Pathway Of Blood Coagulation Research Articles

Protein Composition of the Subretinal Fluid Suggests Selective Diffusion of Vitreous Proteins in Retinal Detachment.

PurposeTo study the proteome of the subretinal fluid (SRF) from rhegmatogenous retinal detachment (RRD) in search for novel markers for improved diagnosis and prognosis of RRD.MethodsHuman undiluted SRF obtained during vitrectomy for primary RRD using a 41-gauge needle (n = 24) was analyzed and compared to vitreous humor from 2-day postmortem eyes (n = 20). Sample preparation underwent nanoflow liquid chromatography–tandem mass spectrometry. Label-free quantification (LFQ) using MaxQuant was used to determine differentially expressed proteins between SRF and vitreous humor. The intensity-based absolute quantification (iBAQ) was used to rank proteins according to their molar fractions within groups. Identification of proteins beyond the quantitative level was performed using the Mascot search engine.ResultsThe protein concentration of the control vitreous humor was lower and more consistent (1.2 ± 0.4 mg) than that of the SRF (17.9 ± 22 mg). The iBAQ analysis showed high resemblance between SRF and vitreous humor, except for crystallins solely identified in vitreous humor. The LFQ analysis found 38 protein misregulations between SRF and vitreous humor of which the blood coagulation pathway was found to be enriched using the PANTHER Classification System. Combined, the iBAQ, LFQ, and Mascot analysis found an overlap only in chitinase-3-like protein 1 and galectin-3-binding protein unique to the SRF.ConclusionsThe proteome of the SRF was highly represented by proteins involved in proteolysis. Such proteins can possibly serve as targets in modulating the effects of SRF in RD.Translational RelevanceTo identify potential novel biomarkers for therapeutic targeting in RD.

Changes in the blood coagulation system and non-specific plasma proteinases in ischemia-reperfusion injury

The aim of this study was to determine the general patterns of pathogenetic changes in the blood coagulation system and in non-specific proteinases and their inhibitors during the development of experimental ischemiareperfusion injury. Materials and methods. The study was conducted on 48 male Wistar rats (180–200 g). We used a model of ischemia-reperfusion injury achieved by applying rubber tourniquets to both hind limbs at the inguinal fold level for 6 hours. Revascularization was performed for 6, 12, or 24 hours following the application of tourniquets, after which we examined the state of the internal and external blood coagulation pathways and the activity of nonspecific proteinases and their inhibitors. Results. Indicators of blood coagulation system change show the development of blood hypocoagulation changes as the reperfusion time increases. By the 6th hour of reperfusion, the prothrombin time (PT) was lengthened by 112.0% ( p = 0.0142) and the activated partial thromboplastin time (APTT) by 170.0% ( p = 0.0147) compared with values in the control group. By the 12th reperfusion hour, the PT was lengthened by 174.2% ( p = 0.0389), and the APTT increased 4.9-fold ( p = 0.0002). When the reperfusion period was increased to 24 hours, it was characterized by lengthened PT and APTT, accompanied by an increase in antithrombin III by 11.5% ( p = 0.0371) and a decrease in protein C by 71.4% ( p = 0.0071). Changes in the non-specific proteinases and their inhibitors were characterized by a 2.8-fold increase in the trypsin-like proteinase activity ( p < 0.001) relative to the control, as well as a 2.2-fold decrease in antitrypsin activity and acid-stable inhibitors ( p < 0.001), which reached a maximum after 24 hours of reperfusion. A direct correlation was found between indicators characterizing the deficiency of coagulation system factors and a decrease in antiproteinase potential. Conclusion. Hemostatic system disorders are characterized by the development of hypocoagulation during ischemia-reperfusion injury as the result of an increase in the trypsin-like proteinase activity and a decrease in the levels of inhibitors. The established changes may be associated with the deficiency of coagulation factors and proteinase inhibitors and share common pathogenic mechanisms.

Особенности плазменного звена гемостаза у пациентов с Ph-негативными миелопролиферативными новообразованиями

Введение: Тромбозы являются одним из основных осложнений Ph-негативных миелопролиферативных новообразований (МПН), таких как истинная полицитемия (ИП), эссенциальная тромбоцитемия (ЭТ) и первичный миелофиброз (ПМФ). Имеются многочисленные свидетельства активации тромбоцитов, лейкоцитов, эндотелия и коагуляции при МПН; однако результаты определения показателей гемостаза, применимых в клинической практике для выявления протромботических состояний при МПН, неоднозначны. Цель исследования: оценить состояние плазменного звена гемостаза у пациентов с Ph-негативными МПН и выявить показатели, отражающие протромботическую направленность изменений в системе гемостаза при данных патологиях. Материалы и методы: В исследование было включено 116 пациентов с МПН (43 с ИП, 35 с ЭТ, 38 с ПМФ). Пациенты находились на антиагрегантной, циторедуктивной и таргетной терапии ингибиторами янус-киназ. Контрольная группа состояла из 43 практически здоровых лиц. В плазме крови всех обследованных определяли индекс активированного парциального тромбопластинового времени (АПТВ) — отношение АПТВ исследуемого образца к АПТВ нормальной плазмы, протромбиновый тест по Квику (ПТ), содержание фибриногена, активности фактора VIII (FVIII) и естественных антикоагулянтов антитромбина (АТ) и протеина С (PC), уровень свободного протеина S (PS), а также показатели теста генерации тромбина (ТГТ), в том числе с добавлением тромбомодулина (ТМ). Результаты: У пациентов с МПН по сравнению с контрольной группой наблюдали сдвиг АПТВ и ПТ в сторону гипокоагуляции, было удлинено время инициации генерации тромбина, снижена скорость и количественные показатели ТГТ без добавления ТМ. При проведении ТГТ с добавлением ТМ у больных МПН выявлено снижение чувствительности к ТМ, которое коррелировало с падением активности PC и уровня свободного PS. Содержание фибриногена и активность FVIII у обследованных пациентов были повышены. Заключение: Для пациентов с МПН характерны разнонаправленные изменения показателей плазменного звена гемостаза. Важной детерминантой развития протромботических состояний при МПН выступает недостаточная эффективность естественных антикоагулянтов системы протеина С, приводящая к выраженному дисбалансу в системе гемостаза. Background: Thromboses are one of the main complications of Ph-negative myeloproliferative neoplasms (MPN), such as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). There are abundant evidences of platelet, leukocyte, endothelial, and coagulation activation in MPN; however, studies of hemostatic parameters applicable in clinical practice for revealing of prothrombotic states in MPN are not identical. Objectives: to assess plasma hemostasis in patients with Ph-negative MPN and to identify parameters reflecting the prothrombotic tendency of hemostatic changes in these pathologies. Patients/Methods: The study included 116 patients with MPN (43 with PV, 35 with ET, 38 with PMF). Patients were treated with antiplatelet, cytoreductive and targeted therapy with Janus kinases inhibitors. The control group consisted of 43 healthy individuals. In blood plasma of all examined persons we determined the index of activated partial thromboplastin time (APTT) — the ratio of APTT of the test sample to APTT of normal plasma, Quick prothrombin test (PT), fi brinogen content, activities of factor VIII (FVIII) and natural anticoagulants — antithrombin (AT) and protein C (PC), free protein S (PS) level, as well as thrombin generation assay (TGA) parameters, including thrombomodulin (TM) addition. Results: As compared with control group, in patients with MPN a shift in APTT and PV to hypocoagulation was observed, the initiation time of thrombin generation was prolonged, the rate and quantitative parameters of TGA without TM addition were decreased. In patients with MPN TGA with TM addition showed reduced sensitivity to TM that correlated with decreased PC activity and reduction of free PS level. Fibrinogen content and FVIII activity in the examined patients were increased. Conclusions: Patients with MPN are characterized by multidirectional changes of plasma hemostasis parameters. The important determinant of prothrombotic states development in MPN is insufficient effectiveness of the natural anticoagulants of protein C system that leads to expressed hemostasis imbalance.

Abstract 4900: An ALK fusion gene regulates different signaling pathways in mortal versus immortalized normal human cells for cellular senescence and transformation

Abstract Accumulating results of clinical trials lead targeted therapies to be the first choice for unresectable or recurrent lung cancer with driver mutations. Echinoderm Microtubule Associated Protein Like 4 (EML4) - Anaplastic lymphoma kinase (ALK) fusion is known as such a driver mutation. It presents in 3-6% of non-small cell lung carcinoma (NSCLC). EML4-ALK fusion protein generate the constitutive ALK kinase activity in NSCLC. The basic understanding of EML4-ALK remains insufficient due to the lack of functional studies using normal human cells. We investigated the role of EML4-ALK in mortal and immortalized normal human cells. The expression of EML4-ALK in normal, mortal human fibroblasts caused accumulated DNA damage, telomere shortening and the early induction of cellular senescence with senescence-associated beta-galactosidase activity and upregulation of p16INK4A and p21WAF1. In contrast, when EML4-ALK was expressed in telomerase reverse transcriptase (hTERT)-immortalized normal human fibroblasts and bronchial epithelial cells, the cells showed accelerated proliferation in vitro and anchorage-independent growth in soft agar, revealing its transformation activity. No chromosome aberrations, no mutations or loss of p53, nor impairment of the p16INK4A response was associated with this in vitro transformation, likely reflecting certain clinical features of EML4-ALK-positive NSCLC. In both mortal and immortalized cells, EML4-ALK induced the phosphorylation of STAT3, which is involved in both cellular senescence and transformation. Our data validate that EML4-ALK functions as an oncogene, although an additional oncogenic event(s) seems to be required for full tumorigenicity in vivo. This study also suggests that telomerase-mediated immortalization manifests the oncogenic activity of EML4-ALK, switching from its senescence-inducing activity. The RNA-seq analysis showed that the STAT3-induced cytokine/interferon signaling pathways were most significantly upregulated by EML4-ALK in mortal fibroblasts, consistent with accumulated DNA damage and senescence induction. The blood coagulation pathway activated by EML4-ALK in hTERT-immortalized cells may contribute to increased risk of disseminated intravenous coagulation in patients with EML4-ALK-positive cancer. These results suggest that EML4-ALK regulates the different signaling pathways in mortal versus immortalized normal human cells to induce the different cellular outcomes. Citation Format: Masaru Matsumoto, Akihiko Miyanaga, Jessica Beck, Izumi Horikawa, Mohammed Khan, Delphine Lissa, Masahiro Seike, Akihiko Gemma, Hiroyuki Mano, Curtis Harris. An ALK fusion gene regulates different signaling pathways in mortal versus immortalized normal human cells for cellular senescence and transformation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4900.

P-selectin blockade in COVID-19-related ARDS

P-selectin blockade in COVID-19-related ARDS

Annexin A4 inhibits sulfatide‐induced activation of coagulation factor XII

BackgroundFactor XII (FXII) is a plasma serine protease that initiates the intrinsic pathway of blood coagulation upon contact with anionic substances, such as the sulfated glycolipid sulfatide. Annexins (ANXs) have been implicated in the regulation of the blood coagulation reaction by binding to anionic surfaces composed of phospholipids and sulfated glycoconjugates, but their physiological importance is only partially understood. ObjectiveTo test the hypothesis that ANXs are involved in suppressing the intrinsic pathway initiated by sulfatide, we examined the effect of eight recombinant ANX proteins on the intrinsic coagulation reaction and their sulfatide binding activities. MethodsRecombinant ANXs were prepared in Escherichia coli expression systems and their anticoagulant effects on the intrinsic pathway initiated by sulfatide were examined using plasma clotting assay and chromogenic assay. ANXA4 active sites were identified by alanine scanning and fold deletion in the core domain. Results and ConclusionsWe found that ANXA3, ANXA4, and ANXA5 strongly inhibited sulfatide‐induced plasma coagulation. Wild‐type and mutated ANXA4 were used to clarify the molecular mechanism involved in inhibition. ANXA4 inhibited sulfatide‐induced auto‐activation of FXII to FXIIa and the conversion of its natural substrate FXI to FXIa but showed no effect on the protease activity of FXIIa or FXIa. Alanine scanning showed that substitution of the Ca2+‐binding amino acid residue in the fourth fold of the core domain of ANXA4 reduced anticoagulant activity, and deletion of the entire fourth fold of the core domain resulted in complete loss of anticoagulant activity.

Endotoxin Potentiates The Procoagulant Effects Of Tissue Factor

Tissue Factor is the initiator of the extrinsic pathway of blood coagulation. It is&#x0D; generated in blood as a result of a variety of diseases and conditions and is, in part, responsible for the majority of morbidity and mortality in humans. In spite of this potential release during major trauma or diseases, there are few rapid clinical whole blood coagulation tests that can monitor the early generation of tissue factor. This study will evaluate whether endotoxin enhances the procoagulant effects of tissue factor on human blood and plasma.

Nucleic acid-based drug delivery strategies

Nucleic acids have not been widely considered as an optimal material for drug delivery. Indeed, unmodified nucleic acids are enzymatically unstable, too hydrophilic for cell uptake and payload encapsulation, and may cause unintended biological responses such as immune system activation and prolongation of the blood coagulation pathway. Recently, however, three major areas of development surrounding nucleic acids have made it worthwhile to reconsider their role for drug delivery. These areas include DNA/RNA nanotechnology, multivalent nucleic acid nanostructures, and nucleic acid aptamers, which, respectively, provide the ability to engineer nanostructures with unparalleled levels of structural control, completely reverse certain biological properties of linear/cyclic nucleic acids, and enable antibody-level targeting using an all-nucleic acid construct. These advances, together with nucleic acids' ability to respond to various stimuli (engineered or natural), have led to a rapidly increasing number of drug delivery systems with potential for spatiotemporally controlled drug release. In this review, we discuss recent progress in nucleic acid-based drug delivery strategies, their potential, unique use cases, and risks that must be overcome or avoided.

Functional identification of factor VIII B domain regions in hepatocyte cells

Factor VIII (FVIII) functions as a cofactor within the intrinsic pathway of blood coagulation in process of FX activation by FIXa, for which deficiency results in the bleeding disorder hemophilia A. The gene of FVIII contains 26 exons that code for a 19 amino acid signal peptide and a 2332 amino acid polypeptide with a domain structure designated A1-A2-B-A3-C1-C2, of which the A domains are homologous with each other, as are the C domains. It has been well-documented that both the domains are the necessary elements for FVIII activities. The B domain is highly glycosylated and has a variable sequence, even among FVIIIs from different species. The B domain plays versatile roles in FVIII lifespan except for coagulation activity, but the functional characteristics of its specific regions remain still obscure. A series of recombinant FVIIIs (rFVIIIs) with B domain truncated were constructed and transiently expressed in hepatocyte cells. Media and cell lysates were collected after 72 h for the analyses of FVIII biosynthesis, secretion, activity and stability in ex vivo plasma relative to the full length wild-type FVIII. Unexpectedly, various regions in B domain exhibited different contribution to these functionalities. The discovery might facilitate the bioengineered rFVIIIs and gene therapeutics.

MP08-11 RENAL VEIN OSTIUM WALL INVASION OF RENAL CELL CARCINOMA WITH AN INFERIOR VENA CAVA TUMOR THROMBUS: COMPREHENSIVE MOLECULAR CHARACTERIZATION AND ITS IMPLICATIONS FOR CLINICAL PRACTICE

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (MP08)1 Apr 2020MP08-11 RENAL VEIN OSTIUM WALL INVASION OF RENAL CELL CARCINOMA WITH AN INFERIOR VENA CAVA TUMOR THROMBUS: COMPREHENSIVE MOLECULAR CHARACTERIZATION AND ITS IMPLICATIONS FOR CLINICAL PRACTICE Cheng Liu*, Jianjun Yu, Mengyao Tan, Shidong Jia, and Lulin Ma Cheng Liu*Cheng Liu* More articles by this author , Jianjun YuJianjun Yu More articles by this author , Mengyao TanMengyao Tan More articles by this author , Shidong JiaShidong Jia More articles by this author , and Lulin MaLulin Ma More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000828.011AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Renal cell carcinoma (RCC) is a common cancer worldwide with up to 10% of patients accompanying intravascular tumor thrombus (TT). Patients with RCC may develop phenotypically distinct TT, some of which adhere densely to the venous wall (DATT, densely adherent TT) while some others do not adhere to the endothelium (NATT, non-adherent/minimal adherence TT). To explore whether patients with NATT or DATT can benefit more from the surgical resection and characterize molecular difference between the two groups for patient stratification and prognosis. METHODS: Clinical information and overall survival of 77 non-metastatic patients who went caval thrombectomy was retrospectively analyzed. Whole exome sequencing (WES) and whole transcriptome sequencing (WTS) were performed to profile genomic and transcriptomic landscape of matched primary and TT samples. Genomic alterations and transcriptomic profiling data were processed using public and in-house bioinformatics tools and analyzed for enriched mutations and pathways. Differentially mutated and expressed genes between DATT and NATT groups were assessed by Fisher exact test and DESeq2 respectively. TGCA expression dataset was analyzed through unsupervised hierarchical clustering. RESULTS: Patients with DATT showed worse survival after the surgical operation (Hazard Ratio: 5.1, P = 0.03). WES of tumor samples from prospectively enrolled patients revealed that VHL, PBRM1, BAP1, ARID1A and SETD2 are the most frequent mutated genes, consistent with public TCGA RCC data. Notably, SETD2, PTEN and TP53 mutations are significantly enriched in NATT patients relative to DATT. Analogously, transcriptomic profiling revealed P53 signaling pathway, DNA damage repair pathways and genes involved in tumor metastasis are upregulated in NATT. By contrast, genes related to cell-cell adhesion, blood coagulation, hypoxia, and VEGF signaling pathway are enriched in DATT samples. Interestingly, expression data from public primary RCC samples uncovered a subgroup of samples that are NATT-like harbor enriched TP53, PTEN and SETD2 mutations and have poorer progression-free survival and overall survival, suggesting that NATT may intrinsically represent a more aggressive subgroup of patients. CONCLUSIONS: Patients with non-adherent/minimal adherence TT (NATT) benefit more from surgery than that with densely adherent TT. Interestingly, molecular characterization by whole-exome and whole-transcriptomic sequencing delineated that patients with NATT harbor significantly enriched mutations in SETD2, PTEN and TP53 and may represent a more aggressive subtype. Such genes may serve as predictive biomarkers to stratify patients who can benefit most from the intervention of surgery. Source of Funding: The study was funded by Peking University Medicine Fund of Fostering Young Scholars’ Scientific & Technological Innovation BMU2018PY003; the National Natural Science Foundation of China (Nos.: 81711530048, 81572515, 81672522); Peking University Third Hospital Clinical Research Fund BYSY2018062, BYSY2018012. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e110-e110 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Cheng Liu* More articles by this author Jianjun Yu More articles by this author Mengyao Tan More articles by this author Shidong Jia More articles by this author Lulin Ma More articles by this author Expand All Advertisement PDF downloadLoading ...

Serine protease in a bred variety of oriental melon (Cucumis melo L. var. makuwa) curtails vascular thrombosis by balancing hemostasis and fibrinolysis in a rodent model

Maintaining proper blood flow is critical in promoting good health. This study examined whether oral consumption of a bred variety of oriental melon for high serine protease (OMS) dampened collagen/epinephrine (CE)-induced vascular thrombosis in rats in comparison with a regular cultivar (OM). Freeze-dried OM or OMS were administered by oral gavage for 4 weeks. In a CE-induced thrombotic condition, OMS significantly reduced thrombogenesis by inhibiting both cyclooxygenase-1-mediated thromboxane B2 production and intrinsic blood coagulation pathways. It also stimulated fibrin clot lysis by activating tissue-type plasminogen activator. Besides, OMS restored CE-mediated alterations in adhesion molecules and endothelial nitric oxide synthase, thereby resulting in vasodilation. OM showed similar tendencies, but not statistically equivalent. The current findings, for the first time, suggest that plant SP from OMS may have the potential to protect endothelial and vascular dysfunctions that can be encountered by unintentional exposure to thrombotic risk factors in daily life.

Evaluation of the Antihemostatic and Antithrombotic Effects of Lowering Coagulation Factor VII Levels in a Non-human Primate.

Tissue factor (TF) and factor (F) VII, components of the extrinsic pathway of blood coagulation, are essential for hemostatic plug formation in response to injury; less clear are their roles in propagating thrombosis, as observational data in humans with congenital FVII deficiency suggests persistent thrombotic and bleeding risk even at significantly decreased FVII levels. We aimed to define the contribution of FVII to thrombus formation and hemostasis using a non-human primate model. We treated baboons with a FVII antisense oligonucleotide (ASO) and measured platelet and fibrin deposition inside and distal to collagen- or TF-coated vascular grafts. We assessed hemostasis by measuring bleeding time (BT) and prothrombin time (PT). Enoxaparin and vehicle treatments served as controls. FVII-ASO treatment reduced FVII levels by 95% and significantly increased both the PT and BT. Lowering FVII levels did not decrease platelet deposition in collagen- or TF-coated grafts, in thrombi distal to the grafts, or fibrin content of either collagen- and TF-coated grafts. Lowering FVII levels were associated with a modest 25% reduction in platelet deposition at 60min in the distal thrombus tail of TF-coated grafts only. FVII inhibition by way of ASO is feasible yet significantly impairs hemostasis while only exhibiting antithrombotic effects when thrombosis is initiated by vessel wall surface-associated TF exposure.

Identification of a Plasma MicroRNA Profile Associated With Venous Thrombosis.

Venous thrombosis (VT) is a complex condition with a highly heritable genetic component that predisposes one to its development. Certain microRNAs (miRNAs) might be used as biomarkers of VT, but few studies have examined miRNA expression in this respect. The aim of the present work was to identify a plasma miRNA profile associated with VT. Approach and Results: miRNAs were analyzed by quantitative polymerase chain reaction in plasma samples from members of the GAIT-2 (Genetic Analysis of Idiopathic Thrombophilia 2) population (n=935). A discovery phase involving the screening of 752 miRNAs from a subset of 104 GAIT-2 subjects was followed by an internal validation phase in which the selected miRNAs were quantified in the whole GAIT-2 population. In the discovery phase, 16 miRNAs were selected, including 9 associated with VT and 7 that correlated with an intermediate phenotype of VT. In the next phase, 4 miRNAs were validated as differentially expressed (false discovery rate, <0.1) in VT: hsa-miR-126-3p, hsa-miR-885-5p, hsa-miR-194-5p, and hsa-miR-192-5p. The 4 miRNAs each returned a significant (P<0.05) odds ratio for VT (range of 1.3-1.8). A risk model including the 4 miRNAs, age, and sex returned an area under the receiver operating characteristic curve of 0.77. Moreover, all 4 miRNAs showed significant correlations with intermediate phenotypes of VT (eg, protein S and factor VII). The targets of the miRNAs in the blood coagulation pathway and their interactions are also discussed. The present results suggest a 4-miRNA plasma profile associated with VT is of potential use in predicting the risk of this condition.

Comparison of acid and water-soluble chitosan doped fibrous cellulose hemostat wet laid nonwoven web for hemorrhage application

Fibrous hemostat was produced using wet laying technique comprising of acid and water soluble chitosan with fibrous cellulose. The hemostatic efficiency and their structural properties were evaluated using blood clotting time, whole blood adhesion and analytical characterization techniques. The maximum concentration of chitosan utilised for the production of nonwoven was 1.5 w/v % upon which increased the viscosity of the solution. The absorption of phosphate buffer solution was found to be 1117% for acid soluble chitosan based nonwoven whereas for water soluble chitosan based nonwoven was 997%. Increased red blood cells and platelets were found to be higher in acid soluble chitosan compared to water soluble chitosan based cellulose nonwoven. The blood clotting time for acid soluble chitosan nonwoven was found to be 166 s and water soluble chitosan nonwoven was 170 s. The developed hemostat have potential application in reducing blood loss and also inducing the blood coagulation pathway which was confirmed by the adhesion of platelets on to the surface of nonwoven web.

Thrombin generation test with the calibrated automated thrombogram and anticoagulant activity of Mentha crispa.

: Evaluate the in-vitro effect of Mentha crispa extract on blood coagulation, compare the conventional coagulometric tests with thrombin generation test (TGT), and study the qualitative micromolecular composition of M. crispa. Extract of M. crispa was incubated with plasma and used in the coagulometric tests: prothrombin and activated partial thromboplastin times, fibrinogen, and TGT. A phytochemical prospection was performed to evaluate the chemical composition of this extract. The extract was efficient in prolonging prothrombin time and activated partial thromboplastin time, and reducing fibrinogen levels and TGT parameters, indicating that the extract of M. crispa inhibited the intrinsic and extrinsic pathways of blood coagulation. The results obtained in TGT are in agreement with the results of conventional coagulometric tests and the in-vitro anticoagulant activity of M. crispa suggests that its use by patients using oral anticoagulants deserves caution.

THROMBOSE-PREVENTIVE INFLUENCE OF QUERCETIN IN RATS ON THE HYPER-COAGULATION DIET

Relevance. Hyper-coagulation processes increase the risk of coronary heart disease.&#x0D; Objective of the study was to study the effect of quercetin on the hemostatic system when using hyper-coagulation diet in rats.&#x0D; Materials and methods. The study included 17 male rats weighing 240-270 g. The rats of the control group (n = 6) were fed with conventional compound feed; Group I rats (n = 5) - mixed feed; and group II rats (n = 6) mixed with meat and quercetin (1.33 g/l). After 21 days, sterile blood was collected. The Amellung Coagulometer KS 4A studied the external and internal blood coagulation pathways, the activity of their own anticoagulant and fibrinolytic blood systems. The data obtained were statistically processed using Excel 2010 and Statistica v.10 (StatSoft, USA).&#x0D; Results. In rats of group 1, the external pathway of blood coagulation underwent the most significant changes, as indicated by an almost four-fold acceleration of prothrombin time and a decrease in the international normalized ratio. From the side of the internal pathway, the greatest changes were recorded in the last phase of clot formation: thrombin time decreased by 39.8% with an almost constant rate of activated partial thromboplastin time (APTT), which confirmed the activation of the fibrin formation phase. In addition, the content of soluble fibrin-monomer complexes (RFMC) increased 1.7 times and the fibrinogen decreased. At the same time, depletion of the anticoagulant potential of the blood was observed (a 42.2% decrease in the activity of the protein C system with a tendency to a decrease in antithrombin III (AT III). The fibrinolytic system was significantly suppressed, as indicated by more than three times inhibition of XII-dependent fibrinolysis. In animals Group 2 compared with the indicator in group 1, APTT was 2 times longer; the concentration of RFMC was also doubled, fibrinolysis was activated by 25%.&#x0D; Conclusion: The use of quercetin bioflavonoids in animals with simulated coronary heart disease slows down blood coagulation and potentiates the anticoagulant system, including due to a 40% increase in AT III activity, can be considered as a thrombosis-preventive effect

ET-05 PRECLINICAL STUDY OF AN ANTI-HUMAN TISSUE FACTOR ANTIBODY-DRUG CONJUGATE IN A MALIGNANT GLIOMA XENOGRAFT MODEL

Whereas macromolecules such as antibody hardly extravasate from normal blood vessels compared with low molecular agents, macromolecules leak from tumor vasculature because of the enhanced permeability. As a result, macromolecules selectively accumulate at tumor sites. We apply this phenomenon, known as the enhanced permeability and retention effect (EPR effect), to drug delivery for cancer therapy. Drug delivery system (DDS) based on the EPR effect is called the passive targeting. On the other hand, DDS based on antigen-antibody or ligand-receptor interaction is the active targeting. Antibody-drug conjugate (ADC), antibody conjugated with antitumor agents, retains both of the passive and active targeting functions. Tissue factor (TF), an initiator in the extrinsic pathway of blood coagulation, is overexpressed in various cancers including malignant glioma. To target the molecule in tumor sites, we have produced several anti-TF monoclonal antibodies. Previously, we evaluated tumor accumulation of an indium-111-labeled anti-TF antibody in an orthotopic glioma xenograft model with high expression of TF by single photon emission computed tomography/computed tomography (SPECT/CT). The imaging study showed that anti-TF antibody significantly accumulated in the tumor compared with control antibody (P < 0.01). The finding suggests that blood-brain barrier in brain tumors is broken and antibodies accumulate in tumors by utilizing both of the passive and active targeting. In this study, to prepare anti-TF ADC, we conjugated monomethyl auristatin E (MMAE), a microtubule inhibitor, to humanized anti-TF antibody. The anti-TF ADC recognized TF expressed in glioma cells and showed potent cytocidal activity against human glioma cell lines depending on TF expression. In addition, we evaluated in vivo antitumor effect of the ADC in a mouse model subcutaneously inoculated with TF-overexpressing glioma cells. Anti-TF ADC showed a significant higher antitumor effect compared with control ADC (P = 0.015).

Management of bleeding in patients hospitalized in the intensive cardiac care unit: expert opinion of the Association of Intensive Cardiac Care and Section of Cardiovascular Pharmacotherapy of the Polish Cardiac Society in cooperation with specialists in other fields of medicine.

Nowadays, the intensive cardiac care unit (ICCU) provides care for patients with acute coronary syndrome, acute and exacerbated chronic heart failure, cardiogenic shock, sudden cardiac arrest, electrical storm, as well as with indications for urgent cardiac surgical treatment. Most of these patients require the use of 1, 2, or frequently even 3 drugs that act on the blood coagulation pathway. While antithrombotic drugs prevent thromboembolic events, they are associated with a higher risk of bleeding. In this population of patients, bleeding may often have a worse impact on prognosis than the primary disease. In this expert opinion of the Association of Intensive Cardiac Care, we presented practical guidelines on the management of bleeding in patients hospitalized at the ICCU, including bleeding risk reduction and treatment recommendations. Because of multiple comorbidities and diverse organs that may be the source of bleeding, we provided also recommendations from specialists in other fields of medicine. We hope that this document will facilitate the management of one of the most challenging populations at the ICCU.

Nomenclature of factor XI and the contact system

Nomenclature of factor XI and the contact system

Preparation and characterization of inorganic radioactive holmium-166 microspheres for internal radionuclide therapy.

Microspheres with high specific activities of radionuclides are very interesting for internal radiotherapy treatments. This work focuses on the formulation and characterization of inorganic microspheres with a high content of holmium and therefore a high specific radioactivity of holmium-166. Two novel formulations of inorganic microspheres were obtained by dispersing solid holmium acetylacetonate microspheres (Ho2(AcAc)3-ms) in NaH2PO4 or NaOH solutions followed by 2 h incubation at room temperature. By exchange of acetylacetonate with phosphate or hydroxyl ions, holmium phosphate microspheres (HoPO4-ms) and holmium hydroxide microspheres (Ho(OH)3-ms) were formed respectively. The inorganic microspheres had a significantly smaller diameter (28.5 ± 4.4 μm (HoPO4-ms) and 25.1 ± 3.5 μm (Ho(OH)3-ms)) than those of Ho2(AcAc)3-ms (32.6 ± 5.2 μm). The weight percentage of holmium-165 in the microspheres increased significantly from 47% (Ho2(AcAc)3-ms) to 55% (HoPO4-ms) and 73% (Ho(OH)3-ms). After preparation of both HoPO4-ms and Ho(OH)3-ms, the stable holmium-165 isotope was partly converted by neutron activation into radioactive holmium-166 to yield radioactive microspheres. High specific activities were achieved ranging from 21.7 to 59.9 MBq/mg (166HoPO4-ms) and from 28.8 to 79.9 MBq/mg (166Ho(OH)3-ms) depending on the neutron activation time. The structure of both microspheres was preserved up to neutron activations of 6 h in a thermal neutron flux of 4.72 × 1016 n m-2 s-1. After activation, both microspheres revealed excellent stability in administration fluids (saline and phosphate buffer) having less than 0.05% of holmium released after 72 h incubation. Finally, the hemocompatibility of these inorganic microspheres was evaluated and it was shown that the microspheres did cause neither hemolysis nor depletion or inhibition of the coagulation factors of the intrinsic blood coagulation pathway meaning that the microspheres have a good hemocompatibility. Overall, this work shows that radioactive inorganic microspheres with high specific activities of holmium-166 can be prepared which potentially can be used for internal radionuclide therapy.