Thrombin Generation in Vitreous and Subretinal Fluid of Patients with Retinal Detachment.

PURPOSE: To measure prothrombin fragments (F1+2) and thrombin-antithrombin complex (TAT) in vitreous and subretinal fluid (SRF) of rhegmatogenous retinal detachment (RRD) patients and to validate and further specify our earlier finding of increased thrombin activity in patients with proliferative vitreoretinopathy (PVR). METHODS: F1+2 and TAT were measured in 31 vitreous and 16 SRF samples using the Enzygnost® immunoassays. RESULTS: We found significant levels of F1+2 and TAT in the vitreous of all patients with RRD compared to patients with macular hole or macular pucker. However, there was no significant difference between patients who would develop PVR in the future, had established PVR, and patients with uncomplicated RRD both in vitreous concentrations of F1+2 (Kruskal-Wallis p = 0.963) and TAT (p = 0.516). CONCLUSION: The analysis of F1+2 and TAT confirmed significant thrombin generation in both vitreous and SRF of patients with RRD. An imbalance between the thrombin regulation mechanisms TAT and α2-macroglobulin possibly explains the difference from our previous findings.

Postoperative aqueous humour flare as a surrogate marker for proliferative vitreoretinopathy development.

PURPOSE: As some surgical procedures have been shown to increase postoperative flare values and thus contribute to blood-ocular barrier breakdown, retinal reattachment surgery might influence the risk of developing proliferative vitreoretinopathy (PVR). Therefore, we investigated whether postoperative aqueous flare values are a surrogate marker for the development of postoperative PVR. METHODS: We prospectively included 195 patients with primary rhegmatogenous retinal detachment (RRD) and measured aqueous laser flare preoperatively, and at 2 and 6 weeks postoperatively. Postoperative PVR was defined as reoperation for redetachment due to PVR membranes, within 6 months of initial surgery. Logistic regression and receiver operating characteristic (ROC) analysis determined whether higher postoperative flare values were associated with an increased risk of developing PVR later on. RESULTS: Reoperation for postoperative PVR was needed in 12 (6.2%) patients; in 18 (9.2%), reoperation was not related to PVR. The median flare value for patients who would develop PVR was significantly higher than that of patients who would not develop PVR, both at 2 weeks (p = 0.001) and 6 weeks (p < 0.001) postoperatively. Logistic regression analyses showed that a higher flare value significantly increased the odds of developing PVR, either at 2 weeks [odds ratio (OR) 1.027; 95% CI: 1.010-1.044] or 6 weeks (OR 1.076; 95% CI: 1.038-1.115). CONCLUSION: Flare values both at 2 and 6 weeks postoperatively seem a good surrogate marker in terms of sensitivity and specificity for the development of postoperative PVR but have only a modest positive predictive value. The 2-week value would be more useful in terms of early recognition of high-risk patients and hence give the possibility to better study effects of treatment methods.

Dabigatran inhibits intravitreal thrombin activity.

PURPOSE: Proliferative vitreoretinopathy (PVR) is a vitreoretinal disorder in which retinal pigment epithelial (RPE) cell activation contributes to both formation of fibrotic retinal membranes and inflammation. Vitreous of patients with PVR contains increased thrombin activity which induces profibrotic and proinflammatory programs in RPE cells. Inhibition of intravitreal thrombin activity may thus represent a therapeutic option for PVR. In this study, we examined the capacity of the clinically available direct thrombin inhibitor dabigatran to inhibit thrombin activity in vitreous fluids. METHODS: ARPE-19 cells were cultured with the following: (i) thrombin, (ii) vitreous without thrombin activity and (iii) vitreous with elevated thrombin activity (PVR samples and thrombin spiked vitreous) either in the presence or absence of dabigatran (range: 10-5 to 10-7  M). Subsequently, CCL2, CXCL8, GMCSF, IL6 and PDGFB mRNA expression levels were determined by RQ-PCR and protein levels of 27 cytokines, chemokines and growth factors were detected in culture supernatants using a multiplex approach. In addition, the capacity of vitreous fluids obtained from patients after oral dabigatran intake was tested in an in vitro thrombin activity assay. RESULTS: Thrombin and vitreous fluids containing thrombin activity induced CCL2, CXCL8, GM-CSF, IL-6 and PDGF-BB expression by ARPE-19 cells, which was inhibited by dabigatran. In addition, dabigatran that reached the vitreous after repeated oral intake did inhibit thrombin activity in the in vitro activity assay. CONCLUSION: Proliferative vitreoretinopathy (PVR) is associated with increased intravitreal thrombin activity that activates profibrotic and proinflammatory pathways in RPE cells. Our findings provide evidence that this activation pathway can potentially be inhibited by dabigatran.

Preoperative aqueous humour flare values do not predict proliferative vitreoretinopathy in patients with rhegmatogenous retinal detachment.

BACKGROUND/AIMS: Patients with rhegmatogenous retinal detachment (RRD) who develop postoperative proliferative vitreoretinopathy (PVR) have been found to have higher preoperative laser flare values than patients with RRD who do not develop this complication. Measurement of laser flare has therefore been proposed as an objective, rapid and non-invasive method for identifying high-risk patients. The purpose of our study was to validate the use of preoperative flare values as a predictor of PVR risk in two additional patient cohorts, and to confirm the sensitivity and specificity of this method for identifying high-risk patients. METHODS: We combined data from two independent prospective studies: centre 1 (120 patients) and centre 2 (194 patients). Preoperative aqueous humour flare was measured with a Kowa FM-500 Laser Flare Meter. PVR was defined as redetachment due to the formation of traction membranes that required reoperation within 6 months of initial surgery. Logistic regression and receiver operating characteristic analysis determined whether higher preoperative flare values were associated with an increased risk of postoperative PVR. RESULTS: PVR redetachment developed in 21/314 patients (6.7%). Median flare values differed significantly between centres, therefore analyses were done separately. Logistic regression showed a small but statistically significant increase in odds with increasing flare only for centre 2 (OR 1.014; p=0.005). Areas under the receiver operating characteristic showed low sensitivity and specificity: centre 1, 0.634 (95% CI 0.440 to 0.829) and centre 2, 0.731 (95% CI 0.598 to 0.865). CONCLUSIONS: Preoperative laser flare measurements are inaccurate in discriminating between those patients with RRD at high and low risk of developing PVR.

Vitreous and subretinal fluid concentrations of orally administered dabigatran in patients with rhegmatogenous retinal detachment.

PURPOSE: One of the factors that was shown to contribute to the development of proliferative vitreoretinopathy (PVR) is the coagulation factor thrombin. Therefore, a specific oral thrombin inhibitor such as dabigatran might be a possible therapeutic option. An oral drug has the advantage of patient-friendly prolonged administration in contrast to drugs that can only be applied during vitrectomy, on condition that the drug reaches the target site. We tested whether dabigatran reaches the vitreous and subretinal fluid (SRF) after a single oral dose of dabigatran. METHODS: Twenty-eight patients with a retinal detachment received a single dose of 220 mg dabigatran etexilate 2-8 hr prior to surgery. During surgery, we took a blood sample and a vitreous or subretinal fluid sample. The concentration of dabigatran was measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The dabigatran concentration between 2 and 9 hr after administration was higher in SRF than in vitreous (max 8.5 and 3.8 ng/ml). Corresponding plasma concentrations ranged from 15 to 225 ng/ml. There was a significant relationship between SRF levels and plasma levels (rs  = 0.68, p = 0.014); the levels in vitreous fluid showed no such relationship (rs  = 0.20, p = 0.48). In addition, we measured the vitreous concentration of a non-study patient using 150 mg dabigatran twice daily. The concentration was approximately 10 times higher than after a single dosage (25.8 ng/ml). CONCLUSION: We demonstrate that oral intake of dabigatran, a candidate drug to modulate PVR, results in potentially relevant intraocular concentrations. We suggest that repeated dosing may lead to higher concentrations, but this should be further explored.

Clonidine as an adjuvant to prolong local analgesia in conventional scleral buckle surgery.

PURPOSE: The aim of this study was to determine the effect of a single dose of 150 µg of clonidine as an adjuvant to levobupivacaine (Chirocaine(®)) in retrobulbar block on postoperative safety and analgesia. METHODS: This was a prospective, randomized, controlled, double-blind trial. One hundred twenty patients with a rhegmatogenous retinal detachment scheduled to undergo external buckling surgery and cryocoagulation were asked to participate. Participants were randomly assigned either to receive 3-5 mL Chirocaine (22.5-37.5 mg) or 3-5 mL Chirocaine and 1 mL clonidine (150 µg) before surgery. Main outcome measures were postoperative pain, use of analgesics, blood pressure, and plasma clonidine concentration. Nine nonrandomized patients consented to give blood samples for pharmacokinetic analysis. RESULTS: There was no significant difference in pain score between both groups. On average, the use of analgesic medication occurred later in the clonidine group (P=0.0004), but there was no statistical difference in the first time that postoperative medication was taken (P=0.13). Blood pressure was reduced by clonidine (systolic: P=0.02, diastolic: P=0.006). Clonidine levels could be demonstrated during the 24-h postoperative period, with an average half-life of 22 h. CONCLUSIONS: Administration of clonidine as an adjuvant to conventional retrobulbar block is safe, and delays the postoperative use of analgesics. The reduction of postoperative pain and the time of first use of analgesic medication, however, were not significantly different between groups. Further, pain scores in both study groups remained low. Therefore, the beneficial effect of clonidine in conventional scleral buckle surgery appears to be limited.

The role of thrombin in proliferative vitreoretinopathy.

PURPOSE: To determine the role of thrombin in the development of proliferative vitreoretinopathy (PVR). METHODS: Vitreous was collected from patients undergoing a vitrectomy (macular holes and puckers, n = 11 [controls]; retinal detachment without PVR development following vitrectomy, n = 15 [RRD1]; retinal detachment with PVR development within 6 months after vitrectomy, n = 11 [RRD2]; and established PVR, n = 14 [PVR]). Thrombin activity in vitreous was determined using a thrombin-specific chromogenic substrate. ARPE-19 cells were stimulated with 8× diluted vitreous samples in the presence and absence of hirudin. The samples were analyzed at t = 0 and t = 24 hours for the presence of 27 cytokines/chemokines and growth factors using a multiplex approach. In comparable studies, ARPE-19 cells were stimulated for 2 hours, and mRNA expression levels for CCL2, CXCL8, GMCSF, IL6, and PDGFB were determined by real-time quantitative (RQ)-PCR. RESULTS: Thrombin activity was significantly (P < 0.05) higher in vitreous of the PVR group compared to the other groups. Proliferative vitreoretinopathy vitreous stimulated the production of chemokine (C-C motif) ligand (CCL)2, chemokine (C-X-C motif) ligand (CXCL)8, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, and platelet-derived growth factor (PDGF)-BB by ARPE-19 to significantly (P < 0.05) higher levels than vitreous from the RRD1 and RRD2 groups. These effects of PVR vitreous were significantly (P < 0.05) reduced by hirudin. These data were confirmed by mRNA studies. CONCLUSIONS: Thrombin activity is increased in vitreous of patients with established PVR and is involved in the activation of proinflammatory and profibrotic pathways in RPE cells. Inhibition of thrombin activity may therefore represent a potential treatment option for proliferative vitreoretinopathy.

Massive ingestion of cardiac drugs: toxicokinetic aspects of digoxin and sotalol during hemofiltration.

CASE: We present the case of a 75-year-old patient admitted to the emergency department after ingesting a large amount of several cardiac drugs, among which were digoxin and sotalol. Because of renal insufficiency, cardiogenic shock, and high serum digoxin levels, the patient received continuous venovenous hemofiltration (CVVH) and digoxin-specific Fab fragments. Digoxin and the digoxin-specific Fab fragments are normally cleared by the kidneys. METHODS: Serum-free and total digoxin and serum sotalol concentrations were monitored for several days. RESULTS: Less than 10% of the estimated ingested dose of digoxin was cleared by CVVH within 5 days. CONCLUSION: CVVH has little influence on the clearance of Fab-bound digoxin from the body. In contrast, sotalol is efficiently cleared by CVVH.