Fig 1: Shear-Induced VWF Activation and Cleavage(A) ADAMTS-13 antigen (ADAMTS-13:Ag) and (B) its ratio to VWF:Ag in samples from HS and patients sampled at BL, at Post, and at B or T events (sample sizes at the bottom of the panel figures, ∗P < 0.05 and ∗∗∗P < 0.001, HS vs other groups). (C) A2 precipitated VWF from the plasma of LVAD patients who developed bleeding or stroke post-LVAD and the precipitation was blocked by A1 (representative from 26 patients analyzed). A2 precipitated VWF purified from cryoprecipitate only after ristocetin (Risto.) treatment. (D) A2 also blocked SIPA of HS (n = 8/group). (E) Thiol-containing VWF precipitated from HS (n = 13) and patients at BL and Post (n = 26, ∗∗∗P < 0.001). (F) Cleaved VWF (cVWF) and uncleaved VWF (ucVWF) from HS exposed to 5 minutes of high shear stress (lanes 1-5) or in the static condition with 1.5 M urea and 1 mM of BaCl2 (lane 6). (G) A2 cleavage in the static condition. (H) (Top) cVWF and ucVWF from HS exposed to high shear stress for 60 minutes (lanes 1-8), under static incubation of 16 hours (lane 9), or exposed to a constant vortex for 60 minutes (lane 10). (Bottom) Densitometry of 8 independent experiments. All quantitative data were analyzed using 1-way analysis of variance. cA2 = cleaved A2; MM = molecular mark in kDa; RBC = red blood cells; ucA2 = uncleaved A2; ucVWF = uncleaved VWF; other abbreviations as in Figures 1 and 3.
Fig 2: EVs Promoted VWF-Dependent Angiogenesis(A to F) Representative images of angiogenesis from aortic vascular segment (AVSs) cultured for 14 days in growth factor–poor medium (GPM) supplemented with EVFP or EVs from patient plasma (bar = 100 μm, arrowheads indicate microvessels). The (G) number and (H) length of vascular sprouts (10 random viewfields) from AVSs cultured in indicated conditions. The results were from plasma samples of 26 patients collected at discharge and analyzed using 1-way analysis of variance (∗P < 0.05, ∗∗P < 0.01, ∗∗P < 0.001). (I) A cross-section view of a hematoxylin and eosin (HE)–stained AVS (asterisk indicates the vascular lumen, bar = 50 μm). The CD31+ microvessels of AVSs grown in (J) growth factor–rich medium (GRM) and (K) EV-supplemented GPM (bar = 25 μm). These are representative images from 22 AVSs reviewed by a mouse pathologist who was blind to the experimental conditions. Abbreviations as in Figure 1.
Fig 3: VWF in Patients Was Hyperadhesive(A) VWF antigen (VWF:Ag), (B) VWF propeptide (VWF:pp), and (C) the VWF:pp-to-VWF:Ag ratio measured at BL, at Post, and at clinical events of B or T (dotted line indicates levels from HS set as 100% or a ratio of 1). (D) VWF multimers in a patient at BL, Post, and clinical event (large VWF multimers [LM], with representative images from longitudinal samples from patients analyzed). (E) VWF binding to collagen (VWF:CB) and (F) its ratio to VWF:Ag of 13 HSs and 26 patients collected at BL, Post, and the clinical events of B and T. Sample sizes are marked at the bottom of the panel figures. The arrows in panels c and d refer to VWF formed string-like structures to which platelets bound. (G) (a to d) Representative images of platelet thrombi after 10 minutes of perfusing reconstituted blood under 30 and 120 dynes/cm2 of shear stresses (bar = 100 μm). (e) The areas covered by platelet thrombi were quantified for samples from 12 patients and 6 HS. The quantitative data were analyzed using either 1-way analysis of variance or 1-way analysis of variance on ranks (∗P < 0.05, ∗∗P < 0.01, ∗∗P < 0.001). For panel e, ∗P < 0.05 between 30 and 120 dynes/cm2 and ∗∗P < 0.01 vs HS. Abbreviations as in Figure 1.
Fig 4: VWF Mediated EV-Induced Vascular Permeability(A) Endothelial permeability was induced by patient plasma collected at discharge but not by purified von Willebrand factor (VWF) at 30 μg/mL (n = 26 for patient plasma and n = 6 for VWF). The permeability was blocked by the VWF-blocking antibody. (B) Extracellular vesicles (EVs) but not extracellular vesicle–free plasma (EVFP) induced endothelial permeability (n = 26). (C) Phosphatidylserine (PS) expression and (D) VWF released from cultured endothelial cells stimulated with plasma collected before and after left ventricular assist device (LVAD) implants. The post-LVAD data were stratified into bleeding (B) (n = 4), thrombosis (T) (n = 3), and no complication (N) (n = 19) and with baseline values subtracted. Histamine-treated endothelial cells (25 μM, 20 minutes at 37 °C) served as the control samples. EVs stained with anti-VWF antibody together with either (E) annexin V or (F) anti-CD41a antibody in plasma collected from healthy subjects (HS) and patients at baseline (BL), at discharge (Post), and at clinical events of bleeding (B) or thrombosis (T). (G) VWF on platelets from LVAD patients. For E to G, the sample sizes are provided at the bottom of the figures. All data were analyzed using 1-way analysis of variance (∗P < 0.05, ∗∗P < 0.01, ∗∗P < 0.001). MFI = mean fluorescence intensity; OD = optical density.
Fig 5: pEVs Promoted Angiogenesis in a VWF-Dependent MannerNormal platelets in platelet-rich plasma were (A) activated, (B) aggregated, and (C) produced EVs upon exposure to 110 dynes/cm2 of high shear stress for 5 minutes at 37 °C (n = 18 with the samples from each subject tested before and after shear exposure). (D to F) EVs from VWF-activated platelets (pEVs) (1 × 106/μL) from shear-activated platelets but not from unsheared samples promoted angiogenesis from AVSs in GPM (bar = 100 μm, arrow: vascular sprouts). The (G) number and (H) length of vascular sprouts were quantified (n = 18). Representative images of CD31+ microvessels from AVSs cultured in (I) GRM and (J) pEV-supplemented GPM (bar = 10 μm) and (K) the summary of 9 independent experiments. The quantitative data were analyzed using 1-way analysis of variance or 1-way analysis of variance on ranks (∗P < 0.05, ∗∗P < 0.01, ∗∗P < 0.001). SIPA = shear-induced platelet aggregation; other abbreviations as in Figures 1 and 2.
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