calix [ 4 ] arene C-145 Effects on Plasma Haemostasis

Results: C-145 was administrated intravenously in the dose of 7.5 mg/kg of rabbit weight. 4 hours after the injection the thrombin time and activated partial thromboplastin time of rabbit’s blood plasma were prolonged 2 and 1,5 times respectively. Such prolongation was observed after 24 hours as well. However, the total fibrinogen and prothrombin levels did not change. Parameters of fibrinolytic system (PAI-1, clot lysis half-time) and anticoagulant system (protein C) remained unchanged.

The anti-polymerization properties of С-192 and С-145 allowed us to consider them as potential compounds for the design a novel class of antithrombotic agents, which are able to inhibit specifically blood clot formation through the fibrin polymerization inhibition. To introduce a medicinal preparation, however, it is necessary to study its effects not only in vitro, but also in lab animals.
The present work was aimed to study the effect of intravenously injected С-145 on rabbit's haemostasis.

Methods
Sample of Calix [4]arene С-145 was dissolved in 0.9 % NaCl solution to the final concentration 15 mg/ml and was injected in the marginal ear vein of rabbit in the dose of 7.5 mg/kg using Wenflon catheter (Becton Dickinson, USA), G22 (0.8 mm). The animal research protocol for that study was approved by the Institutional Animal and Care Committee of Palladin Institute of Biochemistry of NAS of Ukraine (February 4, 2014, protocol number 1).
Blood samples were collected using Wenflon catheter (Becton Dickinson, USA), G22 (0.8 mm) before the injection and after 2, 4 and 24 hours after injection. 3.2 % Sodium Citrate added immediately after collection to whole blood in 1:9 ratio was used as an anticoagulant.
Blood plasma was prepared from citrated blood by centrifugation at 1200 rpm during 30 min.  Fibrinogen concentration in the blood plasma was determined by the modified spectrophotometric method. Blood plasma (0.2 ml) and PBS (1.7 ml) were mixed in glass tube. Coagulation was initiated by the addition of 0.1 ml of thrombin (2 NIH/ml). To avoid fibrin crosslinking 0.1 ml of 40 mM monoiodacetic acid was added to the sample. Mixture was incubated during 30 min at 37°C. The fibrin clot was removed and re-solved in 5 ml of 1.5 % Acetic acid. The concentration of protein was measured using spectrophotometer SF-2000 (Russia) at 280 nm (ε =1.5).
Prothrombin level was measured using ecamulin test (ET). Ecamulin is the prothrombin activator from Echis multisqumatis snake venom that activates prothrombin and all his inactive forms [5]. So it allowed us to determine total prothrombin level. Thrombin generation was measured by chromogenic substrate assay using thrombinspecific S2238. Results were presented as ecamulin (EI) index, that was calculated by formula: EI = Ap/An; PI= Ap/An, An -normal rabbit's blood plasma thrombin activity; Ap -calix [4]arene-treated rabbit's blood plasma thrombin activity.
Protein C level was determined using the activator of protein C according to [6]. The generation of activated protein C was measured by chromogenic substrate assay using specific chromogenic substrate S2236.
Overall haemostatic potential was studied by monitoring turbidity during clot formation and lysis. The mixture of 12 μl of citrated human plasma in 0.05 М НЕРЕS-buffer рН 7.4 (0.15 М NaCl, 5 mМ СаCl 2 ), 75 NIH/ml of tPA was clotted by 5 μl of thromboplastin and turbidity was monitored at 350 nm at room temperature as described in [7].
PAI-1 was detected using ELISA according to [8]. Mixture was contain 2 μl of blood plasma in 0.2 ml of TBS. Polyclonal anti-PAI-1 antibody and Goat anti-rabbit IgG conjugated to alkaline phosphatise were used for the detection. PNP formed after PNPP cleavage by alkaline phosphatase was determined at 405 nm using multiplate reader Multiscan EX.
Statistical data analysis was performed using Microsoft Excel. All assays were performed in series of three replicates and the data were fitted with standard errors using "Statistica 7".

Results
To evaluate the possible effect of the calix [4] arenes on haemostasis in vivo, healthy rabbits were intravenously injected with 1 ml of С-145 in saline solution (7.5 mg/kg). The quantity amounted to 1.5 % LD50. as determined for rats injected intraperitoneally [9]. As the total volume of an adult rabbit's blood is about 250 ml [10], the level of С-145 was about 0.06 mg/ml (46 μM). In vitro, this prolonged APTT in blood plasma 1.7-fold (Р = 0.05) [9].
Four hours after injection of С-145, APTT of the rabbit's blood plasma grew 1.5-fold and TT twice, and they remained so for 24 hr (Figure 2). TT was affected more because fibrin formation and polymerization are initiated directly by exogenous thrombin, while in APTT blood coagulation happens indirectly through the intrinsic pathway.
Therefore the anti-polymerization effect of С-145 shown in vitro also occurred in the intravenously injected rabbits. C-145 prolonged coagulation time in both basic tests (APTT & TT) with statistical significance, albeit unequally: TT grew 1.33 times more (Р = 0.05). It can be explained by APTT incorporating the whole coagulation cascade, while in TT we tested the influence of C-145 upon polymerization of fibrin originating in the last stage of the cascade: the fibrinogen + thrombin reaction.
We also studied an effect the calix [4]arene had on the other blood coagulation components. Main proteins of the system, fibrinogen and prothrombin, were not influenced by C-145 injection (Figure 3). This supports our conclusion about its selective and focused action only upon the fibrin polymerization stage in the coagulation cascade without any influence upon other protein factors [3].
The state of anti-coagulation branch of haemostasis system following С-145 injection was analyzed by protein C quantification in plasma after 4 and 24 hr.
It was shown that 4 hr after the injection of C-145, protein C level didn't practically change (Figure 4). However, later it decreased by 15-25 %. That the deficit increases after a whole 24 hr, points that C-145 doesn't have a direct impact on the anti-coagulation branch of the system [11].
A number of anti-coagulative preparations are known to coinfluence the fibrinolytic system [12]. It is known that plasminogen is activated on fibrin. As C-145 interacts with monomeric fibrin, preventing its polymerization, we had to study its effect on plasminogen activation, plasmin activity and overall fibrinolysis in plasma [13]. The main direct evidences for the activation of fibrinolytic system are the rise in plasmin activity in blood plasma and the generation of plasminogen activator inhibitors (PAI-1, α2-аntiplasmin) [14]. To reveal possible causes of the fibrinolysis inhibition that was seen after the С-145 injection, we measured PAI-1 in rabbit plasma [15,16]. It remained constant 4 and 24 hr after the injection, there was no significant difference between the control and the experiment ( Figure  5).
For a combined analysis of the blood clot's formation and its hydrolysis we measured haemostasis potential (HP), clotting potential (CP) and fibrinolytic potential (FP) in plasma before the injection and 2 and 4 hr after. The coagulation was initiated by thromboplastin, together with exogenous tPA to determine HP and FP [17].
After the injection, the HP, CP and FP in plasma were 11.0 -, 12.2-and 9.0-fold lower, respectively (Table 1). These data point to the very strong impact C-145 has on the plasma's clotting ability. On the other hand, it does not significantly influence the balance between the coagulative and fibrinolytic components of haemostasis, calculated as the FP/CP ratio (Table 1). This lets us to suppose that the inhibition FP is caused not by C-145's action on fibrinolysis, but by the reduction in size of the hydrolyzed fibrin clot.

Discussion
Antithrombotic preparations act by rapid and controlled decrease of the activity of blood coagulation. The main criteria of their quality are efficiency and lack of contraindications. They can be classified into anti-coagulants, which inhibit clotting on various stages, and profibrinolytics, which speed up the fibrin clot's destruction (t-PA, u-PA, streptokinase) [18,19]. Most anti-coagulants act directly upon thrombin (dabigatran) [20], factor Ха (rivaraxaban) [21], factors VIIIa and V (drotrecogin) [22], blocking the clotting cascade on the stages of factor X activation or prothrombin to thrombin and fibrinogen to fibrin transformations. A possibility of the use of anti-thrombotics directly acting upon the last link of blood clot formation, namely   fibrin polymerization, is a hot topic of research [23,24]. In particular, decreasing of the fibrin polymer density was attempted through adding silver nano-particles [25] or inhibiting peptides (e.g., GPRP), conjugated with albumin [26].
In this respect this calix [4]arene, being low-molecular and potentially non-immunogenic organic molecule may turn out to be potentially valuable highly specific antithrombotic agents, inhibiting fibrin polymerization by blocking the A polymerization site [2]. The effect was revealed to be quite significant in vivo in rabbits intravenously injected with С-145. Meanwhile it had no impact upon prothrombin and fibrinogen levels in plasma and no significant effect upon the activity of the fibrinolysis system and PAI-1 levels.
Therefore, we found a specific anti-polymerisation effect of the calix [4]arene С-145 in vivo comparable to effects in vitro. The substance did not notably influence the total level of prothrombin, fibrinogen, activity of the anti-coagulative and fibrinolytic systems and the balance between the fibrin formation and digestion.
The results allow us to consider calix [4]arene C-145 as a potential anti-thrombotic agent with narrow effect range. The next stage of research in vivo should focus on its impact upon the vascular-platelet link of haemostasis.   Table 1: Clotting potential (CP), haemostasis potential (HP), fibrinolytic potential (FP) and ratios of Clotting and Fibrinolytic potentials (CP/FP) in rabbit plasma before (0) and 2 and 4 hr after an IV injection of C-145.