Virtually same oxidizability of LDL but higher Lp(a) levels in arterial compared to venous plasma.

Plaque formation is confined to the arterial trunk. We assumed that due to the higher aeration of arterial compared to venous blood, higher levels of the atherogenic agent oxidized LDL might be present in arteries, contributing to plaque formation. We aimed to compare (i) the basal oxidative status of LDL in arterial and venous blood and (ii) the susceptibility of arterial and venous LDL to oxidation. The basal oxidative status of LDL was determined by measuring lipid hydroperoxide (LPO) concentrations, plasma levels of auto-antibodies against oxidized LDL, and by measuring oxidation-specific epitopes on LDL particles. The oxidizability of arterial vs. venous LDL (catalyzed by copper) was estimated by monitoring the time-course of conjugated dienes formation. Interestingly, we found the same basal oxidative status of LDL in arterial and venous plasma. LPO concentrations and levels of auto-antibodies against oxidized LDL were similar in arterial and venous plasma and amounts of oxidation-specific epitopes were similar on the respective LDL particles. Moreover, we found similar susceptibilities of arterial and venous LDL to (copper-mediated) oxidation. Lag-times until the onset of conjugated diene formation were slightly shorter in arterial compared to venous LDL in the presence of 5 µM, but not in the presence of 1 µM CuCl2. Additionally, we found significantly higher levels of the atherogenic lipoprotein(a) in arterial plasma. We conclude that not higher oxidizability of arterial LDL but higher arterial lipoprotein(a) levels might help to explain why sclerosis is confined to the arterial trunk.

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).

Stent implantation in the superficial femoral artery: short thrombelastometry-derived coagulation times identify patients with late in-stent restenosis.

INTRODUCTION: The mechanisms of restenosis, the recurrence of luminal narrowing, are complex and incompletely understood to date. Thrombin, the pivotal enzyme in haemostasis, presumably contributes to the formation of in-stent restenosis (ISR). It was therefore the aim of our study to investigate whether blood coagulation/thrombin generation plays a critical role in the formation of ISR in peripheral artery disease patients with stent angioplasty in the superficial femoral artery. MATERIALS AND METHODS: We aimed to examine in this retrospective study whether patients with high-degree restenosis (50-75% lumen diameter reduction, n=20) are in a hypercoaguable state implying enhanced readiness to generate thrombin compared to patients with low-degree restenosis (<50% lumen diameter reduction, n=14). RESULTS: The coagulation tests calibrated automated thrombography, activated partial thromboplastin time, platelet aggregation, platelet adhesion, fibrinogen, and microparticles' procoagulant activity did not indicate a different coagulation status in the two patient groups. However, the thrombelastometry-derived value Coagulation Time (CT) was significantly shorter in the high-degree restenosis group (p=0.012), indicating a hypercoagulable state of patients with high-degree restenosis. Under our experimental conditions, CTs shorter than 444.5s identify patients at high risk (sensitivity=95%) for luminal narrowing. CONCLUSIONS: Our study supports the assumption that blood coagulation/thrombin generation plays a critical role in the development of ISR in peripheral arteries after stent insertion and that the thrombelastometry-derived CT might be a suitable value to identify peripheral artery disease patients at risk for development of high-degree in-stent restenosis in the superficial femoral artery.

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.

Effects of nadroparin, enoxaparin, and unfractionated heparin on endogenous formation of factor Xa and IIa and on thrombelastometry profiles in cord versus adult blood.

BACKGROUND: To date, only few pharmacokinetic studies on low-molecular-weight heparins (LMWHs) in neonates exist not allowing to formally assess pharmacodynamics of LMWHs in neonates. OBJECTIVE: To evaluate the anticoagulant effects of the two LMWHs nadroparin and enoxaparin on endogenous formation of FXa or FIIa in cord versus adult platelet-poor plasma (PPP) and on thrombelastometry profiles in cord versus adult whole blood (WB). Unfractionated heparin (UH) was the reference antithrombotic drug. METHODS: The effects of nadroparin, enoxaparin, or UH on endogenous formation of FXa or FIIa was investigated in tissue factor-activated PPP using a subsampling technique and chromogenic substrates. The anticoagulant efficacy of these drugs was also investigated in WB triggered by the physiological relevant activator collagen/endogenous thrombin using thrombelastometry. RESULTS: The major findings are (i) nadroparin is as efficient as enoxaparin concerning inhibition of the endogenous formation of FXa and FIIa, (ii) cord PPP and WB are significantly more susceptible to the addition of LMWHs or UH than adult PPP or WB, and (iii) compared by equivalent anti-FXa activity, the anticoagulant action of UH is markedly higher than that of the LMWHs in PPP and WB of neonatal or adult origin. CONCLUSIONS: Administration of LMWHs in neonates has to be performed carefully to avoid bleeding side effects due to their high anticoagulant efficacy in cord PPP and WB.

Heparinase-modified thrombelastometry: inactivation of heparin in plasma samples.

BACKGROUND: The heparinase-modified thrombelastometry (HEPTEM) assay is a promising tool to assess the coagulation status of heparinised patients. The aim of our study was to examine the heparin neutralizing capability of the HEPTEM assay in plasma samples. METHODS: In the HEPTEM assay, blood or plasma samples become activated via the intrinsic pathway in the presence of a heparin processing enzyme. RESULTS: We examined coagulation times (CTs) in the presence of increasing amounts (0-4 IU/mL) of heparin. We found that up to a concentration of 0.5 IU/mL, heparin is completely neutralized. However, CTs increased linearly in the presence of heparin concentrations higher than 0.5 IU/mL, indicating incomplete heparin neutralization in the standard HEPTEM assay. CONCLUSIONS: We provide herein a mathematical procedure to correct the misleadingly prolonged CTs (for heparin > 0.5 IU/mL) for the HEPTEM assay performed in plasma samples to allow better estimation of the coagulation status in patients requiring intense anticoagulation (e.g., patients undergoing cardiac surgery).

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.

Effects of nadroparin, enoxaparin, and unfractionated heparin on endogenous factor Xa and IIa formation and on thrombelastometry profiles.

Measurements of anti-FXa or anti-FIIa (thrombin) activities are conventional tests for biological monitoring of low molecular weight heparin (LMWH) or unfractionated heparin treatment. It was the aim of our study to assess the anticoagulant efficacy of the LMWHs nadroparin and enoxaparin and that of unfractionated heparin not by the above-mentioned isolated measurements but in the physiological environment of clotting plasma or whole blood. The effects of increasing amounts of nadroparin, enoxaparin, or unfractionated heparin on the time-course of FXa or FIIa formation were investigated in tissue factor activated platelet-poor plasma using a subsampling technique and chromogenic substrates. Moreover, the anticoagulant efficacy of these drugs was also investigated in whole blood triggered by the physiological relevant activator collagen/endogenous thrombin using thrombelastometry. Nadroparin is as efficient as enoxaparin concerning suppression of endogenous FXa or FIIa formation. The two LMWHs are capable of suppressing the formation of FIIa as efficient as that of FXa. Compared with equivalent anti-FXa activity, unfractionated heparin is markedly more efficient in suppressing the formation of FXa and FIIa than the LMWHs. Corresponding results were obtained in whole blood. The anticoagulant efficacy of nadroparin was comparable with that of enoxaparin and the influence of unfractionated heparin on thrombelastometry parameters was markedly stronger than that of the two LMWHs. We conclude that LMWHs are efficient inhibitors not only of endogenous FXa formation but also of endogenous FIIa formation. Under our experimental conditions, the anticoagulant efficacy of nadroparin was comparable with that of enoxaparin but markedly lower than that of unfractionated heparin.

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.

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.