Rationale: PAD4 (peptidylarginine deiminase type IV), an enzyme essential for neutrophil extracellular trap formation (NETosis), is released together with neutrophil extracellular traps into the extracellular milieu. It citrullinates histones and holds the potential to citrullinate other protein targets. While NETosis is implicated in thrombosis, the impact of the released PAD4 is unknown. Objective: This study tests the hypothesis that extracellular PAD4, released during inflammatory responses, citrullinates plasma proteins, thus affecting thrombus formation. Methods and Results: Here, we show that injection of r-huPAD4 in vivo induces the formation of VWF (von Willebrand factor)-platelet strings in mesenteric venules and that this is dependent on PAD4 enzymatic activity. VWF-platelet strings are naturally cleaved by ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type-1 motif-13). We detected a reduction of endogenous ADAMTS13 activity in the plasma of wild-type mice injected with r-huPAD4. Using mass spectrometry and in vitro studies, we found that r-huPAD4 citrullinates ADAMTS13 on specific arginine residues and that this modification dramatically inhibits ADAMTS13 enzymatic activity. Elevated citrullination of ADAMTS13 was observed in plasma samples of patients with sepsis or noninfected patients who were elderly (eg, age >65 years) and had underlying comorbidities (eg, diabetes mellitus and hypertension) as compared with healthy donors. This shows that ADAMTS13 is citrullinated in vivo. VWF-platelet strings that form on venules of Adamts13 −/− mice were immediately cleared after injection of r-huADAMTS13, while they persisted in vessels of mice injected with citrullinated r-huADAMTS13. Next, we assessed the effect of extracellular PAD4 on platelet-plug formation after ferric chloride-induced injury of mesenteric venules. Administration of r-huPAD4 decreased time to vessel occlusion and significantly reduced thrombus embolization. Conclusions: Our data indicate that PAD4 in circulation reduces VWF-platelet string clearance and accelerates the formation of a stable platelet plug after vessel injury. We propose that this effect is, at least in part, due to ADAMTS13 inhibition.
Enhancing skin allograft longevity lessens the need for new allografts before optimal intervention is available. Reduced activity of ADAMTS13 (an enzyme that cleaves the pro‐thrombotic and proinflammatory von Willebrand factor) and presence of neutrophil extracellular traps (NETs) have been implicated in liver and lung allograft failures. The effect of ADAMTS13 treatment and the impact of NETs on skin allografts, however, remain unexplored. Here, we adopted a murine model of complete mismatch full‐thickness skin transplant by grafting dorsal skin from BALB/c mice to C57BL/6J background mice. Recombinant human ADAMTS13 (rhADAMTS13) treatment of graft recipients increased allograft survival. Western blot and immunofluorescence microscopy revealed the presence of NETs in allografts of vehicle, but surprisingly, not in rhADAMTS13‐treated mice, 3 days after surgery. Recapitulating the observations in mice, NETs were also observed in all the examined allografts from burn patients. Intriguingly, knocking out peptidylarginine deiminase 4 (PAD4, a key enzyme for NET formation) or DNase 1 treatment (which cleaves NETs) also prolonged allograft survival. In summary, rhADAMTS13 lessens inflammation in allografts by reducing NET burden, resulting in enhanced allograft survival. RhADAMTS13 and anti‐NET treatments could be new therapeutic strategies to promote skin allograft longevity and, hence, the survival of patients with severe burns.
Purpose Platelets are known to play an important role in venous thrombogenesis, but their role in thrombus maturation, resolution, and postthrombotic vein wall remodeling is unclear. The purpose of this study was to determine the role that circulating platelets play in the later phases of venous thrombosis. Methods We used a murine inferior vena cava (IVC) stenosis model. Baseline studies in untreated mice were performed to determine an optimal postthrombotic time point for tissue harvest that would capture both thrombus maturation/resolution and postthrombotic vein wall remodeling. This time point was found to be postoperative day 10. After undergoing IVC ultrasound on day 2 to confirm venous thrombus formation, mice were treated with a daily injection of platelet‐depleting antibody (anti‐GP1bα) to maintain thrombocytopenia or with control IgG until postoperative day 10, at which time IVC and thrombi were harvested and thrombus length, volume, fibrosis, neovascularization, and smooth muscle cell invasion analyzed. Vein wall fibrosis and intimal thickening were also determined. Results Mice that were made thrombocytopenic after venous thrombogenesis had thrombi that were less fibrotic, with fewer invading smooth muscle cells. Furthermore, thrombocytopenia in the setting of venous thrombosis resulted in less postthrombotic vein wall intimal thickening. Thrombus volume did not differ between thrombocytopenic mice and their control peers. Conclusions This work suggests that circulating platelets contribute to venous thrombus maturation, fibrosis, and adverse vein wall remodeling, and that that inhibition of platelet recruitment may decrease thrombus and vein wall fibrosis, thus helping thrombolysis and preventing postthrombotic syndrome.
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