Potential value of the calibrated automated thrombogram in patients after a cerebral venous sinus thrombosis; an exploratory study

Background Cerebral venous sinus thrombosis (CVST) is a relatively rare, but potentially lethal condition. In approximately 15% of the patients, the cause of CVST remains unclear. Conventional clotting tests such as prothrombin time and activated partial thromboplastin time are not sensitive enough to detect prothrombotic conditions nor mild haemostatic abnormalities. The calibrated automated thrombogram (CAT) is a physiological function test that might be able to detect minor aberrations in haemostasis. Therefore, we aimed to detect the presence of a prothrombotic state in patients who endured idiopathic CVST with the CAT assay. Methods Five adult patients with an idiopathic, radiologically proven CVST that had been admitted during the past 3 years were included in this study. The control group consisted of five age/gender matched healthy volunteers. Exclusion criteria were known haematological disorders, malignancy (current/past) or hormonal and anticoagulant therapy recipients. We obtained venous blood samples from all participants following cessation of anticoagulation. Using the CAT assay, we determined lag time, normalized endogenous thrombin potential (ETP), ETP reduction and normalized peak height. In addition, prothrombin concentrations were determined. Results We found no significant differences in lag time (4.7 min [4.5–4.9] vs 5.3 min [3.7–5.7], p = 0.691), normalized ETP (142% [124–148] vs 124% [88–138], p = 0.222), ETP reduction (29% [26–35] vs 28% [24–58], p > 0.999), and normalized peak height (155% [153–175] vs 137 [94–154], p = 0.056) between patients and their age/gender matched controls. In addition, prothrombin concentrations did not significantly differ between patients and controls (120% [105–132] vs 127% [87–139], p > 0.999). Conclusion Reasons for absent overt hypercoagulability within this study population may be the small patient sample, long time since the event (e.g. 3 years) and avoidance of acquired risk factors like oral contraception. Given the fact that CVST is a serious condition with a more than negligible risk of venous thrombosis event recurrence, exclusion of clinically relevant hypercoagulability remains a challenging topic to further study at the acute and later time points, particularly in patients with idiopathic CVST.

Potential Value of the Calibrated Automated Thrombogram in Patients After a Cerebral Venous Sinus Thrombosis; an Exploratory Study

Background: Cerebral venous sinus thrombosis (CVST) is a relatively rare, but potentially lethal condition. In approximately 15% of the patients, the cause of CVST remains unclear. Conventional clotting tests such as prothrombin time and activated partial thromboplastin time are not sensitive enough to detect prothrombotic conditions nor mild haemostatic abnormalities. The calibrated automated thrombogram (CAT) is a physiological function test that might be able to detect minor aberrations in haemostasis. Therefore, we aimed to detect the presence of a prothrombotic state in patients who endured idiopathic CVST with the CAT assay. Methods: Adult patients with an idiopathic, radiologically proven CVST that had been admitted during the past 3 years were included in this study. The control group consisted of age/gender matched healthy volunteers. Exclusion criteria were known haematological disorders, malignancy (current/past) or hormonal and anticoagulant therapy recipients. We obtained venous blood samples from all participants following cessation of anticoagulation. Using the CAT assay, we determined lag time, normalized endogenous thrombin potential (ETP), ETP reduction and normalized peak height. In addition, prothrombin concentrations were determined. Results: We found no significant differences in lag time (4.7 min [4.5-4.9] vs 5.3 min [3.7-5.7], p = 0.691), normalized ETP (142% [124-148] vs 124% [88-138], p = 0.222), ETP reduction (29% [26-35] vs 28% [24-58], p >0.999), and normalized peak height (155% [153-175] vs 137 [94-154], p = 0.056) between patients and their age/gender matched controls. In addition, prothrombin concentrations did not significantly differ between patients and controls (120% [105-132] vs 127% [87-139], p>0.999.Conclusion: Reasons for absent overt hypercoagulability within this study population may be the small patient sample, long time since the event (e.g. 3 years) and avoidance of acquired risk factors like oral contraception. Given the fact that CVST is a serious condition with a more than negligible risk of venous thrombosis event recurrence, exclusion of clinically relevant hypercoagulability remains a challenging topic to further study at the acute and later time points, particularly in patients with idiopathic CVST.

Protective Effect of Quercetin Nanoemulsion on 5-Fluorouracil-Induced Oral Mucositis in Mice

The target of this study was to evaluate the efficacy, histopathological, oxidative stress, and molecular effects of quercetin (QRC) in mice with oral mucositis induced by 5-fluorouracil (5-FU). Thirty-six albino male mice with oral mucositis induced by 5-FU as a chemotherapeutic agent were used in this study. The animals were randomly divided into 6 groups: control group, mucositis (MUC) group, pretreatment group, posttreatment group, and two last groups including nanoemulsion form of QRC with a dose of 5 mg/kg in both pretreatment and posttreatment. In the present evaluation, fewer oral lesions were observed in the QRC posttreatment groups compared to the pretreatment and nanoemulsion receiving groups. In the SOD assay, the most significant difference was observed in the posttreatment nanogroup (41.073 ± 1.24) and pretreatment nanogroup (43.453 ± 2.60) in comparison to the 5-FU group (30.897 ± 1.93). The results of CAT assay also showed a significant difference in nano-posttreatment (124.60 ± 10.85), posttreatment (135.4 ± 9.82), and nano-pretreatment groups (128.80 ± 7.20) compared to the 5-FU group (55.07 ± 8.91). The expression of inflammatory genes such as Hif-1α and NfκB in this group was lower than in the other groups, although this difference was not significant. It seems that the use of QRC can improve the treatment process of oral mucositis induced by 5-FU.

Validation of the Role of Thrombin Generation Potential by a Fully Automated System in the Identification of Breast Cancer Patients at High Risk of Disease Recurrence

Background The measurement of thrombin generation (TG) potential by the calibrated automated thrombogram (CAT) assay provides a strong contribution in identifying patients at high risk of early disease recurrence (E-DR). However, CAT assay still needs standardization and clinical validation. Objective In this study, we aimed to validate the role of TG for E-DR prediction by means of the fully automated ST Genesia system. Methods A prospective cohort of 522 patients from the HYPERCAN study with newly diagnosed resected high-risk breast cancer was included. Fifty-two healthy women acted as controls. Plasma samples were tested for protein C, free-protein S, and TG by ST Genesia by using the STG-ThromboScreen reagent with and without thrombomodulin (TM). Results In the absence of TM, patients showed significantly higher peak and ETP compared with controls. In the presence of TM, significantly lower inhibition of ETP and Peak were observed in patients compared with controls. E-DR occurred in 28 patients; these patients had significantly higher peak and endogenous thrombin potential (ETP) in the absence of TM compared with disease-free patients. Multivariable analysis identified mastectomy, luminal B HER2-neg, triple negative subtypes, and ETP as independent risk factors for E-DR. These variables were combined to generate a risk assessment score, able to stratify patients in three-risk categories. The E-DR rates were 0, 4.7, and 13.5% in the low-, intermediate-, and high-risk categories (hazard ratio = 8.7; p < 0.05, low vs. high risk). Conclusion Our data validate the ETP parameter with a fully automated standardized system and confirm its significant contribution in identifying high-risk early breast cancer at risk for E-DR during chemotherapy.

EVALUATION OF ANTIOXIDANT ACTIVITY OF DIFFERENT EXTRACTS OF MEDICINAL PLANTS

Objective: The present study was carried out to evaluate the antioxidant activity of different extracts of two medicinal plants. Methods: In-vitro antioxidant activity of different extracts of Hydrocotyle javanica Thunb and Peristrophe bicalyculata (Retz.) Nees were determined by following methods such as Lipid peroxide assay (LPO), nitric oxide assay (NO), glutathione (GSH), catalase (CAT). Results: Among eight different extracts, n-Hexane H. javanica extract and alcoholic P. bicalyculata extract showed more potent in vitro antioxidant activity in terms of LPO, NO, GSH, SOD, and CAT assay. Conclusion: From the results, it can be concluded that n-Hexane H. javanica extract and alcoholic P. bicalyculata extract showed more potent in vitro antioxidant activity.

Variation in Phytochemical Constituents and Antioxidant Potential of Extracts Derived from Leaves of Sansevieria trifasciata var. Laurentii and Sansevieria trifasciata var. Zeylanica (Asparagaceae)

Sansevieria is an ornamental plant that has many hybrids and varieties make them difficult to distinguish. The most common varieties used for medicinal purposes are Sansevieria trifasciatam which is known for cure of many diseases. However, little attention is given to this plant in proving it medicinal worth and capability as an antioxidant agent. This study was initiated to set up a metabolite classes profile and the potential enzymatic antioxidant of the variations of these plants. Crude extracts of S. trifasciata var. Laurentii and S. trifasciata var. Zeylanica were prepared from their leaves, and solvent used has different polarities. Qualitative phytochemical analysis was carried out using the extracts. Phytochemical screening suggested both of these samples contain carbohydrates in all extracts. It also show that flavonoid was found in hexane and ethyl acetate extracts while did not observed in the methanol extracts for both samples. However, alkaloid, phenol and tannin were positive in all of the methanol extracts except for hexane and ethyl acetate extracts. For the biological activity, all extracts were selected for the determination of enzymatic antioxidant activity test using catalase (CAT) assay and guaiacol peroxidase (gPOD) assay using UV-VIS spectrophotometer. Based on the results, CAT specific activity was the highest in methanol extract of S. trifasciata var. Laurentii (3.15 ± 0.50 units/mg protein) compared to S. trifasciata var. Zeylanica (2.20 ± 0.05 units/mg protein). For gPOD specific activity, ethyl acetate extract of S. trifasciata var. Laurentii shows the highest activity which is 1.46 × 10-2 ± 0.02 units/mg protein compared to the other crude extracts.

Rivaroxaban Effects Illustrate the Underestimated Importance of Activated Platelets in Thrombin Generation Assessed by Calibrated Automated Thrombography

Background: The direct oral anticoagulant rivaroxaban inhibiting specifically activated factor X (FXa) causes delayed thrombin generation (TG) as measured by calibrated automated thrombography (CAT). The implications of these changes for assessing bleeding or residual prothrombotic risks of patients are unclear in the absence of a better understanding of the underlying mechanism. Methods: We compared platelet rich plasma (PRP) without or with prior collagen-induced platelet aggregation (agPRP) in the CAT assay to better characterize TG in the presence of rivaroxaban. Results: In the presence of rivaroxaban, TG curves in agPRP showed a distinct profile with a rapidly ascending phase followed with a protracted phase. Inhibition of tissue factor pathway inhibitor amplified the first phase of the curve which was also modulated by procoagulant phospholipids. Inhibition of FXIIa-dependent FXI activation revealed that aggregated platelets influenced the first phase by a combination of extrinsic and intrinsic coagulation pathway initiations. Thrombin-dependent amplification of TG (even prior collagen activation) was responsible for the second phase of the TG curve. Conclusions: AgPRP fully includes platelet ability to support TG and reveal distinct TG phases in the presence of direct FXa inhibitors highlighting its potential use in an anticoagulated setting.

Antiretroviral Therapy Does Not Correct the Increased Thrombin Generation and Platelet Hyperactivity Associated with HIV

Background: Human Immunodeficiency Virus (HIV) infection is associated with an increased risk of thrombosis, and treatment with antiretroviral therapy (ART) does not decrease this risk. While the mechanism leading to thrombosis is unclear, HIV infection is associated with decreased plasma anticoagulant proteins, most commonly protein S (PS), expression of tissue factor (TF) on monocytes, platelet hyperactivity, and thrombocytopenia. Despite the prevalence of PS deficiency, its pathologic consequences are unclear because PS concentration does not correlate with thrombin generation in the standard calibrated automated thrombography (CAT) assay. As increased plasma thrombin generation is a strong predictor of mortality in these patients, we sought to determine which of the described procoagulant effects of HIV is associated with thrombin generation in this population. Method: Blood was collected from 27 consenting HIV+ patients (16 naive samples collected from patients on first diagnosis and 11 samples from patients on ART) and 10 healthy controls. Results: 63% (17/27) of HIV+ plasma samples were deficient in PS, compared to the control samples, as determined by a total PS ELISA (p=0.034). PS functions as a cofactor for the anticoagulant enzyme activated protein C (APC), whose activation requires endothelial cell thrombomodulin (TM). Thus, to correlate plasma PS with thrombin generation, we established a modified CAT assay that is sensitive to PS concentration, by supplementing plasma samples with 20nM TM. Plasma PS did not correlate with thrombin generation in the absence of TM, consistent with previous reports. In the presence of TM, there was a negative correlation, with decreased PS being associated with increased thrombin peak height (p=0.004) and endogenous thrombin potential (p=0.003). Since 2.5% of total circulating PS is stored in platelets and released upon platelet stimulation, we quantified platelet PS by immunoblotting, and found no correlation with plasma PS concentration, consistent with the physiologically distinct origins of these two pools. HIV is reported to promote TF expression in monocytes, and monocytes can release TF-bearing microvesicles into plasma, so we also measured circulating TF activity, but found no difference between the patients and controls. As anticipated, TF correlated with a shorter lag time for thrombin generation, but only when no exogenous TF was added (p=0.0097). Treatment with ART resulted in undetectable viral load and also decreased plasma markers of inflammation, including TNF-α, IL-6, and IL-1β, to the levels observed in controls. However, there was no effect of ART on PS concentration (p=0.521), and platelet-leukocyte aggregates (PLA) remained elevated in patients on ART (19.66%) compared to controls (4.62%, p=0.0001). Platelet hyperactivity, as measured by PLA, was independent of platelet count, and did not correlate with platelet or plasma PS concentration. Interestingly, the use of ART had no impact on plasma PS or TF, but did correlate with greater thrombin generation in assays performed in the absence of TM, and thus the absence of PS activity (10% higher total thrombin, p=0.039), suggesting a PS-independent procoagulant effect of ART. Conclusion: PS deficiency in HIV patients correlates with higher thrombin generation and likely contributes to the thrombotic risk associated with this condition, while microvesicle TF, which does not differ from that seen in healthy controls, is unlikely to be a contributing factor. ART treatment decreases viral load and inflammation, but does not decrease platelet hyperactivity or restore plasma PS concentration and is not associated with any decrease in thrombin generation. Rather, one or more of the ART drugs appear to be further increasing thrombin generation through a PS-independent mechanism. In summary, it appears that multiple, independent factors contribute to the thrombotic risk associated with HIV, including platelet hyperactivity, plasma PS, and the ART treatment itself. Disclosures No relevant conflicts of interest to declare.

Platelet-Derived Microvesicles Reverse the Lupus Anticoagulant Effect and Are More Potent then Synthetic Phospholipids in Supporting Thrombin Generation in Antiphospholipid Syndrome

Background: Antiphospholipid Syndrome (APS) is associated with clinical thrombosis manifestations. However, it is characterized as prolongation of lag time in phospholipid (PL) dependent laboratory coagulation assays, a reflection of the paradoxical lupus anticoagulant (LAC) effect. In contrast to clinical laboratory testing which almost exclusively utilize synthetic PL (sPL), activated platelets are a major source for PL for in vivo hemostasis. It was recently reported that platelet-derived microvesicles (P-MVs) release is mediated by a positive thrombin generation feedback loop in the process of platelet activation and membrane ballooning, which supports a physiological role for P-MVs in hemostasis (Agbani, et. al., 2017). Objective: To evaluate and compare thrombin generation (TG) supported by P-MVs, Erythroid MVs (E-MVs) and sPL in normal subjects and APS patients. Methods: MVs were prepared by gentle sonication or hypotonic-mechanical disruption of washed whole blood platelet pellets, platelet apheresis units or red blood units; sPL composition used was PS/PE/PC=20/20/60 (Bloemen, et. al., 2017). Particle size distribution and anionic phosphatidylserine (PS) expression on P-MV, E-MVs and sPL by Annexin 5-Oregan green (A5-OG) staining were analyzed by flow cytometry using Apogee A60-Micro Plus. TG measured by calibrated automated thrombinography (CAT) was used to evaluate a procoagulable capacity of sPL, P-MVs, and E-MVs after initiating with tissue factor (TF). Results: P-MVs and sPL were equivalent as a source of PL in supporting TG in normal platelet poor plasmas. TG in pooled normal plasma (PNP) in the presence of 1 pM TF showed that P-MVs in dose-dependent fashion support thrombin generation to a similar degree as standard sPLs dilutions including a dose of 4 µM, validated earlier as a saturated concentration in CAT assay (Gerotziafas GT, et. al., 2005). Increasing concentration of either P-MVs or sPLs had little effect on lag time in PNP. In APS patient plasma, lag time was dose-dependently shortened with P-MVs, from 20 minutes to less than 10 minutes, and peak height was dose-dependently increased to a nearly normal level with the highest P-MVs tested. When sPLs was used, TG more severely affected. Strong thrombin generation occurred only with high concentration sPLs (32 µM) after more than 20 min lag time (Figure 1B). E-MVs showed a similar pattern in generating thrombin as sPL in normal plasma and in APS patient with prolonged lag time. The reversal of LAC phenomenon with correction of lag time in APS patients with P-MVs in TG CAT assay was confirmed by testing another 6 (out of 10) APS patients. Microvesicles size, analyzed on A60-Micro Plus, using large angle light scatter (LALS) demonstrated that all three types of MV were consistently less than 1micrometer in diameter. E-MVs and sPL had a similar size distribution, and the majority were of a diameter comparable to 110nm polystyrene calibration beads. P-MVs had wider distribution ranging up to 500nm. All three types of vesicles showed a PS expression by A5-OG labeling with 88.8%, 69.3% and 43.9% MV positivity for sPL, P-MVs and E-MVs, respectively and equivalent mean fluorescent intensity. Summary: Platelet-derived microvesicles significantly promoted thrombin generation in APS patients. This is a revisiting of the "platelet neutralization effect" that was observed nearly 4 decades ago (Thiagarajan P, et. al., 1980). Our data demonstrate that P-MVs consistently increased TG in APS patient plasma compared to equivalent doses of sPL despite similar level of PS expression. In conclusion, cell-derived membranes from different source expressing PS might have different mechanisms to facilitate assembly of coagulation enzyme-substrate-cofactor complexes with resulting enzymatic activity than sPL and help explain LAC phenomenon. Disclosures No relevant conflicts of interest to declare.

Characterization of the thrombin generation profile in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a multisystemic inflammatory autoimmune disorder. Thrombotic events occur at a higher incidence among SLE patients. The investigation of thrombin generation (TG) with calibrated automated thrombogram (CAT) test as a global hemostasis assay is applicable for the overall functional assessment of the hemostasis. The aim of this study was to characterize the hemostatic alterations observed in SLE by CAT assay. In this study, CAT parameters and basic coagulation parameters of SLE patients (n = 22) and healthy control subjects (n = 34) were compared. CAT area under the curve (i.e., endogenous thrombin potential) was lower than normal in SLE (807 vs. 1,159 nM*min, respectively), whereas other CAT parameters (peak, lag time, time to peak, and velocity index) and the basic coagulation tests were within the normal range. The presence of anti-phospholipid antibodies and the applied therapy was not associated with hemostasis parameters in SLE. We concluded that the reported high risk of thrombosis is not related to TG potential.