Whole Blood Clotting Research Articles

Effect of Albumin in Combination With Mannitol on Whole-blood Coagulation In Vitro Assessed by Thromboelastometry

Albumin and mannitol may interfere with hemostasis, but their coinfluence is unclear. We aimed to determine the effects of albumin alone and in combination with mannitol or Ringer acetate (RAC) on hemostasis in crossover in vitro study. From citrated fresh whole blood withdrawn from 10 volunteers, we prepared 2.5, 5, 10, 15, and 20 vol% dilutions of 4% albumin (Alb group). Each sample was thereafter diluted by 15% mannitol (Alb/Man group) or RAC (Alb/RAC group) at a ratio of 9:1. Using thromboelastometry, FibTEM (fibrinogen ROTEM) and ExTEM (extrinsic ROTEM) tests were performed. A 20 vol%, but not 2.5 to 15 vol% dilution of albumin caused a prolonged clot formation time, α-angle decrease, and maximum clot firmness (MCF) weakening compared with undiluted sample (P<0.05). Clot formation time prolonged more in Alb5/Man than in Alb5 and Alb5/RAC dilution (P<0.05). In Alb2.5/Man, Alb10/Man, and Alb15/Man, dilution α-angle was lower than in corresponding Alb/RAC and Alb-group dilutions (P<0.05). In ExTEM, MCF decreased similarly in every dilution of Alb/Man and Alb/RAC compared with Alb group (P<0.05). In FibTEM, MCF decreased more in Alb10/Man than in Alb10/RAC dilution (P<0.05). In up to 15 vol% dilutions, albumin alone did not impair hemostasis in vitro, but in combination with mannitol or RAC coagulation was disturbed similarly at most concentrations. There was some significant additional effect with mannitol at certain concentrations. Our results indicate that coadministration of mannitol and albumin needs further study in vivo.

Evaluating prodrug characteristics of a novel anticoagulant fusion protein neorudin, a prodrug targeting release of hirudin variant 2-Lys47 at the thrombosis site, by means of in vitro pharmacokinetics

Recombinant neorudin (EPR-hirudin, EH), a low-bleeding anticoagulant fusion protein, is an inactive prodrug designed to be converted to the active metabolite, hirudin variant 2-Lys47 (HV2), locally at the thrombus site by FXa and/or FXIa, following activation of the coagulation system. Our aim was to evaluate the prodrug characteristics of EH by comparing the biotransformation of EH and HV2 in biological matrices, including rat blood, liver, and kidney homogenates, demonstrating the cleavage of EH to HV2 by FXa and FXIa, and comparing the conversion of EH to HV2 between fresh whole blood and whole-blood clot homogenate, using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS). Both EH and HV2 were stable in blood and unstable in the liver and kidney homogenates. Eight EH metabolites and eight HV2 metabolites identified as N-terminal fragments were found in the liver and kidney. C-terminal proteolysis is therefore the major metabolic pathway, with serine/cysteine carboxypeptidases and metallocarboxypeptidases being responsible for the degradation of EH and HV2 in the liver and kidney, respectively. EH was cleaved to release HV2 by FXIa. Higher levels of HV2 were produced from EH in the whole-blood clot homogenate, in which the coagulation system was activated compared with those in fresh whole blood. In conclusion, the metabolism of EH and HV2 shares the same cleavage pattern, and EH is transformed into HV2 when the coagulation system is activated, where FXIa is a specific enzyme. Our in vitro study revealed the anticipated prodrug characteristics of EH newly designed as an inactive prodrug of hirudin.

Staphylococcus aureus‐induced complement activation promotes tissue factor‐mediated coagulation

Background There is extensive cross-talk between the complement system, the Toll-like receptors (TLRs), and hemostasis. Consumptive coagulopathy is a hallmark of sepsis, and is often mediated through increased tissue factor (TF) expression. Objectives To study the relative roles of complement, TLRs and TF in Staphylococcus aureus-induced coagulation. Methods Lepirudin-anticoagulated human whole blood was incubated with the three S. aureus strains Cowan, Wood, and Newman. C3 was inhibited with compstatin, C5 with eculizumab, C5a receptor 1 (C5aR1) and activated factor XII with peptide inhibitors, CD14, TLR2 and TF with neutralizing antibodies, and TLR4 with eritoran. Complement activation was measured by ELISA. Coagulation was measured according to prothrombin fragment 1 + 2 (PTF1 + 2 ) determined with ELISA, and TF mRNA, monocyte surface expression and functional activity were measured with quantitative PCR, flow cytometry, and ELISA, respectively. Results All three strains generated substantial and statistically significant amounts of C5a, terminal complement complex, PTF1 + 2 , and TF mRNA, and showed substantial TF surface expression on monocytes and TF functional activity. Inhibition of C5 cleavage most efficiently and significantly inhibited all six markers in strains Cowan and Wood, and five markers in Newman. The effect of complement inhibition was shown to be completely dependent on C5aR1. The C5 blocking effect was equally potentiated when combined with blocking of CD14 or TLR2, but not TLR4. TF blocking significantly reduced PTF1 + 2 levels to baseline levels. Conclusions S. aureus-induced coagulation in human whole blood was mainly attributable to C5a-induced mRNA upregulation, monocyte TF expression, and plasma TF activity, thus underscoring complement as a key player in S. aureus-induced coagulation.

A commercial soy-based phospholipid emulsion accelerates clot formation in normal canine whole blood and induces hemolysis in whole blood from normal and dogs with inflammatory leukograms.

To compare lipid emulsion-induced hemolysis in blood from dogs with inflammatory leukograms to blood from healthy dogs, and determine the impact of a prototypical soy-based phospholipid emulsion on coagulation in whole blood from healthy dogs. Ex vivo study using EDTA and citrated whole blood from healthy dogs and EDTA anticoagulated whole blood from dogs with inflammatory leukograms. University research laboratory. Healthy dogs (total of 16, 9 for hemolysis assays and 6 for thromboelastography) included student- and staff-owned animals. Blood samples from dogs with inflammatory leukograms (8) were obtained from the clinical pathology laboratory after the complete blood count was performed as part of patient care. For the purposes of this study, an inflammatory leukogram was defined as a neutrophilia with a left-shift (minimum of 3% band neutrophils) and evidence of toxic change. None. Hemolysis was measured via spectrophotometric quantification of released hemoglobin and expressed as a percent of a water-lysed control. The soy emulsion caused hemolysis in blood from healthy dogs, ranging from 3.6% to 16.4% as the dose increased, and 4.1% to 25.0% in blood from dogs with inflammatory leukograms. Hemolysis between these patient groups was significantly different at the highest dose. Coagulation was assessed by native thromboelastography. Treatment of whole blood with the lipid emulsion caused a significant decrease in the time to clot formation (R) and a shorter time to reach a clot amplitude of 20 mm (K). Soy-based lipid emulsions cause hemolysis that is more severe in blood from dogs with inflammatory leukograms and accelerate clot formation in canine blood. The in vivo significance of these findings is not clear at this time, but warrants additional investigation given the use of these emulsions in clinical practice.

Factor (F)VIII/VIIa enhances global haemostatic function in the co‐presence of bypassing agents and FVIII among patients with haemophilia A with inhibitor

Bypassing therapy is essential for the haemostatic management of patients with haemophilia A with inhibitor (PWHA-inh), but the therapeutic effects are inconsistent. We previously reported that activated prothrombin complex concentrates (aPCC) activated factor (F)VIIIinvitro, and was mediated mainly by the activated FVII (FVIIa) contained in aPCC. We have extended those studies to assess global coagulation in whole blood from 18 PWHA-inh in the co-presence of aPCC and FVIII using Ca2+ -triggered rotational thromboelastometry. The clot times (CTs) in the presence of both aPCC (0·05 iu/ml) and recombinant (r)FVIII (1 iu/ml) exvivo were shortened compared to the aPCC alone (P<0·01). These enhancing effects of rFVIII were observed, irrespective of recognizing inhibitor epitopes; however, the clot formation time and 'α'-angle were not significantly different. In samples from 7 PWHA-inh post-infusion of aPCC (70-80 iu/kg), only the CTs were shortened in the presence of rFVIIIexvivo compared to its absence (P<0·05), indicating that the enhanced activity centred on the initiation phase of coagulation. Furthermore, experiments in the co-presence of rFVIIa and rFVIII demonstrated that FVIII accelerated only the CTs. We concluded that FVIII/FVIIa-related coagulation mechanism enhanced global haemostatic function by the co-presence of bypassing agents and FVIII in PWHA-inh.

Erythrocyte compression index is impaired in patients with residual vein obstruction

Defective clot contraction has been postulated to contribute to thrombosis. We aimed to evaluate the association of residual vein obstruction (RVO) with erythrocyte compression within the whole-blood clot. We studied 32 patients with venous thromboembolism (VTE) taking vitamin K antagonists (VKAs) for at least 3 months (median time in therapeutic range 60%), including 12 (37.5%) with RVO, and 32 age- and sex-matched controls. In all study participants we evaluated whole blood clot retraction, expressed as the erythrocyte compression index (ECI), defined as a ratio of mean polyhedrocyte area to mean native erythrocyte area, along with clot area covered by polyhedrocytes, plasma clot permeability (Ks), clot lysis time (CLT), and thrombin generation. In both groups higher ECI, indicating impaired clot contraction, increased with older age, higher body mass index, red blood cell distribution width, and lower platelet count (all p < 0.05), but not with red blood cell count. In VTE patients ECI was 15.8% higher than in controls (median 63.6 vs. 54.9%, p = 0.021). Subjects with RVO had 20% higher ECI and 155% lower clot area covered by polyhedrocytes. RVO patients had also prolonged CLT by 41%, but not Ks, and elevated peak thrombin generation by 33%, as compared to those without RVO (all p < 0.05). This study is the first to show impaired compression of erythrocytes in RVO patients despite VKA anticoagulation. Altered ECI coexisted with hypolysability and increased thrombin generation. ECI might be useful in the diagnostic process of RVO or post-thrombotic syndrome and can help optimize the anticoagulant therapy.

An in vitro proof-of-principle study of sonobactericide

Infective endocarditis (IE) is associated with high morbidity and mortality rates. The predominant bacteria causing IE is Staphylococcus aureus (S. aureus), which can bind to existing thrombi on heart valves and generate vegetations (biofilms). In this in vitro flow study, we evaluated sonobactericide as a novel strategy to treat IE, using ultrasound and an ultrasound contrast agent with or without other therapeutics. We developed a model of IE biofilm using human whole-blood clots infected with patient-derived S. aureus (infected clots). Histology and live-cell imaging revealed a biofilm layer of fibrin-embedded living Staphylococci around a dense erythrocyte core. Infected clots were treated under flow for 30 minutes and degradation was assessed by time-lapse microscopy imaging. Treatments consisted of either continuous plasma flow alone or with different combinations of therapeutics: oxacillin (antibiotic), recombinant tissue plasminogen activator (rt-PA; thrombolytic), intermittent continuous-wave low-frequency ultrasound (120-kHz, 0.44 MPa peak-to-peak pressure), and an ultrasound contrast agent (Definity). Infected clots exposed to the combination of oxacillin, rt-PA, ultrasound, and Definity achieved 99.3 ± 1.7% loss, which was greater than the other treatment arms. Effluent size measurements suggested low likelihood of emboli formation. These results support the continued investigation of sonobactericide as a therapeutic strategy for IE.

Hemostatic porous sponges of cross-linked hyaluronic acid/cationized dextran by one self-foaming process

Effective hemostatic materials are very important for treating trauma cases. Natural polysaccharides have been particularly appealing in the development of new hemostatic materials due to their unique functions in human bodies. In this work, different polysaccharide-based hemostatic porous sponges (SHDP or SHDQ) of cross-linked hyaluronic acid (HA)/cationized dextran were readily prepared by the self-foaming process of HA and poly((2-dimethyl amino)-ethyl methacrylate)-grafted dextran (Dex-PDM) or partially-quaternized Dex-PDM in the presence of sodium trimetaphosphate crosslinkers. SHDP and SHDQ sponges were investigated in terms of liquid-absorption ability, hemolysis, whole-blood clotting and hemostatic activity in hemorrhaging-liver models. Compared with HA/Dex-PDM sponges (HDP) without chemical cross-linking, SHDP and SHDQ sponges displayed higher porosity (>70.0% vs. 48.9%) and swelling ratios (>1000% vs. 520%). Meanwhile, hemolysis assay revealed the good blood compatibility of SHDP and SHDQ with low hemolysis ratio (below 0.5%). Furthermore, in vitro and in vivo hemostatic assay showed that SHDQ possessed better hemostatic properties than SHDP, owing to the higher cationic charges of partially-quaternized Dex-QPDM than Dex-PDM. The present study demonstrated that the self-foaming process of HA/Dex-PDM under a ‘green’ condition is an effective means to produce new hemostatic materials.

Leukocyte filtration lesion impairs functional coagulation in banked whole blood.

Whole blood (WB) transfusion is a promising alternative to component therapy in hemostatic resuscitation. Use of banked WB requires filtration of white blood cells (leukoreduction) and an established shelf life during which WB retains coagulant capacities. The goal of this study was to define the time course of coagulation stability in leukoreduced compared to unfiltered WB under standard refrigeration conditions. Twelve WB units were donated by healthy volunteers after routine screening. Five units underwent standard leukocyte filtration and five did not. Two units were aliquoted into filtered and unfiltered samples, with platelets added to each sample on day 14. Units were stored at 4°C and sampled on days 0, 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, 28, and 35 for immediate thromboelastography (TEG) analysis, and centrifuged and stored at -80°C for later calibrated automated thrombogram and coagulation factor assays. K-dependent factors and fibrinogen were low normal, decreased slightly over 35 days and were similar between unfiltered and filtered units. Labile factors were better preserved in filtered units, although unfiltered units did not show impaired coagulation over 35 days. Filtered blood had delayed clot initiation on days 0, 1, and 2 as measured by TEG R (p < 0.021); slower clot progression (TEG α-angle) on days 0, 1, 2, 3, 4, 5, and 6 (p < 0.023); weaker final clot (TEG MA) on all days (p < 0.0001). Thrombin generation was delayed on day 28 (p = 0.046) and decreased on days 10, 21, 28, and 35 (p < 0.034). Addition of platelets to filtered WB rescued TEG MA. Filtered WB had decreased functional clotting capacity and thrombin generation and may not be suitable for hemostatic resuscitation as the sole blood product. Therapeutic, level IV.

Sonobactericide: An ultrasound-mediated adjunct treatment for bacterial infective endocarditis—In vitro proof-of-principle

Infective endocarditis (IE) is a bacterial infection of the surfaces within the heart, associated with high morbidity and mortality rates. The predominant bacteria causing IE are Staphylococci, which can bind to existing thrombi on heart valves and generate vegetations (biofilms). In this in vitro study, we test a novel strategy called sonobactericide to treat IE using ultrasound and ultrasound contrast agents, in combination with an antibiotic and a thrombolytic. We simulated IE vegetations using human whole-blood clots infected with Staphylococcus aureus. Histology and live-cell imaging revealed a biofilm layer of fibrin-embedded living Staphylococci. Infected clots were treated under flow for 30 min and degradation was assessed by time-lapse microscopy imaging. Clots were exposed to human plasma flow either alone or with different combinations of therapeutics: oxacillin (172 μg/mL), recombinant tissue plasminogen activator (rt-PA; 3.15 μg/mL), intermittent (50 s on, 30 s off) continuous-wave ultrasound (120 kHz, 0.44 MPa peak-to-peak pressure), and Definity (2 μl/ml). Infected clots exposed to the combination of oxacillin, rt-PA, ultrasound, and Definity achieved 99.3 ± 1.7% clot width loss, which was greater than the other treatment arms. These results demonstrate that sonobactericide may have potential as an adjunct therapy for IE.

Tissue factor-dependent coagulation activation by heme: A thromboelastometry study.

Heme has been characterized as potent trigger of inflammation. In hemostasis, although heme has been shown to both induce and inhibit different compartments of hemostasis, its net effect on the hemostatic balance, and the biological relevance of these effects remain to be determined. Herein we evaluated the effect of heme on hemostasis using a global assay able to generate clinically relevant data in several other complex hemostatic diseases. Citrated whole blood samples from healthy participants were stimulated by heme or vehicle and incubated for 4h at 37°C. Rotational thromboelastometry was immediately performed. The participation of tissue factor in coagulation activation was evaluated using inhibitory antibody. Heme was able of inducing ex vivo coagulation activation in whole blood, affecting predominantly parameters associated with the initial phases of clot formation. This activation effect was at least partially dependent on hematopoietic tissue factor, since the effects of heme were partially abrogated by the inhibition of human tissue factor. In conclusion, using a global hemostasis assay, our study confirmed that heme is able to activate coagulation in whole blood, in a tissue factor-dependent way. These findings could explain the disturbance in hemostatic balance observed in conditions associated with the release of heme such as sickle cell disease.

Re: Comment on: A Comparison of the Effects of 20% Mannitol and 3% NaCl on Coagulation Parameters In Vitro using ROTEM: A Prospective Randomized Crossover Study.

OBJECTIVE The aim of the present study is to compare the effect of 20% mannitol and 3% NaCl on blood coagulation in vitro using rotational thromboelastometry (ROTEM). METHODS Twenty-millilitre blood samples were obtained from 15 volunteers. In each group, 2 mL blood samples were collected into both polypropylene tubes and EDTA tubes for ROTEM and hemogram analysis. After sampling, blood samples were diluted with test solutions. Group C (control): Only blood, Group M (mannitol): 7% vol 20% mannitol concentration in the blood, Group hypertonic saline (HS): 7% vol 3% hypertonic saline (NaCl) in the blood, Group M/H (mannitol and hydroxyethyl starch solutions [HES]): 6% vol 20% mannitol concentration and 8% vol HES in the blood and Group HS/H (hypertonic saline and HES): 6% vol 3% hypertonic saline concentration and 8% vol HES in the blood. The following thromboelastometric parameters were measured automatically: clotting time (CT) and clot formation time (CFT) with intrinsic activation by tissue factor (InTEM), CT, CFT and maximum clot firmness (MCF) with extrinsic activation by tissue factor (ExTEM) and MCF with FibTEM. RESULTS The ExTEM CT value was found to be significantly longer in the M/H group than in the controls. The ExTEM CFT median and percentile values were: group C: 85 s (70-95 s), group M: 115 s (94-128 s), group HS: 102 s (84-114 s), group M/H: 128 s (110-144 s) and group HS/H: 118 s (107-132 s). In all the groups, FibTEM MCF values were significantly lower than the control and also there was a significant difference between groups M and HS according to FibTEM MCF values. CONCLUSION Whole-blood coagulation disorder induced by these solutions is mainly dependent on fibrinogen and fibrin interaction. However, 3% HS has much less negative effect on coagulation.

The Association Between Cyanosis and Thromboelastometry (ROTEM) in Children With Congenital Heart Defects: A Retrospective Cohort Study.

Children with congenital heart defects (CHD) have quantitative and qualitative differences in coagulation compared with healthy children. Secondary to polycythemia and increased deformability of red blood cells, cyanosis may be an important confounding factor for altered whole-blood coagulation in this population with potential implications for interpreting intraoperative thromboelastometry (TEM) for children with CHD undergoing major surgery. The primary aim of the study was to evaluate the association between cyanosis in children with CHD and measures of whole-blood coagulation determined using TEM (ROTEM [Tem International, GmbH, Munich, Germany]). In this retrospective cohort study, children who underwent congenital cardiac surgery in a 12-month period between April 2014 and 2015 were investigated. Children who were receiving antiplatelet or anticoagulant medications in the preoperative period were excluded. Eligible children were categorized by the presence of cyanosis, defined as an oxyhemoglobin concentration ≤85%. Multivariable linear regression analyses were used to determine the relationship between cyanosis and TEM outcomes (primary outcome, fibrinogen/fibrin polymerization [FibTEM] maximal clot firmness [MCF]) adjusting for potential confounding factors. Three hundred forty-five TEM profiles from 320 children were included in the cohort for analysis. Twenty-two percent (76/345) of children had cyanotic CHD. Clot firmness measured using the FibTEM assay was decreased in cyanotic children compared with noncyanotic children, median difference (95% confidence interval) interim [2 (0-3) mm; P = .01], and maximal [2 (1-3) mm; P = .01] clot firmness. The association between cyanosis and fibrinogen/fibrin polymerization clot firmness was not significant (A10, P = .7; MCF, P = .7) after adjusting for confounding factors (hematocrit, platelet count, and sex). There was a significant association between cyanosis and intrinsically activated clot firmness (A10, P = .03; MCF, P = .02), but not other TEM outcomes, after adjusting for confounding factors. Cyanotic children had decreased clot firmness in the fibrinogen/fibrin polymerization component of the clot compared with noncyanotic children, but the association between cyanosis and clot firmness was accounted for by differences in hematocrit, platelet count, and sex between groups. These findings will help guide the identification and treatment of coagulopathy in this vulnerable population.

Microfluidic coagulation assay for monitoring anticoagulant therapy in acute stroke patients

Reliable detection of anticoagulation status in patients treated with non-vitamin K antagonist oral anticoagulants (NOACs) is challenging but of importance especially in the emergency setting. This study evaluated the potential of a whole-blood clotting time assay based on Surface Acoustic Waves (SAW-CT) in stroke-patients. The SAW-technology was used for quick and homogenous recalcification of whole blood inducing a surface-activated clotting reaction quantified and visualised by real-time fluorescence microscopy with automatic imaging processing. In 20 stroke or transient ischaemic attack (TIA)-patients taking NOACs kinetics of SAW-CT were assessed and correlated to other coagulation parameters (PT, aPTT) and NOAC-plasma concentration measured by tandem mass spectrometry (LC-MS/MS). In 225 emergency patients with suspicion of acute stroke or TIA, SAW-CT values were assessed. Mean (± SD) SAW-CT in non-anticoagulated stroke patients (n=180) was 124 s (± 21). In patients on dabigatran or rivaroxaban, SAW-CT values were significantly higher 2 and 8 hours (h) after intake rising up to 267 seconds (s) (dabigatran, 2 h after intake) and 250 s (rivaroxaban, 8 h after intake). In patients on apixaban, SAW-CT values were only moderately increased 2 h after intake (SAW-CT 153 s). In emergency patients, SAW-CT values were significantly higher in NOAC and vitamin K antagonist (VKA)-treated as compared to non-anticoagulated patients. In conclusion, the SAW-CT assay is capable to monitor anticoagulant level and effect in patients receiving dabigatran, rivaroxaban and the VKA phenprocoumon. It has a limited sensitivity for apixaban-detection. If specific SAW-CT results were used as cut-offs, SAW-CT yields high diagnostic accuracy to exclude relevant rivaroxaban and dabigatran concentrations in stroke-patients.

Sonobactericide - An Adjunct Therapy for Infective Endocarditis: An In Vitro Proof-of-Concept

Infective endocarditis (IE) is associated with high morbidity and mortality rates. The predominant bacteria causing IE is Staphylococcus aureus (S. aureus), which can bind to existing thrombi on heart valves and generate vegetations (biofilms). In this in vitro study, we evaluated sonobactericide as a novel strategy to treat IE, using ultrasound and an ultrasound contrast agent in combination with an antibiotic and a thrombolytic. We developed a model of IE vegetations using human whole-blood clots infected with S. aureus. Histology and live-cell imaging revealed an outer biofilm layer of fibrin-embedded living Staphylococci around a dense erythrocyte core. Infected clots were treated under flow for 30 minutes and degradation was assessed by time-lapse microscopy imaging. Treatments consisted of either continuous plasma flow alone or with different combinations of therapeutics: oxacillin (antibiotic), recombinant tissue plasminogen activator (rt-PA; thrombolytic), intermittent continuous-wave low-frequency ultrasound (120-kHz, 0.44 MPa peak-to-peak pressure), and an ultrasound contrast agent (Definity®). Infected clots exposed to the combination of oxacillin, rt-PA, ultrasound, and Definity achieved 99.3 ± 1.7% clot width loss, which was greater than the other treatment arms. Size measurements of the effluent suggested low emboli risk. These results demonstrate that sonobactericide could be an effective therapeutic strategy for treating IE.

Hemophilia a Clots Generated with Recombinant Factor VIIa Differed in Structure and Composition from Those Formed with Factor VIII

Patients with severe factor VIII (FVIII) deficiency (hemophilia A [HemA]) develop neutralizing antibodies (inhibitors) against FVIII in up to ~30% of cases. For HemA patients with inhibitors, activated recombinant factor VII (rFVIIa) is a treatment option. High levels of rFVIIa are required for treating HemA patients with inhibitors to induce direct activation of factor X on the surface of activated platelets via a tissue factor (TF)-independent mechanism (Hoffman M, Monroe DM. Thromb Res. 2010;125(suppl 1):S16-S18). To assess how rFVIIa-mediated clot formation in HemA patients with inhibitors may differ from unaffected individuals, we compared the effect of rFVIIa on HemA versus control (or HemA supplemented with 100% FVIII) clot formation in human and/or mouse systems. By TF-induced thrombin generation assay, increasing rFVIIa from 5 nM to 100 nM did not appreciably alter the kinetics or extent of thrombin generation compared with the same human HemA plasma containing 100% FVIII. Confocal microscopy of human HemA plasma clots generated with 75 nM rFVIIa and TF showed few branching fibrin fibers and an open fibrin meshwork. In contrast, TF-induced coagulation of the same HemA plasma containing 100% FVIII formed fibrin clots with numerous branches, interconnecting to form a dense meshwork. To confirm that these findings reflect rFVIIa-mediated clot formation in vivo, we assessed the intrinsic coagulation of mouse HemA whole blood collected without anticoagulant and spiked with rFVIIa. Intrinsic coagulation with rFVIIa was assessed by T2 magnetic resonance (T2MR), a technique capable of monitoring the separation of whole blood into serum, loose-clot, and tight-clot compartments during coagulation (Skewis et al. Clin Chem. 2014;60:1174-1182; Cines et al. Blood. 2014;123:1596-1603). By T2MR, rFVIIa induced the separation of HemA whole blood into the serum and clot compartments, indicating that the reduced fibrin generation with rFVIIa did not interfere with whole blood coagulation. Furthermore, saphenous vein puncture of HemA mice treated with rFVIIa showed a dose-dependent decrease in clot times. Scanning electron microscopy of the clots extracted from these HemA mice indicated markedly different composition than clots extracted from wild-type mice. In wild-type clots, fibrin and polyhedral erythrocytes formed a large proportion of the total structures. In contrast, clots from rFVIIa-treated HemA mice consisted primarily of platelets and erythrocytes with forms intermediate between discoid and polyhedral but, surprisingly, low fibrin content. Taken together, these data suggest that rFVIIa-mediated clot formation may require greater activated platelet involvement, which would be consistent with the TF-independent mechanism of action proposed for rFVIIa in HemA. Finally, the compositional difference between clots from wild-type versus HemA mice dosed with rFVIIa suggest that evaluating HemA therapies for their ability to form more physiologic clots could be an approach to improve treatment options for patients with HemA. DisclosuresLeong:Bayer: Employment. Xu:Bayer: Employment. Mallari:Bayer: Employment. Wong:Bayer: Employment. Sim:Bayer: Employment. Cuker:Stago: Consultancy; Genzyme: Consultancy; Amgen: Consultancy; Biogen-Idec: Consultancy, Research Funding; T2 Biosystems: Research Funding. Marturano:T2 Biosystems: Employment. Lowery:T2 Biosystems: Employment. Kauser:Bayer: Employment. Weisel:Bayer: Research Funding.

Evidence for Anti-Fibrinolytic Activity of Red Cell-Derived Microparticles (RMP): A Novel Hemostatic Action of RMP

BACKGROUND. RMP have been developed as novel hemostatic agents for treatment of bleeding disorders [Thromb Haemost 110:751,2013]. RMP generate thrombin in vitro through activation of contact pathway independent of tissue factor. They correct coagulopathy associated with most factor deficient plasma, indicating promotion of secondary hemostasis. In addition, they promote primary hemostasis by enhancing platelet adhesion and aggregation. Recent literature suggests that excessive levels of tPA are responsible for the uncontrollable bleeding seen in trauma-related coagulopathies [J Trauma and Acute Care Surg 80:1,2016]. In this study, we used thromboelastography (TEG) to investigate the effects of RMP on tPA, a relation hitherto unexplored.METHODS. The RMP were made by high-pressure extrusion of washed red cells. TEG was used to assess coagulation and fibrinolysis in fresh whole blood (diluted to 80%) in presence vs. absence of RMP (2.78x107/mL final conc. based on flow cytometry of PE-labeled mAb to CD235a). To hasten fibrinolysis for ease of measurement, tPA was added in all runs at final conc. 0.4 ug/mL. Calcium chloride (0.2 M) was added to each trial to induce coagulation. Additions to cup were: 325 uL of 80% blood + 10 uL tPA + 10 uL saline or RMP + 20 uL CaCl2. The tPA was stored in 50% glycerol at -20°C.RESULTS. Representative TEG curves of tPA-induced fibrinolysis in the presence or absence of RMP are shown in Figure 1 below. In the presence of tPA without RMP, the MA is small and the amplitude will decline to the baseline in 30 - 60 min, reflecting ongoing fibrinolysis. The MA was increased by about 50% by addition of RMP, from 25mm to 37mm (p=0.016). Judging from fibrinolysis measured by the-built-in LY30 parameter (% lysed at 30 min after MA was achieved), the mean of n=6 paired t-tests was 61.3% lysed in absence of RMP vs. 36.3% with RMP, indicating 40% inhibition of fibrinolysis (p=0.046).To assess the overall anti-fibrinolytic effect of RMP, we used the integral of the 1st derivative of the TEG curve, called TG (thrombus generation), which amounts to the total or net hemostatic-like activity. By this measure, addition of RMP gave TG = 457 vs. 319mm/min in absence of RMP (p=0.0044 by paired t test, P=0.037 by unpaired t test).DISCUSSION. These findings suggest that RMP exert significant anti-fibrinolytic activity, enhancing its hemostatic activity. Uncontrollable bleeding due to fibrinolysis is a major component of many bleeding disorders, including acute coagulopathy of trauma which is seen in about 20% of cases and is associated with poor outcome. The mechanistic basis of this activity is not fully understood. It was suggested that RMP are incorporated into the fibrin network to consolidate its structure thus stabilizing the network and impeding fibrinolysis. Full exploration of this activity is warranted since treatment for excessive fibrinolysis would have great impact in clinical management of many bleeding disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Application effect of ACT monitor cooperated with special nursing during PCI perioperation

Objective To monitor the evaluation of heparin dose in activated clotting time (ACT) during PCI perioperation, and explore its application in clinical care. Methods Totally 327 patients with coronary angiography, PTCA and stent implantation, using heparin to anticoagulating simultaneously in conduit room of the 306th Hospital of PLA from April 2014 to April 2015. By ACT monitoring and preoperative nursing visit, we collected patients′ risk factors, and made operation plans; patients were assigned to five groups by the stage of age, and ten year-old was taken as a stage. ACT was closely observed in the operation, and the risk situation of patients was also observed; after operation, specific guidance was taken. Results There were patients with substandard ACT in each group after operation, and needed additional heparin. 71 cases of patients with preoperative application of tirofiban, their postoperative ACT were within the target range 200-250 s; among 256 patients without preoperative application of tirofiban, there were 41 patients whose ACT value does not reach the required target value and needed additional heparin, wherein the incidence rate of substandard VCT value of 55-64 year-old group was the highest and was 20%. Eight cases without preoperative application of tirofiban, had sheath pipe pulled out because of errhysis in femoral artery sheath pipe puncture after operation, but their VCT value was within the target range of target value. All patients had no intraoperative and postoperative stroke, bleeding and thrombosis, etc. Conclusions ACT monitoring and effective coordination of care, can ensure the safety of heparin used in interventional procedures. Key words: Heparin; Whole blood coagulation time; Nursing; Percutaneous coronary intervention

Factor XIa-specific IgG and a reversal agent to probe factor XI function in thrombosis and hemostasis.

Thrombosis is a major cause of morbidity and mortality. Current antithrombotic drugs are not ideal in that they must balance prevention of thrombosis against bleeding risk. Inhibition of coagulation factor XI (FXI) may offer an improvement over existing antithrombotic strategies by preventing some forms of thrombosis with lower bleeding risk. To permit exploration of this hypothesis in humans, we generated and characterized a series of human immunoglobulin Gs (IgGs) that blocked FXIa active-site function but did not bind FXI zymogen or other coagulation proteases. The most potent of these IgGs, C24 and DEF, inhibited clotting in whole human blood and prevented FeCl3-induced carotid artery occlusion in FXI-deficient mice reconstituted with human FXI and in thread-induced venous thrombosis in rabbits at clinically relevant doses. At doses substantially higher than those required for inhibition of intravascular thrombus formation in these models, DEF did not increase cuticle bleeding in rabbits or cause spontaneous bleeding in macaques over a 2-week study. Anticipating the desirability of a reversal agent, we also generated a human IgG that rapidly reversed DEF activity ex vivo in human plasma and in vivo in rabbits. Thus, an active site-directed FXIa-specific antibody can block thrombosis in animal models and, together with the reversal agent, may facilitate exploration of the roles of FXIa in human disease.

Assessment of the Hemostatic Parameters and Platelet Function on Thromboelastometry and Impedance Aggregometry in Hemodialysis Patients Qualified for Kidney Transplantation: Preliminary Report

BackgroundChronic kidney disease is one of the medical conditions that affect hemostasis. Patients undergoing hemodialysis present both hemorrhagic and prothrombotic tendencies. Platelet adhesion to the artificial surface of the dialyzer's membrane, blood vessel endothelial wall disruption, and quantitative and qualitative changes in clothing factors are thought to be causative agents of the above-mentioned conditions. Thromboelastometry and impedance aggregometry enable precise assessment of clot formation and platelet function abnormalities, including changes related to chronic renal failure in patients undergoing renal replacement therapy. MethodsA prospective study with control group was designed. The study group consisted of 17 adults with diagnosed chronic renal failure undergoing hemodialysis. The control group consisted of 13 healthy volunteers. EXTEM and FIBTEM tests in rotational thromboelastometry and TRAPtest in impedance aggregometry analyzer were performed. ResultsEXTEM parameter test results were comparable between analyzed groups, whereas FIBTEM test results were significantly increased in the study group. Platelet aggregation as measured by the TRAPtests was significantly decreased in patients undergoing hemodialysis. ConclusionsIn end-stage renal disease patients undergoing hemodialysis, whole-blood clot formation is not disturbed, even though platelet dysfunction occurs. Increased fibrin clot formation reflected by FIBTEM results may compensate the observed platelet disorders. The compilation of ROTEM and Multiplate may support appropriate hemostatic control and decision-making during kidney transplantation.