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.

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.

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.

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.

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.

Effects of Exercise and Nutrition on the Coagulation System During Bedrest Immobilization.

Immobilization in hospitalized medical patients or during simulation of spaceflight induced deconditioning has been shown to be associated with loss of muscle mass and bone. Resistance vibrating exercise (RVE) and/or high protein diet are countermeasures, which are capable of mitigating the adverse effects of immobilization. We investigated the effect of these countermeasures on the coagulation system. Two groups of volunteers, each of whom performed such countermeasures, were enrolled in the study. Volunteers, who did nothing while bed rested, served as controls. The berest and the intervention protocols were carried out at Clinique d' Investigation, MEDES, Toulouse, France. Eleven healthy men volunteered for this randomized crossover study. The subjects underwent 21 day of 6° head down bed rest (HDBR) followed by a washout period of 4 months. The first group followed an exercise schedule using resistance-vibrating exercise (RVE group). The second group also used the RVE but complemented it with high-protein supplement diet (NeX group). The third group only did bed rest. The highly sensitive methods calibrated automated thrombography (CAT) and thrombelastometry (TEM) were applied to monitor hemostatic changes. In all 3 groups, the hemostatic system shifted toward hypocoagulability during bed rest. For example, peak and thrombin formation velocity (VELINDEX) reduced in this period. Interestingly, a tendency toward hypercoagulation was observed during re-ambulation. In all 3 groups, ttPeak and StartTail were reduced, and Peak and VELINDEX (except in the RVE group) were significantly higher in relation to baseline values. Influence of bed rest on the coagulation system in the 2 groups performing countermeasures (RVE and NeX group) was the same as in the control bed-rested group. Clotting does not seem to be worsened by prolonged immobilization, or by countermeasures such as RVE/exercise or high-protein supplementation during immobilization. Therefore, only hospitalized medical patients at an elevated risk for thrombosis should be treated with anticoagulants. However, clinicians have to be aware that the re-ambulation period following immobilization might be associated with an elevated risk of thrombotic events.

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.

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.

Prostaglandin E2 levels and platelet function are different in cord blood compared to adults.

Neonatal platelets support primary haemostasis and thrombin generation as well as adult platelets, despite observable hypoaggregability in vitro. High prostaglandin E2 levels at accouchement could account for inhibited platelet function via the EP4 receptor. We set out to determine prostaglandin E2 plasma levels in cord blood of healthy neonates and evaluate the impact of prostaglandin E2 on platelet function in adult and cord blood samples. Prostaglandin E2 plasma levels were measured in cord blood and venous adult blood using GC-MS. Impact of prostaglandin E2 on platelet aggregation was measured by spiking cord blood and adult samples. Contributions of EP3 and EP4 receptors were evaluated using respective antagonists. Intracellular cAMP concentrations were measured using a commercial ELISA-kit. Prostaglandin E2 plasma levels were substantially higher in cord blood than in adult samples. Spiking with prostaglandin E2 resulted in a slight but consistent reduction of platelet aggregation in adult blood, but response to PGE2 was blunted in cord blood samples. Aggregation response of spiked adult samples was still higher than with non-spiked cord blood samples. Blockage of EP4 receptors resulted in improved platelet aggregation in adult platelets upon prostaglandin E2 spiking, while aggregation in cord blood samples remained unaltered. Intracellular cAMP concentrations after preincubation with prostaglandin E2 were only increased in adult samples. In conclusion, very high prostaglandin E2 concentrations in cord blood affect platelet function. This effect may partially explain neonatal platelet hypoaggregability. Peak levels of prostaglandin E2 can potentially protect against birth stress-induced platelet activation.

Low endogenous thrombin potential in trained subjects.

INTRODUCTION: A paradox seems to exist: exercising leads to clotting activation in conventional clotting tests, but exercising persons have a low risk of thrombosis. In this study we tried to evaluate the effect of exercise performance status on in vitro plasma thrombin generation, which represents an overall function test of hemostasis. MATERIALS AND METHODS: We compared 56 trained subjects to 98 healthy age matched sedentary volunteers. Blood samples were analyzed for thrombin generation using calibrated automated thrombography. Microparticles were quantified using ELISA. Additionally prothrombin fragments 1 + 2, thrombin-antithrombin complex, tissue factor pathway inhibitor, antithrombin and prothrombin were measured. The group of the trained subjects performed an incremental cycle-ergometer exercise test after taking the blood sample. RESULTS: A significantly lower endogenous thrombin potential was observed in the group of the trained subjects compared to the sedentary individuals (p = 0.007). Microparticles (ELISA) were significantly lower in the trained subjects compared to the sedentary subjects (p = 0.001). Prothrombin fragments 1 + 2 (p < 0.001) and thrombin-antithrombin complex (p = 0.01) were significant higher in the trained subjects and antithrombin (p = 0.02) as well as prothrombin (p < 0.0001) were significantly lower in this group, whereas tissue factor pathway inhibitor values did not show significant differences. Both maximal and submaximal power output was significantly negatively related to endogenous thrombin potential (r = -0.43, r = -0.45) and thrombin peak (r = -0.44, r = -0.42). CONCLUSIONS: Trained subjects have a lower endogenous thrombin potential than sedentary subjects possibly explaining the lower incidence of thrombosis in this group despite a higher acute clotting activation during strenuous exercise.

Effects of a single bout of walking exercise on blood coagulation parameters in obese women.

Obesity is associated with increased prevalence of thromboembolic events. We aimed to investigate whether obese women might benefit from vigorous aerobic exercise. Forty-two overweight and obese women performed a 30-min walking exercise test (treadmill ergometer) at an intensity of 70% of individual peak oxygen uptake. Blood samples were collected before and immediately after exercise. Thrombelastometry and platelet function measurements were performed on whole blood. Standard coagulation times, thrombin generation curves, markers of thrombin generation, fibrinolytic parameters, plasma levels of pro- and anticoagulatory factors, and microparticle procoagulant activity were determined in platelet-poor plasma samples. Thrombelastometry revealed a significant prolongation of clot formation time (P = 0.037) and a significant deceleration of fibrin build up (alpha angle, P = 0.034) after exercise. Calibrated automated thrombography revealed a significant exercise-induced decrease in endogenous thrombin potential (P = 0.039). Moreover, thrombin formation stopped earlier postexercise, reflected in shortened StartTail (P = 0.046). Significantly elevated tissue-plasminogen activator levels (P = 0.001) indicate an exercise-induced activation of the fibrinolytic system. White blood cell count increased significantly from pre- to postexercise (P = 0.045), indicating a mild exercise-induced leukocytosis. The results of this study demonstrate that vigorous aerobic exercise might be a suitable tool to protect obese women from thrombotic events. We show that a single bout of vigorous aerobic exercise is clearly associated with an activation of the fibrinolytic system and a decreased readiness of the postexercise samples to form a clot and to generate thrombin, the pivotal enzyme of hemostasis.

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.

Coagulation changes during presyncope and recovery.

Orthostatic stress activates the coagulation system. The extent of coagulation activation with full orthostatic load leading to presyncope is unknown. We examined in 7 healthy males whether presyncope, using a combination of head up tilt (HUT) and lower body negative pressure (LBNP), leads to coagulation changes as well as in the return to baseline during recovery. Coagulation responses (whole blood thrombelastometry, whole blood platelet aggregation, endogenous thrombin potential, markers of endothelial activation and thrombin generation), blood cell counts and plasma mass density (for volume changes) were measured before, during, and 20 min after the orthostatic stress. Maximum orthostatic load led to a 25% plasma volume loss. Blood cell counts, prothrombin levels, thrombin peak, endogenous thrombin potential, and tissue factor pathway inhibitor levels increased during the protocol, commensurable with hemoconcentration. The markers of endothelial activation (tissue factor, tissue plasminogen activator), and thrombin generation (F1+2, prothrombin fragments 1 and 2, and TAT, thrombin-antithrombin complex) increased to an extent far beyond the hemoconcentration effect. During recovery, the markers of endothelial activation returned to initial supine values, but F1+2 and TAT remained elevated, suggestive of increased coagulability. Our findings of increased coagulability at 20 min of recovery from presyncope may have greater clinical significance than short-term procoagulant changes observed during standing. While our experiments were conducted in healthy subjects, the observed hypercoagulability during graded orthostatic challenge, at presyncope and in recovery may be an important risk factor particularly for patients already at high risk for thromboembolic events (e.g. those with coronary heart disease, atherosclerosis or hypertensives).

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.

Effect of rivaroxaban, in contrast to heparin, is similar in neonatal and adult plasma.

Neonates have lower levels of clotting factors as well as inhibitors. Effects of heparin in neonatal plasma differ from those in adult plasma, and dosage recommendations cannot be extrapolated from adult trials. Riveroxaban is an oral direct factor Xa inhibitor that can achieve an anticoagulant effect without dependence on anti-thrombin. We performed comparative thrombin generation measurements in neonatal cord and adult plasma with different concentrations of unfractionated heparin and rivaroxaban to evaluate the potential of rivaroxaban in neonatal anticoagulation. The impact of heparin or rivaroxaban on the neonatal and adult hemostatic system was determined measuring calibrated automated thrombin generation and activated partial thromboplastin time in platelet-poor plasma pools of 15 adult samples or 15 neonatal cord samples and addition of seven increasing concentrations of heparin or rivaroxaban, respectively, to the pooled samples. Lag time, time to peak and peak height of thrombin generation in neonatal cord samples were significantly less affected by different heparin concentrations than in adult samples, whereas the impact on reduction of endogenous thrombin potential was higher in neonatal cord samples. The impact of rivaroxaban on thrombin generation parameters showed better comparability between neonatal cord and adult samples. Both anticoagulants showed the same differences in activated partial thromboplastin time between adult and neonatal plasma at each concentration. Rivaroxaban shows a very similar pattern in neonatal cord and adult plasma in suppressing thrombin generation and prolonging activated partial thromboplastin time values, suggesting that dose finding may be easier with rivaroxaban in neonates.

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.

Microparticles in newborn cord blood: slight elevation after normal delivery.

INTRODUCTION: Microparticles formed during delivery may add to the well functioning hemostasis, but also to hypercoaguability in the newborn. We wanted to investigate whether microparticles in newborn cord plasma differ from those in adult plasma in terms of concentration, procoagulant activity, and effect on thrombin generation. MATERIALS AND METHODS: Three different techniques were used to analyze microparticles. To enumerate and characterize microparticles, flow cytometry and ELISA, based on the prothrombinase reaction, were used. The effect of microparticles derived tissue factor on thrombin generation was measured indirectly by calibrated automated thrombography in newborn cord and adult platelet free plasma. RESULTS: The flow cytometric measurements of microparticles showed no significantly increased microparticle concentration in newborn cord compared with adult plasma. By the use of ELISA a significantly increased procoagulant activity of microparticles was found in newborn cord plasma as compared to adult plasma. Initiation of thrombin generation by adding phospholipids alone suggested a higher microparticle activity in newborn cord plasma than in adult plasma. CONCLUSIONS: Our results show a higher impact of microparticles on the hemostatic system in newborn cord plasma than in adult plasma in terms of activity, but not concentration. calibrated automated thrombography and ELISA suggest an increased microparticle activity in newborn cord plasma, but comparable results in microparticle number as determined by flow cytometry argue against strong platelet activation during birth.

Thrombin generation in pediatric patients with Crohn's disease.

BACKGROUND: Patients with inflammatory bowel disease (IBD) have an increased risk of thromboembolic complications. The pathogenesis of IBD is not really clear and a high thrombin activity might contribute to the pathogenesis. We measured thrombin generation by means of calibrated automated thrombography (CAT), a new tool better reflecting overall hemostasis, in children with Crohn's disease (CD) during active and inactive disease and compared it to conventional markers of activity. We wanted to see whether children with CD have a higher potential for thrombin generation and if there is a correlation between hypercoagulability and disease activity. METHODS: Plasma samples were collected from 22 patients with CD and from 61 healthy children. Thrombin generation was measured by means of CAT. The disease activity was estimated using the Pediatric Crohn's Disease Activity Index (PCDAI). In addition, F1+2, TAT, tissue factor pathway inhibitor (TFPI), fibrinogen, prothrombin (FII), antithrombin (AT), erythrocyte sedimentation rate (ESR), platelet count, α2-globulin, and orosomucoide were measured. RESULTS: In all patients we found a significantly higher endogenous thrombin potential (ETP) and higher peak values during active disease. In accordance with this we also found significantly higher mean ETP values during active disease compared with the control group. We observed a significantly positive correlation between PCDAI and thrombin generation parameters. CONCLUSIONS: Our study clearly shows that the active state of CD in children is associated with the potential for high thrombin generation, but this seems to be caused mainly by the inflammatory process and not by a preexisting propensity for high thrombin generation.