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.

Simvastatin Efficiently Lowers Small LDL-IgG Immune Complex Levels: A Therapeutic Quality beyond the Lipid-Lowering Effect.

We investigated a polyethylene glycol non-precipitable low-density lipoprotein (LDL) subfraction targeted by IgG and the influence of statin therapy on plasma levels of these small LDL-IgG-immune complexes (LDL-IgG-IC). LDL-subfractions were isolated from 6 atherosclerotic subjects and 3 healthy individuals utilizing iodixanol density gradient ultracentrifugation. Cholesterol, apoB and malondialdehyde (MDA) levels were determined in each fraction by enzymatic testing, dissociation-enhanced lanthanide fluorescence immunoassay and high-performance liquid chromatography, respectively. The levels of LDL-IgG-IC were quantified densitometrically following lipid electrophoresis, particle size distribution was assessed with dynamic light scattering and size exclusion chromatography. The influence of simvastatin (40 mg/day for three months) on small LDL-IgG-IC levels and their distribution among LDL-subfractions (salt gradient separation) were investigated in 11 patients with confirmed coronary artery disease (CAD). We demonstrate that the investigated LDL-IgG-IC are small particles present in atherosclerotic patients and healthy subjects. In vitro assembly of LDL-IgG-IC resulted in particle density shifts indicating a composition of one single molecule of IgG per LDL particle. Normalization on cholesterol levels revealed MDA values twice as high for LDL-subfractions rich in small LDL-IgG-IC if compared to dominant LDL-subfractions. Reactivity of affinity purified small LDL-IgG-IC to monoclonal antibody OB/04 indicates a high degree of modified apoB and oxidative modification. Simvastatin therapy studied in the CAD patients significantly lowered LDL levels and to an even higher extent, small LDL-IgG-IC levels without affecting their distribution. In conclusion simvastatin lowers levels of small LDL-IgG-IC more effectively than LDL-cholesterol and LDL-apoB levels in atherosclerotic patients. This antiatherogenic effect may additionally contribute to the known beneficial effects of this drug in the treatment of atherosclerosis.

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.

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.

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.

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.

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.

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.