A novel quantitative method to evaluate the contribution of platelet products to white thrombus formation in reconstituted blood under flow conditions.

BACKGROUND AND OBJECTIVES: Currently, the quality of platelet (PLT) products is evaluated using a series of in vitro tests, which only analyse PLTs as an inspection material. However, it would be ideal to assess the physiological functions of PLTs under conditions similar to the sequential blood haemostatic process. In this study, we attempted to establish an in vitro system where the thrombogenicity of PLT products was evaluated in the presence of red blood cells (RBCs) and plasma using a microchamber under constant shear stress (600/s). MATERIALS AND METHODS: Blood samples were reconstituted by mixing PLT products, standard human plasma (SHP) and standard RBCs. Each component was serially diluted keeping the other two components fixed. The samples were applied onto a flow chamber system (Total Thrombus-formation Analysis System [T-TAS]), and white thrombus formation (WTF) was assessed under large arterial shear conditions. RESULTS: We observed a good correlation between the PLT numbers in the test samples and WTF. The WTF of samples containing ≦10% SHP was significantly lower than those containing ≧40% SHP, and no difference was observed in WTF among samples containing 40%-100% SHP. WTF significantly declined in the absence of RBCs, whereas no change in WTF was observed in the presence of RBCs, over haematocrit range of 12.5%-50%. CONCLUSION: The WTF assessed on the T-TAS using reconstituted blood may serve as a new physiological blood thrombus test to quantitatively determine the quality of PLT products.

Cardiovascular risk factors are associated with augmented thrombogenicity in healthy individuals: analysis using the Total Thrombus-formation Analysis System.

BACKGROUND: Rupture of an atherosclerotic plaque and subsequent exposure of the subendothelial prothrombotic matrix to blood cause arterial thrombosis. Circulating platelets play an indispensable role in the growth of arterial thrombi partially owing to their unique ability to adhere to the subendothelial matrix and to aggregate to each other under flow conditions. Recently, the Total Thrombus-formation Analysis System (T-TAS) was developed for ex vivo analysis of the thrombogenic potential of whole blood samples under flow conditions. Despite the potential clinical utility of the T-TAS in assessing the risk for thrombosis and bleeding, reference intervals for T-TAS analysis in healthy individuals have not been determined. METHODS: In total, 122 whole blood samples were collected from healthy volunteers ranging in age from 25 to 45 years. T-TAS analysis and hematological, physiological, and lifestyle assessments were conducted in these subjects. Whole blood samples anticoagulated with hirudin were perfused into a collagen-coated microchip (PL chip). The time to 10 kPa and the area under the flow pressure curve up to 10 min (AUC10) were analyzed as representative variables for thrombogenic potential. Reference intervals, which were defined as 2.5-97.5 percentiles, were determined. Additionally, univariate and multivariate analyses were performed to identify factors associated with the AUC10 in the T-TAS. RESULTS: The time to 10 kPa and the AUC10 widely varied, even in healthy volunteers. The reference intervals were 1.50-4.02 min and 223.4-456.8, respectively, at a shear rate of 1500 s- 1. Univariate and multivariate analyses showed that platelet counts were most significantly associated with the AUC10 of the T-TAS. The presence of one or more cardiovascular risk factors of a high body mass index, a high pulse pressure, high fasting serum glucose levels, high low-density lipoprotein-cholesterol levels, a history of smoking, and no habitual exercise, had the second largest effect on the AUC10 of the T-TAS. CONCLUSIONS: Healthy volunteers who had any cardiovascular risk factors showed augmented thrombogenicity, even in artificial uniform capillaries, compared with those without any risk factors in the T-TAS.

Analysis of blood clotting with the total thrombus analysis system in healthy dogs.

To date, coagulation tests are unable to reflect in vivo coagulation status in the same system, including platelet function, fibrin clot formation, and whole blood flow. The Total Thrombus Analysis System (T-TAS), which is a microfluidic assay that simulates conditions in vivo, measures whole blood flow at defined shear rates under conditions designed to assess platelet function (PL-chip) or coagulation and fibrin clot formation (AR-chip). The T-TAS records occlusion start time, occlusion time, and area under the curve. We evaluated this test in healthy control dogs. We also investigated the effect in vivo of acetylsalicylic acid (ASA), and the effect in vitro of an anticoagulation drug (dalteparin; low-molecular-weight heparin; LMWH). The CV of the AUC of both chips was good (CVs of 6.45% [PL] and 1.57% [AR]). The inhibition of platelet function by ASA was evident in the right-shift in the PL test pressure curve. The right-shift in the AR test pressure curves showed that the administration of LMWH inhibited both platelets and the coagulation cascade. The T-TAS may be useful in the evaluation of canine blood coagulation.

A modified microchip-based flow chamber system for evaluating thrombogenicity in patients with thrombocytopenia.

BACKGROUND: In the intensive care unit (ICU), patients with thrombocytopenia are at high risk for bleeding and should be assessed for their thrombogenic potential. However, the analytical conditions of conventional hemostatic tests are unsuitable for the evaluation of low-platelet samples. Here we aimed to establish suitable analytical conditions with the Total Thrombus-formation Analysis System (T-TAS) for quantitative assessment of thrombogenic potential in patients with thrombocytopenia and to investigate how T-TAS values relate to bleeding symptoms and the effects of platelet transfusion. METHODS: Modified chips with a different chamber depth were developed for the analysis of low-platelet samples in the T-TAS. We included 10 adult patients admitted to the ICU of Kagoshima University Hospital who required platelet transfusion. Patients were divided into major and minor bleeding groups according to their bleeding scale before platelet transfusion. The thrombogenic potential of these patients before and after platelet transfusion was assessed with hemostatic function tests, including rotational thromboelastometry, multiplate aggregometry, and the T-TAS. RESULTS: Analysis of low-platelet samples revealed that, compared with the conventional chip (80-µm-deep chamber), the modified chip (50-µm-deep chamber) achieved higher sensitivity in detecting elevation of flow pressure caused by growth of an occlusive thrombus in the T-TAS analytical chamber. All patients in the minor bleeding group retained thrombogenic potential that occluded the modified chip (occlusion time 16.3 ± 3.3 min), whereas most patients in the major bleeding group were unable to occlude the modified chip during the 30-min measurement (P <  0.01). The recovery of thrombogenic potential after platelet transfusion was confirmed with the T-TAS and correlated with the function, rather than the count, of transfused platelets. Among all evaluated parameters in hemostatic function tests, only the T-TAS showed significant differences in occlusion time and area under the curve both between the minor and major bleeding groups and between pre- and post-platelet transfusion. CONCLUSIONS: We developed a modified microchip-based flow chamber system that reflects the hemostatic function of patients with thrombocytopenia.

Association Between HMGB1 and Thrombogenesis in a Hyperlipaemia-induced Microminipig Model of Atherosclerosis.

BACKGROUND/AIM: An appropriate animal model is essential to investigate the relationship between inflammation, atherosclerosis, and thrombogenesis, and the development of preventive measures and therapies for atherosclerosis. MATERIALS AND METHODS: Atherosclerosis was induced in Microminipigs (MMPs) using a high-fat diet. We assessed high mobility group box 1 (HMGB1) expression levels and measured thrombus formation using a Total Thrombus Formation Analysis System (T-TAS). MMPs were divided into a normal diet (control) group and four high-fat diet groups, with differing amounts of cholesterol. After 8 weeks, blood was collected for analysis. RESULTS: HMGB1 levels increased with increasing dietary cholesterol, and a negative correlation was found between HMGB1 levels and thrombus formation time. CONCLUSION: T-TAS is useful in the assessment of thrombogenesis in MMPs and HMGB1 is associated with thrombus formation.

New methodological approaches for assessing thrombus formation in cardiovascular disease.

Antiplatelet therapy is the mainstay preventive strategy for cardiovascular diseases, and dual antiplatelet therapy comprising aspirin and a P2Y12 inhibitor is the standard treatment for patients who underwent percutaneous coronary intervention. The Total Thrombus­Formation Analysis System (T­TAS) is a microchip flow­chamber system developed to evaluate overall thrombus formation under flow conditions, which is reportedly able to assess single and combined antithrombotic therapy. Here, we focus on this new system, T­TAS, and review its characteristics together with those of the conventional systems available for evaluation of antithrombotic therapies for cardiovascular diseases.

Recombinant Thrombomodulin Suppresses Histone-Induced Neutrophil Extracellular Trap Formation.

Histones, the major protein components of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and subsequently mediate organ failure. Extracellular histones can promote endothelial damage and platelet aggregation, which can be suppressed by administration of recombinant thrombomodulin (rTM). The present study aimed to clarify whether histones can activate neutrophils to induce NET formation and whether rTM can prevent histone-induced NET formation. NET formation was analyzed in vitro by stimulating human neutrophils with histones in the absence or presence of rTM. NET formation was further analyzed in vivo by intravenous infusion of histones into rats with or without rTM. Histones induced NET release in a dose-dependent manner in vitro and NET release was induced as early as 1 h after stimulation. Histone-induced NET release was independent of NADPH oxidase. rTM suppressed histone-induced NET release in vitro as well as in vivo. The suppression might be mediated by rTM binding to histones, as suggested by analysis using a quartz crystal microbalance system. The present findings suggest that histones can activate neutrophils to form NETs and that rTM can inhibit histone-induced NET formation.

Effects of glycemic control and hypoglycemia on Thrombus formation assessed using automated microchip flow chamber system: an exploratory observational study.

BACKGROUND: Thrombus formation is an important factor affecting cardiovascular events and venous thromboembolism in type 2 diabetes. However, it is unclear whether glycemic control reduces thrombogenicity. We investigated the effect of short-term glycemic control (STUDY 1) and hypoglycemia (STUDY 2) on thrombus formation using an automated microchip flow chamber system. METHODS: For STUDY 1, we recruited 10 patients with type 2 diabetes. Before and after 2 weeks of treatment, blood glucose was analyzed with a continuous glucose monitoring system, and thrombogenicity was analyzed with an automated microchip flow chamber system. For STUDY 2, we recruited 10 subjects without diabetes who underwent an insulin tolerance test. We evaluated the change in thrombogenic potential with hypoglycemia. RESULTS: STUDY1: The mean blood glucose level reduced from 10.1 ± 2.6 to 6.9 ± 0.97 mM (P < 0.01). T10, an indicator of thrombogenicity, significantly attenuated after glycemic control (338 ± 65 vs. 425 ± 117 s, P < 0.05). The attenuation in T10 was significantly correlated with changes in mean blood glucose level after treatment (r = - 0.718, P < 0.05). STUDY 2: Platelet function was enhanced with decreasing blood glucose; increased platelet function was strongly correlated with an increase in epinephrine. CONCLUSIONS: We demonstrated attenuation in thrombogenicity with short-term comprehensive diabetes care and enhancement in thrombogenicity with hypoglycemia, using a new flow chamber system. TRIAL REGISTRATION: UMIN-CTR UMIN 000019899, registered 26-Jan-2015 (STUDY 2).

Comparison between blood coagulability in the intra-atrial and peripheral regions during the acute phase after rapid atrial pacing.

The changes in intra-atrial blood coagulability of acute phase after development of atrial fibrillation (AF) have not been elucidated in human. In the present study, blood coagulability were examined in the intra-atrial and peripheral regions during the acute phase after development of rapid atrial pacing (RAP) in experimentally created model dog similar to AF, using Total Thrombus-formation Analysis System (T-TAS) that is capable of comprehensively evaluating thrombogenicity in the bloodstream in the microvascular channel. According to the results, both the coagulating function-evaluating time to +10 kPa (T10) and occlusion time (OT) of the AR chip (chip for thrombus analysis mixed with coagulation and platelet) were significantly shortened in the atrial blood as early as 30 min after pacing (T10, 150.5 ± 40.5 s; OT, 212.4 ± 44.3 s) compared to the pre-pacing levels (T10, 194.5 ± 47.5 s; OT, 259.9 ± 49.5 s) (P<0.05). The OT of PL chip (chip for platelet thrombus analysis) was significantly shortened 30 min after pacing (231.8 ± 57.6 s), compared to the pre-pacing level (289.5 ± 96.0 s) (P<0.05). Meanwhile, none of T10 and OT of AR and PL chips showed any significant changes in the peripheral blood. The study demonstrated increase of blood coagulability 30 min after development of RAP. While no significant changes were observed in the peripheral blood in the present study, the outcome suggested that the anti-thrombus treatments are better to be started early after AF even if coagulability of the peripheral blood shows no change.

Uric acid enhances alteplase-mediated thrombolysis as an antioxidant.

Uric acid (UA) therapy may prevent early ischemic worsening after acute stroke in thrombolysis patients. The aim of this study was to examine the influence of UA on the thrombolytic efficacy of alteplase in human blood samples by measuring thrombolysis under flow conditions using a newly developed microchip-based flow-chamber assay. Human blood samples from healthy volunteers were exposed to UA, alteplase, or a combination of UA and alteplase. Whole blood and platelet-rich plasma were perfused over a collagen- and thromboplastin-coated microchip, and capillary occlusion was monitored with a video microscope and flow-pressure sensor. The area under the curve (extent of thrombogenesis or thrombolysis) at 30 minutes was 92% lower in the UA-alteplase-treated group compared with the alteplase-treated group. D-dimers were measured to evaluate these effects in human platelet-poor plasma samples. Although hydrogen peroxide significantly decreased the elevation of D-dimers by alteplase, UA significantly inhibited the effect of hydrogen peroxide. Meanwhile, rat models of thromboembolic cerebral ischemia were treated with either alteplase or UA-alteplase combination therapy. Compared with alteplase alone, the combination therapy reduced the infarct volume and inhibited haemorrhagic transformation. UA enhances alteplase-mediated thrombolysis, potentially by preventing oxidative stress, which inhibits fibrinolysis by alteplase in thrombi.

Comparison of chronological changes in blood characteristics in the atrium and peripheral vessels after the development of non-valvular atrial fibrillation.

INTRODUCTION: Changes in blood characteristics in the atrium and peripheral vessels in patients with non-valvular atrial fibrillation (NVAF) are unclear. We investigated chronological changes in blood characteristics in the atrium and peripheral vessels in a dog model of NVAF by using a total thrombus-formation analysis system (T-TAS). MATERIALS AND METHODS: In NVAF model dogs (n = 8, 390 bpm rapid atrial pacing), atrial and peripheral blood samples were collected. Using this blood, T-TAS was performed before and 1, 2, and 3 weeks after the onset of rapid atrial pacing. RESULTS: Occlusion time (OT: time to +80 and +60 kPa in the AR and PL chips, respectively) and area under the flow pressure curve (AUC) were measured using the AR chip (for mixed white thrombus analysis) and PL chip (for platelet thrombus analysis). OT of the AR chip showed shortening as early as 1 week after NVAF onset, which continued for 3 weeks. OT of the PL chip showed significant shortening in atrium blood only 3 weeks after NVAF onset. By contrast, peripheral blood showed no significant changes in OT or AUC with both AR and PL chips. CONCLUSIONS: In our dog model of NVAF, thrombus formation accelerated in the atrium as early as 1 week after NVAF onset and continued for 3 weeks, but no significant changes were found in peripheral blood. We conclude that antithrombotic therapy should be started early after NVAF onset even if no changes in coagulation activity are observed in peripheral blood.

Edaravone, a Synthetic Free Radical Scavenger, Enhances Alteplase-Mediated Thrombolysis.

The combination of alteplase, a recombinant tissue plasminogen activator, and edaravone, an antioxidant, reportedly enhances recanalization after acute ischemic stroke. We examined the influence of edaravone on the thrombolytic efficacy of alteplase by measuring thrombolysis using a newly developed microchip-based flow-chamber assay. Rat models of embolic cerebral ischemia were treated with either alteplase or alteplase-edaravone combination therapy. The combination therapy significantly reduced the infarct volume and improved neurological deficits. Human blood samples from healthy volunteers were exposed to edaravone, alteplase, or a combination of alteplase and edaravone or hydrogen peroxide. Whole blood was perfused over a collagen- and thromboplastin-coated microchip; capillary occlusion was monitored with a video microscope and flow-pressure sensor. The area under the curve (extent of thrombogenesis or thrombolysis) at 30 minutes was 69.9% lower in the edaravone-alteplase- than alteplase-treated group. The thrombolytic effect of alteplase was significantly attenuated in the presence of hydrogen peroxide, suggesting that oxidative stress might hinder thrombolysis. D-dimers were measured to evaluate these effects in human platelet-poor plasma samples. Although hydrogen peroxide significantly decreased the elevation of D-dimers by alteplase, edaravone significantly inhibited the decrease. Edaravone enhances alteplase-mediated thrombolysis, likely by preventing oxidative stress, which inhibits fibrinolysis by alteplase in thrombi.

HMGB1 Promotes Intraoral Palatal Wound Healing through RAGE-Dependent Mechanisms.

High mobility group box 1 (HMGB1) is tightly connected to the process of tissue organization upon tissue injury. Here we show that HMGB1 controls epithelium and connective tissue regeneration both in vivo and in vitro during palatal wound healing. Heterozygous HMGB1 (Hmgb1+/-) mice and Wild-type (WT) mice were subjected to palatal injury. Maxillary tissues were stained with Mallory Azan or immunostained with anti-HMGB1, anti-proliferating cell nuclear antigen (PCNA), anti-nuclear factor-κB (NF-κB) p50 and anti-vascular endothelial growth factor (VEGF) antibodies. Palatal gingival explants were cultured with recombinant HMGB1 (rHMGB1) co-treated with siRNA targeting receptor for advanced glycation end products (RAGEs) for cell migration and PCNA expression analysis. Measurement of the wound area showed differences between Hmgb1+/- and WT mice on Day 3 after wounding. Mallory Azan staining showed densely packed of collagen fibers in WT mice, whereas in Hmgb1+/- mice weave-like pattern of low density collagen bundles were present. At three and seven days post-surgery, PCNA, NF-κB p50 and VEGF positive keratinocytes of WT mice were greater than that of Hmgb1+/- mice. Knockdown of RAGE prevents the effect of rHMGB1-induced cell migration and PCNA expression in gingival cell cultures. The data suggest that HMGB1/RAGE axis has crucial roles in palatal wound healing.

Plasminogen activator inhibitor type 1 in platelets induces thrombogenicity by increasing thrombolysis resistance under shear stress in an in-vitro flow chamber model.

INTRODUCTION: Despite the proven benefits of thrombolytic therapy with tissue plasminogen activator (t-PA) for peripheral thromboembolism, perfusion failure frequently occurs, particularly in arterial circulation. We evaluated how the modification of fibrinolytic activity affects thrombus formation under flow and static conditions. MATERIALS AND METHODS: t-PA-treated human whole-blood samples (n=6) were perfused over a microchip coated with collagen and tissue thromboplastin at different shear rates, and thrombus formation was quantified by measuring flow pressure changes. For comparison, rotational thromboelastometry (ROTEM) was used to evaluate fibrinolytic activity under static conditions. RESULTS: At a shear rate of 240s-1, t-PA (200-800IU/ml) concentration-dependently delayed capillary occlusion, whereas at 600s-1, capillary occlusion was significantly faster and t-PA had limited effects, even at a supra-pharmacological concentration (800IU/ml). In contrast, 200IU/ml t-PA efficiently prevented clot formation in the ROTEM assay. The combined treatment of blood with a specific PAI-1 inhibitor (PAI-039) moderately enhanced the efficacy of t-PA, but only under flow conditions. In addition, 1:1-diluted blood samples of PAI-1-deficient (-/-) mice showed a significant delay of capillary occlusion at 240s-1, compared with those from wild-type mice (1.55 fold; P<0.001). This delayed occlusion was reproduced in samples containing platelets from PAI-1-/- and plasma from wild type, but was not observed by the opposite combination of blood components. CONCLUSIONS: The present results suggest that the anti-thrombotic efficacy of t-PA is sensitive to arterial shear flow, and that PAI-1 secreted from activated platelets plays an essential role in thrombolytic resistance.

Recombinant thrombomodulin protects mice against histone-induced lethal thromboembolism.

INTRODUCTION: Recent studies have shown that histones, the chief protein component of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and act as major mediators of the death of an organism. This study was designed to elucidate the cellular and molecular basis of histone-induced lethality and to assess the protective effects of recombinant thrombomodulin (rTM). rTM has been approved for the treatment of disseminated intravascular coagulation (DIC) in Japan, and is currently undergoing a phase III clinical trial in the United States. METHODS: Histone H3 levels in plasma of healthy volunteers and patients with sepsis and DIC were measured using enzyme-linked immunosorbent assay. Male C57BL/6 mice were injected intravenously with purified histones, and pathological examinations were performed. The protective effects of rTM against histone toxicity were analyzed both in vitro and in mice. RESULTS: Histone H3 was not detectable in plasma of healthy volunteers, but significant levels were observed in patients with sepsis and DIC. These levels were higher in non-survivors than in survivors. Extracellular histones triggered platelet aggregation, leading to thrombotic occlusion of pulmonary capillaries and subsequent right-sided heart failure in mice. These mice displayed symptoms of DIC, including thrombocytopenia, prolonged prothrombin time, decreased fibrinogen, fibrin deposition in capillaries, and bleeding. Platelet depletion protected mice from histone-induced death in the first 30 minutes, suggesting that vessel occlusion by platelet-rich thrombi might be responsible for death during the early phase. Furthermore, rTM bound to extracellular histones, suppressed histone-induced platelet aggregation, thrombotic occlusion of pulmonary capillaries, and dilatation of the right ventricle, and rescued mice from lethal thromboembolism. CONCLUSIONS: Extracellular histones cause massive thromboembolism associated with consumptive coagulopathy, which is diagnostically indistinguishable from DIC. rTM binds to histones and neutralizes the prothrombotic action of histones. This may contribute to the effectiveness of rTM against DIC.

Coagulation activity and white thrombus formation in the microminipig.

Swine are becoming increasingly attractive as animal models for clinical research and the recently developed Microminipig (MMPig) has emerged as a possible experimental animal model. In this study, we demonstrated age-dependent changes in hematological parameters and coagulation activity in healthy MMPigs (58 male and 67 females, aged 0-34 months), and investigated white thrombus formation (WTF) using an in vitro microchip flow-chamber system (four males and four females, aged 22-23 months). There was no clear sex or age-dependent differences in any hematological parameters. While activated partial thromboplastin time (APTT) was shorter than prothrombin time (PT), with APTT:PT of 0.88:1, microchip flow-chamber system analysis showed that WTF time was shorter than that in humans, suggesting a possible thrombotic tendency in the MMPig. These results could be useful to life science researchers in the use of the MMPig as an experimental model animal for thrombus formation.

1,5-Anhydro-D-fructose: A natural antibiotic that inhibits the growth of gram-positive bacteria and microbial biofilm formation to prevent nosocomial infection.

Nosocomial infections caused by microbial opportunistic infections or microbial biofilms may occur during hospitalization and increase patient morbidity, mortality and health care costs. Artificial antibiotic agents were initially used to prevent infection; however, the high prevalence of nosocomial infections has resulted in their excessive use, which has led to microbial resistance to these agents. The increase in microbial resistance to antibiotics and the development of antibiotic agents may be the cause of the production of other microbial resistance. Thus, natural compounds that have no adverse side effects would be a preferred treatment modality. Recently, the monosaccharide 1,5-anhydro-D-fructose (1,5-AF), a natural plant compound derived from starch, has been found to have multifunctional properties, including antioxidant, antiplatelet aggregation by thrombin and anti-inflammatory activities. The results of the present study demonstrate that 1,5-AF suppressed the growth of coagulase-negative staphylococci on the hands as well as the growth of Staphylococcus epidermidis, which is a cause of opportunistic infections. Furthermore, 1,5-AF suppressed biofilm formation by the methicillin-resistant Staphylococcus aureus. In conclusion, 1,5-AF is a natural compound that may be effective in preventing nosocomial infections, without causing adverse side effects.