Process Of Clot Formation Research Articles

Ultrasmall Nanoparticles Bind to Fibrinogen and Impair Normal Clot Formation

AbstractThe formation of a proper fibrin clot is essential during blood coagulation, as abnormal clots can predispose individuals to bleeding or thrombosis. Despite these concerns, there is currently limited understanding of the potential adverse effects of engineered nanomaterials on fibrin clot formation. This is surprising, given that fibrinogen is highly concentrated in plasma and has a large surface area, making it prone to unintended interactions with nanomaterials. In this study, the impact of ultrasmall gold nanoparticles (usGNPs) on fibrin clot formation is investigated. UsGNPs have gained significant interest in biomedical applications due to their unique physicochemical properties and favorable behavior in complex biofluids. It is found that the usGNPs interacted with fibrinogen, delayed the onset of clot formation, and became physically trapped within the forming fibrin matrix. Confocal microscopy showed that the usGNPs disrupted the normal architecture of the fibrin clot, resulting in a less dense network structure. This disruption led to larger clot pore sizes and increased clot permeability to liquid. Considering the potential health risks associated with abnormal clot formation, a detailed examination of the clot formation process should be included in the standard safety assessment of usGNPs and other nanomedicines.

Transformational features of the vein wall during a long-term period of endovenous laser ablation using 1910 nm laser radiation. Results of in-vivo experiments.

In this paper, results of in-vivo experiments on the animals of endovenous laser ablation (EVLA) using laser radiation with the wavelength 1910 nm are reported. The results of histological studies of the vein segments removed immediately after the procedure and in a long-term period (30 days and 3 months) are presented. Their structural transformation and the obliteration degree of the vein lumen using different values of the linear energy density of laser radiation (LEED=7.5; 15; 20 J/cm) are estimated. Edilbay breed of sheep (males) were used as experimental animals. Laser radiation with a wavelength of λ=1910 nm and power of Р<inf>rad</inf> = 1.5, 3, 4 W was used for EVLA experiments, and speed of fiber traction (v) was 2 mm/s. 8 days after EVLA stitches and an elastic bandage were removed. Animals were observed for 3 months in the vivarium. Animals have duplex ultrasound scanning of coagulation veins under anesthesia, after analysis vein segments were excised for histological examination. As a result, the damage degree to the vein wall tissues (intima, media, adventitia) and peri-venous tissues was revealed. It is shown that in the long-time period, the intima and partial muscle layer transformed to connective tissue. EVLA using laser radiation with a higher value of LEED led to the growth of connective tissue, oedema of all vein layers and peri-venous tissue. The lumen closure occurred due to clot and the vein wall transformation, a maximum value was 25% using LEED=20 J/cm. The connective-tissue transformation of the coagulated vein occurs in a long-term period and more pronounced for higher LEED. However, features of vein hemodynamics of animals and differences between the clot formation process of human varicose veins and healthy animal veins lead to incomplete occlusion. These features should be taken into account during extrapolation results of experiments on animals in clinical practice.

Comparative Evaluation of Serum Lithium Estimation Using Plain Glass Vial and Serum Clot Activator Vacutainer by Reflectance Photometry.

Introduction The collection of blood samples in different vacutainers can affect the result of serum lithium estimation due to the presence of distinct additives in the blood collection vacutainer for enhancing the clot formation process. Due to the low therapeutic index and threat of toxicity of lithium, it is imperative to correctly report the test result. Thus, it has become a challenge for the laboratory physician to estimate lithium in any clinical laboratory setup. Materials and Methods Sample of 100 patients were collected and paired into clot activator vacutainers and plain glass vials. After centrifugation, samples from the paired collection tubes were processed immediately for serum lithium estimation by VITROS 4600 analyzer working on the principle of reflectance photometry. Both the paired tubes were stored at 2 to 8°C and were further analyzed, at 24 and 48 hours, respectively, from the time of their collection. The statistical analysis was done in IBM SPSS software version 23. Results There was a statistically significant differences between the mean of lithium values when processed within 1st hour of collection, obtained from clot activator vacutainers in comparison to glass vials. However, within tube comparison, there was no statistical difference in the lithium values estimated at 1st hour, 24 hours, and 48 hours of collection. Conclusion In this study, lithium values measured by clot-activated vacutainers are found to be lower as compared with values measured through glass vials.

Manejo intraoperatório do sangramento em cirurgias de grande porte com métodos viscoelásticos

Perioperative monitoring of blood clotting is essential to estimate the risk of bleeding, to diagnose coagulopathy, and to guide hemostatic therapies during surgical procedures. Assessment of hemostasis and the choice of blood products have traditionally been made based on conventional coagulation tests. However, these tests have limitations, as they have high turnaround times and evaluate only the beginning of the whole clot formation process. Viscoelastic tests are an adequate alternative to the use of traditional methods of hemostasis monitoring, as they allow a global assessment of blood clotting, from the beginning of thrombus generation to its dissolution, with fast point-of-care results. Rotational thromboelastometry is a viscoelastic test that has gained importance in the perioperative setting. The test shows a graphical representation of clot formation in real-time and provides a range of parameters about the structure and kinetics of clot formation. The main recommendations of use of this method include diagnosing complex coagulopathy and guiding the hemostatic therapy in the perioperative setting of major surgery, such as cardiac surgery, liver transplant, major trauma, and bleeding in critical patients. The objective of this study is to present an update on viscoelastic methods (focusing on rotational thromboelastometry) and their applicability in different clinical scenarios.

Performance and Interpretation of Clot Waveform Analysis.

The prothrombin time (PT) and the activated partial thromboplastin time (aPTT) are two basic tests for routine purposes, which are widely used in the clinical screening of coagulopathies. PT and aPTT are useful tests for detecting both symptomatic (hemorrhagic) and asymptomatic defects, but they are unsuitable for studying hypercoagulable states. However, these tests are available for studying the dynamic process of clot formation by means of the detection of the clot waveform analysis (CWA), which has been introduced several years ago. CWA can provide useful information on both hypocoagulable and hypercoagulable states. Nowadays it is possible to detect the whole clot formation both in the PT and aPTT tubes starting from the initial step of fibrin polymerization by means of specific and dedicated algorithm implemented in a coagulometer. In particular, CWA provides information on the velocity (first derivative), acceleration (second derivative), and density (delta) of clot formation. CWA has been applied to several pathologic conditions such as coagulation factor deficiency (including congenital hemophilia from factor VIII, IX, or XI deficiency), acquired hemophilia, disseminated intravascular coagulation (DIC), sepsis, replacement therapy management, chronic spontaneous urticarial, and liver cirrhosis, in patients with high venous thromboembolic risk before LMWH prophylaxis, and in patients with different hemorrhagic patterns along with an electron microscopy evaluation of the clot density. We report here materials and methods used for detecting the additional clotting parameters available in both PT and aPTT.

Evaluation of hemostasis disorders using the thrombodynamic test in patients with chronic glomerulonephritis with nephrotic syndrome

Nephrotic syndrome (NS) is accompanied by a risk of thrombotic complications due to hypercoagulability. Routine laboratory tests are not sensitive enough to detect these disorders, and therefore the use of integral coagulation tests, including a new thrombodynamic test (TT) in patients with NS, is of high relevance. Using a TT to determine hemostasis disorders in patients with chronic glomerulonephritis (CGN) with NS. The study included 49 patients with CGN, mean age 37 years, of which 25 (51%) women and 24 (49%) men. Of all the examined patients, 20 (40.8%) of people had NS, 29 (59.2%) had no NS. The process of clot formation was assessed by TT. According to TT, 30% (6/20) of patients with NS and 13.7% (4/29) of patients without NS have hypercoagulation with changes in parameters that go beyond the reference values. In patients with NS, an increase in clot density (D), clot formation rate (V) and clot size (CS) was found, especially when albumin decreased below 25 g/l. Negative correlations were found between the levels of albumin, creatinine and clot density (D), which reflects the level of hyperfibrinogenemia, the rate of clot formation (V) and the integral index of coagulation (CS). The results indicate mainly the activation of the plasma hemostasis due to the internal coagulation pathway. However, the correlation of Tlag (delay time for the onset of clot formation after contact of blood plasma with the insert-activator) with serum cholesterol levels may also indicate activation of the extrinsic coagulation pathway. In CGN patients with NS, activation of the plasma hemostasis is noted, as evidenced by an increase in the rate of formation (V) and size of the clot (CS) after 30 minutes, as well as the density of the formed clot (D).

Effect of melatonin on selected parameters of platelet activation, clot formation and overall potential of fibrinolysis

Introduction: Melatonin produced in the pineal gland plays a key role in regulating sleep and wake hours. Synthetic melatonin is used as an adjunct to treat sleep disorders, regulate the sleep-wake rhythm and prevent ailments related to changing time zones or shift work. Its other applications are more widely described, including antioxidant and immunomodulatory properties – therefore melatonin supplementation may be beneficial in alleviating symptoms associated with the occurrence of COVID-19. However, reports on the influence of exogenous melatonin on the platelet, plasma and vascular hemostasis are ambiguous. Aim: The aim of the study was to evaluate the in vitro influence of melatonin on spontaneous and ADP-induced adhesion of platelets to fibrinogen, kinetic parameters of ADP-induced aggregation and selected elements of plasma haemostasis: general potential for clot formation and fibrinolysis, as well as kinetic parameters of the clot formation process, its stabilization and fibrinolysis. Material and methods: The study were performed with the use of the previously described research model, which includes the method of assessing platelet adhesion, a multi-parameter test for assessing platelets aggregation and a test that enables kinetic assessment of the clot formation process, the period of fibrin stabilization and its lysis. Results: Our preliminary studies indicated that melatonin at concentrations: 0.2-10 nmol/L does not show a significant and direct impact on the assessed kinetic parameters of the studied processes, important for platelet and plasma hemostasis. Conclusions: The pleiotropic effects of melatonin are increasingly applied, especially its antioxidant and immunomodulating properties, therefore further and in-depth in vitro as well as in vivo hemostasis studies followed by clinical observations of patients using melatonin are needed.

Sorghum Protein Extract Protects RBC from Sodium Nitrite-Induced Oxidative Stress and Exhibits Anticoagulant and Antiplatelet Activity

Oxidative stress plays a critical role in the progression of diabetes, arthritis, cancer, eryptosis, cardiovascular disease, and thrombosis. Currently, antioxidants from natural sources are in high demand due to their beneficial role in the management of said diseases. The purpose of the study was to evaluate the protective effect of sorghum protein buffer extract (SBE) on sodium nitrite-induced oxidative stress and thrombosis. Protein characterization of SBE was done using SDS-PAGE. Oxidative stress in RBC was induced using sodium nitrite (NaNO2) and the key stress markers such as lipid peroxidation (LPO), protein carbonyl content (PCC), and the level of antioxidant enzymes (SOD and CAT) were measured. The anticoagulant effect of SBE was identified by employing in-vitro plasma recalcification time, activated partial thromboplastin time (APTT), prothrombin time (PT), and in-vivo mouse tail bleeding time. SBE antiplatelet activity was examined using agonist adenosine diphosphate (ADP) and epinephrine-induced platelet aggregation. Non-toxic property of SBE was identified using in-vitro direct haemolytic, haemorrhagic, and edema forming activities using experimental mice. SBE revealed similar protein banding pattern under both reduced and non-reduced conditions on SDS-PAGE. Interestingly, SBE normalized the level of LPO, PCC, SOD, and CAT in stress-inducedRBCs. Furthermore, SBE showed anticoagulant effect in platelet rich plasma by enhancing the clotting time from the control 250 s to 610 s and bleeding time from the control 200 s to more than 500 s (p<0.01) in a dose dependent manner. In addition, SBE prolonged the clot formation process of only APTT but not PT. SBE inhibited the agonists ADP and epinephrine induced platelet aggregation. SBE did not hydrolyze RBC cells, devoid of edema and haemorrhage properties. This study demonstrates for the first time the anticoagulant, antiplatelet, and antioxidant properties of SBE. Thus, the observed results validate consumption of sorghum as good for health and well-being.

Long-term post-discharge coagulation abnormalities in patients with moderate to severe COVID-19

IntroductionCoagulopathy plays a significant role in COVID-19 pathogenesis. Benefit from anticoagulation is well established in hospitalized patients. But since there is a lack of data on coagulopathy resolution: there is no consensus in guidelines if extended anticoagulation is required.PurposeThe purpose of our work was to analyze coagulation abnormalities at 2 to 5 months after moderate to severe COVID-19.MethodsCOVID-19 reconvalescents (CR), discharged from our hospital, were called for follow-up at 2–3 (CR1 group, 21 patients) or 5–6 (CR2 group, 26 patients) months after discharge. All CR were not on the anticoagulation therapy by that time. In addition to clinical examination and standard lab tests, we performed an FMD-test to analyze endothelial function, impedance aggregometry to analyze platelet aggregation, and a thrombodynamics test to assess thrombogenesis and fibrinolysis. The control group was recruited before the pandemic started.ResultsAll CR were free from thrombotic complications after discharge from the hospital.Endothelial function was not significantly impaired in CR compared with control, and was still in the normal range (7,07, IQR (3,36; 11,56) vs. 7,87 (5,42; 13,45)).Platelet aggregation was significantly lower in CR1 than in the control group in ADP-induced mode (37, IQR (19; 47) vs. 46, IQR (41; 50), p=0,02) and didn't differ in other groups and other modes (Asa, TRAP-induced).Thrombodynamics tests revealed suppression of the clot formation process in both CR1 and CR2 compared with control. There were decreased clot growth rates (μm/min) (CR1/CR2: 27,1, IQR (26,1; 29,2)/27,6, IQR (26,4; 30,0) vs. 32,2, IQR (30,0; 35,1), both p<0,001); decreased clot size (μm) (CR1/CR2: 1099, IQR (1069; 1194)/1199, IQR (1058; 1221) vs. 1304, IQR (1164; 1380), both p<0,001), and decreased optical density (arb units) (CR1/CR2: 21'607, IQR (20'363; 24'545)/22'741, IQR (21'344; 25'961) vs. 26'556, IQR (24'672; 29'387, p<0,001 and p=0,09 respectively.Fibrinolysis was enhanced in CR groups compared with control (lysis progression was significantly higher for CR2 only, CR1/CR2: 2,9, IQR (2,5; 3,8)/3,8, IQR (2,6; 5,4) vs. 2,5, IQR (1,1; 3,4) %/min, p=0,087 and p=0,007 respectively; expected clot lysis time was shorter in both CR1 and CR2: 36,5, IQR (29,8; 44,2)/31,7, IQR (24,3; 42,7) vs. 65,9. IQR (36,3; 95,5) min, p=0,019 and p=0,016 respectively).There was no statistical difference in clot formation and in fibrinolysis between CR1 and CR2.ConclusionIn the deferred period (2–5 months) of COVID-19 the fibrinolysis process remains still active whereas the process of clot formation is mostly suppressed. Endothelial function assessed via the FMD test is within the normal range in the post COVID period.FUNDunding AcknowledgementType of funding sources: None.

Is endothelial dysfunction a driving force of COVID-19 induced coagulopathy?

Background/IntroductionThere are numerous reports regarding the direct endothelial damage by the SARS-CoV-2 that can lead to activation of both plasma hemostasis and platelet aggregation. However, the mechanism of interaction between endothelium and haemostasis in COVID-19 remains unclear.PurposeThe aim of our study was to assess the relationship between each link of clot formation process (endothelial function, plasma coagulation, platelet aggregation) with the severity of the disease.Methods58 COVID-19 patients were included in our study. Patients were divided into moderate (n=39) and severe (n=18) subgroups. All patients underwent a flow-mediated dilation (FMD) test, impedance aggregation, rotational thromboelastometry, thrombodynamics and von Willebrand factor antigen (vWF: Ag) quantification. All measurements were repeated on days 3 (point 2) and 9 (point 3) of hospitalization.ResultsCOVID-19 patients demonstrated the enhanced plasma coagulation (clotting time, s 613,0 [480; 820], clot growth rate, μm/min 32,75 [29,3; 38,7]). At point 1 no significant difference in parameters of plasma coagulation between patients' subgroups was noted. At point 2 a significant decrease in the size (CS, μm 1278.0 [1216.5; 1356.5] vs 965.0 [659.8; 1098.0], p<0,01) and clot growth rate (μm/min 32,4 [29,2; 35,0] vs 17,7 [10,3; 24,4], p<0,01) under the influence of anticoagulants in the moderate subgroup compared with point 1 was observed. We didn't observe such phenomenon in severe subgroup.There was no significant difference in platelet aggregation between subgroups at point 1. During the course of the disease the patients in the moderate and severe subgroups demonstrated a significant increase in platelet aggregation induced by arachidonic acid and ADP (severe: AUC ARA 48,0 [25,0; 59,0] vs 77,5 [55,8; 92,7], p=0,04; AUC ADP 44,0 [41,0; 56,0] vs 58,0 [45,5; 69,0], p=0,04; moderate: AUC ARA 31,5 [19,8; 50,7] vs 56,0 [39,0; 76,0], p=0,01; AUC ADP 43,0 [20,0; 59,0] vs 56,6 [50,3; 70,5], p=0,04;), in moderate subgroup the significant increase in TRAP-induced aggregation was also noted (AUC TRAP 58,0 [41,0; 69,5] vs 76,0 [58,3; 81,5], p=0,048).There were no significant differences in the FMD-test results between the patient subgroups. FMD-test results were predominantly within the reference ranges (7,1 [4,0; 8,8]).Patients in the severe subgroup had significantly higher levels of vWF: Ag (228,0 [205,3; 240,7] vs 232,0 [226,0; 423,0], p=0,03).ConclusionSARS-CoV-2 infection was characterized by increased levels of vWF:Ag, that could represent the local endothelial damage, meanwhile there was no generalized endothelial dysfunction assessed via FMD-test in moderate to severe patients. At the same time the enhanced plasma coagulation in COVID-19 patients was observed.FUNDunding AcknowledgementType of funding sources: None.

Dynamic assessment of plasma clotting in samples with distinct fibrinogen concentrations using impulsive acoustic radiation force

While some diseases reduce fibrinogen concentration, others increase the amount of this clotting factor in the blood. Some studies have shown that the fibrinogen concentration in the blood is related to the stiffness of the formed clot. Hence, the aim of this study was to employ an ultrasonic method based on impulsive acoustic radiation force (IARF) to identify the fibrinogen concentration (coagulation factor I) in a plasma sample by means of peak-displacement (PD), time of peak-displacement (TPD), and shear modulus (μ) as well as to identify the change of plasma samples during the clot formation process. The IARF-based ultrasonic system transmitted bursts with a frequency of 2.03 MHz, duration of 246.31 µs, amplitude of 118 VPP, and pulse with 1.25 Hz repetition frequency to generate an IARF on a glass sphere (2.99 mm in diameter and 2500 kg/m3 in density) embedded in a plasma sample, causing a displacement that was monitored by a pulse-echo system with a center frequency of 4.89 MHz. The values of the shear moduli were 124.14 ± 3.02, 556.99 ± 11.76, and 670.39 ± 9.77 Pa, for fibrinogen concentrations of 1.2, 2.4, and 3.6 g/L 20 to 36 min after the beginning of the coagulation process. The TPD values obtained in the same period were 5.28 ± 0.09, 3.03 ± 0.02, and 2.83 ± 0.01 s. The results indicate that an IARF-based ultrasonic system can be used clinically because it uses small amounts of plasma and has the ability to detect differences in PD, TPD, and μ as a function of fibrinogen concentrations.

Clot Formation Under Physiological Flow Conditions Is Altered in Sickle Cell Disease As Measured By a Multiparameter Microfluidic System

Background: While the hallmark of sickle cell disease (SCD) is vaso-occlusion, it is now clear that hypercoagulation plays a prominent role in the pathophysiology of SCD (Sparkenbaugh 2017). Consistent with this prothrombotic state, SCD patients remain at increased risk for thromboembolic events (Naik 2014) such as pulmonary embolism and deep vein thrombosis. In addition, sickle red blood cells (RBC) are known to biochemically contribute to the hypercoaguable state through hemolysis or enhanced PS exposure (Whelihan 2016, Litvinov 2017). However, no studies have examined the biophysical contribution of RBCs to clot formation in SCD, especially under physiologic flow conditions. As both the increased stiffness and adhesion of the sickle RBC (Kasschau 2016) biophysically amplify the frequency and duration of interactions with the developing clot, we hypothesize that enhanced RBC incorporation occurs in sickle cell clots under physiologic flow conditions. Furthermore, as the altered shape and stiffness of sickle RBCs decreases the packing density in clots (Tutwiler 2015, Strauss 2015), we also hypothesize that the application of fluidic forces to the clots likely causes instability potentially leading to thromboemboli. This highlights the need for an assay that studies both clot formation and stability under flow conditions.Innovation: While studying these events in vivo remains the most physiologically relevant approach, the inability to control the cellular and physical parameters renders mechanistic studies to be extremely challenging. Moreover, while in vitro microfluidics offer the unique ability to precisely control parameters known to affect clot formation, including shear stress, hematocrit, platelet count, and oxygen tension, these devices typically cannot be used for extended time studies due to confounding systemic clotting throughout the fluidic. To that end, we developed a novel multi-parameter microfluidic that precisely controls the spatial location of clot initiation and formation for extended (>30 mins) in vitro studies of developing clots using recalcified citrated whole blood (Figure 1a). Our easy-to-use system combines on-chip calcification (Lehmann 2015) to reduce systemic clotting, simple assembly methods (Myers 2017), and protein micropatterning using collagen and tissue factor. Brightfield and fluorescent microscopy was used to examine the flow of blood over this patch to observe the clot formation process, structure, and stability.Results: The device performed as designed and maintained perfusion for at least 45 minutes with each trial for healthy AA donors (Figure 1b). Our preliminary images qualitatively show that clots from SS patients on hydroxyurea are larger than AA clots (Figure 1b-c). Quantitative measurements of the clot areas indicate that the clot size is significantly higher in SCD blood (n=3 patients, Mann-Whitney, p= 0.007) than in blood from healthy AA subjects (n=21) (Figure 1d). This suggests that even in the modern age of hydroxyurea therapy, patients remain in a prothrombotic state. Furthermore, after SCD whole blood clots were formed in our microfluidic system, force applied via continuous exposure to high shear stress of ~3000 dynes/cm2 in the microfluidic created clot fragments composed primarily of RBCs. The creation of these fragments suggests decreased clot stability and has implications for micro-thromboemboli (Figure 1e). These results support our hypothesis that enhanced RBC incorporation in SCD alters the structure and stability of whole blood clots. Ongoing studies are focused on defining the compositional variability and susceptibility to embolism.Conclusion and Relevance: Ongoing studies involve integrating and varying shear stress, hematocrit, platelet count, and oxygen tension to determine how these multiple parameters interact to affect the incorporation of sickle RBCs in thrombi, as our novel microfluidic enables real time visualization of clot formation and clot stability under physiologic flow conditions (Figure 2). Our device allows us to examine the stability of these clots under shear stress and the propensity for emboli formation. Collectively, these studies will lead to a better understanding of the prothrombotic state of SCD and how the sickle RBCs interacts with platelets and the coagulation cascade. [Display omitted] DisclosuresLam:Sanguina, LLC: Equity Ownership.

Long-term differences in clot formation and endothelial function between STEMI patients with occlusion of infarct-related artery and those with spontaneous reperfusion

Abstract Introduction Enhanced activation of endogenous fibrinolysis in ST-elevation myocardial infarction (STEMI) patients is the main cause of spontaneous reperfusion of the infarct-related artery (IRA) in the acute period. Whether this enhanced activation remains in the deferred period of myocardial infarction remains unclear. Purpose The main purpose of our work was to analyze the process of clot formation and endogenous fibrinolysis in patients with a history of myocardial infarction. Methods We included 51 patients with a history of myocardial infarction (median, 35 months). Of these patients, 19 had a spontaneous reperfusion of the IRA (SR group) during the index event, and 32 had a total occlusion of the IRA (occlusion group). We assessed parameters of clot formation and endogenous fibrinolysis using thromboelastometry and a thrombodynamics assay. We analyzed endothelial function using the flow-mediated dilation test (FMD). Results The process of clot formation was more active in the occlusion group than in the SR group. Thus, the initial, mean, and total clot velocity times were significantly higher in the occlusion group than in the SR group: 57.2 mcm/min vs. 60.25 mcm/min; 28,1 mcm/min vs. 31.9 mcm/min; and 28.7 mcm/min vs. 31.9 mcm/min, respectively; p&amp;lt;0.01. The clot size was much bigger in the occlusion group than in the SR group (1,190.5 mcm vs. 1,315 mcm; p&amp;lt;0.01). No differences in endogenous fibrinolysis parameters were observed between the two groups. Endothelium function was better in the SR-group than in the occlusion group (FMD 17.4% vs 11.3%, p&amp;lt;0.01). Conclusion STEMI patients with occlusion of the IRA and those with spontaneous reperfusion remain different in clot formation and endothelial function even after many years. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): This study was supported by the Russian Science Foundation grant

Point-of-Care Diagnostics in Coagulation Management.

This review provides a comprehensive and up-to-date overview of point-of-care (POC) devices most commonly used for coagulation analyses in the acute settings. Fast and reliable assessment of hemostasis is essential for the management of trauma and other bleeding patients. Routine coagulation assays are not designed to visualize the process of clot formation, and their results are obtained only after 30–90 m due to the requirements of sample preparation and the analytical process. POC devices such as viscoelastic coagulation tests, platelet function tests, blood gas analysis and other coagulometers provide new options for the assessment of hemostasis, and are important tools for an individualized, goal-directed, and factor-based substitution therapy. We give a detailed overview of the related tests, their characteristics and clinical implications. This review emphasizes the evident advantages of the speed and predictive power of POC clot measurement in the context of a goal-directed and algorithm-based therapy to improve the patient’s outcome. Interpretation of viscoelastic tests is facilitated by a new visualization technology.

Borrelia outer surface protein C is capable of human fibrinogen binding.

Outer surface protein C (OspC) is one of the most abundant surface lipoproteins produced during early infection by the Borrelia spirochete, the causative agent of Lyme disease. The high sequence variability of the ospC gene results in the production of several and strongly divergent OspC types. One of the known roles of OspC is the recruitment of blood components, including complement regulators, to facilitate the bloodstream survival of Borrelia at an essential stage of host infection. Here, we identify and describe a new interaction between OspC and human fibrinogen. To test the ability of OspC to bind fibrinogen, we developed a microscale thermophoresis assay using four fluorescently labeled types of OspC. We show that OspC binds fibrinogen tightly, with nanomolar Kd , and that the binding depends on the OspC type. The binding assays combined with SAXS studies allowed us to map the OspC-binding site on the fibrinogen molecule. Spectrometric measurements of fibrinogen clotting in the presence of OspC indicate that OspC negatively influences the clot formation process. Taken together, our findings are consistent with the hypothesis that OspC interacts with blood protein partners to facilitate Borrelia spreading by the hematogenous route.

Synthesis of C[sbnd]C, C[sbnd]N coupled novel substituted dibutyl benzothiazepinone derivatives and evaluation of their thrombin inhibitory activity

The formation of a thrombus is a key event in thromboembolic disorders, that contribute to high mortality and morbidity in affected patients. In the present study, we synthesized a library of novel substituted 3,3-dibutyl-8-methoxy-2,3-dihydrobenzo [b] [1,4] thiazepin-4(5H)-one derivatives which were tested for their platelet aggregation and thrombin inhibitory activity. Among the tested compounds, 3,3-dibutyl-7-(2-chlorophenyl)-8-methoxy-2,3-dihydrobenzo[b] [1,4]thiazepin-4(5H)-one (DCT) displayed the maximum thrombin inhibition with an IC50 value of 3.85 μM and thus DCT was chosen for further studies. Next, the effect of DCT on primary hemostasis was evaluated using agonist-induced platelet aggregation model. The lead compound inhibited the collagen- or ADP- or thrombin-induced platelet aggregation in a dose-dependent manner. Furthermore, DCT prolonged the process of clot formation when evaluating plasma re-calcification time (320 ± 11 sec at 5 µg DCT), activated partial thromboplastin time (58.0 ± 0.01 sec at 2 µg), and prothrombin time (14.7 ± 0.01 sec at 5 µg). Molecular docking studies suggested that the benzothiazepinones evaluated here consistently display hydrogen bonding with Ser214 of thrombin, which is similar to that of the co-crystallized ligand (1-(2R)-2-amino-3-phenyl-propanoyl-N-(2,5dichlorophenyl)methylpyrrolidine-2-carboxamide). DCT displayed additional hydrogen bonding to Ser195 and π-π interactions between its methoxyphenyl groups and Trp60, thereby providing a structural rationale for the observed biological effect.

Effect of Simvastatin on Thrombopoiesis in Porcine Bone Marrow.

IntroductionStatins are pharmacological agents commonly used to lower serum cholesterol level. The aim of the experiment was to investigate the effect of the statin simvastatin on thrombopoiesis in the porcine model because it is the closest to the human one regarding physiological and genetic similarities.Material and MethodsThe study was conducted on a group of 32 pigs randomly divided into two equal groups: control and experimental. The pigs were treated for 28 and 56 days with simvastatin in a dose of 40 mg per day per animal. Cytological evaluation of bone marrow smears was performed to assess the average number of all types of cells during thrombopoiesis as was analysis of haematological parameters to assess PLT and MPV.ResultsDuring the course of the experiment statistically significant changes in the number of promegakaryocytes were observed. Other parameters also showed some fluctuations during the study. However, these changes were not statistically significant.ConclusionThe obtained results clearly indicate a toxic influence of simvastatin on the process of thrombopoiesis and prove that statins reduce mean platelet volume, thus affecting the process of clot formation through the period of administration in a duration-dependent manner.

A Review of the Current Role of Blood Clotting Analyzers in Clinical Practice

Viscoelastic tests (VETs) such as thromboelastography, rotational thromboelastography, and the Sonoclot Analyzer assess the entire process of clot formation through dissolution in real-time, and may provide additional therapeutic value to conventional laboratory coagulation tests. With the ability to obtain rapid results and identify specific coagulopathies, VETs has been examined in a variety of clinical scenarios, including cardiac surgery, trauma, obstetric emergencies, and liver transplant. This review provides a summary of clinical trials utilizing VETs in the aforementioned clinical scenarios, and suggests that VETs have demonstrated a capability to identify coagulopathies, utility in guiding algorithms to reduce the amount of transfusions, and a limited ability to predict bleeding events or mortality.

Utility of Clot Waveform Analysis in Russell's Viper Bite Victims with Hematotoxicity.

Introduction:In Russell's viper bites, due to the lack of a better alternative, whole blood clotting test (WBCT) remains the standard test even though its reliability and sensitivity has been shown to be low. Activated partial thromboplastin time (aPTT)-based clot waveform analysis (CWA) is an optic absorbance assay that can be used as a global clotting test. In this study, the objective was to assess the changes in CWA and to compare CWA to WBCT and aPTT in patients with Russell's viper envenomation.Methods:The datum was collected prospectively over 2 months as a pilot observational study in a tertiary care center. All proven cases of Russell's viper-envenomated individuals with preliminary CWA data and WBCT were included in the study. The clot wave (CW) of the five individuals, which met all the stringent inclusion criteria, was analyzed and interpreted.Results:CW absorbance sigmoid waveform was deranged in all 5 cases, of which 4 showed a change in CWA even before an abnormal aPTT. Three of the 5 had a normal WBCT but showed early changes in CWA. Atypical biphasic waveform reported in disseminated intravascular coagulation in other prior studies is seen in venom-induced consumptive coagulopathy also. In all patients where a second derivative was plotted, the second (lower) phase of the second derivative showed a slow rise to baseline.Conclusion:CWA showed changes which provided information earlier than the conventional coagulation studies in the snakebite victims studied. While aPTT or WBCT reflects clotting time, CWA conveys the dynamic process of clot formation and stabilization. CWA may reveal disorders of clotting in snakebite victims before the conventional tests become abnormal. Future research should assess the speed and accuracy of the test in diagnosing hemotoxic envenomation and its potential role in guiding antivenom therapy.

Synthesis and Biocompatibility Studies of New Iminodiacetic Acid Derivatives.

Background: Iminodiacetic acid (IDA) derivatives can be used as ligands to form complexes with technetium, with potential application as hepatobiliary diagnostic agents. The aim of this study was to synthesize five novel IDA derivatives and to compare their effects on plasma haemostasis with clinically approved ligands for technetium complexation. Methods: The influence of synthesized IDA derivatives on plasma haemostasis was evaluated spectrophotometrically by clot formation and lysis test (CL-test), coagulation assay, Prothrombin Time and Activated Partial Tromboplastin Time. The effects of the tested compounds on erythrocytes were assessed using haemolysis assays, microscopy and flow cytometry studies. Results: Despite their significant influence on the kinetic parameters of the process of clot formation and fibrinolysis, the tested ligands, at potential diagnostic concentrations, did not alter the overall potential of clot formation and lysis (CLAUC). At potential diagnostic concentrations (0.4 μmol/mL) all the tested compounds showed no adverse effects on the membranes of RBCs (Red Blood Cells). Conclusion: IDA derivatives with methoxy substituents in aromatic ring, exert multidirectional effects on plasma haemostasis and should be considered safe as their significant impacts were mostly observed at 4 μmol/mL, which is about 10-fold higher than the theoretical plasma concentrations of these compounds.