Urokinase plasminogen activator released by alveolar epithelial cells modulates alveolar epithelial repair in vitro

Intra-alveolar fibrin is formed following lung injury and inflammation and may contribute to the development of pulmonary fibrosis. Fibrin turnover is altered in patients with pulmonary fibrosis, resulting in intra-alveolar fibrin accumulation, mainly due to decreased fibrinolysis. Alveolar type II epithelial cells (AEC) repair the injured alveolar epithelium by migrating over the provisional fibrin matrix. We hypothesized that repairing alveolar epithelial cells modulate the underlying fibrin matrix by release of fibrinolytic activity, and that the degree of fibrinolysis modulates alveolar epithelial repair on fibrin. To test this hypothesis we studied alveolar epithelial wound repair in vitro using a modified epithelial wound repair model with human A549 alveolar epithelial cells cultured on a fibrin matrix. In presence of the inflammatory cytokine interleukin-1beta, wounds increase by 800% in 24 hours mainly due to detachment of the cells, whereas in serum-free medium wound areas decreases by 22.4 +/- 5.2% (p < 0.01). Increased levels of D-dimer, FDP and uPA in the cell supernatant of IL-1beta-stimulated A549 epithelial cells indicate activation of fibrinolysis by activation of the plasmin system. In presence of low concentrations of fibrinolysis inhibitors, including specific blocking anti-uPA antibodies, alveolar epithelial repair in vitro was improved, whereas in presence of high concentrations of fibrinolysis inhibitors, a decrease was observed mainly due to decreased spreading and migration of cells. These findings suggest the existence of a fibrinolytic optimum at which alveolar epithelial repair in vitro is most efficient. In conclusion, uPA released by AEC alters alveolar epithelial repair in vitro by modulating the underlying fibrin matrix.

The effect of natural habituation on coagulation responses to acute mental stress and recovery in men

Blood coagulation activation might be one mechanism linking acute mental stress with coronary events. We investigated the natural habituation of coagulation responses and recovery to short-term mental stress. Three times with one-week intervals, 24 men (mean age 47 +/- 7 years) underwent the same 13-min stressor (preparation, job interview, mental arithmetic). During each visit venous blood was obtained four times (baseline, immediately post-stress, 45 min of recovery, 105 min of recovery). Eight blood coagulation parameters were measured at weeks one and three. Acute stress provoked increases in von Willebrand factor antigen, fibrinogen, clotting factor FVII activity (FVII:C), FVIII:C, FXII:C (p's < or = 0.019), and D-dimer (N.S.). All coagulation parameters experienced full recovery except FVIII:C (p = 0.022). Stress did not significantly affect activated partial thromboplastin time and prothrombin time. At all time points FVIII:C and FXII:C levels were significantly higher at week one compared to week three (p's < or = 0.041). Before catheter insertion, systolic blood pressure (p = 0.001) and heart rate (p = 0.026) were relatively higher at week one. Unlike the magnitude of systolic blood pressure response to stress (p = 0.007) and of cortisol recovery from stress (p = 0.002), the magnitude of all coagulation responses to stress and the recovery from stress were similar in week one and week three. Sympathetic activation with anticipatory stress best explained increased baseline activity in FVIII and FXII at week one. An incapacity of the coagulation system to adapt to stress repeats is perhaps a consequence of evolution, but might also contribute to increased coronary risk in some individuals, particularly in those with cardiovascular diseases.

Cell-based therapy facilitates venous thrombus resolution

Endothelial progenitor cells (EPC) are involved in many healing processes in cardiovascular diseases and can be found in spontaneously resolving venous thrombi. The purpose of the present study was to investigate whether the therapeutic administration of EPC might enhance the resolution of venous thrombi. For this purpose, venous thrombosis was induced in the infrarenal inferior vena cava (IVC) in 28 athymic nude rats. Culture expanded EPC derived from human peripheral blood mononuclear cells were injected intravenously two and four days after thrombus induction. Recanalisation of the IVC and thrombus organisation were assessed by laser Doppler measurements of the blood flow and immunohistochemical detection of endothelialised luminal structures in the thrombus. EPC transplantation resulted in significantly enhanced thrombus neovascularisation (capillary density: 186.6 +/- 26.7/HPF vs. 78 +/- 12.3/HPF, p<0.01; area covered by capillaries: 8.9 +/- 1.7 microm(2) vs. 2.5 +/- 1.3 microm(2), p<0.01) and was accompanied by a substantial increase in intra-thrombus blood flow (perfusion ratio: 0.7 +/- 0.07 vs. 0.3 +/- 0.08, p<0.02). These results were paralleled by augmented macrophage recruitment into resolving thrombi in the animals treated with EPC (39.4 +/- 4.7/HPF vs. 11.6 +/- 1.9/HPF, p<0.01). Our data suggest that EPC transplantation might be of clinical value to facilitate venous thrombus resolution in cases where current therapeutic options have limited success.

The absence of angiopoietin-2 leads to abnormal vascular maturation and persistent proliferative retinopathy

Angiopoietin-2 (Ang-2) antagonises the maturing effect of angiopoietin-1 (Ang-1) on blood vessels, and cooperates with VEGF to induce neovascularisation. In knockout mice, Ang-2 displayed a specific role in postnatal angiogenic remodelling. Here, we demonstrate that mice deficient in Ang-2 fail to form a proper spatial retinal vascular network. The retinal vasculature was characterised by reduced large vessel numbers and defects forming the superficial periphery mostly on the arteriolar site, and the secondary and tertiary deep capillary network. Hypoxia in the retinal periphery induced a four-fold VEGF upregulation and active endothelial proliferation for up to 60 days. Concomitantly, retinal digest preparations showed increased arteriolar (+33%) and capillary diameters (+90%), and fluorescein angiograms revealed leakiness of neovascular front. At one year of age, persistent preretinal vessels were non-leaky in accordance with a relative increase in the ratio of Ang-1 to VEGF. Taken together, the data suggest that Ang-2 has an important function in the spatial configuration of the three-dimensional retinal vasculature. Secondarily, prolonged VEGF activity results in a model of persistent proliferative retinopathy.

Transapical access closure: the TA PLUG device

OBJECTIVES Percutaneous closure of the transapical (TA) access site for large-calibre devices is an unsolved issue. We report the first experimental data on the TA PLUG device for true-percutaneous closure following large apical access for transcatheter aortic valve implantation. METHODS The TA PLUG, a self-sealing full-core closure device, was implanted in an acute animal study in six pigs (60.2 ± 0.7 kg). All the pigs received 100 IU/kg of heparin. The targeted activated clotting time was left to normalize spontaneously. After accessing the left ventricular apex with a 39 French introducer, the closure plug device was delivered with a 33 French over-the-wire system under fluoroscopic guidance into the apex. Time to full haemostasis as well as rate of bleeding was recorded. Self-anchoring properties were assessed by haemodynamic push stress under adrenalin challenge. An additional feasibility study was conducted in four pigs (58.4 ± 1.1 kg) with full surgical exposure of the apex, and assessed device anchoring by pull-force measurements with 0.5 Newton (N) increments. All the animals were electively sacrified. Post-mortem analysis of the heart was performed and the renal embolic index assessed. RESULTS Of six apical closure devices, five were correctly inserted and fully deployed at the first attempt. One became blocked in the delivery system and was placed successfully at the second attempt. In all the animals, complete haemostasis was immediate and no leak was recorded during the 5-h observation period. Neither leak nor any device dislodgement was observed under haemodynamic push stress with repeated left ventricular peak pressure of up to 220 mmHg. In the feasibility study assessing pull-stressing, device migration occurred at a force of 3.3 ± 0.5 N corresponding to 247.5 mmHg. Post-mortem analyses confirmed full expansion of all devices at the intended target. No macroscopic damage was identified at the surrounding myocardium. The renal embolic index was zero. CONCLUSIONS True-percutaneous left ventricular apex closure following large access is feasible with the self-sealing TA PLUG. The device allows for immediate haemostasis and a reliable anchoring in the acute animal setting. This is the first report of a true-percutaneous closure for large-calibre transcatheter aortic valve implantation access.

Design and establishment of a biobank in a multicenter prospective cohort study of elderly patients with venous thromboembolism (SWITCO65+)

In the field of thrombosis and haemostasis, many preanalytical variables influence the results of coagulation assays and measures to limit potential results variations should be taken. To our knowledge, no paper describing the development and maintenance of a haemostasis biobank has been previously published. Our description of the biobank of the Swiss cohort of elderly patients with venous thromboembolism (SWITCO65+) is intended to facilitate the set-up of other biobanks in the field of thrombosis and haemostasis. SWITCO65+ is a multicentre cohort that prospectively enrolled consecutive patients aged ≥65 years with venous thromboembolism at nine Swiss hospitals from 09/2009 to 03/2012. Patients will be followed up until December 2013. The cohort includes a biobank with biological material from each participant taken at baseline and after 12 months of follow-up. Whole blood from all participants is assayed with a standard haematology panel, for which fresh samples are required. Two buffy coat vials, one PAXgene Blood RNA System tube and one EDTA-whole blood sample are also collected at baseline for RNA/DNA extraction. Blood samples are processed and vialed within 1 h of collection and transported in batches to a central laboratory where they are stored in ultra-low temperature archives. All analyses of the same type are performed in the same laboratory in batches. Using multiple core laboratories increased the speed of sample analyses and reduced storage time. After recruiting, processing and analyzing the blood of more than 1,000 patients, we determined that the adopted methods and technologies were fit-for-purpose and robust.

Platelet count and outcome in patients with acute venous thromboembolism

The relationship between platelet count and outcome in patients with acute venous thromboembolism (VTE) has not been consistently explored. RIETE is an ongoing registry of consecutive patients with acute VTE. We categorised patients as having very low- (<80,000/µl), low- (80,000/µl to 150,000/µl), normal- (150,000/µl to 300,000/µl), high- (300,000/µl to 450,000/µl), or very high (>450,000/µl) platelet count at baseline, and compared their three-month outcome. As of October 2012, 43,078 patients had been enrolled in RIETE: 21,319 presenting with pulmonary embolism and 21,759 with deep-vein thrombosis. In all, 502 patients (1.2%) had very low-; 5,472 (13%) low-; 28,386 (66%) normal-; 7,157 (17%) high-; and 1,561 (3.6%) very high platelet count. During the three-month study period, the recurrence rate was: 2.8%, 2.2%, 1.8%, 2.1% and 2.2%, respectively; the rate of major bleeding: 5.8%, 2.6%, 1.7%, 2.3% and 4.6%, respectively; the rate of fatal bleeding: 2.0%, 0.9%, 0.3%, 0.5% and 1.2%, respectively; and the mortality rate: 29%, 11%, 6.5%, 8.8% and 14%, respectively. On multivariate analysis, patients with very low-, low-, high- or very high platelet count had an increased risk for major bleeding (odds ratio [OR]: 2.70, 95% confidence interval [CI]: 1.85-3.95; 1.43 [1.18-1.72]; 1.23 [1.03-1.47]; and 2.13 [1.65-2.75]) and fatal bleeding (OR: 3.70 [1.92-7.16], 2.10 [1.48-2.97], 1.29 [0.88-1.90] and 2.49 [1.49-4.15]) compared with those with normal count. In conclusion, we found a U-shaped relationship between platelet count and the three-month rate of major bleeding and fatal bleeding in patients with VTE.

Practical use of dabigatran etexilate for stroke prevention in atrial fibrillation

Atrial fibrillation (AF) is associated with an increased risk of thromboembolism, and is the most prevalent factor for cardioembolic stroke. Vitamin K antagonists (VKAs) have been the standard of care for stroke prevention in patients with AF since the early 1990s. They are very effective for the prevention of cardioembolic stroke, but are limited by factors such as drug-drug interactions, food interactions, slow onset and offset of action, haemorrhage and need for routine anticoagulation monitoring to maintain a therapeutic international normalised ratio (INR). Multiple new oral anticoagulants have been developed as potential replacements for VKAs for stroke prevention in AF. Most are small synthetic molecules that target thrombin (e.g. dabigatran etexilate) or factor Xa (e.g. rivaroxaban, apixaban, edoxaban, betrixaban, YM150). These drugs have predictable pharmacokinetics that allow fixed dosing without routine laboratory monitoring. Dabigatran etexilate, the first of these new oral anticoagulants to be approved by the United States Food and Drug Administration and the European Medicines Agency for stroke prevention in patients with non-valvular AF, represents an effective and safe alternative to VKAs. Under the auspices of the Regional Anticoagulation Working Group, a multidisciplinary group of experts in thrombosis and haemostasis from Central and Eastern Europe, an expert panel with expertise in AF convened to discuss practical, clinically important issues related to the long-term use of dabigatran for stroke prevention in non-valvular AF. The practical information reviewed in this article will help clinicians make appropriate use of this new therapeutic option in daily clinical practice.

Procoagulant reactivity to laboratory acute mental stress in Africans and Caucasians, and its relation to depressive symptoms: the SABPA study.

The risk of cardiovascular disease is dramatically increasing in Africans (black). The prothrombotic stress response contributes to atherothrombotic disease and is modulated by depressive symptoms. We examined coagulation reactivity to acute mental stress and its relation to psychological well-being in Africans relative to Caucasians (white). A total of 102 African and 165 Caucasian school teachers underwent the Stroop Color-Word Conflict test. Circulating levels of von Willebrand factor (VWF) antigen, fibrinogen, and D-dimer were measured before and after the Stroop. Cardiovascular reactivity measures were also obtained. All participants completed the Patient Health Questionnaire-9 and the General Health Questionnaire-28 for the assessment of depressive symptoms and total psychological distress, respectively. After controlling for covariates, resting levels of VWF, fibrinogen, and D-dimer were higher in Africans than in Caucasians (all p-values ≤0.006). Depressive symptoms and psychological distress were not significantly associated with resting coagulation measures. Stress reactivity in VWF (p<0.001) and fibrinogen (p=0.016), but not in D-dimer (p=0.27), were decreased in Africans relative to Caucasians with Africans showing greater reactivity of total peripheral resistance (p=0.017). Depressive symptoms, but not general psychological distress, were associated with greater VWF increase (p=0.029) and greater fibrinogen decrease (p=0.030) in Africans relative to Caucasians. In conclusion, Africans showed greater hypercoagulability at rest but diminished procoagulant reactivity to acute mental stress when compared with Caucasians. Ethnic differences in the vascular adrenergic stress response might partially explain this finding. Depressive symptoms were associated with exaggerated VWF reactivity in Africans relative to Caucasians. The clinical implications of these findings for Africans need further study.

Consistency of thromboelastometry analysis under scrutiny: Results of a systematic evaluation within and between analysers.

While the use of thromboelastometry analysis (ROTEM®) in evaluation of haemostasis is rapidly increasing, important validity parameters of testing remain inadequately examined. We aimed to study systematically the consistency of thromboelastometry parameters within individual tests regarding measurements between different analysers, between different channels of the same analyser, between morning and afternoon measurements (circadian variation), and if measured four weeks apart. Citrated whole blood samples from 40 healthy volunteers were analysed with two analysers in parallel. EXTEM, INTEM, FIBTEM, HEPTEM and APTEM tests were conducted. A Bland-Altman comparison was performed and homogeneity of variances was tested using the pitman test. P-value ranges were used to classify the level of homogeneity (p<0.15 - low homogeneity, p = 0.15 to 0.5 - intermediate homogeneity, p>0.5 high homogeneity). Less than half of all comparisons made showed high homogeneity of variances (p>0.5) and in about a fifth of comparisons data distributions were heterogeneous (p<0.15). There was no clear pattern for homogeneity. On average, comparisons of MCF, ML and LI30 measurements tended to be better, but none of the tests assessed outperformed another. In conclusion, systematic investigation reveals large differences in the results of some thromboelastometry parameters and lack of consistency. Clinicians and scientists should take these inconsistencies into account and focus on parameters with a higher homogeneity such as MCF.

Fixed low-dose ultrasound-assisted catheter-directed thrombolysis followed by routine stenting of residual stenosis for acute ilio-femoral deep-vein thrombosis.

Patients with ilio-femoral deep-vein thrombosis (DVT) are at high risk of developing the post-thrombotic syndrome (PTS). In comparison to anticoagulation therapy alone, extended venography-guided catheter-directed thrombolysis without routine stenting of venous stenosis in patients with ilio-femoral DVT is associated with an increased risk of bleeding and a moderate reduction of PTS. We performed a prospective single-centre study to investigate safety, patency and incidence of PTS in patients with acute ilio-femoral DVT treated with fixed-dose ultrasound-assisted catheter-directed thrombolysis (USAT; 20 mg rt-PA during 15 hours) followed by routing stenting of venous stenosis, defined as residual luminal narrowing >50%, absent antegrade flow, or presence of collateral flow at the site of suspected stenosis. A total of 87 patients (age 46 ± 21 years, 60% women) were included. At 15 hours, thrombolysis success ≥50% was achieved in 67 (77%) patients. Venous stenting (mean 1.9 ± 1.3 stents) was performed in 70 (80%) patients, with the common iliac vein as the most frequent stenting site (83%). One major (1%; 95% CI, 0-6%) and 6 minor bleedings (7%; 95%CI, 3-14%) occurred. Primary and secondary patency rates at 1 year were 87% (95% CI, 74-94%) and 96% (95% CI, 88-99%), respectively. At three months, 88% (95% CI, 78-94%) of patients were free from PTS according to the Villalta scale, with a similar rate at one year (94%, 95% CI, 81-99%). In conclusion, a fixed-dose USAT regimen followed by routine stenting of underlying venous stenosis in patients with ilio-femoral DVT was associated with a low bleeding rate, high patency rates, and a low incidence of PTS.

Multicentre validation of the Geneva Risk Score for hospitalised medical patients at risk of venous thromboembolism. Explicit ASsessment of Thromboembolic RIsk and Prophylaxis for Medical PATients in SwitzErland (ESTIMATE).

There is a need to validate risk assessment tools for hospitalised medical patients at risk of venous thromboembolism (VTE). We investigated whether a predefined cut-off of the Geneva Risk Score, as compared to the Padua Prediction Score, accurately distinguishes low-risk from high-risk patients regardless of the use of thromboprophylaxis. In the multicentre, prospective Explicit ASsessment of Thromboembolic RIsk and Prophylaxis for Medical PATients in SwitzErland (ESTIMATE) cohort study, 1,478 hospitalised medical patients were enrolled of whom 637 (43%) did not receive thromboprophylaxis. The primary endpoint was symptomatic VTE or VTE-related death at 90 days. The study is registered at ClinicalTrials.gov, number NCT01277536. According to the Geneva Risk Score, the cumulative rate of the primary endpoint was 3.2% (95% confidence interval [CI] 2.2-4.6%) in 962 high-risk vs 0.6% (95% CI 0.2-1.9%) in 516 low-risk patients (p=0.002); among patients without prophylaxis, this rate was 3.5% vs 0.8% (p=0.029), respectively. In comparison, the Padua Prediction Score yielded a cumulative rate of the primary endpoint of 3.5% (95% CI 2.3-5.3%) in 714 high-risk vs 1.1% (95% CI 0.6-2.3%) in 764 low-risk patients (p=0.002); among patients without prophylaxis, this rate was 3.2% vs 1.5% (p=0.130), respectively. Negative likelihood ratio was 0.28 (95% CI 0.10-0.83) for the Geneva Risk Score and 0.51 (95% CI 0.28-0.93) for the Padua Prediction Score. In conclusion, among hospitalised medical patients, the Geneva Risk Score predicted VTE and VTE-related mortality and compared favourably with the Padua Prediction Score, particularly for its accuracy to identify low-risk patients who do not require thromboprophylaxis.