-FABP, TNF-α, IL-6, and IL-1β. An elevated miR-377 level can be used as a diagnostic marker in patients with ACS. A reduction of miR-377 may alleviate ACS by improving myocardial damage such as endothelial injury and the inflammatory response.

Neutrophils Protect Against Staphylococcus aureus Endocarditis Progression Independent of Extracellular Trap Release.

Infective endocarditis (IE) is characterized by an infected thrombus at the heart valves. How bacteria bypass the immune system and cause these thrombi remains unclear. Neutrophils releasing NETs (neutrophil extracellular traps) lie at this interface between host defense and coagulation. We aimed to determine the role of NETs in IE immunothrombosis. We used a murine model of Staphylococcus aureus endocarditis in which IE is provoked on inflamed heart valves and characterized IE thrombus content by immunostaining identifying NETs. Antibody-mediated neutrophil depletion and neutrophil-selective PAD4 (peptidylarginine deiminase 4)-knockout mice were used to clarify the role of neutrophils and NETs, respectively. S. aureus mutants deficient in key virulence factors related to immunothrombosis (nucleases or staphylocoagulases) were investigated. Neutrophils releasing NETs were present in infected thrombi and within cellular infiltrates in the surrounding vasculature. Neutrophil depletion increased occurrence of IE, whereas neutrophil-selective impairment of NET formation did not alter IE occurrence. Absence of S. aureus nuclease, which degrades NETs, did not affect endocarditis outcome. In contrast, absence of staphylocoagulases (coagulase and von Willebrand factor binding protein) led to improved survival, decreased bacteremia, smaller infiltrates, and decreased tissue destruction. Significantly more NETs were present in these vegetations, which correlated with decreased bacteria and cell death in the adjacent vascular wall. Neutrophils protect against IE independent of NET release. Absence of S. aureus coagulases, but not nucleases, reduced IE severity and increased NET levels. Staphylocoagulase-induced fibrin likely hampers NETs from constraining infection and the resultant tissue damage, a hallmark of valve destruction in IE.

Polyphosphate Activates von Willebrand Factor Interaction with Glycoprotein Ib in the Absence of Factor VIII In Vitro.

Polyphosphate (polyP), a phosphate polymer released by activated platelets, may modulate various stages of hemostasis by binding to blood proteins. In this context, we previously reported that polyP binds to the von Willebrand factor (VWF). One of the most significant functions of VWF is to bind to and protect the blood circulating Factor VIII (FVIII). Therefore, here, we study the role of polyP in the VWF-FVIII complex in vitro and suggest its biological significance. Surface plasmon resonance and electrophoretic mobility assays indicated that polyP binds dynamically to VWF only in the absence of FVIII. Using the VWF Ristocetin Cofactor assay, the most accepted method for studying VWF in platelet adhesion, we found that polyP activates this role of VWF only at low levels of FVIII, such as in plasmas with chemically depleted FVIII and plasmas from severe hemophilia A patients. Moreover, we demonstrated that FVIII competes with polyP in the activation of VWF. Finally, polyP also increases the binding of VWF to platelets in samples from patients with type 2 and type 3 von Willebrand disease. We propose that polyP may be used in designing new therapies to activate VWF when FVIII cannot be used.

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

Application of microfluidic chip technology to study the inhibitory effect of tetramethylpyrazine on platelet aggregation, activation, and phosphatidylserine exposure mediated by pathological high shear rate.

In order to study the antithrombotic effect and mechanism of tetramethylpyrazine (TMA). In this study, we developed a microfluidic chip model that can mimic normal arteries and stenotic arterial vessels, and studied the inhibitory effects of TMA on platelet aggregation, activation (P-selectin, GPIIb/IIIa, monocyte-platelet aggregates) and phosphatidyl serine (PS) exposure. In addition, we also investigated the effect of TMA on ADP and ristocetin-induced platelet aggregation by turbidimetry. The results showed that TMA significantly inhibited the platelet aggregation, activation and PS exposure induced by pathological high shear rate. Under static conditions, TMA can inhibit ADP and ristocetin-induced platelet aggregation. The results indicated that TMA mainly inhibited platelet aggregation, activation and PS exposure by inhibiting the binding of von Willebrand factor (vWF) to the GPIb/IX/V complex, and partially inhibited platelet aggregation through the platelet P2Y 12 -ADP receptor pathway.

Von Willebrand factor links primary hemostasis to innate immunity

The plasma multimeric glycoprotein von Willebrand factor (VWF) plays a critical role in primary hemostasis by tethering platelets to exposed collagen at sites of vascular injury. Recent studies have identified additional biological roles for VWF, and in particular suggest that VWF may play an important role in regulating inflammatory responses. However, the molecular mechanisms through which VWF exerts its immuno-modulatory effects remain poorly understood. In this study, we report that VWF binding to macrophages triggers downstream MAP kinase signaling, NF-κB activation and production of pro-inflammatory cytokines and chemokines. In addition, VWF binding also drives macrophage M1 polarization and shifts macrophage metabolism towards glycolysis in a p38-dependent manner. Cumulatively, our findings define an important biological role for VWF in modulating macrophage function, and thereby establish a novel link between primary hemostasis and innate immunity.