thrombin-AT Complex Research Articles

Synthetic Pentasaccharide Fondaparinux (Arixtra®) Reduces Sepsis Induced Coagulopathy and Promotes Survival in the Baboon Model of E. coli Sepsis

Bacterial sepsis triggers profound dysfunction of the coagulation and fibrinolytic systems leading to disseminated intravascular coagulation (DIC), characterized by consumptive coagulopathy and bleeding. DIC may contribute to multiple organ failures due to occlusion of microvasculature and tissue hypoperfusion. Bacteria and/or bacteria-derived molecules initiate the activation of both the intrinsic/contact phase and extrinsic pathways of coagulation that activate factor X (FX). Active FX (FXa) is the enzyme of the prothrombinase complex that leads to thrombin generation and subsequent formation of fibrin clots. Arixtra® (Fondaparinux Sodium), a synthetic pentasaccharide, binds to antithrombin and thereby selectively inhibits FXa. Here, a baboon model of E. coli sepsis was used to investigate the effect of FXa inhibition on sepsis-induced activation of coagulation and fibrinolytic pathways.Two experimental baboon groups were studied:(i) E. coli challenge (n=4) , (ii) E. coli + Arixtra®(n=4). E. coli (1-2 x 1010 CFU/kg, LD100 dose) was infused intravenously over a 2 hour period. Arixtra® (0.08mg/kg) was administered subcutaneously to achive therapeutic concentration at the time of E. coli challenge (T0). The activation of coagulation was studied by measuring plasma levels of inhibitory complexes of antithrombin (AT) with activated factors: FXa-AT, FXIa-AT, thrombin-AT (TAT) and FVIIa-AT.E. coli challenge induced the activation of coagulation pathway as early as 2 hours post-challenge as evident by increase in plasma level of FXa-AT and TAT complexes. Contact pathway activation (FXIa-AT), as well as FVII activation (FVIIa-AT), were significantly reduced in Arixtra® treated animals. FXa-AT and TAT complexes were maximally present at 4-8 hours post-challenge and were reduced by more than 50% in Arixtra® treated animals (Fig. 1, A and B). Similarly, Arixtra® reduced activated partial thromboplastin time and prothrombin time as well as fibrinogen consumption following E. coli challenge. In line with the above observation, the plasma levels of fibrinolysis markers, fibrinogen/fibrin degradation products, D-Dimer and plasmin-antiplasmin complex (PAP) were significantly reduced in the Arixtra® treated group (Fig. 1, C and D). Platelets consumption/thrombocytopenia was similar in both the Arixtra® treated and the untreated groups. Moreover, neutrophil activation as measured by myeloperoxidase activity in plasma was reduced in the Arixtra® treated group. Two out of four animals survived in Arixtra® treated groups compared to 100% mortality in untreated group [Log-rank (Mantel-Cox) test P=0.0037]. Notably, Arixtra® treated non-survivor animals demonstrated prolonged survival times.Overall, the data suggests that Arixtra® reduced both coagulation and fibrinolytic pathway activation, thereby preventing DIC and providing survival benefits in E. coli sepsis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Single Low Dose of Human Recombinant Antithrombin (ATryn) has no Impact on Endotoxin-Induced Disseminated Intravascular Coagulation: An Experimental Randomized Open Label Controlled Study.

Antithrombin (AT) III physiological levels are decreased during septic shock and supplementation therapy could therefore be beneficial. We hypothesized that the use of recombinant human AT could reduce disseminated intravascular coagulation (DIC) occurrence. We conducted a randomized open label controlled experimental study. Ten female "Large White" pigs were challenged with i.v. infusion of Escherichia coli endotoxin. Two groups of 5 pigs were randomly assigned to receive either recombinant human AT 100 U/kg over 30 min (ATryn group) or 0.9% saline (control group). AT III levels, coagulation, hemostasis, inflammation parameters, hemodynamics, and microcirculatory parameters were measured over a 5-h period. Immediately after euthanasia, kidneys were withdrawn for histology evaluation. Statistical analysis was performed with nonparametric tests and Dunn's test for multiple comparisons. AT III activity was significantly higher in the ATryn group than in the control group from 60% (213% [203-223] vs. 104% [98-115], P = 0.008, respectively) to 300 min (115% [95-124] vs. 79% [67-93], P = 0.03). Recombinant human AT supplementation had no impact on hemodynamics, microcirculatory parameters, and sequential changes of coagulation parameters (platelet count, fibrinogen level, thrombin-AT complexes, and von Willebrand factor). Interleukin 6 and tumor necrosis factor α values were statistically the same for both groups throughout the study. Percentage of thrombosed glomeruli and percentage of thrombosed capillary in glomerulus were not significantly different between both groups. In our model of endotoxic shock, a single low dose of recombinant human AT did not prevent DIC occurrence, severity, inflammatory profile, or hemodynamic alterations.

Coagulation and Fibrinolysis Imbalance Demonstrated By Novel Simultaneous Thrombin and Plasmin Generation Assay during L-Asparaginase Treatment Phase of Induction Therapy in Pediatric Acute Lymphoblastic Leukemia

Introduction: L-asparaginase (L-Asp) is one of the risk factor of thromboembolism during ALL chemotherapy. Although the pathogenesis has been still unclarified, L-Asp may have profound effects on hepatic synthesis of pro-, anti-coagulant and fibrinolytic factors. In addition, recent studies demonstrated that the age of over 10-years might be a risk factor for this L-Asp associated coagulopathy. In this study, we hypothesized that change of balance between coagulation and fibrinolysis contributes to hyper-coagulation condition during chemotherapy including L-Asp. In order to clarify our hypothesis, we investigated the global coagulation and fibrinolytic function during the ALL induction therapy with simultaneous thrombin and plasmin generation assay (T/P-GA). Patients: Seventy-two pediatric patients aged 1.0 to 15.2 years (43 males and 29 females) with newly diagnosed ALL were enrolled from Aug. 2014 to Mar. 2018 at four hospitals in Japan. All patients and their families had no thrombotic predisposition. Fifty-five cases (76.4%) (BCP-ALL; n=47, T-ALL; n=7, MPAL; n=1) received BFM-95 oriented protocol consisting of a total of 8 doses of E.coli L-Asp 5,000 U/m2, whilst the others (BCP-ALL; n=13, T-ALL; n=2, MPAL; n=1, Ph-ALL; n=1) received Japan Association of childhood Leukemia Study (JACLS)-ALL02-protcol consisting of a total of 6 doses of E.coli L-Asp 6,000 U/m2. Fifty-two cases (72.2%) were categorized to younger group (1-10 years; median, 4.3 years) and the other cases to older group (>10 years; median, 12.6 years). The percentage of patients of each category were similar in the two treatment protocols (p=0.764). Methods: The global functions of coagulation and fibrinolysis were evaluated using T/P-GA [Matsumoto et al. TH 2013]. This clotting was initiated by the mixture of recombinant human tissue factor (Innovin®, f.c. 1 pM), phospholipid vesicles (f.c. 4 μM) and tissue-type plasminogen activator (f.c. 3.2 nM). Thrombin and plasmin generation were monitored simultaneously using thrombin- and plasmin-specific fluorogenic substrate (Z-Gly-Gly-Arg-AMC and BOC-Glu-Lys-Lys-MAC, respectively) in separate microtiter wells. The first derivatives (velocity) of thrombin and plasmin generation were utilized to derive the parameters, lag time (LT), endogenous potential (EP), peak levels (peak), and time to peak (ttPeak). EP of thrombin generation (T-EP) and plasmin-peak (P-peak) were selected as parameters for evaluation in this study. A ratio of T-EP and P-peak of patients' plasmas to those of control normal plasma were calculated. The conventional laboratory markers of coagulation and fibrinolysis were also monitored by fibrinogen (Fbg), fibrin-Fbg degradation products (FDP), antithrombin (AT), thrombin-AT complex and plasmin-α2 plasmin inhibitor complex. Plasmas were collected at T0; pre-phase of L-Asp, T1; intermittent phase of L-Asp, T2; post-phase of L-Asp, and T3; post induction phase. Results: None of the cases developed L-Asp associated coagulopathy, and two cases (2.8%) developed induction failure. Thirteen cases (18.1%) received fresh frozen plasma transfusion for low Fbg level, whilst 52 (72.2%) cases received AT supplement for low AT level. Fbg showed a median of 165, 99.0, 97.0 and 316 mg/dl at T0, T1, T2 and T3, respectively, and AT showed a median of 143, 80.9, 80.0 and 105%, respectively, whilst the value of other conventional markers remained within reference value. T-EP revealed a median of 1,134, 1,135, 1,221, 1,277 and 1,102 nM, whilst P-peak showed a median of 5.33, 3.75, 3.63, 4.91 and 5.51 nM for T0, T1, T2, T3 and control plasma, respectively, indicating the elevated T-EP ratios and reduced P-peak ratios (Fig. 1). T-EP ratios showed significantly higher at T2 and T3 than at T0 and T1 (p<0.05), whilst P-peak ratios showed significantly lower at T1 and T2 than at T0 and T3 (p<0.01) (median ratios of T-EP/P-peak; T0; 1.06/0.97, T1; 1.04/0.65, T2; 1.12/0.65, T3; 1.17/0.87, respectively). P-peak ratios of the older group showed significantly lower than the younger group at T1 (median; 0.58 vs 0.69, p=0.016). Conclusion: The results from T/P-GA showed that their hemostatic dynamics appear likely to shift to hyper-coagulation and hypo-fibrinolysis at L-Asp treatment phase. These results suggest that the imbalance of coagulation and fibrinolysis might cause thrombotic complication during the L-Asp treatment for ALL, especially in patients aged 10-years older. [Display omitted] DisclosuresNogami:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau.

Asymptomatic Dysprothrombinemia (Prothtombin Himi) with p.M380T and p.R431H Shows Severely Reduced Clotting Activity, Moderate Antithrombin Resistance and Severe Thrombomodulin Binding Defect

Background and purpose:Congenital prothrombin (PT) deficiency is categorized into two phenotypes, true hypoprothrombinemia and dysprothrombinemia. A strong discrepancy between observed values from coagulation tests and bleeding severity can be found in some forms of dysprothrombinemia. Some dysprothrombinemias, such as the PT Himi that we reported previously (Morishita E, et al: Blood 1992), are asymptomatic despite low PT activity. PT Yukuhashi disturbs the interaction between thrombin and antithrombin (AT), causing a paradoxical thrombotic phenotype, called "AT resistance (ATR)". To investigate the mechanisms by which patient with PT Himi is asymptomatic, mutant PTs reflecting each of the two different gene sites identified in gene analysis were prepared, and functional analyses of fibrinogen (Fbg) degradation, ATR, and thrombomodulin (TM)-binding capacity were conducted. This study was approved by the ethics committee at Kanazawa University School of Medicine.Case report: The proband was a 26-year-old Japanese woman who had been diagnosed with congenital dysprothrombinemia at the time of first pregnancy, based on PT activity and PT antigen levels of 10% and 88%, respectively. Although cesarean section was performed for the second delivery, abnormal bleeding did not develop. Genomic DNA analysis revealed compound heterozygosity for two missense mutations in the F2gene (c.1139T>C, p.M380T and c.1292G>A, p.R431H).Methods: We established stable transformants of Chinese hamster ovary cells that expressed wild-type and two mutant recombinant PTs (rPTs), M380T PT and R431H PT. Conditioned media of stable transformants expressing rPTs in serum-free medium were collected, concentrated, and stored at -80°C until use. 1) Fbg clotting assays: We performed a one-stage clotting assay and a chromogenic assay. 2) Kinetic analysis of thrombin inactivation using AT and formation of thrombin-AT complex (TAT): After incubation with PT activator components, samples were treated with excess AT in the absence of heparin for various durations and changes in absorbance per minute were measured. Formed TAT was measured using an enzyme-linked immunosorbent assay (ELISA) kit. 3) TM-binding assay: To evaluate the ability of wild-type and mutant rPTs to bind to TM, after activating PT, we detected TM-binding thrombin using a TM ELISA method.Results:1) M380T PT activity in the one-stage assay was below the threshold of sensitivity (clot not detected), whereas R431H PT activity was decreased to 10% of that of wild-type rPT (100%). On the other hand, both mutant PT activities in the chromogenic assay were slightly decreased compared to wild-type rPT. 2) After 30 min without heparin, relative residual thrombin activity of M380T PT (74%) was higher than that of wild-type (10%), indicating severe ATR, whereas that of R431H PT was slightly increased (21%), indicating mild ATR. TAT formation by wild-type PT increased over time. TAT formation by R431H PT was about 40% of that of wild-type PT, but that by M380T PT was almost negligible after 120 min. 3) TM-binding thrombins of both mutant rPTs were barely detected.Discussion: M380T PT has high ATR and fails to bind with TM, so a large amount of thrombin with greater clotting activity than normal is thought to be present. With this abnormal thrombin, however, fibrin-forming ability is greatly decreased and almost no clotting ability is thought to exist in the body. Hence, R431H PT, which made up about half of the PT protein in the proband, was considered responsible for hemostasis in the body. The mutant thrombin activated from the R431H PT shows decreased clotting activity, but is resistant to inactivity from AT and thus maintains activity longer than ordinary thrombin. At the same time, the activation of protein C is also decreased due to failure to bind with TM, so ultimately the clotting ability necessary for hemostasis was conjectured to have been maintained, resulting in the proband being asymptomatic.Conclusion:Although neither PT position M380 nor R431 are AT binding sites, M380T PT and R431H PT showed ATR. These mutants are the first AT-resistant PTs seen from mutations other than at the Arg596 site. In recent years, TM resistance has been newly proposed as a mechanism producing thrombotic tendencies, and may also apply to the present patient. The reason the proband did not show a bleeding tendency may have been the combined contributions of AT and TM resistance. DisclosuresNo relevant conflicts of interest to declare.

Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance.

Antithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0 %, in contrast to values of all variants were maintained at above 80 %. The thrombin-AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12 %, whereas that of tested variants was 37 %-56 %. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin-TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin-TM affinity-dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM-mediated anticoagulation.

Evaluation of Modified Japanese Ministry Health and Labor Welfare Diagnostic Criteria for DIC to Detect Early Phase of DIC

Evaluation of the modified Japanese Ministry Health, Labour and Welfare (JMHLW) diagnostic criteria for DIC to detect the early phase of DICIntroduction: Disseminated intravascular coagulation (DIC) is a syndrome characterized by the systemic activation of blood coagulation, which generates intravascular thrombin and fibrin, resulting in the thrombosis of small-to medium-sized vessels and ultimately organ dysfunction and severe bleeding. Three different diagnostic criteria incorporating similar global coagulation tests have been established by the International Society of Thrombosis and Haemostasis (ISTH), Japanese Ministry Health, Labour and Welfare (JMHLW) and Japanese Association of Acute Medicine (JAAM). The JMHLW score is well correlated with the severity of DIC and can be used to predict the outcome of the disease, however, it has a relatively low sensitivity for the early phase of DIC (pre-DIC).Material and Methods: The Modified JMHLW criteria are shown in the Table 1. From 1996 to 2014, 282 patients with hematopoietic disorder type, 220 patients with infection type and 249 patients with other type were retrospectively diagnosed with DIC according to the JMHW criteria. There were 184 patients with pre-DIC and 256 patients without DIC.Table 1Modified Japanese Ministry Health, Labor and Welfare criteria for DICHematopoietic disorder typeOthertypeInfectiontypePlatelet count(X107/ml)none8 < < 12: 1 point, 5 < < 8: 2 points,(> 30% decrease: plus 1 point)< 5: 3 pointsFDP (ƒÊg/ml)10 < < 20: 1 point, 20 < < 40; 2 points, 40< : 3 pointsFibrinogen (g/L)1 < < 1.5 : 1 point, < 1: 2 pointsnonePT ratio1.25 < < 1.67: 1 point, 1.67 < : 2 pointsAT (%)< 70 : 1 pointTAT or SF2X (upper limit of normal range): 1 pointDIC4 points = or <6 points = or <5 points = or <AT: antithrombin, TAT: thrombin AT complex, SF: soluble fibrinResults: The agreement rate between the JMHLW and modified JMHLW criteria was 95.9%. The diagnosis rate of pre-DIC was 0% using the JMHLW criteira, 8.3% using the ISTH diagnostic criteria, 63.0% using the JAA criteria and 38.6% using the modified JMHLW criteira for DIC. The mortality of DIC was 42.5% using the JMHLW criteria, 45.5% using the ISTH criteria, 41.3% using the JAA criteria and 42.6% using the modified JMHLW criteria.Discussion and conclusion: The agreement rate between the JMHLW and modified JMHLW criteria is high. As the modified JMHLW criteria adds the score for antithrombin (AT) activity, thrombin-AT complex (TAT) or soluble fibrin (SF), these criteria increase the sensitivity for pre-DIC. DisclosuresNo relevant conflicts of interest to declare.

Laboratory-monitored fondaparinux and coagulation activity in association with total hip replacement

Fondaparinux, indirect factor Xa (FXa) inhibitor, is recommended for thromboprophylaxis for high-risk patients undergoing major orthopedic surgery. We evaluated the prothrombotic state and anticoagulant intensity of fondaparinux (2.5 mg daily) after total hip replacement (THR). Twenty patients underwent THR - seven bilateral and 13 unilateral. Blood samples were collected preoperatively and at 6 h, 8 h (2 h after fondaparinux), 1 day (12-14 h after fondaparinux), and 4 weeks (12-14 h after fondaparinux) postoperatively. Antithrombin (AT), fibrinogen, factor VIII activity, coagulation times, thrombin-AT (TAT) complex, D-dimer, C-reactive protein, prothrombinase-induced clotting time (PiCT) and anti-Xa activity were measured. The latter two were also tested after plasma spiking with fondaparinux 0-1.25 μg/ml. In spiked prophylactic fondaparinux samples (0-0.25 μg/ml), PiCT and anti-Xa activity correlated (r = 0.84) better than in the patient samples (r = 0.35). On the first day, anti-Xa activity and PiCT dissociated, and PiCT lost sensitivity for fondaparinux. AT decreased but stayed within the normal range, whereas TAT complex and D-dimer peaked at 6 h as signs of thrombin generation. On the first postoperative day, TAT and D-dimer halved. Bilateral THR associated with higher TAT and D-dimer levels up to 4 weeks. Perioperative FVIII levels were not affected, but were elevated in both groups (range 191-211%) after 4 weeks. Anti-Xa activity detected prophylactic fondaparinux with higher sensitivity than PiCT in vitro, but even more so in vivo. Thus, PiCT is not the method of choice to assess fondaparinux at least in association with THR. THR, bilateral more than unilateral, increased thrombin generation and D-dimer 7-11-fold early after surgery. Factor VIII activity and D-dimer remained elevated even after 4 weeks despite the compliant thromboprophylaxis with fondaparinux.

Nebulized Anticoagulants Limit Coagulopathy But Not Inflammation in Pseudomonas aeruginosa-Induced Pneumonia in Rats

Disturbed alveolar fibrin turnover is a characteristic feature of pneumonia. Inhibitors of coagulation could exert lung-protective effects via anticoagulant (inhibiting fibrin deposition) and possibly anti-inflammatory pathways, but could also affect host defense. In this randomized controlled in vivo laboratory study, rats were challenged intratracheally with Pseudomonas aeruginosa, inducing pneumonia, and randomized to local treatment with normal saline (placebo), recombinant human activated protein C (rh-APC), plasma-derived antithrombin (AT), heparin, or danaparoid. Induction of P. aeruginosa pneumonia resulted in activation of pulmonary coagulation and inhibition of pulmonary fibrinolysis, as reflected by increased pulmonary levels of thrombin-AT complexes and fibrin degradation products and decreased pulmonary levels plasminogen activator activity. Pseudomonas aeruginosa pneumonia was accompanied by systemic coagulopathy, since systemic levels of thrombin-AT complexes increased, and systemic levels of plasminogen activator activity decreased. Although rh-APC and plasma-derived AT potently limited pulmonary coagulopathy, neither heparin nor danaparoid affected net pulmonary fibrin turnover. Recombinant human APC also displayed systemic anticoagulant effects. Neither bacterial clearance nor pulmonary inflammation was affected by anticoagulant therapy. Nebulization of rh-APC or plasma-derived AT attenuated pulmonary coagulopathy, but not bacterial clearance or inflammation, in a rat model of P. aeruginosa pneumonia.

Evaluation of Non Overt DIC Diagnostic Criteria.

Abstract 1297Poster Board I-319 IntroductionSeveral diagnostic criteria for disseminated intravascular coagulation (DIC) have been proposed by Colman RW et al, Japanese Ministry Health and Welfare (JMHW) and International Society of Thrombosis and Haemostasis (ISTH) etc. The efficacy of treatment in relation to the DIC score when the treatment was begun showed that greater efficacy was achieved in pre-DIC than in DIC patients, suggesting that early diagnosis and early treatment are important. Then, the diagnostic criteria for non-overt DIC was proposed by ISTH/SSC subcommittee in order to diagnose of early phase DIC but it was still not established. In this study, modified non-overt DIC diagnostic criteria using molecular hemostatic markers and antithrombin (AT) has been prospectively evaluated.Total 613 patients suspected to be associated with DIC in nine institutes were registered in this prospective study for DIC diagnostic criteria from January 1, 2005 to December 31, 2008. There were 219 patients with infectious disease, 142 with solid cancer, 115 with hematopoietic tumor, 29 with aneurysm, 10 with obstetrics disease, 23 with trauma, 5 with liver disease, 70 with other disease. Materials and MethodsOvert-DIC or non-overt DIC was diagnosed by modified overt-DIC diagnostic criteria or modified non-overt DIC diagnostic criteria using abnormalities or changes of platelet count, prothrombin time (PT), fibrinogen, fibrin and fibrinogen degradation products (FDP), AT, soluble fibrin monomer complex (SFMC), D-dimer and thrombin-AT complex (TAT). Pre-DIC was considered the state within a week before onset of DIC. Measurement of PT, fibrinogen, platelet count, FDP were carried out in each institutes based on numerous previous reports. TAT, SFMC, D-dimer and AT activity were measured in SRL Inc. TAT was measured by enzyme immunoassay (EIA) using. SFMC and D-dimer were measured by latex immune agglutination test using T-test, respectively. AT activity was measured by heparin cofactor activity. ResultsFrequency of overt-DIC was 29.5 % in 613 patients suspected to be associated with DIC, and the highest frequency of overt-DIC was observed in the patients with obstetrics disease or liver disease. Pre-DIC state was detected in 44 of 613 patients (7.2 %) and the highest frequency of pre-DIC was observed in the patients with hematopoetic tumor (12.2 %). Frequency of non-overt DIC in the patients with overt-DIC, with pre-DIC or without overt-DIC was 97.8 %, 97.7 % or 17.0%, respectively. The mortality of 28 days was the highest in the patients with overt DIC (37.6%), and it was also high in those with non-overt DIC (32.9%) and with pre-DIC (27.3%) in comparison to the patients without overt DIC (15.2%) and those without non-overt DIC (13.8%). DiscussionModified non-overt DIC diagnostic criteria is more sensitive for DIC than overt DIC diagnostic criteria and is related to the outcome. Non overt DIC diagnostic criteria might be able not only to diagnose DIC but also to predict early phase of DIC. DisclosuresNo relevant conflicts of interest to declare.

Antithrombin effect on coagulation and fibrinolytic profiles after living donor liver transplantation: a pilot study

Early after liver transplantation, patients are in a hypercoagulable state because of an imbalance between coagulation and fibrinolysis because of the slow recovery of depleted anticoagulant proteins. Antithrombin (AT) is used in anticoagulant protocols to prevent thrombosis. The subjects of the present study were 17 men and eight women that underwent living donor liver transplantation. The initial 15 cases were administered AT concentrate (1500 U/day) on postoperative days (POD) 1 through 3 (AT group) and the following 10 consecutive cases were not administered AT (control). AT, thrombin-AT complex, plasmin-alpha2 plasmin inhibitor complex, thrombomodulin, fibrin degradation product D-dimer (FDP-DD) level, prothrombin time international normalized ratio, activated partial thromboplastin time, and platelet counts were measured. In the AT group, AT activity was maintained at levels >80% for 5 days after transplantation. In the control group, AT activity did not return to normal during the first 2 weeks after the operation. FDP-DD levels were significantly higher in the control group than in the AT group (P < 0.05). Six patients in the control group and three patients in the AT group required transfusions with platelet concentrate (P < 0.05). AT supplementation might reduce FDP-DD levels and prevent decreased platelet counts in the early stages after liver transplantation.

The N-terminal 1-16 peptide derived in vivo from protein seminal vesicle protein IV modulates α-thrombin activity: potential clinical implications

We have previously shown that seminal vesicle protein IV (SV-IV) and its 1-70 N-terminal fragment have anti-inflammatory activity and modulate anti-thrombin III (AT) activity. Moreover, mass spectrometry analysis of purified SV-IV has shown that the protein was found to be highly heterogeneous and 14% of the total SV-IV molecules are truncated forms, of particular interest the 1-16, 1-17, and 1-18 peptides. In this work we report experimental data which demonstrate that the 1-16 peptide (P1-16) possesses a marked effect on the AT activity by preventing the formation of the thrombin-AT complex. We found that the formation of thrombin-AT complex is markedly decreased in the presence of P1-16 used at equimolar concentration with thrombin as evaluated with SDS-PAGE. We also monitored the conformational changes of thrombin in the presence of different P1-16 concentrations, and calculated the K(d) of thrombin/P1-16 system by circular dichroism technique. The probable interaction sites of P1-16 with thrombin have been also evaluated by molecular graphics and computational analyses. These results have potential implications in the treatment of sterility and thrombotic diseases.

Predicting the Severity of Systemic Inflammatory Response Syndrome (SIRS)-Associated Coagulopathy With Hemostatic Molecular Markers and Vascular Endothelial Injury Markers

The changes in biomarkers of coagulation or fibrinolysis, anticoagulation, inflammation, and endothelial damage occur in patients with systemic inflammatory response syndrome (SIRS). The purpose of this study is to assess the prognostic value of these markers in patients with SIRS-associated hypercoagulopathy. Sixty-six SIRS patients with a platelet count less than 15.0 x 10(4)/mm3 in three university hospital intensive care units were enrolled in this prospective, comparative study. Blood samples were obtained on day 0 and day 2. Twelve hemostatic, inflammatory, and vascular endothelial indices were measured and the data were compared between the severe group (patients with a total maximum Sequential Organ Failure Assessment score of 10 or more and nonsurvivors; n = 25) and the less-severe group (Sequential Organ Failure Assessment score <10; n = 41). Significant changes between the groups were observed in platelet count, fibrin or fibrinogen degradation products, interleukin-6, soluble thrombomodulin, antithrombin (AT) activity, and protein C activity, both on day 0 and on day 2. In contrast, the d-dimer, soluble fibrin, plasmin-[alpha]2-antiplasmin complex, and E-selectin levels were higher in the severe group only on day 2. No significant difference was seen regarding the thrombin-AT complex and total plasminogen activator inhibitor on both days. A comparison of the areas under the receiver operating characteristic curve revealed the AT activity to be the best predictor of a progression of organ dysfunction. The changes in some hemostatic molecular markers and vascular endothelial markers were conspicuous in patients with organ dysfunction. The AT activity is considered to be the most useful predictor of organ dysfunction.

Natural anticoagulants limit lipopolysaccharide-induced pulmonary coagulation but not inflammation

Pulmonary coagulopathy and hyperinflammation may contribute to an adverse outcome in sepsis. The present study determines the effects of natural inhibitors of coagulation on bronchoalveolar haemostasis and inflammation in a rat model of endotoxaemia. Male Sprague-Dawley rats were randomised to treatment with normal saline, recombinant human activated protein C (APC), plasma-derived antithrombin (AT), recombinant human tissue factor pathway inhibitor (TFPI), heparin or recombinant tissue plasminogen activator (tPA). Rats were intravenously injected with lipopolysaccharide (LPS), which induced a systemic inflammatory response and pulmonary inflammation. Blood and bronchoalveolar lavage were obtained at 4 and 16 h after LPS injection, and markers of coagulation and inflammation were measured. LPS injection caused an increase in the levels of thrombin-AT complexes, whereas plasminogen activator activity was attenuated, both systemically and within the bronchoalveolar compartment. Administration of APC, AT and TFPI significantly limited LPS-induced generation of thrombin-AT complexes in the lungs, and tPA stimulated pulmonary fibrinolytic activity. However, none of the agents had significant effects on the production of pulmonary cytokines, chemokines, neutrophil influx and myeloperoxidase activity. Natural inhibitors of coagulation prevent bronchoalveolar activation of coagulation, but do not induce major alterations of the pulmonary inflammatory response in rat endotoxaemia.

The cleaved and latent forms of antithrombin are normal constituents of blood plasma: a quantitative method to measure cleaved antithrombin.

Antithrombin (AT), a member of the serine protease inhibitor family, is the key regulator of thrombin activity in vivo. Thrombin inhibition is accomplished by the formation of covalent thrombin-AT (TAT) complex. The rate of inhibition is accelerated by heparin, which also leads to the formation of a substantial amount of cleaved AT. We produced a murine monoclonal antibody (mAb) (M9) that is specific for the two forms of AT, in which the reactive center loop is inserted into beta-sheet A, i.e. cleaved and latent AT. The antibody has no measurable affinity for native AT. Using M9 as a catcher antibody in conjunction with a mAb (M27) that does not bind latent AT, we developed a sandwich assay that measures cleaved AT without interference from latent and native AT. The concentration in healthy subjects was determined to be 1.3 mg L(-1) (range: 1.0-1.9), which was about 100-fold lower than the plasma concentration of native AT and 1000-fold higher than the concentration of the TAT complex. The cleaved AT concentration is higher than what would be expected from the rate of formation of cleaved AT in vitro in conjunction with TAT complex formation in the presence of heparin. The concentration of cleaved AT did not correlate with the TAT concentration in plasma from patients with venous thrombosis.

Plasma levels of heparin cofactor II (HCII) and thrombin-HCII complex in patients with disseminated intravascular coagulation.

Plasma levels of heparin cofactor II (HCII), thrombin-HCII complex (THC), antithrombin (AT), and thrombin-AT complex (TAT) were evaluated in patients with disseminated intravascular coagulation (DIC) associated with several underlying diseases. Plasma levels of AT were significantly reduced in almost all underlying diseases associated with DIC, but the plasma levels of HCII and HCII/AT ratio were significantly reduced only in patients with infections. While the plasma level of TAT was significantly increased in patients with all underlying diseases associated with DIC, the increase of THC was not significant. Plasma levels of AT were significantly reduced in DIC and pre-DIC associated with almost all underlying diseases, but those of HCII were significantly reduced only in DIC and pre-DIC patients with inflammatory diseases. The plasma levels of TAT were significantly increased in DIC, pre-DIC, and non-DIC patients with all underlying diseases, and those of THC were significantly increased in DIC and pre-DIC patients with inflammatory diseases. The plasma levels of THC were not significantly increased in non-DIC patients of any disease group. The decrease of AT may be caused by thrombin generation or inflammatory reaction that occurs in DIC associated with underlying diseases, while the decrease of HCII might be caused by both thrombin generation and inflammatory reaction. Finally, AT inhibits thrombin more strongly than HCII in several underlying diseases associated with DIC except for inflammatory diseases. In inflammatory diseases, HCII might play an important role in preventing the onset of DIC.

Influence of heparin and hirudin on endothelial binding of antithrombin in experimental thrombinemia.

During the last decade, experimental and clinical evidence has accumulated that antithrombin (AT) exerts anti-inflammatory effects when given in high doses. Meanwhile, AT substitution has been shown to significantly increase prostacyclin release. However, the link between endothelial AT binding and anti-inflammatory AT effects remains to be established in vivo, although heparin has been shown to counteract anti-inflammatory AT effects. We hypothesized that the administration of heparin in endotoxin-challenged rats would decrease endothelial AT binding and systemic prostacyclin concentrations. Prospective, randomized, controlled experimental in vivo study. Research laboratory of a university hospital. Fifty-six Wistar rats. Baseline values of coagulation variables were measured in six animals. Forty of 50 Wistar rats in the study groups were given endotoxin (50 mg x kg(-1) iv) and were treated with saline (group LPS), AT (15 units x kg(-1) x hr(-1)) (LPS+AT), AT and heparin (80 IU x kg(-1) x hr(-1)), or AT and hirudin (0.12 mg x kg(-1) x hr(-1)); the other 10 received saline instead of endotoxin and were treated with AT alone. Before endotoxin application, a tracheostomy was performed, and venous and arterial catheters were inserted for blood sampling and infusion. Intravital endothelial AT binding was studied by using fluorescence isothiocyanate-marked antibodies during intravital microscopy of intestinal submucosal venules. Systemic prostacyclin, thrombin-AT complex, and fibrinogen concentrations were measured after 4 hrs. Intergroup differences were tested by Kruskal-Wallis analysis of variance on ranks. AT and AT + heparin were equally effective in inhibiting systemic procoagulant turnover as reflected by fibrinogen concentrations. Only the administration of AT + hirudin significantly prevented fibrinogen consumption (p < .05). In contrast with all other treatments, the administration of heparin significantly reduced intravital endothelial AT binding (p < .05). However, prostacyclin concentrations were similarly increased in all endotoxin-challenged study groups irrespective of the anticoagulatory treatment. There is evidence that heparin in contrast with hirudin prevents AT from being bound to the endothelial cell surface in this experimental model. Under low-dose AT substitution, systemic prostacyclin concentrations do not depend on whether heparin or hirudin is used for thrombin inhibition. These results support the view that heparin may counteract anti-inflammatory AT effects by keeping AT away from its endothelial binding sites; however, the results question the view that decreased endothelial prostacyclin release is solely responsible.

Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of vitronectin with thrombin-antithrombin.

In this study, purified preparations of platelet protein disulfide isomerase (PDI), vitronectin, alpha-thrombin, and antithrombin (AT) were used to demonstrate that PDI catalyzes formation of vitronectin-thrombin-AT complexes. Complex formation requires reduced glutathione (GSH) and can be prevented by N-ethymaleimide, and the formed complex is dissociated by reducing agents such as mercaptoethanol. No vitronectin-thrombin complex formed in the absence of AT, indicating that the thrombin-AT complex is an obligate intermediate in the reaction. Under optimal conditions, the majority of the thrombin-AT is incorporated into the complex in 60 min. Thrombospondin-1, known to form disulfide-linked complexes with thrombin-AT [Milev, Y., and Essex, D. W. (1999) Arch. Biochem. Biophys. 361, 120-126], competes with vitronectin for thrombin-AT in the low-Ca(2+) environment that favors the active form of thrombospondin. The results presented here may also explain previous studies showing that vitronectin-thrombin-AT complexes form better in plasma (which contains PDI) than with purified proteins (where PDI was not used). We were able to purify a PDI from plasma that was immunologically identical to the platelet enzyme. We used the scrambled RNase assay to show that added purified PDI can function in a plasma environment. Complex formation in plasma was inhibited by inhibitors of PDI. PDI was released from the platelet surface in a soluble form at high pH (around the physiologic range), suggesting a source of the plasma PDI. In summary, these studies indicate that PDI functions to form disulfide-linked complexes of vitronectin with thrombin-AT.

Protein Disulfide Isomerase Catalyzes the Formation of Disulfide-Linked Complexes of Thrombospondin-1 with Thrombin–Antithrombin III

The recent demonstration of a protein disulfide isomerase (PDI) on the surface of and secreted from blood platelets raises the possibility that proteins involved in hemostasis and wound healing are also substrates of this enzyme. In this study purified preparations of platelet PDI, thrombospondin-1 (TSP), α-thrombin, and antithrombin III (AT) were used to demonstrate that PDI catalyzes formation of a TSP–thrombin–AT complex consistent with previous results with supernatant platelet activation. Concentrations of 1.25 μg/ml of PDI were sufficient to convert almost 50% of thrombin to TSP–thrombin–AT complex. Complex formation requires low concentrations of a reduced thiol and the reaction can be prevented byN-ethymaleimide. The complex is dissociated by reducing agents such as mercaptoethanol. Absence of Ca2+and the addition of EDTA increased the rate of complex formation, indicating that TSP in the Ca2+-free form is most effective. In the absence of AT a small amount of TSP–thrombin complex formed which was only 0–13% of maximal complex formation in the presence of AT. This result, in combination with kinetic studies showing rapid formation of thrombin–AT complex followed by conversion to ternary complex, suggests that the thrombin–AT complex is an obligatory intermediate in the reaction. Under optimal conditions over 70% of the thrombin is incorporated into the complex in 60 min. Heparin accelerated the reaction largely by enhancing formation of thrombin–AT complexes and had little effect on TSP. PDI coprecipitated with TSP from the supernatant solution of activated platelets, suggesting an association between PDI and its substrate. In summary, these data are consistent with a role for PDI-catalyzed formation of disulfide-linked complexes of TSP with other proteins.

Humoral coagulation and early complications after allogeneic bone marrow transplantation.

Alterations of the coagulation system that may lead to coagulation activation and thrombosis are common sequelae after allogeneic bone marrow transplantation (BMT). We performed prophylactic anticoagulation by low dose heparin (50 units/kg/day) and substitution of antithrombin (AT) concentrate to sustain plasma levels above 90% of pooled normal human plasma. Conventional tests for plasmatic hemostasis and substitution of AT concentrate were recorded for 50 patients until day +50 after BMT. Incidence of sepsis, graft-versus-host-disease [GVHD], capillary leakage syndrome [CLS] and veno-occlusive disease of the liver [VOD] were investigated and compared with the results of patients without any of these complications. Patients with proven sepsis (n = 6) showed decreased activity of AT, and a prolonged activated partial thromboplastin time (aPTT), while fibrinogen levels were slightly increased. This constellation was interpreted as mild to moderate activation of the humoral coagulation cascade. Patients with VOD (n = 10) showed an increased consumption of AT concentrate at day +7 followed by a decrease of prothrombin time, of clotting factors II and VII, and a prolongation of aPTT at days +11 to +18 after BMT. This suggests, that activation of coagulation precedes decreased synthesis of coagulation factors. Patients with CLS (n = 15) or GVHD > or = II degree (n = 14) showed no major alterations of coagulation parameters. In conclusion, after BMT, two types of coagulopathy were observed: (i) an activation of the coagulation cascade (i.e. sepsis and VOD) which was followed by (ii) a diminished synthesis of coagulation factors (VOD). In order to perform timely therapeutic interventions in the coagulation system in patients with sepsis and/or VOD it appears to be important to assess the clinical value of parameters for early detection of coagulation activation as thrombin-AT complexes, D-dimers and F1 + 2 fragments.

A TRIPLE-BLIND PLACEBO-CONTROLLED RANDOMIZED TRIAL OF ANTITHROMBIN THERAPY IN NEONATAL RESPIRATORY DISTRESS SYNDROME † 1035

Background: Although neonatal respiratory distress syndrome (RDS) is associated with decreased plasma activity of antithrombin (AT) and increased formation of thrombin, the benefits of therapy with AT have been uncertain. Objective: To test whether infusion of AT reduces thrombin formation, improves gas exchange and decreases the duration of mechanical ventilation and supplemental oxygen. Methods: 122 premature infants with RDS were randomized within 12 hours of life to pasteurized AT concentrate (Behringwerke AG, Germany) or placebo (1% albumin). A loading dose of 2 mL/kg (equivalent to 100 I.U. AT/kg) was followed by 1 mL/kg (50 I.U./kg) every 6 hours for 48 hours. Plasma AT activity. Thrombin-AT(TAT) complex and prothrombin fragment (F1+2) were measured every 12 hours during treatment. The arterial/alveolar oxygen tension (a/A) ratio and the ventilator efficiency index (VEI) were calculated every 6 hours during the 48 hr treatment phase and daily thereafter until day 7. Mechanical ventilation and oxygen therapy were considered terminated once they had been discontinued for at least 24 hours. The same exogenous surfactant was used in all study infants. Results: In the AT group (n=61), mean (SD) birthweight was 1198 (301) g, mean (SD) GA 28.3 (2.0) wks, 54% were male. In the placebo group(n=61), mean (SD) birthweight was 1201 (315) g, mean (SD) GA 28.8 (2.3) wks, 51% were male. In treated infants, AT activity was raised to means of 1.69 and 2.25 U/mL at 24 and 48 hrs respectively. Corresponding means in controls were 0.37 and 0.44 U/mL (p<0.0001). TAT and F1+2, however, were not significantly reduced by AT. VEI and a/A ratio were similar in both groups throughout the first week of life. Median days on initial mechanical ventilation were 6.3 (AT) versus 4.8 (placebo), p=0.27. Median days on supplemental oxygen were 7.4 (AT) versus 5.5 (placebo), p=0.30. There were 7(11.5%) deaths in the AT group and 3 (4.9%) deaths in the placebo group. Conclusion: Treatment with AT does not reduce biochemical markers of thrombin formation and does not improve gas exchange in neonatal RDS. Funded by the Physicians' Services Incorporated Foundation, Toronto, and Behringwerke AG.