ADMA plasma levels correlate with the severity of pulmonary vascular disease and predict outcome in patients with CTEPH. Measurement of ADMA plasma levels may be useful for estimating the degree of small-vessel arteriopathy in CTEPH.

Acute pulmonary embolism generally resolves within 6 months. However, if thrombus is infected venous thrombi transform into fibrotic vascular obstructions leading to chronic deep vein thrombosis and/or chronic thromboembolic pulmonary hypertension (CTEPH), but precise mechanisms remain unclear. Neutrophils are crucial in sequestering pathogens, therefore, the role of neutrophil extracellular traps (NETs) in chronic thrombosis was investigated. Since chronic pulmonary thrombotic obstructions are biologically identical to chronic deep venous thrombi, the murine inferior vena cava ligation model was used to study the transformation of acute to chronic thrombus. Mice with staphylococcal infection presented with larger thrombi containing more neutrophils and NETs, but less resolution. Targeting NETs with DNase1 diminished fibrosis, and promoted thrombus resolution. For translational studies in humans, we focused on patients with CTEPH, a severe type of deep venous and pulmonary artery fibrotic obstruction following thrombosis. Neutrophils, markers of neutrophil activation, and NET formation were increased in CTEPH patients. NETs promoted the differentiation of monocytes to activated fibroblasts with the same cellular phenotype as fibroblasts from CTEPH vascular occlusions. RNA sequencing of fibroblasts isolated from thrombo-endarterectomy specimens and pulmonary artery biopsies revealed transforming growth factor-β (TGF-β) as the central regulator, a phenotype which was replicated in mice with fibroblast-specific TGF-β overactivity. Our findings uncover a role of neutrophil-mediated inflammation to enhance TGF-β signaling which leads to fibrotic thrombus remodeling. Targeting thrombus NETs with DNases may serve as a new therapeutic concept to treat thrombosis and prevent its sequelae.

BackgroundSplenectomy is a clinical risk factor for complicated thrombosis. We hypothesized that the loss of the mechanical filtering function of the spleen may enrich for thrombogenic phospholipids in the circulation, thereby affecting the vascular remodeling of thrombosis.Methods and ResultsWe investigated the effects of splenectomy both in chronic thromboembolic pulmonary hypertension (CTEPH), a human model disease for thrombus nonresolution, and in a mouse model of stagnant flow venous thrombosis mimicking deep vein thrombosis. Surgically excised thrombi from rare cases of CTEPH patients who had undergone previous splenectomy were enriched for anionic phospholipids like phosphatidylserine. Similar to human thrombi, phosphatidylserine accumulated in thrombi after splenectomy in the mouse model. A postsplenectomy state was associated with larger and more persistent thrombi. Higher counts of procoagulant platelet microparticles and increased leukocyte–platelet aggregates were observed in mice after splenectomy. Histological inspection revealed a decreased number of thrombus vessels. Phosphatidylserine‐enriched phospholipids specifically inhibited endothelial proliferation and sprouting.ConclusionsAfter splenectomy, an increase in circulating microparticles and negatively charged phospholipids is enhanced by experimental thrombus induction. The initial increase in thrombus volume after splenectomy is due to platelet activation, and the subsequent delay of thrombus resolution is due to inhibition of thrombus angiogenesis. The data illustrate a potential mechanism of disease in CTEPH.

Key Points• PECAM-1 deficiency misguides venous thrombus resolution.• PECAM-1 cell surface shedding occurs at the site of venous thrombosis.Platelet endothelial cell adhesion molecule 1 (PECAM-1) is involved in leukocyte migration and angiogenesis, which are key components of venous thrombus resolution. This study investigated the effect of PECAM-1 deficiency on thrombus resolution in FVB/n mice and the extent to which levels of soluble PECAM-1 (sPECAM-1) correlate with delayed thrombus resolution in humans after acute symptomatic deep vein thrombosis (DVT). In a mouse stagnant flow venous thrombosis model Pecam-1 2/2 thrombi were larger, persisted for longer periods of time, and displayed attenuated macrophage invasion and decreased vessel formation in the presence of increased fibrosis. In humans, higher levels of truncated plasma sPECAM-1 possibly cleaved from cell surfaces, were found in patients with delayed thrombus resolution (assessed via duplex-based thrombus scoring) relative to those whose thrombi resolved (median, 25th/75th percentile): 92.5 (87.7/103.4) ng/mL vs 71.5 (51.1/81.0) ng/mL; P < .001. Furthermore, unresolved human deep vein thrombus specimens stained positively with antibodies specific for the extracellular, but not the cytoplasmic domain of PECAM-1, consistent with accumulation of cleaved PECAM-1. Our data suggest a regulatory role of PECAM-1 in venous thrombus resolution and suggest a predictive value of sPECAM-1 for postthrombotic syndrome (PTS) after acute DVT. (Blood. 2013;122(19):3376-3384)