Vitamin K2-MK-7 improves nitric oxide-dependent endothelial function in ApoE/LDLR-/- mice.

Although, vitamin K2 displays vasoprotective effects, it is still not known whether K2 treatment improves endothelial function. In ApoE/LDLR-/- mice at the stage prior to atherosclerosis development, four-week treatment with K2-MK-7, given at a low dose (0.05 mg/kg), improved acetylcholine- and flow-induced, endothelium-dependent vasodilation in aorta or in femoral artery, as assessed by MRI in vivo. This effect was associated with an increased NO production, as evidenced by EPR measurements in ex vivo aorta. Treatment with higher doses of K2-MK-7 (0.5; 5 mg/kg) resulted in a dose-dependent increase in plasma K2-MK-7 and K2-MK-4 concentration, without further improvement in endothelial function. In ApoE/LDLR-/- mice with developed atherosclerotic plaques, treatment with a low (0.03 mg/kg) or high (10 mg/kg) dose of K2-MK-7 resulted in a similar degree of endothelium-dependent vasodilation improvement and increase in plasma nitrate concentration, what was not associated with changes in thrombin generation as measured by CAT. Both doses of K2-MK-7 also reduced media thickness in the brachiocephalic artery, but did not modify atherosclerotic plaque size. In conclusion, K2-MK-7 improves NO-dependent endothelial function in ApoE/LDLR-/- mice. This study, identifies the endothelial profile of the pharmacological activity of vitamin K2, which has not been previously described.

Comparison of Effects of Anti-thrombin Aptamers HD1 and HD22 on Aggregation of Human Platelets, Thrombin Generation, Fibrin Formation, and Thrombus Formation Under Flow Conditions.

HD1 and HD22 are two of the most-studied aptamers binding to thrombin exosite I and exosite, respectively. To complete of their pharmacological profiles, the effects of HD1 and HD22 on thrombin-, ristocetin-, and collagen-induced human platelet aggregation, on thrombin generation and fibrin formation in human plasma, as well as on thrombus formation in human whole blood under flow conditions were assessed. The dissociation constants for HD1 and HD22 complexes with thrombin in simulated plasma ionic buffer were also evaluated. HD1 was more potent than HD22 in terms of inhibiting thrombin-induced platelet aggregation in platelet-rich plasma (PRP; 0.05-3 µM) and in washed platelets (WPs; 0.005-3 µM): approximately 8.31% (±6.99% SD) and 89.53% (±11.38% SD) for HD1 (0.5 µM) and HD22 (0.5 µM), respectively. Neither HD1 nor HD22 (3 µM) did influence platelets aggregation induced by collagen. Both of them inhibited ristocetin-induced aggregation in PRP. Surprisingly, HD1 and HD22 aptamers (3 µM) potentiated ristocetin-induced platelet aggregation in WP. HD1 reduced thrombin generation in a concentration-dependent manner [ETP at 3 µM: 1677.53 ± 55.77 (nM⋅min) vs. control 2271.71 ± 423.66 (nM⋅min)], inhibited fibrin formation (lag time at 3 µM: 33.70 min ± 8.01 min vs. control 7.91 min ± 0.91 min) and reduced thrombus formation under flow conditions [AUC30 at 3 µM: 758.30 ± 344.23 (kPa⋅min) vs. control 1553.84 ± 118.03 (kPa⋅min)]. HD22 (3 µM) also delayed thrombin generation but increased the thrombin peak. HD22 (3 µM) shortened the lag time of fibrin generation (5.40 min ± 0.26 min vs. control 7.58 min ± 1.14 min) but did not modify thrombus formation (3, 15 µM). K d values for the HD1 complex with thrombin was higher (257.8 ± 15.0 nM) than the K d for HD22 (97.6 ± 2.2 nM). In conclusion, HD1 but not HD22 represents a potent anti-thrombotic agent, confirming the major role of exosite I in the action of thrombin. HD22 aptamer blocking exosite II displays weaker anti-platelet and anti-coagulant activity, with surprising activating effects on thrombin and fibrin generation most likely induced by HD22-induced allosteric changes in thrombin dynamic structure.

Hypobaric Hypoxia Causes Elevated Thrombin Generation Mediated by FVIII that is Balanced by Decreased Platelet Activation.

INTRODUCTION: Epidemiological studies suggest that hypobaric hypoxia at high altitude poses a risk for developing venous thromboembolism. The cause of this observed hypercoagulability remains unclear. Therefore, this study aimed to investigate the effect of hypobaric hypoxia at 3,883 m above sea level on thrombin generation and platelet activation. METHODS: After complying with medical ethical procedures, 18 participants were recruited, of whom 1 had to leave the study prematurely due to mild acute mountain sickness. Blood was drawn first at 50 m above sea level and second at 3,883 m altitude after gradual acclimatization for 6 days. Thrombin generation was measured in whole blood, platelet-rich plasma and platelet-poor plasma. Platelet activation was assessed using a whole blood flow-cytometric assay. Coagulation factor levels, D-dimer levels and markers of dehydration and inflammation were measured. RESULTS: Hypobaric hypoxia at 3,883 m altitude caused increased thrombin generation, measured as peak height and endogenous thrombin potential, in whole blood, platelet-rich and platelet-poor plasma without or at low tissue factor concentration. The elevated thrombin generation was mediated by increased factor VIII levels and not caused by dehydration or inflammation. In contrast, spontaneous and agonist-induced platelet activation was decreased at high altitude. CONCLUSION: Hypobaric hypoxia causes increased factor VIII-mediated thrombin generation. The hypercoagulability was balanced by decreased platelet activation. These findings may explain why venous, and not arterial thrombotic events occur more frequently at high altitude.

Near-Patient Thrombin Generation in Patients Undergoing Elective Cardiac Surgery.

BACKGROUND: Measuring thrombin generation (TG) in plasma increasingly gained attention as a diagnostic tool in the field of thrombosis and hemostasis. To include the contribution of all blood cells, recently, the whole blood TG method was developed. METHODS: We changed the calculation method of the standard calibrated automated thrombography (CAT) to a method only taking into account the data until the peak of TG, thereby considerably reducing the time from blood draw to result. By redesigning the method, the blood volume per test was reduced to 15 µL. RESULTS: For all TG parameters, the interassay variation proved to be below 15%. The interindividual variation of all parameters was comparable to the CAT method. Thirty-three patients undergoing cardiothoracic surgery were included to investigate whether our assay correlates with postoperative blood loss. On dividing patients into severe and mild bleeders, significant differences between both groups were found for the peak endogenous thrombin potential (peakETP) and peak values determined by our near-patient device. Importantly, patients with a peakETP below the median experienced significantly more blood loss compared to those with a peakETP above the median. A similar division based on the peak as well as the body mass index of the patient yielded similar significant differences. A combination of the peakETP, the body mass index, and the lag time even resulted in a better predictor of blood loss compared to each parameter separately. CONCLUSIONS: Our adapted whole blood TG assay can be used near patients and is indicative for the amount of blood loss post cardiothoracic surgery.

Thrombin Generation in Zebrafish Blood.

To better understand hypercoagulability as an underlying cause for thrombosis, the leading cause of death in the Western world, new assays to study ex vivo coagulation are essential. The zebrafish is generally accepted as a good model for human hemostasis and thrombosis, as the hemostatic system proved to be similar to that in man. Their small size however, has been a hurdle for more widespread use in hemostasis related research. In this study we developed a method that enables the measurement of thrombin generation in a single drop of non-anticoagulated zebrafish blood. Pre-treatment of the fish with inhibitors of FXa and thrombin, resulted in a dose dependent diminishing of thrombin generation, demonstrating the validity of the assay. In order to establish the relationship between whole blood thrombin generation and fibrin formation, we visualized the resulting fibrin network by scanning electron microscopy. Taken together, in this study we developed a fast and reliable method to measure thrombin generation in whole blood collected from a single zebrafish. Given the similarities between coagulation pathways of zebrafish and mammals, zebrafish may be an ideal animal model to determine the effect of novel therapeutics on thrombin generation. Additionally, because of the ease with which gene functions can be silenced, zebrafish may serve as a model organism for mechanistical research in thrombosis and hemostasis.