Rate Of Clot Lysis Research Articles

Impaired fibrinolysis in JAK2V617F-related myeloproliferative neoplasms

BackgroundMyeloproliferative neoplasms (MPNs) are characterized by a high rate of thrombotic complications that contribute to morbidity and mortality. MPN-related thrombogenesis is assumed to be multifactorial, involving both procoagulant and proinflammatory processes. Whether impaired fibrinolysis also participates in the prothrombotic phenotype of MPN has been poorly investigated. ObjectivesWe determined whether MPN, particularly JAK2V617F-positive MPN, is associated with fibrinolytic changes. MethodsTissue-type plasminogen activator (tPA)–mediated fibrinolysis was evaluated both in whole blood and plasma from mice with a hematopoietic-restricted Jak2V617F expression compared with wild-type (WT) mice (Jak2WT) using (1) halo clot lysis, (2) front lysis, and (3) plasmin generation assays. tPA clot lysis assay was performed in the plasma from 65 MPN patients (JAK2V617F mutation, n = 50; CALR mutations, n = 9) compared with 28 healthy controls. ResultsIn whole blood from Jak2V617F mice, we observed a decreased fibrinolysis characterized by a significantly lower halo clot lysis rate compared with Jak2WT (95 ± 22 vs 147 ± 39 AU/min; P < .05). Similar results were observed in plasma (halo clot lysis rate, 130 ± 27 vs 186 ± 29 AU/min; front lysis rate, 2.8 ± 1.6 vs 6.1 ± 1.2 μm.min−1; P < .05). Plasmin generation was significantly decreased both in plasma clots and standardized fibrin clots from Jak2V617F mice compared with Jak2WT mice. Among MPN patients, impaired tPA-related fibrinolysis with prolonged clot lysis time was observed in JAK2V617F and CALR patients. Plasminogen activator inhibitor-1 and α2-antiplasmin were significantly increased in plasma from JAK2V617F patients compared with controls. ConclusionOur results suggest that impaired tPA-mediated fibrinolysis represents an important prothrombotic mechanism in MPN patients that requires confirmation in larger studies.

Ultrasound combined with urokinase under key-shaped bone window enhances blood clot lysis in an in vitro model of spontaneous intracerebral hemorrhage.

Minimally invasive surgery for spontaneous intracerebral hemorrhage is impeded by inadequate lysis of the target blood clot. Ultrasound is thought to expedite intravascular thrombolysis, thereby facilitating vascular recanalization. However, the impact of ultrasound on intracerebral blood clot lysis remains uncertain. This study aimed to explore the feasibility of combining ultrasound with urokinase to enhance blood clot lysis in an in vitro model of spontaneous intracerebral hemorrhage. The blood clots were divided into four groups: control group, ultrasound group, urokinase group, and ultrasound + urokinase group. Using our experimental setup, which included a key-shaped bone window, we simulated a minimally invasive puncture and drainage procedure for spontaneous intracerebral hemorrhage. The blood clot was then irradiated using ultrasound. Blood clot lysis was assessed by weighing the blood clot before and after the experiment. Potential adverse effects were evaluated by measuring the temperature variation around the blood clot in the ultrasound + urokinase group. A total of 40 blood clots were observed, with 10 in each experimental group. The blood clot lysis rate in the ultrasound group, urokinase group, and ultrasound + urokinase group (24.83 ± 4.67%, 47.85 ± 7.09%, 61.13 ± 4.06%) was significantly higher than that in the control group (16.11 ± 3.42%) (p = 0.02, p < 0.001, p < 0.001). The blood clot lysis rate in the ultrasound + urokinase group (61.13 ± 4.06%) was significantly higher than that in the ultrasound group (24.83 ± 4.67%) (p < 0.001) or urokinase group (47.85 ± 7.09%) (p < 0.001). In the ultrasound + urokinase group, the mean increase in temperature around the blood clot was 0.26 ± 0.15°C, with a maximum increase of 0.38 ± 0.09°C. There was no significant difference in the increase in temperature regarding the main effect of time interval (F = 0.705, p = 0.620), the main effect of distance (F = 0.788, p = 0.563), or the multiplication interaction between time interval and distance (F = 1.100, p = 0.342). Our study provides evidence supporting the enhancement of blood clot lysis in an in vitro model of spontaneous intracerebral hemorrhage through the combined use of ultrasound and urokinase. Further animal experiments are necessary to validate the experimental methods and results.

Pharmacological Investigation of In-vitro Anti-inflammatory, Antimicrobial, Thrombolytic, Cytotoxic and In vivo Analgesic Activities of Ethanolic Leaf Extract of Diospyros malabarica

Aims: This study was designed to investigate the effects of leaf extracts of Diospyros malabarica plant (EEDML) on a number of in vitro activities, including antibacterial, anti-inflammatory, cytotoxic, and thrombolytic activities, as well as in vivo analgesic activities.&#x0D; Place and Duration of Study: The Laboratory of Phytochemistry and Pharmacology at the Department of Pharmacy and the Laboratory of Microbiology at Stamford University Bangladesh and the Bangladesh Council of Scientific and Industrial Research, Dhaka, conducted this investigation from January 2022 to June 2023.&#x0D; Methodology: The study employed the Egg Albumin Protein Denaturation Assay to evaluate the anti-inflammatory effects of various doses. The Disk Diffusion Method was employed for conducting an antimicrobial assay. Different tests were conducted to evaluate the effects of thrombolytic and cytotoxic substances. These tests included the Clot Lysis assay and the Brine Shrimp Lethality Assay. In-vivo analgesic testing was conducted using the hot plate method and the acetic acid-induced writhing test.&#x0D; Results: The experimental tests showed significant pain-relieving effects, with inhibition percentages of 46.2% and 66.61% observed at doses of 200 and 400 mg/kg, respectively. The anti-inflammatory test showed a significant inhibition of EEDML at a concentration of 1000μg/mL, with an inhibition rate of 85.45%. The antimicrobial test showed a moderate antimicrobial effect, with a zone of inhibition ranging from 07-15, depending on the microorganism and dosage. The thrombolytic test showed a clot lysis rate of 85.37%, while the cytotoxic test indicated an LC50 value of 1.47.&#x0D; Conclusion: EEDML's phytochemical displays strong pharmacological properties, suggesting its potential for drug discovery in diverse areas.

An in vitro Model for Experimental Evaluation of Sonothrombolysis under Tissue-mimicking Material Conditions.

The mechanical properties of therapeutic ultrasound (US) have attracted scientific interest for thrombolysis enhancement in combination with thrombolytic agents and microbubbles (MBs). The aim of the study was to develop an in vitro model to observe how the effects of sonothrombolysis change in the case where a tissue-mimicking material (TMM) is placed in the path of the US beam before the clot. Fully retracted blood clots were prepared and pulse sonicated for 1 h under various conditions. The system was in a state of real circulating flow with a branch of an open bypass and an occluded tube containing a blood clot, thus mimicking the case of ischemic stroke. The effectiveness of thrombolysis was quantified in milligrams of clots removed. An agar-based TMM was developed around the occluded tube. The clot breakdown in a TMM was found to be more pronounced than in water, presumably due to the retention of the acoustic field. A higher level of acoustic power was required to initiate clot lysis (>76 W acoustic power) using only focused US (FUS). The greatest thrombolysis enhancement was observed with the largest chosen pulse duration (PD) and the use of MBs (150 mg clot mass lysis). The synergistic effect of FUS in combination with MBs on the enzymatic fibrinolysis enhanced thrombolysis efficacy by 260% compared to thrombolysis induced using only FUS. A reduction in the degree of clot lysis was detected due to the attenuation factor of the intervening material (30 mg at 1 and 4 ms PD). In vitro thrombolytic models including a TMM can provide a more realistic evaluation of new thrombolytic protocols. However, higher acoustic power should be considered to compensate for the attenuation factor. The rate of clot lysis is slow and the clinical use of this method will be challenging.

Association of the metabolic syndrome with PAI 1act and clot lysis time over a 10-year follow up in an African population

Background and aimsThe association between the metabolic syndrome (MetS) and plasminogen activator inhibitor-1 (PAI-1) has been well established in cross-sectional studies. It is less clear whether this translates into decreased clot lysis rates and very little information is available on non-European populations. Little is known regarding prospective associations and whether clot lysis progressively worsens in MetS individuals over time. We determined the prospective association of MetS with PAI-1 activity (PAI-1act) and clot lysis time (CLT) over a 10-year period. Methods and resultsAs many as 2010 African men and women aged ≥30 years were stratified according to MetS status and number of MetS criteria (0–5). We also determined the contribution of the PAI-1 4G/5G polymorphism to these associations and identified which MetS criteria had the strongest associations with PAI-1act and CLT. Both PAI-1act and CLT remained consistently elevated in individuals with MetS throughout the 10-year period. PAI-1act and CLT did not increase more over time in MetS individuals than in controls. The 4G/5G genotype did not influence the association of PAI-1act or clot lysis with MetS. Increased waist circumference, increased triglycerides and decreased HDL-C were the main predictors of PAI-1act and CLT. ConclusionsBlack South Africans with MetS had increased PAI-1act and longer CLTs than individuals without MetS. The inhibited clot lysis in MetS did, however, not deteriorate over time compared to controls. Of the MetS criteria, obesity and altered lipids were the main predictors of PAI-1act and CLT and are thus potential targets for prevention strategies to decrease thrombotic risk.

Assessment of fibrinolytic status in whole blood using a dielectric coagulometry microsensor

Rapid assessment of the fibrinolytic status in whole blood at the point-of-care/point-of-injury (POC/POI) is clinically important to guide timely management of uncontrolled bleeding in patients suffering from hyperfibrinolysis after a traumatic injury. In this work, we present a three-dimensional, parallel-plate, capacitive sensor – termed ClotChip – that measures the temporal variation in the real part of blood dielectric permittivity at 1 MHz as the sample undergoes coagulation within a microfluidic channel with <10 μL of total volume. The ClotChip sensor features two distinct readout parameters, namely, lysis time (LT) and maximum lysis rate (MLR) that are shown to be sensitive to the fibrinolytic status in whole blood. Specifically, LT identifies the time that it takes from the onset of coagulation for the fibrin clot to mostly dissolve in the blood sample during fibrinolysis, whereas MLR captures the rate of fibrin clot lysis. Our findings are validated through correlative measurements with a rotational thromboelastometry (ROTEM) assay of clot viscoelasticity, qualitative/quantitative assessments of clot stability, and scanning electron microscope imaging of clot ultrastructural changes, all in a tissue plasminogen activator (tPA)-induced fibrinolytic environment. Moreover, we demonstrate the ClotChip sensor ability to detect the hemostatic rescue that occurs when the tPA-induced upregulated fibrinolysis is inhibited by addition of tranexamic acid (TXA) – a potent antifibrinolytic drug. This work demonstrates the potential of ClotChip as a diagnostic platform for rapid POC/POI assessment of fibrinolysis-related hemostatic abnormalities in whole blood to guide therapy.

Abstract TP243: Treatment With Vwf Inhibitor Dtri-031 In Combination With Rtpa Increases Thrombolytic Efficacy Compared To Rtpa Or Aspirin In Mouse, Canine, And Human Blood

Introduction: We demonstrated that VWF inhibition thrombolyses stabilized clots in and decreases infarct size after middle cerebral artery occlusion (MCAO) in pre-clinical models of stroke. Objective: To compare the thrombolytic efficacy of von Willebrand Factor (VWF) inhibition by DTRI-031 versus rTPA and aspirin utilizing an ex vivo Halo assay. Hypothesis: We hypothesized that DTRI-031 treatment, would result in greater combinatorial efficacy with aspirin or rTPA than any one treatment alone. Methods: Ex vivo Halo assay was performed with whole blood from male and female, 15-month-old wild-type C57BL/6J adult mice, 1-year-old hounds, and healthy control patients 40-60 years of age). Results: Healthy patients The mean CLR max of the negative control group (n=6) was 0.024 ± 0.17 min -1 . Addition of 250 nM DTRI-031 (n=8) or 0.7 nM rtPA (n=8) increased the CLR max to 1.67 ± 1.56 min -1 and 1.80 ± 1.41 min -1 respectively. Addition of 500 nM DTRI-031 and 0.7 rtPA (n=4) resulted in a synergistic effect of both compounds with a CLR max of 10.15 ± 2.4 min -1 (p=.0003). Mice The CLR max of the negative control (n=6) was 0.013 ± 0.011 min -1 . Addition of 250 nM DTRI-031 (n=6) increased the CLR max to 1.19 ± 1.27 min -1 . Addition of 7 nM rtPA (n=6) increased the CLR max to 0.13 ± 0.13 min -1 , which was considerably muted compared to the assay with human blood. The synergistic effect of DTRI-031+rtPA was seen at doses of 250 nM DTRI + 7 nM rtPA (n=4) with a CLR max of 2.08 ± 1.45 min -1 and 500 nM + 7 nM rtPA (n=6) with a CLR max of 4.3 ± 2.5 min -1 compared to monotherapy (p&lt;0.0001). Canine The CLR max of the negative control (n=33) was 2.611 ± 2.659 min -1 . The addition of 250 nM DTRI-031 or 500 nM DTRI-031 and 0.7 rtPA (n=18) resulted in a synergistic effect of both compounds which were significantly greater than all monotherapies with a CLR max of 13.52 ± 8.985 min -1 and 9.152± 6.943 min -1 , respectively. Conclusion: VWF inhibition by DTRI-031 aptamer + rTPA significantly improves clot lysis rate compared to monotherapy with rTPA, DTRI-031, and aspirin in mouse, canine, and human blood.

Blood loss and coagulation profile in pregnant and non-pregnant queens undergoing elective ovariohysterectomy.

ObjectivesThe aims of this study were to determine if there is increased risk of intraoperative bleeding in pregnant cats undergoing elective ovariohysterectomy (OHE), and to compare coagulation in queens in various stages of estrus and pregnancy subjected to elective OHE using a whole-blood viscoelastic assay.MethodsIntraoperative blood loss was compared between non-pregnant and pregnant cats undergoing elective OHE. Viscoelastic evaluations of whole blood drawn pre- and postoperatively were performed using a point-of-care device measuring clot time (CT), clot formation time (CFT), alpha angle, maximum clot formation (MCF), amplitude at 10 and 20 mins (A10 and A20, respectively), and lysis index at 30 and 45 mins after MCF (LI30 and LI45, respectively).ResultsOne hundred and ninety-three cats underwent OHE by a ventral midline approach. Median blood loss was greater for pregnant cats (2.0 ml, range <0.5–13 ml) than non-pregnant cats (<0.5 ml, range <0.5–15 ml; P <0.0001). Preoperatively, pregnant cats had a shorter median CFT (165 s vs 190.5 s), increased median A10 (31 from 25.5 VCM units) and A20 (38 from 35 VCM units), and a lower median LI45 (99% from 100%) than non-pregnant cats. Postoperatively, A10 and A20 increased, and LI30 and LI45 decreased in both non-pregnant and pregnant queens. In pregnant queens, mean CT also increased postoperatively.Conclusions and relevancePregnant cats were relatively hypercoagulable and had an increased rate of clot lysis than non-pregnant cats. Intraoperative blood loss was increased in pregnant vs non-pregnant cats, but no clinically relevant bleeding conditions occurred.

PB2432 POSSIBLE GENETIC RISK FACTORS OF PULMONARY EMBOLISM IN YOUNG PATIENTS FROM THE NORTH‐WESTERN REGION OF RUSSIA

Background:Pulmonary embolism (PE) and deep‐vein thrombosis (DVT) are the most common clinical manifestations of venous thromboembolism (VTE) which associated with high rates of morbidity and mortality in population. To date, little is known about the factors which could predict PE development in VTE patients. Several studies have shown that fibrin clot structure as well as the rate of clot lysis could influence the risk of PE development. Moreover, nucleotide variations in genes coding for components involved in processes of thrombus formation or its lysis are well‐described. Nevertheless, association between patient's genotype and features of clinical VTE manifestation, in particular, risk of PE development, remains debatable.Aims:To reveal possible association between the alpha‐fibrinogen (FI‐A), factor XIII A‐subunit (FXIII‐A), tissue plasminogen activator (TPA) and plasminogen activator inhibitor type I (PAI‐1) gene polymorphisms and the features of clinical VTE manifestation in patients with early‐onset disease from the North‐Western region of Russia.Methods:We analyzed 243 young patients under 45 years old (119 men and 124 women, mean age – 37.4 years) with at least one confirmed episode of acute VTE. Isolated DVT (“DVT only”) was diagnosed in 154 (63.4%), DVT complicated by PE (“DVT + PE”) – in 53 (21.8%), and isolated PE (“PE only”) – in 36 (14.8%) patients. Genotyping for the FI‐A Thr312Ala, FXIII‐A Val34Leu, PAI‐1 ‐675 4G/5G and TPA 311 bp Ins/Del variations was performed by PCR‐RFLP. To assess intergroup differences in genotype frequencies, the odds ratios (OR), their 95% confidence intervals (CI) and p‐values were calculated by Fisher's exact method.Results:Distributions of the FI‐A, FXIII‐A, PAI‐1 and TPA genotypes in patients with different clinical manifestations of VTE have shown several differences. Frequency of the TPA Del/Del genotype was significantly increased in VTE patients with the signs of PE when compared to those with isolated DVT (31.5% vs. 18.8%, respectively, OR = 2.0; 95% CI: 1.1‐3.6; p = 0.029). Homozygosity for the TPA Del allele was most prevalent in patients with isolated PE (38.9% vs. 18.8% in the group “DVT only”, OR = 2.7; 95% CI: 1.3‐6.0; p = 0.014). In this group, the frequency of the FI‐A 312 Ala/Ala variant was 2‐fold increased when compared to patients with isolated DVT (22.2% vs. 11.0%, respectively, OR = 2.3; 95% CI: 0.9‐5.9; p = 0.098). In patients with the signs of PE, homozygosity for the FXIII‐A 34Leu variant was observed more frequently than in the “DVT only” group (11.2% vs. 6.5%, respectively, OR = 1.8; 95% CI: 0.7‐4.6; p = 0.23). At the same time, the frequency of heterozygous FXIII‐A genotype was significantly increased in patients with isolated DVT when compared to “DVT + PE” group (48.7% vs. 30.2%, respectively, OR = 2.2; 95% CI: 1.1‐4.3; p = 0.024).Summary/Conclusion:TPA Del/Del genotype is associated with the risk of PE development in young VTE patients from the North‐Western region of Russia, whereas heterozygous FXIII‐A variant (34 Val/Leu) could protect from PE in patients with DVT. Role of the FI‐A 312 Ala/Ala and FXIII‐A 34 Leu/Leu variants as possible determinants of different clinical course of VTE needs further clarification.

Carbon monoxide releasing molecule enhances coagulation and decreases fibrinolysis in canine plasma exposed to Crotalus viridis venom in vitro and in vivo.

Carbon monoxide releasing molecule-2 (CORM-2), an emerging therapeutic in human medicine, enhances plasmatic coagulation and attenuates fibrinolysis in vitro in human, rabbit and horse plasma and ameliorates hypocoagulation and hyperfibrinolysis secondary to venom exposure in human plasma in vitro. Fibrinogenases in rattlesnake venom cause decreased clot strength, and in the presence of tissue plasminogen activator (tPA) in vitro, a markedly increased rate of clot lysis. CO interacts with a haem group on fibrinogen, changing its configuration so that the fibrin clot is strengthened and more resistant to fibrinolysis. We hypothesized that CORM-2 enhances coagulation and attenuates fibrinolysis in canine plasma exposed to Cviridis venom. We measured the effects of Cviridis venom on clot strength, rates of coagulation and fibrinolysis in both pooled canine plasma and plasma from individual naturally envenomed dogs, with and without CORM-2, using thromboelastography (TEG). We tested venom effects on coagulation using tissue factor (TF) activated TEG and on both coagulation and fibrinolysis using TF-activated TEG with added tPA. We found that 17.9µg/mL of venom causes a mean 26.4% decrease in clot strength, a 61.8% decrease in maximum rate of thrombus generation, 75% faster clot lysis, a 226% increase in maximum rate of lysis and a 92% decrease in total clot life span (CLS). CORM-2 ameliorated these effects, increasing CLS in the presence of venom by 603%. Additionally, we showed that CORM-2 has similar effects in vitro on plasma from naturally envenomed dogs, showing promise as an adjunct therapy for snake envenomation.

Physico-Chemical and Biological Properties of Biosimilar and Reference Tissue Plasminogen Activator Products

Recombinant tissue plasminogen activator (international nonproprietary name — alteplase) which was developed by «GENERIUM» (Russia) and received a marketing authorisation in Russia is completely analogous to Actilyse® which is used to treat medical conditions accompanied by thrombosis, such as acute myocardial infarction, pulmonary embolism, and ischemic stroke. The aim of the study was to carry out a comprehensive comparison of physico-chemical and biological properties of Revelyse® and the reference product Actilyse® in order to assess their biosimilarity. Materials and Methods: comparative peptide mapping and determination of comparability of chromatographic profiles of tryptic hydrolysates was performed using RP-HPLC and massspectrometry; the molecular weight distribution was determined by mass-spectrometry and polyacrylamide gel electrophoresis (Laemmli method). The purity and homogeneity of products as well as the content of related impurities (oligomers and fragments) were determined using gel filtration; N-glycosylation profile was analysed by hydrophilic HPLC, total sialic acid was quantified by the Svennerholm resorcinol method. Protein binding to fibrin and human fibrinogen was assessed by surface plasmon resonance, and the specific activity was compared by fibrin clot lysis. Results: the research demonstrated a complete overlap of the products’ peptide maps, which indicates the identity of аlteplase amino acid sequences in the two medicines being compared. The authors of the study also determined the molecular weight and the content of the intact single-stranded form of the protein, and quantified post-translational modifications, the content of sialic acids and neutral sugars. The analysis of the N-glycosylation profile revealed insignificant differences in the percentage of multiantenna complex glycans. The specificity of alteplase was evaluated by analysing the formation of protein complexes with natural alteplase ligands – fibrin and plasminogen activator inhibitor-1, but no significant differences were found. The comparison of specific activation of plasminogen fibrinolytic activity was performed based on the results of the assay analysing the fibrin clot lysis rate, and it demonstrated comparability of Revelyse® and Actilyse®. Conclusions: comparative experimental studies have shown no differences in the structure, charge distribution heterogeneity, impurities content, and specific activity of alteplase as a component of Revelyse® and the reference product Actilyse®, which leads to the conclusion that they are similar in terms of physicochemical and biological properties.

Sonothrombolysis with Magnetically Targeted Microbubbles

Microbubble-enhanced sonothrombolysis is a promising approach to increasing the tolerability and efficacy of current pharmacological treatments for ischemic stroke. Maintaining therapeutic concentrations of microbubbles and drugs at the clot site, however, poses a challenge. The objective of this study was to investigate the effect of magnetic microbubble targeting upon clot lysis rates in vitro. Retracted whole porcine blood clots were placed in a flow phantom of a partially occluded middle cerebral artery. The clots were treated with a combination of tissue plasminogen activator (0.75 µg/mL), magnetic microbubbles (∼107 microbubbles/mL) and ultrasound (0.5 MHz, 630-kPa peak rarefactional pressure, 0.2-Hz pulse repetition frequency, 2% duty cycle). Magnetic targeting was achieved using a single permanent magnet (0.08–0.38 T and 12-140 T/m in the region of the clot). The change in clot diameter was measured optically over the course of the experiment. Magnetic targeting produced a threefold average increase in lysis rates, and linear correlation was observed between lysis rate and total energy of acoustic emissions.

Abstract 80: The Experimental Neuroprotective Drug Na1 Does Not Interfere With the Fibrinolytic Activities of Alteplase or Tenecteplase

Introduction: Tissue plasminogen activator (Alteplase; tPA) is the most commonly used thrombolytic medication for treatment of acute ischemic stroke, and recent research suggests that Tenecteplase (TNKase) is also effective. NA-1 is a novel neuroprotective drug intended to reduce disability when administered after stroke that is currently being studied in two late stage clinical trials, FRONTIER (NCT02315443) and ESCAPE-NA1 (NCT02930018). Given the overlap in the patient populations and the likelihood that subjects receiving NA-1 could receive thrombolysis, studies were performed to evaluate the effects of NA-1 on the fibrinolytic activity of tPA and TNKase. Methods: A spectrophotometric method of measuring clot formation and lysis in citrated human plasma was developed and used to assess the fibrinolytic activity of tPA and of TNKase. Aprotinin, a known protease inhibitor capable of blocking tPA and TNKase activity, was used as a positive control to demonstrate that the assay system was appropriately sensitive to detect inhibition of thrombolytic activity. Varying concentrations of NA-1 were added to the reaction to assess whether NA-1 could affect the rate of clot formation or the rate of clot lysis in the presence of tPA or TNKase. Results: The sensitivity of this assay system was demonstrated to enable detection of small changes in the fibrinolytic activity of tPA and TNKase on human plasma in the presence of thrombin. NA-1 did not display any fibrinolytic activity on its own nor did it affect the rate of clot formation in the system. NA-1 also did not affect the rate of clot formation or of fibrinolysis in the presence of tPA or TNKase. Conclusions: NA-1 has no intrinsic fibrinolytic activity and does not modulate the fibrinolytic activity of tPA or TNKase. Therefore, NA-1 can be given safely to stroke victims receiving tPA or TNKase without negatively impacting the potential benefit of thrombolysis. This method could also be used to assess other drugs that may have the potential to interact with or be concurrently administered to patients receiving thrombolytic medications.

Разработка и апробация нового метода определения параметров гемостаза у больных с психической патологией

Introduction: hemostasis is one of the most important protective systems of the organism, and its deviations from the norm can lead to fatal results. Changes in hemostasis are observed in many diseases, including mental pathology. In addition, parameters of this system may also change during antipsychotic therapy. Objective: Development and testing of a new method and device for estimating plasma hemostasis parameters in patients with mental pathologies in order to optimize treatment and eliminate fatal outcomes. Materials and methods: in the study we used frozen (-80 °C) platelet-free blood plasma (PFP) samples from patients with schizophrenia (16 patients), taken at admission to the clinic FSBSI «Mental Health Research Centre». Plasma coagulation of patients and healthy donors was assessed with the device of our design. Hemostasis assessment is based on the determination of the clot size in proportion to the brightness of the image, which changes as a result of clot formation and lysis. Results: optimal concentrations of coagulation and fibrinolysis activators, as well as concentrations of calcium ions were determined during the first stage of testing the method and device. The median values of all parameters of coagulation and fibrinolysis except «time before the formation of a clot» in patients upon admission to the clinic were reduced relative with combined plasma donors. The rates of clot formation and lysis in patients are significantly reduced at discharge compared with these parameters at admission. Consequently, these parameters can serve as an estimate of the change in the state of the coagulation and fibrinolysis systems during treatment. Conclusion: during approbation of the device and method in the process of coagulation and fibrinolysis research in patients with schizophrenia, lower levels of parameters were detected compared to donors, and a decrease in the activity of plasma coagulation and fibrinolysis during treatment with antipsychotics was shown.

Efficacy of percutaneous mechanical thrombectomy combined with percutaneous transluminal angioplasty in treatment of iliofemoral deep vein thrombosis

To evaluate the clinical efficacy of percutaneous mechanical thrombectomy (PMT) combined with percutaneous transluminal angioplasty (PTA) in the treatment of iliofemoral deep vein thrombosis. Ninety-four patients with iliofemoral deep vein thrombosis were identified in this retrospective study in our institution from November 2015 through December 2017. Patients were divided into two groups:PMT+PTA group (n=50) and PMT only group (n=44). Clot lysis rates, the retrieving and the embolism of the interior vena cava filter, as well as the incidence of complications were compared between two groups. The changes of serum creatinine, lactate dehydrogenase and hemoglobin were also measured perioperatively. All procedures were completed successfully. The average clot lysis rate was higher and the procedure time was shorter in PMT+PTA group than those in PMT group (both P<0.01). No significant differences were found in the rates of venous stenosis, catheter-directed thrombolysis and stent placement between two groups (all P>0.05). The interior vena cava filter was taken out in 82.0% (41/50) patients of PMT+PTA group and 81.8% (36/44) patients of PMT group (P>0.05). There were no significant differences of the changes in serum creatinine, lactate dehydrogenase and hemoglobin (all P>0.05). In PMT+PTA group, symptomatic pulmonary embolism and puncture bleeding occurred in 1(2.0%) and 2(4.0%) patients, while in PMT group, it was 0 and 2(4.5%), respectively (P>0.05). PMT combined with PTA is more effective than PMT alone for the treatment of iliofemoral deep vein thrombosis with less procedure time and without more incidence of pulmonary embolism.

331 - ProliNONOate Alters Fibrin Clot Formation Through Peroxynitrite Formation and Protein Oxidation

When a blood clot forms, the protein thrombin cleaves fibrinogen into fibrin that polymerizes into a branched network of fibers that forms the structure of the clot. The kinetics of fiber formation affect fiber diameter, fiber network density, rate of clot lysis and potentially clot stiffness. We previously showed that exposure to nitric oxide, an important regulator of blood flow, alters the characteristics of these fibrin clots. The addition of ProliNONOate to fibrinogen or thrombin together or separately lead to a decreased the rate of fiber formation, increased the time the clot took to gel, increased fiber diameters, and decreased clot density. We hypothesize the effects of nitric oxide on the fibrin clots are due to oxidation of the proteins due to the creation of peroxynitrite. Peroxynitrite is created when nitric oxide reacts with superoxide anion and is known to cause nitration of tyrosine resides and oxidation of methionine and cysteine residues. Therefore, we measured carbonyl formation and tyrosine nitration upon exposure to ProliNONOate. Here we show the addition of the peroxynitrite scavenging ferric porphyrin complex, Iron(III)-5,10,15,20-tetrakis94-sulonatophenyl) porphyrinato chloride (FeTPPS) to the samples immediately prior to the addition of proliNONOate reduces protein oxidation. The addition of FeTPPS reduced or eliminated protein carbonyl formation. Previously we reported the formation of 880 ± 480 mmol of carbonyls per mole of fibrinogen after the addition of ProliNONOate however, with the addition of FeTPPS no carbonyls were detected. Carbonyls detected on thrombin exposed to ProliNONOate were reduced from 390 ± 80 mmol/mol thrombin to 18 mmol/mole thrombin with the addition of FeTPPS. FeTPPS abrogated the changes to the rate of fibrin formation. When FeTPPS was added to fibrinogen before the addition of ProliNONOate there was no significate change in the rate of fiber formation. Without FeTPPS we previously reported a decreased rate of clot formation. Here we show the exposure of blood clot proteins fibrinogen and thrombin to NO donor ProliNONOate lead to peroxynitrite formation which caused the previously reported protein oxidation and altered fibrin fiber formation kinetics and thereby altered various fibrin clot properties.

Thromboelastography as a Surrogate Marker of Perisurgical Hemostasis in Gaucher Disease.

Thromboelastography (TEG) has long been available for routine monitoring of perisurgical and postpartum hemostasis, especially at point of care. The purpose of this study is to retrospectively compare TEG parameters to concomitant standard clotting test results in an unselected cohort of patients with Gaucher disease to ascertain whether TEG values are specific and sensitive enough to substitute for classic coagulation tests for decision making. This remains a cogent concern because of high incidence of thrombocytopenia in patients with Gaucher disease. Thromboelastography values were compared to concomitant platelet counts, partial thromboplastin time, international normalization ratio, and plasma fibrinogen. Demographic characteristics were collected from patients' files. There were 22 patients with Gaucher disease (2 children; 12.5%) for whom there were 24 TEG results at the same time as classic coagulation test results and 30% performed platelet function tests. The current study shows linear and/or monotonic relationships between platelet counts and several TEG values that were significant over a range of platelet counts including severe thrombocytopenia. The fibrinogen component, correlating only with the rate of clot lysis, played a lesser role. Based on these preliminary results albeit in a small cohort with only 1 case of hemorrhage, there is putative support for the intention to treat patients with Gaucher disease based on TEG results using the same TEG protocol as for other patients undergoing comparable procedures in our institution.

Left Ventricular Assist Device-Associated Carbon Monoxide and Iron-Enhanced Hypercoagulation: Impact of Concurrent Disease.

Left ventricular assist device (LVAD) therapy is associated with thrombophilia despite anticoagulation. Of interest, LVAD patients have increased carboxyhemoglobin, a measure of upregulated heme oxygenase (Hmox) activity that releases carbon monoxide (CO) and iron. Given that CO and iron enhance plasmatic coagulation, we determined if LVAD patients had hypercoagulability and decreased fibrinolytic vulnerability with measurable CO and iron-mediated effects. Blood samples were obtained a month or more after implantation of the LVAD. Thrombelastographic methods to assess coagulation kinetics, fibrinolytic kinetics, formation of carboxyhemefibrinogen, and iron-mediated enhancement of clot growth were utilized. Coagulation and fibrinolytic parameter normal individual (n = 30) plasma values were determined. Sixteen LVAD patients were studied. CO and iron enhancement of coagulation were observed in the majority of LVAD patients, contributing to hypercoagulation. However, most patients demonstrated abnormally increased rates of clot lysis. Critically, hemolysis as assessed by circulating lactate dehydrogenase activity was small in this cohort, and only four patients without comorbid states (e.g., obesity, diabetes, sleep apnea) were hypercoagulable with evidence of Hmox upregulation. However, seven patients with comorbidities were hypercoagulable with Hmox upregulation. Future investigation of CO and iron-related thrombophilia and comorbid disease is warranted to define its role in LVAD-related thrombosis.

Platelet rich clots are resistant to lysis by thrombolytic therapy in a rat model of embolic stroke.

BackgroundEarly recanalization of occluded vessels in stroke is closely associated with improved clinical outcome. Microbubble-enhanced sonothrombolysis is a promising therapy to improve recanalization rates and reduce the time to recanalization. Testing any thrombolytic therapy requires a model of thromboembolic stroke, but to date these models have been highly variable with regards to clot stability. Here, we developed a model of thromboembolic stroke in rats with site-specific delivery of platelet-rich clots (PRC) to the main stem of the middle cerebral artery (MCA). This model was used in a subsequent study to test microbubble-enhanced sonothrombolysis.MethodsIn Study 1 we investigated spontaneous recanalization rates of PRC in vivo over 4 hours and measured infarct volumes at 24 hours. In Study 2 we investigated tPA-mediated thrombolysis and microbubble-enhanced sonothrombolysis in this model.ResultsStudy 1 demonstrated stable occlusion out to 4 hours in 5 of 7 rats. Two rats spontaneously recanalized at 40 and 70 minutes post-embolism. Infarct volumes were not significantly different in recanalized rats, 43.93 ± 15.44% of the ischemic hemisphere, compared to 48.93 ± 3.9% in non-recanalized animals (p = 0.7). In Study 2, recanalization was not observed in any of the groups post-treatment.ConclusionsSite specific delivery of platelet rich clots to the MCA origin resulted in high rates of MCA occlusion, low rates of spontaneous clot lysis and large infarction. These platelet rich clots were highly resistant to tPA with or without microbubble-enhanced sonothrombolysis. This resistance of platelet rich clots to enhanced thrombolysis may explain recanalization failures clinically and should be an impetus to better clot-type identification and alternative recanalization methods.Electronic supplementary materialThe online version of this article (doi:10.1186/s13231-014-0014-y) contains supplementary material, which is available to authorized users.

Microparticles Modulate Formation, Structure, and Properties of Fibrin Clots

Microparticles (MPs) are phospholipid vesicles about 0.1-1 μm in size released into blood from vascular and blood cells upon activation and apoptosis. The mechanism of MP formation by the exocytic budding of the outer cell membrane provides them with procoagulant activity, mainly due to phosphatidylserine exposure and tissue factor expression. MPs are present in the peripheral blood of healthy subjects but their level is elevated in a variety of vascular, infectious, and immune-mediated pathologies. While there are extensive studies on multiple roles of MPs in various diseases, including thrombotic disorders, the question remains open as to whether MPs are involved in the formation and properties of fibrin, the scaffold of hemostatic clots and obstructive thrombi. To investigate this problem, we studied in vitro the effects of MPs on the kinetics of fibrin polymerization, fibrin clot structure and susceptibility to fibrinolysis.Blood was drawn from healthy donors according to an IRB protocol. MPs were removed from citrated platelet-free plasma (PFP) by filtration through 0.1-μm pores, yielding microparticle-depleted plasma (MDP). To restore the phospholipids eliminated, MDP was replenished with cephalin (MDP-C). Fresh samples of PFP, MDP, and MDP-C from the same donor were analyzed in parallel. Concentrations of MPs and expression of platelet-specific CD61 were studied by flow cytometry and confocal microscopy. A thrombin generation test in recalcified defibrinated plasma was performed using a chromogenic substrate. Fibrin polymerization induced by recalcification of PFP, MDP, and MDP-C was studied by dynamic turbidimetry. tPA-induced fibrinolysis was also followed optically as a decrease of a plasma clot turbidity. Fibrin ultrastructure was studied with scanning electron microscopy (SEM) in a FEI Quanta 250 microscope. Each test was performed with plasma samples from at least 3 donors and the results were averaged.Enumeration of MPs in PFP vs. MDP showed that about 90% of total MPs were removed by filtration, including 99.6% of CD61-positive platelet-derived MPs. The rate of thrombin generation was significantly reduced in MDP vs. PFP and was fully restored in MDP-C, confirming an essential role of MPs in the intrinsic coagulation pathway. Based on the lag times and slopes of the turbidimetric curves, fibrin formation in recalcified plasma samples was significantly delayed in the absence of MPs (MDP) and was much faster in their presence (PFP and MDP-C). The maximum optical density was consistently and significantly higher in MDP than in PFP and MDP-C, suggesting that fibrin clots formed in the absence and presence of MPs have fibers of different thickness. This presumption was confirmed by SEM of fibrin clots formed upon recalcification of the plasma samples. Clots formed in PFP were less porous and had thinner fibers (170 ± 38 nm), while clots from MDP had larger pores and were built of thicker fibers (214 ± 53 nm, p<0.01). Addition of phospholipids to MDP (MDP-C) resulted in the formation of densely packed thin fibrin fibers (141 ± 34 nm), similar to the initial clots from PFP. Consistent with the dissimilarity in ultrastructure, the clots displayed a distinct susceptibility to fibrinolysis: the rate of clot lysis was significantly higher in MDP than in PFP and MDP-PL. A novel and important finding from the SEM of fibrin clots is that fibers formed in MDP were quite smooth, while fibers formed in the presence of MPs (PFP and MDP-C) had relatively rough surfaces with multiple sub-micron size particles attached, suggesting that MPs might directly bind fibrin. Consistent with the SEM of the appearance of MPs on the fibers, confocal microscopy of PFP-clots stained for CD61 with FITC-labeled antibodies, unlike MDP-clots, revealed fluorescent spots associated with fibrin, indicating that MPs were adsorbed on fibrin fibers.In conclusion, MPs have profound effects on fibrin formation and on the final structure and characteristics of fibrin clots via at least two mechanisms. One is an indirect kinetic effect based on the MP-dependent rate of thrombin generation. The other is direct binding of MPs to fibrin fibers during and after fibrin assembly. Both mechanisms underlie a previously underappreciated potential role of MPs in hemostasis and thrombosis as modulators of fibrin formation and properties.(Research supported by NIH grant HL090774 and the Program of Competitive Growth of Kazan Federal University) DisclosuresNo relevant conflicts of interest to declare.