654 Original article

Intravitreal thrombin activity is elevated in retinal vein occlusion Thomas Bertelmanna, Thomas Stiefb, Walter Sekundoa, Stefan Mennelc, Nauke Nguyend and Michael J. Kosse To evaluate whether intravitreal thrombin activity is elevated in eyes with branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO) in comparison to healthy controls. Prospective clinical case series of 19 patients with BRVO, 13 patients suffering from CRVO and nine participants serving as controls. Vitreous taps were extracted from the central vitreous body, 200 ml frozen/ thawed sample was immediately stabilized with 200 ml 5% human albumin, and 200 ml mixture thereof was stabilized with 200 ml 2.5 mol/l arginine, pH 8.6. Thrombin activity was determined chromogenically. Intravitreal levels of vascular endothelial growth factor (VEGF) as a marker for blood– retina barrier (BRB) breakdown were measured by a commercial chemiluminescent enzyme immuno assay (R&D). Intravitreal thrombin activity and VEGF levels were 1.6 W 1.2 mIU/ml (mean value W SD; range: 0.2–4.2 mIU/ml) and 554 W 568 pg/ml (range: 20–2005 pg/ml) in BRVOaffected eyes, 2.6 W 1.2 mIU/ml (range: 0.8–5.2 mIU/ml) and 1332 W 1350 pg/ml (range: 58–3943 pg/ml) in eyes suffering from CRVO as well as 0.8 W 0.8 mIU/ml (range: 0.2–2.7 mIU/ml) and 115 W 120 pg/ml (range: 32–431 pg/ ml) in controls. There are significant differences of intravitreal thrombin activity and intravitreal VEGF levels between eyes with BRVO, CRVO, and controls (P U 0.007 and P U 0.003, Kruskal–Wallis test). Intravitreal thrombin

activity is significantly correlated with intravitreal VEGF levels (r U 0656; P < 0.001, Pearson correlation). Intravitreal thrombin activity might serve as a new marker for BRB breakdown or macular fibrin deposition in ophthalmology. Significant differences of intravitreal thrombin activity between eyes with BRVO, CRVO, and healthy controls might offer new therapeutic strategies for RVO-affected eyes. The effect of oral and intravitrealy injected direct thrombin inhibitors needs to be evaluated in further investigations. Blood Coagul Fibrinolysis 25:654–659 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Introduction

the severity of macular edema, the amount of ischemic retinal area, and the extent of the blood–retina barrier (BRB) breakdown in both RVO types [9–11]. Further studies detected a variety of different cytokines to be elevated in RVO-affected eyes as well [10,12–14]. This in turn provided new therapeutic options, and today intravitreally applied anti-VEGF substances (Lucentis, Novartis Pharma AG/Eylea, and Bayer Pharma AG) as well as corticosteroids (Ozurdex and Allergan) have advanced to be the treatment of choice beside laser photocoagulation therapy [15–18].

Until today, branch retinal vein occlusions (BRVOs) and central retinal vein occlusions (CRVOs) are the second and third most common retinal vascular disorders behind diabetic retinopathy that can cause a sustainable decrease in visual acuity [1,2]. The latter is particularly attributed to the consecutive appearance of intraretinal hemorrhages, macular hypoxia, the development of a cystoid macular edema, or neovascularizations in different compartments of the eye [1,3–5]. Although initially described more than 150 years ago by Liebreich [6] and Leber [7], the understanding of the pathophysiological processes as well as the therapeutic options for the patients involved has still been limited and/or controversial [2,8]. To get a better understanding of both aspects intraocular fluids and their contents of different proteins, enzymes and cytokines got more and more into the focus of interest within the last decades and revolutionized the pathophysiologic knowledge as well as the treatment strategies for both retinal vein occlusion (RVO) subtypes. Vascular endothelial growth factor (VEGF) was found to be significantly elevated as well as significantly correlated with 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins

Blood Coagulation and Fibrinolysis 2014, 25:654–659 Keywords: branch retinal vein occlusion, central retinal vein occlusion, direct thrombin inhibitor, thrombin, vitrectomy a Department of Ophthalmology, Philipps-University, bDepartment of Laboratory Medicine, Philipps-University, Marburg, Germany, cDepartment of Ophthalmology, Feldkirch Regional Hospital, Feldkirch, Austria, dDepartment of Ophthalmology, Goethe University, Frankfurt/Main, Germany and eDoheny Eye Institute, Los Angeles, California, USA

Correspondence to Thomas Bertelmann, MD, Department of Ophthalmology, Philipps-University Marburg, Baldingerstraße, 35043 Marburg, Germany Tel: +49 6421 5862600; e-mail: [email protected] Received 19 November 2013 Revised 17 January 2014 Accepted 18 January 2014

Recently, intravitreal thrombin activity has been described for the first time in healthy eyes that is, in eyes without BRB breakdown, using an innovative stabilization regimen for extracted vitreous fluid [19]. Thrombin is the central enzyme of human hemostasis, and thus plays a crucial role in (retinal) vein occlusion development [20]. It might furthermore play an essential role in preventing BRB breakdown (‘leaky eyes’) [20]. The purpose of our investigation was to detect intravitreal thrombin activity in eyes with recent onset of BRVO and DOI:10.1097/MBC.0000000000000109

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Intravitreal thrombin in retinal vein occlusion Bertelmann et al. 655

CRVO and to demonstrate higher levels of intravitreal thrombin activity in RVO-affected eyes in comparison to healthy controls.

Material and methods This study was performed in accordance with the European Guidelines for Good Clinical Practice and the Helsinki Declaration of 1975 (sixth revision, 2008). The institutional review boards of Goethe-University of Frankfurt and Philipps-University of Marburg approved the protocol for collection and testing of acquired vitreous samples. In a prospective approach, 19 consecutive patients with recent onset of BRVO, 13 successive patients with recent onset of CRVO and nine consecutive patients with macular diseases [macular hole (n ¼ 2), macular pucker (n ¼ 3), or vitreal floaters (n ¼ 4)] serving as controls were included into our series after written consent was obtained from each individual following an explicit explanation of the purpose and potential adverse side-effects of the procedure. Patients with recent BRVO or CRVO not older than 6 weeks were included into the study group if typical clinical signs of one of both RVO entities were apparent and confirmed with fundus photography and fluorescein angiography. Furthermore, a cystoid macular edema involving the fovea and a central macular thickness (CMT) of less than 1000 mm had to be present as measured with standard SD-OCT (SD-OCT; 3D OCT-2000; Topcon, Tokyo, Japan). All scans were performed with a scan depth of 2.3 mm with a horizontal resolution of 20 mm and a longitudinal resolution of 5–6 mm as well as an A-scan speed of 27.000 A scans/s. CMT was thus calculated as the distance of the internal limiting membrane to the basal membrane of the retinal pigment epithelium, including all compartments in between. Patients scheduled for standard 23-gauge three-port pars-plana vitrectomy (ppV) for macular surgery (macular hole and macular pucker) or vitreous floater removal served as controls [21]. Exclusion criteria for both groups included the presence of vitreomacular traction or neovascular complications defined as any neovascularization of the anterior (e.g. rubeosis iridis) as well as posterior segment of the eye (neovascularization of the disc or elsewhere); previous intravitreal drug injections; laser photocoagulation; previous vitrectomy; other intraocular surgery on both eyes, including cataract surgery on the fellow eye in the last 6 months; signs of glaucoma, diabetic retinopathy, intraocular inflammation, or trauma; participation in any clinical trial; and the use of immunosuppressive drugs or history of malignant tumors of any location.

which has two separate channels for aspiration and infusion. After conjunctival displacement, an oblique sclerotomy was performed. To visualize the tip of the vitrector in the central vitreous cavity, a headset and a magnifying 28-diopter lens were used. All procedures were performed in an antiseptic operating room environment. An assistant then aspirated a total of 500 –700 ml of undiluted fluid from the central vitreous as instructed by the surgeon, who controlled against clinically relevant perioperative hypotonia. Thus, a minimum sample volume of 500 ml of central vitreous was aspirated from all patients. At the end of the limited cppV, subsequent isovolumetric substitutions of balanced salt solution (BSS, Alcon, Freiburg, Germany), 1.25 mg (100 ml) of bevacizumab (Avastin, Genentech, San Francisco, California, USA) and 0.8 mg (200 ml) of dexamethasone (Dexaratiopharm, Ulm, Germany) were injected in adherence with the principle of intravitreal combination therapy [22]. In the control group, nine consecutive patients with an intact BRB [21] with different macular diseases [macular hole (n ¼ 2), macular pucker (n ¼ 3), or vitreal floaters (n ¼ 4)] were included, and undiluted samples of the central vitreous body were taken during ppV performed for macular peeling due to idiopathic epiretinal membranes, macular hole surgery, or vitreal floater removal. A volume of 800 ml was aspirated before the infusion line was set active. The undiluted vitreous samples of both groups were transferred into a 1.5 ml Eppendorf cup and immediately frozen in a – 208C freezer. A maximum volume of 200 ml of each vitreous tap was available for our investigations, as the residual capacity of the aspirated samples was used for another research project. After thawing at 238C, 100 or 200 ml samples were instantly stabilized with 5% human albumin (CSL Behring, Marburg, Germany) (one part sample to one part albumin) and consecutively 1þ1 mixed with 2.5 mol/l arginine, pH 8.6 (Sigma, Deisenhofen, Germany). To detect thrombin activity, 50 ml arginine-stabilized samples were incubated in duplicate with 25 ml 0 mmol/l (turbidity control) or 1.5 mmol/l HD-CHG-Ala-Arg-pNA (a fast chromogenic thrombin substrate from Pentapharm, Basel, Switzerland) in 1.25 mol/l arginine, pH 8.7, in polystyrene half area wells (Greiner, Frickenhausen, Germany; article nr. 675101) for 3 h at 378C. The 5.5 mIU/ml thrombin standard was pooled normal EDTA-plasma, stabilized with 1.25 mol/l arginine [23]. To measure VEGF levels, 10 ml of arginine-stabilized samples were assayed in the R&D Chemiluminescence Elisa Quantiglo-VEGF kit (article nr: QVE00B) with 400 mmol/l arginine, pH 8.7, in the first incubation period with the capture antibody.

Sample collection and preparation

In regard to the study group, a limited core pars plana vitrectomy (cppV) was performed in all patients with recent onset of BRVO as well as CRVO and a consecutive BRB breakdown [5] using a single-site 23-gauge vitrector (Intrector; Insight Instruments, Stuart, Florida, USA),

Statistical analysis

Statistical analysis was performed using SPSS 14.0 for windows (IBM, Ehningen, Germany), tables and figures were designed using Office Word 2007, Excel 2007 (Microsoft), and SigmaPlot 12.0 (Systat GmbH, Erkrath,

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656 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 7

Results Overall, 41 eyes were included into our investigation, thereof 24 eyes (58%) from female and 17 eyes (42%) from male participants, respectively. In the BRVO group (n ¼ 19), mean patient age was 67  13 years (range: 41–86 years), and intravitreal thrombin activity exhibited to be 1.6  1.2 mIU/ml (mean value  SD; range: 0.2– 4.2 mIU/ml). Members of the CRVO group (n ¼ 13) were 69  15 years (range: 40–89 years), and intravitreal thrombin activities were detected at levels of 2.6  1.2 mIU/ml (range: 0.8–5.2 mIU/ml). Attendees‘ age’ in the control group (n ¼ 9) was 58  15 years (range: 33–78 years), and intravitreal thrombin activity revealed to be 0.8  0.8 mIU/ml (range: 0.2–2.7 mIU/ml). Although patients in the control group were younger than those in the BRVO and CRVO group, no significant age differences exist between the three groups (P ¼ 0.193, Kruskal–Wallis test). There is a significant difference of intravitreal thrombin activity between eyes with BRVO, CRVO, and controls (P ¼ 0.007, Kruskal–Wallis test) (Fig. 1). Intravitreal VEGF levels were 554  568 pg/ ml (range: 20–2005 pg/ml) in eyes with BRVO, 1332  1350 pg/ml (range: 58–3943pg/ml) in CRVOaffected eyes, and 115  120 pg/ml (range: 32–431 pg/ ml) in controls. There is a significant difference of intravitreal VEGF levels between eyes with BRVO, CRVO, and controls (P ¼ 0.003, Kruskal–Wallis test) (Fig. 2). Intravitreal thrombin activity is significantly correlated with intravitreal VEGF levels (r ¼ 0656; P < 0.001, Pearson correlation) (Fig. 3).

Fig. 2 5000

VEGF concentration [pg/ml]

Germany). For testing statistical significance, Kruskal– Wallis test was conducted. The association between intravitreal thrombin activity and intravitreal VEGF levels was calculated by means of Pearson correlation. Significant results were assumed if P values were < 0.05.

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Intravitreal VEGF concentrations differ significantly between eyes with CRVO (1332  1350 pg/ml), BRVO (  568 pg/ml), and controls (115  120 pg/ml) (P ¼ 0.003, Kruskal–Wallis test). BRVO, branch retinal vein occlusion; CRVO, central retinal vein occlusion; VEGF, vascular endothelial growth factor.

Discussion Sampling vitreous specimens

In our series, vitreous specimen was collected using Intrector technology to perform a cppV in patients with BRVO and CRVO as well as a ppV to collect vitreous samples in the control group. No adverse events or serious side-effects occurred, and sampling vitreous fluid with either technique revealed to be a safe procedure. When collecting vitreous samples, it is important to aspirate the specimen at the same intravitreal location. Particular attention is needed in regard to the stabilization regimen of extracted vitreous fluid to avoid artificial thrombin generation as well. Both aspects were recently described and apply to this investigation as well [19]. Fig. 3

Fig. 1

6

6 P = 0.007

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Thrombin activity [mIU/ml]

P = 0.003 4000

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r = 0.656; P < 0.001 0

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Intravitreal thrombin activity differs significantly between eyes with CRVO (2.6  1.2 mIU/ml), BRVO (1.6  1.2 mIU/ml), and controls (0.8/ml  0.8 mIU/ml) (P ¼ 0.007, Kruskal–Wallis test). BRVO, branch retinal vein occlusion; CRVO, central retinal vein occlusion.

Correlation of intravitreal thrombin activity (mIU/ml) and intravitreal VEGF concentrations (pg/ml) (r ¼ 0.656; P < 0.001, Pearson correlation). VEGF, vascular endothelial growth factor.

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Intravitreal thrombin in retinal vein occlusion Bertelmann et al. 657

Retinal vein occlusion, intravitreal thrombin, and vascular endothelial growth factor

In accordance with the Virchow‘s triad, RVO develops due to hemodynamic alterations (venous stasis) and changes of the vessel walls (endothelial cell damage due to compression and/or turbulent blood flow), thus initiating the coagulation cascade and its critical player, thrombin [24]. RVO can cause retinal ischemia and increases hydrostatic pressure anterior to the obstruction site, both of which in turn lead to a BRB breakdown, leakage of the retinal vein involved, and macular edema development [25]. Former investigations demonstrated the crucial role of VEGF in respect to these aspects, as VEGF is correlated with the extend of ischemic retinal area and the severity of macular edema in both RVO entities [9–11]. Thus, intravitreal thrombin activity and intravitreal VEGF levels are fundamental players in the complex pathophysiological sequences in RVO-affected eyes. A basal intravitreal thrombin activity was recently reported [19] and was linked to guarantee vascular integrity and to avoid leaky vessels, thus ensuring sealing of the BRB [20,26]. Herein, we demonstrate intravitreal thrombin activity in RVO-affected eyes for the first time, and the activity levels differ significantly between eyes suffering from BRVO or CRVO and healthy controls (Fig. 1). This is an interesting new finding indicating that the extent of retinal tissue involved roughly adheres with intravitreal thrombin activity. There were significant differences in intravitreal VEGF levels between the groups investigated as well (Fig. 2), and these findings herein are in accordance with former reports of intravitreal VEGF levels in RVO-affected eyes [27]. Furthermore, intravitreal thrombin activity and VEGF levels in eyes suffering from RVO correlate, which was so far unknown (Fig. 3). Intravitreal VEGF levels correlate with the severity of BRB breakdown, intravitreal thrombin activity is significantly correlated with intravitreal VEGF levels, and thus intravitreal thrombin activity correlates with the extent of BRB breakdown. Further studies using retinal leakage analysis [28] to prove this interrelation are indicated though. Intravitreal thrombin activity might be a new marker for BRB breakdown, because an intraocular source of thrombin has not been demonstrated so far, and thus thrombin should pass over from the blood stream at the occlusion site due to the severity of BRB damage. Thrombin itself and its interactions with VEGF influences clot formation, the extent of BRB breakdown, macular edema development, and collateral vessel growth. Thrombin and clot formation

Thrombin activity designates the structure of the resulting fibrin clot, which in turn determines the rate of fibrinolysis [29]. Low thrombin results in clots that are prone to fibrinolysis, whereas high thrombin will generate

clots that are relatively resistant to fibrinolysis and prone to further thrombotic action [29]. Furthermore, a high thrombin activity or uncontrolled thrombin generation (prothrombotic role of thrombin) can lead to further thrombotic events [20], whereas low thrombin activity is linked to a decreased risk of thrombosis development and may prevent the development of the latter downstream of the injury site [29]. Thus, knowing the local thrombin activity will help to estimate whether the patient is at high risk for a sustainable and long-lasting retinal ischemia. This in turn offers the involved ophthalmologist to schedule further appointments appropriately. Thus, thrombin deserves a new marker in ophthalmology for patients with RVO. Former investigations demonstrated that nonischemic RVO can turn into ischemic RVO in up to 34% of affected eyes [30]. The pathophysiological reasons for this conversion are still vague [31]. The level of intravitreal thrombin activity might be a key player in this respect. Further investigations have to explore the levels of intravitreal thrombin activity in eyes turning from nonischemic to ischemic RVO and compare these levels with nonischemic and ischemic-typed RVO. Thrombin, blood–retina barrier breakdown, and macular edema development

Macular edema is a consequence of BRB breakdown and exhibits a direct and sustainable impact on visual acuity in eyes suffering from each RVO entity [27]. The intravitreal detection of VEGF and its correlation with CMT and VA development has revolutionized the treatment options for RVO-affected eyes [9,32,33]. VEGF is an excellent marker for BRB breakdown [10,32,34]. Intravitreally injected anti-VEGF or corticosteroids stabilize the BRB and are to date the standard treatment for RVOdependent macular edema. This highlights the need for further exploration of intraocular fluid to learn more about the pathophysiological sequences within the eye as well as to establish new treatment substances and/or strategies. Intravitreal thrombin is significantly elevated, and thus may play an important role in RVO-affected eyes with a consecutive macular edema as well. Thrombin was addressed to guarantee vascular integrity and to avoid leaky vessels, thus ensuring sealing of the BRB [20,26]. Furthermore, intravitreal thrombin activity herein is significantly correlated with intravitreal VEGF levels. This in turn might demonstrate that VEGFinduced BRB damage and leakage is counteracted by thrombin, which stabilizes the BRB (repair mechanism), and thus hampers a passover from more plasma enzymes and cytokines into the vitreous body [20,26], which in turn would increase the severity of macular edema, and might lead to more severe damage of the retinal structure due to (serin-)protease activity. Thrombin and collateral vessel growth

Thrombin plays an essential role in mature blood vessel growth (angiogenesis) as demonstrated in occlusive and

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658 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 7

ischemic cardiovascular diseases [35]. Thrombin initiates collateral vessel growth with vascular stability in a dose– dependent manner that can provide sufficient blood flow to the ischemic tissue [35]. The detection of intravitreal thrombin activity in RVO-affected eyes herein is an interesting finding, as it might demonstrate the importance of elevated intravitreal thrombin as one repair mechanism after thromboembolic damage of the retinal vasculature to constitute mature collateral vessels that help to provide a sufficient blood supply to the infarcted retinal tissue. Thrombin has been injected intravitreally in former studies to prevent intraocular bleeding during vitrectomy, and no adverse effects occurred [36–38]. This might offer a new therapeutic option to support RVO-affected eyes in developing collateral vessels for a sufficient blood supply. Furthermore, thrombin upregulates VEGF, the most important angiogenic factor, and potentiates the mitogenic activity of the latter on endothelial cells due to an increased VEGFR-2 expression [35]. In contrast to thrombin-generated collaterals, VEGF promotes neovascularization development, which in turn are immature and leaky, and can lead to devastating damage of the eye. Thus, thrombin in this respect might act as a VEGF antagonist. This could be of importance, because neovascularizations can lead to major intravitreal bleedings and tractional retinal detachments. In summary, the shortcoming of our investigation is the limited number of vitreal samples analyzed, which in turn makes correlations of intravitreal thrombin activity with the severity of macular edema, the extend of BRB breakdown (‘leaky eyes’), and the area of involved retinal tissue impossible. These aspects will be the scope of further research projects to evaluate if the degree of retinal damage can be predicted by the detection of intravitreal thrombin activity. However, the strength of our study is the first detection and quantification of intravitreal thrombin activity in eyes suffering from BRVO and CRVO. Intravitreal thrombin activity can serve as a new marker for BRB breakdown. This in turn might offer new therapeutic strategies for RVO-affected eyes. Oral thrombin inhibitors, such as dagibatran, are available today [26,39], and their effect on RVO-affected eyes should be addressed in further research projects. They might have the potential to reduce intravitreal thrombin activity, and thus might help to prevent the development of and the conversion into ischemic-typed RVO. Intravitreally injected argobatran, a specific thrombin inhibitor, shown to inhibit intraocular fibrin formation after vitrectomy [40]. If the latter is of benefit for eyes suffering from RVO needs to be addressed in further investigations as well. Intravitreally applied thrombin [36–38] might help to reestablish an intact BRB. The impact of intravitreal thrombin in RVO-affected eyes might be like a double-edged sword. On the one

hand, high intravitreal thrombin activities are needed to stabilize the fragile BRB, and thus diminish macular edema development, which causes a decline in visual acuity. On the other hand, high intravitreal thrombin activities might assist in the development of and the progression into ischemic RVO.

Acknowledgements Source of funding: This research project was granted by Novartis Pharma AG, Nuremberg, Germany. Conflicts of interest

There are no conflicts of interest.

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Intravitreal thrombin activity is elevated in retinal vein occlusion.

To evaluate whether intravitreal thrombin activity is elevated in eyes with branch retinal vein occlusion (BRVO) and central retinal vein occlusion (C...
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