Collagen/endogenous thrombin-induced platelet aggregation in cord versus adult whole blood.

BACKGROUND: In previous studies, neonatal platelets have been shown to be hypoaggregable to various agonists when compared with adult platelets. OBJECTIVES: It was the aim of this study to investigate the aggregability of neonatal versus adult platelets when the physiological relevant agonist collagen/endogenous thrombin is used. METHODS AND RESULTS: Whole blood (WB) aggregation experiments employing the impedance method revealed the same responsiveness of neonatal and adult platelets to collagen/endogenous thrombin. Maximum aggregation (13.5 +/- 3.2 vs. 13.6 +/- 3.2 Omega; p = 0.94), slope (5.8 +/- 1.8 vs. 6.2 +/- 2.6 Omega/min; p = 0.79) and lag time until the onset of platelet aggregation (38.7 +/- 8.9 vs. 42.6 +/- 16.5 s; p = 0.59) were similar in cord and adult WB. However, the rise in serotonin plasma levels due to platelet activation was significantly lower in neonates versus adults (227.57 +/- 57.65 vs. 473.34 +/- 155.75 ng/ml; p = 0.0001). Furthermore, we found a fast capability of cord plasma to generate (the efficient platelet agonist) endogenous thrombin: thrombin generation started significantly earlier in cord compared with adult plasma (215 +/- 19 vs. 247 +/- 21 s; p = 0.01). Moreover, thrombelastometry revealed significantly shorter coagulation times in cord versus adult WB activated with collagen/endogenous thrombin (229.8 +/- 12.5 vs. 256.3 +/- 25.3 s; p = 0.003). CONCLUSIONS: The efficient platelet aggregation in cord WB provoked by collagen/endogenous thrombin might help to explain the clinically observed well-functioning primary hemostasis of neonates.

Clot strength: a comparison between cord and adult blood by means of thrombelastometry.

OBJECTIVE: To evaluate the clot strength in cord versus adult blood. METHOD: Thrombelastometry (TEM) was the method of choice as it provides information on the clot strength in terms of the maximum clot firmness (MCF) and on the fibrin polymerization process in terms of the clot formation time and the alpha angle. RESULTS: The MCFs were significantly lower in cord versus adult platelet rich plasma (PRP, 63.0+/-3.8 vs. 67.0+/-3.9 mm, P=0.006) and in cord versus adult whole blood (WB, 55.3+/-3.8 vs. 59.3+/-3.6 mm, P=0.001) employing the thrombelastometry with extrinsic activator assay. We suggest that the diminished clot strength in cord versus adult blood and plasma samples is attributable to an impaired polymerization of neonatal fibrin: (i) the thrombelastometry with extrinsic activator and inactivated platelets (FIBTEM) assay revealed significantly lower MCFs in cord versus adult PRP (23.0+/-3.1 mm vs. 27.3+/-3.9 mm, P=0.002) and in cord versus adult WB (11.6+/-2.3 mm vs. 15.3+/-3.3 mm, P<0.001); (ii) the alpha angle in the FIBTEM assay was significantly lower in cord versus adult WB (39.0+/-12.8 degrees vs. 55.5+/-12.3 degrees, P=0.02); (iii) the clot formation times in the FIBTEM assay were significantly longer in cord versus adult PRP (248.0+/-143.5 s vs. 81.5+/-39.8 s, P=0.001). CONCLUSIONS: Neonatal fibrin shows impaired polymerization properties under our experimental conditions resulting in reduced clot strength compared with fibrin of adult origin.

Collagen/endogenous thrombin-induced platelet aggregation in whole blood samples.

The aim of the study was to investigate the individual and combined effects of collagen (3.5 microg/ml) and endogenously generated thrombin (due to addition of 0.35 pmol/l tissue factor) on platelet aggregation in the physiological environment of whole blood by means of the impedance method. Lag times were significantly shorter when a combination of collagen and endogenous thrombin was used to provoke platelet aggregation (41.9 +/- 16.3 s) compared with collagen (173.8 +/- 52.1 s, P < 0.0001) or endogenous thrombin (94.3 +/- 43.6 s, P < 0.001). Amplitudes and slopes were the lowest in collagen-induced experiments (2.83 +/- 1.59 Omega and 1.79 +/- 0.45 Omega/min, respectively), whereas they were approximately the same in endogenous thrombin-induced experiments whether collagen was present or not (13.7 +/- 3.1 versus 11.2 +/- 4.0 Omega and 6.3 +/- 2.8 versus 5.6 +/- 2.3 Omega/min, respectively). No synergistic effect of collagen and endogenous thrombin on the clot formation process was observed by means of thrombelastometry. Moreover, thrombin potentials in tissue factor-activated plasma samples were approximately the same whether collagen was present or not (834 +/- 67 versus 809 +/- 63 nmol/l.min). In conclusion, endogenously generated thrombin is a potent platelet agonist in whole blood, and a combination of collagen and endogenous thrombin synergistically shortens the lag time until the onset of platelet aggregation.

High availability of intravascular tissue factor in neonates.

In the present study, we compared the levels of intravascular tissue factor (TF) present in cord versus adult whole blood (WB) prior and after lipopolysaccharide (LPS) stimulation. High levels of intravascular TF might help to explain the clinically observed efficient clotting of cord blood despite low levels of procoagulatory factors. Quantitative reverse transcription-polymerase chain reaction revealed same (basal) TF mRNA expression levels in both native cord and adult WB, and approximately same increase in TF mRNA expression owing to LPS incubation in both cord and adult WB (normalized to the housekeeping gene beta-actin). Flow-cytometric (fluorescence activated cell sorting) analysis revealed significantly higher surface TF antigen exposure on the neonatal monocyte membrane in native WB samples, and approximately same ability of neonatal and adult monocytes to express TF upon LPS-stimulation. Thrombelastography revealed significantly shorter clotting times of native cord versus adult WB (527+/-41 vs. 592+/-23 s, P<0.05). Moreover, shortening of clotting times owing to LPS-stimulation was significantly more pronounced in cord versus adult WB (29.65+/-3.35% vs. 12.03+/-6.23%, P<0.05). Because both quantitative reverse transcription-polymerase chain reaction and fluorescence activated cell sorting analysis revealed same capability of both neonatal and adult monocytes to express TF upon LPS-stimulation, this efficient shortening effect in cord WB might be explained by the constitutively high number of monocytes present in neonates. We suggest that the high levels of intravascular TF present in neonates (prior and after LPS-stimulation) might help to explain the clinically observed efficient clotting of cord blood despite low levels of procoagulatory factors.

Effects of beta2-glycoprotein-I on platelet aggregation in cord versus adult whole blood.

We present a peculiarity of the neonatal hemostatic system that might contribute to establish a procoagulant readiness in neonatal blood by sensitizing neonatal platelets for ADP stimulation. beta2-glycoprotein-I (beta2-GP-I) is a plasma constituent capable of suppressing ADP-induced platelet aggregation. We found significant lower levels of beta2-GP-I in cord vs. adult plasma (120 +/- 27 vs. 180 +/- 37 microg/mL, P<0.001). We demonstrate dose-dependent inhibition of ADP-induced platelet aggregation in cord whole blood (WB) in the presence of increasing amounts of beta2-GP-I, evaluated by means of WB aggregometry employing the impedance method. Particularly, raising the beta2-GP-I concentration in cord WB from neonatal level up to the respective adult value caused significant reduction of amplitude (from 9.5 +/- 2.7 to 2.8 +/- 0.9 Omega, P<0.001) and of slope (from 5.9 +/- 2.4 to 1.89 +/- 0.9 Omega/min, P<0.001), and a significant prolongation of the aggregation time (from 51.8 +/- 22.9 to 110.8 +/- 60.3 s, P<0.001). In conclusion, physiological low levels of beta2-GP-I in cord WB cause enhanced responsiveness of neonatal platelets to ADP stimulation. This mechanism might help to explain the clinically observed well-functioning hemostasis in neonates.

Anticoagulant action of melagatran: a comparison between neonates and adults using calibrated automated thrombography (CAT).

In the present study, we comparatively evaluated the anticoagulant efficacy of the new direct thrombin inhibitor melagatran in cord vs. adult plasma. In contrast to heparin, melagatran does not require antithrombin as a cofactor. Thus, anticoagulant treatment with melagatran is of special interest in neonatal patients, whose plasma is relatively deficient in antithrombin. We evaluated the anticoagulant action of increasing amounts of melagatran (0.1-2.0 micromol/l) in both cord and adult plasma by means of calibrated automated thrombography (CAT) with respect to the lag time until the onset of thrombin formation, time to thrombin peak maximum (TTP), endogenous thrombin potential (ETP), and thrombin peak height. Melagatran exhibited approximately the same ability to prolong lag times or TTPs in both cord and adult plasma. Similar concentrations (IC(50)) of melagatran were required to double the lag times (0.44+/-0.04 micromol/l vs. 0.52+/-0.05 micromol/l) or to double the TTPs (0.91+/-0.08 micromol/l vs. 1.06+/-0.09 micromol/l) in cord vs. adult plasma. Melagatran exhibited a higher ability to suppress ETPs or thrombin peak heights in cord vs. adult plasma. Markedly lower concentrations (IC(50)) of melagatran were required to suppress ETPs (0.27+/-0.03 micromol/l vs. 0.70+/-0.06 micromol/l) or thrombin peak heights by 50% (0.29+/-0.03 micromol/l vs. 0.53+/-0.04 micromol/l) in cord vs. adult plasma. We conclude that our results suggest a higher ability of melagatran to suppress thrombin formation in cord vs. adult plasma. Thus, lower amounts of melagatran might be required in neonates undergoing antithrombotic therapy.

Minor neurological dysfunction, cognitive development and somatic development at the age of 3 to 11 years in very-low-birthweight infants with transient periventricular echodensities.

AIM: To determine, using strict exclusion criteria, whether transient periventricular echodensities (TPE) in very-low-birthweight infants lead to minor neurological dysfunction and problems in cognitive and somatic development in children without major neurological impairments. METHODS: 23 children with TPE were matched to 23 children without TPE. Exclusion criteria were small for gestational age, microcephaly at birth, diplegia, asphyxia, psychomotor retardation, intraventricular haemorrhage grade III/IV, major surgical interventions and malformations. The Kaufman Assessment Battery for Children, Draw-a-Man Test and neuropaediatric examination were used for evaluation. RESULTS: There were no differences in demographic data, growth and socio-economic status. Significant differences with lower results in the TPE group were found in fine motor skills and in the Draw-a-Man Test. In the Kaufman Assessment Battery for Children, all subscales were below average in the TPE group, except the sequential processing scale. In the control group, all subscales were within the average range. CONCLUSION: By using strict exclusion criteria to eliminate other risk factors for minimal neurological dysfunction and poor cognitive development, we were able to focus on the effect of TPE. TPE seem to affect cognitive development and cause minor neurological dysfunction.

Effects of melagatran on activated partial thromboplastin time and on ecarin clotting time in cord versus adult plasma.

Melagatran is the active form of the oral direct thrombin inhibitor ximelagatran. Melagatran does not require antithrombin as a cofactor. Its administration is therefore of special interest in neonatal patients, whose plasma is relatively deficient in antithrombin. We investigated the effects of increasing amounts of melagatran (0.05-1 micromol/l) on the activated partial thromboplastin time (APTT) and ecarin clotting time (ECT) in cord versus adult plasma. Both the APTT and ECT were dose-dependently prolonged in the presence of increasing amounts of melagatran. Furthermore, the ECT revealed a higher susceptibility of cord plasma to addition of melagatran than adult plasma. Whereas similar amounts of melagatran were required in cord and adult plasma samples to double the APTT (IC(50), 0.47 vs 0.46 micromol/l), significantly less melagatran was required in cord versus adult plasma to double the ECT (IC(50), 0.26 vs 0.56 micromol/l). Based on APTT measurements, similar plasma levels of melagatran might be required in neonates and in adults to treat thromboembolic complications. The APTT, however, is relatively insensitive to plasma melagatran concentrations. When the sensitive indicator ECT is used, results suggest that lower amounts of melagatran might be required in neonates than in adults. This has to be scrutinized in future clinical studies.

Bladder voiding in sleeping infants is consistently accompanied by a cortical arousal.

The aim of the study was to find out whether bladder voiding in healthy sleeping infants was accompanied by any arousal reaction. Polygraphic recordings were performed in 21 healthy infants (11 female) born at term. The infants' age at study entry was 42 +/- 4 days and actual body weight was 4852 +/- 689 g (mean +/- SD). Bladder voiding was recorded by an adapted enuresis detector which was connected to the polygraphic computer unit. Arousals were defined as suggested by the 'International Paediatric Work Group on Arousals'. Awakenings were excluded from the study. Bladder voiding was recorded at a mean time of 68 +/- 7 min after the infant had fallen asleep and occurred during quiet sleep (QS). Electroencephalogram frequency (P < 0.01) and heart rate (P < 0.05) were higher during the 5-s period before and after bladder voiding when compared with a 30-s interval before voiding. Furthermore, bladder voiding was accompanied by body movements in all infants. Respiratory frequency did not change significantly. We could demonstrate for the first time in sleeping infants, that bladder voiding during QS was accompanied by a cortical arousal.

An evaluation of the procoagulant action of recombinant activated factor VII in cord whole blood versus adult whole blood using thromboelastography.

Recombinant activated factor VII (rFVIIa) has been reported to be effective in adult patients in various clinical situations and might be beneficial in neonates with bleeding tendency. In the present study we compared the procoagulant action of increasing amounts of rFVIIa in both cord whole blood and adult whole blood with respect to changes in the values of the clotting time, clot formation time, and maximum clot firmness by means of thromboelastography. Thromboelastography allows evaluation of the effects of rFVIIa on haemostasis in whole blood. When increasing amounts of rFVIIa were added in vitro to whole blood samples, significant decreases in the values of the clotting time and clot formation time and a significant increase in the maximum clot firmness were observed. Cord whole blood was significantly more sensitive to rFVIIa addition than adult whole blood, an effect probably attributable to the low anticoagulant capacity of the neonatal plasma. Maximum clot firmness values were significantly lower in cord whole blood than in adult whole blood, an effect mainly attributable to the hypofunctional state of neonatal platelets. Since cord whole blood exerted a significantly higher sensitivity to addition of rFVIIa, we speculate that lower doses of rFVIIa might be required to treat neonates with bleeding tendency compared with the adult rFVIIa administration strategy.

Protein S modulates the anticoagulant action of recombinant human activated protein C: a comparison between neonates and adults.

Recombinant human-activated protein C (rhAPC, Drotrecogin alpha (activated), Xigris) has been shown to reduce organ damage and decrease mortality in severe sepsis. Since protein S (PS) serves as a potentiating cofactor of activated protein C and since PS levels are low in neonatal plasma, we hypothesized that the anticoagulant effect of rhAPC would be decreased in cord plasma compared to adult plasma. We demonstrate that the anticoagulant action of 0.3 microg ml(-1) rhAPC (5 nmol l(-1)) was decreased in cord plasma compared to adult plasma, and dose dependently increased in cord plasma in the presence of increasing activities of PS. Correspondingly, the anticoagulant action of rhAPC decreased in adult plasma in the presence of decreasing activities of PS. The low anticoagulant action of rhAPC in cord compared to adult plasma is attributable to low neonatal levels of PS, and as previously shown, to low neonatal levels of TFPI and AT. Our laboratory experiments do not allow definite conclusions for clinical situations. However, we speculate that the anticoagulant efficacy of rhAPC is impaired in neonates and in clinical situations associated with consumption and/or inhibition of PS, AT, and TFPI, such as severe sepsis.

Recombinant human activated protein C, heparin and melagatran in umbilical cord versus adult plasma.

AIM: We investigated the anticoagulant effects of recombinant human activated protein C (rhAPC), unfractionated heparin (UH) and melagatran (a new direct thrombin inhibitor [DTI]), when administered individually and in combinations of rhAPC with either UH or melagatran, in umbilical cord and adult plasma. rhAPC is a promising candidate treatment to improve the outcome of severe sepsis in neonates and adults; the DTI melagatran represents a potential advance in antithrombotic therapy. METHODS: The anticoagulant efficacy of these drugs was measured using the standard coagulation assays activated partial thromboplastin time (aPTT) and prothrombin time (PT). RESULTS: Administered individually, rhAPC, UH and melagatran dose-dependently prolonged aPTT to a significantly greater extent in umbilical cord than in adult plasma. Melagatran alone, but not rhAPC or UH alone, dose-dependently prolonged the PT in both umbilical cord and adult plasma. Combining rhAPC with either UH or melagatran significantly augmented aPTT prolongation in both umbilical cord and adult plasma. CONCLUSION: Our results, which facilitate estimation of rhAPC and melagatran dose requirements in umbilical cord plasma, may be of benefit in critically sick newborns with severe sepsis.

Alpha 2-macroglobulin enhances prothrombin activation and thrombin potential by inhibiting the anticoagulant protein C/protein S system in cord and adult plasma.

Protein S (PS) is a vitamin K-dependent plasma protein and serves as a cofactor for the anticoagulant activities of activated protein C (APC). We investigated the effects of different PS concentrations on prothrombin activation and thrombin generation in cord and adult plasma containing APC and different amounts of alpha 2-macroglobulin (a2-M). Prothrombin activation was assessed by monitoring the time-course of prothrombin fragment 1+2 (F1+2) generation. Thrombin generation curves were determined by means of a subsampling technique using the chromogenic substrate S-2238. We demonstrate a dose-dependent inhibition of the anticoagulant action of PS by a2-M: suppression of F1+2 and thrombin generation due to addition of PS was stronger in plasma containing low amounts of a2-M than in plasma with elevated a2-M levels. Since no complex formation between a2-M and PS was observed by means of SDS-PAGE, we attribute decreased anticoagulant action of PS at high a2-M levels to enhanced complex formation between APC and a2-M. Thereby, APC is subtracted from its cofactor PS, resulting in suppressed formation of the anticoagulant APC/PS complex. Thus, our data suggest that a2-M, besides its well-known anticoagulant effects, also acts as a procoagulant by suppressing the formation of the anticoagulant APC/PS complex. Our findings have implications particularly on thrombin generation and inhibition in cord plasma, since a2-M levels in newborns are elevated over adult values and the antithrombotic APC/PS pathway is up-regulated at birth. Therefore, elevated levels of a2-M might restrict the up-regulation of the APC/PS pathway.