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 mu M) and in washed platelets (WPs; 0.005-3 mu M): approximately 8.31% (+/- 6.99% SD) and 89.53% (+/- 11.38% SD) for HD1 (0.5 mu M) and HD22 (0.5 mu M), respectively. Neither HD1 nor HD22 (3 mu M) did influence platelets aggregation induced by collagen. Both of them inhibited ristocetin-induced aggregation in PRP. Surprisingly, HD1 and HD22 aptamers (3 mu M) potentiated ristocetin-induced platelet aggregation in WP. HD1 reduced thrombin generation in a concentration-dependent manner [ETP at 3 mu M: 1677.53 +/- 55.77 (nM +/- min) vs. control 2271.71 +/- 423.66 (nM +/- min)], inhibited fibrin formation (lag time at 3 mu M: 33.70 min +/- 8.01 min vs. control 7.91 min +/- 0.91 min) and reduced thrombus formation under flow conditions [AUC(30) at 3 mu M: 758.30 +/- 344.23 (kPa +/- min) vs. control 1553.84 +/- 118.03 (kPa +/- min)]. HD22 (3 mu M) also delayed thrombin generation but increased the thrombin peak. HD22 (3 mu M) 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 mu M). 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.</p

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). Kd values for the HD1 complex with thrombin was higher (257.8 ± 15.0 nM) than the Kd 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

A Keplerian method for Collision Detection in Protoplanetary Disk Simulations: Calculating the computational efficiency and the consecutive collision times in N-body systems as a function of Keplerian parameters

This report has been made to gain more insight and knowledge of the Keplerian method for collision detection simulations by simplifying N-body systems to a list or particles with orbital parameters. Collision detection is an essential part in modeling the evolution of protoplanetary disk becoming planetary systems by the merging of planetesimal objects. Presumably Kep- lerian systems allow for a computationally efficient algorithm, having its computational time influenced by orbital parameters, unlike the efficient octree method which only scales with the number of particles.The aim of this research was to create a collision detection model for a simplified proto- planetary disk system using Kepler systems and analyse the results of the model to check the reliability of this simulation method, i.e. if the quality of the results are trustworthy, perform- ing consistently well, and are accurate. In addition, the method was tested on its computational efficiency by investigating the run time of separate parts of the algorithm.In the initialisation of the algorithm a particle list is created and sorted by increasing apoap- sis. Then by a sweep and prune filter the list is checked for possible collision pairs. For pairs that have a small enough minimal orbital intersection distance that they can collide, the colli- sion time is calculated and added to a time-ranked collision list. The algorithm keeps merging the pairs on top op the list, creating a new particle with new possible collision pairs and new collision times, and updates the time-ranked collision list after each merge. An empty collision list implies the end of the simulation. Various parameters as well as length of the lists are saved within the algorithm for subsequent examination.The results of the Keplerian model found showed similarities to other simulation studies, and include the perceived critical eccentricity of 0.02, preservation of shape of body radius distribution, and the formation of large bodies. Simulations showed three stages of collisions, characterised by collisions occurring homogeneously throughout the disk, large body colli- sions, a final stage of small body collisions. The performance of the algorithm could be sum- marised by the number of checks by the sweep and prune filter #checks ∝ N 2 and #checks ∝ ε for eccentricity ε &lt; 0.1, the number of pairs in the collision pair list was found to be propor- tional to the number of starting particles N, the body radius s, the inverse of the inclination 1/I, and the eccentricity ε, so #pairs ∝ N2, s, 1/I, ε. For N &lt; 1000 the computation time of the algorithm appeared to be proportional to N2, but for N &lt; 10.000 proportional to to a higher power. For ε &lt; 10−9 and N &lt; 10.000 the computation time was found to be runtime ∝ s.The conclusion of this research is that the Keplerian model could be used to model N-body systems for collision detection because of its behavioural resemblance to observable astrophys- ical systems. The computation time of the Keplerian system scales with a higher order per N than for instance the three code algorithm, but has showed to be influenced by orbital parame- ters in which this method differentiates itself from other simulation methods, with a remarkable influence on the total computation time by the body radius s of the system. More research could be done for higher orders of N to better model reality as well as providing more insight on the proportionality of the computation time. Recommendations to the algorithm consist of adding planetary spin as a particle parameter, and methods to incorporate apsidal precession in a com- petent manner.Applied Mathematics | Applied Physic

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

Near Patient Thrombin Generation in Patients Undergoing Elective Cardiac Surgery

Background: Measuring thrombin generation (TG) in plasma increasingly gained attention as a diag-nostic 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 deter-mined 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. IMPACT STATEMENT The near patient thrombin generation device allows for a fast

Inhibition of thrombin generation in zebrafish by thrombin or FXa inhibition.

<p>Zebrafish were treated with melagatran <b>(A)</b> or rivaroxaban <b>(B)</b> at the indicated doses for 30 min, followed by thrombin generation measurements. <b>(C-D)</b> Results of two independent experiments are expressed as the percentage inhibition compared to a control group of vehicle-treated fish (n = 8). The percentage CV is indicated for all parameters. Statistical differences between treatment and control groups were performed with *Mann-Whitney U-test or ^$1-way ANOVA. (ns = not significant).</p

Scanning electron microscopy (SEM) of blood clots.

<p>Representative image of SEM analysis of blood clots formed during thrombin generation measurements. Sequential enlargements of the fibrin network with entrapped red blood cells are depicted. <b>(A)</b> Zebrafish blood clot. <b>(B)</b> Human blood clot without added tissue factor (TF). <b>(C)</b> Human blood clot with addition of a high dose (100 pM) of TF.</p

Inhibition of thrombin generation in zebrafish by inhibition of platelet aggregation.

<p>Zebrafish were treated with 30 μg/g aspirin or vehicle for 30 min <b>(A-B)</b> or were allowed to swim in aspirin-supplemented water (250 mg/ml) for 24 hours <b>(C-D)</b>, followed by thrombin generation measurements. <b>(A,C)</b> Thrombin generation curves are shown. <b>(B,D)</b> Results are expressed as the percentage inhibition compared to a control group of vehicle-treated fish (n = 5). The percentage CV is indicated for all parameters. *Mann-Whitney U-test as compared to controls.</p