It is well-known that ischemic heart disease and ischemic strokes are the world leaders among the cardiovascular diseases. The changes in the vascular wall caused by atherosclerosis promote the activation of platelets and coagulation hemostasis, which ultimately lead to the formation of a thrombus substantially reducing or completely blocking the blood flow. Affection of vascular wall, activation of platelets and coagulation hemostasis caused by atherosclerosis lead to formation of thrombus. Adhesion and aggregation of platelets promoted by the activation of plasmatic pro-coagulants on phospholipid cell surface contacting with blood cause thrombosis. Notably, cardiovascular diseases are connected with increased sensitivity of platelets to adhesion and aggregation to inductors. Activation of specific platelet receptors leads to a reorganization of their membrane, a disruption in the asymmetry of the location of phospholipids, and the appearance of mesomorphic structures (Finegold et al., 2013; Krudysz-Amblo et al., 2015).

Drugs inhibiting platelet activity are used to treat and prevent this group of diseases. Currently used drugs do not guarantee sufficient prevention and treatment of acute cardiovascular diseases. E.g., although the exact prevalence is unknown, up to 60% of patients taking acetylsalicylic acid might have different degrees of so-named “aspirin resistance” (Gasparyan et al., 2008). Therefore, the investigation of the substances that are capable to affect and normalize the processes indicated are the main objects of creating new antithrombotic drugs.

On the other hand, in some cases it is necessary to preserve the functional activity of platelets. Platelets concentrate is utilized to treat the patients with various types of thrombocytopenia. Clinical effectiveness of donor platelets depends on functional activity of platelets. The damage of vascular wall during the donor blood donation procedure and stress hormone adrenaline level increasing lead to platelet adhesion and activation, and result in appearing of the microvesicles. In addition, storage of platelets decreases their biological activity. Despite sodium citrate or heparin are commonly used as the anticoagulants of blood products preservation, the stabilizing effects of these compounds are insufficient due to formation of the platelets microvesicles. This fact also remains a driving force to find out the novel effective blood preserving agents.

It has been found that about 80% of people use traditional medicine for their primary health care. Most of these therapies involve the use of plant extracts or their active compounds, such as monoterpenoids. Recently pharmacological activity of some herbal substances has already been studied. Coagulation activities were confirmed for some of the phyto-compounds (Balick et al., 1994). It was also shown that monoterpenes might be the promising agents for prevention and treatment of cardiovascular diseases (Santos et al., 2011). On the other hand, sulfur is biogenic element and sulfur-containing compounds are widely represented both in natural products and synthetic biologically active substances. Combination of terpene fragment and biogenic sulfur-containing functional groups into one molecule allows to obtain new compounds with biologically active properties. The most available natural mono- and bicyclic monoterpenes such as camphene, α- and β-pinenes, limonene, carvone, 3-carene, and their derivatives—epoxides, epysulfides, alcohols, cyclopropenes, and allenes are usually used as initial compounds for our synthesis. We have recently created a series of sulfur-containing terpenoids of varied structures with the large set of the functional groups (Ishmuratov et al., 2014). Moreover, we also demonstrated the anti-fungal, anti-inflammatory, anti-helicobacter, antimicrobial and other types of activities of sulfur-containing monoterpenoids (Nikitina et al., 2009, 2010, 2012a,b; Gavrilov et al., 2010). Coagulation activity of these compounds has already been described in our previous publications (Kiselev et al., 2017; Nikitina et al., 2017a,b). We observed that all synthesized sulfur-containing monoterpenoids have a low toxicity, whereas mutagenic and genotoxic effects were not found (Nikitina et al., 2011).

Sulfoxides are known to be very attractive tools in organic synthesis as chiral auxiliaries or ligands for catalysis and in the pharmaceutical industry for their biological activity (Anderson et al., 2006; Alfonsov et al., 2008; Carreno et al., 2009; Wojaczynska and Wojaczynski, 2010).

Previously we have ascertained a stable “racemic compound-like” behavior of diastereomeric mixture of sulfoxides 2a+2b obtained by the oxidation of enantio-pure sulfide derivative of (–)-β-pinene (Startseva et al., 2014). The oxidation of a β-hydroxysulfide 1 using m-chloroperbenzoic acid resulted in the formation of the corresponding β-hydroxysulfoxide 2 as a mixture of two diastereomers in 4:5 ratio (see Scheme 1).

Attempts to separate diastereomers 2a and 2b using column chromatography or fractional crystallization have failed. It turned out that the sample of 2 crystallized as an asymmetric dimer containing a supramolecular centro-symmetric moiety formed through S = O··H–O interactions between the present two independent molecules. The β-pinene skeleton keeps its natural configuration, while the configurations of the sulfur atoms are opposite. The sample of 2 is interesting as the first example of the co-crystallization of chiral sulfur compounds. Moreover, the dimer 2a+2b can crystallize in two so-called “packing polymorphs” (Grant, 1999). The instances of polymorphism in a system with conserved hydrogen bonded synthons are rare (Fucke et al., 2010). In addition triclinic and monoclinic modifications of diastereomeric sulfoxides cocrystal, remarkable alterations in unit cell parameters by transition from 293 to 150 were ascertained (Lodochnikova et al., 2015; Nikitina et al., 2017c).

The importance of pure optical isomer forming in synthesis of potential biologically active compounds is widely accepted. In this article we present the synthesis of enantiopure sulfoxide 2a and study the influence of this compound on hemostasis.

According to results of previous researches, optically active sulfoxides of the terpene series were prepared by the oxidation of the corresponding sulfides using various oxidizing agents, including m-chloroperbenzoic acid (Binns et al., 1985; Annunziata et al., 1987; Eschler et al., 1988; Pyne et al., 1989; Arai et al., 1991), sodium periodate (Vargas-Díaz et al., 2005), air oxygen (Nikitina et al., 2001), as well as by the Kagan-Modena and Bolm asymmetric oxidation methods (Yang et al., 1994; Aversa et al., 2002; Demakova et al., 2012a,b). We also reported that the crystal of the diastereomer 2a might be obtained as a result of the crystallization after a careful separation of the dimer crystals (Startseva et al., 2014).

In our case sulfoxide 2a was prepared by oxidizing of sulfide 1 according to Uemura method, using Ti(Oi-Pr)₄, (R)-C₆H₅CH(OH)COOH/t-BuOOH as the oxidation system (Scheme 2) (Komatsu et al., 1993).

The structure of the sulfoxide 2a was determined by X-ray structural analysis at room temperature. According to X-ray structural data, sulfoxide 2a was presented as a single diastereomer form with the S-configuration of the sulfinyl group located relatively to the pinane framework of the molecule (Figure 1).

According to the NMR spectroscopy data the enantiomeric excess is close to 100%. Some of sulfoxide molecules would be in the form of dimer in the case of diastereomer formation, and that fact confidently proves the absence of second diastereomer. This, in turn, would have influenced the ¹³C NMR spectrum, in which two sets of signals would have been observed (Anderson et al., 2006), whereas there is only one set in the spectrum in this case (Table 1).

Complete assignment of the ¹H NMR spectrum of sulfoxide 2a was accomplished by 2D ¹H-¹H COSY, ¹H-¹³C HSQC and¹H-¹³C HMBC NMR experiments (Figures 2–4) and based on the analysis of the signal multiplicities, the integral values and the characteristic chemical shifts. CH₃-8 and CH₃-9 protons are observed in the spectrum as two singlets with chemical shifts of 1.07 and 0.88 ppm respectively (Figure 7A). Geminal protons of CH₂-12 resonate in the spectrum as AB-quadruplet at 3.87 ppm. According to ¹H-¹H COSY (Figure 2) and ¹H-¹³C HSQC (Figure 3) correlations all protons signals and directly bonded to them carbon signals were assigned. It was established that geminal protons H6 and H6′ related to CH₂-6 were highly non-equivalent. They were observed in the ¹H NMR spectrum as two multiplets with chemical shifts 2.27 and 0.89 ppm. A similar pattern was observed for geminal H3 and H3′ protons related to CH₂-3 (δ 1.99 and 1.54 ppm). CH₂-4 protons signals overlapped with CH-1 and CH-5 resonance lines, but from 2D spectra it was established that this methylene group signals had chemical shifts 1.86 and 1.76 ppm. Geminal protons CH₂-10 and CH₂-11 were also pairwise non-equivalent. Corresponding multiplets were overlapped and had resonances in the ¹H NMR spectrum in the area of 2.8…3.0 ppm. CH-1 and CH-5 chemical shifts were also defined according to 2D NMR experiments. They were observed in the spectrum as overlapped multiplets at 1.81 ppm. All bonded to protons carbon signals were assigned according to ¹H-¹³C HSQC (Figure 3) spectrum. Finally, chemical shift of the C-7 signal was defined based on the results of ¹H-¹³C HMBC experiment (Figure 4). All assignment of ¹H and ¹³C NMR signals was also confirmed by ¹H-¹³C HMBC correlations. Chemical shifts of all ¹H and ¹³C NMR signals are shown in the Table 1.

Recent studies have shown that sulfur-containing monoterpenoids have membranotropic properties (Kiselev et al., 2017). Therefore, in present study we examined the possibility of using sulfoxide 2a as a platelet concentrate stabilizer. Two mmol concentration of sulfoxide 2a was used as the most effective minimal concentration with anti-aggregative activity and low cytotoxicity (in this concentration ~100%-viability of the BJ fibroblasts was observed) (Table 2, Figure 5). Sulfoxide 2a almost completely inhibited platelet aggregation induced by arachidonic acid, collagen and adrenaline in comparison with acetylsalicylic acid that decreased only ADP-induced aggregation in this concentration.

According to results of flow cytometry analysis, we observed almost 2.5-fold decrease in the numbers of microvesicles in platelet concentrates supplemented with sulfoxide 2a when compared to the control samples. Important the numbers of microvesicles increased ~11-fold by the end of the storage period when compared to the initial time-point (Table 2). The microvesicles indicated above have the thrombogenic properties which were proved by activation of blood coagulation after 48 h of storage with adding 0.1 ml of supernatant from platelet concentrate to 0.9 ml of plasma from healthy donors. Hypercoagulation was confirmed by decreasing of aPTT and prothrombin time, significant increase of fibrin formation rate (V, Vi, Vst), fibrin clot size (Cs) and density (D), appearance of spontaneous clots (Tsp) in the test of thrombodynamics. Of note, all the parameters indicated above approached to the parameters of the control plasma in the presence of sulfoxide 2a: aPTT and prothrombin time increased, and the rates of fibrin formation decreased. Removal of microvesicles from the donor plasma leaded to similar results, but fibrin clot formation rate decreased in more significant extent (Figure 6, Table 3).

In order to ascertain the possible interaction between sulfoxide 2a and cell membranes, we utilized NMR spectroscopy. There are some difficulties in studying of interaction of sulfoxide with model phospholipid cell membrane by NMR. Proton transverse relaxation time of phospholipid aggregates is too short relative to the NMR chemical shifts time-scale and leads to significant broadening of the signals in the spectra. For this reason, sodium dodecyl sulfate micelles were used as model of cell membrane, which are extensively applied in similar NMR studies (Henry and Sykes, 1994; Lubecka et al., 2010; Galiullina et al., 2012, 2015; Usachev et al., 2013; Rakhmatullin et al., 2016). Head polar groups of SDS can be used to physically mimic a surface of biological membrane.

¹H NMR spectrum of the compound is significantly changed after addition of SDS micelles (Figure 7B).

The signals are broadened and some of them are shifted if compared with the spectrum of the compound in pure D₂O (see Table 1). Noticeable changes are observed for the signals CH₂-10 and CH₂-11. The signals are broadened and a number of lines in multiplet are decreased. H-11 and H-11′ geminal protons are become almost equivalent. Unfortunately, line shape changes of the signal CH₂-12 cannot be analyzed because it is overlapped with SDS signal CH₂-12^SDS. However, the resonances of H-2, H-3, H-6,6′ and CH₃-8,9 are shifted to lower fields. These differences between ¹H NMR spectra of the compound in pure D₂O and in D₂O+ SDS micelles can be explained by interaction of terpene with model membrane. Dramatic changes of the signals CH₂-10 and CH₂-11 allow assuming that sulfoxide group of the terpene is responsible for binding of the compound with SDS micelle.

To clarify the mechanism of the complex formation between the terpene and SDS micelle 2D NOESY experiment was carried out (Figure 8). Several non-trivial intermolecular nuclear Overhauser effects (NOE) indicating close spatial location of the corresponding chemical groups of the terpene and SDS micelle were observed in the spectrum. The cross peaks between the signal CH₂-11 of SDS (11^SDS) and the signals CH-2 and CH₂-10 of the compound showed that the compound slightly penetrated into the surface of the micelle. Probably, polar group S = O is a binding site of the compound in this complex.

To examine the detailed atomistic picture of sulfoxide 2a/SDS micelle interactions, classical molecular dynamics simulations were performed. Figure 9 shows the snapshot of the system after 20-ns MD simulation. Visual examination of the sulfoxide 2a/micelle complex indicates that sulfoxide is embedded with its bicyclic fragment inside the SDS micelle, whereas the -SO(CH₂)₂OH fragment of sulfoxide is located on the outer part of SDS micelle and is in contact with solvent.

Our present data confirms the anti-coagulation properties of sulfoxide 2a are associated with their ability to stabilize the cellular membrane by van-der-Waals interactions with phospholipids of the external layer. This is proved by changes in platelet aggregation induced by arachidonic acid. Arachidonic acid, in contrast to other inducers, activates platelets directly penetrating into the cell, and reduces cyclic adenine monophosphate (cAMP) concentration. Therefore, the loss of this property in the presence of sulfoxide 2a might be due to the changes in permeability of platelet membranes associated with formation of additional intermolecular interactions between hydrophobic parts of the molecules and the phospholipids of the membrane. Decrease of spontaneous aggregation in the presence of thioterpenoid is associated with increased intracellular cAMP formation due to membrane lipids peroxidation blocking. Integration of the hydrophobic part of sulfoxide 2a molecule into the cellular membrane induces its stabilization and thereby prevents the utilization of the phosphatidylcholine molecules from the external layer as a formation source of lipid peroxidation products, which in turn will trigger the of platelet aggregation mechanisms. Sulfoxide 2a also reduced coagulation activity of the plasma: aPTT and prothrombin time became similar to normal values in presence of this compound. These facts indicate suppression of the internal way protrombinaze activation. Platelet activation and significant increase in microvesiculation during storage have been described in several studies. These processes are caused by membrane phospholipids reallocation of and exposure of negatively charged phosphatidylserine both on platelets and on the surface of microvesicles (Lawrie et al., 2009; Lhermusier et al., 2011). The results of our study show that the number of microvesicles changes is an informational marker of platelets activation process (Figure 6). In addition, this reallocation of platelets membrane phospholipids leads to initiation of coagulation hemostasis. Therefore, activation of these processes reduces both the number of functionally active platelets and pro-coagulant reserve of the blood plasma associated with accumulation of thrombogenic particles and activation of the plasma clotting enzyme complexes factors on the surface of membranes. This activity depends on the presence of microvesicles. Coagulation properties of plasma changes in presence of microvesicles from the platelet concentrate: APTT, prothrombin time reduces, and excess thrombin generation leads to acceleration of fibrin clots formation (increased speed of fibrin formation, size, density, and spontaneous clots appearance). Thrombogenic properties of microvesicles are confirmed by the results of their removal from plasma, leading to blood coagulation reduction (increasing fibrin formation initiation time and decreasing of other thrombodynamics indicators, Table 3). The results of our study also demonstrate incomplete stabilization of blood products in case of sodium citrate using. Extracellular calcium ions binding with sodium citrate exclude the possibility of their participation in the activation of platelets and coagulation factors. However, mobilization of intracellular calcium is sufficient for platelet initiating. Intracellular calcium concentration increases after the platelets receptors interaction with inductors—epinephrine, ADP, collagen, and others (Siess, 1989; Shitikova, 2000). The inhibition of receptor activity by sulfoxide 2a effectively decreased the platelet-derived microvesicles content (Figure 6). Platelet receptors function blocking property of sulfoxide is combined with the anticoagulant effects—aPTT, prothrombinc time and indicators of thrombodynamics reach normal plasma values. The observed hypocoagulative effect of sulfoxide 2a is induced by reduction of coagulation activity of microvesicles present in solution, because the same changes are caused by their removal from plasma (Table 3). Thrombogenic microvesicles are mainly associated with the presence of phosphatidylserine (Lhermusier et al., 2011) promoting Ca²⁺-dependent binding of its negative charge with vitamin K-dependent coagulation catalytic complexes on the surface of membrane (Cutsforth et al., 1989).

The results of our study revealed that coagulation activity of thioterpenoids is associated with selective inhibition of platelet aggregation and coagulation factors inhibition. Considering the low toxicity, the ability to block spontaneous- and induced-aggregation, sulfur-containing terpene compounds might be considered as the promising agents with perspective use for platelet blood products stabilization, treatment and prevention of thrombophilia.

LN: General leadership, results discussion, writing the article. SK: Biochemical assays, results discussion. VS: Synthesis. AB: Synthesis, article design. ZA: Biochemical assays. OS: Synthesis. IF: Synthesis. SB: Biochemical assays, statistic analysis. OL: Single-crystal X-ray. VK: NMR spectroscopy. LG: NMR spectroscopy. AK: Molecular dynamics simulations.