New Blood Coagulation Factor XIIa Inhibitors: Molecular Modeling, Synthesis, and Experimental Confirmation

In the modern world, complications caused by disorders in the blood coagulation system are found in almost all areas of medicine. Thus, the development of new, more advanced drugs that can prevent pathological conditions without disrupting normal hemostasis is an urgent task. The blood coagulation factor XIIa is one of the most promising therapeutic targets for the development of anticoagulants based on its inhibitors. The initial stage of drug development is directly related to computational methods of searching for a lead compound. In this study, docking followed by quantum chemical calculations was used to search for noncovalent low-molecular-weight factor XIIa inhibitors in a focused library of druglike compounds. As a result of the study, four low-molecular-weight compounds were experimentally confirmed as factor XIIa inhibitors. Selectivity testing revealed that two of the identified factor XIIa inhibitors were selective over the coagulation factors Xa and XIa

Synthesis, Docking, and In Vitro Anticoagulant Activity Assay of Hybrid Derivatives of Pyrrolo[3,2,1-ij]Quinolin-2(1H)-one as New Inhibitors of Factor Xa and Factor XIa

Coagulation factor Xa and factor XIa are proven to be convenient and crucial protein targets for treatment for thrombotic disorders and thereby their inhibitors can serve as effective anticoagulant drugs. In the present work, we focused on the structure–activity relationships of derivatives of pyrrolo[3,2,1-ij]quinolin-2(1H)-one and an evaluation of their activity against factor Xa and factor XIa. For this, docking-guided synthesis of nine compounds based on pyrrolo[3,2,1-ij]quinolin-2(1H)-one was carried out. For the synthesis of new hybrid hydropyrrolo[3,2,1-ij]quinolin-2(1H)-one derivatives, we used convenient structural modification of both the tetrahydro- and dihydroquinoline moiety by varying the substituents at the C6,8,9 positions. In vitro testing revealed that four derivatives were able to inhibit both coagulation factors and three compounds were selective factor XIa inhibitors. An IC50 value of 3.68 μM for was found for the best factor Xa inhibitor and 2 μM for the best factor XIa inhibitor

New Hybrid Tetrahydropyrrolo[3,2,1-<i>ij</i>]quinolin-1-ylidene-2-thioxothiazolidin-4-ones as New Inhibitors of Factor Xa and Factor XIa: Design, Synthesis, and In Silico and Experimental Evaluation

Despite extensive research in the field of thrombotic diseases, the prevention of blood clots remains an important area of study. Therefore, the development of new anticoagulant drugs with better therapeutic profiles and fewer side effects to combat thrombus formation is still needed. Herein, we report the synthesis and evaluation of novel pyrroloquinolinedione-based rhodanine derivatives, which were chosen from 24 developed derivatives by docking as potential molecules to inhibit the clotting factors Xa and XIa. For the synthesis of new hybrid derivatives of pyrrolo[3,2,1-ij]quinoline-2-one, we used a convenient structural modification of the tetrahydroquinoline fragment by varying the substituents in positions 2, 4, and 6. In addition, the design of target molecules was achieved by alkylating the amino group of the rhodanine fragment with propargyl bromide or by replacing the rhodanine fragment with 2-thioxoimidazolidin-4-one. The in vitro testing showed that eight derivatives are capable of inhibiting both coagulation factors, two compounds are selective inhibitors of factor Xa, and two compounds are selective inhibitors of factor XIa. Overall, these data indicate the potential anticoagulant activity of these molecules through the inhibition of the coagulation factors Xa and XIa

Hysteresis-like binding of coagulation factors X/Xa to procoagulant activated platelets and phospholipids results from multistep association and membrane-dependent multimerization.

International audienceBinding of coagulation factors X (fX) and Xa (fXa) to activated platelets is required for the formation of membrane-dependent enzymatic complexes of intrinsic tenase and prothrombinase. We carried out an in-depth characterization of fX/fXa binding to phospholipids and gel-filtered, thrombin-activated platelets. Flow cytometry, surface plasmon resonance, and computational modeling were used to investigate interactions of fX/fXa with the membranes. Confocal microscopy was employed to study fXa binding to platelet thrombi formed in flowing whole blood under arterial conditions. Binding of fX/fXa to either vesicles or procoagulant platelets did not follow a traditional one-step reversible binding model. Their dissociation was a two-step process resulting in a plateau that was up to 10-fold greater than the saturation value observed in the association experiments. Computational modeling and experimental evidence suggested that this was caused by a combination of two-step association (mainly for fX) and multimerization on the membrane (mainly for fXa). Importantly, fX formed multimers with fXa, thereby improving its retention. The same binding/dissociation hysteresis was observed for annexin V known to form trimers on the membranes. Experiments with platelets from gray syndrome patients showed that alpha-granular factor Va provided an additional high-affinity binding site for fXa that did not affect the hysteresis. Confocal microscopy observation of fXa binding to platelet thrombi in a flow chamber and its wash-out confirmed that this phenomenon persisted under physiologically relevant conditions. This suggests its possible role of "locking" coagulation factors on the membrane and preventing their inhibition in plasma and removal from thrombi by flow

Computer simulation of C1-inhibitor washout.

<p>(<b>A</b>) We used a scheme that depicts the platelet aggregate to test the C1-INH washout hypothesis. The platelet concentration inside the thrombi was considered 1 per 15 fl. The initial C1-inhibitor distribution in the aggregate for the simulations is shown in grey, and at values ranging from 0.1 to 10 μm/s, the flow penetrated the aggregate. At t = 0, C1-INH at 100 μM (estimated from ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.ref033" target="_blank">33</a>]) and fXII at 450 nM appeared simultaneously. Factor XII could be activated on the platelet surface, and C1-INH could diffuse through the aggregate (D = 10 μm²/s, based on the molecular weight) and move due to the flow. The fXIIa diffusion was assumed negligible because fXII activation is surface-associated ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.ref010" target="_blank">10</a>] and this study), and typically, fXIIa is tightly bound to the activation surface [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.ref039" target="_blank">39</a>]. The C1-INH action was described using a mass action equation with the reaction constant [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.ref034" target="_blank">34</a>] 0.00366 μM^-1s^-1. (<b>B</b>) Time-course of the distance-averaged [C1-INH] for various flow velocities. (<b>C</b>) Time-course of the surface-averaged [fXIIa] for various flow velocities. The C1-INH and fXIIa spatial distributions were governed by a set of differential equations, which were solved using the finite volume solver available within the Virtual Cell environment.</p

Comparison of fXII activation by platelets from a normal donor and GPS patients.

<p><b>(A)</b> Comparison of the fXIIa-generating capacities for A23187-activated platelets (at 2×10⁷/mL) from a normal donor (labelled “normal plt”) and three grey platelet syndrome patients (labelled GPS-1, GPS-2, and GPS-3, respectively). <b>(B)</b> Flow-cytometry dot plots demonstrating the significant PS exposure in the analysed platelet preparations upon activation with A23187.</p

FXII activation model.

<p><b>(A)</b> Computational model schematic for fXII activation on the platelet surface. Soluble fXII first binds the surface and is then activated; fXIIa can inhibit further activation through an auto-inhibition mechanism [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.ref026" target="_blank">26</a>]. The reaction rate constants (<i>k</i>_i) for the model were estimated based on the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.g001" target="_blank">Fig. 1C</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.s004" target="_blank">S4C Fig.</a>, and the surface area of an activated platelet was considered 50 μm². <b>(B)</b> Platelet-dependent fXIIa formation in buffer as a function of the fXII concentration added. The black circles correspond to the experimental data (±SD for three independent experiments, n = 3); the curve corresponds to the computational model. The consistency between the data and model curve confirms the fXII auto-inhibition hypothesis. <b>(C)</b> The dose-dependence of fXIIa formation in 20% plasma for A23187-activated platelets after platelet secretion removal. The platelets were activated at 2×10⁸/mL and diluted to the indicated concentrations for further reaction with plasma. The black squares represent the experimental data (n = 3), and the curve represents the computational model. The consistency between the data and model curve confirms the reliability of the model.</p

The role of PS and poly-P in surface-associated fXII activation by platelets.

<p><b>(A)</b> The effect of 20 μg/mL annexin V or 1.5 μM prothrombin on 280 nM fXII activation (in the presence of 2.5 mM CaCl₂) by A23187-activated (secretion-depleted) platelets (n = 3). <b>(B)</b> Comparison of the fXIIa-generating capacities for A23187-activated platelets (at 2×10⁶/mL) and 4 μM PS- or PC-liposomes (depicted as PS and PC, respectively) in 20% chelated plasma. The mean values (±SD) were calculated from three independent experiments (n = 3). <b>(C)</b> Confocal microscopy analysis of poly-P localisation in activated platelets with 2.5 mM calcium. DAPI was used as poly-P marker [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.ref027" target="_blank">27</a>], and AlexaFluor-647-annexin V was used as a marker for procoagulant PS-positive platelets (labelled “PS+”). PS-negative activated platelets are labelled “PS-” (n = 3). <b>(D)</b> Confocal microscopy analysis of poly-P localisation in activated platelets without 2.5 mM calcium. FITC-anti-fibrin(ogen) antibody was used as an activated platelet marker (n = 3). The images in (C) and (D) were collected at an ∼2 μm z-depth over the fibrinogen surface to reduce non-specific fibrinogen immunofluorescence.</p

Platelet activation of purified and plasma fXII.

<p><b>(A)</b> Flow-cytometry dot-plots demonstrating predominant FITC-fXII binding for PS-positive platelet subpopulations (labelled “PS+”). The result corresponds to 1000 nM FITC-fXII binding. <b>(B)</b> The effect of platelets on activation of purified 200 nM fXII. Platelets were activated by 10 nM thrombin (n = 3). <b>(C)</b> Dose-dependence for platelet-dependent fXIIa formation upon a reaction between thrombin-activated platelets and purified fXII (n = 3). A hyperbolic curve for a representative, typical experiment was fit using <i>K</i>_m^fXII = 64 and [<i>fXIIa</i>]_max = 0.126 nM. The given <i>K</i>_m and [<i>fXIIa</i>]_m values are mean values (±SD) calculated using data from three independent experiments. <b>(D)</b> The effect of different platelet stimulation methods on platelet-dependent fXIIa formation in 20% plasma. The asterisks symbols (*) correspond to p<0.05. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.t001" target="_blank">Table 1</a> represents the p values for comparing fXII activating capacities between activated and non-activated platelets.</p

Platelet Surface-Associated Activation and Secretion-Mediated Inhibition of Coagulation Factor XII

<div><p>Coagulation factor XII (fXII) is important for arterial thrombosis, but its physiological activation mechanisms are unclear. In this study, we elucidated the role of platelets and platelet-derived material in fXII activation. FXII activation was only observed upon potent platelet stimulation (with thrombin, collagen-related peptide, or calcium ionophore, but not ADP) accompanied by phosphatidylserine exposure and was localised to the platelet surface. Platelets from three patients with grey platelet syndrome did not activate fXII, which suggests that platelet-associated fXII-activating material might be released from α-granules. FXII was preferentially bound by phosphotidylserine-positive platelets and annexin V abrogated platelet-dependent fXII activation; however, artificial phosphotidylserine/phosphatidylcholine microvesicles did not support fXII activation under the conditions herein. Confocal microscopy using DAPI as a poly-phosphate marker did not reveal poly-phosphates associated with an activated platelet surface. Experimental data for fXII activation indicates an auto-inhibition mechanism (<i>k</i>_i/<i>k</i>_a = 180 molecules/platelet). Unlike surface-associated fXII activation, platelet secretion inhibited activated fXII (fXIIa), particularly due to a released C1-inhibitor. Platelet surface-associated fXIIa formation triggered contact pathway-dependent clotting in recalcified plasma. Computer modelling suggests that fXIIa inactivation was greatly decreased in thrombi under high blood flow due to inhibitor washout. Combined, the surface-associated fXII activation and its inhibition in solution herein may be regarded as a flow-sensitive regulator that can shift the balance between surface-associated clotting and plasma-dependent inhibition, which may explain the role of fXII at high shear and why fXII is important for thrombosis but negligible in haemostasis.</p></div