by Changjie Zhang, Anju Kelkar, and Sriram Neelamegham von Willebrand factor self-association is regulated by the shear-dependent unfolding of the A2 domain by Changjie Zhang, Anju Kelkar, and Sriram Neelamegham BloodAdv Volume 3(7):957-968 April 9, 2019 © 2019 by The American Society of Hematology

Changjie Zhang et al. Blood Adv 2019;3:957-968 © 2019 by The American Society of Hematology

Role of VWF-A2 in self-association. Role of VWF-A2 in self-association. (A) Schematic of VWF proteins. A1 domain is deleted in ΔA1-VWF. WT protein (WT-VWF) has vicinal Cys in the A2 domain. The disulfide bond links the N and C terminus of VWF-A2 in Lock-VWF. (B) Protein multimer distribution of VWF variants expressed in HEK293T-furin cells. (C) Time course of VWF cleavage by 1 U/mL ADAMTS13 in the presence of 1.6 M urea at 37°C. Minimal cleavage of Lock-VWF is noted. (D-E) Five × 107/mL washed platelets were shear mixed at 9600/s in a cone-plate viscometer along with 10 μg/mL ΔA1-488, either in the absence of VWF or upon addition of 10 μg/mL WT-VWF or Lock-VWF. Buffer calcium concentration was varied from 0 to 1.5M CaCl2 (EDTA was not added). VWF self-association (D) and percentage platelet activation (E) were measured using flow cytometry for samples withdrawn at 5 minutes. VWF self-association quantifies the percentage of platelets binding more than basal (t = 0) levels of Alexa 488-conjugated ΔA1-VWF (ΔA1-488). Platelet activation measures the percentage of platelets binding PE-conjugated Annexin V. (F-G) VWF self-association (F) and platelet activation (G) triggered by WT-VWF was blocked by 20 µg/mL mAbs AK2 (anti-GpIbα) and AVW-3 (anti-VWF A1 domain). Data in panels D-G are from 3 to 4 independents runs, each containing 3 technical replicates. *P < .05 with respect to all other treatments at that calcium concentration. VWF self-association and platelet activation are higher for runs performed with WT-VWF compared with Lock-VWF. Changjie Zhang et al. Blood Adv 2019;3:957-968 © 2019 by The American Society of Hematology

Microfluidics thrombus formation assay. Microfluidics thrombus formation assay. (A-C) Washed blood containing fluorescent-labeled platelets and 10 µg/mL WT-VWF/Lock-VWF were perfused at a wall shear rate of 500/s over substrates adsorbed with 40 µg/mL ΔA1-VWF. (A) Thrombus formation was measured based on fluorescence intensity in the field of view. (B) Representative images of platelet accumulation on ΔA1-VWF substrate shows greater accumulation in the case of WT-VWF. (C) Platelet accumulation on ΔA1-VWF in runs containing WT-VWF was blocked by mAbs against GpIbα (AK2), VWF-A1 (AVW-3) and VWF-D’D3 (clone DD3.1). (D-E) Thrombus formation on collagen substrate in the presence of 10 µg/mL WT-VWF or Lock-VWF. (D) Wall shear rate was varied: 400/s, 1000/s, and 2000/s. Thrombus formation was blocked by mAbs AK2, AVW-3, and DD3.1, at 1000/s. (E) Representative images of thrombus formation at 3 minutes at different shears. All microfluidics assays were performed 3 to 4 times, each with 2 to 4 repeats. *P < .05 with respect to all other treatments. (B,E) Scale bars, 100 µm. Platelets stained using BCECF-AM. Changjie Zhang et al. Blood Adv 2019;3:957-968 © 2019 by The American Society of Hematology

VWF fiber formation on collagen in stenosed channel. VWF fiber formation on collagen in stenosed channel. (A) Schematic design of flow chamber used for fiber formation assay. (B) Citrated human plasma, supplemented with either 1.5 M CaCl2 or 10 mM EDTA, was perfused over type I collagen substrates for 10 minutes at 100 000/s. A FITC-coupled anti-VWF Ab was added postperfusion to visualize VWF fibers. Representative images in different parts of the flow chamber are show. VWF fiber formation was promoted upon calcium chelation. (C) Fluorescence intensity of fibers formed was measured in different parts of the flow device: inlet transition from 100 µm to 50 µm width, 50 μm middle section, and outlet from 50 μm to 100 μm as shown in panel B. (D-F) VWF variants (WT and Lock) were created with a Cerulean (CFP)-insert prior to the A1 domain in WT and Lock-VWF (D). Protein multimer distribution following expression in HEK293T-furin cells (E), and susceptibility to proteolysis by 1 U/mL ADAMTS13 overnight at 37°C in the presence of 1.6 M urea (F). (G-H) Human plasma was mixed with 5 μg/mL WT-Cer or Lock-Cer VWF variants in the presence of 10 mM EDTA, and perfused over collagen at 100 000/s. Fiber formation was measured in different regions of the flow device. FITC-conjugated anti-VWF Ab measured total VWF deposits, and cerulean fluorescence assayed only recombinant-protein accumulation. Representative images (G) and quantitative analysis (H) are presented. (I) Approximately10 µg/mL WT- or Lock-VWF were repeatedly perfused in the collagen coated stenosed flow chamber at a maximum wall shear rate of 100 000/s for 10 minutes. This allowed 20 passages of VWF through the flow constriction. Solution concentration of VWF was measured before and after shear application. Greater amounts of WT-VWF was lost compared with Lock-VWF. All microfluidics assays were performed 3 times, each with 2 to 4 repeats. *P < .05 with respect to all other treatments. VWF self-association was diminished in the presence of calcium, and upon use of Lock-variants. (B,G) Scale bars, 50 µm; VWF labeled with FITC (green) or Ceruelan (cyan). Changjie Zhang et al. Blood Adv 2019;3:957-968 © 2019 by The American Society of Hematology

Self-association of VWD type 2A and calcium-binding–deficient mutants. Self-association of VWD type 2A and calcium-binding–deficient mutants. (A) Multimer distribution of WT- and mutant-VWF produced in HEK293T-furin cells. (B) A total of 1 U/mL ADAMTS13 was added to various proteins for 4 hours in the absence or presence of 1.6 M urea. VWF proteolysis was greater for the mutant molecules, and this was evident even in the absence of urea. (C-D) PRP from normal human donors was diluted 50-fold and shear mixed with 10 μg/mL ΔA1-488 at 9600/s in a viscometer in the absence of supplemented calcium, along with either 5 μg/mL WT-VWF, D1498A, or R1597W. VWF self-association (C) and platelet activation (D) were measured identically to Figure 1. (E) Thrombus formation on collagen type I was measured at 1000/s following methods in Figure 2D-E. Greater thrombus formation was noted upon supplementing washed blood with D1498A and R1597W (either 2 or 4 µg/mL) compared with WT-VWF. (F) Representative images of thrombus formation at 3 minutes. Assays were performed 3 to 4 times, each with 2 to 4 repeats. *P < .05 with respect to all other treatments. Scale bars in panel F, 100 µm; BCECF platelet stain. Changjie Zhang et al. Blood Adv 2019;3:957-968 © 2019 by The American Society of Hematology

Molecular mechanism of VWF self-association. Molecular mechanism of VWF self-association. (A) Four VWF mutants lacking the A1 domain were produced, and each was labeled with Alexa 647. (B-E) PRP was diluted 50-fold into HEPES buffer containing one of the ΔA1-Alexa 647 variants (2.5 µg/mL), along with either 5 µg/mL WT-VWF (B-C) or Lock-VWF (D-E). The mixture was sheared at 9600/s using a viscometer in buffer containing either no exogenous calcium (B,D) or physiological calcium (C,E). VWF self-association was measured based on ΔA1-Alexa 647 binding to platelets using flow cytometry. VWF self-association was observed even when the ΔA1-proteins were sheared in the presence of Lock-VWF (lower panels), indicating that the unfolded A2 domain may bind other VWF regions also, in addition to homotypic association. *P < .05 with respect to all other treatments at indicated times. Data are from 3 repeats. Changjie Zhang et al. Blood Adv 2019;3:957-968 © 2019 by The American Society of Hematology

Inhibitors of VWF self-association. Inhibitors of VWF self-association. (A-B) Studies similar to Figure 5B-C were performed, by shearing 1:50 diluted PRP with 2.5 µg/mL ΔA1-647 and 5 µg/mL WT-VWF. HEPES buffer was replaced by VWF-deficient plasma in one case. In other cases, 500µg/mL ApoA-I or HDL was added. Studies were performed in the absence (A) and presence (B) of exogenous calcium. VWF self-association was reduced by lipoproteins in panel A. *P < .05 with respect to other treatments at indicated times. Changjie Zhang et al. Blood Adv 2019;3:957-968 © 2019 by The American Society of Hematology

Conceptual model of VWF self-association. Conceptual model of VWF self-association. The A2 domain of WT/native VWF is in a closed conformation. The unfolding of this domain is promoted by fluid shear, and enhanced upon depleting calcium and mutating A2 to prevent calcium binding. Such shear mediated A2-unfolding promotes VWF self-association on platelet GpIbα thus enhancing SIPAct. This also augments VWF binding to collagen, promoting thrombus growth. The introduction of a disulfide bond across A2 locks/seals the domain, preventing A2-unfolding and VWF self-association. Changjie Zhang et al. Blood Adv 2019;3:957-968 © 2019 by The American Society of Hematology