COAGULATION SYSTEM CHANGES AND THROMBOSIS PROPHYLAXIS DURING LAPAROSCOPIC FUNDOPLICATIONS

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

Indrė Žoštautienė

COAGULATION SYSTEM CHANGES

AND THROMBOSIS PROPHYLAXIS

DURING LAPAROSCOPIC

FUNDOPLICATIONS

Dissertation has been prepared at the Department of Radiology of Lithua-nian University of Health Sciences during the period of 2013–2018.

Scientific Supervisor

Prof. Dr. Mindaugas Kiudelis (Lithuanian University of Health Sciences, Biomedical Sciences, Medicine – 06B)

Dissertation is defended at the Medical Research Council of the Lithua-nian University of Health Sciences.

Chairperson

Prof. Dr. Brigita Šitkauskienė (Lithuanian University of Health Sciences, Biomedical Sciences, Medicine – 06B).

Members:

Prof. Dr. Saulius Lukoševičius (Lithuanian University of Health Scien-ces, Biomedical ScienScien-ces, Medicine – 06B);

Prof. Dr. Daimantas Milonas (Lithuanian University of Health Sciences, Biomedical Sciences, Medicine – 06B);

Prof. Dr. Nomeda Rima Valevičienė (Vilnius University, Biomedical Sciences, Medicine – 06B);

Prof. Dr. Mindaugas Andrulis (Institute of Pathology University of Ulm (Germany), Biomedical Sciences, Medicine – 06B).

Dissertation will be defended at the open session of the Medical Research Council of the Lithuanian University of Health Sciences on the 27ͭ ͪ of June 2018, at 11 a.m. in Big Auditorium of the Department of Endocrinology of the Hospital of Lithuanian University of Health Sciences Kauno Klinikos.

LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS

Indrė Žoštautienė

KOAGULIACINĖS SISTEMOS

KITIMAI IR PRIEŠTROMBOTINĖ

PROFILAKTIKA LAPAROSKOPINIŲ

FUNDOPLIKACIJŲ METU

Disertacija rengta 2013–2018 metais Lietuvos sveikatos mokslų universiteto Radiologijos klinikoje.

Mokslinis vadovas

Prof. dr. Mindaugas Kiudelis (Lietuvos sveikatos mokslų universitetas, biomedicinos mokslai, medicina – 06B)

Disertacija ginama Lietuvos sveikatos mokslų universiteto medicinos mokslo krypties taryboje:

Pirmininkė

Prof. dr. Brigita Šitkauskienė (Lietuvos sveikatos mokslų universitetas, biomedicinos mokslai, medicina – 06B).

Nariai:

Prof. dr. Saulius Lukoševičius (Lietuvos sveikatos mokslų universitetas, biomedicinos mokslai, medicina – 06B);

Prof. dr. Daimantas Milonas (Lietuvos sveikatos mokslų universitetas, biomedicinos mokslai, medicina – 06B);

Prof. dr. Nomeda Rima Valevičienė (Vilniaus universitetas, biome-dicinos mokslai, medicina – 06B);

Prof. dr. Mindaugas Andrulis (Patologijos institutas, Ulmo universitetas (Vokietija), biomedicinos mokslai, medicina – 06B).

Disertacija ginama viešajame medicinos mokslo krypties tarybos posė-dyje.

2018 m. birželio 27 d. 11 val. Lietuvos sveikatos mokslų universiteto ligoninės Kauno klinikų Endokrinologijos klinikos Didžiojoje auditorijoje.

CONTENT

ABBREVIATIONS ... 7

1. INTRODUCTION ... 8

1.1. Aim of the study ... 9

1.2. Tasks of the study ... 9

1.3. Novelty and originality of the study ... 10

1.4. Practical significance of the study ... 10

2. LITERARY REVIEW ... 11

2.1. Postoperative thrombosis – common surgery problem ... 11

2.2. The role of blood coagulation system in the development of thrombosis in surgical patients ... 17

2.3. Modern thrombosis diagnosis ... 19

2.4. Prophylaxis of DVT in surgical patients ... 27

3. STUDY POPULATION AND METHODS ... 33

3.1. Study population and its general characteristics ... 33

3.2. Study protocol ... 36

3.3. Blood collection and processing ... 37

3.4. Test method of thrombin-antithrombin complex in the venous blood ... 38

3.5. Test method of plasma prothrombin fragment F1+2 in the venous blood ... 38

3.6. Test method of free tissue factor pathway inhibitor in the venous blood ... 39

3.7. Test method for an activity of tissue factor micro-particles in the plasma ... 39

3.8. Thromboelastography ... 40

3.9. Lower extremity deep vein ultrasound ... 42

3.10. Computed Tomography Venography ... 43

3.11. Statistical analysis ... 44

4. STUDY RESULTS ... 46

4.1. Effect of the antithrombotic prophylaxis in thromboelastographic parameters changes ... 46

4.2. Effect of the antithrombotic prophylaxis in blood coagulation parameters changes ... 51

4.3. Evaluation of correlation between studied parameters ... 55

5. DISCUSSION ... 58

5.1. Influence of prophylactic measures on the changes in thromboelastogram indicators ... 58

5.2. Effects of Prophylaxis on the Changes in TAT and F1+2 in the Plasma ………...60

5.3. Influence of Prophylactic Measures on the Changes of MP-TF in the Plasma ... 62

5.4. Influence of Prophylactic Measures on the Changes of Free TFPI in the Plasma ... 63

5.5. Influence of Preventive Measures on the Development of DVT ………65 6. CONCLUSIONS ... 69 7. BIBLIOGRAPHY LIST ... 70 8. LIST OF PUBLICATIONS ... 89 SANTRAUKA ... 110 ATTACHMENTS ... 127 CURRICULUM VITAE ... 130 ACKNOWLEDGEMENTS ... 132

ABBREVIATIONS

CDDUS – Color and duplex Doppler ultrasonography CTV – Computed tomography venography

DVT – Deep vein thrombosis

ELISA – Enzyme like immunosorbent assay

Free-TFPI – Free plasma tissue factor pathway inhibitor GBP – Gastric bypass surgery

GCS – Graduated compression stockings GERD – Gastroesophageal reflux disease IAP – Intra-abdominal pressure

IPC – Intermittent pneumatic compression

LMWH – Low molecular weight heparin MP-TF – Tissue factor micro-particles MPs – Microparticles

MRPA – Magnetic resonance pulmonary angiography MRV – Magnetic resonance venography

NFD – Nephrogenic systematic dermopathy NSF – Nephrogenic systematic fibrosis OC – Open cholecystectomy

PE – Pulmonary embolism

ROTEM – Rotational thromboelastometry

SAGES – Society of American Gastrointestinal and Endoscopic Surgeons SD – Standard deviation

TAT – Thrombin-antithrombin complex TEG – Thromboelastography

TFPI – Tissue factor pathway inhibitor t-PA – Tissue plasminogen activator US – Ultrasonography

1. INTRODUCTION

Venous thromboembolism (VTE) is one of the following two interrelated pathologies: pulmonary embolism (PE) and deep vein thrombosis (DVT). 1/3 of VTE cases are reported as pulmonary embolism, 2/3 of cases – as deep vein thrombosis. DVT remains an important issue in health system as it is associated with complications related to high morbidity and mortality rates: acute and chronic pulmonary embolism, pulmonary hypertension and post-thrombotic syndrome. The cause of pulmonary embolism is usually a thrombus formed in proximal veins and pelvic deep veins.

The estimated average annual incidence rate of overall VTE among per-sons of European ancestry ranges from 104 to 183 per 100,000 person-years [78, 126, 177, 176, 185]. VTE recurs frequently; about 30% of patients de-velop recurrence within the next 10 years [78, 176, 178, 204]. Venous thromboembolism is high incidence among patients who undergo surgery: 20-30% after general surgical operations, 50-75% after orthopedic pro-cedures [41], 0.2-9.46% after laparoscopic surgery [41, 81, 122, 130, 173, 213]. Patients with VTE are at increased risk of dying, especially within the first year after diagnosis, but also during the entire 30-years of follow-up with VTE as an important cause of death [174]. While 30-day mortality after DVT remained fairly constant over the last three decades, it improved markedly for PE [174]. Pulmonary thromboembolism is a feared com-plication of DVT. The mortality rate in untreated cases is 25-30%, whereas the mortality rate in treated cases decreased to 5-8% [111]. In the existing literature the magnitude of long-term mortality after VTE varies substan-tially [174]. A recent study reported an eight-year mortality risk of 12%, while in an earlier study mortality risk reached 50% after eight years of follow-up [174]. Previous studies were limited by short follow-up time (maximum 10 years) [174].

Currently, laparoscopic operations account for about 50-60% of all sur-gical interventions; moreover, their range has already expanded rapidly and continues to grow.

1. During the surgery, the peritoneal cavity is inflated with CO2 gas, which results in the appearance of pneumoperitoneum of up to 12-14 mm Hg. The increased intra-abdominal pressure exerts a v. cava inferior in the retroperitoneal space and veins of the hips – v. illiaca dextr. et sin., thereby aggravating venous blood flow from the legs;

2. Operations are performed with patients’ legs directed downwards (even at an angle of 45° - in the reverse Trendelenburg direction). It also

complicates venous blood flow from the legs. This is especially true during laparoscopic fundoplications.

As a number of patients undergoing laparoscopic gastrofundoplications is growing, there is an increased likelihood of the incidence of both DVT and PE. To prevent these complications an optimal preventive measure or combinations of them is necessary.

Research work was performed at the Clinics of Radiology and Surgery of the Hospital of Lithuanian University of Health Sciences. It analyzed in the coagulation changes system during and after laparoscopic gastrofun-doplications, evaluated the efficacy of medical prophylaxis assigned to dif-ferent treatment regimens, and used the radiological examination methods to monitor the state of deep veins concerning possible thromboembolic complications. The dissertation summarizes the results of this work.

1.1. Aim of the study

To evaluate the optimal coagulability state and deep vein thrombosis prophylaxis according during laparoscopic fundoplications.

1.2. Tasks of the study

1. To determine a hypercoagulation state by thromboelastography, throm-bin-antithrombin complex, levels of prothrombin fragment F1+2, tissue factor micro-particles in patient’s plasma during and after laparoscopic fundoplications.

2. To determine a hypocoagulation state by free plasma tissue factor path-way inhibitor in patient’s plasma during and after laparoscopic fundop-lications.

3. To find out changes of these factors in patient’s, receiving different deep vein thrombosis prophylaxis regimes.

4. To estimate the sensitivity and specificity of Doppler ultrasound in determining postoperative deep vein thrombosis and assess the rate of thrombosis in studied subjects.

5. To identify the possible correlation between the most sensitive hipo-coagulation (free plasma tissue factor pathway inhibitor) and hiper-coagulation (tissue factor micro-particles) markers in the plasma.

1.3. Novelty and Originality of the study

With the onset of an era of laparoscopic surgeries, specialists soon started to take interest in their effects on various body systems, in particular changes in the coagulation system. The majority of the previous studies have monitored coagulation status only in the early postoperative period, most often within 72 hours after the surgery. We focused on an assessment of coagulation status in patients during and after laparoscopic gastrofun-doplication and one month after the surgery, when these patients were already predominantly discharged.

In our study, the efficacy evaluations of intraoperative deep vein throm-bosis prophylaxis were performed using advanced scientific research metho-dology – random sampling. The research was performed using the latest equipment and technologies: double computed thomography scanning and color Doppler; moreover, the changes in the coagulation system were mea-sured by accurate laboratory tests that are not used in everyday practice.

1.4. Practical significance of the study

The 9th American College of Chest Physicians guidelines provide no recommendation regarding the duration of venous thromboembolism prophylaxis in surgery for benign diseases; moreover, no definite consensus exists because of the absence in the sufficient numbers of valid data. Routine pharmacologic prophylaxis of venous thromboembolism in general surgery is recommended only for moderate and high risk patients [70]. Some authors demonstrate the feasibility of short postoperative venous thromboembolism prophylaxis without higher rates of venous thrombo-embolism. They recommend individualised duration of venous thromboem-bolism prophylaxis administration based on the venous thromboemthromboem-bolism risk stratification in concrete patients [112, 191].

This study was useful in objectively determining changes in the rates of the coagulation system during laparoscopic gastrofundoplications, and in showing that they are statistically significantly increasing during the surgeries – that a hypercoagulable state develops. Based on these findings, it can be argued that these surgeries are attributable to the middle or high risk of deep vein thrombosis development, while intraoperative measures of deep vein thrombosis prophylaxis should be applied during these operations. All this allowed the motivated recommendation of low-molecular-weight-heparin and multi-chamber stockings as the most effective intraoperative measures of deep vein thrombosis prophylaxis used during laparoscopic gastrofundoplications.

2. LITERARY REVIEW

2.1. Postoperative thrombosis - common surgery problem

The reasons for venous thrombosis have been captivating scientists for over 100 years. In 1856, Virchow described triad of factors employed in the development of venous thrombosis:

1. Venous stasis – abnormalities in the blood flow. 2. Endothelial damage – injuries to vascular endothelium.

3. Hypercoagulable state (activation of coagulation) – biochemical im-balance between circulating factors.

Laparoscopic surgery may lead to high rate of postoperative thromboem-bolic complications because of longer operating time, placement patients in the reverse Trendelenburg position increased intraabdominal pressure, which causes venous stasis in legs [122]. Venous stasis has long been recognized as one of the factors that may increase the risk of deep vein thrombosis and pulmonary embolism [97, 168]. Venous stasis is caused by the slowed blood flow, the increased blood volume in veins of the legs and the appearance of turbulence near the valves. This gives rise to thromboem-bolic complications due to venous stasis in legs, which is induced during a laparoscopic surgery by pneumoperitoneum [7, 28, 118, 144]. Scientists are increasingly interested in the role of pneumoperitoneum for the blood coagulation system during a laparoscopic surgery. The aim of Topal et al.‘s study [192] was to determine the influence of pneumoperitoneum at 10, 13, and 16 mmHg using thromboelastograph (TEG) in laparoscopic cholecys-tectomy. They have shown that 16 mm Hg intra-abdominal pressure (IAP) during laparoscopic surgery alter the TEG values and increase thrombotic activation. This may be due to the fact that high intra-abdominal pressure causes more stasis in the blood flow. For this reason, low intra-abdominal pressure must be used for peritoneal insufflation during a laparoscopic sugery to prevent thrombotic complications. Hasukic reported that in case of the patient in reverse Trendelenburg position present in the majority of laparoscopic procedures, the raised IAP increases venous stasis in the lower extremities and reduces the return of blood from the lower extremities by more than 40% [77]. Potential risk of deep vein thrombosis is present in these patients [66].

Short-term increased activity of the blood coagulation system (hyper-coagulation) can be noticed during an early postoperative period, and can be determined by different laboratory tests. The most accurate diagnosis of blood clotting disorders can be guaranteed by the following sensitive criteria

of coagulation and fibinolysis: tissue factor, prothrombin fragment F1 + 2 and TAT correspond to the formed volume of thrombin and can be regarded as coagulation activity markers [16, 122]. Fibrinogen is a marker of acti-vated coagulation and fibrinogen also belongs to the group of acute phase proteins. Zezos et al. found a significant activation of coagulation and fibri-nolysis during the immediate postoperative period in all patients, supporting the increased thromboembolism risk following a laparoscopic surgery [218, 219]. F1+2 and TAT plasma levels show an increase in the immediate postoperative period (1 hour postoperatively) with a drop after 24 hours, whereas D-dimers are increased 24 hours postoperatively. Data from the studies [167,196] with more extended time points of postoperative samples has shown a continuous decline and final normalization of F1+2 and TAT plasma levels in the next 48 to 72 hours after laparoscopic cholecystectomy, while at the same time points D-dimers may decline to normal values after 48 hours [122, 167] or may stay elevated even after 72 hours [118, 196], indicating on going fibrinolysis.

Experimental evidence suggests that venodilatation caused by venous stasis in surgical treatment may increase the mechanical stress on a con-nective tissue of the vein walls, leading to microtears in the endothelial lining of peripheral veins and exposing the blood to thrombogenic material [40, 192]. In case of endothelial dysfunction, subendothelial collagen in a blood vessel stimulates the release of clotting factors – thromboplastin and Willebrand factor.

Surgery is associated with an organism’s stressful reaction, which is called a systemic inflammatory response or acute phase reaction. Systemic inflammatory response is a normal reaction of the body to any trauma. Several authors have already published the results of the systemic inflam-matory response in patients after an abdominal hernia surgery during the early postoperative period (within 48 h after the surgery) [9, 71, 89, 152, 184, 201, 202]. Di Vita et al. and Sista et al. have measured the systemic inflammatory response after open and laparoscopic cholecystectomy till the 7th and 12th postoperative day, respectively [44, 171]. According to the results of the study by Ostrowski et al., glycocalyx found in the endothelium of a blood vessel, as well as inflammatory mediators and stress hormones, play a very important role in regulating the coagulation system during severe tissue damage [136]. Postoperative hypercoagulation and the level of blood clotting markers may vary depending on the systemic inflammatory response, type of a surgery, postoperative complications and postoperative medication use [199].

Lower extrimity deep vein thrombosis is a serious medical condition that can result in death or major disability to pulmonary embolism (PE) or

post-thrombotic syndrome. Clinical expression of acute lower extremity DVT depends on an anatomical position of the thrombus, its size, and a degree of vascular occlusion. It can start from asymptomatic DVT and end with severe oedema, pain, erythema and cyanosis with imminent venous gangrene, fever, as well as the appearance of a superficial venous network and pain with passive dorsiflexion of the foot (Homan’s sign) [123]. Unfortunately, the diagnosis of DVT based on clinical signs and symptoms is certainly inaccurate. DVT symptoms and signs are non-specific and may be related to other lower extremity disorders, including lymphedema, superficial vein thrombosis, cellulitis, muscle, bone trauma and Baker‘s cysts. None of the signs or symptoms is sufficiently sensitive or specific, either alone or in combination, to accurately diagnose or exclude thrombosis [138]. The most common areas for lower extremity vein thrombosis are the following: iso-lated veins of the calf (distal), femoropopliteal, and iliofemoral thrombosis, and symptoms tend to be more severe as thrombosis extends more proximally [123]. Although there are no precise epidemiological data, it is determined that the frequency of PE is approximately 60-70 cases per 100,000 inhabitants and that of venous thrombosis is approximately 124 cases per 100,000 inhabitants [134, 211]. The European guidelines for the diagnosis and management of PE state that the frequency of annual vein thrombosis and PE is about 0.5-1.0 cases per 1,000 inhabitants [193]. However, the actual numbers are likely to be significantly higher as silent PE can develop in up to 40-50% of patients with deep vein thrombosis. In addition, autopsy studies have shown that PE was diagnosed prior to the death in 30-45% of patients [145]. Untreated acute PE is associated with a significant mortality rate (up to 30%), while mortality rate for the diagnosed and treated PE is reduced to 8%. Up to 10% of patients with acute PE die suddenly. Two out of three PE patients die within the first 2 hours after the onset of embolism [65,193,211]. The most common sources of PE (in up to 85% of cases) include DVT followed by thrombosis of iliac and renal veins, and the inferior vena cava [90]. Byrne JJ et al. and Kakkar et al. state that more than 90% of pulmonary emboli arise from deep veins of the legs and pelvis, and the primary risk factor for reccurent pulmonary embolism is the presence of residual proximal venous thrombosis [23, 92]. Hull RD, Pineo GF, Huisman MV et al. have established that isolated distal vein thrombosis rarely leads to clinically relevant PATE; however, inappropriate treatment results in the spreading of calf vein thrombosis to the upper arm and thigh (proximal) veins in about 20% of cases [86,88]. Loud PA. et al. analyzed data from their acute PE registry, which included patients with verified acute and subacute PE and showed that 20% of patients belonged to the group with massive PE, 47% – to the group with submassive PE and the

remaining 33% of the patients had small PE [111]. Overall, 67% of cases could be classified as hemodynamically severe PE. Total in-hospital patient mortality was 5.6%, involving 24.5% and 1.6% mortality for massive and submassive PE, respectively [111]. The highest risk of embolism is the first 72 hours after the onset of thrombosis [128]. Complications of DVT are not only PATE but also post-thrombotic syndrome. This is an important late complication of acute lower extremity DVT. Older studies have found post-thrombotic syndrome in 2/3 of patients with acute lower extremity DVT. Newer studies, on the other hand, have shown that PTS develops in 29.6% of patients with acute lower extremity thrombosis and in 30% of patients with isolated calf vein thrombosis [150]. The Austrian Study on Recurrent Venous Thromboembolism was a prospective cohort study, which offered the opportunity to study PTS in a large number of well-defined patients with a first DVT of the leg. Within an average of 3 years, 43% of these patients developed PTS. Severe, disabling PTS with ulcers was, however, a rare event seen in only < 2% of cases. A similar frequency of PTS was reported in other prospective studies as well [20, 58, 91, 150, 151]. The study by Stain M. et al. has shown that older age, male gender and proximal DVT (compared to distal DVT) are associated with an increased risk of PTS [180]. One of the manifestations of post-thrombotic syndrome – trophic ulcers of the lower legs – develops in 4 to 8% of patients with proximal DVT [55]. Development of PTS is also associated with an increased risk of recurrent VTE. An important finding of the study by Stain M. is that the presence of PTS confers an almost threefold increased risk of recurrent VTE [180] (Table 2.1.1).

Table 2.1.1. DVT frequency after an open or laparoscopic surgery

Groups of operations

and patients Used prophylaxis Number of studies of patients Number DVT rate (%) Open GBP

Nguyen et al. 2001 [3]

Brasileiro et al. 2008 [129] IPC Enoxaparin 1 1 34 57 0.79 2.9

In total 2 91 3.89

Open cholecystectomy Milic et al. 2006 [4]

Lord et al. 1998 [130] Without prophyl. LMWH and IPC 1 1 56 41 16.07 2.4

In total 2 97 18.47

Laparoscopic GBP

Wittgrove and Clark 2000 [69] Higa et al. 2000 [70]

Nguyen et al. 2001 [3] Without prophyl. IPC 1 1 1 500 1040 36 0 0.2 0 In total 3 1576 0.2% Laparoscopic cholecystectomy Milic et al. 2006 [4] Ulrych et al. 2016 [30] Compagna et al. 2013 [112] Schaepkens van Riempst et al. 2001 [118]

Schaepkens van Riempst et al. 2001 [118] Without prophyl. IPC and LMWH Without prophyl. Without prophyl. Nadroparin 1 1 1 1 1 58 90 90 133 105 6.9 0.46 0 1.68 0.42 In total 5 476 9.46

Abbreviations: DVT – deep vein thrombosis; IPC – intermittent pneumatic compression; LMWH – low molecular weight heparin; PE – pulmonary embolism; GBP – gastric bypass surgery.

Video-laparoscopic surgery is probably the fastest evolving field of minimally invasive surgery. Currently, laparoscopic surgeries account for approximately 15% of all surgical interventions; moreover, their scope has already expanded rapidly and continues to grow.

Since the publication of the guidelines for venuos thromboembolism (VTE) prophylaxis by the Society of American Gastrointestinal and Endoscopic Surgeons (SAGES) during the laparoscopic surgery in 2007 [173], the American College of Chest Physicians (ACCP) has published their comprehensive guidelines that address VTE prophylaxis for non-ortho-pedic surgery patients [70]. The ACCP guidelines utilize the VTE risk stratification systems by Rogers et al. [160] and Caprini [30]; they also outline prophylactic strategies based on the calculated risk of VTE (Table 2.1.2).

Table 2.1.2. VTE risk levels and reccomendation according to the risk level

after applying the Caprini [30] risk assessment model (RAM)

Risk Incidence of calf DVT (%) Incidence of proximal DVT (%) Incidence of clinical PE (%) Incidence of fatal PE (%) Successful prevention strategies Low Minor surgery in patients with aged <40 years, with no

additional risk factors

2 0.4 0.2 <0.01 No specific prophylaxis

Moderate

Minor surgery in patients with additional risk factors

Surgery in patients aged 40-60 years with no additional risk factors 10-20 2-4 1-2 0.1-0.4 LDUFH every 12 hours, LMWH (3400 U daily), graduated compression stocking or IPC High

Surgery in patients age >60 years or age 40-60 with additional risk factors (cancer, prior VTE, established hypercoagulability disorder) 20-40 4-8 2-4 0.4-1.0 LDUFH every 8 hours, LMWH (>3400 U daily) or IPC Highest

Surgery in patients with multiple risk factors (age >40 years, cancer, prior VTE)

Hip or knee arthroplasty or hip fracture surgery Major trauma or spinal cord injury 40-80 10-20 4-10 0.2-5 LMWH (>3400 U daily), fondaparinux, oral vitamin K antagonist or either IPC or graduated compression stockings plus LDUFH or LMWH Abbreviations: DVT – deep vein thrombosis; IPC – intermittent pneumatic compression; LMWH – low molecular weight heparin; PE – pulmonary embolism;

2.2. The role of blood coagulation system in the development of thrombosis in surgical patients

Any surgical intervention is bigger or smaller tissue damage which acti-vates blood coagulation system and platelets. When applying or maintaining anaesthesia, two major disorders of the coagulation system – clinically manifested by bleeding or thrombosis – may occur during and after a surgery. Blood clotting is a natural process in which blood cells and strands of fibrin clump together to stop bleeding after blood vessel was injured. The clot forms the protective scab over healing wound. The structure of the blood coagulation system is quite complicated and its potential in healthy body is very high. Blood in the vessels stays liquid even after severe tissue damage or local thrombosis. Anticoagulants, on the other hand, cannot gua-rantee the stoppage of thrombotic process, which in its turn indicates that in case of pathology the protective and compensatory possibilities of the coa-gulation system are limited [4, 13, 106]. The blood coacoa-gulation system con-sists of three main components: tissues (vascular and tissue coagulation factors), platelets (platelets and their factors) and humoral (plasma factors, activators and inhibitors of clotting and fibrinolysis) [115].

In 1882, Bizzozero recognized the platelet a cell structure different from red blood cells and white blood cells. It was not until 1970, however, that scientists recognized the relationship of platelets to hemostasis and throm-bosis as significant and extensive [38, 147]. Contemporary understanding of the coagulation system is based on cascading mechanism offered by O. Rat-noff and R.G. MacFarlane [1967], i.e. on the hypothesis of consistent acti-vation of the coagulation factor by its predecessor. Coagulation is divided into two major systems: primary and secondary systems of hemostasis. Pri-mary hemostasis, which includes acute vasoconstriction, platelet adhesion and platelet aggregation, is the most active after the injury. Depending on the size of an infection, secondary hemostasis, which activates coagulation factors, is activated; moreover, fibrin is also formed. In addition to the activation of hemostasis, fibrinolysis, followed by thrombolysis, is activated [102]. The most important moment in all reactions is the generation of thrombin by two pathways, which are called intrinsic and extrinsic path-ways, and which are then merged into a common one (Fig. 2.2.1). The extrinsic pathway is initiated with the release of tissue thromboplastin, which is expressed after vessels damage. The complex formed by factor VII, tissue thromboplastin, and calcium converts factors X and Xa, which in their turn convert prothrombin to thrombin. Thrombin convert fibrinogen to fibrin [38]. This entire process takes between 10 and 15 seconds. The intrinsic system is a contact activation pathway. The intrinsic pathway is

slower and starts with an activation of factor XII. However, factor XIIa not only triggers the coagulation system but also stimulates the kinin–kallikrein, fibrinolytic and complement systems. These two pathways meet at the common pathway, where they both generate factor Xa from X, leading to a common pathway of thrombin from prothrombin and the conversion of fibrinogen to fibrin [38].

The process of hemostasis is completed with fibrinolysis, which destroys the clot made of fibrin and blood cellular components; moreover, it also restores the integrity of a blood vessel and blood flow. This process also occurs on two different paths: on the intrinsic path, when the activation begins together with the clotting system activation; and on the extrinsic path, when in case of tissue damage, tissue plasminogen activator (t-PA) begins its activity. Fibrinolysis is controlled by the plasminogen activator system. Components of this system are found in tissues, urine, plasma, lyso-somal granules, and vascular endothelium [38]. Tissue plasminogen active-tor (t-PA) activates plasminogen to plasmin, resulting in fibrin degradation.

2.3. Modern thrombosis diagnosis

First of all, it is very important to identify risk factors for DVT. It has been shown that between 30% and 60% of patients do not manifest any DVT symptoms [75]. Therefore, special laboratory and instrumental equipment, i.e. special testing using the apparatus that can accurately and timely diagnose DVT, is required. It should be performed when risk factors rather than signs of DVT are present.

Thromboelastography (TEG) is a point-of-care whole blood coagulation monitor which provides an information on the specific aspects of coagu-lation including time of the production of initial fibrin strands (R-time), time to develop a clot (R-time, K-time), rate of fibrin build-up and cross linking (a-angle), the maximum clot strength (maximum amplitude–bMA) and mea-sures of fibrinolysis (decreasing amplitude post-MA) [161]. Thromboelas-tography was developed and described by Helmut Harter in 1948 (Hei-delberg, Germany) [76]; however, in clinical practice, testings began only in 1974 during liver transplantations [84]. More than 40 years after the disco-very of the method, thromboleastography has become more widespread in other areas as well, i.e. in intensive care units, in the fields of cardiosurgery, obstetrics and emergency anaesthesia, as well as in cases of severe trauma-caused massive bleeding [210].

With the help of developing technology and the use of graphy, the following two types of apparatus were offered: thromboelasto-grapher – TEG 5000 Hemostasis Analyzer System (Haemonetics Corp., Braintree, MA) and rotational thromboelastography ROTEM (Tem Inter-national GmbH, Munich, Germany).

TEG analysis is more sensitive to the qualitative defects in fibrin or platelets than standard laboratory tests [105]. The technique of thrombo-elastograph hemostasis analysing provides a measurement of dynamic

changes in viscoelasticity property of the blood clot and gives a permanent graphic documentation [119]. Thromboelastography is also the method to monitor the hemostasis that is easy to use in the operation theatre while the anaesthesiologist is provided with rapid and acurate results. TEG tracing provides all phases of the process of hemostasis from the formation of the first fibrin strands to the fibrinolytic dissolution of the clot [105]. TEG is a technique that gives a global overview of the coagulation cascade including fibrinolysis. Many workers have tried to correlate TEG parameters with conventional coagulation profile but it is not possible to achieve as both techniques are different [127]. Commonly used blood tests are often aspe-cific, whereas the dosage of all plasma factors involved in coagulation and platelet activity is expensive and mostly not useful to individuate the clinical risk of hypercoagulability [53]. However, to some extent, R correlates with APTT, while an angle correlates with fibrinogen (and platelet function), MA correlates with platelet function (and fibrinogen) [33, 93, 135, 140, 195, 197] and a whole blood clot lysis index correlates with euglobulin lysis time, fibrin degradation products (FDP) [127]. Recently, a number of artic-les stating that TEG should become an integral part of the diagnostic equip-ment in each medical institution have been increasing; moreover, according to some researchers, part of clinical situations could lead to the abandon-ment of routine blood coagulation tests and the use of more precise TEG methodology [143].

Prothrombin fragment 1+2 (F1+2) is a short-lived peptide released when prothrombin is converted to thrombin during an activation of the coagu-lation cascade and its plasma levels are used as markers of thrombin gene-ration [219]. The activation of prothrombin takes place in the presence of factor Xa, factor Va, calcium ions and a phospholipid suface (platelets) [72]. For the measurement of prothrombin fragment Fl+2 enzyme-linked immu-nosorbent assay (ELISA) tests are used. The first test was developed in 1982 by the group of Teitel et al. [187] and was a radioimmunoassay using polyclonal antibodies against prothrombin fragment Fl+2 and F2. When the coagulation system is activated under pathological conditions only a small amount of circulating prothrombin is activated to thrombin (1%) [72]. Furthermore, the resulting enzyme is rapidly neutralized by antithrombin. Quantitation of prothrombin fragment F1+2 or thrombin-antithrombin (TAT) complexes should allow monitoring of small degrees of the activa-tion of coagulaactiva-tion with the formaactiva-tion of thrombin [72].

The findings by Ota et al. suggest that high concentrations of the hemo-static molecular markers, especially F1+2, which is also known as a marker for a hypercoagulable state, reflect a high risk of thrombosis [137].

Tissue factor pathway inhibitor (TFPI) is a coagulation inhibitor that modulates initiation of coagulation induced by tissue factor. It is now well established that the prime trigger to blood coagulation in vivo is tissue fac-tor (TF) that is exposed on the surface of fibroblasts as a result of a vessel wall injury [57]. TFPI is synthesized by vascular endothelial cells and part of it is associated with glycosaminoglycans of these cells. In the blood TFPI is found in a free-form (active) and in a nonactive form associated with lipo-protein. TFPI directly inhibits activated factor Xa and then – factor VIIa/TF complex [163]. A decreasing TFPI activity facilitates an activation of blood coagulation and fibrin forming; increasing TFPI activity inhibits these pro-cesses [163].

D-dimer levels have been used as a marker of intravascular clot forma-tion. [142]. D-dimer is a cross-linked fibrin degradation product, which forms as a result of a breakdown of fibrin. D-dimer levels are frequently in-creased after a surgery or trauma and indicate the presence of the intravas-cular clot that has undergone lysis [130]. D-dimers can be measured by an enzyme-linked immunosorbent assay (ELISA) or by the use of coated latex particles that agglutinate in the presence of plasma containing D-dimer, with the degree of agglutination directly proportionate to the concentration of D-dimer in the plasma [189]. A lack of specificity of this study (a high number of falsely positive results) limits its clinical significance. In the majority of cases this test is used to exclude thromboembolic activity and a positive result of this test means that more precise diagnostics of possible thrombosis is required.

Thrombin-antithrombin complexes (TAT) formed following the neutrali-zation of thrombin by antithrombin III (AT) have been used as a surrogate marker for the thrombin generation [45]. An increased plasma level of the thrombin-antithrombin complex (TAT) also reflects with thrombin gene-ration as such, as enhanced F1+2 levels [137].

Microparticles (MPs) are submicron fragments of the cell membrane affecting a number of biological processes, e.g. coagulation [200]. MPs are a normal constituent of blood and can be isolated from the plasma by ultra-centrifugation [206]. The effect of MPs on the coagulation system may be attributed mainly to the two following components: to a tissue factor (TF), i.e. the known initiator of TF-dependent coagulation that can be attached to MPs surface, and to the spectacular properties of phosphatidylserine (PS) – a cell membrane component [200]. The plasma pool of MPs is heteroge-neous, with a few fractions that differ in their origin, structure (proteomic analysis) or biological activity. Platelet-derived MPs constitute the predomi-nant fraction; endothelialcell-derived MPs, monocyte-, leukocyte- and red cell-derived MPs account for 1-5% of the total number [200]. MPs can

affect a number of biological processes, such as coagulation, inflammation, angiogenesis, immune responses and others, as well as participate in an intercellular communication [200].

Venous stasis and ischemia result in the upregulation of P-selectin which localizes prothrombotic MPs to the area of stasis and promotes DVT formation [124, 125, 149, 206]. MPs are not only prothrombotic but also appear to inhibit fibrinolysis [206]. On the activation, MPs shed from platelets express a plasminogen activator inhibitor (PAI-1) and these MPs are localized to the growing thrombus via P-selectin: PSGL-1 interactions [206]. In this manner, platelet MPs are not only prothrombotic but also inhibit fibrinolysis, delaying thrombus resolution and facilitating thrombus growth [148].

Ultrasonography (US) is widely recognized as the most cost-effective and preferred imaging modality for diagnosing proximal DVT [11, 54, 69, 73, 95, 115, 157, 208, 209]. US is a non-invasive and easy-to-perform examination without the effect of ionizing radiation and contrast agent (for example, on the bedside, if necessary) and it can be repeated several times.

A venous ultrasonography examination involves several methods: com-pression ultrasound (images are obtained by applying the simple B-mode and by pressing the region of the visible vein with a transducer) – the best method for the evaluation of proximal leg veins – common femoral, femoral (surface femoral) and popliteal veins; duplex US – the image is obtained by applying the B-mode together with the color Doppler signal (CDDUS); and color Doppler imaging (CDU) alone. In the current state of art, CDDUS is the modality of choice for the diagnosis of DVT. The appropriate examina-tion is compression color duplex ultrasound of the complete venous system, including distal veins [59].

The veins scanned comprise the deep venous system – a femoral vein at the groin and along the thigh, a popliteal vein, and a tibioperoneal trunk at the upper calf and the confluence of a superficial great saphenous vein with the femoral vein. The deep calf veins are usually examined when localized pain or swelling is present.

Color Doppler ultrasonography (CDUS) has become the initial diagnostic tool due to its high sensitivity in the detection of DVT, and some authors now believe that CDUS should be considered the golden standard for DVT diagnosis [67].

The present medical practice diagnosing venous thrombosis still applies the compression method in the majority of cases – DVT is excluded when the venous channel is fully compressed and DVT is confirmed when at least slight changes in the venous channel become obvious. Transversal images are evaluated with a 5- or 7.5-MHz linear transducer; sagittal grayscale

ima-ges supplemented with color and spectral Doppler imaima-ges are evaluated as well.

Doppler color-flow imaging can assist in characterizing a clot as obstruc-tive or partially obstrucobstruc-tive; the uneven color-flow can also help to locate an isoechogenic thrombus. Further information includes the thrombus extent and characterization – fresh or organized, free floating or attached, and par-tial or totally occlusive – that have a prognostic value for the development of pulmonary embolism and post-thrombotic syndrome.

A recent meta-analysis has found US to have high sensitivity (range – 93.2%-95.0%; pooled sensitivity – 94.2%) and high specificity (range – 93.1%-94.4%; pooled specificity – 93.8%) for diagnosing proximal DVT [94]. In the same study, US is determined to have a much lower sensitivity (range – 59.8%-67.0%; pooled sensitivity – 63.5%) for diagnosing distal DVT, which confirms a widely known diagnostic limitation for this techni-que [94], while the calf US examination is not routinely performed in many centers due to its relatively low accuracy. However, if the patient indicates local pain in the calf, the examination of this region should be performed. In a meta-analysis of 100 cohort studies, which compared Duplex US to contrast venography in patients with suspected DVT, the sensitivity for proximal DVT was 96.5%, that for distal calf DVT – 71.2% and specificity of 94.3%; the sensitivity improved in the recent years probably due to the

equipment development, US technique used, and operator expertise [68]. The iliac and pelvic veins are not visible consistently with ultrasound

mostly due to gas in the intestine. The use of US images only makes it difficult to differ between acute and chronic DVT. The findings during the US examination in chronic DVT may include: higher-echogenecity throm-bus, uneven walls of veins, low-caliber veins or collateral veins; however, these symptoms may not occur; the echogenecity of chronic DVT can also change without any previous image data for comparison; it is difficult to differ between acute and chronic DVT. It has been recognized that it is problematic to differ between acute and chronic DVT by applying non-invasive examination methods, including US.

Moreover, US may be limited in obese patients, in patients with marked low extremity (LE) edema and in patients with overlying casts [94]; moreover, US might show falsely negative results in patients with duplicate veins.

Table 2.3.1. Ultrasound for diagnosing proximal (thigh) DVT

B. Ghaye 2007 [63] Katz. D.S et al. 2014 [94] Elias A. et al. 2004 [50]

Sensitivity 92-100% 93.2-95.0% 94% (73-100) Specificity 80-100% 93.1-94.4% 89% (76-96)

Table 2.3.2. Ultrasound for diagnosing distal (calf) and pelvic veins

throm-bosis

B. Ghaye 2007 [63] Katz. D.S et al. 2014 [94] Atri M. et al. 1996 [5]

Sensitivity 40-87% 59.8-67.0% 82-100%

Specificity 83-96% 93.1-94.4% 95-100%

Computed Tomography Venography (CTV) permits a routine evaluation of deep veins of the calves, the iliac veins/IVC, and the deep femoral vein, none of which are routinely well evaluated with US [94]. Katz D.S. et al. [94] believe, that US and CTV are complementary in a subset of patients, and may help to resolve problematic findings encountered on either exami-nation. CTV can be performed either as direct CTV, using a venous inject-tion of iodinated contrast media in a pedal vein similar to that in contrast x-ray venography, or, more commonly, as indirect CTV using an antecubital vein for a contrast media injection and a delayed-imaging acquisition suitable for deep-venous opacification [94]. Many studies have found that the amount of contrast agent used in CTV was lower by about 80% than in case of venography [83]. Studies comparing the findings of CTV with tones of venography showed 100% sensitivity and 96-97% specificity. The most frequent methodology of CTV in leg DVT is between 120-150 ml of a iodi-nated contrast (depending on the patient’s weight); scanning starts 180 s after injecting the contrast agent into the elbow vein; scanning is performed from the tarsi or knee-joints every 5 mm towards the pelvis (involving the ileal wings). CTV enables a comprehensive evaluation of some regions in one examination – i.e. pulmonary CT angiography evaluating pulmonary embolism – and an evaluation of pelvic and deep leg veins. Combined CT venography and pulmonary angiography – CTVPA, is a “one-stop exami-nation”, requiring only a few additional minutes – although the overall radiation dose is higher, there are more images to review, and the dose of IV contrast needs to behigher for the optimal venous enhancement compared with CTPA alone [94]. CTPA is readily available in the majority of hos-pitals. The advantage of CTV like that of MRI is imaging of transversal sec-tions, which can also evaluate extravascular pathology – for example, rea-sons of external vein compression (adenopathy, pelvic masses) which may cause DVT. Moreover, the latest CT scanners really cause a lower radiation

dose during the examination compared to that of the older equipment; moreover, they also enable performing different reconstructions and 3-di-mensional images, which makes a diagnosis easier.

CTV can be repeated in dynamics during the treatment (anticoagulation therapy), if necessary. A recent meta-analysis has found that in case of the patients, who have a suspected pulmonary embolism, CTV has high sensitivity (range – 71%-100%; pooled sensitivity – 95.9%) and high spe-cificity (range – 93%-100%; pooled spespe-cificity – 9.2%), when compared to that of US for diagnosing proximal DVT [190]. The limitations of CTV should also be considered: the examination may not be performed for the patients sensitive to the iodinated contrast agent, as well as for the patients with renal function insufficiency as a considerably high amount of the contrast agent is used; for the pregnant patients and for the patients with claustrophobia, aswell as those unable to stay still during the examination; for children; for the patients with metal implants causing artifacts in surrounding tissues; and for the high-circumference patients. The evaluation of changes in the channels of veins is also hindered by atherosclerotic chan-ges in the walls – it may be difficult to differ from real DVT (Table 2.3.3).

Table 2.3.3. CT evaluation of deep veins of the calves, the iliac veins/IVC,

and the deep femoral vein, none of which are routinely well evaluated with US

Baldt M.M et al. 1996 [6] Katz. D.S et al. 2014 [94] Garg et al. 2000 [62]

Sensitivity 100% 100% 100%

Specificity 96% 96-97% 97%

A satisfactory or good quality CT venography examination was obtained in 97% of studies [106].

Magnetic Resonance Venography (MRV) has high accuracy when com-pared with conventional venography for the pelvic and thigh veins, but is less accurate for the calves.

MRV can differentiate an acute occlusion from chronic thrombus. Ac-cording to Hoffer E.K. et al. [83], MRV is effective and accurate, with sen-sitivity and specificity for iliac and femoral DVT approaching 100% when compared with venography and 92% sensitivity – in detecting isolated calf-vein thrombus. In a meta-analysis to estimate the diagnostic accuracy of MRV for DVT, the pooled estimate of sensitivity was 91.5% and the pooled estimate of specificity was 94.8%. Sensitivity for proximal DVT was higher than sensitivity for distal DVT (93.9% versus 62.1%) [166]. MR veno-graphy seems to be more accurate than color Doppler sonoveno-graphy in detecting the extension of deep venous thrombosis. A variety of sequences

can be utilized, including spin-echo, gradient-recalled echo, and gadoli-nium-enhanced images [26, 74, 108].

MRI is an alternative to CTV for the patients with suspected DVT needing to avoid the effect of ionizing radiation or suffering from the allergy to iodine contrast agents. In the patients with suspected PE, in whom ra-diation should be avoided, or who are allergic to the iodine contrast agents, magnetic resonance pulmonary angiography (MRPA) has a potential as an alternative to CTPA [139, 155, 175, 182].

The advantage of MRV over US is the possibility to evaluate extra-vascular anatomy and to determine a pathology causing the external vein compression which can condition DVT or DVT-imitating pathologies. MRV may be performed for pregnant women that must avoid the radiation effect but need the determination of pelvic deep vein thrombosis. MRV has the advantage over US in evaluating veins above the inguinal ligament, as 20% of DVTs are isolated to the pelvic veins.[44] MRV may be superior to US for determining the chronicity of DVT, although this has not been well studied. [94,179] MRV is contraindicated for the patients with ferromag-netic implants; those with metal foreign bodies; the patients with high-circumference; those with claustrophobia and those unable to stay still during the examination.

Lately, there have been more references about the effect of gadolinium contrast agents on the patients with renal dysfunction – nephrogenic sys-tematic fibrosis (NSF) or nephrogenic syssys-tematic dermopathy (NFD) after the examination which can be manifested with clinical symptoms or even result in death. As long as full evidence is not obtained, there is a common agreement not to use any gadolinium contrast agent for the dialysed patients or the patients with a very limited glomerular filtration speed.

Nuclear medicine venography – the radionuclide investigation of DVT includes such techniques as radionuclide venography and thrombus-avid scintigraphy. With the emergence of nuclear medicine methods, new pers-pectives have opened early on for the diagnosis of DVT [3].

Radiolabeled peptides that bind to various components of a thrombus have been investigated. Apcitide – a technetium-labeled platelet glycopro-tein IIb/IIIa receptor antagonist – is approved for diagnostic studies of DVT [83]. Foci of the increased activity indicate an acute thrombus in that location.

In a multicenter evaluation of technetium-99m-apcitide scintigraphy compared with contrast venography in 243 symptomatic or high-risk pa-tients, 99mTc-apcitide had a sensitivity of 75.5% and a specificity of 72.8%. However, after patients with a history of DVT or PE were excluded, the

sensitivity and specificity were 90.6% and 83.9%, respectively, for 99mTc-apcitide [3, 186].

Phlebography (also called venography, ascending contrast phlebography, or contrast venography) was considered the golden standard in the diagnosis of peripheral DVT; it is the most accurate test with a nearly 100% sen-sitivity and specificity [43]. It is painful; expensive, exposes the patient to a fairly high dose of radiation; and can cause complications related to nephrotoxicity and allergic reactions to iodinated contrast agents. It also carries a risk for post venographic phlebitis [52, 87]. Due to its invasive nature and the risk of complications, it cannot be used either as a routine test for the diagnosis of symptomatic DVT or as a screening tool in asympto-matic patients at high risk for DVT.

2.4. Prophylaxis of DVT in surgical patients

Prevention of DVT and PE is recommended for all patients at risk. Anticoagulants are among the most commonly used drugs in the field of clinical medicine. They are important in preventing and treating arterial and venous thrombosis [8, 100]. It has been shown that typical haemostatic response to a surgery is the development of a hypercoagulable state with an increased fibrinolytic activity [100]. Balance in the hemostatic system can be achieved via balance between endogenous procoagulants and anticoagu-lants [60]. Heparin is one of the oldest antithrombotic biological drugs. From the discovery of heparin in 1916 and the first clinical trials in 1930-1940 it has been the only medicine used for the treatment of venous throm-boembolism. Until 1980, NFH was the main medicine used in general and abdominal surgery for the postoperative prophylaxis of DVT and PE. The laboratory potential of a more efficacious fraction of the whole, unfractio-nated heparin, i.e. LMWH, was recognized in 1979 and a clinical note was made shortly afterwards [15]. Heparin is a natural product present on the cell membrane but when provided as a therapeutic drug, it is a mixture of glycosaminoglycans and polysaccharides composed of long chains of repeated disaccharide units (hexosamine and glucuronic or iduronic acid), although the composition of different macromolecules vary markedly [15]. In its whole unfractionated form, with species of molecular weights ranging from 3 to 30 kDa (although the majority is in the range 12-15 kDa), perhaps only one-third of a standard heparin preparation has anticoagulant activity [79]. Heparin by itself is not an anticoagulant; it is a co-factor in the activity of antithrombin, a 58 kDa single chain polypeptide synthesized in the liver [15, 159]. Heparin binds to antithrombin (AT). An AT-heparin complex inhibits activated coagulation factors IIa, IXa, Xa, XIa and XIIa, the most

important of which are Xa and thrombin factors. An understanding of the relationship between the structure and activityof UFH led to the develop-mentof low molecular weight heparins (LMWHs) [82]. An appearance of LMWH resulted in a number of clinical studies comparing the effectiveness of LMWH and UH worldwide and targeted at the prophylaxis of DVT and PE. Low-molecular-weight heparins have replaced unfractionated heparin [207] because studies show that about half of the patients with venous thrombosis can be safely treated using low-molecular-weight heparins without hospital admission [207], and heparin-induced thrombocytopenia – a dangerous complication of unfractionated-heparin therapy – occurs less frequently with low-molecular-weight heparins [207]. LMWH has a longer subcutaneous half-life and therefore offers a potential for outpatient use, a more predictable anticoagulant response requiring less monitoring; moreo-ver, it also possesses a better antifactor Xa effect [15]. LMWHs inactivate factor Xa but are less effective than UFH in inhibiting factor IIa because the molecules of heparin are not large enough to bind both AT and factor IIa. [82, 207] As a group, LMWHs have a reduced affinity for plasma proteins, endothelial cells and macrophages and also interact less with von Wille-brand factor and platelets than UFH [207]. Bemiparin (bemiparin sodium; Hibor, Ivor, Zibor, Badyket) is a low molecular weight heparin (LMWH) with a lower mean molecular weight (3600D) and a higher anti-Xa/IIa ratio (8 : 1) than other LMWHs [34].

Subcutaneous Bemiparin has been evaluated for the prevention of post-surgical VTE in a number of trials of adult patients undergoing abdominal [34, 51] or orthopaedic surgery [17, 51, 129, 146].

Many studies [17, 34, 37, 51, 80, 129, 146] have shown that Bemiparin: • Bemiparin (Hibor®, Zibor®, Ivor®, Badyket®) is a new second

genera-tion heparin of low molecular weight (LMWH), which has been used in Europe for the last 13 years.

• Bemiparin has the strongest anti-Xa:anti IIa properties (ratio 8:1) when compared to other LMWHs.

• A unique structure of the molecule and a strong effect on the coagulation factor Xa determine a wide range of bemiparin application in practice and a low risk of bleeding.

• The duration of action is longer than 18 hours; thus, taking bemiparin one time a day is enough.

• Bemiparin is rapidly absorbed from the membrane after an injection; longer duration of action – 5.3 hours.

• Having administered prophylactic doses of bemiparin of 2,500 or 3,500 IU, anti-Xa is the most active after 2-3 hours.

Table 2.4.1. Comparative data of the pharmacological and pharmacokinetic

properties of LMWH

Bemiparin Dalteparin Enoxiparin Tinzaparin

Medium molecular weight 3,600 6,000 4,500 6,500

Anti-Xa/anti-IIa ratio 8.0 2.7 3.8 2.8

Bioavailability (%) 96 87 91 87

Duration of action (hr) 5.3 3.5 4.5 3.9

Maximum anti-Xa activity (hr) 3-4 3-4 3-5 4-6 There is a very sparse medical literature examining an effect of aspirin therapy upon the generation of TAT or F1+2 [10]. In the Boston Area Anticoagulation Trail for Atrial Fibrillation, aspirin has no effect on the levels of F1+2 [96]. A meta-analysis of the aspirin therapy for DVT pro-phylaxis demonstrates no or minimal benefit of using aspirin for DVT prophylaxis following total hip or knee arthroplasty [47, 114]. Bern et al. [10] note that there is a slight increase of F1+2 without a concomitant increase of T-AT for patients receiving aspirin after 28±2 days of the therapy. In the studies by Rao [153] and Lubsczyk [113], aspirin does not suppress the release of tissue factor procoagulant activity, with no change of TAT or F1+2. Some authors believe that antiplatelet drugs can be used to-gether with other prophylactic measures.

Mechanical modalities include graduated compression stockings (GCS), intermittent pneumatic compression devices (IPC) and venous foot pump. In general surgery elastic stockings are the most commonly used group of compression stockings for the prevention of CHD. These stockings can be used by applying the same pressure on the ankle, calf and thigh (graduated compression elastic stockings) (Table 2.4.2), or by pressing these anato-mical areas of the leg at intermittent compression (intermittent compression elastic stockings) (Table 2.4.3). Intermittent pneumatic compression (IPC) is an effective form of deep vein thrombosis prophylaxis for general surgery patients [39]. Intermittent pneumatic compression potentially affects two of the three limbs of Virchow‘s triad by increasing venous blood flow velocity, thereby reducing stasis and stimulating fibrinolytic activity, which in its turn alters hypercoagulability [39]. The following two kinds of pneumocom-pressive stockings are mainly used for the prophylaxis of DVT: single-chamber stockings reaching the knee joint and multi-single-chamber stockings reaching the upper third of the thigh. The pressure of the multi-chamber stockings is different in each chamber – the biggest in the ankle area, the smallest – in the thigh area. A. N. Nicolaides, A.V. Pollock conducted a cli-nical study comparing an effect of single-chamber and multi-chamber pneumocompressive stockings on the blood stream in veins [131]. The

results of the study have shown that multi-chamber pneumocompressive stockings are statistically significantly more effective than single-chamber stockings in increasing a blood stream of the leg veins. The different mo-dality of external compression has been studied using a computer-simulated model. It has been found that a sequentially applied wavelike compression provides the most efficient method of venous emptying, suggesting that a sequential compression device may be more efficient in preventing DVT [35].

The improved emptying of lower extremity veins and lowered venous pressure led to an increase in the A–V pressure gradient. An increased dis-parity of the A–V pressure also caused an increase in the lower extremity arterial blood [35].

Table 2.4.2. Intermittent pressure elastic stockings (comparative

characte-ristics of randomized clinical trials)

The author, year _GCS DVT rate _{Control gr.}

Sachdeva et al. 2014 [ 165 ] 126 of 1391 (9%) 282 of 1354 (21%) Sacheda et al. 2017 [ 165 ] 7 of 517 (1%) 28 of 518 (5%) Torngren 1980 [ 194 ] 4 of 98 (4%) 12 of 98 (12%) Chin et al. 2009 [ 36 ] 14 of 110 (13%) 24 of 110 (21%) Allan et al. 1983 [ 2 ] 15 of 97 (15%) 37 of 103 (40%) Hui et al. 1996 [ 85 ] 38 of 86 (44%) 30 of 54 (55%) Wille-Jorgensen ir kt. 1991 [ 212 ] 2 of 83 (2%) 12 of 83 (14%) In total 206 of 2382 (9%) 425 of 2320 (18%)

DVT – deep vein thrombosis; GCS – graduated compression stockings.

Table 2.4.3. Intermittent pneumocompression elastic stockings (comparative

characteristics of randomized clinical trials)

The author, year DVT rate

IPC Control gr. Domeij-Arverud et al. 2015 [ 46 ] 15 of 74 (21%) 27 of 74 (37%) Ryan et al. (2002) [ 164 ] 1 of 50 (2%) 5 of 50 (10%) Eisele et al. (2007) [ 49 ] 4 of 901 (0.4%) 15 of 902 (2%) Chin et al. (2009) [ 36 ] 9 of 110 (8%) 24 of 110 (2%) Yang et al. (2009) [ 215 ] 4 of 47 (8%) 10 of 48 (2%) Zhang et al. (2011) [ 220 ] 3 of 79 (4%) 16 of 83(19%) Vignon et al. (2013) [ 205 ] 13 of 205 (6%) 16 of 202 (8%) Sobieraj-Teague (2012) [ 172 ] 3 of 75 (4%) 14 of 75 (18%) In total 52 of 1541 (3,4%) 127 of 1544 (8%)

A seminal observation was that when IPC was applied to the upper extre-mities, the incidence of DVT in the lower extremities could be lowered [101]. This finding suggested that the efficacy of IPC in lowering the rate of DVT was not solely the direct result of mechanical compression causing hemodynamic changes. Instead, the compressive forces of IPC might also stimulate the systemic fibrinolytic capacity or other biochemical mecha-nisms of the circulation. Clinical studies do support the notion that the effects of IPC are not purely mechanical and that the release of biochemical mediators may play a role that is at least equally important. The reviewed studies provide strong foundations on which further studies could be de-signed to accurately predict a mechanical outcome of IPC and its biochemi-cal consequences.

Randomized clinical trials have shown that the use of different measures of DVT prophylaxis, i.e. mechanical means (IPC) and medical thrombopro-phylaxis (LMWH) results in a statistically significantly better effect than using these measures of prophylaxis alone.

The vast majority of deep venous thrombosis (DVT) through laparo-scopic operations occur during operations. It has already been shown that in high-risk patients, mechanical pneumocompressive socks during surgery, significantly reduces the risk of DVT. The 2012 ACCP guidelines are easy to use, are more comprehensive, and are based on stronger evidence than the 2007 SAGES VTE prevention guidelines [156]. However, they are not specifically directed at laparoscopic surgery patients. The specific type procedure is not considered in the calculation of VTE risk [156]. Steele [181] et al. a randomized double-blind pilot trial with 198 bariatric surgery patients received either 40mg enoxaparin twice daily or 5 mg fondaparinux sodium once daily. Antifactor Xa activity was measured on all patients in both study arms, 3 hours after the first dose (on the day of the operation), immediately before the second dose (postoperative day one), and 3 hours after the second dose. At the routine 2 week postoperative visit, patients underwent magnetic resonance venography (MRV) to detect DVT. Nearly half of the patients (47.4%) did not attain target prophylactic antifactor Xa levels. Adequate antifactor Xa levels were more common with fondaparinux (74.2%) than with enoxaparin (32.4%). 4 of the 175 patients who underwent MRV developed DVT, 2 in each arm of the study. They concluded that fondaparinux was much more likely to produce target prophylactic anti-factor Xa levels than enoxaparin. Both regimens appear to be equally effect-tive at reducing the risk of DVT. They say that further prospeceffect-tive studies are needed to determine the optimal DVT prophylaxis regimen in the ba-riatric surgical population. Kozina [103] et al. examined 200 patients, de-pending on the type and mode of thromboprophylaxis they were divided

into 6 groups: group 1 – unfractionated heparin (UFH), 5,000 IU in standard mode, group 2 – nadroparin calcium, 9,500 antiXa IU 2 hours before the operation, group 3 – enoxaparin sodium, 2,500 IU 2 hours before surgery, group 4 – enoxaparin sodium, 2,500 IU 8 hours prior to surgery, group 5 – bemiparin sodium, 2,500 IU 2 hours before the operation, group 6 – bemi-parin sodium, 2,500 IU 6 hours after surgery and then 1 time a day for 7 days after it. Bemiparin sodium, regardless of the start time of thrombo-prophylaxis, as well as pre-operative start of nadro parin calcium, reduces intraoperative blood loss by 26.1%. Intraoperative blood loss on the back-ground of enoxaparin sodium and UFH with the start of thromboprophylaxis 2 hours before the operation is the same, whereas in thromboprophylaxis with enoxaparin sodium, started 8 hours before, increased by 4.6%. Enoxa-parin sodium in the preoperative period significantly increases the incidence of wound bleeding and increases the incidence of bleedings in the postope-rative period. Thromboprophylaxis using bemiparin sodium, regardless of its start, provides the least number of ecchymosis in the place of drug injection, reduces wound bleeding in the total absence of thromboembolic complications. It’s still not known when it’s the best way to use low-mo-lecular-weight heparin for prophylaxis of DVT – before or after surgery. Even these days, the question remains unanswered; based on this uncer-tainty our study was conducted; the main goal was to determine what is the optimal time to use LMWH in order to avoid DVT and to get the maximal hypocoagulant effect.

3. STUDY POPULATION AND METHODS

A prospective, randomized clinical trial was performed at the Hospital of Lithuanian University of Health Sciences Kaunas Clinics, Department of Radiology, Unit of Computed Tomography and Department of Surgery, Unit of Abdominal Surgery; laboratory tests for the coagulation system – at the HLUHS KK Departments of Laboratory Medicine, Laboratory of Hematology and General Cytology and the HLUHS KK Department of Anesthesiology. The study protocol was evaluated and approved by Kaunas Regional Committee on Biomedical Research Ethics (8th _{of October, 2014,} No. BE-2-13). This clinical trial was also registered at BioMed Center of the International Center for Clinical Trials ISRCTN on 29th of December, 2014 (ID ISRCTN62203940).

The study population consisted of patients scheduled to undergo laparoscopic fundoplication because of GERD at the Surgery Department of HLUHS KK in the year 2013-2016. 121 patients were examined; among them 41 were males and 80 – females.

Including and excluding criteria were established for the randomized clinical trial.

The study included patients who met the following criteria:

• patients with clinicaly significant gastroesophageal reflux disease, whom laparoscopic fundoplication was indicated;

• patients 18-80 years of age, having read an information letter, familiar with the protocol, having signed the form for consent to participate in the study.

Excluding criteria:

• subjects with somatic symptom disorder: renal or hepatic insuffi-ciency, acute peptic ulcer, stroke, aneurysm, acute or chronic bacterial endocarditis;

• a coagulation system disorder, antithrombin deficiency, and protein C and S deficiency;

• reported allergic reactions to heparin;

• reported hepatitis-associated thrombocytopenia in anamnesis;

• mental or nervous disorders that may interfere with self-concept and correct assessment of subject‘s own state of health;

• pregnancy and/or nursing;

• absence of possibility to monitore subject after the treatment; • were participating in another clinical trial.

The patients were excluded from the study if:

• they refused to continue the trial after the surgery;

• laparoscopic surgery was not performed or an open surgery was car-ried out for some reason.

In case the patient complied the criteria for study participation, they were introduced with the purpose of the study, intended medical treatment, planned use of intra-operative measures for DVT prophylaxis, further re-search and treatment in advance.

Anesthesiologists, who had examined the patient before surgery, eva-luated their operational risk according to the Classification of American Society of Anesthesiologists (ASA) [56]:

I – patients without organic and mental illnesses II – patients with mild systemic diseases

III – patients with severe systemic diseases that are not incapacitating IV – patients with life-threatening systemic diseases that limit vital ac-tivity.

Patients were randomly divided (according to the distribution of random numbers by line) into two groups according different DVT prophylaxis regimes. Study scheme is shown in Fig. 3.1.1.

First group

Intra-operative pneumocompressive stockings (IPC) and LMWH were assigned to the first group of patients 12 hours prior to the surgery, as well as 6 hours and 30 hours after the surgery; moreover, the antithrombotic agent Bemiparin (Zibor) of 2,500 IU – 0.2 ml. was also injected subcuta-neously.

Second group

This group included the patients who also received intra-operative IPC and Bemiparin (Zibor) of 2,500 IU – 0.2 ml. subcutaneously 1 hour prior surgery.

3.2. Study protocol

Before the surgery, patients‘ data were collected and their comorbidity were registered. An ultrasound test (US) of DVT was performed (described later). In the course of the study (four times), venous blood was taken for future analysis (thrombin-antithrombin complex, plasma prothrombin frag-ment F1+2, free tissue factor pathway inhibitor, and MP-TF activity in plasma) for an assessment of thromboelastogram – blood clotting para-meters (for an activation of the coagulation system):

1 – before the injection of MMMH (Bemiparin); 2 – 1 hour from the start of the surgery;

3 – after the extubation of the patient in the postoperative ward; 4 – at the third day after surgery.

Before the start of surgery, pneumocompressive stockings, which were connected to a compressor, were placed on both patient‘s legs. A special system Kendall SCD™ 700 Series Controller was used for this purpose (Fig. 3.2.1). The System consisted of the controller, the tubing sets (pro-vided with the controller) and both leg sleeves and foot cuffs, used to compress the limbs to enhance venous blood movement. After the compres-sion cycle reaches the set pressure, the Controller measures the time it takes