Percentage Of Lysis Research Articles

The effect of hydroxychloroquine on haemostasis, complement, inflammation and angiogenesis in patients with antiphospholipid antibodies.

HCQ has been described as having a beneficial effect in patients with APS but its mechanism of action is unclear. We hypothesized that HCQ may have effects on subnormal angiogenesis, inflammation and haemostatic biomarkers seen in APS. The aim of our study was to assess laboratory markers [annexin A5 (AnxA5) anticoagulant activity, tissue factor (TF) levels, thromboelastography (TEG), CRP, Bb, C3a and VEGF] in HCQ-naïve patients with aPL at baseline and after commencing HCQ. Twenty-two patients with aPL [20 female, 2 male, median age 55 (range 18-70) years] had blood taken pre- and 3 months after starting HCQ 200 mg daily. Soluble TF levels were significantly reduced comparing baseline and 3 months after HCQ commencement [401.8 (152.8) vs 300.9 (108) pg/ml (P = 0.010)]. No significant changes were found in the following [reported as pre- and post-HCQ commencement, mean (s.d.)]: AnxA5 anticoagulant ratio [187.1 (29.5) vs 193 (31) (P = 0.157)], anti-domain1 β2 glycoprotein1 IgG activity [1.8 (2) vs 1.2 (1.4) μg/ml (P = 0.105)], complement C3a-des-Arg [147.8 (84.5) vs 154.4 (88.1) ng/ml (P = 0.905)], complement Bb [1.3 (0.7) vs 1.1 (0.7) μg/ml (P = 0.422)], VEGF [68.8 (40) vs 59.4 (19.6) pg/ml (P = 0.454)] and CRP [7 (3.5) vs 7 (3.9) μg/ml (P = 0.917)]. TEG results including TEG reaction time, achievement of clot firmness, TEG maximum amplitude and TEG percentage lysis 30 and 60 min after maximum amplitude showed no significant difference. HCQ significantly reduced soluble TF levels in patients with aPL. No significant change was observed in AnxA5 activity, anti-domain 1 IgG activity, TEG, CRP, complement Bb and C3a-des-Arg, and VEGF. Further studies of a larger patient cohort are needed.

Formation of functional nanofibrous electrospun polyurethane and murivenna oil with improved haemocompatibility for wound healing

Electrospinning is an emerging tool and promising method to fabricate polymer nanofibers. The aim of this work was to fabricate electrospun polyurethane mats reinforced with murivenna oil for wound dressings. The scanning electron microscopy (SEM) micrographs showed the fiber diameter of nanocomposites in the range of 740 ± 160 nm and found to be decreased compared to pure polyurethane. Surface of nanocomposites was analyzed by Fourier transform infrared spectroscopy (FTIR) insinuated the interactions between PU and murivenna oil by the formation of hydrogen bond and changes in the characteristics peaks. Contact angle of the PU incorporated murivenna oil showed a decrease in its value compared to pure PU indicating the increased wettability and hydrophilic nature. The thermal degradation and stability of fabricated composites was found be enhanced compared to pure PU. The surface morphology through atomic force microscopy (AFM) analysis showed a change in surface roughness due to presence of murivenna oil in the polymer matrix. In blood compatibility results, both activated partial thromboplastin time (APTT) and prothrombin time (PT) were delayed due to improved surface properties and the addition of murivenna oil in the PU matrix. Compared to pure PU, the hemolysis assay of the PU incorporated murivenna oil showed a significant decrease in the percentage of lysis of red blood cells indicating better blood compatibility. Following the results, it was confirmed that fabricated novel scaffolds having better physicochemical and enhanced blood compatibility properties may be utilized for wound dressing.

Efficient Synthesis of Novel Pyridine-Based Derivatives via Suzuki Cross-Coupling Reaction of Commercially Available 5-Bromo-2-methylpyridin-3-amine: Quantum Mechanical Investigations and Biological Activities.

The present study describes palladium-catalyzed one pot Suzuki cross-coupling reaction to synthesize a series of novel pyridine derivatives 2a–2i, 4a–4i. In brief, Suzuki cross-coupling reaction of 5-bromo-2-methylpyridin-3-amine (1) directly or via N-[5-bromo-2-methylpyridine-3-yl]acetamide (3) with several arylboronic acids produced these novel pyridine derivatives in moderate to good yield. Density functional theory (DFT) studies were carried out for the pyridine derivatives 2a–2i and 4a–4i by using B3LYP/6-31G(d,p) basis with the help of GAUSSIAN 09 suite programme. The frontier molecular orbitals analysis, reactivity indices, molecular electrostatic potential and dipole measurements with the help of DFT methods, described the possible reaction pathways and potential candidates as chiral dopants for liquid crystals. The anti-thrombolytic, biofilm inhibition and haemolytic activities of pyridine derivatives were also investigated. In particular, the compound 4b exhibited the highest percentage lysis value (41.32%) against clot formation in human blood among all newly synthesized compounds. In addition, the compound 4f was found to be the most potent against Escherichia coli with an inhibition value of 91.95%. The rest of the pyridine derivatives displayed moderate biological activities.

Thromboelastographic evaluation of hemostatic functionin dogs with dilated cardiomyopathy

This study aimed to evaluate the hemostatic function by thromboelastography (TEG) and determine if there was an association between the changes in hemostatic parameters and serum cardiac troponin I (cTnI) in dogs with dilated cardiomyopathy (DCM). DCM was diagnosed by echocardiography in 19 dogs. Ten healthy dogs were selected as controls. Myocardial injury was confirmed by increased serum cTnI levels. Coagulation was assessed by TEG evaluating clot kinetics (reaction time [R] and kinetic time [K]), clot strengthening (alpha [α] angle and G value), platelet function (maximum amplitude [MA] and coagulation-index [CI]), clot stability (LY30, the percentage of lysis 30 min after MA), and global clotting times (activated partial thromboplastin time [aPTT] and prothrombin time [PT]). Dogs with DCM had higher R and K times but lower MA, α-angle, G, and CI compared to controls (P < 0.05). LY30 and PT did not statistically differ between groups, but the aPTT in dogs with DCM was higher than that of the controls. Serum cTnI (median [range], ng/mL) was higher in dogs with DCM (0.25 [0.13-3.1]) than in healthy dogs (0.03 [0.01-0.06]). There was no statistical relation between cTnI and coagulation parameters. This study showed that alterations of the coagulation status in dogs with DCM could develop independently of serum cTnI elevations.

Impact of environment on Red Blood Cell ability to withstand mechanical stress.

Susceptibility of red blood cells (RBC) to hemolysis under mechanical stress is represented by RBC mechanical fragility (MF), with different types or intensities of stress potentially emphasizing different perturbations of RBC membranes. RBC membrane mechanics were shown to depend on cell environment, with many details not yet understood. Here, stress was applied to RBC using a bead mill with oscillation up to 50 Hz, over durations up to 50 minutes. MF profiles plot percent lysis upon stresses of progressive durations. Supplementing media with polyethylene glycol (PEG) which interacts with the cell membrane, but not Dextran which does not, resulted in higher resistance to hemolysis. Albumin, and to a lesser extent fibrinogen and globulins (at physiological concentrations), significantly increased cell ability to withstand mechanical stress versus with un-supplemented buffer solution and with PEG. This is partly due to changes in rheology, per tests done including (PEG) and Dextran, but is mostly due to cell-protein interaction, noting the effect of pH on RBC MF with albumin but not with buffer. Presence of lipids reduced RBC resistance to potentially hemolytic stress with lypemic plasma effecting lower "protection" from induced hemolysis than essentially fatty-acid free plasma. This effect was less dependent on incubation than on fatty-acid presence during stressing. The reduced propensity for hemolysis afforded by plasma proteins also depended markedly on the speed of the bead, potentially reflecting changes from a predominantly Von Karman trail at lower frequencies to an increasingly disorganized turbulent wake at higher frequencies.

Coagulation Profile of Patients with Adolescent Idiopathic Scoliosis Undergoing Posterior Spinal Fusion.

Blood loss and transfusion requirements during posterior spinal fusion for adolescent idiopathic scoliosis remain a concern. The mechanism of bleeding in these patients is poorly characterized. Thromboelastography is a comprehensive test of a patient's coagulation system commonly used in cardiac surgical procedures. It has not been well studied for use in patients with adolescent idiopathic scoliosis. A prospective, observational study of the coagulation profile of patients with adolescent idiopathic scoliosis undergoing posterior spinal fusion is presented. Healthy patients with adolescent idiopathic scoliosis without a bleeding abnormality were analyzed during posterior spinal fusion. Standard coagulation laboratory and thromboelastogram measures were obtained at the time of the incision and at 1-hour intervals during the surgical procedure. Laboratory values were analyzed in relation to outcomes such as bleeding, transfusion, and a fibrinolysis score. Fifty-eight patients were observed. Eighty-one percent of patients were female, the mean age was 13.5 years, a mean of 11.1 levels were fused, the median estimated blood loss was 645 mL, and 47% of patients received blood products. Overall, laboratory values remained stable throughout the surgical procedure. Mild increases in prothrombin time and partial thromboplastin time were observed, and platelets remained stable. From thromboelastogram analysis, an acceleration of clot formation (decreased reaction time) and a slight increase in clot lysis (increased lysis percentage at 30 minutes) were observed. A fibrinolysis score compiled from the presence of fibrin degradation products, the presence of D-dimers, and increased prothrombin time rose steadily over surgical time. The fibrinolysis score was predictive of both transfusion and greater estimated blood loss per level. The stress of posterior spinal fusion induces a hypercoagulable state in patients with adolescent idiopathic scoliosis. Over the first 2 hours of a surgical procedure, varying degrees of fibrinolysis develop. Platelets and coagulation factors are not depleted. Our data support the use of antifibrinolytic therapy for patients with adolescent idiopathic scoliosis.

An in vitro study of the effects of t-PA and tranexamic acid on whole blood coagulation and fibrinolysis

AimsAcute traumatic coagulopathy is characterised by fibrinolysis and low fibrinogen. It is unclear how much fibrinogenolysis contributes to reduce fibrinogen levels. The study aim was to: investigate in vitro the...

Natural killer cells and single nucleotide polymorphisms of specific ion channels and receptor genes in myalgic encephalomyelitis/chronic fatigue syndrome.

AimThe aim of this paper was to determine natural killer (NK) cytotoxic activity and if single nucleotide polymorphisms (SNPs) and genotypes in transient receptor potential (TRP) ion channels and acetylcholine receptors (AChRs) were present in isolated NK cells from previously identified myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS) patients.Subjects and methodsA total of 39 ME/CFS patients (51.69±2 years old) and 30 unfatigued controls (47.60±2.39 years old) were included in this study. Patients were defined according to the 1994 Centers for Disease Control and Prevention criteria. Flow cytometry protocols were used to examine NK cytotoxic activity. A total of 678 SNPs from isolated NK cells were examined for 21 mammalian TRP ion channel genes and for nine mammalian AChR genes via the Agena Bioscience iPlex Gold assay. SNP association and genotype was determined using analysis of variance and Plink software.ResultsME/CFS patients had a significant reduction in NK percentage lysis of target cells (17%±4.68%) compared with the unfatigued control group (31%±6.78%). Of the 678 SNPs examined, eleven SNPs for TRP ion channel genes (TRPC4, TRPC2, TRPM3, and TRPM8) were identified in the ME/CFS group. Five of these SNPs were associated with TRPM3, while the remainder were associated with TRPM8, TRPC2, and TRPC4 (P<0.05). Fourteen SNPs were associated with nicotinic and muscarinic AChR genes: six with CHRNA3, while the remainder were associated with CHRNA2, CHRNB4, CHRNA5, and CHRNE (P<0.05). There were sixteen genotypes identified from SNPs in TRP ion channels and AChRs for TRPM3 (n=5), TRPM8 (n=2), TRPC4 (n=3), TRPC2 (n=1), CHRNE (n=1), CHRNA2 (n=2), CHRNA3 (n=1), and CHRNB4 (n=1) (P<0.05).ConclusionWe identified a number of SNPs and genotypes for TRP ion channels and AChRs from isolated NK cells in patients with ME/CFS, suggesting these SNPs and genotypes may be involved in changes in NK cell function and the development of ME/CFS pathology. These anomalies suggest a role for dysregulation of Ca2+ in AChR and TRP ion channel signaling in the pathomechanism of ME/CFS.

Abstract A50: Chemoimmunotherapy with PD-1 blockade and paclitaxel induce a potent antitumor immunity in ovarian cancers.

Abstract Some chemotherapeutic agents are known to enhance both systemic immunosuppressive changes and local immunogenicity of tumor cells at the tumor site. To evaluate how chemotherapeutic agents commonly used for ovarian cancer could influence on the immune system, we analyzed the number of CD8+ TILs in ovarian cancer tissues from 21 patients who received operation before and after chemotherapy by immunohistochemistry and found that CD8+ TILs significantly increased after chemotherapy (p&amp;lt;0.05). Because the expansion of CD8+ TILs is generally related to the overexpression of PD-L1 (programmed cell death ligand 1) on cancer cells, we collected cancer cells from massive ascites of a stage IIIc ovarian cancer patient both before and after paclitaxel (PTX) plus carboplatin (CBDCA), and analyzed the expression PD-L1 expression on the tumor cells. These ascites cells expressed about 5-fold higher expression of PD-L1 after chemotherapy than before chemotherapy. To confirm overexpression of PD-L1 by chemotherapy, we next conducted in vitro experiments using four human ovarian cancer cell lines and two mouse ovarian cancer cell lines. We treated the cell lines with several dose of paclitaxel (PTX) or gemcitabine (GEM) for 24 hours, each. These chemotherapeutic agents increased protein expression of PD-L1, detected by Western blotting, in all the cell lines tested to a varying level in dose dependent manner. Increased expression of PD-L1 was also confirmed with flow cytometry in HM1 and ID8 treated with PTX or GEM. We next assessed alterations in CD8+ TILs following their encounter with PTX-induced PD-L1 using cytotoxic T cell assay. Following co-culture with CD8+ T cells, PTX-pretreated ID8-control cells show a lower lysis percentage than non-pretreated ID8-control cells, indicating that T-cell activation may be inhibited by PTX (p&amp;lt;0.05). Additionaly, PTX-pretreated ID8-pdl1KO, co-cultured with CD8+ T cells show the highest lysis percentage (p&amp;lt;0.01). This suggests that depletion of PD-L1 in ovarian cancer cells induces activation of CD8+ T cells. Lastly, to evaluate whether blocking PD-L1 in tumor cells reversed the decline in T cell immunity and promoted tumor rejection after chemotherapy, we conducted a combined immunochemotherapy in a mouse ovarian cancer model. We injected ID8 cells intraperitoneally into syngeneic mice following treatment with PTX alone, anti-PD-1 antibody (αPD-1) alone, the combination of PTX and αPD-1, or saline and IgG intraperitoneal injection (control). Combined treatment of PTX with αPD-1 resulted in the best survival among the four groups. ID8 tumor-bearing mice that received either αPD-1 or PTX alone showed significantly better survival than the no treatment control group (p&amp;lt;0.05). In place of anti-PD-1 antibody, we also treated these mice model with anti-PD-L1 antibody (αPD-L1) and found that the combined treatment of PTX with αPD-L1 also resulted in the best survival (p&amp;lt;0.05). In summary, combined chemoimmunotherapy with PD-1 signal blocking may improve the antitumor response and offer a promising new treatment against ovarian cancer. Citation Format: Junzo Hamanishi, Jin Peng, Noriomi Matsumura, Kaoru Abiko, Tsukasa Baba, Ken Yamaguchi, Ikuo Konishi, Masaki Mandai. Chemoimmunotherapy with PD-1 blockade and paclitaxel induce a potent antitumor immunity in ovarian cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A50.

CD38 Protects Tumor Cells from NK Lysis

Expression of the type II glycoprotein CD38 in hematological malignancies is abnormally high compared to that in normal cells of hematopoietic origin. Across all malignancies, the highest CD38 expression levels are detected on the surface of multiple myeloma (MM) cells. The high CD38 level has prompted the development of anti-CD38 antibody-based therapies, like daratumumab and isatuximab (SAR650984), that are promising therapeutics for the treatment of patients with MM. Cellular functions associated with CD38 include co-receptor mediated signaling, cell adhesion and ecto-enzymatic conversion of NAD to modulate calcium signaling and adenosine synthesis; but other than allowing antibody-mediated engagement of innate immunity, the role of CD38 in MM is not fully understood.In order to investigate a potential immune-protective role for CD38, we evaluated a panel of 15MM and 6 diffuse large B-cell lymphoma (DLBCL) cell lines with varying levels of CD38 for their susceptibility to NK-mediated lysis. Briefly, calcein AM pulsed target cells were incubated with NK-92.CD16 NK cells (E:T=3:1) for 1 hr and cell lysis measured as a function of the fluorescent calcein released into the supernatant. In an initial experiment, we observed that the percentage of cell lysis for all lines with >100,000 CD38s per cell was low, below 10%. The cell lines with <100,000 CD38s per cell were more susceptible to NKs, with a median percentage cell lysis of around 25%. The observed levels of cell lysis were not correlated with the levels of released IFNγ or TNFα.The differential lytic activity against the target cells led to the hypothesis that CD38 might have a role in protecting the tumor cells from NK-mediated lysis. To investigate this, we first knocked down CD38 in SUDHL-8 cells using shRNA, decreasing the levels of CD38 from 246,000 to 51,000 per cell. This resulted in an increase in the percentage of lysed cells from 25% to more than 50%, likely reflecting a loss of protection against the NK cells. We next overexpressed CD38 in U266 cells, increasing the CD38 levels from 9,000 to 327,000 per cell. As a result, the percentage of lysis decreased from more than 60% to around 30%, indicating that U266 cells are now more resistant to the lysis by NK cells. The protection that CD38 seems to confer to CD38-overexpressing U266 cells is completely lost by addition of isatuximab which triggers CD16-mediated lysis (antibody dependent cellular toxicity, ADCC).Whether the protective function of CD38 described here is a direct or an indirect protection mechanism is currently under investigation. DisclosuresOff Label Use: isatuximab (SAR650984, anti-CD38 antibody). Song:Sanofi: Employment. Adrian:Sanofi: Employment.

Ofatumumab Exhibits Enhanced In Vitro and In Vivo Activity Compared to Rituximab in Preclinical Models of Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is a mature B-cell lymphoma considered to be incurable with current treatments, including first-line rituximab in combination with multiagent chemotherapy and for those eligible, high-dose chemotherapy and stem cell support or rituximab maintenance. On the other hand, achieving a complete remission by high-sensitive flow cytometry is associated with prolonged duration of remission, stressing the need to develop and/or incorporate novel agents into the management of MCL. To this end, we examined the activity of ofatumumab, an anti-CD20 monoclonal antibody with distinct binding and immunologic properties compared to rituximab, in MCL preclinical models. MCL cells were labeled with (51)Cr before incubation with rituximab or ofatumumab (10 μg/mL) plus human serum or effector cells. (51)Cr-release was measured and the percentage of lysis was calculated. Surface CD20, CD55, and CD59 were measured by Imagestream analysis. SCID mice inoculated subcutaneously with Z138 cells were assigned to control versus four doses of ofatumumab or rituximab (10 mg/kg/dose). Ofatumumab exhibited enhanced in vitro complement-dependent cytotoxicity activity compared with rituximab in MCL cell lines, despite a high degree of in vitro resistance to rituximab associated with low CD20 levels and/or high expression of complement inhibitory proteins. Ofatumumab also delayed tumor progression and prolonged survival in a murine model of MCL. Our results demonstrate that ofatumumab is more effective than rituximab in MCL preclinical models, including in the presence of rituximab resistance, and support the clinical investigation of ofatumumab in combination with standard systemic chemotherapy in MCL (NCT01527149).

Thromboelastography Values in Hispaniolan Amazon Parrots ( Amazona ventralis ): A Pilot Study.

Thromboelastography (TEG) provides a global assessment of coagulation, including the rate of clot initiation, clot kinetics, achievement of maximum clot strength, and fibrinolysis. Thromboelastography (TEG) is used with increasing frequency in the field of veterinary medicine, although its usefulness in avian species has not been adequately explored. The purpose of this preliminary study was to assess the applicability of TEG in psittacine birds. Kaolin-activated TEG was used to analyze citrated whole blood collected routinely from 8 healthy adult Hispaniolan Amazon parrots ( Amazona ventralis ). The minimum and maximum TEG values obtained included time to clot initiation (2.6-15 minutes), clot formation time (4.3-20.8 minutes), α angle (12.7°-47.9°), maximum amplitude of clot strength (26.3-46.2 mm), and percentage of lysis 30 minutes after achievement of maximum amplitude (0%-5.3%). The TEG values demonstrated comparative hypocoagulability relative to published values in canine and feline species. Differences may be explained by either the in vitro temperature at which TEG is standardly performed or the method of activation used in this study. Although TEG may have significant advantages over traditional coagulation tests, including lack of need for species-specific reagents, further evaluation is required in a variety of avian species and while exploring various TEG methodologies before this technology can be recommended for use in clinical cases.

Role of coexisting contralateral primary venous disease in development of post-thrombotic syndrome following catheter-based treatment of iliofemoral deep venous thrombosis.

It has been reported that early clot removal benefits patients with iliofemoral deep venous thrombosis (DVT) by removing obstruction and preserving valve function. However, a substantial number of patients who had successful clot removal develop post-thrombotic syndrome (PTS). Residual thrombus and rethrombosis play a part in this phenomenon, but the role of coexisting primary chronic venous disease (PCVD) in these patients has not been studied. All patients who underwent catheter-based techniques of thrombus removal for symptomatic acute iliofemoral DVT during a 5-year period compose the study group. These patients were assessed for PTS by the Villalta scale, the Venous Clinical Severity Score (VCSS), and the Venous Insufficiency Epidemiological and Economic Study on Quality of Life (VEINES-QOL) questionnaire. The presence of coexisting PCVD was determined by clinical and duplex ultrasound findings in the contralateral leg at the time of the initial DVT diagnosis. Patients who had coexisting PCVD were compared with those without PCVD. Forty patients (40 limbs) were included in the study group. At initial diagnosis, 15 patients (38%) had coexisting symptomatic primary valve reflux in the unaffected limb. After thrombolysis, 9 of 40 limbs (22%) had complete lysis, 29 (73%) had ≥ 50% to 99% lysis, and 2 (5%) had <50% lysis. The mean percentage of lysis in patients with or without PCVD was similar (78% vs 86%; P = .13). Patients without coexisting PCVD had significantly better Villalta score and VCSS compared with those with coexisting PCVD (Villalta score, 2.52 vs 3.27, P = .014; VCSS, 2.96 vs 3.29, P = .005). Forty-five percent of patients (18 of 40) developed PTS. Patients who developed PTS had less clot lysis than those without PTS. This was true for patients with coexisting PCVD (60% vs 85%; P = .025) and in patients without PCVD (75% vs 89%; P = .013). There was no significant difference in the VEINES-QOL score between those with or without PCVD (79.5 vs 80.5; P = .9). Patients who had reflux in the treated limb after lysis had a five times greater chance for development of PTS compared with those who retained normal valve function during follow-up (odds ratio, 5.3; 95% confidence interval, 1.6-17.045). However, in patients with normal veins in the contralateral leg, the chance of development of PTS was 1.5 times higher if reflux was present in the treated limb (odds ratio, 1.49; 95% confidence interval, 0.043-10.253). Coexisting PCVD is a contributing factor to development of PTS after treatment of iliofemoral DVT with thrombus removal techniques.

Cytidine 5'-diphosphocholine differentially affects hemostatic parameters in diverse conditions in rats: an investigation via thromboelastography.

Cytidine 5'-diphosphocholine (CDP-choline) has several physiological and pharmacological effects on various bodily functions, including hemostasis. This study determined the impact of CDP-choline on hemostasis in a trauma-hemorrhage (T-H) model in rats or under in vitro conditions or after chronic treatment via thromboelastography. Trauma-hemorrhage resuscitation was induced, and either saline (1 mL/kg) or CDP-choline (50 mg/kg) was injected intravenously just prior to resuscitation in the T-H group and at the same time point in the sham-control group. The effects of CDP-choline on thromboelastogram parameters, coagulation markers, and platelet aggregation were investigated under in vitro conditions (1.5 mM, 30- or 3-min incubation in blood or plasma) and after chronic use (50 mg/kg, i.p., 10 days). Acute CDP-choline treatment was shown to decrease the initial and maximum clot formation time, accelerate clotting rapidity, reduce the lysis percentage, and increase the coagulation index in the T-H resuscitation group, whereas the same treatment in the sham-control rats did not alter any of the thromboelastogram parameters. However, the incubation of whole blood with CDP-choline prolonged the initial and maximum clot formation time, and CDP-choline treatment significantly decreased the slopes of the disaggregation and aggregation curves when platelets were stimulated with ADP and collagen, respectively. Interestingly, the chronic use of this drug did not influence any of these hemostatic parameters. These data implicate that acute but not chronic CDP-choline administration may differentially alter the hemostatic parameters under diverse conditions. The drug may produce a hypercoagulable state in activated situations but cause opposite effects under normal in vitro conditions.

Study of acute hemocoagulation changes in a porcine endotoxemic shock model using thrombelastography

Disseminated intravascular coagulation and fibrinolysis have been associated with lipopolysaccharide (LPS)-induced endotoxemic sepsis. It has been well established by point-of-care (POC) thrombelastography (TEG) that pigs have a hemocoagulation pathophysiology that resembles humans. We evaluated the use of TEG during the development of coagulation abnormalities in a porcine model of endotoxemia. After approval by the Animal Welfare Committee, pigs were instrumented to record hemodynamic variables. Ten days after surgical instrumentation, LPS (50 μg/kg) was infused intravenously over a period of 45 minutes in conscious animals. Hemodynamic parameters were recorded before and for 6 hours after LPS infusion was completed. Simultaneously, blood samples were analyzed using TEG to measure reaction time (R), clotting time (K), alpha angle (α), maximum amplitude (MA), coagulation index (CI), percent lysis at 30 minutes, and percent lysis at 60 minutes. LPS induced profound hemodynamic changes associated with the induced endotoxemia. Concomitantly, a progressive consumption coagulopathy characterized by significant increases in R and K and decreases in α, MA, and CI developed. The overall hemocoagulation profile of the 3 nonsurviving animals (27%) was significantly different than that of the survivors. Fibrinolysis was not detected during the 6-hour evaluation period. All stages of clot formation were affected as demonstrated by TEG (increased R and K, decreased α and MA). Our results suggest that TEG is a rapid method for assessing coagulation abnormalities in early stages of endotoxemia in pigs. TEG could have significant clinical applications as a rapid POC method in human patients with sepsis.

Enhanced blood compatibility of metallocene polyethylene subjected to hydrochloric acid treatment for cardiovascular implants.

Blood compatibility of metallocene polyethylene (mPE) was investigated after modifying the surface using hydrochloric acid. Contact angle of the mPE exposed to HCl poses a decrease in its value which indicates increasing wettability and better blood compatibility. Surface of mPE analyzed by using FTIR revealed no significant changes in its functional groups after treatment. Furthermore, scanning electron microscope images supported the increasing wettability through the modifications like pit formations and etching on the acid rendered surface. To evaluate the effect of acid treatment on the coagulation cascade, prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured. Both PT and APTT were delayed significantly (P < 0.05) after 60 min exposure implying improved blood compatibility of the surfaces. Hemolysis assay of the treated surface showed a remarkable decrease in the percentage of lysis of red blood cells when compared with untreated surface. Moreover, platelet adhesion assay demonstrated that HCl exposed surfaces deter the attachment of platelets and thereby reduce the chances of activation of blood coagulation cascade. These results confirmed the enhanced blood compatibility of mPE after HCl exposure which can be utilized for cardiovascular implants like artificial vascular prostheses, implants, and various blood contacting devices.

Thrombin generation and fibrin clot formation under hypothermic conditions: An in vitro evaluation of tissue factor initiated whole blood coagulation

BackgroundDespite trauma-induced hypothermic coagulopathy being familiar in the clinical setting, empirical experimentation concerning this phenomenon is lacking. In this study, we investigated the effects of hypothermia on thrombin generation, clot formation, and global hemostatic functions in an in vitro environment using a whole blood model and thromboelastography, which can recapitulate hypothermia. MethodsBlood was collected from healthy individuals through venipuncture and treated with corn trypsin inhibitor, to block the contact pathway. Coagulation was initiated with 5pM tissue factor at temperatures 37°C, 32°C, and 27°C. Reactions were quenched over time, with soluble and insoluble components analyzed for thrombin generation, fibrinogen consumption, factor (f)XIII activation, and fibrin deposition. Global coagulation potential was evaluated through thromboelastography. ResultsData showed that thrombin generation in samples at 37°C and 32°C had comparable rates, whereas 27°C had a much lower rate (39.2 ± 1.1 and 43 ± 2.4 nM/min vs 28.6 ± 4.4 nM/min, respectively). Fibrinogen consumption and fXIII activation were highest at 37°C, followed by 32°C and 27°C. Fibrin formation as seen through clot weights also followed this trend. Thromboelastography data showed that clot formation was fastest in samples at 37°C and lowest at 27°C. Maximum clot strength was similar for each temperature. Also, percent lysis of clots was highest at 37°C followed by 32°C and then 27°C. ConclusionsInduced hypothermic conditions directly affect the rate of thrombin generation and clot formation, whereas global clot stability remains intact.

Impairment of proliferation and NK activity of spleen leucocytes in young rats treated with a leptin antagonist during the physiological leptin surge through postnatal days 5–10

It is known that there is a physiological increase of leptin in rodents during the early postnatal (PND) period, from PND 5 to PND 10, with a peak at PND9‐10, which seems to be related to brain developmental processes. We have previously shown that rats submitted to 24 h of maternal deprivation (MD) at PND 9 showed an impairment of several immune functions that were evident in the neonatal and adolescent periods and persisted into adulthood (1) . Since conditions with a reduced production of leptin, a very important factor in modulating immune and inflammatory reactions (2) , are associated with increased susceptibility to infection (3) , it is likely that the decrease in circulating leptin levels in MD rats is related with their impaired immune response. The aim of the present study was to determine if the use of a leptin antagonist during the period corresponding to the physiological surge of leptin (PND5‐10) could affect several relevant functions of the immune cells in young rats. Young female and male rats were divided into two groups: 1) Treated with rat “mono-pegylated super active leptin antagonist” (mutant D23L/L39A/D40A/F41A), injected subcutaneously for 6 days (PND5‐10) (5 mg/kg/day) (T) and 2) Injected with the corresponding vehicle for the same period (Controls: C). When males and females reached 43 days and 33 days of age, respectively, the animals were sacrificed and their spleens were removed. In the spleen leucocytes the proliferation (basal and in the response to two mitogens, namely ConA and LPS) as well as the NK activity (percentage of lysis of murine tumour cells) were analysed. The results showed that in young male and female rats treated with mono-pegylated leptin antagonist, the anti-tumour NK activity was significantly lower than in their controls counterparts (P < 0.001 and P < 0.05, respectively). The proliferative response of lymphocytes to ConA decreased significantly (P < 0.05) in the female T group. These results suggest that the physiological leptin surge during the neonatal PN5-PND 10 period is involved in the establishment of an adequate immune response and support the notion that the impaired immune response of MD rats is related to the MD induced leptin deficiency in a critical period for developmental (programming) leptin actions. This work was supported by MICINN: BFU2011‐30336 and BFU2009‐10109, UCM (910379ENEROINN and UCM 951579), RETICEF (RD06/0013/ 0003) and RTA (RD06/0001/1013) from ISCIII.

Effects of oral prednisone administration with or without ultralow-dose acetylsalicylic acid on coagulation parameters in healthy dogs

To determine the effects of oral prednisone administration with or without ultralow-dose acetylsalicylic acid on coagulation parameters in healthy dogs and to assess intraindividual variation in thromboelastography results. 14 healthy research dogs and 10 healthy client-owned dogs. In a randomized controlled trial, research dogs underwent thromboelastography twice (3 days apart), and intraindividual variation in test results was calculated. Dogs were given prednisone (2 mg/kg/d, PO) plus acetylsalicylic acid (0.5 mg/kg/d, PO) or prednisone (2 mg/kg/d, PO) plus a placebo for 14 days, after which thromboelastography and other tests were repeated. Differences from preadministration (baseline) test results between and within groups were compared. In a separate trial, client-owned dogs also underwent thromboelastography twice 2 days apart to assess intraindividual variation in untreated dogs. Intraindividual variation in thromboelastography results for research dogs was ≤ 10% for maximum amplitude (MA) and α angle. In the research dogs, MA and fibrinogen values significantly increased from baseline, whereas percentage lysis 30 minutes after attainment of the MA as well as antithrombin activity significantly decreased within each group. In the dogs that received prednisone plus a placebo, percentage lysis 60 minutes after attainment of the MA was significantly lower than at baseline. For all parameters for research dogs, there was no difference between groups for change from baseline. Intraindividual variation in findings for client-owned dogs was similar to the variation for research dogs. Prednisone administration resulted in hypercoagulability in healthy dogs as indicated by an increase in MA and plasma fibrinogen concentration and a decrease in antithrombin activity. Concurrent ultralow-dose acetylsalicylic acid use had no effect on measured thromboelastography values. The high intraindividual variation in some thromboelastography parameters may preclude routine use of this technique in clinical practice.

Safety and efficacy of catheter directed thrombolysis in children with deep venous thrombosis

Safety and efficacy of catheter directed thrombolysis in children with deep venous thrombosis