Abstract In the present study, we investigated the capabilities of a novel ultrasonic approach for real-time control of fibrinolysis under flow conditions. Ultrasonic monitoring was performed in a specially designed experimental in vitro system. Fibrinolytic agents were automatically injected at ultrasonically determined stages of the blood clotting. The following clots dissolution in the system was investigated by means of ultrasonic monitoring. It was shown, that clots resistance to fibrinolysis significantly increases during the first 5 minutes since the formation of primary micro-clots. The efficiency of clot lysis strongly depends on the concentration of the fibrinolytic agent as well as the delay of its injection moment. The ultrasonic method was able to detect the coagulation at early stages, when timely pharmacological intervention can still prevent the formation of macroscopic clots in the experimental system. This result serves as evidence that ultrasonic methods may provide new opportunities for real-time monitoring and the early pharmacological correction of thrombotic complications in clinical practice.

Citation: Ivlev DA, Shirinli SN, Guria KG, Uzlova SG, Guria GT (2019) Control of fibrinolytic drug injection via real-time ultrasonic monitoring of blood coagulation. PLoS ONE 14(2): e0211646. https://doi.org/10.1371/journal.pone.0211646 Editor: Alexander V. Panfilov, Universiteit Gent, BELGIUM Received: October 15, 2018; Accepted: January 17, 2019; Published: February 27, 2019 Copyright: © 2019 Ivlev et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: In accordance with the requirements we uploaded the minimal (anonymized) data set necessary to replicate our study findings to stable public repository "Harvard Dataverse". Relevant DOI is: https://doi.org/10.7910/DVN/H00MOE. Funding: The present work was partially supported by the Russian Foundation of Basic Research (www.rfbr.ru) grants 14-04-01193 (DAI, SNS, SGU, GTG) & 16-34-01180 (KGG, DAI) and the Federal Program “5top100” (www.5top100.ru) (KGG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Monitoring and timely correction of hemostasis is a crucial medical task [1, 2]. A number of severe thrombotic pathologies, such as myocardial infarction and stroke, might occur suddenly and develop very rapidly [3, 4]. In these cases large thrombi occluding blood flow in major arteries can be formed during several minutes [4]. That is why prompt and efficient techniques for hemostasis monitoring are needed. Over the past two decades turnaround times of clotting tests were substantially reduced by introduction of so-called point-of-care techniques [5]. Novel methods for on-line ex vivo monitoring of hemostasis are actively developed [6]. A logical step towards real-time control of hemostasis would be creation of the technique for direct in vivo monitoring of intravascular blood coagulation. One of the possible approaches to creation of such a technique is the use of ultrasonic methods. The idea for applying ultrasonic methods to detect blood coagulation was proposed quite long ago, at first for in vitro measurements [7–9]. In recent years, due to developments in modern ultrasonic equipment, this area of research has become active again [10]. Various research teams have offered several ultrasonic techniques for the registration of blood coagulation in vitro [11–19]. More recently capabilities of ultrasonic methods for in vivo detection of blood coagulation were demonstrated in animal experiments [20–22]. It is essential that ultrasonic methods can detect blood coagulation under flow conditions similar to those that take place in major arteries of human body [23, 24]. This fact reveals the possible application of ultrasonic methods for non-invasive monitoring of coagulation processes in clinical practice [25]. Efficient control of hemostasis implies both its monitoring and means for its pharmacological correction. Usually monitoring can be performed with routine coagulation tests and correction can be achieved by use of anticoagulant drugs [1, 2]. But in acute situations, then formation of arterial thrombi has already started and progress rapidly, coagulation tests are already late and anticoagulants are not capable of thrombi dissolution. In these situations the last line of defense remaining is thrombolytic therapy [26, 27]. Its efficiency drastically depends on the delay after the onset of coagulation processes [28, 29]. A method for real-time monitoring of the onset of intravascular blood coagulation might be very useful in these acute situations for the reduction of onset-to-treatment time. In the present work we investigated possible benefits of ultrasonic detection of early stages of blood coagulation for fibrinolytic dissolution of forming thrombi. To do so, we designed a special experimental setup for the ultrasonic monitoring of blood coagulation under intensive flow conditions in vitro. This setup allowed us to monitor blood coagulation in real time and to perform an automated injection of a fibrinolytic drug at precisely determined stages of the coagulation process. Our experiments showed the following: The ultrasonic method used enables the reliable registration of blood coagulation and following fibrinolytic dissolution of clots. The method facilitates the qualitative evaluation of the efficiency of various fibrinolytic influences and enables the comparison of different fibrinolytic drugs; The fibrinolytic resistance of clots formed under flow conditions increases significantly over the first few minutes of their formation; An immediate injection of a fibrinolytic drug after the ultrasonic registration of the onset of coagulation is able to prevent the formation of large clots in the experimental system.

Materials and methods Ethics statement This study was approved by the Institutional Committee of Blood Donation and Blood Processing Problems at the National Research Center for Hematology (Permit number: 5/2016). This study was performed with blood received from healthy donors who provided written informed consent before blood collection in accordance with Russian Federal Law No 125 on July 20, 2012. All methods were carried out in accordance with relevant guidelines and regulations (The Order of Russian Health Care Ministry No 183n on April 02, 2014). Materials Whole blood and blood plasma were used in the experiments. The blood and fresh frozen plasma were provided by the Division of Blood and Blood Components Collection and Storage of the National Research Center for Hematology. Blood was preserved in Imuflex (Terumo Europe NV, Belgium) containers with citrate phosphate dextrose (CPD) anticoagulant solution. Plasma was separated from whole blood by centrifugation at 5000 g for 7 minutes. To initiate coagulation several types of activators were used: 50 μl of 1% kaolin suspension (NPO-Renam, Russia), 50 μl of thromboplastin solution, diluted by 12 times with normal saline (NPO-Renam, Russia) or 10% calcium chloride solution (Mapichem AG, Switzerland). Unless otherwise specified, activation of coagulation was initiated by injection of 600–800 μl of 10% calcium chloride solution. Three different types of fibrinolytic drugs were used in the experiments: streptokinase (Streptokinaza, Belmedpreparaty, Belarus), tissue-type plasminogen activator (Actilyse, Boehringer Ingelheim International, Germany) and urokinase (Urokinase, Medac GmbH, Germany). The dosages of the fibrinolytic drug were varied in different experiments, while the volume of the fibrinolytic solution injected into the experimental system (0.5 ml) was kept constant. Experimental setup The principal scheme of the experimental setup is shown in Fig 1. A closed system of flexible transparent silicone tubes (1 in Fig 1) was filled with either blood or blood plasma. The inner diameter of the tube was 4 mm, and the total volume of the experimental system was 18 ml. The flow of liquid in the system was generated by a peristaltic pump, Elpan type 372.1 (2 in Fig 1). The mean velocity of the flow was kept at a rate of 20 cm/sec (shear rate up to 400 s-1). The activators of coagulation were injected in flowing blood directly when experiment started. All experiments were performed at the room temperature, 24 ± 2°C. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. The layout of the experimental set-up. 1 –system of transparent flexible silicone tubes; 2 –peristaltic pump; 3 –digital camcorder; 4 –ultrasonic scanner; 5 –ultrasonic sensor; 6 –personal computer; and 7 –automated drug injector. https://doi.org/10.1371/journal.pone.0211646.g001 In the experiments with blood plasma, the processes of coagulation and fibrinolysis were registered both optically and acoustically. In the experiments with whole blood, due to its optical opacity, the registration was conducted only through the acoustic channel. Optical registration was performed in the transmitted light with a digital camcorder, GoPro HERO 3 (Woodman Labs, Inc., USA) (3 in Fig 1). A macro lens with an optical power of 21 diopters was used to focus the camcorder on the tube. The tube was held within the focal plane of the camera by a special screw clamp. The same screw clamp was used to create an area of local narrowing in the tube, beyond which a stagnation zone appeared in the flow. In several experiments, such a stagnation zone was created to facilitate the optical registration of fibrin microemboli, which form at an early stage of coagulation. Acoustic registration was performed via an ultrasonic scanner Vingmed SD50 (Vingmed Sound; Norway) working in a Doppler mode at a frequency of 5 MHz (4 in Fig 1). To reduce the signal loss, the ultrasonic sensor (5 in Fig 1) together with a section of the tube system, was immersed in a bath filled with degassed water. The data from the optical and acoustical registration were recorded on a personal computer (6 in Fig 1). A custom automated drug-injector (7 in Fig 1) was designed to perform the infusion of fibrinolytic drugs into the experimental system. The injector was connected to the computer via a Bluetooth channel. Following a signal from the computer, the injector delivered a fibrinolytic agent into the system in a precisely controlled and reproducible manner. The injection was performed gradually over 6 seconds, which was roughly equal to the turnover time of the liquid within the experimental system. A special computer program was written in Python for real-time data analysis and the control of drug-injector operations. The Doppler shift of the ultrasonic signal was transferred from the scanner to the computer, digitized in a format of 44100 Hz, 16 bit and subjected to filtration by a second-order Butterworth filter with a passband from 200 to 1600 Hz [30]. Subsequently, the modulus of the amplitude of the filtered acoustic signal was averaged for 2-second time intervals. This value, indicated below as the averaged modulus of amplitude (AMA), was used for the monitoring of blood coagulation and fibrinolysis in the system. Upon the increase in AMA above a certain threshold, the program sent a command signal to the drug-injector to perform the injection. The threshold value of AMA was defined basing on a series of preliminary experiments as the background level of AMA in the beginning of the experiment multiplied by a predefined coefficient (equal to 2 for blood plasma and 1.3 for whole blood). Calculation of fibrinolysis efficiency index The efficiency of fibrinolytic processes was assessed basing on the data from the acoustic registration after the end of each experiment. The area between the upper and the lower envelopes of the AMA curve was calculated for a time period of 60 minutes after the registration of the coagulation onset. This value calculated for the particular experiment was denoted as S exp , while S ref stands for the respective value calculated for a reference experiment with the blood (blood plasma) of the same donor, but with normal saline instead of a fibrinolytic drug injected. Finally, fibrinolysis efficiency index (FEI) was calculated with the following formula: It should be noted that S exp is proportional to the integrated intensity of the acoustic signal reflected by macroscopic clots in the system during the experiment. The faster the dissolution of fibrin clots, the smaller the value of S exp . Accordingly, FEI tends to one in cases of the immediate dissolution of all fibrin clots and is close to zero in cases of the complete absence of lysis in the experimental system.

Discussion Presently, ultrasonic methods are already used rather widely in the field of thrombosis and hemostasis, for instance, in the diagnostics of deep vein thrombosis [31, 32], the detection of thrombi in the left atrial appendage [33] and the monitoring of intravascular emboli [34]. Taking into account the recent achievements in the development of implantable ultrasonic sensors [35] it seems quite likely that, eventually, an ultrasonic technique for the monitoring of blood coagulation and thrombi formation inside the human body will be created. In our previous works, we have shown the applicability of ultrasonic methods for the non-invasive registration of coagulation processes occurring under intensive blood flow conditions [24, 25]. Further development of these methods seems to be very promising because they may enable coagulation monitoring in the areas of the vascular system where thrombus formation poses the greatest threat to the patient’s life and health, specifically, the large vessels of heart and brain. In the present study, it has been shown that ultrasonic methods enable the registration of coagulation processes at the stage when timely pharmacological intervention can still prevent the formation of macroscopic clots in the experimental system. Thus, it was shown that real-time ultrasonic registration of coagulation processes, in principle, provides the facility to control thrombi formation. The results presented in this paper may open prospects for creating portable or even implantable devices, which would be somewhat similar to insulin pumps currently used in clinical practice [36]. By means of ultrasound, such a device could provide not only the monitoring of blood clotting and fibrinolysis, but also active control of these processes. The miniature portable injector with several Doppler sensors on critical human arteries could timely inject fibrinolytics directly at the early stage of clotting when hemostasis could be corrected faster and more efficiently. In our experiments the resistance of clots to fibrinolysis increased drastically in the first few minutes of clots formation. The increase in the resistance of clots to the action of fibrinolytic agents with time is well known in clinical practice [28, 37]. Although our results show a similar trend to that of clinical observations, the time period within which the clots remained sensitive to the action of fibrinolytic agents turned out to be at least ten fold shorter in our experiments. The particular mechanisms underlying such a rapid increase in the fibrinolytic resistance of the clots are still unclear. However, it may be assumed that the effect observed in our work is the result of chemical stabilization of the clots on one hand [38], and on the other hand, changes in the structure of the clots, leading to a decrease in the permeation of fibrinolysis activators to the inner areas of the clots [39]. Concerning chemical stabilization of fibrin clots it is generally known that the action of coagulation factor XIII [38] and thrombin activatable fibrinolysis inhibitor (TAFI) [40] substantially increase the fibrinolytic resistance of forming clots. Both of the factors are converted to their active forms by thrombin. Activated factor XIII restrain fibrinolysis by covalent linking of α2-antiplasmin to fibrin [41] as well as by cross-linking of α- and/or γ-chains of fibrin [42]. Activated TAFI down-regulates fibrinolysis by removal of C-terminal lysines from fibrin, preventing in that way binding and activation of plasminogen [43]. Moreover it is worth to mention that blood flow itself can influence fibrinolytic resistance of forming clots in a bidirectional manner. On one hand flow influences the structure of the fibrin network [44, 45], making it more dense and less permeable to lytic agents, thus impeding the fibrinolytic process [46]. On the other hand, the flow substantially influences the character of the mass transfer inside the clot and near its surface, thus accelerating the fibrinolytic dissolution of the clots [47, 48]. Keeping this in mind, within the present work, it was essential to create the experimental conditions of flow to mimic thrombi formation taking place in large arteries. The experimental scheme chosen for this purpose is in a way, analogous to the well-known Chandler system [49], which is widely used up to date to create artificial clots mimicking arterial thrombi [50, 51]. Despite some differences in the setups, the development of coagulation processes in our experimental system was, in many aspects, similar to that observed in a classical Chandler system. For instance, the stage of multiple microemboli formation in the flow, resembling a “snow-storm”, which was observed in our experiments, was previously described for the Chandler system in experiments by McNicol et al [52]. Of course no in vitro experimental system could completely reproduce in vivo formation of arterial thrombi. Certainly, further in vivo investigations are required to answer a general question: whether the monitoring of the early stages of blood coagulation can increase the real clinical facilities for the prevention of thrombotic complications. Until recently, practically all research on the ultrasonic registration of blood coagulation has been carried out with in vitro model systems [7–18, 23–25, 53]. A few novel studies in this research field that have employed in vivo experiments have been published just recently [20–22]. The small number of such works may be attributed to the necessity of the convergence of several branches of modern science to carry out this type of research. We hope that the present work will attract additional interest and attention among researchers to further address the problems of ultrasonic monitoring of blood coagulation in vivo.