PAR1 Agonists Stimulate APC-Like Endothelial Cytoprotection and Confer Resistance to Thromboinflammatory Injury

Stimulation of protease-activated receptor 1 (PAR1) on endothelium by activated protein C (APC) is protective in several animal models of disease, and APC has been used clinically in severe sepsis and wound healing. Clinical use of APC, however, is limited by its immunogenicity and its anticoagulant activity. We show that a class of small molecules termed “parmodulins” that act at the cytosolic face of PAR1 stimulates APC-like cytoprotective signaling in endothelium. Parmodulins block thrombin generation in response to inflammatory mediators and inhibit platelet accumulation on endothelium cultured under flow. Evaluation of the antithrombotic mechanism showed that parmodulins induce cytoprotective signaling through Gβγ, activating a PI3K/Akt pathway and eliciting a genetic program that includes suppression of NF-κB–mediated transcriptional activation and up-regulation of select cytoprotective transcripts. STC1 is among the up-regulated transcripts, and knockdown of stanniocalin-1 blocks the protective effects of both parmodulins and APC. Induction of this signaling pathway in vivo protects against thromboinflammatory injury in blood vessels. Small-molecule activation of endothelial cytoprotection through PAR1 represents an approach for treatment of thromboinflammatory disease and provides proof-of-principle for the strategy of targeting the cytoplasmic surface of GPCRs to achieve pathway selective signaling

Parmodulins Inhibit Thrombus Formation Without Inducing Endothelial Injury Caused by Vorapaxar

Protease-activated receptor-1 (PAR1) couples the coagulation cascade to platelet activation during myocardial infarction and to endothelial inflammation during sepsis. This receptor demonstrates marked signaling bias. Its activation by thrombin stimulates prothrombotic and proinflammatory signaling, whereas its activation by activated protein C (APC) stimulates cytoprotective and antiinflammatory signaling. A challenge in developing PAR1-targeted therapies is to inhibit detrimental signaling while sparing beneficial pathways. We now characterize a novel class of structurally unrelated small-molecule PAR1 antagonists, termed parmodulins, and compare the activity of these compounds to previously characterized compounds that act at the PAR1 ligand–binding site. We find that parmodulins target the cytoplasmic face of PAR1 without modifying the ligand-binding site, blocking signaling through Gαq but not Gα13 in vitro and thrombus formation in vivo. In endothelium, parmodulins inhibit prothrombotic and proinflammatory signaling without blocking APC-mediated pathways or inducing endothelial injury. In contrast, orthosteric PAR1 antagonists such as vorapaxar inhibit all signaling downstream of PAR1. Furthermore, exposure of endothelial cells to nanomolar concentrations of vorapaxar induces endothelial cell barrier dysfunction and apoptosis. These studies demonstrate how functionally selective antagonism can be achieved by targeting the cytoplasmic face of a G-protein–coupled receptor to selectively block pathologic signaling while preserving cytoprotective pathways

ADAMTS13 meets von Willebrand factor strings: a fluorescence microscopy approach

ADAMTS13 and its substrate von Willebrand factor (VWF) circulate together in our blood where they exert crucial roles in hemostasis. The multimeric VWF protein captures platelets at sites of vascular injury thereby limiting blood loss. Upon endothelial perturbation, however, ultra-large (UL) VWF multimers are released that can spontaneously bind platelets and cause thrombus formation. ADAMTS13 regulates the size of these hyper-reactive VWF multimers by degrading them into smaller, less thrombogenic ones. Secreted UL-VWF multimers can assemble into long strings on endothelial cells that catch circulating platelets and are rapidly digested by ADAMTS13. Detailed information about the proteolysis of these platelet-decorated VWF strings is however lacking. In this thesis we aimed at elucidating how ADAMTS13 digests platelet-decorated VWF strings anchored to the endothelial surface by performing studies in bulk as well as on a single molecule level.In vitro flow chamber experiments were implemented to follow the digestion of platelet-decorated VWF strings by ADAMTS13 in real-time. Individual VWF string characteristics, like the number and location of cleavage sites, and additional features influencing proteolysis, were evaluated in detail using customized imaging software that tracks VWF string length in function of time. Experiments were performed in the presence of active ADAMTS13, catalytically inactive ADAMTS13E225Q or in the absence of ADAMTS13 to determine whether the observed string characteristics were specific for ADAMTS13. Our data revealed that ADAMTS13 cleaves platelet-decorated VWF strings at a discrete number of sites thereby progressively shortening the strings as a function of time. Although cleavage sites were evenly distributed over the strings released VWF fragments, remained UL in size, suggesting that further proteolysis in circulation might occur. Interestingly, local elongations were observed at the cleavage site preceding ADAMTS13 proteolysis in the majority (89%) of cleavage events. Local elongation occurred at different sites and was a characteristic behavior of VWF strings under flow, independent of the presence of active ADAMTS13. VWF strings could elongate locally up to 40 µm, corresponding to more than 500 monomers if fully extended. The occurrence of such large elongations might imply that multiple ADAMTS13 cleavage sites become exposed simultaneously by further unfurling of the VWF string, hence facilitating rapid clearance of endothelium-anchored UL-VWF strings. In the second part of the thesis single molecule fluorescence microscopy (SMFM) was used to track single ADAMTS13 enzymes on the platelet-decorated VWF strings and hence evaluate how ADAMTS13 locates its binding sites on these strings. A customized single molecule microscope set up, using high magnifications, bright dyes and powerful lasers, was developed allowing the visualization of single ADAMTS13 enzymes under static conditions. Further adaptation of the microscope using total internal reflection (TIRF)-like illumination, two simultaneously acquiring CCD cameras and different magnification for ADAMTS13 and VWF string detection, enabled observation of single enzymes binding to the long platelet-decorated VWF strings in real-time. Single particle tracking software was next developed to identify single ADAMTS13 enzymes and to follow their trajectories in function of time. A comprehensive analysis of all detected enzymes revealed difference in the motion of individual enzymes. Most enzymes (80 %) slowed down or made very short contacts with the platelet-decorated VWF string. This behavior appeared to be a general characteristic of molecules passing in flow, as it was also observed with a control IgG antibody. The remainder of enzymes (20 %) were observed to arrive and attach on the VWF strings for longer periods of time. Once bound no further movement of the enzymes was observed on the platelet-decorated VWF strings. Together, these data suggest that ADAMTS13 bumps into the string and binds to one of the (many) available sites.status: publishe

Unwinding the von Willebrand factor strings puzzle

von Willebrand factor (VWF) is amongst others synthesized by endothelial cells and stored as ultra-large (UL) VWF multimers in Weibel-Palade bodies. Although UL-VWF is proteolysed by ADAMTS13 (a disintegrin-like and metalloprotease domain with thrombospondin type-1 motif, number 13) on secretion from endothelial cells, in vitro experiments in the absence of ADAMTS13 have demonstrated that a proportion of these UL-VWF multimers remain anchored to the activated endothelium. These multimers unravel, bind platelets, and wave in the direction of the flow. These so-called VWF "strings" have also been visualized in vivo, lining the lumen of activated mesenteric veins of Adamts13(-/-) mice. Various studies have demonstrated the extraordinary length of these VWF strings, the availability of their platelet binding and ADAMTS13 cleavage sites, and the possible nature of their endothelial attachment. VWF strings are also capable of tethering leukocytes and parasite-infected red blood cells. However, the majority of studies have been performed in the absence of ADAMTS13, a condition only experienced in thrombotic thrombocytopenic purpura. A normal functional role of VWF strings in healthy persons or in other disease pathologies remains unclear. In this review, we discuss some of the puzzling characteristics of VWF strings, and we debate whether the properties of VWF strings in the absence of ADAMTS13 might be relevant for understanding (patho)physiologic mechanisms.status: publishe

Shear-Stress-Induced Conformational Changes of von Willebrand Factor in a Water-Glycerol Mixture Observed with Single Molecule Microscopy

The von Willebrand factor (VWF) is a human plasma protein that plays a key role in the initiation of the formation of thrombi under high shear stress in both normal and pathological situations. It is believed that VWF undergoes a conformational transition from a compacted, globular to an extended form at high shear stress. In this paper, we develop and employ an approach to visualize the large-scale conformation of VWF in a (pressure-driven) Poiseuille flow of water-glycerol buffers with wide-field single molecule fluorescence microscopy as a function of shear stress. Comparison of the imaging results for VWF with the results of a control with λ-phage double-stranded DNA shows that the detection of individual VWF multimers in flow is feasible. A small fraction of VWF multimers are observed as visibly extended along one axis up to lengths of 2.0 μm at high applied shear stresses. The size of this fraction of molecules seems to exhibit an apparent dependency on shear stress. We further demonstrate that the obtained results are independent of the charge of the fluorophore used to label VWF. The obtained results support the hypothesis of the conformational extension of VWF in shear flow.status: publishe

Local elongation of endothelial cell-anchored von Willebrand factor strings precedes ADAMTS13 protein-mediated proteolysis

Platelet-decorated von Willebrand factor (VWF) strings anchored to the endothelial surface are rapidly cleaved by ADAMTS13. Individual VWF string characteristics such as number, location, and auxiliary features of the ADAMTS13 cleavage sites were explored here using imaging and computing software. By following changes in VWF string length, we demonstrated that VWF strings are cleaved multiple times, successively shortening string length in the function of time and generating fragments ranging in size from 5 to over 100 mu m. These are larger than generally observed in normal plasma, indicating that further proteolysis takes place in circulation. Interestingly, in 89% of all cleavage events, VWF strings elongate precisely at the cleavage site before ADAMTS13 proteolysis. These local elongations are a general characteristic of VWF strings, independent of the presence of ADAMTS13. Furthermore, large elongations, ranging in size from 1.4 to 40 mu m, occur at different sites in space and time. In conclusion, ADAMTS13-mediated proteolysis of VWF strings under flow is preceded by large elongations of the string at the cleavage site. These elongations may lead to the simultaneous exposure of many exosites, thereby facilitating ADAMTS13-mediated cleavage

Local Elongation of Endothelial Cell-anchored von Willebrand Factor Strings Precedes ADAMTS13 Protein-mediated Proteolysis

Platelet-decorated von Willebrand factor (VWF) strings anchored to the endothelial surface are rapidly cleaved by ADAMTS13. Individual VWF string characteristics such as number, location, and auxiliary features of the ADAMTS13 cleavage sites were explored here using imaging and computing software. By following changes in VWF string length, we demonstrated that VWF strings are cleaved multiple times, successively shortening string length in the function of time and generating fragments ranging in size from 5 to over 100 mu m. These are larger than generally observed in normal plasma, indicating that further proteolysis takes place in circulation. Interestingly, in 89% of all cleavage events, VWF strings elongate precisely at the cleavage site before ADAMTS13 proteolysis. These local elongations are a general characteristic of VWF strings, independent of the presence of ADAMTS13. Furthermore, large elongations, ranging in size from 1.4 to 40 mu m, occur at different sites in space and time. In conclusion, ADAMTS13-mediated proteolysis of VWF strings under flow is preceded by large elongations of the string at the cleavage site. These elongations may lead to the simultaneous exposure of many exosites, thereby facilitating ADAMTS13-mediated cleavage.status: publishe

Single Particle Tracking of ADAMTS13 ( A Disintegrin and Metalloprotease with Thrombospondin Type-1 Repeats) Molecules on Endothelial von Willebrand Factor Strings

von Willebrand factor (VWF) strings are removed from the endothelial surface by ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type-1 repeats)-mediated proteolysis. To visualize how single ADAMTS13 molecules bind to these long strings, we built a customized single molecule fluorescence microscope and developed single particle tracking software. Extensive analysis of over 6,000 single inactive ADAMTS13(E225Q) enzymes demonstrated that 20% of these molecules could be detected in at least two consecutive 60-ms frames and followed two types of trajectories. ADAMTS13(E225Q) molecules either decelerated in the vicinity of VWF strings, whereas sometimes making brief contact with the VWF string before disappearing again, or readily bound to the VWF strings and this for 120 ms or longer. These interactions were observed at several sites along the strings. Control experiments using an IgG protein revealed that only the second type of trajectory reflected a specific interaction of ADAMTS13 with the VWF string. In conclusion, we developed a dedicated single molecule fluorescence microscope for detecting single ADAMTS13 molecules (nm scale) on their long, flow-stretched VWF substrates (μm scale) anchored on living cells. Comprehensive analysis of all detected enzymes showed a random interaction mechanism for ADAMTS13 with many available binding sites on the VWF strings.status: publishe

The novel ADAMTS13-p.D187H mutation impairs ADAMTS13 activity and secretion and contributes to thrombotic thrombocytopenic purpura in mice

Summary: Background: Congenital thrombotic thrombocytopenic purpura (TTP) is characterized by mutations in the ADAMTS13 gene, which either impair protein secretion or influence ADAMTS13 (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type-1 motif, member 13) activity. Phenotypic consequences of these mutations have not yet been evaluated in animal models for TTP. Objectives: To identify the in vitro effect of a novel ADAMTS13 mutation and to investigate whether this mutation induces TTP in vivo. Methods: All 29 ADAMTS13 exons with exon-intron boundaries of a patient with pregnancy-onset TTP were sequenced. Wild-type and mutant ADAMTS13 proteins were both transiently and stably expressed in human embryonic kidney cells, and their activity was evaluated in vitro using fluorescence resonance energy transfer and flow assays. Molecular dynamics simulations were performed to study Ca2+ stability. Adamts13-/- mice were hydrodynamically injected with wild-type and mutant expression plasmids and triggered with recombinant human von Willebrand factor. Results: We identified a novel heterozygous c.559G>C mutation in exon 6 of the proposita's ADAMTS13 gene. This mutation resulted in a p.Asp187His substitution (p.D187H), which was located in the high affinity Ca2+-binding site in the metalloprotease domain of ADAMTS13. The homozygous p.D187H mutation down-regulated ADAMTS13 activity in vitro. Impaired proteolytic activity was linked to unstable Ca2+ binding as visualized using a molecular dynamics simulation. In addition, the p.D187H mutation affects protein secretion in vitro. In Adamts13-/- mice, the homozygous p.D187H mutation reduced ADAMTS13 secretion and activity and contributed to TTP when these mice were triggered with recombinant human von Willebrand factor. Conclusions: Our data indicate that the p.D187H mutation impairs ADAMTS13 activity and secretion and is responsible for TTP onset in mice