New insights into neonatal coagulation: normal clot formation despite lower intra-clot thrombin levels.

BACKGROUND: Healthy neonates exhibit no bleeding tendencies, but exhibit longer partial thromboplastin times than adults. Lower clotting factor levels may be balanced by lower inhibitor levels, which is not reflected in routine coagulation assays, but could result in normal clot formation in vivo. The novel thrombodynamics assay simulates a damaged vessel with tissue factor immobilized to a surface. We hypothesized that intra-clot thrombin levels and spatial fibrin clot formation with this assay are comparable in neonates and adults. METHODS: Coagulation was tested in plasma from venous neonatal blood (N = 12), cord blood (N = 30), and adult blood (N = 20) using thrombodynamics and calibrated automated thrombography. RESULTS: Neonates exhibited a higher initial rate of clot formation than adults (adult: 60.7 ± 3.9 µm/min; neonatal: 66.8 ± 3.9 µm/min; cord: 68.1 ± 3.3 µm/min; P < 0.001) and a comparable stationary rate of clot formation (adult: 35.8 ± 8.5 µm/min; neonatal: 37.0 ± 4.6 µm/min; cord: 36.0 ± 5.2 µm/min; P = 0.834). Intra-clot thrombin levels were lower in neonates (adult: 41.9 ± 11.2 AU/l; neonatal: 22.6 ± 10.2 AU/l; cord: 23.6 ± 9.7 AU/l; P < 0.001), but the longitudinal rate of thrombin propagation was comparable (adult: 27.2 ± 4.2 µm/min neonatal; 27.9 ± 2.9 µm/min; cord: 27.6 ± 3.4 µm/min; P = 0.862). CONCLUSIONS: Despite lower intra-clot thrombin levels, neonates exhibit normal spatial fibrin clot growth, which concurs with clinically well-functioning hemostasis in healthy neonates.

Bed rest does not induce hypercoagulability.

BACKGROUND: Although there is no direct evidence, it is generally believed that bed rest shifts the haemostatic system towards hypercoagulability; thus, immobilized patients are commonly treated with anticoagulants. We therefore aimed to investigate whether long-term bed rest actually leads to an elevated risk for thromboembolic events. MATERIALS AND METHODS: Eleven healthy men were enrolled in our study (bed rest campaign in MEDES Clinique d'Investigation, Toulouse, France). Besides various standard laboratory methods, we used calibrated automated thrombography (CAT) and thrombelastometry (TEM). Activation of samples with minute amounts of relipidated tissue factor allowed sensitive detection of hyper- or hypocoagulable states. RESULTS: CAT and TEM values were not indicative of bed rest-induced hypercoagulability. On the contrary, several parameters were indicative of a tendency towards a hypocoagulable state. Peak and thrombin formation velocity (VELINDEX) were significantly decreased during bed rest compared to baseline. Coagulation times were significantly increased and alpha angles were significantly decreased, indicating attenuated clot formation. Moreover, F1 + 2 and thrombin/antithrombin complex (TAT) values were significantly decreased during bed rest, indicating suppressed coagulation activation. FVII plasma levels were also significantly decreased during the first week of bed rest. CONCLUSIONS: Our data indicate that the re-ambulation period is associated with a tendency towards hypercoagulability: ttPeak and StartTail were significantly shorter, Peak and VELINDEX were significantly higher compared to baseline. Moreover, plasma levels of F1 + 2, TAT, FVII and FVIII were significantly higher compared to baseline. The results from our study suggest that bed rest by itself is not associated with hypercoagulable states in healthy subjects.

Onset of Thrombin Generation Occurs More Rapidly in Pediatric Patients With Celiac Disease.

OBJECTIVE: Celiac disease (CD) is a risk factor for venous thromboembolism (VTE) and stroke, but the mechanisms are unclear. Continuous measurement of thrombin generation in plasma is a feasible way to detect hypercoagulable changes. The aim of this pilot study was to investigate thrombin generation in pediatric patients with CD compared with pediatric controls. METHODS: Plasma samples were collected from 19 pediatric patients with CD and 20 healthy controls. In each patient diagnosed as having CD, thrombin generation was determined twice by means of calibrated automated thrombography. The first measurement was undertaken when CD was diagnosed; the second measurement was undertaken after normalization of their IgA antitissue transglutaminase antibody (tTG-Ab) titers following a gluten-free diet. In the controls, measurement for TTG-Ab and thrombin generation was undertaken once during recruitment. RESULTS: Patients with CD at diagnosis showed a significantly shorter lag time compared with controls (P < 0.001) and a shorter time-to-peak compared with controls (P < 0.02). These differences were no longer detectable after normalization of TTG-Ab values. The overall amount of generated thrombin, represented by the endogenous thrombin potential (ETP), showed no significant difference between the study groups. CONCLUSIONS: Our results show that alterations in coagulation can be found in untreated CD that may help to explain the described increased risk of stroke or VTE. A shorter lag time in patients with untreated CD indicates a more rapid onset of thrombin generation as a sign of hypercoagulability. ETP, the best predictive parameter for thromboembolic disease, however, was not altered.

Increased procoagulant function of microparticles in pediatric inflammatory bowel disease: role in increased thrombin generation.

OBJECTIVES: Patients with inflammatory bowel disease (IBD) have a higher risk for venous thromboembolism compared with non-IBD subjects. The pathogenic mechanisms of the thrombotic events are not fully understood. We investigated levels of circulating microparticles and their influence on thrombin generation in pediatric patients with IBD during active and quiescent disease compared with healthy controls. METHODS: Plasma samples were collected from 33 pediatric patients with Crohn disease (CD), 20 pediatric patients with ulcerative colitis (UC), and 60 healthy controls. Microparticles' procoagulant activity was measured by enzyme-linked immunosorbent assay, and the dependency of thrombin generation on microparticles-derived tissue factor was determined by means of calibrated automated thrombography. RESULTS: The procoagulant function of microparticles was significantly increased in patients with active and inactive CD, and active UC compared with controls. Endogenous thrombin potential was significantly higher in patients with CD and UC compared with controls. A minor influence of microparticles on thrombin generation was only observed for patients with active UC. CONCLUSIONS: Our study shows increased procoagulant function of microparticles in pediatric patients with active and quiescent CD and active UC compared with controls, but demonstrates that they are not a major cause for the higher thrombin generation in pediatric patients with IBD.

Thrombin receptor levels in platelet concentrates during storage and their impact on platelet functionality.

BACKGROUND: Quality control of platelet (PLT) concentrates is challenging, due to PLT lesions, which are difficult to detect with routine methods. The search for reliable PLT lesion biomarkers is focused on the role of PLTs in primary hemostasis. PLT transfusions also have a significant impact on secondary hemostasis. In this phase, responsiveness of PLTs to small amounts of thrombin is crucial. PAR1 and PAR4 are protease-activated receptors and are responsible for thrombin reactivity of human PLTs. This study should elucidate if levels of those two receptors are changing in PLT concentrates during storage and if those changes have an impact on PLT aggregation and support of thrombin generation. STUDY DESIGN AND METHODS: PLT concentrates from buffy coat preparations were stored in SSP+ solution for 9 days at 22±2°C on a horizontal flatbed agitator, and samples were taken daily for analysis. PAR1 and PAR4 levels were evaluated using Western blot analysis. PLT aggregation was measured using Born aggregometry and specific PAR1 or PAR4 agonists. Thrombin generation was measured using calibrated automated thrombography. RESULTS: Levels of both receptors (PAR1 and PAR4) started to decrease after 5 days of storage. PAR1-mediated PLT aggregation remained constant, whereas PAR4-mediated PLT aggregation decreased with storage time. Rate of thrombin generation was accelerated after 5 days of storage. CONCLUSION: Decreasing levels of PARs in PLT concentrates after 5 days of storage influenced PAR4-mediated, but not PAR1-mediated, aggregation. Thrombin generation with senescent PLTs was increased, which may be attributed to other mechanisms promoting increased phosphatidylserine exposure.

Cardiac catheterization: haemostatic changes in pediatric versus adult patients.

Thrombophilic or haemorrhagic complications are possible adverse events following cardiac catheterization particularly in pediatric patients. It was therefore the aim of our study to compare the cardiac catheterization-related haemostatic changes in children with that in adults. The total of 50 patients was subdivided into Gr I (1-6 years), Gr II (7-18 years), and Gr III (19-58 years). Parameters of coagulation activation, plasma levels of various clotting factors and heparinase-modified thrombelastometry parameters were determined prior and immediately after cardiac catheterization. The haemostatic system of pediatric patients was markedly more affected by the procedure than that of adults. Levels of thrombin/antithrombin complex and prothrombin fragment 1+2 in the post-catheter plasma samples were significantly increased in Grs I and II, not in Gr III. The catheter-related decrease in fibrinogen and F II levels was higher in Gr I than in Grs II and III. F VII levels were significantly decreased in Grs I and II, not in Gr III. The catheter-related prolongation of Coagulation times was highest in Gr I, followed by Gr II and finally Gr III. A significant catheter-related decrease of maximum clot firmness was observed solely in Gr I. Our results show that cardiac catheterisation perturbs the haemostatic system of adults, and, even more pronounced, that of pediatric patients. Thus, our results indicate that children might be at a higher risk for either thrombotic complications or post-operative bleeding events than adults.

Neonatal thrombocytopenia: Thrombin generation in presence of reduced platelet counts and effects of rFVIIa in cord blood.

Healthy neonates exhibit a well-functioning haemostatic system despite peculiarities regarding composition of clotting factors and inhibitors as well as impaired platelet aggregation. Thrombocytopenia and severe bleeding events are feared in sick infants. Recombinant factor VIIa (rFVIIa) is a haemostatic agent used as a last resort in neonates with refractory bleedings. Aim of this study was to investigate in-vitro (i) changes in thrombin generation with different platelet counts, (ii) effects of rFVIIa under conditions of thrombocytopenia and (iii) potentially differing dose-response of rFVIIa in cord blood as a surrogate for neonatal blood compared to adult blood. Thrombin generation parameters were observed in cord blood plasma and adult plasma with various platelet counts, with or without addition of rFVIIa, respectively. Low platelet counts did not influence thrombin generation in cord blood in contrast to adult blood. RFVIIa primarily affected lag time throughout all platelet concentrations. Interestingly, peak height was reduced exclusively in cord blood plasma after addition of rFVIIa. No significant differences regarding dose-response were observed between cord blood and adult blood. In contrast to adult blood, thrombocytopenia in cord blood does not significantly influence thrombin generation. Even at very low platelet counts there is enough negatively charged surface to support rFVIIa action in plasma from cord blood and adult blood in-vitro.

Calibrated automated thrombin generation in normal uncomplicated pregnancy.

Pregnancy is associated with substantial changes in the haemostatic system and a six-fold higher incidence of venous thromboembolism. Conventional global tests, such as prothrombin time and activated partial thromboplastin time, do not definitely detect this hypercoagulable condition. We investigated whether the changes in haemostatic system during pregnancy are reflected in the calibrated automated thrombography (CAT). Thrombin generation was measured in platelet-poor plasma (PPP) of 150 healthy pregnant women without any pregnancy associated diseases by means of CAT. In addition, prothrombin (FII), antithrombin (AT), protein S, protein C, tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), thrombin-antithrombin complex (TAT), and prothrombin fragments 1+2 (F1+2) were measured. Endogenous thrombin potential (ETP) and peak of thrombin generation increased significantly with gestational weeks, while lag time and time to peak remained unchanged. A significant increase of PAI-1, TFPI, F1+2 and TAT as well as a significant decrease of free protein S, protein S antigen, and protein S activity was observed. Levels of AT and protein C remained stable during pregnancy. Division of population in trimester of pregnancy and analysis of differences between the trimesters showed rather similar results. Our study shows that endogenous thrombin potential does increase with duration of normal uncomplicated pregnancy. Whether parameters of continuous thrombin generation will correlate with thrombembolic disease remains to be shown.

Thrombin generation determined by calibrated automated thrombography (CAT) in pediatric patients with congenital heart disease.

INTRODUCTION: Thrombin generation was studied in pediatric patients with congenital heart disease (CHD) undergoing cardiac surgery using the calibrated automated thrombography (CAT) in terms of the lag time until the onset of thrombin formation, time to thrombin peak maximum (TTP), endogenous thrombin potential (ETP), and thrombin peak height. The possible suitability to determine the coagulation status of these patients was investigated. MATERIALS AND METHODS: CAT data of 40 patients with CHD (age range from newborn to 18 years) were compared to data using standard coagulation parameters such as prothrombin (FII), antithrombin (AT), tissue factor pathway inhibitor (TFPI), prothrombin fragment 1.2 (F 1.2), thrombin-antithrombin (TAT), activated partial thromboplastin time (aPTT), and prothrombin time (PT). RESULTS: A significant positive correlation was seen between ETP and FII (p<0.01; r=0.369), as well as between peak height and F II (p<0.01; r=0.483). A significant negative correlation was seen between ETP and TFPI values (p<0.05; r=-0.225) while no significant correlation was seen between peak height and TFPI. A significant negative correlation was seen between F 1.2 generation and ETP (p<0.05; r=-0.254) and between F 1.2 generation and peak height (p<0.05; r=-0.236). No correlation was seen between AT and ETP or peak. CONCLUSIONS: Our data indicate that CAT is a good global test reflecting procoagulatory and inhibitory factors of the hemostatic system in pediatric patients with CHD.

Pediatric patients with congenital heart disease: thrombin generation measured by calibrated automated thrombography.

The aim of this study was to investigate the possible suitability of the calibrated automated thrombography to determine the coagulation status of pediatric patients with congenital heart disease. Thrombin generation was measured in 60 patients with congenital heart disease using the calibrated automated thrombography and compared to data using standard coagulation parameters such as prothrombin, antithrombin, tissue factor pathway inhibitor, prothrombin fragment 1.2 (F 1.2), and activated partial thromboplastin time. A significant positive correlation was observed between prothrombin and the endogenous thrombin potential (P < 0.01; r = 0.295) as well as between prothrombin and peak height (P < 0.01; r = 0.581). A significant negative correlation was seen between tissue factor pathway inhibitor and endogenous thrombin potential (P < 0.01; r = -0.480) and between tissue factor pathway inhibitor and peak height (P < 0.01; r = -0.234). No statistically significant correlation was found between antithrombin and parameters of continuous thrombin generation. Significant correlation was seen neither between activated partial thromboplastin time and F1.2 nor between activated partial thromboplastin time and prothrombin. The data presented here indicate that calibrated automated thrombography measurements determine thrombin generation more accurately and therefore reflect better the coagulation status of pediatric patients with congenital heart disease then standard global coagulation assays such as activated partial thromboplastin time.

Reassessment of autoreactivity of the broadly neutralizing HIV antibodies 4E10 and 2F5 and retrospective analysis of clinical safety data.

BACKGROUND: The broadly neutralizing recombinant human HIV-1 antibodies 4E10, 2F5 and Igh1b12 are reported to have autoreactive potential, which is significant for HIV-1 vaccine development and passive immunotherapy using these antibodies. OBJECTIVE: To investigate the clinical relevance of these findings in subjects receiving passive immunotherapy with these antibodies. METHODS: Four types of investigations were performed: (1) Investigation of clotting parameters in an ongoing clinical study with 4E10, 2F5 and 2G12. (2) Mixing experiments of pooled plasma with the same antibodies. (3) Retrospective analysis of serum from patients who received passive immunotherapy with 4E10, 2F5 and 2G12 either alone or in combination. (4) Assessment of clinical safety data obtained after 418 infusions with these antibodies. RESULTS: Standard clinical assays confirmed that 4E10 showed low-level cross-reactivity with cardiolipin, while previously reported cardiolipin cross-reactivity for 2F5 could not be confirmed. High serum titers of 4E10 induced mild prolongation of the activated partial thromboplastin time, which resolved with the wash out of 4E10. Neither 2F5 nor 2G12 affected coagulation. Repeated high-dose infusions of the monoclonal antibody combination were well tolerated with no incidence for thrombotic complications after 418 infusions in 39 subjects. CONCLUSIONS: Monoclonal antibody 4E10 but not 2F5 or 2G12 showed autoreactive binding specificities. Infusion of 4E10 resulted in transient low anticardiolipin titers. Although an increased thromboembolic risk cannot definitely be excluded, this risk appears to be low and likely depend on underlying disorders.

PFA-100 closure times in preoperative screening in 500 pediatric patients.

Three to five percent of patients undergoing surgery have either an acquired or congenital platelet defect or von Willebrand disease (vWD). The predictive value of preoperative coagulation screening is questionable. PFA-100 is now routinely used in preoperative screening in our pediatric outpatient service. We wanted to assess whether the PFA-100 would help to identify patients with primary haemostatic defects or if the additional use of PFA-100 would add to the problem of unnecessary pathologic preoperative laboratory values resulting in delay of surgical procedure. We investigated 500 children consecutively seen in our outpatient service before surgery. Blood cell count, aPTT, PFA-100 closure times (CT) were done in all patients. If abnormalities were found, the patient was presented to a haemostatic expert. vWF:AG, R:Cof and factor VIII were analysed in all patients with prolonged closure times and APTT values. One hundred twenty-six patients (25.2%) showed abnormalities in APTT and/or PFA-100. Further investigations in 89 of these 126 patients did not yield a specific diagnosis; neither diagnostic criteria for impaired haemostasis were found by questionnaire. None of these 89 patients had a bleeding complication during surgery. Forty-eight patients showed prolonged CTs. Twelve patients with low vWF:AG were detected, 10 of these patients were found by PFA-100. Four of these patients did present with normal APTT values. Our study shows that similar to the APTT the PFA-100 is probably only a good screening method when a haemostatic defect in a patient is clinically likely, especially to screen forVWD, and the test should not be used in general unselective screening.

The respective and combined anticoagulant effects of recombinant human activated protein C, melagatran and heparins using CAT.

INTRODUCTION: Combinations of anticoagulants might be beneficial in some patients with sepsis, but most anticoagulants require specific clotting assays for monitoring. Thrombin generation assay, however, is a global function test of hemostasis. MATERIALS AND METHODS: We performed an in vitro investigation of the respective effects of recombinant human activated protein C (rhAPC) alone and in combination with either melagatran (a new direct thrombin inhibitor), unfractionated heparin (UH) or low molecular weight heparin (LMWH) in varying concentrations on the thrombin generation (TG) using the calibrated automated thrombography. RESULTS: RhAPC, UH, LMWH and melagatran dose-dependently prolonged the lag time and the time to peak, and significantly suppressed the endogenous thrombin potential (ETP). Combined application of rhAPC with either melagatran, UH or LMWH induced an additive prolongation of the lag time; this effect was more pronounced in a combination of rhAPC with UH or LMWH. CONCLUSION: In our in vitro study adding either melagatran, UH or LMWH augmented the capacity of rhAPC to suppress thrombin generation in human plasma. These findings suggest that patients with severe sepsis might benefit from a treatment with combinations of anticoagulant agents.

Polyphosphate in Neonates: Less Shedding from Platelets and Divergent Prothrombotic Capacity Due to Lower TFPI Levels.

Background: The neonatal hemostatic system exhibits a fragile balance featuring lower levels of clotting factors as well as inhibitors. Neonatal platelets show in-vitro hypoaggregability, but neonates exhibit well-functioning primary and secondary hemostasis despite this impairment. Recently, polyphosphate shed by activated platelets has been shown to induce a prothrombotic shift on the plasmatic coagulation system of adults. The impact of platelet derived polyphosphate might differ in neonates due to aforementioned peculiarities. Aims: We aimed to comparatively determine polyphosphate content and release from adult and neonatal platelets and to determine its impact on thrombin generation in plasma from adult and cord blood. Methods: Polyphosphate was extracted from adult and neonatal platelet lysates and releasates using silica spin-columns and quantified with a DAPI based fluorescence assay. The impact of exogenous polyphosphate in various concentrations (208-0.026 µg/ml) on thrombin generation was evaluated in plasma from adult and cord blood as well as in adult plasma with reduced tissue factor pathway inhibitor (TFPI) levels using calibrated automated thrombography. Results: Polyphosphate content was comparable in both groups, but the fraction of released polyphosphate upon stimulation with thrombin receptor activating peptide was lower in neonatal samples (adult: 84.1 ± 12.9%; cord: 58.8 ± 11.2%). Relative impact of polyphosphate on lag time of thrombin generation was higher in adult samples compared to samples from cord blood (adult: 41.0% [IQR: 35.2-71.8%] of vehicle; cord: 73.4% [IQR: 70.2-91.4%] of vehicle). However, in samples from cord blood, lower concentrations of polyphosphate were required to obtain maximal impact on thrombin generation (adult: 26 µg/ml; cord: 0.814 µg/ml). PolyP affected thrombin generation in adult plasma similarly to cord plasma, when the TFPI concentration was reduced to neonatal levels. Conclusion: Differences in the impact of polyphosphate on adult and neonatal coagulation are largely caused by differences in TFPI levels. Lower polyphosphate release from neonatal platelets, but lower optimum concentration to drive neonatal plasmatic hemostasis emphasizes the well-matched, but fragile interplay between platelets and coagulation in newborns. A potential developmental mismatch should be considered when transfusing adult platelets into neonates.

Anticoagulant action of low, physiologic, and high albumin levels in whole blood.

Albumin is the most abundant plasma protein. Critical illness is often associated with altered, predominately decreased, serum albumin levels. This hypoalbuminaemia is usually corrected by administration of exogenous albumin. This study aimed to track the concentration-dependent influence of albumin on blood coagulation in vitro. Whole blood (WB) samples from 25 volunteers were prepared to contain low (19.3 ± 7.7 g/L), physiological (45.2 ± 7.8 g/L), and high (67.5 ± 18.1 g/L) levels of albumin. Haemostatic profiling was performed using a platelet function analyzer (PFA) 200, impedance aggregometry, a Cone and Platelet analyzer (CPA), calibrated automated thrombogram, and thrombelastometry (TEM). Platelet aggregation-associated ATP release was assessed via HPLC analysis. In the low albumin group, when compared to the physiological albumin group, we found: i) shortened PFA 200-derived closure times indicating increased primary haemostasis; ii) increased impedance aggregometry-derived amplitudes, slopes, ATP release, as well as CPA-derived average size indicating improved platelet aggregation; iii) increased TEM-derived maximum clot firmness and alpha angles indicating enhanced clot formation. TEM measurements indicated impaired clot formation in the high albumin group compared with the physiological albumin group. Thus, albumin exerted significant anticoagulant action. Therefore, low albumin levels, often present in cancer or critically ill patients, might contribute to the frequently occurring venous thromboembolism.

Orthostatic Challenge Shifts the Hemostatic System of Patients Recovered from Stroke toward Hypercoagulability.

Aims: The objective of our study was to assess the effects of orthostatic challenge on the coagulation system in patients with a history of thromboembolic events and to assess how they compared with age-matched healthy controls. Methods: Twenty-two patients with histories of ischemic stroke and 22 healthy age-matched controls performed a sit-to-stand test. Blood was collected prior to- and at the end of- standing in the upright position for 6 min. Hemostatic profiling was performed by determining thrombelastometry and calibrated automated thrombogram values, indices of thrombin generation, standard coagulation times, markers of endothelial activation, plasma levels of coagulation factors and copeptin, and hematocrit. Results: Orthostatic challenge caused a significant endothelial and coagulation activation in patients (Group 1) and healthy controls (Group 2): Plasma levels of prothrombin fragment F1+2 were increased by approximately 35% and thrombin/antithrombin-complex (TAT) increased 5-fold. Several coagulation variables were significantly altered in Group 1 but not in Group 2: Coagulation times (CTs) were significantly shortened and alpha angles, peak rate of thrombin generation (VELINDEX), tissue factor (TF) and copeptin plasma levels were significantly increased (comparison between standing and baseline). Moreover, the shortening of CTs and the rise of copeptin plasma levels were significantly higher in Group 1 vs. Group 2 (comparison between groups). Conclusion: The coagulation system of patients with a history of ischemic stroke can be more easily shifted toward a hypercoagulable state than that of healthy controls. Attentive and long-term anticoagulant treatment is essential to keep patients from recurrence of vascular events.

Age-dependency of thrombin generation measured by means of calibrated automated thrombography (CAT).

Many coagulation parameters, such as PT or aPTT, show age-dependency. In this study we investigated if the generation of thrombin, possibly better reflecting overall haemostasis, shows an age-dependency. Thrombin generation was measured in platelet poor plasma of 121 children and 86 adults at different ages by means of calibrated automated thrombography (CAT). Correlation analysis shows that endogenous thrombin potential (ETP) (r = 0.702), lag time (r = -0.266), peak (r = 0.533) and time to peak (r = -0.214) are significantly correlated with age (p < 0.01). 'Younger' (age limit 35 years) and 'older' adults were compared with groups of children and adolescent aged between 0.5 and 6 years, 7 and 11 years and 12 to 17 years by means of the Mann-Whitney-U-Test. ETP values of all children and adolescents were significantly lower than those of adults. In the group of the youngest children, additionally shorter lag times and times to peak and lower peak levels differed significantly from those of adults. In the group of 7- to 11-year-old children, lag times were significantly longer than those of both groups of adults, while lower peaks and longer times to peak differed only from the group of the 'older' adults. In the group of the 12- to 17-year-olds, the values of ETP were lower than those of adults. In addition, both adult groups differed significantly in all studied parameters. Our results show an age-dependency of thrombin generation even beyond the juvenile period. If thrombin generation measurement is to be used as a routine method, age has to be considered. Assuming that thrombin potential is an indicator for the risk of thrombosis, our findings are in accordance with the observation of an increased incidence of thrombembolic disease with higher age.

Non-enzymatic quantification of polyphosphate levels in platelet lysates and releasates.

Inorganic polyphosphate has been shown to be shed upon platelet activation inducing prothrombotic stimuli on the coagulation system. Several methods have been published to detect and quantify polyphosphate in various cells and tissues, but evaluation of platelet content has only been achieved by indirect detection of orthophosphate after enzymatic digestion, thus, relying heavily on specificity of an exopolyphosphatase that is not commercially available. We present a non-enzymatic method for quantification of platelet-derived polyphosphate featuring optimized extraction on silica spin-columns, followed by specific fluorescence detection using DAPI. This allowed us to quantify polyphosphate in platelet lysates, but also in releasates of TRAP-activated platelets for the first time. Extraction of exogenous polyphosphate from buffer and sample matrices resulted in quantitative yields while removing matrix effects observed with direct fluorescence detection. Treatment of eluted fractions with phosphatase completely abrogated polyphosphate-specific fluorescence arguing for no additional compounds influencing the fluorescence detection. This was confirmed by no change in fluorescence intensity in samples previously treated with DNase and RNase. Taken together, we developed a robust and easily standardizable method to quantify polyphosphate in platelet lysates and releasates that will facilitate polyphosphate related investigations of platelet physiology and coagulation.

Only minor changes in thrombin generation of children and adolescents with type 1 diabetes mellitus - A case-control study.

BACKGROUND: Micro- and macrovascular diseases are frequent complications in patients with diabetes. Hypercoagulability may contribute to microvascular alterations. OBJECTIVE: In this study, we investigated whether type 1 diabetes in children is associated with a hypercoagulable state by performing a global function test of coagulation - the thrombin generation assay. SUBJECTS: 75 patients with type 1 diabetes aged between 2 and 19years were compared to an age-matched healthy control group. Diabetes patients were divided into high-dose and low-dose insulin cohorts with a cut-off at 0.8Ukg-1d-1. METHODS: Measurements were performed with platelet poor plasma using Calibrated Automated Thrombography and 1 pM or 5 pM tissue factor. Additionally, we quantified prothrombin fragments F1+2, thrombin-antithrombin complex, prothrombin, tissue factor pathway inhibitor, and antithrombin. RESULTS: Patients with type 1 diabetes exhibited a significantly shorter of lag time as well as decreased thrombin peak and endogenous thrombin potential compared to control subjects with 5 pM but not with 1 pM tissue factor. In high-dose insulin patients peak thrombin generation was higher and time to peak shorter than in low-dose patients. Thrombin-antithrombin complex was decreased in patients with type 1 diabetes, whereas prothrombin fragments F1+2 was comparable in both groups. Thrombin generation parameters did not correlate with parameters of metabolic control and the duration of diabetes. CONCLUSIONS: Taken together, we found only minor changes of thrombin generation in children and adolescents with type 1 diabetes which - in contrast to type 2 diabetes - do not argue for a hypercoagulable state.

Additive effects of anticoagulants: recombinant human activated protein C and heparin or melagatran, in tissue factor-activated umbilical-cord plasma.

Severe sepsis in children or adults may cause a life-threatening coagulopathy, with widespread consumption of activated protein C (APC); recombinant human APC (rhAPC) is a promising candidate anticoagulant treatment. We investigated the effects of rhAPC and other anticoagulants on coagulation triggered by adding small quantities of lipidated tissue factor to human umbilical-cord plasma in vitro. rhAPC, unfractionated heparin (UH), and melagatran (a direct thrombin inhibitor) were studied individually, and in combinations of rhAPC with either UH or melagatran. rhAPC alone dose-dependently prolonged the activated partial-thromboplastin time (aPTT) but not the prothrombin time (PT), and dose-dependently suppressed two indices of thrombin generation, namely prothrombin fragment F 1.2 (F 1.2) generation and thrombin-antithrombin (TAT) complex formation. UH alone dose-dependently prolonged the aPTT but not the PT, while melagatran alone dose-dependently prolonged both the aPTT and the PT. Adding either UH or melagatran dose-dependently augmented the capacity of rhAPC to suppress F 1.2 generation (with addition of UH showing a greater effect) and TAT formation (with addition of melagatran showing a greater effect). Both the capacity of UH to prolong the aPTT and the capacity of melagatran to prolong the aPTT and the PT were augmented by adding rhAPC. In our in-vitro study, adding either UH or melagatran augmented the capacity of rhAPC to suppress thrombin generation in human umbilical-cord plasma, with the anticoagulant effect of melagatran being more predictable than that of UH. Hence, combining rhAPC with melagatran might be a valuable therapeutic option in patients with severe sepsis.