Simultaneous thrombin and plasmin generation capacities in normal and abnormal states of coagulation and fibrinolysis in children and adults

Introduction

Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis, respectively. Plasma coagulative and fibrinolytic potentials in normal children and adults, and in representative pathologically altered hemostatic states, were evaluated via simultaneous assessment of thrombin and plasmin generation.

Materials and Methods

An assay of Simultaneous Thrombin and Plasmin generation (STP) was developed to measure thrombin and plasmin in plasma using individual fluorometric substrates. Coagulation is initiated with dilute tissue factor, phospholipid, and calcium in platelet-poor plasma; fibrinolysis is accelerated via tissue plasminogen activator (tPA). Abnormal states of hemostasis were investigated.

Results

STP assay reproducibility and normal adult and pediatric values for measured and calculated parameters have been established. Onset of both thrombin and plasmin generation was significantly delayed in children relative to adults (p < 0.001) and the maximum amplitudes of thrombin and plasmin generation were less in children than adults (p < 0.01). No significant differences were measured among pediatric age groups. The most profound impairments in thrombin generation were observed for extrinsic and common pathway factor deficiencies, with the exception of afibrinogenemia. Plasmin generation was severely impaired in deficiencies of fibrinogen and plasminogen as well as with decreased tPA reagent concentration and addition of aminocaproic acid. Plasmin generation was greatly enhanced by alpha-2-antiplasmin deficiency and excess tPA reagent.

Conclusion

Simultaneous assessment of thrombin and plasmin generation in plasma shows promise for affording an enhanced understanding of overall coagulative and fibrinolytic functions in physiological and pathologically altered states of hemostasis in children and adults.     

Coagulation parameters in patients receiving dabigatran etexilate or rivaroxaban: two observational studies in patients undergoing total hip or total knee replacement

Introduction

Dabigatran and rivaroxaban have recently been added to the armamentarium for thromboprophylaxis in orthopedic surgery. Although this is their first licensed indication, others will soon follow. Owing to their claimed predictable anticoagulant response that dispenses with the need for monitoring coagulation, their effects are poorly described in routine cases. However, interpreting blood coagulation results and evaluating whether a treatment is properly targeted in the case of untoward incidents will become a common concern for clinicians.

Methods

Eighty patients undergoing total hip or knee replacement were included in two studies. Forty of them received dabigatran (study 1) and 40 rivaroxaban (study 2). Blood samples (n = 176 and 166) were taken preoperatively and twice a week from the first postoperative day.

Results

Dabigatran increased aPTTr about two-fold and PT about 1.2-fold, and it was mostly an initiation-phase modulator of thrombin generation. Mean circulating concentrations as measured by a diluted thrombin time were 105±85 ng/mL at T_max in samples from patients receiving the full dosing. They depended significantly on renal function, body weight and gender.Rivaroxaban increased aPTTr and PTr around 1.5 fold and modified the initiation and amplification phases of thrombin generation, with a lowered and prolonged thrombin burst. Mean circulating concentrations as measured by an antiXa test were 117 ± 78 ng/mL at T_max.With both drugs, routine coagulation tests, thrombin generation curves and functionally determined concentrations exhibited high interindividual variability.

Conclusion

Routine coagulation tests are altered in patients receiving dabigatran or rivaroxaban, but their alterations poorly reflect the circulating concentrations as determined by functional approaches.     

1,10-Phenanthroline stabilizes mRNA of the carcinogen-metabolizing enzyme,cytochrome P450 1a1

1,10-phenanthroline (phen), flufenamic acid, and indomethacin are inhibitors of aldo-keto reductases 1C1 (AKR1C1), but only phen decreased the benzo[a]pyrene (BaP)-induced cytochrome P450 1a1 (Cyp1a1) protein level. Therefore the decrease in the BaP-induced Cyp1a1 protein level was not due to inhibition of Akr1c1, but to phen itself. Phen decreased the BaP-induced Cyp1a1 promoter activity and protein expression, and in contrast, it increased Cyp1a1 mRNA, resulting from an increase in mRNA stability. Phen is also known as a transition metal ion-chelator. Along with the phen study, we also found that Zn²⁺, Fe²⁺ and Cu²⁺ increased Cyp1a1 mRNA and protein stability. Our results show that phen stabilized the mRNA of Cyp1a1, although it decreased cell viability. In addition, Zn²⁺ and Fe²⁺ highly neutralized phen's suppression of Cyp1a1 protein expression, but they only slightly neutralized phen's promotion of mRNA stability and suppression of cell viability, and had no effect on phen's suppression of promoter activity. Phen's effect on Cyp1a1 expression was reversible, which indicates that phen is non-covalently linked to its target. This report elucidates a new role for phen of stabilizing Cyp1a1 mRNA, and provides information for further studies on mRNA stabilization.     

Subchronic toxicity study of ulvan from Ulva pertusa (Chlorophyta) in Wistar rats

Ulvan extracted from Ulva pertusa (Chlorophyta) is a group of sulfated heteropolysaccharide, for simplicity, the sulfated polysaccharide is referred to as ulvan in this paper. To our knowledge, there is no detailed report investigating the toxicity of ulvan. In this study, the subchronic (6 months) toxicity of varying levels of ulvan extracted from U. pertusa was investigated in Wistar rats after oral administration. ALT, ALB, ALP, WBC, PLT, and liver relative organ weigh of female rats showed significantly difference at 3000 mg/kg body weight per day, compared with control group. On the other hand, TG, T-CHO concentrations of female rats (6 months) were significantly decreased at 600, 1200 and 3000 mg/kg body weight per day. This result proved that ulvan had antihyperlipidemic activity. Beside, ulvan showed anticoagulant activity in this study. Overall, our findings indicated that ulvan had affected specific hematology, serum biochemistry parameters and liver, and had great differences between males and females rats.     

Immunological effects of transglutaminase-treated gluten in coeliac disease

Coeliac disease pathogenesis is characterized by an immune response triggered, in genetically predisposed subjects, by ingested gluten and its withdrawal from the diet is the only available therapy. However, enzymatic modification of gluten through the insertion of lysine to avoid antigen presentation could represent a new therapeutical approach for patients. Sixty-six duodenal biopsies from 17 coeliac patients were cultured for 48h with gluten or enzymatically-modified gluten (treated with human recombinant transglutaminase type 2 or bacterial transglutaminase, with or without lysine). Interferonγ, anti endomisium and anti transglutaminase IgA antibodies, lactate dehydrogenase and transglutaminase activity were measured in the culture medium. Transglutaminase type 2 expression was evaluated on biopsies by immunohistochemistry. Gluten and transglutaminase-treated gluten increased by 13-15 fold interferon γ release, as well as antibodies, transglutaminase activity, and the immunohistochemical expression of transglutaminase type 2. Addition of lysine to the enzymatic modification of gluten normalized interferon γ, antibodies, transglutaminase activity and immunohistochemical expression of transglutaminase type 2. Lactate dehydrogenase did not differ among the studied groups. Enzymatic modification of gluten by transglutaminase plus lysine prevents the immunologic effects on cultured duodenal biopsies from coeliac patients and could be tested as an alternative therapy in coeliac disease.     

An investigational tetravalent meningococcal serogroups A, C, W-135 and Y-tetanus toxoid conjugate vaccine co-administered with Infanrix™ hexa is immunogenic, with an acceptable safety profile in 12-23-month-old children

Tetravalent meningococcal serogroups ACWY conjugate vaccines will provide an advantage to those at most risk of invasive meningococcal disease; namely young children. Co-administration of ACWY-TT with DTaP-HBV-IPV/Hib was assessed in a randomized trial in 793 children aged 12-23 months. Pre-specified criteria for non-inferiority of immunogenicity following co-administration versus separate ACWY-TT and DTaP-HBV-IPV/Hib administration were reached. One month post-vaccination, ≥97.3% of ACWY-TT vaccinees had rSBA titres ≥1:8 (all serogroups). Seroprotection/seropositivity rates against DTaP-HBV-IPV/Hib antigens were ≥98.2%. The safety profile of co-administration was similar to that of DTaP-HBV-IPV/Hib alone. ACWY-TT and DTaP-HBV-IPV/Hib co-administration during the second year would facilitate introduction of ACWY-TT into routine toddler vaccination schedules.     

实验条件对PT和APTT测定值的影响

目的 了解实验因素或者人为因素所造成的如溶血对血浆凝血酶原时间测定(PT),活化部分凝血活酶时间测(APTT)的影响,改变和指导实验室检测及加强与临床的沟通.方法 用半自动血凝仪仪器法(武汉中泰生物)检测,严格按标准操作规程操作.结果 溶血对测定结果的影响,无溶血与溶血组之间PT结果有显著性差异(P<0.05),不同溶血程度之间无显著性差异.APTT无溶血组与溶血组之间有显著性差异,不同溶血程度之间有显著性差异(PPT和APTT有显著性差异(P＜0.05),轻度乳糜血和中度乳糜血之间无差异,重度乳糜血标本测定不出结果.结论 对于凝血项目的 测定时,严格按标本采集的标准进行标本采集,避免溶血的发生,得到更加可靠的检测结.