A Case of Recurrent Bilateral Central Retinal Vein Occlusion in an Otherwise Healthy Adolescent Male

Introduction: Central retinal vein occlusion (CRVO) is a rare finding in children. Well-known risk factors include hypertension and diabetes in older patients. CRVO has also been attributed to local causes such as compression or inflammation secondary to trauma, as well as systemic causes, such as thrombophilia or hyperviscosity etiologies. Here, we describe a case of recurrent bilateral CRVO in an otherwise healthy child. Design/Methods: This is a retrospective case report reviewing a patient with recurrent bilateral CRVO. Ophthalmological and pediatric records were reviewed. Results: A 10-year-old previously healthy male presented with sudden onset blurry vision of his left eye and was found to have CRVO with venous engorgement of the retina. He had a grandfather with polycythemia vera, but no other contributing family history. Initial coagulopathy workup was normal with a PTT of 29 sec, PT INR of 1.1 sec, fibrinogen of 291 mg/dL, Factor VIII of 174%, Von Willebrand Factor Antigen of 139%, Von Willebrand Factor Activity of 132%, Factor IX of 101%, and Factor XI of 112%. Computed tomography (CT) of the head was unremarkable. At the age of 13, he developed acute onset headache with blurry vision of his right eye. He had evidence of macular edema and CRVO of his right eye. Ophthalmology treated him three injections of vascular endothelial growth factor inhibitor, aflibercept, over four months with improvement in both macular edema and visual acuity. Daily aspirin prophylaxis was also initiated pending further hypercoagulability workup. Laboratory investigation for an inherited or acquired hypercoagulability including prothrombin mutation, AT III mutation, hyperhomocysteinemia, protein C & S, and antiphospholipid were negative. He initially had a positive cryoglobulin found to be negative on repeat evaluation. He underwent a comprehensive rheumatological and infectious workup that was unrevealing. Additionally, he had an elevated RBC of 6.09 mill/cmm, hemoglobin of 18.2 g/dL, and hematocrit of 52.0% but a targeted panel for inherited erythrocytosis and polycythemia was normal. The patient remained symptomatic with persistent episodes of blurred vision and lightheadedness. Further extensive hematologic workup revealed reduced thrombin inhibitor and elevated rates of thrombin formation. Lipoprotein (a) was 168 mg/dL. Interestingly, his mother had elevated rates of thrombin formation and a lipoprotein (a) level of 153 mg/dL. The patient is currently being treated with therapeutic rivaroxaban and whole exome sequencing (WES) for the patient and both parents are pending. Conclusion: This case demonstrates a novel cause of recurrent bilateral CRVO secondary to reduced thrombin inhibition resulting in accelerated rates of thrombin formation and elevated lipoprotein (a) overall supporting a hypercoagulable state. Perhaps, there is a familial component predisposing recurrent thrombosis in this patient. Whole exome sequencing is pending to evaluate for a genetic predisposition to his findings. After completing a full course of therapeutic anticoagulation, he will likely require lifelong prophylaxis to reduce the risk of future thrombotic events and preserve his vision. Disclosures No relevant conflicts of interest to declare.

Sphingosine-1-phosphate receptors modulate platelet activation and thrombin formation

Background The bioactive lipid sphingosine-1-phosphate (S1P) is known as a modulator of various cell functions, including cell proliferation, migration, adhesion and survival. Hence, S1P plays a crucial role in both inflammatory and hematopoietic regulations. It is also known that S1P affects key processes of hemostasis, as platelets store and upon activation release large amounts of the phospholipid. S1P acts through binding to its five G-coupled receptors (S1PR1–5). Accumulating evidence suggests that S1P is involved in mechanisms of atherogenesis linking platelet activation and thrombus formation to inflammatory responses. However, the precise role of S1P and its receptors during platelet activation and hemostasis are still under debate. Purpose Until today, the effects of S1P in hemostasis are poorly understood. The aim of this study is to investigate the functions of specific agonists and antagonists for each of the S1PRs 1–5 on platelet aggregation and thrombin generation. Methods Blood was obtained from healthy volunteers after written informed consent. For both platelet aggregation studies using light transmission aggregometry (LTA) and thrombin generation measurements quantified by calibrated automated thrombography (CAT), platelet-rich-plasma (PRP) was used. PRP was generated by centrifugation from whole blood. PRP was incubated with either 10 μM S1PR-agonists or -antagonists for 15 minutes. Subsequently, platelets were activated with adenosine diphosphate (ADP) or collagen to evaluate potential synergistic or antagonistic effects. Results Platelet aggregation weakens after 2–3 hours. As described in the literature, this is presumably due to activation effects and partial release of platelet granules. Determined by LTA, we found that pre-incubation with the S1PR1-agonist CYM5442 maintains and even elevates ADP- but not collagen-induced platelet aggregation for up to 1.5 h. This effect was reversed by co-incubation with the S1PR1-antagonist Ex26. We only observed this findings for S1PR1. No effect on platelet aggregation was detected when PRP was incubated with S1PR1–5-agonists or -antagonists (10 μM) alone. In comparison, none of the S1PR-agonists or -antagonists did directly affect thrombin formation in PRP measured by CAT. However, co-incubation with a S1PR4-agonist and ADP (5 μM) shortened the lag time to thrombin formation significantly. Furthermore, co-incubation with collagen (5 μg/mL) and agonists for S1PR1, 3 or 4 did reduce the time-to-peak (ttpeak) of thrombin formation. Conclusion Activation of S1PR1 maintains the ADP-induced platelet aggregation. In addition, co-activation of S1PR1, 3 or 4 and collagen enhances and accelerates thrombin generation. Consequently, S1P and its receptors appear to play an important role in different processes of hemostasis. Thus, S1PRs may be promising future therapeutic targets for diseases involving elevated platelet activation such as coronary heart disease or diabetes. FUNDunding Acknowledgement Type of funding sources: None.

Sulfated agaran with 4,6-pyruvated form from red seaweed Acanthophora muscoides attenuates thrombin formation: in vitro and ex vivo studies

In vitro studies have described the sulfated agaran from Acanthophora muscoides as an intrinsic inhibitor of thrombin generation (TG), but not in ex vivo assay. This investigation partially characterized a pyruvate fraction with in vitro and ex vivo effects on an intrinsic/extrinsic pathway-induced thrombin generation (TG) continuous model using 36 or 60-fold diluted mice or defibrinated, normal human plasma. Fraction separated by DEAE-cellulose chromatography exhibited charge homogeneity and non-sulfated polysaccharides (<100 kDa) by agarose and polyacrylamide gel electrophoresis, respectively, using Stains-all alone. Fourier Transform Infrared and Nuclear Magnetic Resonance studies indicated a 4,6-pyruvated agaran-structure. The fraction and heparin had no effect on prothrombin time, but there was a preponderant intrinsic rather than extrinsic pathway inhibition in TG assay; themselves, acting on both free and fibrin bound thrombin activity without chromogenic substrate interaction. Both fractions, desulfated and native, anticipated and induced thrombin formation in activators-devoid or normal plasma. In addition, mice pretreated with fraction (20 mg kg-1, intraperitoneally) reduced intrinsically plasma TG ex vivo after 2h. Heparin suppressed TG in vitro, but induced it ex vivo. Therefore, agaran from A. muscoides blocks TG on in vitro and ex vivo studies, suggesting to evaluate the blood coagulability status.

Chromogenic anti-Xa test: the ratio between heparin activity units and concentration of apixaban and rivaroxaban

Introduction. The direct oral anticoagulants (DOC) therapy does not require alaboratory control; however, it may be required to determine the anticoagulationlevel to choose a treatment strategy if alarge bleeding is developing or emergency surgery is needed.The objective of this experimental study was to investigate the relationship between the residual factor Xa (FXa) activity, anti-Xa activity units oflow molecular weight heparins (LMWH), and the apixaban and rivaroxaban plasma concentrations in a chromogenic anti-Xa assay.Material and methods. Concentrated DOC solutions were prepared by extracting apixaban and rivaroxaban from crushed tablets using methanol and dimethyl sulfoxide, respectively. The resulting solutions were added to the donor plasma pool until final inhibitor concentrations are achieved in the range from 10 to 100 ng/ml plasma. Anti-Xa activity was determined using an STA-compact analyser and the Liquid anti-Xa reagent kit, an analysis protocol, and calibrators designed to control the LMWH therapy. The effect on the thrombin formation dynamics was investigated using the thrombin generation test (TGT) and the PPR reagent as a trigger (final concentrations of tissue factor are 5 pM, and those of phospholipids are 4 μM). TGT curves were analysed using the Thrombinoscope program.Results. It was shown that in the anti-Xa activity test version designed to control the LMWH therapy, there is a high correlation (R2 > 0.98) between thelogarithm of the residual factor Xa activity and the content of apixaban and rivaroxaban in the range from 10 to 80 ng/ml. Rivaroxaban shows about 1.5 times more anti-Xa activity than apixaban at equal concentrations. It was also shown that apixaban and rivaroxaban at doses equal both in concentration and in anti-Xa activity differ in their effect on the thrombin formation dynamics and thrombin inactivation in the TGT.Conclusion. In the LMWH anti-Xa activity test version, the measured range of apixaban and rivaroxaban includes 30 ng/ml and 50 ng/ ml concentrations taken as “cut-off points” to determine the treatment tactics in emergency cases. However, thelack of certified DOC calibratorslimits the use of this test in clinical practice.

Menstrual Phase Affects Coagulation and Hematological Parameters during Central Hypovolemia

Background: It has been reported that women have a higher number of heart attacks in the “follicular phase” of the menstrual cycle. We, therefore, tested the hypothesis that women in the follicular phase exhibit higher coagulability. As lower body negative pressure (LBNP) has been used previously to assess coagulation changes in whole blood (WB) samples in men and women, effects of menstrual phase on coagulation was assessed during LBNP. Methods: Seven women, all healthy young participants, with no histories of thrombotic disorders and not on medications, were tested in two phases of the menstrual cycle (early follicular (EF) and mid-luteal (ML)). LBNP was commenced at −10 mmHg and increased by −10 mmHg every 5 min until a maximum of −40 mmHg. Recovery up to 10 min was also monitored. Blood samples were collected at baseline, at end of LBNP, and at end of recovery. Hemostatic profiling included comparing the effects of LBNP on coagulation values in both phases of the menstrual cycle using standard coagulation tests, calibrated automated thrombogram, thrombelastometry, impedance aggregometry, and markers of thrombin formation. Results: LBNP led to coagulation activation determined in both plasma and WB samples. During both phases, coagulation was affected during LBNP, as reflected in their decreased partial thromboplastin time (PTT) and elevated coagulation factor VIII FVIII, F1 + 2, and thrombin-antithrombin (TAT) levels. Additionally, during the ML phase, greater PT [%] and shorter time to peak (ttPeak) values (implying faster maximum thrombin formation) suggest that women in the ML phase are relatively hypercoagulable compared to the early follicular phase. Conclusions: These results suggest that thrombosis occurs more during the midluteal phase, a finding with substantial medical implications.