Peter G. Chiles

High prevalence of dysfibrinogenemia among patients with chronic thromboembolic pulmonary hypertension

The mechanism by which chronic thromboembolic pulmonary hypertension (CTEPH) develops after acute pulmonary thromboembolism is unknown. We previously reported that fibrin from CTEPH patients is relatively resistant to fibrinolysis in vitro. In the present study, we performed proteomic, genomic, and functional studies on fibrin(ogen) to investigate whether abnormal fibrin(ogen) might contribute to the pathogenesis of CTEPH. Reduced and denatured fibrinogen from 33 CTEPH patients was subjected to liquid chromatography-mass spectrometry analysis. Fibrinogen from 21 healthy controls was used to distinguish atypical from commonly occurring mass peaks. Atypical peaks were further investigated by targeted genomic DNA sequencing. Five fibrinogen variants with corresponding heterozygous gene mutations (dysfibrinogenemias) were observed in 5 of 33 CTEPH patients: Bbeta P235L/gamma R375W, Bbeta P235L/gamma Y114H, Bbeta P235L, Aalpha L69H, and Aalpha R554H (fibrinogens(San Diego I-V)). Bbeta P235L was found in 3 unrelated CTEPH patients. Functional analysis disclosed abnormalities in fibrin polymer structure and/or lysis with all CTEPH-associated mutations. These results suggest that, in some patients, differences in the molecular structure of fibrin may be implicated in the development of CTEPH after acute thromboembolism.

PULMONARY VASCULAR REMODELING DISTAL TO PULMONARY ARTERY LIGATION IS ACCOMPANIED BY UPREGULATION OF ENDOTHELIN RECEPTORS AND NITRIC OXIDE SYNTHASE

There is increasing evidence that the pathogenesis and progression of many forms of pulmonary vasculopathy are related to abnormalities in endothelial mediators, including endothelin-1 (ET-1) and nitric oxide (NO). Using a rat model of chronic unilateral pulmonary artery ligation, we investigated the role of ET-1 and NO in postobstructive pulmonary vasculopathy (POPV). Eight months after a left thoracotomy with either left main pulmonary artery ligation (ligated group) or no ligation (sham group), rat lungs, including those contralateral to the ligation (hyperperfused group), were fixed and mounted for histologic sectioning. Morphometric measurements were carried out by computer-assisted image analysis and immunohistochemical staining was performed using specific antibodies against ET-1, ETA, and EBB receptors, and endothelial NO synthase (eNOS). Compared to sham lungs, the ligated lungs showed (1) an increase in muscular, adventitial, and intimal thickness of pulmonary artery; (2) increase in external diameter of the bronchial artery (39.8 +/- 2.2 microns vs. 16.8 +/- 0.9 microns in sham group; P < .005) and number of bronchial arteries per bronchiole (3.21 +/- mu 0.26 vs. 1.86 +/- mu 0.21 in sham group; P < .001); and (3) increase in the intensity of eNOS and ETA, B receptor immunoreactivity. No morphometric or immunohistochemical differences were observed between the hyperperfused and sham lungs. These findings suggest that increased synthesis of endothelial NO may serve as a compensatory mediator in ET-1-mediated vascular remodeling.There is increasing evidence that the pathogenesis and progression of many forms of pulmonary vasculopathy are related to abnormalities in endothelial mediators, including endothelin-1 (ET-1) and nitric oxide (NO). Using a rat model of chronic unilateral pulmonary artery ligation, we investigated the role of ET-1 and NO in postobstructive pulmonary vasculopathy (POPV). Eight months after a left thoracotomy with either left main pulmonary artery ligation (ligated group) or no ligation (sham group), rat lungs, including those contralateral to the ligation (hyperperfused group), were fixed and mounted for histologic sectioning. Morphometric measurements were carried out by computer-assisted image analysis and immunohistochemical staining was performed using specific antibodies against ET-1, ETA, and EBB receptors, and endothelial NO synthase (eNOS). Compared to sham lungs, the ligated lungs showed (1) an increase in muscular, adventitial, and intimal thickness of pulmonary artery; (2) increase in external diameter of the bronchial artery (39.8+/-2.2 mum vs.16.8+/-0.9 mum in sham group; P <. 005) and number of bronchial arteries per bronchiole (3.21+/-mu0.26vs.1.86+/-mu0.21 in sham group; P <. 001); and (3) increase in the intensity of eNOS and ETA, B receptor immunoreactivity. No morphometric or immunohistochemical differences were observed between the hyperperfused and sham lungs. These ndings suggest that increased synthesis of endothelial NO may serve as a compensatory mediator in ET-1 mediated vascular remodeling.

Suppression of thrombolysis in a canine model of pulmonary embolism.

BackgroundThe brisk fibrinolytic response of canines has impaired efforts to develop a canine model of chronic thromboembolic pulmonary hypertension. Difficulties in retaining chronic embolic residuals were partially overcome by administration of tranexamic acid (TXA) (Circulation. 1991;83:1272–1279.). In this study, we used type 1 plasminogen activator inhibitor (PAI-1), a major inhibitor of the endogenous fibrinolytic system, to determine its efficacy in the suppression of thrombolysis in canines. Methods and ResultsThrombus was induced in the inferior vena cava of anesthetized mongrel dogs with thrombin and a special double-balloon catheter; 2 hours later, the thrombus was embolized. In one group of dogs, activated type 1 plasminogen activator inhibitor (PAI-1) (130 μg) was delivered directly into the forming thrombus; in another, TXA (110 mg/kg) was given intravenously before thrombus formation; in controls, thrombus was induced without inhibitors. Cross-linked fibrin degradation product (D-dimer) appeared in the blood of control animals within 1 hour of thrombus induction (176 ± 62.5 versus 1.02 ± 0.39 ng/mL baseline; mean ± SEM), was maximal by 4 hours (413 ± 110 ng/mL) and remained elevated at 24 hours (90.8 ± 19.5 ng/mL). Compared with controls, PAI- 1 and TXA suppressed D-dimer release by 80% and 85%, respectively, over the first 24 hours. One week later, animals were killed, and residual emboli were harvested. Perfusion scan defects persisted in all animals at this time, but there were no scan defect differences among groups. However, emboli recovered from animals receiving PAI-1 still harbored immunoreactive PAI-1 and were, on average, more than twofold greater in mass (393 ± 56 mg) than emboli recovered from either controls (183 ± 76 mg) or animals receiving TXA (180 ± 80 mg). ConclusionsIntravenous TXA and intrathrombus PAI-1 effectively suppress thrombolysis for 24 hours in canines. Thromboemboli enriched with PAI-1 appear to resist lysis for longer periods of time (up to 1 week). These findings are consistent with the hypothesis that PAI-1 remains associated with the embolus, where it continues to inhibit lysis, whereas TXA eventually diffuses out of the embolus, allowing lysis to ensue.

Chronic thromboembolic pulmonary hypertension-associated dysfibrinogenemias exhibit disorganized fibrin structure

INTRODUCTION Mechanisms contributing to the pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH) are poorly understood. This disorder is characterized by incomplete resolution of pulmonary perfusion defects resulting from acute venous thromboembolism. We previously identified several dysfibrinogenemias in some patients with CTEPH. The purpose of this study was to determine whether fibrin clot architecture might be implicated in the thrombolytic resistance in patients with these CTEPH-associated dysfibrinogenemias. MATERIALS AND METHODS Purified fibrinogen from patients and healthy controls was clotted with thrombin in the presence of calcium. Clot turbidity, porosity, and susceptibility to fibrinolysis were evaluated by spectrophotometric and permeation analyses. Fibrin network structure was assessed by laser-scanning confocal microscopy. RESULTS Compared to normal fibrinogen, CTEPH-associated dysfibrinogenemias exhibited low clot turbidity, decreased porosity, and fibrinolytic resistance. In addition, the dysfibrinogenemias exhibited a more disorganized fibrin network structure characterized by thinner fibers, greater network dispersal and more extensive fiber branching. CONCLUSIONS Abnormal clot architecture and fibrinolytic resistance may contribute to incomplete clot resolution following acute venous thromboembolism in patients with CTEPH-associated dyfibrinogenemia.

Chronic exposure to fibrin and fibrinogen differentially regulates intracellular Ca2+ in human pulmonary arterial smooth muscle and endothelial cells

Acute pulmonary embolism occurs in more than half a million people a year in the United States. Chronic thromboembolic pulmonary hypertension (CTEPH) develops in approximately 4% of these patients due to unresolved thromboemboli. CTEPH is thus a relatively common, progressive, and potentially fatal disease. One currently proposed theory for the poor resolution advocates that modification of fibrinogen in CTEPH patients causes resistance of emboli to fibrinolysis. The current study investigated the regulation of cytosolic Ca(2+) ([Ca(2+)](cyt)), central to the control of cell migration, proliferation, and contraction, by chronic exposure of pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells to fibrinogen and fibrin. Basal [Ca(2+)](cyt) was substantially elevated in PAEC after culture on fibrinogen, fibrin, and thrombin and in PASMC on fibrinogen and fibrin. In PAEC, fibrinogen significantly decreased the peak [Ca(2+)](cyt) transient (P <0.001) without a change in the transient peak width (at 50% of the peak height). This response was independent of effects on the proteinase-activated receptor (PAR) 1. Furthermore, chronic exposure to thrombin, an activator of PAR, significantly reduced the peak agonist-induced Ca(2+) release in PAEC, but increased it in PASMC. The recovery rate of the agonist-induced [Ca(2+)](cyt) transients decelerated in PASMC chronically exposed to fibrin; a small increase of the peak Ca(2+) was also observed. Substantial augmentation of PASMC (but not PAEC) proliferation was observed in response to chronic fibrin exposure. In conclusion, chronic exposure to fibrinogen, fibrin, and thrombin caused differential changes in [Ca(2+)](cyt) in PAEC and PASMC. Such changes in [Ca(2+)](cyt) may contribute to vascular changes in patients who have CTEPH where the pulmonary vasculature is persistently exposed to thromboemboli.

Transvenous procurement of pulmonary artery smooth muscle and endothelial cells using a novel endoarterial biopsy catheter in a canine model

OBJECTIVES The aim of this study was to evaluate the performance of a new arterial biopsy catheter in obtaining pulmonary endovascular samples in a canine model. BACKGROUND Percutaneous endomyocardial biopsy is a widely used and valuable procedure in the management of posttransplant rejection and selected cardiomyopathies. A similar method of obtaining endoarterial biopsy samples would aid in the study, diagnosis and management of arterial diseases. METHODS Catheterization was performed in 19 dogs, each weighing 20 to 30 kg, through an 8F sheath in the external jugular vein to obtain pulmonary endoarterial samples. The catheter consists of two sliding tubes: an inner one with a beveled opening that accommodates endoarterial tissue by means of a vacuum and an outer tube with a sharp distal edge that cuts the tissue when activated. RESULTS Overall, a total of 266 separate biopsy attempts were performed, and 161 tissue samples were obtained (success rate 61%). With modifications in technique in the last nine dogs, 54 (93%) of 58 attempts were successful. There were no deaths, extravasation of contrast material on angiography or thrombi. Of 20 vessels with prebiopsy and postbiopsy angiograms, 1 developed transient spasm (5%). On microscopic examination of cross sections of 50 separate pulmonary endoarterial biopsy samples, all had smooth muscle cells and 30 contained endothelial cells (60%). The arteries of origin showed small intimal and medial tears and mild perivascular hemorrhage. Angiographic and pathologic examination of previously biopsied arterial segments 2 weeks (two dogs) and 8 weeks (two dogs) after the procedure showed patent vessels and no thrombi. Histologically, the biopsy sites revealed mild neointimal and medial proliferation. CONCLUSIONS This new endoarterial biopsy catheter is safe and effective in obtaining pulmonary artery samples in normotensive dogs.

Improved imaging of deep venous thrombi during anticoagulation using radiolabelled anti-D-dimer antibodies.

ObjectivesRadiolabelled anti-fibrin antibodies have not yet enabled reliable and practical diagnosis of venous thromboembolism. However, previous unsuccessful clinical trials were performed with anti-fibrin &bgr;-chain antibodies that do not optimally bind to thrombi during anticoagulation. The current experiments were performed to determine if radiolabelled anti-D-dimer antibodies more reliably allowed nuclear medicine imaging of deep venous thrombi during anticoagulation than anti-&bgr;-chain antibodies. MethodsDogs with pre-existing unilateral femoral vein thrombi were anticoagulated with heparin (300 units·kg−1 bolus followed by 90 units·kg−1·h−1 continuous infusion). They were then allocated to receive one of three 111In labelled antibodies: anti-D-dimer, anti-&bgr; or a non-specific control antibody. Gamma scans of the legs were performed at regular intervals for 24 h. Scans were interpreted in a blinded fashion and the number of gamma counts from the femoral area on the thrombosed side was compared to the contralateral side. Clot/blood isotope density ratios were determined post-mortem. ResultsLeg thrombi were 100% detectable in the anti-D-dimer group, but only 60% detectable in the anti-&bgr; group. Mean±SD relative counts in the thrombosed femoral area was 137±10% compared to the contralateral side in the anti-D-dimer group, but only 116±20% in the anti-&bgr; group. The clot/blood ratio was 24.5±2.8 in the anti-D-dimer group, but only 7.8±2.0 in the anti-&bgr; group. Conclusions111In labelled anti-D-dimer provides superior nuclear medicine images of thrombi during intensive anticoagulation compared to anti-&bgr;. Clinical results with radiolabelled anti-D-dimer may be more promising than those previously observed with other anti-fibrin antibodies.

Antibiotics against Pseudomonas aeruginosa for COPD exacerbation in ICU: a 10-year retrospective study

Summary Chronic obstructive pulmonary disease (COPD) is a frequent source of hospitalization. Antibiotics are largely prescribed during COPD exacerbation. Our hypothesis is that large broad-spectrum antibiotics are more and more frequently prescribed. Our results confirm this trend and highlight that the increase in large broad-spectrum use in COPD exacerbation is largely unexplained. Background Acute COPD exacerbation (AECOPD) is frequently due to respiratory tract infection, and the benefit of antipseudomonal antibiotics (APA) is still debated. Health care–associated pneumonia (HCAP) was defined in 2005 and requires broad-spectrum antibiotherapy. The main objectives are to describe the antibiotic use for AECOPD in intensive care unit and to identify factors associated with APA use and AECOPD prognosis. Methods We conducted a monocentric, retrospective study on all AECOPDs in the intensive care unit treated by antibiotics for respiratory tract infection. Treatment failure (TF) was defined by death, secondary need for mechanical ventilation, or secondary systemic steroid treatment. A multivariate analysis was used to assess factors associated with APA prescription and TF. Results From January 2000 to December 2011, 111 patients were included. Mean age was 69 years (±12), mean forced expiratory volume 38% of theoretic value (±13). Thirty-five (31%) patients were intubated, and 52 (47%) were treated with noninvasive ventilation. From 107 patients, 8 (7%) cases of Pseudomonas aeruginosa were documented. APAs were prescribed in 21% of patients before 2006 versus 57% after (P=0.001). TF prevalence was 31%. Risk factors for P. aeruginosa in COPD and HCAP diagnosis did not influence APA, whereas the post-2006 period was independently associated with APA prescription (odds ratio 6.2; 95% confidence interval 1.9–20.3; P=0.0013). APA did not improve TF (odds ratio 1.09; 95% confidence interval 0.37–3.2). Conclusion HCAP guidelines were followed by an increase in APA use in AECOPD, without an improvement in prognosis. HCAP prevalence cannot account for the increasing APA trend. Time effect reveals a drift in practices. The microbiological effect of such a drift must be evaluated.

Fibrinogen and the Prediction of Residual Obstruction Manifested after Pulmonary Embolism Treatment

Residual pulmonary vascular obstruction (RPVO) and chronic thromboembolic pulmonary hypertension (CTEPH) are both long-term complications of acute pulmonary embolism, but it is unknown whether RPVO can be predicted by variants of fibrinogen associated with CTEPH. We used the Akaike information criterion to select the best predictive models for RPVO in two prospectively followed cohorts of acute pulmonary embolism patients, using as candidate variables the extent of the initial obstruction, clinical characteristics and fibrinogen-related data. We measured the selected models’ goodness of fit by analysis of deviance and compared models using the Chi-squared test. RPVO occurred in 29 (28.4%) out of 102 subjects in the first cohort and 46 (25.3%) out of 182 subjects in the second. The best-fit predictive model derived in the first cohort (p=0.0002) and validated in the second cohort (p=0.0005) implicated fibrinogen Bβ-chain monosialylation in the development of RPVO. When the derivation procedure excluded clinical characteristics, fibrinogen Bβ-chain monosialylation remained a predictor of RPVO in the best-fit predictive model (p=0.00003). Excluding fibrinogen characteristics worsened the predictive model (p=0.03). Fibrinogen Bβ-chain monosialylation, a common structural attribute of fibrin, helped predict RPVO after acute pulmonary embolism. Fibrin structure may contribute to the risk of developing RPVO. Fibrinogen helps predict residual pulmonary vascular obstruction after pulmonary embolism: results from the PROMPT study http://ow.ly/G7pY30m2HU6