[Is the DDAVP-test helpful to diagnose children?]. / Trägt ein DDAVP-Test bei Kindern zur Diagnose bei?

UNLABELLED: It is very difficult to determine if patients with a moderate low level of VWF parameters have mild disease or if they are just low normal (so called grey area of VWD). This applies particularly to pediatrics, because it is difficult to evaluate the bleeding history of children. Al our centres every child diagnosed with vWD gets DDAVP to test the response for it. This study was done to evaluate the DDAVP- test as a diagnostic tool. PATIENTS, METHODS: A retrospective analysis of data obtained with routine DDAVP administration for test purposes in 52 patients with borderline von Willebrand disease at the haemophilia centre Graz was done. The increase of VWF:Ag, VWF:RiCof and FVIII:C has been document and compared. RESULTS: All of our patients had a very good response after application of DDAVP. The increase of VWF:Ag, VWF:RiCof and FVIII:C was compared in patients with positive and negative bleeding anamneses. The patients with positive anamneses had significantly lower parameters at the beginning. The increase of VWF parameters did not differ significantly between the groups at the different time-points. These results demonstrate that a positive anamnesis is not significantly associated with a lower increase. On the other side a high increase is not associated with a negative anamnesis. CONCLUSION: It is not possible to use the DDAVP test as a diagnostic tool for patients within the diagnostic grey area of VWD.

High microparticle concentration in cord plasma.

UNLABELLED: We investigated if differences in the microparticle concentration and activity between newborn cord plasma and adult plasma exist. METHODS: To enumerate and characterize microparticles (MP) FACS and ELISA were used.The effect of microparticles derived tissue factor (TF) on thrombin generation was measured indirectly by CAT (calibrated automated thrombography). RESULTS: The flow cytometric measurements revealed an 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 resulted in a significant lower prolongation of the lag time, time to peak in cord plasma, while the decrease of endogenous thrombin potential (ETP) and peak was comparable between newborns and adults. CONCLUSION: Our results show a higher impact of microparticles on the haemostatic system of newborns than on that of adults. The three methods suggest a somewhat increased microparticle activity in newborn cord plasma, but argue against strong platelet activation during birth.

Comparative evaluation of PAR1, GPIb-IX-V, and integrin αIIbß3 levels in cord and adult platelets.

BACKGROUND: Newborn platelets show hyposensitivity in vitro to many important agonists. However, sensitivity of platelets to these agonists is crucial for a functional clot formation. Nevertheless newborns have an excellent hemostasis. Hence, we examined levels of PAR1 thrombin receptor, GPIb-IX-V (CD42b), and Integrin αIIbß3 in newborn and adult platelets using Western blot analysis. Materials, methods: Platelets of adult and cord blood were isolated, washed, and lysed. Protein samples were separated by SDS-PAGE and blotted on nitrocellulose membranes. Receptors were visualized using immunodetection and evaluated densitometrically. Statistical analysis was performed using SPSS 16.0. RESULTS: We found significantly lower levels of PAR1-receptors and higher levels of CD42b in newborn platelets as compared to adult platelets. Levels of Integrin αIIbß3 in newborn platelets were comparable to adult platelets. CONCLUSION: A lower content of PAR1-receptors explains very well the hyposensitivity of cord platelets to thrombin. Higher levels of CD42b may additionally support the effect of larger more adhesive multimeric vWF in newborn plasma.

Relationship between thrombin generation and carotid intima-media thickness.

UNLABELLED: Increasing evidence indicates that thrombin plays a role not only in thrombosis but also in the progression of atherosclerosis. AIM: The relationship between thrombin generation and intima-media thickness (IMT) as an index of subclinical atherosclerosis was investigated. Participants, material, methods: We examined 163 asymptomatic middle-aged persons free of overt clinical atherosclerotic disease. They underwent ultrasonography of the common carotid arteries. In addition, thrombin generation was measured by means of CAT (calibrated automated thrombography). For our study we divided the healthy study participants into three age groups (<45, 45-60 and >60 years). RESULTS: A significant positive correlation was seen between endogenous thrombin potential (ETP) (p = 0.012), time to peak (TTP) (p = 0.033) start tail (p = 0.007) and carotid IMT in the group of healthy volunteers younger than 45 years. CONCLUSION: We demonstrated that in adults younger than 45 years without clinically overt atherosclerotic disease ETP was significantly associated with carotid IMT. It is tempting to speculate that ETP may serve as an index for subclinical atherosclerosis in persons below 45 years.

Newborn platelets: lower levels of protease-activated receptors cause hypoaggregability to thrombin.

Newborn platelets show in vitro hypoaggregability to thrombin. Sensitivity of platelets to such a potent agonist is crucial for a functional clot formation. Nevertheless, newborns have an excellent hemostasis. We wanted to investigate the reason for this impairment by comparatively analysing levels of receptors known to be involved in thrombin signaling in newborn and adult platelets. Platelets of adult and cord blood were isolated, washed, and lysed. Resulting protein samples were separated by SDS-PAGE and blotted on nitrocellulose membranes. Receptors were visualized using immunodetection and evaluated densitometrically. Thrombin receptor activating peptide induced platelet aggregation was measured in citrated whole blood on a Multiplate analyzer. Statistical analysis was performed using SPSS 16.0. Significantly lower levels of protease-activated receptors (PAR1, PAR4) and higher levels of glycoprotein Ibα (GPIbα) were found in newborn platelets as compared to adult platelets. Platelet aggregation was lower in newborn samples than in adult controls and values correlated with the corresponding PAR levels. Our results suggest that lower levels of protease-activated receptors contribute to the poor thrombin induced aggregation observed with newborn platelets, which can not be compensated by higher levels of GPIbα.

Calibrated automated thrombin generation in paediatric patients with inflammatory bowel disease.

UNLABELLED: In adults, inflammatory bowel disease (IBD) is associated with an increased risk of thromboembolic complications. The pathogenesis of IBD is not really clear and a high thrombin activity might contribute to disease progression. We wanted to see whether children with IBD have a higher thrombin generation (TG). PATIENTS, MATERIAL, METHODS: Plasma samples were collected of 20 patients with IBD and of 60 healthy controls (age range from 10 to 19). TG was measured by means of Calibrated automated thrombography (CAT). The disease activity was estimated, using the Pediatric Crohn's Disease Activity Index (PCDAI) for Crohn's disease and the Pediatric Ulcerative Colitis Disease Activity Index (PUCAI) for Ulcerative Colitis. In addition, we investigated F1+F2, TAT, TFPI and fibrinogen. RESULTS: There was a significant increase of endogenous thrombin potential (ETP), lag time and time to peak in patients with IBD, while peak showed no difference to healthy controls. ETP and F1+F2 in children with IBD also showed a significant correlation with PCDAI (PUCAI) and fibrinogen. CONCLUSION: IBD in children is associated with high TG, but this seems to be caused mainly by the inflammatory process and not by any individual disposition.

No differences in support of thrombin generation by neonatal or adult platelets.

UNLABELLED: Newborns have, despite low clotting factors and poor in vitro platelet function, a well functioning hemostasis. The reason for this is still not completely clear. The aim of our study was to investigate whether phospholipids in neonatal platelets differ from those in adult platelets in their total amount, in their exposure on the platelet surface, and their effect on thrombin generation (TG). METHODS: Clotting times of newborn and adult platelet-rich plasma were measured. Effect of newborn and adult platelets on TG was measured by means of CAT (calibrated automated thrombography). In addition, the effect of newborn and adult platelets with or without stimulation by ionophor on TG was measured in a purified prothrombinase complex. Phosphatidylserine-exposure (PS) of newborn and adult platelets was measured by flow cytometry of annexin V binding. The amount of phospholipids (PL) was determined by means of mass spectrometry. RESULTS: Clotting times of platelet-rich plasma (PRP) of newborns stimulated with ionophor showed a significant lower reduction of clotting time than in adult PRP. No differences in the support of TG between neonatal and adult platelets were found in neonatal or adult plasma by means of CAT. In the purified system TG was increased by adding ionophor stimulated platelets but no difference was evident between stimulated newborn and adult platelets. Flow cytometric analysis showed no difference in annexin V binding between adult and newborn platelets. The results of mass spectrometry showed a very similar amount and pattern of PL of adult and newborns platelets. CONCLUSION: Our results do not provide any evidence that a different PL content or expression of neonatal platelets may alter TG in neonates.

Thrombin generation in paediatric patients with congenital heart disease. Determination by calibrated automated thrombography.

UNLABELLED: Thrombin generation was studied in paediatric 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 suitability to determine the coagulation status of these patients was investigated. PATIENTS, MATERIAL, 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 FII (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: CAT is a good global test reflecting procoagulatory and inhibitory factors of the haemostatic system in paediatric patients with CHD.

Thrombin generation before and after multicomponent blood collection.

The development of apheresis technology has increased efficiency in donor blood use by collecting specific blood components in several combinations. The question of donor safety raised by the contact of donor blood with foreign, only in part biocompatible surfaces remains. The aim of this study was to estimate the effect of multicomponent blood collection on thrombin generation performing an overall function test of coagulation. DONORS, METHODS: 26 blood donors were included. Per apheresis two units of platelets and one unit of RBCs were collected by two cell separators (Amicus and Trima Accel). Each donor underwent the procedure on both apheresis systems. Samples were collected before, immediately after, and 48 hours after apheresis. Thrombin generation was measured by means of calibrated automated thrombography (CAT). RESULTS: CAT-data changed only slightly and no significant changes were seen before, immediately after, and 48 hours after apheresis. The parameters did not differ significantly between the two different apheresis devices. CONCLUSION: No change in parameters of continuous thrombin generation occurred, suggesting that apheresis did not lead to severe alterations in the haemostatic system.

Thrombin generation in factor VIII-depleted neonatal plasma: nearly normal because of physiologically low antithrombin and tissue factor pathway inhibitor.

BACKGROUND: Bleeding in hemophilic neonates has a low incidence. A possible explanation for this could be the peculiarities of the neonatal hemostatic system, especially low levels of the inhibitors tissue factor pathway inhibitor (TFPI) and antithrombin (AT). OBJECTIVE: We investigated the influence of an elevation of these inhibitors to adult levels on the thrombin generation (TG) in normal neonatal plasma and factor (F) VIII-depleted neonatal plasma by means of incubation with anti-FVIII-antibodies. PATIENTS/METHODS: TG was measured after activation with low amounts of tissue factor (TF) by using Calibrated Automated Thrombography. RESULTS: TG in FVIII-depleted neonatal plasma was nearly as high as in normal neonatal plasma. TG decreased after elevation of AT in both neonatal plasmas. After elevation of TFPI TG decreased much more in FVIII-depleted neonatal plasma than in normal neonatal plasma. After elevation of both inhibitors their synergistic effect led to a stronger decrease of TG in FVIII-depleted neonatal plasma. TG measured in plasma of one hemophilic newborn showed the same pattern as in FVIII-depleted neonatal plasma. CONCLUSION: Our observation provides a biochemical basis for the rare bleeding in hemophilic neonates and shows the important role of the natural inhibitors in the hemostatic system of hemophilic patients.

Low tissue factor pathway inhibitor (TFPI) together with low antithrombin allows sufficient thrombin generation in neonates.

Neonates have an excellent hemostasis despite, in comparison to adults, markedly decreased and delayed ability to generate thrombin. Only 30-50% of peak adult thrombin activity can be produced in neonatal plasma by means of conventional in vitro assays. We show that in contrast to conventional activation, activation with small amounts of lipidated tissue factor (<10 pmol L(-1)) results in shorter clotting times and faster activated factor X- and thrombin generation in neonates compared with adults due to the concomitant action of low tissue factor pathway inhibitor and antithrombin. The concentrations of both inhibitors in cord plasma are approximately 50% of the respective adult values. After addition of 2.5 pmol L(-1) lipidated tissue factor, cord plasma clotted approximately 90 s earlier than adult plasma and the amount of free thrombin generated was approximately 90% of adult value (291 +/- 14 vs. 329 +/- 16 nmol L(-1) min(-1), P < 0.01). Our results might help to explain the clinically observed excellent hemostasis of neonates despite low levels of procoagulant factors.

Respective roles of factors II, VII, IX, and X in the procoagulant activity of FEIBA.

Activated prothrombin complex concentrates (APCCs) are effective in the therapy of bleeding episodes in hemophilic patients with inhibitors. We investigated the respective roles of factor II, factor VII, factor IX, and factor X in the procoagulant activity of the APCC FEIBA. Factor II, factor VII, factor IX, and factor X were reduced in platelet-poor plasma, and the thrombin potential (TP) was determined using a chromogenic substrate in the absence or presence of FEIBA. Reduction of factor II resulted in a significant decrease of the TP without influencing the lag phase until the onset of thrombin generation. The reduction of factor VII showed no effect on the TP, but resulted in a prolongation of the lag phase. Changes of factor IX or factor X concentrations showed neither an effect on the TP nor on lag phases. Our study demonstrates that thrombin generation in the presence of FEIBA mainly depends on prothrombin.

Inverse correlation between thyroid function and hemostatic markers for coronary heart disease in obese children and adolescents.

BACKGROUND: Childhood obesity may be associated with thyroid dysfunction. Both obesity and hypothyroidism are related to increased risk of coronary heart disease (CHD) in adults through high levels of serum lipids and/or hemostatic abnormalities. OBJECTIVE: To investigate a possible relationship between thyroid function and hemostatic markers for CHD in obese children and adolescents. STUDY DESIGN: Thirty-nine obese children and adolescents were investigated for thyroid function and markers for CHD after overnight fast. Thyroid hormones were measured by radioimmunoassay. Factor VII coagulant activity (VIIc) and factor VIII coagulant activity (VIIIc) were determined using one stage clotting assays; fibrinogen was measured according to the method of Clauss; von Willebrand factor antigen (vWF-Ag), tissue type plasminogen activator antigen (tPA-Ag), and plasminogen activator inhibitor-1 antigen (PAI-1-Ag) were determined by ELISA. RESULTS: We found a significant inverse correlation between fT4 and factor VIIc (r = -0.33, p = 0.03) and fibrinogen (r = -0.35, p = 0.02), which remained significant after adjustment for body fat mass. Factor VIIIc (r = -0.26, p = 0.066) and vWF-Ag (r = -0.28, p = 0.053) tended to be correlated negatively to fT4. fT4 did not correlate with tPA-Ag and PAI-1-Ag. fT3 was inversely related to factor VIIc (r = -0.3, p = 0.039), which was not independent of body fat mass, and showed a less impressive negative correlation with fibrinogen (r = -0.27, p = 0.058). fT3 did not correlate with vWF-Ag, tPA-Ag, or PAI-1-Ag. There was no relationship between TSH and the determined hemostatic markers. CONCLUSION: Our study demonstrates a close relationship between thyroid function and hemostatic markers for CHD in obese children and adolescents and suggests that thyroid dysfunction is associated with an unfavorable hemostatic state even in pediatric patients.

Longer aPTT values in healthy children than in adults: no single cause.

We have shown that activated partial thromboplastin time values in children are considerably longer than in adults, but the causes for this observation remained unclear. Therefore, we investigated the correlation between activated partial thromboplastin time values and concentrations of clotting factors, quotients and titers of the tissue thromboplastin inhibition test, and antiphospholipid antibodies in healthy children, children with recurrent infections, and adults. Concentrations of factors VIII, IX, and HMWK were significantly lower in children than in adults. Simple linear regression analysis failed to show a correlation between the concentration of a single clotting factor and the activated partial thromboplastin time values. No significant correlation was found between activated partial thromboplastin time and elevation of the tissue thromboplastin inhibition test quotients or titers, or antiphospholipid antibodies values. The determined activated partial thromboplastin time was best described by a function including all measured coagulation factors. Our study suggests, that no single clotting factor or lupus anticoagulants are responsible for the longer activated partial thromboplastin time in healthy children, but that activated partial thromboplastin prolongation is caused by the combination of several slightly lower clotting factors.

Activation of the clotting system: heparin-coated versus non coated systems for extracorporeal circulation.

UNLABELLED: The purpose of this experimental study was to compare heparin-coated versus non-coated systems for extracorporeal membrane oxygenation (ECMO), to investigate the dynamic course of clotting activation in both groups. METHODS: Eight pigs weighing 19.7 (+/- 1.3) kg, each underwent ECMO for 24 hours. Two groups were formed: in group 1, heparin-coated circuits were used with low dose heparinization (10 IU/kg/hr), whereas in group 2 non-coated circuits with high dose heparinization (60 IU/kg/hr) were used. Coagulation was monitored by measuring prothrombin time, partial thromboplastin time, fibrinogen, antithrombin III (AT III) and specific markers of clotting activation (thrombin-antithrombin III complexes (TAT) and D-dimer). Furthermore, platelet count, hematocrit, activated clotting time (ACT), and plasma heparin concentration were determined regularly RESULTS: The dynamic course of the specific coagulation activation markers showed some differences: whereas TAT and D-dimer increased quickly in group 2, the increase in group 1 was delayed. Activation marker values tended to be lower in group 1 during the first six hours, after which no more differences between the groups were seen. After 24 hours of ECMO, TAT and D-dimer had nearly returned to baseline values. Platelets showed a continuous decrease throughout the experiment, which was very similar in both groups. CONCLUSIONS: The heparin coated system showed a distinct delay in clotting activation during the first six hours of ECMO. After six hours there were no more differences between the groups.