Abstract:Von Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2-domain. We aimed to isolate nanobodies distinguishing between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3-domain, the epitope of which overlaps the collagen-binding site. While KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysi… Show more
“…Furthermore, this assay does not recognize the presence of VWF (∼10 μg/mL) or endogenous ADAMTS13-cleaved VWF (7% of the plasma pool by estimation) in citrated plasma. 8 In control experiments, we confirmed the strong preference of this assay for plasmin-cleaved VWF over ADAMTS13-cleaved VWF ( Figure 2 C) and VWF cleavage products resulting from other proteases that are prevalent within the intravascular compartment ( supplemental Figure 3 ). Figure 2.…”
Section: Resultssupporting
confidence: 65%
“…ADAMTS13-cleaved plasma-derived VWF was kindly provided by Peter Lenting, INSERM, and characterized as previously described. 8 …”
Von Willebrand factor (VWF) is an essential contributor to microvascular thrombosis. Physiological cleavage by ADAMTS13 limits its prothrombotic properties, explaining why ADAMTS13 deficiency leads to attacks of microthrombosis in patients with thrombotic thrombocytopenic purpura (TTP). We previously reported that plasminogen activation takes place during TTP attacks in these patients. Furthermore, stimulation of plasminogen activation attenuates pathogenesis in preclinical TTP models in vivo. This suggests that plasmin is an endogenous regulator of VWF thrombogenicity, in particular when ADAMTS13 falls short to prevent microvascular occlusions. VWF cleavage by plasmin is biochemically distinct from cleavage by ADAMTS13. We hypothesized that plasmin-cleaved VWF (cVWF) holds value as a biomarker of microvascular thrombosis. We here describe the development of a VHH-based bioassay that can distinguish cVWF from intact and ADAMTS13-cleaved VWF in plasma. We validate this assay by tracking cVWF release during degradation of microthombi in vitro. We demonstrate that endogenous cVWF formation takes place in TTP patients during acute attacks of thrombotic microangiopathy, but not in remission. Finally, we show that therapeutic plasminogen activation in a mouse model for TTP amplifies cVWF formation, which is accompanied by VWF clearance. Our combined findings indicate that cVWF is released from microthrombi in the context of microvascular occlusion.
“…Furthermore, this assay does not recognize the presence of VWF (∼10 μg/mL) or endogenous ADAMTS13-cleaved VWF (7% of the plasma pool by estimation) in citrated plasma. 8 In control experiments, we confirmed the strong preference of this assay for plasmin-cleaved VWF over ADAMTS13-cleaved VWF ( Figure 2 C) and VWF cleavage products resulting from other proteases that are prevalent within the intravascular compartment ( supplemental Figure 3 ). Figure 2.…”
Section: Resultssupporting
confidence: 65%
“…ADAMTS13-cleaved plasma-derived VWF was kindly provided by Peter Lenting, INSERM, and characterized as previously described. 8 …”
Von Willebrand factor (VWF) is an essential contributor to microvascular thrombosis. Physiological cleavage by ADAMTS13 limits its prothrombotic properties, explaining why ADAMTS13 deficiency leads to attacks of microthrombosis in patients with thrombotic thrombocytopenic purpura (TTP). We previously reported that plasminogen activation takes place during TTP attacks in these patients. Furthermore, stimulation of plasminogen activation attenuates pathogenesis in preclinical TTP models in vivo. This suggests that plasmin is an endogenous regulator of VWF thrombogenicity, in particular when ADAMTS13 falls short to prevent microvascular occlusions. VWF cleavage by plasmin is biochemically distinct from cleavage by ADAMTS13. We hypothesized that plasmin-cleaved VWF (cVWF) holds value as a biomarker of microvascular thrombosis. We here describe the development of a VHH-based bioassay that can distinguish cVWF from intact and ADAMTS13-cleaved VWF in plasma. We validate this assay by tracking cVWF release during degradation of microthombi in vitro. We demonstrate that endogenous cVWF formation takes place in TTP patients during acute attacks of thrombotic microangiopathy, but not in remission. Finally, we show that therapeutic plasminogen activation in a mouse model for TTP amplifies cVWF formation, which is accompanied by VWF clearance. Our combined findings indicate that cVWF is released from microthrombi in the context of microvascular occlusion.
“…Knowing that increased VWF degradation (eg, in VWD-type 2A) is associated with increased risks of angiodysplasia [ 27 ], we tested if binding of Ang-1 and Ang-2 to VWF is modified upon its degradation by ADAMTS-13. We therefore examined the binding of plasma-derived VWF (referred to as intact VWF) and degraded VWF (referred to as VWF-degr, ie, >85% proteolyzed by recombinant ADAMTS-13) [ 19 ] to immobilized Ang-1 or Ang-2. With maximal binding for intact VWF being arbitrarily set at 100%, it appeared that VWF-degr displayed slightly enhanced binding to Ang-1 (Bmax 151% (95% CI: 133%-176%) vs 100%; P = .0008; Figure 6 D).…”
Section: Resultsmentioning
confidence: 99%
“…Specific radioactivity varied from 3 to 6 μCi/μg. Degraded VWF (VWF-degr) was prepared as described [ 19 ]. Recombinant VWF fragments D’-D3-HPC4, A1/Fc, A2/Fc, A3/Fc, and D4/Fc have been described previously [ 17 , 20 ].…”
Activation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify two groups of nanobodies. One group, represented by clone 6D12, is conformation-insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1. 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation-sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and that binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.
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