Thrombin-antithrombin Complex Formation Research Articles

A 3-O-sulfated heparan sulfate dodecasaccharide (12-mer) suppresses thromboinflammation and attenuates early organ injury following trauma and hemorrhagic shock.

Dysregulated inflammation and coagulation are underlying mechanisms driving organ injury after trauma and hemorrhagic shock. Heparan sulfates, cell surface glycosaminoglycans abundantly expressed on the endothelial surface, regulate a variety of cellular processes. Endothelial heparan sulfate containing a rare 3-O-sulfate modification on a glucosamine residue is anticoagulant and anti-inflammatory through high-affinity antithrombin binding and sequestering of circulating damage-associated molecular pattern molecules. Our goal was to evaluate therapeutic potential of a synthetic 3-O-sulfated heparan sulfate dodecasaccharide (12-mer, or dekaparin) to attenuate thromboinflammation and prevent organ injury. Male Sprague-Dawley rats were pre-treated subcutaneously with vehicle (saline) or dekaparin (2 mg/kg) and subjected to a trauma/hemorrhagic shock model through laparotomy, gut distention, and fixed-pressure hemorrhage. Vehicle and dekaparin-treated rats were resuscitated with Lactated Ringer's solution (LR) and compared to vehicle-treated fresh-frozen-plasma-(FFP)-resuscitated rats. Serial blood samples were collected at baseline, after induction of shock, and 3 hours after fluid resuscitation to measure hemodynamic and metabolic shock indicators, inflammatory mediators, and thrombin-antithrombin complex formation. Lungs and kidneys were processed for organ injury scoring and immunohistochemical analysis to quantify presence of neutrophils. Induction of trauma and hemorrhagic shock resulted in significant increases in thrombin-antithrombin complex, inflammatory markers, and lung and kidney injury scores. Compared to vehicle, dekaparin treatment did not affect induction, severity, or recovery of shock as indicated by hemodynamics, metabolic indicators of shock (lactate and base excess), or metrics of bleeding, including overall blood loss, resuscitation volume, or hematocrit. While LR-vehicle-resuscitated rodents exhibited increased lung and kidney injury, administration of dekaparin significantly reduced organ injury scores and was similar to organ protection conferred by FFP resuscitation. This was associated with a significant reduction in neutrophil infiltration in lungs and kidneys and reduced lung fibrin deposition among dekaparin-treated rats compared to vehicle. No differences in organ injury, neutrophil infiltrates, or fibrin staining between dekaparin and FFP groups were observed. Finally, dekaparin treatment attenuated induction of thrombin-antithrombin complex and inflammatory mediators in plasma following trauma and hemorrhagic shock. Anti-thromboinflammatory properties of a synthetic 3-O-sulfated heparan sulfate 12-mer, dekaparin, could provide therapeutic benefit for mitigating organ injury following major trauma and hemorrhagic shock.

Neural Network Diagnoses the Antiphospholipid Syndrome without Interrupting Anticoagulant Therapy

Background The antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies (aPL) and is associated with an increased risk of thrombosis and pregnancy morbidity. The Laboratory diagnosis of APS is difficult because most patients already use anticoagulants due to a thrombotic event, resulting in interference with the clotting assays used for lupus anticoagulant (LAC) testing. Nevertheless, the aPL profile defines patient management, making aPL testing warranted during anticoagulant therapy. Sometimes, cessation of anticoagulant treatment is necessary to perform the lupus anticoagulant (LAC) test, which is unfavorable. The thrombin generation (TG) test is a functional and global hemostatic test. Clinical studies have shown that increased TG is associated with thrombosis, also in thrombotic APS patients. We have previously shown that the artificial intelligence method neural networks can be used to diagnose untreated APS patients from a group of healthy, auto-immune disease, and thrombosis controls with high accuracy. At the moment of aPL testing, many patients are already on anticoagulants, hampering the detection of LAC. Therefore, we developed a neural net (NN) for diagnoses APS in a cohort of anticoagulated subjects, based on the TG test. Methods Antiphospholipid antibodies positivity was diagnosed according to the Sapporo-Sydney guidelines. TG was measured using the calibrated automated thrombogram assay (CAT) on platelet poor plasma with PPP reagent (intermediate concentration of tissue factor) in 48 APS patients and 64 control non-APS patients, both treated with vitamin K antagonists (VKAs) TG was explored in more depth by quantifying parameters of prothrombin conversion and thrombin inactivation, using thrombin dynamics (TD) analysis, and assessing the sensitivity of the activated protein C pathway through the addition of thrombomodulin (TM). Five NNs were developed and the most accurate NN was selected for clinical validation based on its sensitivity and specificity. Input parameters for the NN were TG parameters lag time, peak, endogenous thrombin potential, time-to-peak, and velocity index, and TD parameters total prothrombin conversion, maximum prothrombin conversion rate, thrombin-antithrombin complex formation, and thrombin-α2-macroglobulin complex formation, and the inhibition of TG and TD by TM. The top performing NN was clinically validated using the TG and TD data acquired in the validation cohort, consisting of 33 APS patients and 62 controls, both on VKA therapy, 49 auto-immune disease controls, 38 thrombosis controls, 92 patients visiting the hospital for other indications and 37 healthy subjects. All control subjects were negative for aPL. Results TG was slightly increased in APS patients compared to controls. VKA therapy abolished the typical lag time prolongation seen in non-anticoagulated APS patients due to the LAC effect. The rate of prothrombin conversion was significantly increased in anticoagulated APS patients compared to controls, indicating a more prothrombotic phenotype. The selected NN classified APS patients with a sensitivity of 92% and a specificity of 95% in the developmental cohort consisting of APS patients and controls on VKA antagonists (ROCAUC = 0.9805; 0.9542-1.000; p<0.0001). On average, controls and APS patients were assigned an APS likelihood score of 12% and 89%, respectively. The NN was clinically validated in a separate validation cohort of 311 individuals, including 33 anticoagulated APS patients, 62 anticoagulated controls, 38 thrombosis patients, 49 auto-immune disease patients, 92 hospital controls, and 37 healthy controls. The sensitivity of the NN for the detection of APS was 85%. The overall specificity of the NN was 93%, and 100%, 96%, 96%, 92% and 76% respectively in healthy controls, auto-immune disease controls, hospital controls, anticoagulated controls, and thrombosis controls. Conclusions We developed a NN that accurately classifies APS under anticoagulant treatment. Therefore, this NN could be an alternative for diagnosing APS patients already treated with VKAs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The importance of α1AMP-activated protein kinase (α1AMPK) in hemostatic dysfunctions induced by sepsis

Sepsis is a pathological circumstance whose outcome is associated with organ dysfunction and death. Vascular hyperpermeability and hemostasis impairment both play a key role in the onset of organ failure. Our previous work highlighted the protective role of α1AMPK against sepsis-induced hyperpermeability. However, the impact of α1AMPK on hemostasis has never been investigated in this condition. This study aims to characterize the interaction of α1AMPK with hemostasis in sepsis. α1AMPK wild type (WT) and knockout (KO) mice were challenged with intraperitoneal injection of lipopolysaccharide (LPS) from Escherichia coli O55B5 (10 mg/kg) during 6 and 18 hours. Plasmatic markers of coagulation, NETosis and platelet activation were measured by ELISA. To identify cell type responsible for the dysregulation, same analyses were conducted on endothelial-, leucocyte- or platelet- specific α1AMPK knockout mouse models. Hemostasis was altered at 6 hours and 18 hours after LPS administration, in both α1AMPK WT and KO mice. Thrombin generation, reflected by thrombin-antithrombin complex formation, was significantly increased in α1AMPK KO mice compared to WT mice, after 18 hours of treatment with LPS. In contrast, fibrinolysis was more inhibited in α1AMPK KO than in WT mice, with a higher level of plasminogen activator inhibitor-1 measured at 6 and 18 hours, while tissue plasminogen activator was not different. This exacerbated procoagulant phenotype in α1AMPK KO mice was associated with an increase of sCD62P in plasma, reflecting an overactivation of platelets under septic conditions. Septic α1AMPK KO mice also showed an increase of Neutrophil Elastase and plasma DNA with no variation of MPO-DNA complexes, suggesting that NETosis was not altered but that exacerbated cellular damages occurred in these mice compared to WT animals. Platelet- and leucocyte- specific α1AMPK knockout mouse models did not recapitulate the phenotype observed in total α1AMPK knockout mice after LPS administration. However, endothelial specific α1AMPK knockout mice showed an increase of thrombin generation with a tendency to higher platelet activation. Altogether, these results demonstrate that α1AMPK plays a crucial role in the regulation of hemostasis disturbances during sepsis. While α1AMPK from platelets and leucocytes does not contribute to the septic damage, endothelialα1AMPK might be a brake for hemostatic dysregulation.

The importance of alpha-1 AMP-activated protein kinase (α1AMPK) in hemostatic dysfunctions induced by sepsis

Sepsis is a pathological circumstance whose outcome is associated with organ dysfunction and death. Vascular hyperpermeability and hemostasis impairment, both play a key role in the onset of organ failure. Our previous work highlighted the protective role of α1AMPK against sepsis-induced hyperpermeability. However, the impact of α1AMPK on hemostasis has never been investigated in this condition. This study aims to characterize the interaction of α1AMPK with hemostasis in sepsis. α1AMPK wild type (WT) and knockout (KO) mice were challenged with intraperitoneal injection of lipopolysaccharide (LPS) from Escherichia coli O55B5 (10 mg.kg) during 6 and 18 hours. Plasmatic markers of coagulation, NETosis and platelet activation were measured by ELISA. In order to identify the origin of the dysregulation, same analyses were conducted on endothelial, leucocyte or platelet specific deletion model. In both α1AMPK WT and KO mice, hemostasis was altered at 6 hours and 18 hours after LPS administration. Thrombin generation, reflected by thrombin-antithrombin complex formation, was significantly increased in α1AMPK KO mice compared with WT mice, after 18 hours of treatment by LPS. In contrast, fibrinolysis was more inhibited in α1AMPK KO than in WT mice, with a higher level of plasminogen activator inhibitor-1 measured at 6 and 18 hours, while tissue plasminogen activator was not different. This exacerbated procoagulant phenotype in α1AMPK KO mice was associated with an increase in sCD62P in plasma, showing an overactivation of platelets under septic conditions. Septic α1AMPK KO mice showed an increase of Neutrophil Elastase and plasmatic DNA with no variation of MPO-DNA complex suggesting a stronger cellular damage than WT mice with no increase of NETosis. Platelet and leucocyte specific models did not have the phenotype observed in the total deletion model after LPS administration. However, endothelial specific model showed an increase of thrombin generation with a tendency of more platelet activation. Altogether, these results demonstrate that α1AMPK plays an important role in the regulation of hemostasis disturbances during sepsis. Specific model showed that α1AMPK from platelet and leucocyte does not contribute directly to the septic damage but endothelial α1AMPK seems to be important to contain hemostasis dysregulation.

Cytokine storm induced coagulopathy in septic shock and critical Covid-19: head-to-head comparison

BackgroundSeptic shock generates an important inflammatory reaction, endothelial activation and a procoagulant state leading to microvascular thrombosis and subsequent organ impairment [1].Similarly, a severe inflammatory reaction and a coagulopathy with pulmonary micro-thrombosis eventually leading to acute lung injury, is a typical feature of critical form of Coronavirus disease 2019 (Covid-19) [2].Our aim was to compare coagulation, platelet activation and platelets-neutrophils interplay between control, septic shock and critical Covid-19 patients.Methods/MaterialsA total of 118 patients were included in our prospective, monocentric, observational study between February 2019 and June 2020. Septic shock (n=48) and Covid-19 (n=22) patients were consecutively included at admission in our ICU department. Control patients (n=48) with matched gender and co-morbidities were recruited at central lab consultation.ResultsSeptic shock patients had worse severity scores due to multiple organ failure (assessed by APACHE II and SOFA score) whereas Covid-19 patients had more severe respiratory failure and a longer ICU length-of-stay (Table 1).At the time of inclusion, CRP and lymphocyte count were comparable between septic shock and Covid-19 patients. White cell count ad neutrophil count was higher for septic shock patients.Analysis of coagulation showed a prolonged INR, TT and aPTT in septic shock although only INR was prolonged in Covid-19. Thrombin antithrombin complex (TATc) formation was similar in both pathologies, whereas consumption of antithrombin III (ATIII) and D-dimers formation was more pronounced in septic shock.Platelet count was lower in septic shock and platelet activation, assessed via plasmatic levels of soluble P-selectin (sCD62P) and Trem-like transcript 1 (sTLT-1), was more important in septic shock.Neutrophil activation and NETosis, evaluated by levels of circulating myeloperoxidase (MPO) and citrullinated histone 3 (H3-Cit), was similarly increased in both groups (Figure 1).ConclusionsThis study confirmed an activation of coagulation cascade, platelet activation and NETosis in both septic shock and critical Covid-19, compared with control patients. Importantly, the extent of these changes was similar or less pronounced in critical COVID-19 compared with septic shock.Funding AcknowledgementType of funding sources: Foundation. Main funding source(s): Fondation Saint Luc Figure 1

The main pathogenetic mechanisms of hypercoagulation in diabetes and the possibility of its drug correction

Disorders in the blood coagulation system play an important role in the development of cardiovascular pathology in diabetes. Factors that cause them are hyperglycemia, insulin deficiency, insulin resistance, dyslipidemia, oxidative stress. The most significant changes are observed in the vascular-platelet link of hemostasis. Diabetes is characterized by morphological and functional changes in the endothelium of blood vessels. The activity of platelets increases, which is manifested by their high level of spontaneous aggregation and increased sensitivity to the action of activating factors. The role in the disturbance of hemostasis is played by increasing the activity of the von Willebrand factor, reflecting damage to endothelial cells. Diabetes is characterized by an increase in the activity of plasma clotting factors (I, II, III, VII, VIII, IX, XI, XII and XIII), activation of the callicrein-kinin system. In some cases, this correlates with the development of complications of diabetes. Characteristic disorders in the coagulation inhibition system are a decrease in the activity of antithrombin III, reduced formation of thrombin-antithrombin complexes, reduction of thrombomodulin and protein C. In diabetes, there is a decrease in fibrinolysis, due to a decrease in the expression of tissue activator plasminogen and an increase in the level of the inhibitor of the activator plasminogen. The possibilities of drug correction of hypercoagulation factors in diabetes are to achieve glycemic control with sugar-reducing drugs and elimination of dyslipidemia through hypolipidemic therapy. The most well-studied sugar-lowering drug that improves the state of the blood clotting system is metformin. The system of hemostasis in diabetic patients is positively affected by statins both due to the direct hypolipidemic effect, and by improving endothelial function and increasing fibrinolysis.

Pluronic Micelle-Mediated Tissue Factor Silencing Enhances Hemocompatibility, Stemness, Differentiation Potential, and Paracrine Signaling of Mesenchymal Stem Cells.

Mesenchymal stem/stromal cells (MSCs) evoke great excitement for treating different human diseases due to their ability to home inflamed tissues, suppress inflammation, and promote tissue regeneration. Despite great promises, clinical trial results are disappointing as allotransplantation of MSCs trigger thrombotic activity and are damaged by the complement system, compromising their survival and function. To overcome this, a new strategy is presented by the silencing of tissue factor (TF), a transmembrane protein that mediates procoagulant activity. Novel Pluronic-based micelles are designed with the pendant pyridyl disulfide group, which are used to conjugate TF-targeting siRNA by the thiol-exchange reaction. This nanocarrier design effectively delivered the payload to MSCs resulting in ∼72% TF knockdown (KD) without significant cytotoxicity. Hematological evaluation of MSCs and TF-KD MSCs in an ex vivo human whole blood model revealed a significant reduction in an instant-blood-mediated-inflammatory reaction as evidenced by reduced platelet aggregation (93% of free platelets in the TF-KD group, compared to 22% in untreated bone marrow-derived MSCs) and thrombin–antithrombin complex formation. Effective TF silencing induced higher MSC differentiation in osteogenic and adipogenic media and showed stronger paracrine suppression of proinflammatory cytokines in macrophages and higher stimulation in the presence of endotoxins. Thus, TF silencing can produce functional cells with higher fidelity, efficacy, and functions.

Role of Autophagy in Von Willebrand Factor Secretion by Endothelial Cells and in the In Vivo Thrombin-Antithrombin Complex Formation Promoted by the HIV-1 Matrix Protein p17.

Although the advent of combined antiretroviral therapy has substantially improved the survival of HIV-1-infected individuals, non-AIDS-related diseases are becoming increasingly prevalent in HIV-1-infected patients. Persistent abnormalities in coagulation appear to contribute to excess risk for a broad spectrum of non-AIDS defining complications. Alterations in coagulation biology in the context of HIV infection seem to be largely a consequence of a chronically inflammatory microenvironment leading to endothelial cell (EC) dysfunction. A possible direct role of HIV-1 proteins in sustaining EC dysfunction has been postulated but not yet investigated. The HIV-1 matrix protein p17 (p17) is secreted from HIV-1-infected cells and is known to sustain inflammatory processes by activating ECs. The aim of this study was to investigate the possibility that p17-driven stimulation of human ECs is associated with increased production of critical coagulation factors. Here we show the involvement of autophagy in the p17-induced accumulation and secretion of von Willebrand factor (vWF) by ECs. In vivo experiments confirmed the capability of p17 to exert a potent pro-coagulant activity soon after its intravenous administration.

Analysis of thrombin-antithrombin complex formation using microchip electrophoresis and mass spectrometry.

Preterm birth (PTB) related health problems take over one million lives each year, and currently, no clinical analysis is available to determine if a fetus is at risk for PTB. Here, we describe the preparation of a key PTB risk biomarker, thrombin-antithrombin (TAT), and characterize it using dot blots, MS, and microchip electrophoresis (µCE). The pH for fluorescently labeling TAT was also optimized using spectrofluorometry and spectrophotometry. The LOD of TAT was measured in µCE. Lastly, TAT was combined with six other PTB risk biomarkers and separated in µCE. The ability to make and characterize TAT is an important step toward the development of an integrated microfluidic diagnostic for PTB risk.

FXa-α2-Macroglobulin Complex Neutralizes Direct Oral Anticoagulants Targeting FXa In Vitro and In Vivo.

Increasing number of patients are treated with direct oral anticoagulants (DOAC). An antidote for dabigatran inhibiting thrombin (idarucizumab) is available but no antidote is yet approved for the factor Xa (FXa) inhibitors (xabans). We hypothesized that a complex between Gla-domainless FXa and α2-macroglobulin (GDFXa-α2M) may neutralize the xabans without interfering with normal blood coagulation.Purified α2M was incubated with GDFXa to form GDFXa-α2M. Affinity of apixaban and rivaroxaban for GDFXa-α2M was only slightly decreased compared to FXa. Efficacy and harmlessness of GDFXa-α2M were tested in vitro and in vivo. Stoichiometric excess of GDFXa-α2M neutralized rivaroxaban and apixaban as attested by clot waveform assay and rotational thromboelastometry, whereas GDFXa-α2M alone had no effect on these assays. Efficacy and pro-thrombotic potential of GDFXa-α2M were also assessed in vivo. Half-life of GDFXa-α2M in C57BL6 mice was 4.9 ± 1.1 minutes, but a 0.5 mg/mouse dose resulted in uptake saturation such that 50% persistence was still observed after 170 minutes. Single administration of GDFXa-α2M significantly decreased the rivaroxaban-induced bleeding time (p < 0.001) and blood loss (p < 0.01). GDFXa-α2M did not increase D-dimer or thrombin-antithrombin complex formation, suggesting a lack of pro-thrombotic potential.GDFXa-α2M is therefore an attractive candidate for xaban neutralization neither pro- nor anticoagulant in vitro as well as in vivo.

Myeloid but not epithelial tissue factor exerts protective anti‐inflammatory effects in acid aspiration‐induced acute lung injury

Essentials Tissue factor (TF) represents a central link between hemostasis and inflammation.We studied the roles of myeloid and airway epithelial TF in acid‐caused acute lung injury (ALI).TF on myeloid cells displays a non‐coagulatory role regulating the inflammatory response in ALI.Airway epithelial TF contributes to hemostatic functions, but is dispensable in ALI pathogenesis. SummaryIntroductionAcute lung injury (ALI) is a life‐threatening condition characterized by damaged alveolar–capillary structures and activation of inflammatory and hemostatic processes. Tissue factor (TF) represents a crucial link between inflammation and coagulation, as inflammatory mediators induce myeloid TF expression, and TF initiates extrinsic coagulation.ObjectiveAs pulmonary inflammation stimulates TF expression and TF modulates immune responses, we aimed to elucidate its impact on ALI. In particular, we wanted to distinguish the contributions of TF expressed on airway epithelial cells and TF expressed on myeloid cells.MethodsMice with different cell type‐specific TF deficiency and wild‐type littermates were intratracheally treated with hydrochloric acid, and leukocyte recruitment, cytokine levels, thrombin–antithrombin (TAT) complexes and pulmonary protein‐rich infiltrates were analyzed.ResultsOur data demonstrate that a lack of epithelial TF did not influence acute responses, as bronchoalveolar neutrophil accumulation 8 h after ALI induction was unaltered. However, it led to mild, prolonged inflammation, as pulmonary leukocyte and erythrocyte numbers were still increased after 24 h, whereas those in wild‐type mice had returned to basal levels. In contrast, myeloid TF was primarily involved in regulating the acute phase of ALI without affecting local coagulation, as indicated by increased bronchoalveolar neutrophil infiltration, pulmonary interleukin‐6 levels, and edema formation, but equal TAT complex formation, 8 h after ALI induction. This augmented inflammatory response associated with myeloid TF deficiency was confirmed in vitro, as lipopolysaccharide‐stimulated TF‐deficient alveolar macrophages released increased levels of chemokine (C‐X‐C motif) ligand 1 and tumor necrosis factor‐α as compared with wild‐type macrophages.ConclusionWe conclude that myeloid TF dampens inflammation in acid‐induced ALI.

Platelet-derived microparticles regulates thrombin generation via phophatidylserine in abdominal sepsis.

Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL-6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL-6, and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet-derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild-type recombinant annexin V abolished PMP-induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild-type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP-induced formation of thrombin-antithrombin complexes were reduced in platelet-depleted mice and in animals pretreated with annexin V. PMP-induced TG attenuated in FXII- and FVII-deficient plasma. These findings suggest that sepsis-induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure, and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis.

Preclinical Pharmacokinetics, Pharmacodynamics, Tissue Distribution, and Interspecies Scaling of Recombinant Human Coagulation Factor XaI16L

FXaI16L is a recombinant human FXa variant which is currently being evaluated in the clinic for treating intracerebral hemorrhage. The aim of our studies is to investigate overall pharmacokinetics, pharmacodynamics, and distribution of FXaI16L in preclinical species, and to understand its potential implication in human. Pharmacokinetics of FXaI16L was examined using active site probes and the results showed that FXaI16L displayed fast clearance, low volume of distribution, and a very short plasma resident time in mice, rats, and monkeys. When pharmacodynamics was examined in monkeys, concentration effects of FXaI16L on shortening of active partial prothrombin time and formation of thrombin-antithrombin complex were observed. Furthermore, biodistribution study was conducted in mice using radiolabeled FXaI16L, and showed that 125I-FXaI16L has high plasma protein binding and significant liver and kidney distribution. Human pharmacokinetic prediction for first-in-human dosing was evaluated using allometric scaling, liver blood flow, and a fixed coefficient method, and single species allometric scaling using monkey data was most predictive for human pharmacokinetics of FXaI16L.

Efficacy of A Collagen Hemostat Versus A Carrier-bound Fibrin Sealant

Aim: A fast activation time of hemostatic agents with blood must be sufficient to effectively stop bleeding within surgical procedures. However, there are no functional in-vitro tests of hemostatic agents which mimick such a clinical application. Method: The efficacy of two common hemostatic agents was examined with heparinised human whole blood (0.7 IU/ml) and a contact time of only three minutes between blood and hemostatic agents. Traditional biochemical assays were compared with a new rheometric method for measuring clot formation. Results: Blood without previous material contact (negative control) induced a basal thrombin-antithrombin (TAT, 240 ± 85 μg/l) or s-thromboglobulin (TG, 1000 ± 216 U/ml) complex formation. Stainless steel (positive control) or a thrombin coated equine collagen fleece failed to increase TAT or s-TG. However, a bovine collagen fleece significantly increased formation of TAT (1426 ± 378 μg/l) or s-TG (3829 ± 857 U/ml). In rheometric measurements of the negative control the clotting time (CT) was 17 ± 4 min and the clot strength (CS) was 71 ± 45Pa. In the positive control CT (stainless steel) was 9 ± 3 min and CS was 298 ± 68Pa. The equine collagen fleece caused no detectable stimulation of CT and CS whereas the bovine collagen fleece (CT 13 ± 3 min, CS 186 ± 86Pa) was almost as effective as stainless steel. Conclusion: Traditional biochemical parameters fail to indicate thrombogenicity under the tested conditions but oscillatory shear rheometry is a sensitive tool to analyse blood coagulation in vitro. Furthermore, mimicking the clinical relevant application times, the rheometric method detects functional differences of hemostatic agents. Since these differences correlate with in vivo data, the rheometric method is a valuable tool during the development of hemostatic agents.Aim: A fast activation time of hemostatic agents with blood must be sufficient to effectively stop bleeding within surgical procedures. However, there are no functional in-vitro tests of hemostatic agents which mimick such a clinical application. Method: The efficacy of two common hemostatic agents was examined with heparinised human whole blood (0.7 IU/ml) and a contact time of only three minutes between blood and hemostatic agents. Traditional biochemical assays were compared with a new rheometric method for measuring clot formation. Results: Blood without previous material contact (negative control) induced a basal thrombin-antithrombin (TAT, 240 ± 85 μg/l) or s-thromboglobulin (TG, 1000 ± 216 U/ml) complex formation. Stainless steel (positive control) or a thrombin coated equine collagen fleece failed to increase TAT or s-TG. However, a bovine collagen fleece significantly increased formation of TAT (1426 ± 378 μg/l) or s-TG (3829 ± 857 U/ml). In rheometric measurements of the negative control the clotting time (CT) was 17 ± 4 min and the clot strength (CS) was 71 ± 45Pa. In the positive control CT (stainless steel) was 9 ± 3 min and CS was 298 ± 68Pa. The equine collagen fleece caused no detectable stimulation of CT and CS whereas the bovine collagen fleece (CT 13 ± 3 min, CS 186 ± 86Pa) was almost as effective as stainless steel. Conclusion: Traditional biochemical parameters fail to indicate thrombogenicity under the tested conditions but oscillatory shear rheometry is a sensitive tool to analyse blood coagulation in vitro. Furthermore, mimicking the clinical relevant application times, the rheometric method detects functional differences of hemostatic agents. Since these differences correlate with in vivo data, the rheometric method is a valuable tool during the development of hemostatic agents.

Multifunctional Hyaluronic Acid and Chondroitin Sulfate Nanoparticles: Impact of Glycosaminoglycan Presentation on Receptor Mediated Cellular Uptake and Immune Activation.

Hyaluronic acid (HA) and chondroitin sulfate (CS) polymers are extensively used for various biomedical applications, such as for tissue engineering, drug delivery, and gene delivery. Although both these biopolymers are known to target cell surface CD44 receptors, their relative cellular targeting properties and immune activation potential have never been evaluated. In this article, we present the synthesis and characterization of novel self-assembled supramolecular HA and CS nanoparticles (NPs). These NPs were developed using fluorescein as a hydrophobic component that induced amphiphilicity in biopolymers and also efficiently stabilized anticancer drug doxorubicin (DOX) promoting a near zero-order drug release. The cellular uptake and cytotoxicity studies of these NPs in different human cancer lines, namely, human colorectal carcinoma cell line HCT116 and human breast cancer cell line MCF-7 demonstrated dose dependent cytotoxicity. Interestingly, both NPs showed CD44 dependent cellular uptake with the CS-DOX NP displaying higher dose-dependent cytotoxicity than the HA-DOX NP in different mammalian cells tested. Immunological evaluation of these nanocarriers in an ex vivo human whole blood model revealed that unlike unmodified polymers, the HA NP and CS NP surprisingly showed platelet aggregation and thrombin-antithrombin complex formation at high concentrations (0.8 mg/mL). We also observed a clear difference in early- and late-stage complement activation (C3a and sC5b-9) with CS and CS NP triggering significant complement activation at high concentrations (0.08-0.8 mg/mL), unlike HA and HA NP. These results offer new insight into designing glycosaminoglycan-based NPs and understanding their hematological responses and targeting ability.

Human α1-antitrypsin improves early post-transplant lung function: Pre-clinical studies in a pig lung transplant model

The translation of novel drugs in lung transplantation is challenged by different physiologic conditions between small animals and humans. Large-animal models provide important pre-clinical evidence and the next step that best informs clinical trials. In the present study, we used a pig lung transplant model to determine whether human α1-antitrypsin (A1AT), a medication shown to prevent pulmonary ischemia-reperfusion injury in rats, could attenuate reperfusion injury after prolonged hypothermic preservation in a large-animal lung transplant model. Donor lungs were preserved for 24 hours at 4°C, followed by lung transplantation. In a randomized and blinded fashion, intravenous A1AT (240 mg/kg; n = 5) or human albumin (n = 5) was administered to the recipient before reperfusion. Allograft gas exchange function and lung mechanics were monitored during a 4-hour reperfusion period. Microscopic lung injury, inflammatory response, coagulation activity, and cell death were assessed. Pulmonary gas exchange was significantly better during the 4-hour reperfusion period in the A1AT group. Treatment with A1AT improved static pulmonary compliance and significantly reduced pulmonary edema and lung permeability. A1AT treatment inhibited inflammatory mediators in the circulation, with reduced activation of nuclear factor-κB and inflammasome, reduced formation of thrombin-antithrombin complex in plasma, and reduced apoptosis in the allografts. Administration of human A1AT before reperfusion in recipients improved immediate post-transplant lung function in pigs. A large-animal survival model should be considered to support further advancement toward a clinical trial of A1AT to prevent primary graft dysfunction in lung transplantation.

Rebalanced Thrombin Generation in Liver Cirrhosis Calls for Tailored Transfusion Protocols

IntroductionMost coagulation factors are produced by the liver, and therefore in liver cirrhosis both pro- and anticoagulant factor levels are reduced. Cirrhosis patients show no clear systemic bleeding or thrombotic phenotype and thrombin generation (TG) is (almost) unaffected, therefore it has previously been proposed that coagulation is rebalanced in cirrhosis because both pro- and anticoagulant processes are affected to a similar extent. Nevertheless, clinical parameters such as the prothrombin time indicate a bleeding risk in cirrhosis patients, and based on these tests patients often receive blood product transfusion during surgery.In this study we investigated thrombin generation and its main underlying pro- and anticoagulant processes (prothrombin conversion and thrombin inactivation) to provide empirical evidence for the hypothesis of rebalanced thrombin generation in liver cirrhosis. In addition, we used in silico experimentation to study the consequences of rebalanced TG for current transfusion practices.Methods25 cirrhosis patients and 25 healthy subjects were enrolled in our study. The group of cirrhosis patients consisted of 22 Child-Pugh A and 3 Child-Pugh B patients. Prothrombin, antithrombin (AT) and α2Macroglobulin (α2M) levels were determined and TG was measured at 5 pM tissue factor by calibrated automated thrombinography. Prothrombin conversion and thrombin inactivation were quantified from each TG curve, and thrombin-antithrombin and thrombin-α2Macroglobulin complex formation was determined.The effect of transfusion of prothrombin complex concentrate (PCC) was simulated by an in silico increase of prothrombin conversion. The increase of prothrombin conversion was simulated by substituting the prothrombin conversion curve of a cirrhosis patient by the average healthy subject prothrombin conversion curve, to normalize prothrombin conversion in patients. Thrombin generation curves were calculated based on this normalized prothrombin conversion curve and the original thrombin inactivation parameters of each cirrhosis patient.ResultsProthrombin and antithrombin are significantly decreased in cirrhosis patients (74 % and 70 %, p<0.001), whereas the α2M level is significantly increased (p<0.001). Thrombin generation in healthy subjects and patients is similar (figure), although prothrombin conversion (65 %, p<0.001) and thrombin inactivation (73 % p<0.001) are markedly reduced in cirrhosis patients. Acquired AT deficiency results in a reduction of thrombin inactivation by AT (64 %, p<0.001), but the substitution of AT by α2M leads to increased thrombin inactivation by α2M (220 %, p<0.001).The effect of prothrombin complex concentrate transfusion on thrombin generation in cirrhosis patients was determined by computational modeling (figure). In silico normalization of prothrombin conversion, as would be achieved by PCC transfusion, causes TG to rise to levels that have been shown to be highly prothrombotic (ETP 2878 ± 1323 nM∙min, p<0.001).ConclusionsDespite large differences in prothrombin conversion and thrombin inactivation, TG in liver cirrhosis patients remains within the normal range (rebalanced), in contrast to the prothrombin time that predicts a bleeding risk in these patients. The transfusion of PCC based on a prolonged prothrombin time would result in a substantial elevation of thrombin generation. This indicates that standard transfusion protocols need to be tailored to the specific needs of cirrhosis patients. [Display omitted] DisclosuresKremers:Synapse bv: Employment. De Laat:Synapse bv: Employment. Wagenvoord:Synapse bv: Employment. Hemker:Synapse bv: Employment.

Antithrombin resistance: a new mechanism of inherited thrombophilia

Venous thromboembolism is a multifactorial disease resulting from complex interactions among genetic and environmental factors. To date, numerous genetic defects have been found in families with hereditary thrombophilia, but there may still be many undiscovered causative gene mutations. We investigated a possible causative gene defect in a large Japanese family with inherited thrombophilia, and found a novel missense mutation in the prothrombin gene (p.Arg596Leu) resulting in a variant prothrombin (prothrombin Yukuhashi). The mutant prothrombin had moderately lower activity than wild type prothrombin in clotting assays, but formation of the thrombin-antithrombin (TAT) complex was substantially impaired resulting in prolonged thrombin activity. A thrombin generation assay revealed that the peak activity of the mutant prothrombin was fairly low, but its inactivation was extremely slow in reconstituted plasma. The Leu596 substitution caused a gain-of-function mutation in the prothrombin gene, resulting in resistance to antithrombin and susceptibility to thrombosis. We also showed the effects of the prothrombin Yukuhashi mutation on the thrombomodulin-protein C anticoagulation system, recent development of a laboratory test detecting antithrombin resistance in plasma, and another antithrombin resistant mutation found in other thrombophilia families.

A Novel &lt;em&gt;In vitro&lt;/em&gt; Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel. We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNFα) and found the presence of CD16+ cells to be significantly higher on TNFα stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A novel in vitro model for studying the interactions between human whole blood and endothelium.

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNFα) and found the presence of CD16+ cells to be significantly higher on TNFα stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.