Fibrinolytic Process Research Articles

A novel ex vivo approach for measuring plasminogen activation upon established plasma clots

BackgroundThe fibrinolytic system plays a critical role in maintaining hemostasis. Central to fibrinolysis is the degradation of fibrin by plasmin, produced in the circulation following the activation of plasminogen by plasminogen activators (PAs). Accurately measuring the plasminogen activation rate is vital for the understanding of fibrinolytic processes, particularly in the context of thrombolysis. Yet, due to the insoluble nature of fibrin, in vitro and ex vivo investigations of PA‐mediated plasminogen activation have proven challenging. As researchers frequently adopt soluble fibrinogen fragments and/or alter the experimental system beyond what is physiologically relevant, they limit the validation and interpretation of their findings. Here, we present a novel, high‐throughput assay for measuring plasminogen activation rates on natural, plasma‐derived fibrin that optimally simulates in vivo conditions. MethodHuman plasma was used as the source of plasmin(ogen) and fibrin(ogen). “Halo‐shaped” plasma clots were produced in a 96‐well plate using a thrombin‐containing clotting mixture, ensuring that an optically compatible and plasma‐free center is maintained in each well. Subsequent additions of a plasmin chromogenic substrate and different PAs were followed by absorbance measurements over time to extract the corresponding enzyme kinetics information. Results and DiscussionValidation experiments demonstrated the capability of our approach to accurately model fibrin‐dependent and ‐independent plasminogen activation as well as sensitively detect variations in plasminogen and fibrinogen plasma levels. ConclusionThis assay allows a straightforward, yet powerful, measurement of plasminogen activation rates on established plasma clots, with the capability of properly assessing fibrin‐ and non–fibrin‐dependent plasminogen activation by various therapeutic PAs.

Microscale structural changes of individual fibrin fibers during fibrinolysis

Fibrinolysis is the enzymatic digestion of fibrin, the primary structural component in blood clots. Mechanisms of fibrin fiber digestion during lysis have long been debated and obtaining detailed structural knowledge of these processes is important for developing effective clinical approaches to treat ischemic stroke and pulmonary embolism. Using dynamic fluorescence microscopy, we studied the time-resolved digestion of individual fibrin fibers by the fibrinolytic enzyme plasmin. We found that plasmin molecules digest fibers along their entire lengths, but that the rates of digestion are non-uniform, resulting in cleavage at a single location along the fiber. Using mathematical modeling we estimated the rate of plasmin arrival at the fiber surface and the number of digestion sites on a fiber. We also investigated correlations between local fiber digestion rates, cleavage sites, and fiber properties such as initial thickness. Finally, we uncovered a previously unknown tension-dependent mechanism that pulls fibers apart during digestion. Taken together these results promote a paradigm shift in understanding mechanisms of fibrinolysis and underscore the need to consider fibrin tension when assessing fibrinolytic approaches. Statement of significanceWe developed a method for interrogating lysis of individual fibrin fibers, enabling the time-resolved observation of individual fiber digestion for the first time. Our results resolve longstanding disagreements about fibrinolytic processes and reveal previously unknown mechanisms that also play a role. Also, we developed the first microscale mathematical model of plasmin-fibrin interaction, which predicts the number of plasmin molecules on each fiber and can serve as a framework for investigating novel therapeutics.

Angiotensin-Converting Enzyme (ACE) Insertion/Deletion (I/D) Polymorphism as a Conjoint Regulator of Coagulation, Fibrinolytic, and RAAS Pathway in Infertility and Associated Pregnancy Complications.

Despite the increase in assisted reproductive technologies, the high rates of infertility and pregnancy complications are a major concern to infertility specialists worldwide. Infertility may be attributed to pregnancy complications like thrombophilia, preeclampsia and fibrin-induced recurrent pregnancy loss (RPL). Renin-angiotensin-aldosterone system (RAAS) directly or indirectly causes preeclampsia and thrombophilia through the fibrinolytic pathway that ultimately leads to RPL or infertility. The underlying mechanisms of this interaction are still unclear. The present comprehensive review is intended to demonstrate the role and interaction of RAAS and fibrinolytic pathways in pregnancy complications. How this interaction can induce pregnancy complications, and ultimately infertility, is also discussed in the light of current evidence. This study also presents common markers that link RAAS and fibrinolytic processes in developing thrombophilia, preeclampsia and RPL. The common link in these pathways is ACE gene I/D polymorphism. Apart from ACE, PAI-1, VIIa, XIIa, AT1R, AT1AA, and TF are common molecules that can delineate the underlying causes of pregnancy complications and infertility.

The Difference Of PT, aPTT, Fibrinogen, D-Dimer Activities In HIV Patients With CD4 Count Of <200 uL And >200 uL

ABSTRACT
 Introduction: HIV is a developing disease that has been a global problem. The progress of HIV infection is characterized by decreased CD4 count. Hemostasis disorder is often found in patients with HIV, where the formed virus-antibody complex can activate the coagulation system, beginning from the activation of the Hageman factor (Factor XII) into the active form (Factor XIIa). This factor will activate the fibrinolysis process. Fibrin polymer is broken down into fragments X and Y. Fragment Y is further broken down into Fragment D and E, which is known as D-dimer.
 Objective: To determine the difference of PT, aPTT, Fibrinogen, and D-Dimer in HIV patients with a CD4 lymphocyte count of < 200/µL and > 200/µL in H. Adam Malik General Hospital Medan.
 Hypothesis: There is a difference of PT and aPTT activities in HIV patients with a CD4 count of < 200 cells/µL and ≥ 200 cells/µL and a difference of D-dimer and fibrinogen levels in HIV patients with a CD4 count of < 200/µL and > 200/µL in H. Adam Malik General Hospital Medan.
 Methods: This study was conducted in the Clinical Pathology Laboratory, Department of Internal Medicine, H. Adam Malik General Hospital. Samples were collected with a consecutive sampling method which included patients diagnosed with HIV in H. Adam Malik General Hospital Medan from September 2019 to July 2020 who fulfilled the inclusion and exclusion criteria. Thirty-eight patients were divided into two groups, i.e., HIV patients with a CD4 count of < 200/µL and HIV patients with a CD4 lymphocyte count of > 200/ µL.
 Results: Mann-Whitney test was used to assess the comparison of PT and aPTT values between HIV patients with a CD4 count of < 200 cells/µL and ≥ 200 cells/µL. The result was significant with a p-value = 0.002, which means that there is a significant difference in PT and aPTT values between HIV patients with a CD4 count of < 200 cells/µL and ≥ 200 cells/µL. An Independent T-test was used to assess the difference in fibrinogen level between HIV patients with a CD4 count of < 200/µL and > 200/µL, which resulted in p-value = 0.032. This means that there is a significant difference in fibrinogen levels between HIV patients with a CD4 count of < 200/µL and > 200/µL. Mann-Whitney test was used to determine the comparison in D-dimer level between HIV patients with a CD4 count of < 200/µL and > 200/µL, which showed a p-value = 0.002. This indicated a significant difference in D-dimer level between HIV patients with a CD4 count of < 200/µL and > 200/µL.
 Conclusion: The lower the CD4 lymphocyte count, the higher the activities of PT, aPTT, fibrinogen, and D-dimer in HIV patients.
 
 Keywords: HIV, PT, aPTT, D-Dimer, Fibrinogen, Hemostasis

Imbalance of the hemocoagulation and fibrinolysis systems under conditions of diabetes mellitus complicated by acute circulatory disturbance in the pool of carotid arteries of rats

Introduction. Both cerebral ischemia and diabetes mellitus (DM) are evidenced to be associated with hypercoagulation state, though today the facts concerning hemocoagulation condition with diabetes mellitus complicated by cerebral ischemia-reperfusion are lacking. 
 The aim of the study. To examine the dynamics of interrelations between the pro-, anticoagulant and fibrinolytic parameters in rats with diabetes mellitus complicated by acute cerebral circulatory disturbance. 
 Results. At the early ischemic-reperfusion period activation of the pro-coagulant potential (decrease of prothrombin and thrombin time and increase of fibrinogen content) is found to be balanced by a reduced activity of XIII factor and an increase activity of antithrombin III in rats without diabetes mellitus. At the same period in rats with diabetes increase of the pro-coagulant potential is accompanied by the activation of XIII factor and a reduced activity of antithrombin III promoting clot formation. On the 12th day of the observation the parameters of the coagulation chain in the hemostasis system and antithrombin III return to the level of the control animals in rats without diabetes, but in rats with diabetes the factors of intensification of a thrombotic risk remain activated.
 At the early and late ischemic-reperfusion periods the parameters of blood fibrinolytic activity increase in rats without diabetes; in animals with diabetes mellitus the parameters of fibrinolytic activity remain without changes at the early period of observation (except decrease of a potential plasminogen activity), and they decrease on the 12th day of the post-ischemic period, which deteriorates conditions of thrombolysis. 
 Conclusions. Complication of diabetes mellitus by ischemic-reperfusion lesion of the brain results in imbalance in the hemocoagulation system at the expense of intensification of pro-coagulant mechanisms, and promotes inhibition of fibrinolytic processes with advanced changes in the dynamics of observation.

FIBRINOLYTIC ACTIVITY IN HEART TISSUE IN HYPOTHYROID AND HYPERTHYROID RATS UNDER THE INFLUENCE OF EXOGENOUS MELATONIN AND BLINDING CONDITIONS (ENUCLEATION)

The aim of our scientific work was to study the effect of exogenous melatonin on the indicators of fibrinolytic processes in the heart tissues and to analyze the changes in fibrinolytic activity occurring in the heart tissues of enucleated hyper- and hypothyroid white rats. The experiments were carried out on white nonlinear male rats weighing 0.12-0.14 kg. 4 experimental groups of animals were formed. The control group consisted of 11 normothyroid rats, which were injected with a melatonin solution in appropriate volumes. The animals were euthanized under light ether anesthesia. Samples of the examined heart tissue were ground in a glass homogenizer with borate buffer (pH 9.0). The homogenate was used in biochemical analysis. Determination of the total, enzymatic and non-enzymatic fibrinolytic activity (TFA, EFA, NFA, respectively) in the heart tissues was carried out by the method of azofibrin lysis (LLC "Simko", Ukraine). Statistical processing of the results was carried out by the method of variation statistics using the Student's test. Experimental studies on nonlinear male white rats have shown that the introduction of exogenous melatonin, as well as modeling the conditions of endogenous melatonin overproduction by blinding (enucleation) cause an increase in the intensity of enzymatic and non-enzymatic fibrinolysis in heart tissue in normotyroid animals. At other hand, enucleation causes an increase in the intensity of fibrinolysis in the tissues of the heart in hypothyroid rats, but not in hyperthyroid.

The inclusion of leukocytes into platelet rich plasma reduces scaffold stability and hinders extracellular matrix remodelling.

BackgroundScaffolds should have controllable degradation rate and allow cells to produce their own extracellular matrix. Platelet rich plasma (PRP) is a source of autologous growth factors and proteins embedded in a 3D fibrin scaffold. There is no consensus regarding the obtaining conditions and composition of PRPs. The aim of this study was to evaluate how the inclusion of leukocytes (L-PRP) in plasma rich in growth factors (PRGF) may alter the process of fibrinolysis. The effect of different combinations of cellular phenotypes with PRGF and L-PRP clots on both the fibrinolysis and matrix deposition process was also determined. MethodsPRGF and L-PRP clots were incubated for 14 days and D-dimer and type I collagen were determined in their conditioned media to evaluate clots’ stability. For remodelling assays, gingival fibroblasts, alveolar osteoblasts and human umbilical vein endothelial cells (HUVEC) were seeded onto the two types of clots for 14 days. D-dimer, type I collagen, and laminin α4 were measured by ELISA kits in their conditioned media. Morphological and histological analysis were also performed. Cell proliferation was additionally determined ResultsPRGF clots preserved their stability as shown by the low levels of both D-dimer and collagen type I compared to those obtained for L-PRP clots. The inclusion of both gingival fibroblasts and alveolar osteoblasts stimulated a higher fibrinolysis in the PRGF clots. In contrast to this, the degradation rates of both PRGF and L-PRP clots remained unchanged after culturing with the endothelial cells. In all cases, type I collagen and laminin α4 levels were in line with the degree of clots’ degradation. In all phenotypes, cell proliferation was significantly higher in PRGF than in L-PRP clots. ConclusionThe inclusion of leukocytes in PRGF scaffolds reduced their stability, decreased cell number and slowed down cell remodelling.

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.

Dysregulation of Fibrinolytic Process Contributes to the Thrombotic and Bleeding Complications in ESRD Patients

Dysregulation of Fibrinolytic Process Contributes to the Thrombotic and Bleeding Complications in ESRD Patients

New and Off-Label Uses of Tranexamic Acid.

New and Off-Label Uses of Tranexamic Acid.

State of proteolytic and fibrinolytic processes in the brain of rats with experimental diabetes mellitus complicated by cerebrovascular accident in the basin of the carotid arteries

Introduction. The imbalance in the protease-antiprotease system is an integral part of the pathogenesis of acute disorder of cerebrovascular circulation and diabetes mellitus (DM), but its manifestations in the complication of diabetes by ischemia-reperfusion of the brain have not been investigated yet.&#x0D; The objective of the work – to study the dynamics of carotid ischemia-reperfusion effect on the proteo- and fibrinolytic activity in brain structures of rats with experimental DM.&#x0D; Rats with the four-month streptozotocin DM were modeled bilateral carotene ischemia during 20 minutes. In homogenates of brain structures, indicators of proteo- fibrinolytic activity were determined after 1 hour from the beginning of reperfusion and during the 12th day of the post-ischemic period. In the absence of DM, the proteolytic activity of all or individual indicators il increased in both periods of the post-ischemic period in the cortex of the frontal and occipital lobes, the fields of hippocampus CA2 and CA3, and during the 12th day in the field CA1.&#x0D; Results. In rats with diabetes, in all brain structures, there are no changes in the lysis of azo-albumin and azo-casein in both terms of observation and lysis of collagen progressively decreases. In rats without DM in the cortex of the studied particles, fields of the hippocampus CA1 and CA2, all or separate indices of fibrinolytic activity are increased in both periods of the post-ischemic period, in the field CA3 – all indices at the 12th day. In the presence of diabetes in the cortex of both studied lobes and the field CA1, the fibrinolytic activity decreases in the late post-ischemic period, in the fields of CA2 and CA3 – during both observation periods.&#x0D; Conclusion. In the brain structures under investigation at both time intervals, the DM eliminates the reaction of low and high molecular weight proteins to the ischemia-reperfusion and suppresses fibrinolytic activity.

Fibrinogen and a Triad of Thrombosis, Inflammation, and the Renin-Angiotensin System in Premature Coronary Artery Disease in Women: A New Insight into Sex-Related Differences in the Pathogenesis of the Disease.

Coronary artery disease (CAD) is the leading cause of morbidity and mortality in women worldwide. Its social impact in the case of premature CAD is particularly devastating. Many differences in the presentation of the disease in women as compared to men, including atypical symptoms, microvascular involvement, and differences in pathology of plaque formation or progression, make CAD diagnosis in women a challenge. The contribution of different risk factors, such as smoking, diabetes, hyperlipidemia, or obesity, may vary between women and men. Certain pathological pathways may have different sex-related magnitudes on CAD formation and progression. In spite of the already known differences, we lack sufficiently powered studies, both clinical and experimental, that assess the multipathogenic differences in CAD formation and progression related to sex in different age periods. A growing quantity of data that are presented in this article suggest that thrombosis with fibrinogen is of more concern in the case of premature CAD in women than are other coagulation factors, such as factors VII and VIII, tissue-type plasminogen activator, and plasminogen inhibitor-1. The rise in fibrinogen levels in inflammation is mainly affected by interleukin-6 (IL-6). The renin–angiotensin (RA) system affects the inflammatory process by increasing the IL-6 level. Unlike in men, in young women, the hypertensive arm of the RA system is naturally downregulated by estrogens. At the same time, estrogens promote the fibrinolytic path of the RA system. In young women, the promoted fibrinolytic process upregulates IL-6 release from leukocytes via fibrin degradation products. Moreover, fibrinogen, whose higher levels are observed in women, increases IL-6 synthesis and exacerbates inflammation, contributing to CAD. Therefore, the synergistic interplay between thrombosis, inflammation, and the RA system appears to have a more significant influence on the underlying CAD atherosclerotic plaque formation in young women than in men. This issue is further discussed in this review. Fibrinogen is the biomolecule that is central to these three pathways. In this review, fibrinogen is shown as the biomolecule that possesses a different impact on CAD formation, progression, and destabilization in women to that observed in men, being more pathogenic in women at the early stages of the disease than in men. Fibrinogen is a three-chain glycoprotein involved in thrombosis. Although the role of thrombosis is of great magnitude in acute coronary events, fibrinogen also induces atherosclerosis formation by accumulating in the arterial wall and enabling low-density lipoprotein cholesterol aggregation. Its level rises during inflammation and is associated with most cardiovascular risk factors, particularly smoking and diabetes. It was noted that fibrinogen levels were higher in women than in men as well as in the case of premature CAD in women. The causes of this phenomenon are not well understood. The higher fibrinogen levels were found to be associated with a greater extent of coronary atherosclerosis in women with CAD but not in men. Moreover, the lysability of a fibrin clot, which is dependent on fibrinogen properties, was reduced in women with subclinical CAD compared to men at the same stage of the disease, as well as in comparison to women without coronary artery atherosclerosis. These findings suggest that the magnitude of the pathological pathways contributing to premature CAD differs in women and men, and they are discussed in this review. While many gaps in both experimental and clinical studies on sex-related differences in premature CAD exist, further studies on pathological pathways are needed.

Intra-abdominal propylene suture fragment leading to complete small bowel obstruction: A case report.

IntroductionPropylene sutures are non-absorbable sterile surgical sutures, which when present intra-abdominally act as a foreign body leading to adhesion formation. Post-operative adhesions are a common cause of bowel obstruction, with complete closed loop obstruction presenting as a surgical emergency.Case detailsA 66-year-old man who had undergone a laparotomy for bowel obstruction two years back, presented to our emergency department with features of complete bowel obstruction. Midline laparotomy revealed propylene suture and intra peritoneal adhesions acting as a constricting band around the jejunum to be the cause.DiscussionAdhesions are common perturbing problem after surgeries. The presence of a foreign body further incites the process of adhesion formation by causing hindrance to the fibrinolysis process. In this case, the adhesive bands acted synergistically with the encircling prolene suture material from the previous surgery in causing a complete loop obstruction.ConclusionPropylene sutures used in routine surgical procedures can migrate intra-abdominally, and lead to adhesion formation. Adhesions along with the non-absorbable suture can cause complete bowel obstruction.

Biochemical and Pharmacological Differentiation of APC and Recombinant Thrombomodulin with Reference to the Effect on the Hemostatic and Fibrinolytic Processes

Introduction:Both thrombomodulin and activated protein C play an important role in the regulation of hemostasis. Recombinant versions of both the thrombomodulin (rTM) and activated protein C (rAPC) have been developed for clinical applications. Marked differences in their biochemical and pharmacological profiles exist. Such differences in their effects may be due to target sites and other interactions and may have therapeutic and safety implications.Aim:The aim of this study is to investigate the biochemical and pharmacological differences between a clinically developed rTM (ART-123, Asahi Kasei Pharma, Tokyo, Japan) and a plasma derived APC (Haematologic Technologies, Burlington, VT, USA).Materials and Methods:The anticoagulant profile of both agents was investigated in native whole blood using the ACT, TEG and global anticoagulant profile using PT and APTT assays at a concentration range of 0-10 ug/ml. The global anticoagulant profile in plasma based systems in terms of PT and APTT was also investigated. The effect of both of these agents was studied in the antiprotease (Xa and IIa) inhibitory assays and thrombin generation inhibition methods. A modified TEG system was used to study the modulation of tissue plasminogen activator mediated fibrinolysis by these agents.Results:In the ACT assays APC produced a much stronger anticoagulant effect in a concentration range of 0-10 ug/ml with a doubling time at 3 ug/ml, whereas rTM produced marginal effects on this parameter. The TEG profile of APC exhibited much stronger anticoagulant effects in comparison to rTM. At 3 ug/mL, APC produces much longer prolongation of the R and K time parameters in comparison to rTM. APC also facilitated tPA-mediated fibrinolysis in contrast to rTM. In the anti-IIa assays, rTM produced concentration dependent inhibition of thrombin and no inhibition of factor Xa. APC did not have any effect on either Factor Xa or IIa. In the thrombin generation assays, APC produced a stronger inhibition of thrombin generation (IC50=0.4 ug/mL) whereas rTM was relatively weaker (IC50 = 1.2 ug/mL). In the APTT assay APC produced a much stronger anticoagulant effect whereas rTM produced relatively weaker effects. Both agents did not produce any modification of agonist induced platelet aggregation. In terms of anticoagulant potency the APC exhibited 70 + 6 USP U/mg whereas the rTM was much weaker < 10 USP U/mg.Conclusions:These studies demonstrate that APC is a much stronger anticoagulant in comparison to rTM. In addition both agents produced different modulation of fibrinolytic and hemostatic processes. These observations suggest that in clinical indications such as DIC, these agents may have differential safety/efficacy implications. DisclosuresTanaka:Asahi Kasei Pharma America Corporation: Employment. Tsuruta:Asahi Kasei Pharma America Corporation: Employment.

Differential Modulation of Plasminogen Activator Mediated Thrombolysis By Recombinant Thrombomodulin and Activated Protein C

Introduction:Urokinase (UK) and tissue plasminogen activator (tPA) mediate thrombolytic actions by activating endogenous plasminogen. Thrombomodulin (TM) complexes with thrombin to activate Protein C and thrombin activatable fibrinolysis inhibitor (TAFI). Activated Protein C (APC) modulates coagulation by digesting factors V and VIII and activates fibrinolysis by decreasing PAI-1 functionality. Since the thrombin/thrombomodulin complex regulates both TAFI functionality and protein C activation, rTM modulates the fibrinolytic process by multiple mechanisms.Aim:To compare the effects of rTM and APC on urokinase and tPA mediated thrombolysis utilizing thromboelastography.Materials and methods:Native whole blood was activated using a diluted intrinsic activator (APTT reagent, Triniclot). The modulation of thrombolysis by tPA and UK (Abbott, Chicago, USA) was studied by supplementing these agents to whole blood and monitoring TEG profiles for 3 hours. To investigate effect of APC (Haematologic Technologies, VT, USA) and rTM (Asahi Kasai Pharma, Tokyo, Japan) these agents were also supplemented to the activated blood at a concentration range of 0.02 - 3.0 ug/ml. The modulation of tPA and UK induced thrombolysis by APC and rTM was studied in terms of thromboelastograph patterns. In addition, the effect of both APC and rTM on plasma based systems supplemented with tPA was also investigated.Results:In comparison to rTM, APC produced a stronger anticoagulant effect in terms of r time, k time, angle and MA. Fibrinolysis was assessed in terms of LY30 (%) and LY60 (%). At concentrations of up to 3.0ug/ml rTM and APC did not produce any direct fibrinolytic effects. APC also produced strong augmentation of the lytic of effects of tPA and urokinase in a concentration dependent fashion. rTM at lower concentrations produced stabilization of clot resisting fibrinolysis. In the plasma based system, APC exhibited a stronger interaction with tPA in comparison to rTM.Conclusion:These studies demonstrate the differential anticoagulant and procoagulant effects of APC and rTM. APC is a stronger anticoagulant than rTM and facilitates thrombolysis. rTM is a weaker anticoagulant and modulates clot stability branding it resistant to fibrinolysis. These observations suggest that while APC may impair hemostasis, rTM restores hemostasis via TAFI activation. The dual mode of action of the rTM may contribute to the regulation of fibrinolysis in facilitating hemostatic balance in both physiologic and pathologic states. [Display omitted] DisclosuresTanaka:Asahi Kasei Pharma America Corporation: Employment. Tsuruta:Asahi Kasei Pharma America Corporation: Employment.

PAI-1 in Diabetes: Pathophysiology and Role as a Therapeutic Target.

Hypofibrinolysis is a key abnormality in diabetes and contributes to the adverse vascular outcome in this population. Plasminogen activator inhibitor (PAI)-1 is an important regulator of the fibrinolytic process and levels of this antifibrinolytic protein are elevated in diabetes and insulin resistant states. This review describes both the physiological and pathological role of PAI-1 in health and disease, focusing on the mechanism of action as well as protein abnormalities in vascular disease with special focus on diabetes. Attempts at inhibiting protein function, using different techniques, are also discussed including direct and indirect interference with production as well as inhibition of protein function. Developing PAI-1 inhibitors represents an alternative approach to managing hypofibrinolysis by targeting the pathological abnormality rather than current practice that relies on profound inhibition of the cellular and/or acellular arms of coagulation, and which can be associated with increased bleeding events. The review offers up-to-date knowledge on the mechanisms of action of PAI-1 together with the role of altering protein function to improve hypofirbinolysis. Developing PAI-1 inhibitors may form for the basis of future new class of antithrombotic agents that reduce vascular complications in diabetes.

Spontaneous fibrinolysis and possibilities of its acceleration in pulmonary embolism

This review contains the data concerning the mechanisms of spontaneous fibrinolysis in pulmonary vessels and possibilities of its acceleration in pulmonary embolism. The spontaneous fibrinolysis system is known to be sequential and multifactorial, with the interaction of accelerators (t-PA and u-PA) and inhibitors (alpha-2-antiplasmin, PAI-1, TAFI). The fibrinolytic processes take place in case of prevailing reactions of accelerating factors over inhibiting ones. The endothelium of pulmonary vessels possesses pronounced antithrombogenic and profibrinolytic properties, therefore, the processes of fibrinolysis in the pulmonary vascular bed normally occur more intensively than in the vessels of the systemic circulation. The membrane proteins of the endothelium annexins A2 activate plasminogen, whereas thrombomodulin inhibits the activity of PAI-1. The main approaches to increase the fibrinolysis intensity in conditions of pulmonary embolism may be aimed at elevating the activity of fibrinolytic enzymes (enhancing the synthesis of annexins A2, the use of NMDA-receptor antagonists) and suppressing its inhibitors (the use of monoclonal antibodies to alpha-2-antiplasmin, as well as plasminogen activator inhibitor-1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI). Promising directions for future research can be the synthesis of a new generation of tissue-type plasminogen activators, and investigations of the possibility of clinical application of antithrombin and thrombomodulin, angiotensin converting enzyme inhibitors and cortisol antagonists. To meet these challenges, it is necessary to develop new models of venous thrombosis and acute pulmonary embolism in different animal species, with the assessment of the changes in the venous haemodynamics and pulmonary microcirculation on the background of administration of a new class of fibrinolytic agents.

TEG's Utility to Detect Hypercoagulability in Adult Patients at Post-Cardiac Surgery Using Cardiopulmonary Bypass in ICU

The use of Cardiopulmonary Bypass (CPB) in adult patients of cardiac surgery disrupts the coagulation system. The mostcommon complication of the coagulation system is bleeding; however, that does not rule out the possibility of a dangeroushypercoagulation condition. A quick and precise coagulation test can provide clues for clinicians to predict futurehemostatic disorders or determine interventional therapy. aPTT and PT are standard laboratory tests, which are limited todetect a deficiency of coagulation factors. Thromboelastography (TEG) test (R time, K time, α angle, MA, and LY30) providesan overview of the entire coagulation and fibrinolysis process with faster results. A 2.7 mL citrate blood sample was takenand tested in a TEG®5000 device, then centrifuged. The plasma was then tested for aPTT and PT using the Sysmex CS-2100idevice. Bleeding volume was measured from chest drain 1-2 hours in the ICU after chest closure in the operating room.Bleeding criteria were as follows: &gt; 1.5 mL/kg/hour for 6 hours consecutively in 24 hours or &gt; 100 mL/hour. The resultsshowed 30 patients with no clinically significant bleeding. A significant correlation was found between PT and bleedingvolume at IV hour (p=0.008, r= 0.472). There was no correlation between aPTT and TEG (R time, K time, α angle, MA, andLY30) with the bleeding volume at I, II, III, and IV hours. There was a hypercoagulation indication of the TEG test of 56.7%,which showed clinical importance for the patient. PT can be used to analyze changes in bleeding volume at IV hour and TEGis more superior to detect hypercoagulability of adult patients after cardiac surgery with CPB.

Repurposing of Plasminogen: An Orphan Medicinal Product Suitable for SARS-CoV-2 Inhalable Therapeutics.

The SARS-CoV-2 infection is associated with pulmonary coagulopathy, which determines the deposition of fibrin in the air spaces and lung parenchyma. The resulting lung lesions compromise patient pulmonary function and increase mortality, or end in permanent lung damage for those who have recovered from the COVID-19 disease. Therefore, local pulmonary fibrinolysis can be efficacious in degrading pre-existing fibrin clots and reducing the conversion of lung lesions into lasting scars. Plasminogen is considered a key player in fibrinolysis processes, and in view of a bench-to-bedside translation, we focused on the aerosolization of an orphan medicinal product (OMP) for ligneous conjunctivitis: human plasminogen (PLG-OMP) eye drops. As such, the sterile and preservative-free solution guarantees the pharmaceutical quality of GMP production and meets the Ph. Eur. requirements of liquid preparations for nebulization. PLG-OMP aerosolization was evaluated both from technological and stability viewpoints, after being submitted to either jet or ultrasonic nebulization. Jet nebulization resulted in a more efficient delivery of an aerosol suitable for pulmonary deposition. The biochemical investigation highlighted substantial protein integrity maintenance with the percentage of native plasminogen band > 90%, in accordance with the quality specifications of PLG-OMP. In a coherent way, the specific activity of plasminogen is maintained within the range 4.8–5.6 IU/mg (PLG-OMP pre-nebulization: 5.0 IU/mg). This is the first study that focuses on the technological and biochemical aspects of aerosolized plasminogen, which could affect both treatment efficacy and clinical dosage delivery. Increasing evidence for the need of local fibrinolytic therapy could merge with the availability of PLG-OMP as an easy handling solution, readily aerosolizable for a fast translation into an extended clinical efficacy assessment in COVID-19 patients.

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