Potential Mechanism of Platelet GPIIb/IIIa and Fibrinogen on Retinal Vein Occlusion

Abstract Purpose To explore the role of coagulation and fibrinolytic factors, and the potential mechanism of platelet aggregation in the pathogenesis of retinal vein occlusion. Methods Coagulation and fibrinolytic parameters in patients with retinal vein occlusion were determined using hemagglutinin and HISCL-5000. Relationships between these elevated parameters and factors representing typical clinical manifestations of retinal vein occlusion were examined, and these parameters were analyzed using a STRING database to indicate the potential role of platelet aggregation. Platelet glycoprotein IIb/IIIa (GPIIb/IIIa) levels were evaluated by flow cytometry after antiplatelet treatment in patients and mouse models. Furthermore, the GPIIb/IIIa ligand fibrinogen in peripheral blood and retina of mouse models was assessed by the turbidimetric method and real-time PCR, respectively. Results In patients, significant increases in peripheral blood fibrinogen and GPIIb/IIIa levels were observed (p = 0.0040, p < 0.0001, respectively). A positive correlation was observed between macular thickness (MT) and both fibrinogen and GPIIb/IIIa (r = 0.4528, p = 0.0063; r = 0.3789, p = 0.0427, respectively). After intravitreal injections of anti-vascular endothelial growth factor drugs, a significant reduction in fibrinogen levels was observed (p = 0.0072). In addition, the use of antiplatelet drugs resulted in a significant decrease in GPIIb/IIIa (p < 0.0001). In a mouse model, antiplatelet therapy significantly reduced both peripheral blood and retina fibrinogen levels and the overall rate of vein occlusion 3 days after occlusion (p < 0.0005). In addition, the reduction in GPIIb/IIIa levels after antiplatelet therapy was remarkable. Conclusion Fibrinogen and GPIIb/IIIa may be involved in retinal vein occlusion and blocking platelet aggregation may be a new therapeutic approach for retinal vein occlusion.


Introduction
Retinal vein occlusion (RVO) is the second most common ocular vascular disease in elderly patients, after diabetic retinopathy. 1 RVO can be divided into two major types based on the location of the occlusion: central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO).Both BRVO and CRVO can lead to severe visual impairment due to macular edema and retinal neovascularization. 2 Although the etiology of RVO has not been fully elucidated, it is believed to be multifactorial and dependent on abnormal mechanisms of thromboresistance.
Thrombosis is a disease with an unclear incidence, slow onset, high mortality, and high disability rate that has been observed on histopathological examination of patients with CRVO. 3 The pathology involves three main processes: vascular endothelial damage, coagulation, and fibrinolysis.Research has confirmed that these three major systems are altered when the body is in the prethrombotic state.The combination of these three systemic changes is known as Virchow's triad (venous stasis, endothelial injury, and a hypercoagulable state). 4The latter two factors interact and influence each other, leading to thrombosis.Although it is unclear whether thrombosis is primary or secondary, once it occurs, it clearly leads to or exacerbates blood flow obstruction, resulting in retinal vascular sequelae. 5When the body is in a prethrombotic state, coagulation and fibrinolysis indicators are significantly abnormal, and patients with RVO are often at high risk for thrombosis, especially those with CRVO, who are often at higher risk for serious cardiovascular and cerebrovascular diseases.Atherosclerosis and hypertension are systemic factors that lead to RVO at arteriovenous crossing . 6Atherosclerosis and hypertension can cause the arterial wall to become stiff and lose elasticity.Narrow arteries compress the veins, causing narrowing of the venous lumen and slowing blood flow velocity. 7In addition, venous compression can cause endothelial damage to the venous vessels, leading to increased local platelet aggregation and further exacerbating thrombosis.
Platelet activation and aggregation play an important role in thrombosis.Although some studies have found that platelet monitoring parameters, such as mean platelet volume, platelet distribution width, and platelet large cell ratio are increased in patients with RVO, [8][9][10] and platelet-derived microvesicles promote RVO disease progression, 11 few studies have clarified the role of platelets in RVO disease.
Although coagulation, fibrinolysis, and platelets are all involved in the process of thrombosis and are closely related to the pathogenesis of RVO, it remains unclear which mechanism is most important in the pathogenesis of RVO.In addition, no recent reports have observed the relationship between these coagulation and fibrinolysis indicators and the progression of RVO disease, such as the degree of macular edema, arm retinal circulation time (ARCT), and best corrected visual acuity (BCVA).Furthermore, due to the complexity of the pathogenesis of RVO, the current clinical treatment methods for this disease are limited and most of them focus on the complications of the disease, while the etiology of the disease cannot be completely alleviated.Therefore, it remains unclear whether the degree of coagulation and fibrinolysis parameters of peripheral blood is higher in eyes with BRVO or CRVO.Hence, in this study, the degree of coagulation and fibrinolytic factors from peripheral blood was determined in several groups and the effects of these parameters on the characteristic factors of RVO were compared in order to screen the important influencing factors.In addition, we are trying to find appropriate ways of intervention to further elucidate the possible mechanisms of thrombosis formation both in the mouse model and in RVO patients.

Ethical statement
The study adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Ethics Committee of the First Affiliated Hospital of Soochow University (No. 2020.193).Informed consent was obtained from the participants before their inclusion in the study.All animal experiments were conducted in accordance with the Guideline for the Animal Ethics Committee of Soochow University for Studies on Animals (SUDA20221124A16) and the requirements of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Participant enrollment
After biochemical and physiological examinations (medical history, drug use and other treatment history, routine blood tests, blood glucose tests, blood coagulation function tests) and ophthalmological examinations (fundus photography (FP), optical coherence tomography (OCT), and fundus fluorescence angiography (FFA)), patients with RVO and controls (age-and sex-matched participants for evaluation of eye conditions, mainly cataract) were enrolled at the First Affiliated Hospital of Soochow University from June 2020 to December 2022.Inclusion criteria were as follows: (1) age ≥ 18 years and onset time ≤ 2 weeks; (2) no refractive medium turbidity affecting fundus examination, and the pupil could be dilated ≥ 6 mm, meeting the requirements for thorough fundus examination; (3) meeting the diagnostic criteria for RVO; and (4) signing the informed consent form.Exclusion criteria were as follows: (1) other ocular diseases that would affect the determination of experimental results; (2) conditions that seriously affect blood components, such as leukemia, coronary heart disease, upper respiratory tract infection, obesity, and pregnancy; (3) use of drugs that affect blood composition; and (4) inability to cooperate or mental disorders.Neither hemicentral nor hemispheric eyes with RVO were included in the study.MT: macular thickness.Macular edema is a serious complication of RVO disease that affects vision, and its extent is measured by MT.The early treatment diabetes retinopathy research grid divides the macula into nine parts, uses the algorithm of the OCT device to automatically identify the inner limiting membrane and the retinal pigment epithelium line, and calculates the average macular thickness of nine parts of the macular area to determine a central macular thickness of one millimeter. 12RCT: Arm Retinal Circulation Time, measured from FFA video images, which reflects the time from administration of sodium fluorescein from the antecubital vein to its arrival at the retinal artery. 13BCVA: Best-corrected visual acuity, which is one of the most commonly used factors in the assessment of ocular conditions.It is measured with corrective lenses and requires patients to recognize letters or graphics on a chart at a given distance to assess their ability to distinguish object shapes and details.The decimal acuity on the chart was converted to logMAR for analysis. 14

Assays of hemostatic parameters
Plasma samples were obtained by centrifugation (3,000 × g for 15 min) within 1 h to remove all platelets.Activated partial thromboplastin time (APTT), prothrombin time (PT), international normalized ratio (PT-INR), and fibrinogen (FIB) were measured using a hemagglutinin device (Diagnostica Stago, Asnieres, France).Von Willebrand factor (vWF), thrombomodulin (TM), tissue plasminogen activator inhibitor complex (tPAIC), and plasmin inhibitor complex (PIC) were detected using the HISCL-5000 (Sysmex Corporation, Kobe, Japan).Reagents and calibrators were supplied by the original manufacturer and the entire procedure was performed in strict accordance with the manufacturer's instructions.

Animals and RVO model
Wild-type C57Bl/6J mice (8-week old), weighing 20-25 g, were purchased from Gempharmatech Co., Ltd.(Nanjing, Jiangsu, China) and maintained our animal facility under specific pathogen-free conditions with a 12-h light/dark cycle.To minimize the influence of direct retinal injury by laser burn, we combined laser irradiation with an intravenous injection of rose bengal to induce local vascular endothelial injury and platelet aggregation. 15,16Briefly, mice were randomly assigned to receive tirofiban (0.5 µg/g BW, 5 mg/100 ml) 17 or an equal volume of normal saline (NS) by tail vein injection after laser irradiation, and rose bengal (6 µl/g BW, 5 mg/ml) was administered intravenously 5 min before laser irradiation.Some mice were included in the normal control (NC) group without undergoing any procedures.Immediately before all imaging and RVO procedures, mice were anesthetized with Avertin (0.2 ml/10 g) and their eyes were dilated with tropicamide and phenylephrine chloride eye drops.The major retinal veins (n = 2-3) of each eye were irradiated with VISULAS (532 nm) (Carl Zeiss AG, Oberkochen, Germany).Three adjacent laser pulses (power 100 mW, spot size 50 µm, duration 1 s, total energy 0.3 J) were delivered to each vein, and the average distance from the optic nerve center was 375 µm. 18,19SPECTRALIS HAR (Heidelberg lnc., Germany), SLM-4ER (Kanghuaruiming Science Technology Co., Ltd., Chongqing, China), and RTVueXR100 (Optovue, Inc., USA) were used for ophthalmic examinations.

Hematoxylin-eosin (HE) staining
The model mice were euthanized, and their eyes were collected.The cornea was punctured with a 1-ml syringe, and then, the eyes were fixed in 4% paraformaldehyde for 24 h at 4 °C, 20 dehydrated through a serial gradient of alcohol concentrations, and embedded in paraffin.After paraffin embedding, the slices (4 µm-thick sections) were stained with hematoxylin and eosin, and the staining results were observed under a microscope.

RNA isolation and real-time PCR
Total RNA was extracted from mouse retinas using an Axygen Total RNA Miniprep Kit (TaKaRa Bio, Otsu, Shiga, Japan) and stored at −80 °C.The resulting RNA preparations were pretreated with ribonuclease-free deoxyribonuclease (DNase) I (Corning Inc., Corning, NY, USA) to remove genomic DNA.According to the instructions of the reverse transcription kit (TaKaRa), 500 ng of total RNA (concentration range of 800-900 ng/µl) was reverse transcribed in 10 µl reaction mixture containing reverse transcriptase and hexanucleotide random primers.The program was set at 37 °C for 15 min, 85 °C for 5 s and then lowered to 4 °C to obtain cDNA.The polymerase chain reaction (PCR) solution contained 1.6 µl cDNA, 0.4 µl for each specific primer set (0.2 µM final concentration), and 10 µl of TB green (2 ×) in a final volume of 20 µl.The PCR was performed in duplicate.The sequences of the PCR primer pairs are listed in Table S1.Real-time PCR was performed using the CFX96 Real-Time PCR Detection System (BioRad, Hercules, CA, USA) with SYBR Premix Ex Taq (TaKaRa Bio).The PCR procedure was as follows: initial denaturation at 95 °C for 1 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Relative gene expression levels were calculated using the 2 -ΔΔCt method, and GAPDH was used as a reference.

Flow cytometry
Human peripheral blood platelets were collected in tubes containing sodium citrate anticoagulant.After gentle mixing, 10 µl of each blood sample was transferred into a flow tube.Modified Tyrode's buffer (MTB) was added to a total volume of 50 µl, followed by the addition of 2.5 µl of FITC-labeled PAC-1 (25 µg/ml, diluted 1:100) as a specific marker for activated GPIIb/IIIa.This was kept in the dark for 15 min, and then, MTB was added to a total volume of 300 µl.Approximately 1 ml of blood was collected from the orbit of each anesthetized mouse, and acid-citrate-dextrose (ACD)-anticoagulated whole blood was washed in MTB and centrifuged at 180 × g for 10 min.ACD was added to the top layer and centrifuged at 700 relative centrifugal force for 10 min.MTB was resuspended after the supernatant was discarded.Platelets were stained at 37 °C for 20 min.Finally, 2.5 µl of FITC-labeled GPIIb/IIIa antibody (diluted 1:100) was added and kept for 15 min.All samples were analyzed by flow cytometry (Cytomics FC 500, Beckman Coulter).

Data processing
The protein-protein interaction (PPI) network was analyzed using the STRING database (http://www.string-db.org) and produced using RStudio software (The R Project for Statistical Computing, Vienna, Austria).Results are expressed as mean ± standard deviation.All analyses were conducted using GraphPad Prism 8.0 software (GraphPad, San Diego, CA, USA).For all comparisons, p < 0.05 was considered statistically significant.

Plasma coagulation and fibrinolytic parameters in RVO patients and their correlation with macular thickness (MT), ARCT, BCVA, and anti-VEGF therapy
The demographic characteristics of 63 patients with RVO and 27 controls are shown in Table S2.Among the RVO patients, BRVO accounted for 58.73% (37/63) and CRVO accounted for 41.27% (26/63) of the eyes.The RVO, BRVO and CRVO groups were all age and gender matched with the control group (p > 0.05).
The fibrinolytic parameters of PIC and tPAIC were significantly higher in the RVO group than in the control group (p = 0.0001 and p = 0.0029, respectively) (Figure 1(A-B)).As coagulation parameters, APTT, PT and PT-INR were also significantly lower in the RVO group than in the controls (p = 0.0038, p = 0.0459, p = 0.0464, respectively) (Figure 1(C-E)).These indicators showed a hypercoagulable state in the blood of RVO patients, which was consistent with the FIB levels (p = 0.0040) (Figure 1(F)).Statistical analysis showed that vWF and TM were significantly higher in the RVO group than in the control group (p = 0.0169 and p = 0.0049, respectively), indicating injury to the vascular endothelium (Figure 1(G-H)).In addition, there was no statistically significant difference in thrombin-antithrombin complex (TAT) between the and control groups (Supplementary Figure 1).After dividing the RVO group into BRVO and CRVO subgroups, PIC, tPAIC, FIB, vWF, and TM levels were still increased in the BRVO and CRVO subgroups, whereas APTT, PT, and PT-INR levels were decreased in the BRVO and CRVO subgroups compared to the control group (Figure 1).
As we observed a positive relationship between FIB and MT, we continued to collect new RVO patient data according to the described standards.Of the 75 enrolled patients with RVO, 21 were receiving intravitreal injections (IVT) of anti-VEGF drugs, and 54 were not.No significant differences in age or sex were found between the IVT and non-IVT groups (p = 0.7696 and p = 0.1995, respectively; Table S3).The FIB level decreased significantly (p = 0.0072) after intravitreal injection of anti-VEGF drugs (Figure 3(A)).However, as shown in Figure 3(B-H), there were no significant differences between the two groups in the other parameters (PIC, p = 0.0622; tPAIC, p = 0.7406; TM, p = 0.8381; vWF, p = 0.0794; APTT, p = 0.1718; PT, p = 0.5455; PT-INR, p = 0.2500).

Potential role of GPIIb/IIIa in the process of RVO disease
Although prothrombin (F2) had the highest score (Figure 4(A-B)) after protein-protein interaction network (PPI) analysis of indicators that are significantly increased in RVO patients, other researchers have found no significant correlation between F2 and the risk of retinal vein occlusion disease, whether at age ≤ 50 or > 50 years. 21In the STRING analysis, GPIIb/IIIa (ITGA2B), as a common receptor of vWF, FIB (FGA, FGB and FGG), and VTN, may act as a pivotal regulator of thrombus formation and play an important role in the pathogenesis of RVO (Figure 4(B)).More importantly, GPIIb/IIIa receptors expressed on activated platelets, together with FIB, form the final pathway of platelet aggregation. 22,23gure 1.statistically significant elevated coagulation and fibrinolytic parameters in rVO patients compared with control group.(a-h) the expressed level of PIC, tPaIC, aPtt, Pt, Pt-Inr, FIB, vWF and tM in rVO groups and controls.Independent samples t-test or Mann-Whitney u test was used for statistics.FIB: fibrinogen, PIC: plasmin-alpha2-antiplasmin, tPaIC: tissue plasminogen activator-inhibitor complex, tM: thrombomodulin, vWF: von Willebrand factor, aPtt: activated partial thromboplastin time, Pt: prothrombin time, Pt-Inr: international normalized ratio.

Increased activated GPIIb/IIIa antibody in RVO patients and inhibitory effect of panax notoginseng saponins
To further explore the practical role of activated GPIIb/IIIa, 36 new patients with RVO and 26 controls were enrolled to detect the expression of the activated GPIIb/IIIa biomarker (PAC-1) in peripheral blood (Table S3).These patients were divided according to whether they were taking the antiplatelet aggregation drug Panax notoginseng saponins (PNS).PAC-1 expression was significantly higher in the RVO group without treatment with PNS than in the control group (p < 0.0001).In contrast, after treatment with PNS, PAC-1 decreased significantly (p < 0.0001) in comparion with the RVO group without PNS (Figure 5(A-D)).PAC-1 levels were positively correlated with MT in these patients (r = 0.3789, p = 0.0427) (Figure 5(E)).

GPIIb/IIIa inhibitor tirofiban promotes vascular recanalization in an RVO mouse model
To further elucidate the pathological mechanism of thrombosis in RVO by studying the relationship between GPIIb/IIIa and FIB, we established an RVO model using C57BL/6J mice.  Figure 6(A,B) showed the normal venous sinus structure without venous dilatation or erythrocyte aggregation.Figure 6(C,D) showed a typical thrombus in a laser-treated vein with a vein protrusion filled with red blood cells.Endothelial cell wall structures can be seen in the enlarged images of both groups (blue dye).Using fundus photography and fluorescein angiography, we found that compared with the control group, the proximal vessels in the RVO group were occluded, and the distal vessels were tortuous and dilated, accompanied by subretinal hemorrhages (Figure 6(E-H)).Most of the veins were occluded at the time of laser treatment, decreasing to approximately 80% occlusion 4 h after laser irradiation.The occlusion of most of these vessels lasted for 3 days and completely reopened after 7 days (Figure 6(I)).If these vessels did not reopen after 7 days, laser cauterization injury was indicated.TRF, a specific antagonist of platelet GPIIb/IIIa, reversibly inhibits platelet aggregation by blocking the combination of FIB and activated GPIIb/IIIa receptor. 24Importantly, the rate of vein occlusion in the TRF group rapidly decreased to 16.67% ± 5.77% (Figure 6(J)) on day 3 compared with the NS group, demonstrating the role of TRF in accelerating vascular recanalization.

Reduction in FIB expression after TRF treatment in RVO mouse model
FIB from retro-orbital venous plexus blood was significantly higher in the NS group than in the TRF group at 12 h after laser irradiation (p < 0.0001), and it decreased rapidly in the TRF group at day 7 compared with the NS group (p < 0.0001) (Figure 7(A)).The NC group was the sham laser treatment group that did not receive any treatment.
To further observe changes in FIB levels in local tissues, we extracted mouse retinal tissues for real-time PCR (Table S1).FGA and FGG in the NS group were highly prevalent at 4 h and gradually decreased at day 3 or day 7 compared with the TRF group (p < 0.0001); FGA in the NS group continued to peak at day 7 (Figure 7(B)).

Reduction in GPIIb/IIIa expression after TRF treatment in RVO mouse model
Compared with the NS group at 12 h after laser irradiation, the level of GPIIb/IIIa in the retro-orbital venous plexus blood was lower in the TRF group, but slightly higher than that in the normal group (Figure 8(A-B)).

Discussion
Multiple studies have reported that coagulation and fibrinolytic factors play an important role in retinal vein thrombosis.Bertelmann et al. found increased intravitreal thrombin activity, plasminogen, and PIC in patients with RVO, and these were significantly related to the level of vascular endothelial growth factor (VEGF) in the vitreous humor. 3,66][27] As a key mediator of vascular endothelial injury, the role of vWF in CRVO has been controversial based on previous findings; however, it has been implicated in endothelial cell injury due to its ability to aggregate platelets and mediate their adhesion to the endothelium. 28,29Less is known about TM in RVO, although its mRNA expression has been found to be upregulated in human retinal microvascular endothelial cells (HRMECs). 30TAT, PIC, TM, and tPAIC are effective indicators of early changes in the vascular endothelium, coagulation, and fibrinolysis systems. 31These indicators are applicable for early diagnosis, risk assessment, and evaluation of therapeutic efficacy of thrombosis in high-risk groups in various clinical disciplines, as well as for risk screening for thrombosis in healthy individuals. 32In our study, the levels of PIC and tPAIC were higher in patients with RVO than in controls, indicating the process of thrombosis and activation of the fibrinolytic system.Increased vWF levels indicate endothelial cell injury in patients with RVO.Conventional coagulation tests such as APTT and PT are the most commonly used tests to evaluate endogenous and exogenous coagulation function. 33A decrease in APTT and PT with an increase in FIB levels a hypercoagulable state 34 in patients with RVO.In summary, these results suggest that patients with early stage RVO are in a state of hypercoagulable state, and that fibrinolysis is activated, accompanied by some degree of vascular endothelial damage.
Fibrinogen (FIB), a vital fibrin protein involved in coagulation and hemostasis, plays an important role in retinal vein occlusion. 35Plasma viscosity was significantly correlated with plasma FIB. 36FIB chains (FGA, FGB and FGG) in the aqueous humor are increased in BRVO and correlate with central retinal thickness. 37FIB was also significantly increased in the aqueous humor of patients with CRVO, and the level of the FGA chain was correlated with the severity of macular edema. 38FIB chains are a component of cystoid lesions in macular edema with BRVO after pars plana vitrectomy. 39The significance of the phenomenon that the peripheral blood indicators with relatively small changes in Figure 1 showed a significant correlation with RVO disease characteristic factors is indeed worth further investigation.We speculated that this may be because when there were significant changes in local indicators in the retina, the response in the peripheral blood may have been smaller.FGA knockdown reduced VEGFA levels via VEGFA-VEGFR-FAK signaling and inhibited human umbilical vein endothelial cell (HUVEC) migration and tube formation in vitro and in vivo. 40In our study, FIB was observed to have a high increase in FGA and FGB chains at 4 h in the NS group compared with the TRF group.In contrast, the FGB content in all groups after laser irradiation was too low to be detected in this experiment, which is consistent with a previous finding of pulmonary embolism in patients with deep vein thrombosis. 41Furthermore, FIB binds to the   GPIIb/IIIa receptor.The GPIIb/IIIa receptor has two forms in platelets: active or inactive; only the active form can bind to other ligands, such as vWF, FIB, and VTN. 42The PlA2 allele in GPIIb/IIIa is a risk factor for venous thromboembolism (VTE). 43In our study, we found increased levels of FIB and GPIIb/IIIa in both RVO patients and mouse models.After administration of the antiplatelet aggregation drugs PNS or TRF in RVO patients and mouse models, respectively, the levels of FIB and GPIIb/IIIa decreased.
PNS can reduce the level of PAC-1 in patients with coronary artery disease or chronic gastritis, 44 and has a therapeutic effect on retinal lesions in RVO mouse models. 24TRF is a specific antagonist of platelet GPIIb/IIIa with a half-life of approximately 1.5 h, and has been shown to be effective in reducing ischemic events.It can reversibly inhibit platelet aggregation by blocking the combination of FIB and activated GPIIb/IIIa receptor. 23,45It is widely used in the treatment of acute cardiovascular disease.TRF has been in animal models of deep vein thrombosis labeled with technetium-99. 46,47Currently, clinical treatment of RVO focuses on improving the symptoms of retinal ischemia and hypoxia, such as macular edema and secondary glaucoma. 48,49ew treatments target the cause of the venous obstruction. 50revious studies have found that tPA can effectively reduce MT in patients with RVO to achieve better long-term vision. 51,52However, due to the difficulty of surgery and retinal toxicity of drugs, it has not been widely used. 18herefore, the vascular recanalization effect of TRF via intravenous injection in our study may provide a promising treatment option for RVO by inhibiting platelet aggregation.
As far as we know, platelet aggregation is the most common cause of thrombosis in cardiovascular diseases, which can produce fresh white thrombus with fibrin. 53It is well known that platelets, as immune cells, have multiple functions and are actively involved in inflammation via immune thrombosis or thromboinflammation. 54 A meta-analysis of 1059 patients with RVO investigated a significantly elevated platelet-lymphocyte ratio and neutrophil-lymphocyte ratio and found that there is a cross-talk existed between monocytes, neutrophils and platelets related to inflammation and venous thrombosis. 55More importantly, the pathological sections of human or animal RVO model eyes contain fresh thrombi with platelets, especially in the early stages of RVO. 56,57 eyes with RVO, venous obstruction leads to blood flow disorder, increased venous pressure, and overloaded drainage capacity, which may cause small blood vessels and capillaries to expand, become tortuous, and curl. 58In our mouse model, distally dilated vessels and narrowed proximal veins led to retinal swelling and aggregation of red blood cells in the vessels, compared with normal veins.The degree of retinal swelling and occlusion was most evident 24 h after laser irradiation.Therefore, we removed the eyeball and observed the accumulation of erythrocytes in the dilated vessels.Rose bengal was used in our RVO animal model because it releases active oxygen species when irradiated with green light, leading to vascular endothelial damage, platelet activation, and formation of intraluminal emboli in the irradiated vein. 59here are several limitations in our study, other than those mentioned above.First, we only included patients with early-stage RVO; larger prospective studies are needed, especially for other stages of RVO.Second, the pathological sections were not stained for platelets or examined by electron microscope.Third, an animal model may not fully represent human conditions, and further clinical trials are needed to confirm the function of TRF in RVO.
In conclusion, the results of this study provide evidence that FIB is significantly elevated in both RVO patients and mouse models, while platelet GPIIb/IIIa levels show an increasing trend.Platelets may also be involved in the pathogenesis of RVO.Considering that both FIB and platelets are present in RVO tissue and peripheral blood, the mechanism may be similar to that of promoting arterial thrombosis.These findings expand our understanding of TRF and may help in the development of new treatment options for patients with RVO.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 2 .
Figure 2. Correlations of coagulation and fibrinolytic parameters with Mt, BCVa and arCt in patients with rVO.(a-B) FIB was significantly positive associated with the severity of macular edema in both BrVO and rVO patients.(C) vWF was significantly positively correlated with BCVa (BCVa was LogMar transferred) in BrVO.(D) aPtt was significantly negatively correlated with BCVa (BCVa was LogMar transferred) in CrVO.(e-F) tPaIC was positively associated with arCt in both BrVO and rVO.(G-h) Pt and Pt-Inr were negatively associated with arCt in BrVO.statistical significance was determined by Pearsons correlation test.Mt: macular thickness, arCt: arm retinal circulation time, BCVa: best corrected visual acuity.

Figure 3 .
Figure 3.Comparison of levels of coagulation and fibrinolytic parameters in rVO patients between intravitreal injection and non-injection groups.(a) FIB was significantly decreased after intravitreal injection of anti-VeGF drugs.(B-h) Other parameters (PIC, tPaIC, tM, vWF, aPtt, Pt, Pt-Inr) that were elevated in rVO patients were not significantly changed compared with the non-IVt group,.Independent sample t-test or Mann-Whitney u-test was used to compare parameters.IVt: intravitreal injection.

Figure 4 .
Figure 4. strInG clusters of regulated proteins in rVO patients compared to controls by protein-protein interaction network analysis.(a) analysis conducted using the strInG database (high confidence 0.7).(B) scores of the most significant 18 proteins related to rVO displayed by rstudio software.

Figure 5 .
Figure 5. PaC-1 expression on platelet in peripheral blood of rVO patients.(a-C) Flow cytometry for PaC-1 from peripheral blood of controls and rVO patients.rVO-untreated, without any treatment; rVO-treated, with Pns treatment.(D) PaC-1 level increased in rVO patients (n = 18) compared to control group (n = 26) and significantly decreased after Pns treatment (n = 18).(e) PaC-1 level has a significant positive relationship with severity of macular edema in rVO patients.Data were statistically analyzed by one-way anOVa and Pearson correlation tests (**** p < 0.0001).Pns, Panax notoginseng saponins.

Figure 6 .
Figure 6. he staining of mouse retinal sections and tirofiban reduced the rate of retinal vein occlusion compared with ns group.(a) Control mice without treatment.scale bar: 100 µm.(B) Control mice.normal structure of vein lumen without expansion (blue arrow).scale bar: 20 µm.(C) retinal sections from rose bengal-treated mice 24 h after laser irradiation.scale bar: 100 µm.(D) Mouse rVO model.thrombosis in vein vessel and filling with erythrocytes (yellow arrow).scale bar: 20 µm.n = 3-4 mice/group; the experiment was repeated three times.(e-F) Fundus photography and FFa of control mice.(G-h) Fundus photography and FFa of rVO model showed distal vascular tortuosity and proximal vascular occlusion.accompanied by subretinal hemorrhage.(I) retinal fundus imaging or fluorescence angiography photos at 0 h, 4 h, 3 d, and 7 days post-rVO in ns (upper left) or trF group (lower left) from three independent replicates.ns or trF was injected into the tail vein 10 min after laser irradiation.retinal occlusion and distal vasodilation (green arrow) were seen on fundus imaging.(J) Vein occlusion fractions at 4 h, 3 d and 7 d post-rVO in trF (n = 8) and ns groups (n = 10).each mouse had 2-3 retinal veins irradiated.Chi-squared test for comparison.*** p < 0.0005 vs. ns group.FFa: fundus fluorescein angiography.trF: tirofiban, ns: normal saline.

Figure 7 .
Figure 7. trF induced the decrease of fibrinogen in the rVO mouse model.(a) the levels of plasmatic fibrinogen from peripheral blood at 30-min, 4-h, 12-h and 7-d post-rVO were shown (n = 6, 6, 9, 9 mice/time point in each group).(B) the levels of fibrinogen chains at 4 h, 3 d, or 7 d post-rVO (n = 15, 15, 15 mice/time point in each group) in the retinal tissue of mice.each value is presented as mean ± seM of three independent experiments.One-way anOVa was used for statistical analysis.**** p < 0.0001 between ns group and trF group at 12 h and 7d after laser irradiation, respectively.**** p < 0.0001 between ns group and trF group at 4 h or 7 d post-rVO, respectively.** p < 0.001between ns group and trF group at 3 d post-rVO.
This work was supported by the National Natural Science Foundation of China [81970830], Jiangsu Provincial Medical Innovation Team [CXTDA2017039]; and the Suzhou Municipal Natural Science Foundation [SKJY2021056].

Figure 8 .
Figure 8. trF induced the decrease of GPIIb/IIIa at 12 h post-rVO in mouse model (a) Flow cytometry for GPIIb/IIIa from peripheral blood of three groups of mice.nC group was not treated.(B) GPIIb/IIIa level increased in ns group compared with nC group and decreased after trF treatment.n = 15, the experiment was repeated three times.One-way anOVa was used for statistical analysis.trF: tirofiban, ns: normal saline.