Thrombin Formation Research Articles

PF801 DASATINIB ATTENUATES THROMBIN GENERATION AND CLOT RETRACTION OF CONVULXIN ACTIVATED HUMAN PLATELETS

Background:Tyrosine kinase inhibitors (TKI) are a very effective group of drugs that considerably prolong survival of patients with chronic myeloid leukemia. Several lines of evidence suggest that dasatinib an inhibitor of BCR‐ABL may induce bleeding due to its effect on platelets. Previously it was shown that dasatinib induced impaired platelet adhesion, aggregation and secretion. In our in vitro studies we investigated the effect of dasatinib on the procoagulant activity and on clot retraction of human platelets.Aims:We hypothesized that dasatinib treatment may impair various platelet functions in activated platelets.Methods:Platelet‐rich plasmas (PRPs) were prepared from citrate‐anticoagulated blood samples of healthy volunteers using low speed centrifugation. Subsequently PRPs were incubated with or without dasatinib, then platelets were activated by a GPVI agonist, the snake venom convulxin. Phosphatidylserine (PS) expression of activated platelets was examined by flow cytometry. The thrombin generation test (TGT) of PRPs was performed in the presence of 1pM Tissue factor (PRP reagent) by a fluorimetric assay. Fluorescence was detected by a Fluoroskan Ascent fluorimeter (Thermo Fischer Scientific), and the thrombin generation curves were analyzed by the Thrombinoscope software (Thrombinoscope BV). The activated form of GPIIb/IIIa was detected by measuring PAC1 expression by flow cytometry. Clot retraction of platelets was elicited by high calcium concentration in PRP and results were expressed as retracted percent of the original volume and was followed up to 1 hour. Sarcoma Family Kinases (SFK) play role in platelet activation, thus their phosphorylation levels were examined from dasatinib pretreated platelet lysates by Western‐blot.Results:Convulxin resulted in a dose‐dependent increase of platelet PS expression peaking at 51% when 12.5 ng/mL convulxin was used. The lower end of the therapeutic range of dasatinib (10 nM) showed no effect on the rate of this activation marker while the high therapeutic dasatinib concentration (100 nM) considerably prevented this activating effect. PS expressed on the platelet surface may serve as an anchor for the multiprotein complexes of the coagulation cascade. Thus, we investigated the effect of dasatinib on thrombin generation in PRP and found that 100 nM dasatinib attenuated thrombin formation as it reduced the peak thrombin values from 322 ± 31 nM thrombin to 204 ± 62 nM. Similarly, PAC1 expression on convulxin activated platelets was significantly prevented by preincubation with 100 nM dasatinib (8% versus 81%). Clot retraction was 80% in non activated samples and this was decreased to 53% upon convulxin activation that was completely prevented by dasatinib preincubation. We found that dasatinib pretreatment downregulated the phosphorylation levels of Fyn, Lyn and Src kinases in a concentration dependent manner.Summary/Conclusion:Dasatinib is an effective TKI that alters platelet reactions to activation via the collagen receptor GPVI. This effect may be mediated via its effect on phosphorylation status of SFK and can contribute to bleeding symptoms observed during dasatinib treatment.

PB2257 USE OF ARGATROBAN AS AN ANTICOAGULANT TREATMENT ALTERNATIVE IN CRITICAL PATIENTS WITH HEPARIN‐INDUCED THROMBOCYTOPENIA. EXPERIENCE IN A THIRD LEVEL HOSPITAL.

Background:Heparin is a widely used drug. The most frequent complication derived from its use is the haemorrhage. However, an uncommon but potentially serious adverse effect is heparin‐induced thrombocytopenia (HIT). The goal of treatment of HIT is to reduce platelet activation and thrombin formation to decrease the risk of thrombosis. Argatroban is the drug of choice in HIT patients as an alternative anticoagulant treatment in critically ill patients with high risk of haemorrhage, postoperative cardiac events or renal failure, since it allows close control of anticoagulation.Aims:To analyze the clinical, laboratory and therapeutic characteristics of patients treated with Argatroban by HIT in our hospitalMethods:Retrospective analysis or patients treated with Argatroban for diagnosed HIT during the last year.Results:Four patients have required treatment with Argatroban for HIT since they required maintenance of anticoagulation due to mitral prosthesis, unstable angina, DVT in the right lower extremity and acute ischemia in the left lower extremity. Median age 68 years (43‐79). All of them were diagnosed with HIT according to the 4T score during their admission to the Intensive Care Unit. PF4/Heparin antibody testing was positive in the four patients.The median number of days from the start of Heparin treatment (2 with unfractioned and 2 with low molecular weight) to the onset of thrombocytopenia was 6 days (1‐8). Median platelets at diagnosis were 73x109/L (49‐82). Median number of days from the start of Argatroban to platelet recovery was 6 days (3‐12). Median number of days of treatment with Argatroban was 9.5 days (2‐18). Argatroban dose was adjusted to maintain aPTT at 1.5‐3.0 times baseline. 3 patients presented thrombotic events, 2 directly related to HIT (1 ischemic stroke and 1 acute ischemia of right upper extremity) and 1 with worsening of reperfusion of the limb. 2 patients required amputation of the limb. No adverse events associated with Argatroban, including haemorrhage, were observed.Summary/Conclusion:HIT is a potentially catastrophic complication of heparin treatment which requires urgent detection and treatment with a non‐heparin anticoagulant such as Argatroban should be initiated. Even in critically ill patients, Argatroban anticoagulation improves clinical outcomes in patients who have HIT, without increasing bleeding risk.

Nonhemostatic Activities of Factor Xa: Are There Pleiotropic Effects of Anti-FXa Direct Oral Anticoagulants?

Factor Xa (FXa) is the key serine protease of the coagulation cascade as it is the point of convergence of the intrinsic and extrinsic pathways, leading to the formation of thrombin. Factor Xa is an established target of anticoagulation therapy, due to its central role in coagulation. Over the past years, several direct oral anticoagulants (DOACs) targeting FXa have been developed. Rivaroxaban, apixaban, and edoxaban are used in clinical practice for prevention and treatment of thrombotic diseases. Increasing evidence suggests that FXa exerts nonhemostatic cellular effects that are mediated mainly through protease-activated receptors-1 and -2 and are involved in pathophysiological conditions, such as atherosclerosis, inflammation, and fibrosis. Direct inhibition of FXa by DOACs could be beneficial in these conditions. This is a narrative review that focuses on the cellular effects of FXa in various cell types and conditions, as well as on the possible pleiotropic effects of FXa-targeting DOACs.

Pharmacokinetic and Pharmacodynamic Drug Monitoring of Direct-Acting Oral Anticoagulants: Where Do We Stand?

For decades, oral anticoagulation has been based on vitamin K antagonist such as warfarin, which requires pharmacodynamic (PD) drug monitoring to guide the therapy. The drug effect is measured by the clotting test prothrombin time and expressed as international normalized ratio. New direct oral anticoagulants are increasingly used in fixed-dose regimens but are licensed without any therapy monitoring. However, extensive clinical experiences have demonstrated that interindividual variations in the response to the therapy with direct oral anticoagulants do exist. In situations such as bleeding or thrombosis, therapeutic drug monitoring could be useful. Unfortunately, global coagulation assays such as the prothrombin time or the activated partial thrombin time are not suitable for this purpose. To measure drug concentrations, more specific coagulation test can be used if they are externally calibrated with the respective drugs. For the direct thrombin inhibitor dabigatran etexilate, a calibrated diluted thrombin time or ecarin clotting time can be used, whereas for anti-factor Xa drugs such as rivaroxaban, apixaban, edoxaban, and betrixaban, calibrated anti-factor Xa assays are appropriate. However, the gold standard to measure drug concentrations is LC-MS/MS. The variation in bleeding and thrombotic events noted with both drug classes under fixed-dose conditions suggests additional interindividual PD differences. Therefore, PD monitoring to individualize the therapy may be an option. For dabigatran, this is the inhibition of thrombin formation and for anti-factor Xa drugs, the inhibition of factor Xa activity, which can be followed using the functional assays mentioned above but without calibration. Alternatively, thrombin generation assays have been proposed for both drug classes. So far, not many clinical data have been published about the potentially beneficial effects of PD monitoring for dose individualization. The assay platforms for PD monitoring are present in many clinical laboratories, but efforts are needed to validate and standardize available assays to perform appropriate clinical trials.

Elevated plasma levels of hepatocyte growth factor in rats experimentally envenomated with Bothrops jararaca venom: Role of snake venom metalloproteases

The hepatocyte growth factor (HGF)/c-met pathway, which mainly consists of HGF activator (HGFA) and its substrate HGF, protects various types of cells via anti-apoptotic and anti-inflammatory signals. Thrombin is the main physiological activator of such plasmatic pathway, and increased plasma concentrations of HGF have been considered as a molecular marker for some pathological conditions, such as disseminated intravascular coagulation. Since thrombin generation is often linked to tissue injury, and these events are common during snake venom-induced consumption coagulopathies (VICC), our goals were to examine whether Bothrops jararaca venom (Bjv), which induces VICC in vivo: (i) activates the HGF/c-met pathway in vivo and (ii) cleaves zymogen forms of HGFA and HGF (proHGFA and proHGF, respectively) in vitro. Two experimental groups (n = 6, each) of male adult Wistar rats were subcutaneously injected with 500 μL of 0.9% NaCl solution (control) or sub-lethal doses (1.6 mg/kg) of Bjv. Three hours after envenomation, whole blood samples were collected from the carotid arteries to evaluate relevant coagulation parameters using rotational thromboelastometry and fibrinogen level (colorimetric assay). Additionally, the plasma concentration of HGF was assayed (ELISA). Thromboelastometric assays showed that blood clotting and fibrin polymerization were severely impaired 3 h after Bjv injection. Total plasma HGF concentrations were almost 6-fold higher in the Bjv-injected group (410.0 ± 91) compared with control values (68 ± 18 pg/mL, p < 0.05). Western blotting assay showed that Bjv processed proHGFA and proHGF, generating bands resembling those generated by thrombin and kallikrein, respectively. In contrast to the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), the metalloprotease inhibitor ethylenediaminetetraacetic acid disodium salt (Na2-EDTA) strongly reduced the ability of Bjv to process proHGFA and generated one active band similar to that of thrombin. Since Bjv contains prothrombin and factor X activators, increased intravascular thrombin formation might partly explain the increased HGF levels after bothropic envenomation. In conclusion, these findings suggest that snake venom metalloproteases may be determinant for elevation of plasma levels of HGF in rats experimentally envenomated with Bjv.

Utilizing Plasma Composition Data to Help Determine Procoagulant Dynamics in Patients with Thermal Injury: A Computational Assessment.

The development of methods that generate individualized assessments of the procoagulant potential of burn patients could improve their treatment. Beyond its role as an essential intermediate in the formation of thrombin, factor (F)Xa has systemic effects as an agonist to inflammatory processes. In this study, we use a computational model to study the FXa dynamics underlying tissue factor-initiated thrombin generation in a small cohort of burn patients. Plasma samples were collected upon admission (Hour 0) from nine subjects (five non-survivors) with major burn injuries and then at 48 hours. Coagulation factor concentrations (II, V, VII, VIII, IX, X, TFPI, antithrombin (AT), protein C (PC)) were measured and used in a computational model to generate time course profiles for thrombin (IIa), FXa, extrinsic tenase, intrinsic tenase and prothrombinase complexes upon a 5 pM tissue factor stimulus in the presence of 1 nM thrombomodulin. Parameters were extracted from the thrombin and FXa profiles (including max rate (MaxRIIa and MaxRFXa) and peak level (MaxLIIa and MaxLFXa)). Procoagulant potential was also evaluated by determining the concentration of the complexes at select times. Parameter values were compared between survivors and non-survivors in the burn cohort and between the burn cohort and a simulation based on the mean physiological (100%) concentration for all factor levels. Burn patients differed at Hour 0 (p < 0.05) from 100% mean physiological levels for all coagulation factor levels except FV and FVII. The concentration of FX, FII, TFPI, AT and PC was lower; FIX and FVIII were increased. The composition differences resulted in all nine burn patients at Hour 0 displaying a procoagulant phenotype relative to 100% mean physiological simulation (MaxLIIa (306 ± 90 nM vs. 52 nM), MaxRIIa (2.9 ± 1.1 nM/s vs. 0.3 nM/s), respectively p < 0.001); MaxRFXa and MaxLFXa were also an order of magnitude greater than 100% mean physiological simulation (p < 0.001). When grouped by survival status and compared at the time of admission, non-survivors had lower PC levels (56 ± 18% vs. 82 ± 9%, p < 0.05), and faster MaxRFXa (29 ± 6 pM/s vs. 18 ± 6 pM/s, p < 0.05) than those that survived; similar trends were observed for all other procoagulant parameters. At 48 hours when comparing non-survivors to survivors, TFPI levels were higher (108 ± 18% vs. 59 ± 18%, p < 0.05), and MaxRIIa (1.5 ± 1.4 nM/s vs. 3.6 ± 0.7 nM/s, p < 0.05) and MaxRFXa (13 ± 12 pM/s vs. 35 ± 4 pM/s, p < 0.05) were lower; similar trends were observed with all other procoagulant parameters. Overall, between admission and 48 hours, procoagulant potential, as represented by MaxR and MaxL parameters for thrombin and FXa, in non-survivors decreased while in survivors they increased (p < 0.05). In patients that survived, there was a positive correlation between FX levels and MaxLFXa (r = 0.96) and reversed in mortality (r= -0.91). Thrombin and FXa generation are increased in burn patients at admission compared to mean physiological simulations. Over the first 48 hours, burn survivors became more procoagulant while non-survivors became less procoagulant. Differences between survivors and non-survivors appear to be present in the underlying dynamics that contribute to FXa dynamics. Understanding how the individual specific balance of procoagulant and anticoagulant proteins contributes to thrombin and FXa generation could ultimately guide therapy and potentially reduce burn injury-related morbidity and mortality.

Coagulation changes induced by lower-body negative pressure in men and women.

We investigated whether lower-body negative pressure (LBNP) application leads to coagulation activation in whole blood (WB) samples in healthy men and women. Twenty-four women and 21 men, all healthy young participants, with no histories of thrombotic disorders and not on medications, were included. LBNP was commenced at -10 mmHg and increased by -10 mmHg every 5 min until a maximum of -40 mmHg. Recovery up to 10 min was also monitored. Blood samples were collected at baseline, at end of LBNP, and end of recovery. Hemostatic profiling included comparing the effects of LBNP on coagulation values in both men and women using standard coagulation tests, calibrated automated thrombogram, thrombelastometry, impedance aggregometry, and markers of thrombin formation. LBNP led to coagulation activation determined in both plasma and WB samples. At baseline, women were hypercoagulable compared with men, as evidenced by their shorter "lag times" and higher thrombin peaks and by shorter "coagulation times" and "clot formation times." Moreover, men were more susceptible to LBNP, as reflected in their elevated factor VIII levels and decreased lag times following LBNP. LBNP-induced coagulation activation was not accompanied by endothelial activation. Women appear to be relatively hypercoagulable compared with men, but men are more susceptible to coagulation changes during LBNP. The application of LBNP might be a useful future tool to identify individuals with an elevated risk for thrombosis, in subjects with or without history of thrombosis. NEW & NOTEWORTHY LBNP led to coagulation activation determined in both plasma and whole blood samples. At baseline, women were hypercoagulable compared with men. Men were, however, more susceptible to coagulation changes during LBNP. LBNP-induced coagulation activation was not accompanied by endothelial activation. The application of LBNP might be a useful future tool to identify individuals with an elevated risk for thrombosis, in subjects with or without history of thrombosis.

Restenosis is associated with prothrombotic plasma fibrin clot characteristics in endovascularly treated patients with critical limb ischemia

IntroductionHypolysible fibrin clots composed of tightly packed fibers characterize patients with peripheral artery disease (PAD) especially those with critical limb ischemia (CLI). Little is known about the impact of a prothrombotic clot phenotype on restenosis following endovascular revascularization in CLI. The goal of this study was to compare fibrin clot properties and their determinants in CLI patients with restenosis after endovascular treatment (ET) and those free of this complication.Methods85 patients with CLI and restenosis within 1 year after ET on optimal pharmacotherapy and 47 PAD control patients without restenosis were included into the study. Plasma fibrin clot permeability (Ks, a measure of the average pore size in the fibrin network) and clot lysis time (CLT) with its potential determinants were determined. During follow-up, the composite endpoint including re-intervention, amputation and death was assessed.ResultsCompared with the control group, patients with restenosis had reduced Ks (− 9.5%, p < 0.001), prolonged CLT (+ 12.4%, p = 0.003), higher thrombin generation (+ 7.9%, p < 0.001) and elevated von Willebrand factor (vWF) antigen (+ 14.2%, p < 0.001). During a 24 months follow-up the composite endpoint occurred in 54 CLI patients with restenosis (63.5%) and nine control patients (19.1%, p < 0.001) with no association with baseline Ks and CLT.ConclusionThe increased thrombin formation and unfavorable fibrin clot properties occur in patients with CLI who experienced restenosis despite optimal endovascular and pharmacological therapy.

Potential role of activated factor VIII (FVIIIa) in FVIIa/tissue factor-dependent FXa generation in initiation phase of blood coagulation.

Factor VIIa/tissue factor (FVIIa/TF) initiates blood coagulation by promoting FXa generation (extrinsic-Xa). Subsequent generation of intrinsic FXa (intrinsic-Xa) amplifies thrombin formation. Previous studies suggested that FVIIa/TF activates FVIII rapidly in immediate coagulation reactions, and FVIIa/TF/FXa activates FVIII prior to thrombin-dependent feedback. We investigated FVIII/FVIIa/TF/FXa relationships in early coagulation mechanisms. Total FXa generated by FVIIa/TF and FVIIa/TF-activated FVIII (FVIIIaVIIa/TF) was 22.6 ± 1.7nM (1min); total FXa with FVIIa-inhibitor was 3.4 ± 0.7nM, whereas FXa generated by FVIIa/TF or FVIII/TF was 10.4 ± 1.1 or 0.74 ± 0.14nM, respectively. Little Xa was generated by FVIII alone, suggesting that intrinsic-Xa mechanisms were mediated by FVIIIaVIIa/TF and FVIII/TF in the initiation phase. Intrinsic-Xa was delayed somewhat by von Willebrand factor (VWF). FVIII activation by FXa with FVIIa/TF was comparable to activation with Glu-Gly-Arg-inactivated-FVIIa/TF. TF counteracted the inhibitory effects of VWF on FXa-induced FVIII activation mediated by Arg372 cleavage. The FVIII-C2 domain bound to cytoplasmic domain-deleted TF (TF1-243), and VWF blocked this binding by > 80%, indicating an overlap between VWF- and TF1-243-binding site(s) on C2. Overall, these data suggest that FVIII-associated intrinsic-Xa, governed by both FVIIa/TF-induced and FXa-induced FVIII activation mediated by FVIII-TF interactions, together with FVIIa-dependent extrinsic-Xa mechanisms, may be central to the initiation phase of coagulation.

Conditions in the coagulant and anticoagulant activity of blood in patients with coronary heart disease with concomitant diabetes mellitus type 2, depending on the his compensation

The aim: to evaluate and analyze the parameters of coagulant and anticoagulant activity in the blood in different groups of patients with coronary heart disease without and with associated Diabetes Mellitus (DM) type 2 depending on its compensation.Materials: during the study of the coagulant and anticoagulant activity in the blood system, 52 patients were hospitalized in the cardiology and endocrinology department of the Kyiv Clinical Hospital on Railway Transport No. 2 of the Health Protection Center branch of the Ukrainian Railways Public Joint-Stock Company (clinical base of the Department of Internal Medicine Propedeutics No. 1 of the National OO Bogomolets Medical University) with clinical signs of coronary artery disease. Patients were divided into three groups: 27 patients with IHD and with type 2 diabetes in the subcompensation stage, 14th - compensation stage and 11th - decompensation stage.Results: after analyzing the data, at all stages of compensation there was a decrease in all indicators of coagulation hemostasis: APTT - by 6,94 % (compensation stage (I)) (p<0,05) and 13,7 % (decompensation stage (III)) (p<0,01), PTT - by 9,14 % (І) (р<0,01), by 6,43 % (the stage of subcompensation (II) (p<0,05), by 10,3 % (ІІІ ) (p<0,001), TT - by 15,54 % (I) (p<0,001), by 19,2 % (II) (p<0,001), by 20,9 % (III) (p<0,001) and also an increase in the level of fibrinogen by 32,16 % (I) (p<0,001), by 36,04 % (II) (p<0,001), by 48,06 % (III) (p<0,001), which indicates about accelerating the processes of coagulation at once in three links: reduction of the periods of generation of active trombin by internal and external mechanisms with simultaneous activation of fibrogenesis (factor IIA and fibrin). The anticoagulant potential was reduced: AT III by 16,5 % (I) (p<0,001), by 22,7 % (II) (p<0.001) , by 23,6 % (ІІІ) (p<0,001), and PS - by 18,63 % (I and ІІ) (p<0,001). It should be noted that when comparing the groups between themselves, a significant decrease in AT III between the stages of subcompensation and decompensation on 8,5 % (p<0,05).Conclusion: it should be noted that in patients with comorbid IHD with type 2 diabetes, hypercoagulable shifts in the hemostasis system occurred against the background of inhibition of the own anticoagulant blood potential. Changes in the coagulation potential in the combination of type 2 DM were marked by accelerating the processes of coagulation at once in all three stages of coagulation (prothrombinase, thrombin and fibrin formation)

Occlusion of anion-binding exosite 2 in meizothrombin explains its impaired ability to activate factor V

The proteolytic conversion of factor V to factor Va is central for amplified flux through the blood coagulation cascade. Heterodimeric factor Va is produced by cleavage at three sites in the middle of factor V by thrombin, yielding an N terminus-derived heavy chain and a C terminus-derived light chain. Here, we show that light chain formation resulting from the C-terminal cleavage is the rate-limiting step in the formation of fully cleaved Va. This rate-limiting step also corresponded to and was sufficient for the ability of cleaved factor V to bind Xa and assemble into the prothrombinase complex. Meizothrombin, the proteinase intermediate in thrombin formation, cleaves factor V more slowly than does thrombin, resulting in a pronounced defect in the formation of the light chain. A ∼100-fold reduced rate of meizothrombin-mediated light chain formation by meizothrombin corresponded to equally slow production of active cofactor and an impaired ability to amplify flux through the coagulation cascade initiated in plasma. We show that this defect arises from the occlusion of anion-binding exosite 2 in the catalytic domain by the covalently retained propiece in meizothrombin. Our findings provide structural insights into the prominent role played by exosite 2 in the rate-limiting step of factor V activation. They also bear on how factor V is converted into a cofactor capable of assembling into prothrombinase.

Unexpected role of natural killer cell‐derived interferon‐γ as a driver of NETosis and DVT

Unexpected role of natural killer cell‐derived interferon‐γ as a driver of NETosis and DVT

A Prothrombotic State in Patients With a History of Left Ventricular Thrombus

Left ventricular thrombus (LVT) is associated with a hypercoagulable state and occurs most frequently after myocardial infarction (MI). Blood prothrombotic alterations might predispose to LVT formation, its recurrence, and subsequent cerebrovascular events. We investigated 58 patients with a history of LVT unrelated to recent MI or LV ejection fraction <25% and 58 well-matched control subjects. We determined plasma clot permeability, fibrinolytic efficiency, thrombin generation, and endothelial markers after 3 to 6 months of anticoagulant treatment. During follow-up we recorded LVT and thromboembolic events. Patients with LVT more often had LV akinesia, congestive heart failure, and prothrombotic state as evidenced by increased endogenous thrombin potential, lower antithrombin, lower clot permeability, and longer clot lysis time associated with lower antiplasmin, higher plasminogen activator inhibitor-1, thrombin activatable fibrinolysis inhibitor, and von Willebrand factor. During follow-up (57.5 ± 8.1 months for LVT patients and 59.6 ± 5.3 months for controls) strokes, transient ischemic attacks, or LVT occurred in 18 (31%) LVT patients and in 6 (10.3%) control subjects (4.1 vs 1.4% per year, p = 0.006). LVT recurred in 10 (2.3%/year) patients, who had higher risk of stroke/transient ischemic attacks (relative risk = 4.73, 95% confidence interval 1.8 to 40.4). The most compact clot formation at baseline, defined as the lowest quartile of clot permeability (≤5.4 × 10-9 cm2) was a predictor of recurrent LVT (relative risk = 4.67, 95% confidence interval 1.32 to 18.37). This study shows that a persistent prothrombotic state involving enhanced thrombin generation, hypofibrinolysis, and formation of more compact fibrin clots characterizes patients who develop LVT not related to MI and those prone to its recurrence.

Synthesis and characterisation of composite sulphonated polyurethane/polyethersulphone membrane for blood purification application

Polyurethane (PU) with three different functional groups: carboxyl, hydroxyl and sulphonyl group on its molecular structure were synthesised in this work. The synthesised material suppresses blood clotting and exhibits anticoagulant characteristics due to the presence of the important anionic groups. The synthesised PU was blended with polyethersulphone (PES) and fabricated into flat-sheet membrane to study the physico-chemical and biocompatibility properties of the PES membrane for blood purification application. PES-PU flat-sheet membranes were fabricated via the dry-wet phase separation technique. Different loading of PU (0, 1, 2, 3, 4, and 5%) blended with PES was studied and compared. Based on the in-vitro biocompatibility analysis of the membrane, it can be suggested that the membrane incorporated with PU has better anticoagulant properties compared to the pristine PES membrane. PU incorporation prolonged the clotting time, decreased the formation of thrombin, decreased soluble complement component 3a (C3a) generation and suppressed platelet adhesion and aggregation. The anionic groups on the membrane surface might bind to coagulation factors (antithrombin) and the calcium ions, Ca2+ and thus improve anticoagulant ability. Based on both physico-chemical and in-vitro studied, 4% loading of PU is the optimum loading for incorporation with PES membrane. These results suggested that the blended PES-PU membranes with good haemocompatibility allowed practical application in the field of blood purification.

Trombositosis: Faktor Risiko Peningkatan Aterosklerosis pada Perokok Pasif di Kecamatan Pahandut Kota Palangka Raya

Passive smokers that exposure to cigarette smoke in their house has been reached 78,4%. Inhaled of cigarette smoke in the room was more dangerous than in an open place. Every puff of cigarette smoke from active smoker contains 1015 of free radicals which can cause oxidative stress. The presence of free radicals will trigger of activation of inflammatory genes, and then they can be activated of endothelial cells, dysfunction, and injury. Oxidative stress also stimulates large production of platelet (thrombogenesis) and followed by excessive formation of fibrin and thrombin. So that can cause thrombus in endothelial tissue, and it subsequently develops into atherosclerosis. This study aimed to describe thrombocytosis as a risk factor for increased of atherosclerosis in passive smokers in Kecamatan Pahandut, Kota Palangka Raya. This study used a descriptive observational method with a cross-sectional design. The sample obtained by 45 people with purposive sampling technique with criteria such as respondents were passive smokers, housewives, exposed to cigarette smoke more than five years, no history of the disease, and willing to become respondents. Examination of platelet counts was analyzed by an automatic method using Hematology Analyzer. The results showed that 6.7% of passive smokers had high platelet counts (thrombocytosis). High platelet counts in 6.7% of passive smokers caused by exposure of cigarette smoke from active smokers can endanger passive smoking, especially the risk of atherosclerosis. Therefore, a preventive and educative method for both passive and active smokers are needed to prevent the dangers of cigarette smoke to the risk of atherosclerosis

ROLE OF FIBRINOLYTIC ACTIVITY OF BLOOD IN PATHOGENESIS OF NON-ALCOHOLIC FATTY LIVER DISEASE AND CHRONIC KIDNEY DISEASE (ORIGINAL RESEARCH)

The aim of the research − to find out of changes fibrinolytic activity of blood in patients with non-alcoholic fatty liver on the background of obesity, depending on the presence of comorbid chronic kidney disease. Material and methods of research: 444 patients were examined: 84 of them were with NAFLD and class I obesity (group 1), which contained 2 subgroups: 32 patients with non-alcoholic steatosis (NAS) and 52 patients with non-alcoholic steatohepatitis (NASH); 270 patients with NAFLD with comorbid class I obesity and CKD І–ІІІ stage (group 2), including 110 patients with NAS and 160 patients with NASH. The control group consisted of 90 patients with CKD of І–ІІІ stage with normal body weight (group 3). To determine the dependence of the NAFLD course on the form and stage of the CKD, the group of patients was randomized according to age, sex, degree of obesity, and activity of NASH. Research results. The study of fibrinolytic activity of blood showed that total fibrinolytic activity (TFA) of blood plasma in patients of all groups was significantly lower than the control indexes: in patients with NAS – by 7.1%, patients with NAS with CKD – by 14.9%, patients with NASH – by 17.2%, patients with NASH with CKD – by 18.9%, patients with CKD – by 10.6% (p &lt;0.05) with the presence of a probable intergroup difference between groups with comorbidity and isolated course of CKD (p &lt;0.05). The suppression of TFA occurred through the decrease of EF: in patients with NAS the index is significantly lower than that in the controls by 1.2 times, in patients with NAS with CKD – by 1.4 times, in patients with NASH – by 1.7 times, in the group of patients with NASH and CKD – by 1.9 times, while in the group of patients with CKD, the suppression of EF was registered – 1.3 times (p &lt;0.05). At the same time, the NEF in patients of all groups increased in comparison with the AHP group: in patients with NAS – by 1.2 times, in patients with NAS with CKD – by 1.3 times, in patients with NASH – by 1.4 times, in the group of patients with NASH with CKD – 1.5 times, while in the group of patients with CKD the activation of NEF was registered 1.2 times (p &lt;0.05), with the presence of a probable difference between the groups with comorbidity and isolated course of CKD (p &lt;0.05). Conclusion. Analysis of hemostasis and fibrinolysis indices in examined patients with NASH, depending on the stage of CKD showed that with the growth of the CKD stage, the activity of the cohort increases, with the exception of the fibrinogen content (most likely due to coagulopathy consumption), the activity of the anti-coagulants decreases, the total and enzymatic activity of fibrinolysis is reduced, and non-enzymatic compensator increases. Thus, metabolic intoxication, oxidative stress, which accompany the flow of NAFLD with obesity and CKD, promote the activation of the kallikrein-kinin system, the formation of plasma and thrombin, with subsequent disturbance of equilibrium between them, the development of stasis, slag phenomenon, the formation of platelet and erythrocyte aggregates in blood circulation system. The consequence of significant activation of hemocoagulation against the suppression of total fibrinolytic activity (TFA) is the local clotting of blood in the arteries.

A novel hemostatic scaffold material and the importance of scaffold formation on ending hemorrhage: An experimental rat study.

Different pharmacological agents are developed to control bleeding. However, it is critical for these agents to induce thrombin formation and have an effect on vasoconstriction, coagulation, and scaffold. In this study, we aimed to demonstrate the agents' ability to stop bleeding properties on minor and major open bleedings after skin clefts, extracorporal injuries, traumatic cuts, spontaneous or surgical intervention besides scaffold properties. For this purpose, a new and authentic hemostatic agent, processed diatomite (PD) and the most preferred chitosan in the medical area were used to test blood stopping and scaffold effects in a rat femoral bleeding model. The samples were examined by scanning electron microscopy (SEM), and the results on blood stopping were shared. The current experimental study was conducted on rats. The effects of hemostatic agents on our femoral bleeding model were determined. In this study, 22 male Wistar albino rats weighing 158-215 g, were used. The rats were assigned randomly to three groups: control group (n=6), chitosan group (n=8), and PD group (n=8). Bleeding time, scaffold formation, weight differences, histopathological effect and scanning electron microscope (SEM) analyses were performed. In our experimental model, weight loss was 5.0±1.3 g for the control group, 2.9±1.1 g for the chitosan group, and 2.7±1.0 g for the PD group, respectively. When weighed before and after the experiment, there was a significant change in weights of rats in chitosan, and PD groups regarding scaffold formation: it was complete for six rats (75%) and weak for two (25%) rats in chitosan group; however, it was complete for seven rats (87.5%) and weak for one (12.5%) rat in the PD group. Scaffold formation was significant for the chitosan and PD groups versus the control group (p=0.002). In our study, the scaffold formed by PD exerts appropriate porousness and contributes to fibrin formation and prevent re-bleeding. PD had a strong and significant scaffold effect. The effectiveness of PD to stop bleeding was equal to chitosan. Besides being natural, hemostatic agents should not induce cellular damage. We histopathologically demonstrated that PD was harmless for the natural structure of cells and vessels in the femoral site.

Inositol hexakisphosphate increases the size of platelet aggregates

The inositol phosphates, InsP5 and InsP6, have recently been identified as binding partners of fibrinogen, which is critically involved in hemostasis by crosslinking activated platelets at sites of vascular injury. Here, we investigated the putative physiological role of this interaction and found that platelets increase their InsP6 concentration upon stimulation with the PLC-activating agonists thrombin, collagen I and ADP and present a fraction of it at the outer plasma membrane.Cone and plate analysis in whole blood revealed that InsP6 specifically increases platelet aggregate size. This effect is fibrinogen-dependent, since it is inhibited by an antibody that blocks fibrinogen binding to platelets. Furthermore, InsP6 has only an effect on aggregate size of washed platelets when fibrinogen is present, while it has no influence in presence of von Willebrand factor or collagen. By employing blind docking studies we predicted the binding site for InsP6 at the bundle between the γ and β helical subunit of fibrinogen.Since InsP6 is unable to directly activate platelets and it did not exhibit an effect on thrombin formation or fibrin structure, our data indicate that InsP6 might be a hemostatic agent that is produced by platelets upon stimulation with PLC-activating agonists to promote platelet aggregation by supporting crosslinking of fibrinogen and activated platelets.

Evaluation of EC50 of factor VIII as predictor of prophylaxis efficacy in patients with severe haemophilia A

Trough factor (F) VIII level is a not reliable bleeding risk indicator to predict prophylaxis efficacy in severe haemophilia A (SHA), therefore, accurate biomarkers are much needed. Thrombelastography (TEG) monitors both thrombin and clot formation addressing the global haemostatic status but its usefulness to tailor prophylaxis in haemophilia has been poorly evaluated. In this study, correspondence between individual pharmacodynamic/pharmacokinetic profile of FVIII and joint condition, physical activity and bleeding phenotype of SHA patients under prophylactic treatment was assessed. Nineteen SHA patients < 18 years old on long-term prophylaxis treatment with FVIII were studied in an observational cross-sectional study. Whole blood was withdrawn before FVIII administration and at five time-points after infusion for a TEG-based pharmacodynamic- and pharmacokinetic-study. Type of prophylaxis and joint condition at inclusion and physical activity as well as onset of treated spontaneous bleeding events in the previous two years were retrospectively assessed. Six patients had suffered at least one treated spontaneous bleeding event and were named as “bleeders”. The rest were named as “non-bleeders”. Only the half maximal effective concentration of FVIII (FVIII-EC50) for TEG parameters R-time, K-time and α-angle correlated with the bleeding phenotype being significantly higher in bleeders suggestive of a poorer response to FVIII. Poorer joint condition, trough FVIII levels or type of prophylaxis were not definitive predicting variables of bleeding phenotype. In conclusion, this study reveals FVIII-EC50 for the first time as a valuable biomarker to anticipate individual efficacy of prophylaxis in SHA.

Computational Modeling and Microfluidics Reveal Characteristic Patterns of Regulation of Clot Structure and Mechanics By Tissue Factor Localization

The growth of a blood clot is initiated by tissue factor (TF) exposure and is expected to depend on TF localization (i.e., its level and spatial distribution). We sought to understand how the structural and mechanical properties of clots under flow are shaped by simultaneously varying the surface density of TF and its area of exposure. We used a microfluidic device harboring thrombogenic surfaces that differed in length and TF surface density. We perfused human whole blood through this device and continually measured platelet deposition, thrombin formation, and fibrin accumulation by means of fluorescence microscopy. Using our recently developed, detailed mathematical model of spatial clot growth under flow, we performed computational simulations to gain insights into the connection between the structure of a clot and its mechanical properties, such as resistance to blood flow and flow axial velocity.We detected a non-additive, synergistic influence of the thrombogenic surface length and TF surface density on bulk thrombin and fibrin generation. We found that thrombogenic surface length and TF density controlled not only bulk accumulation, but also the occlusivity of the deposited platelet mass, as well as clot resistance to flow. The extent of this control depended on the blood flow velocity. An increase in the TF surface density resulted in condition-dependent differential acceleration of platelet deposition, thrombin formation, and fibrin accumulation. The viscous resistance of the clot was characterized by spatial variations and was higher in the inner clot region. Notably, despite variations in the intraclot structure, variations in the intraclot flow velocity were minor compared to the abrupt decrease in flow velocity at the boundary of the platelet deposition domain.Our analysis revealed characteristic patterns that describe how the shape, size, internal structure, and viscous resistance of a clot depend on the surface density of TF and its area of exposure. Taken together, our results suggest that the structure and mechanics of a growing clot are correlated, but can differ in their regulation by the distinct aspects of TF localization. These findings provide new insights into how initiating signals can temporally and spatially affect thrombus growth under flow. DISCLAIMERThe opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the U.S. Army or of the U.S. Department of Defense. This abstract has been approved for public release with unlimited distribution. DisclosuresNo relevant conflicts of interest to declare.