Antiphospholipid Antibodies are Associated with Low Levels of Complement C3 and C4 in Patients with Systemic Lupus Erythematosus.

Complement activation and low complement levels are common in systemic lupus erythematosus (SLE). Antiphospholipid antibodies (aPL) are found in about 30-40% of patients with SLE. This study aimed to investigate the association between aPL and complement levels in patients with SLE. Serum samples were collected from 269 patients with SLE enrolled in the Norwegian Systemic Connective Tissue and Vasculitis Registry (NOSVAR) during 2003-2009, and from 353 controls. All samples were analysed for anti-ß2 glycoprotein 1 (anti-ß2GP1) and anticardiolipin antibodies (aCL), C-reactive protein (CRP) and complement components C3 and C4. Median CRP level was significantly higher in cases than in controls (2.06 versus 0.90 mg/l; P < 0.0001). No significant difference in CRP was found between SLE patients with or without aPL (2.09 versus 1.89; P = 0.665). Median C3 levels were similar in cases (1.03 g/l) and controls (1.00 g/l), whereas median C4 levels were 0.16 g/l in cases versus. 0.19 in controls (P < 0.0001). However, aPL-positive SLE patients had significantly lower median C3 levels (0.92 versus. 1.07 g/l; P = 0.001) and C4 levels (0.11 versus 0.16 g/l; P < 0.0001) compared to aPL-negative patients. Lower C3 and C4 values in aPL-positive SLE patients may reflect a higher consumption of C3 and C4 due to more pronounced complement activation in aPL-positive SLE patients compared to SLE patients without aPL.

Circadian rhythms of hemostatic factors in tetraplegia: a double-blind, randomized, placebo-controlled cross-over study of melatonin.

STUDY DESIGN: This is a double-blind, randomized, placebo-controlled cross-over study of melatonin in complete tetraplegia. OBJECTIVES: Tetraplegic patients have an increased risk of venous thrombosis despite prophylaxis, blunted variations in melatonin and altered circadian variation of several hemostatic markers. To examine whether melatonin could modify the regulation of hemostasis, we measured plasma melatonin and several markers of hemostasis in tetraplegic subjects with or without melatonin supplement. SETTING: The study was conducted in the Section for Spinal Cord Injury, Sunnaas Hospital, Nesoddtangen, Norway. METHODS: Six subjects with long-standing complete tetraplegia were included in this cross-over study with 2 mg of melatonin or placebo given 4 days before sampling. We also included six able-bodied men without any intervention. Plasma samples were then collected frequently during a 24-h awake/sleep cycle. The plasma concentrations of melatonin and the various markers were analyzed using linear mixed models. RESULTS: The 24-h profiles of prothrombin fragment 1+2 and von Willebrand factor, but not D-dimer, activated FVII, tissue factor pathway inhibitor and plasminogen activator inhibitor type 1, differed (P<0.05) between tetraplegic patients and able-bodied subjects. The absolute plasma concentration of activated FVII was higher (P<0.05) among the able-bodied compared with the tetraplegic groups. Supplementation of melatonin had no impact on these findings. CONCLUSIONS: We found differences in circadian variation of several hemostatic markers between able-bodied and tetraplegics. These differences were apparently unrelated to fluctuations in the melatonin concentrations, suggesting little or no role of melatonin in the regulation of hemostasis in tetraplegia. SPONSORSHIP: Financial support was provided from the Throne Holst Foundation.

Increased acquired activated protein C resistance in unselected patients with hematological malignancies.

BACKGROUND: We have previously found that activation of coagulation in patients with various hematological malignancies was apparently not initiated by tissue factor (TF). Acquired activated protein C (APC) resistance may be another mechanism responsible for such hypercoagulation, and has been demonstrated in patients with solid tumors, but not in patients with hematological malignancy. OBJECTIVE: To investigate acquired APC resistance in a hypercoagulable cohort of patients with hematological malignancies. PATIENTS/METHODS: Blood samples from 93 patients with acute myeloid leukemia (AML), chronic lymphatic leukemia, multiple myeloma, or non-Hodgkin's lymphoma, were analyzed before start and after completion of cancer therapy. APC resistance was measured using calibrated automated thrombography. The APC sensitivity ratio (APC-SR) was calculated as the ratio of the endogenous thrombin potential (ETP) determined in plasma probed with either APC or buffer. RESULTS: Untreated patients were found to have higher APC-SR than healthy controls, and patients with AML had higher APC-SR as compared to the other diagnoses, both findings being consistent with acquired APC resistance. The acquired APC resistance was partly ameliorated with cancer treatment. Decreased levels of protein S and TF pathway inhibitor were inversely correlated to APC resistance. CONCLUSIONS: APC resistance may contribute to the hypercoagulable state in hematological malignancies.

Activated protein C resistance determined with a thrombin generation-based test is associated with thrombotic events in patients with lupus anticoagulants.

BACKGROUND: Several studies suggest that antiphospholipid antibodies interfere with the activity of activated protein C (APC). This acquired form of APC resistance has been proposed as a possible pathogenic mechanism underlying hypercoagulability associated with the antiphospholipid syndrome (APS). OBJECTIVES: We wanted to investigate the inhibitory effect of recombinant APC (rAPC) on ex vivo thrombin generation in plasma and the modification of this effect by the presence of lupus anticoagulants (LA). PATIENTS/METHODS: We analyzed plasmas from 81 patients with LA (52 patients fulfilling the criteria for the APS) and 91 controls. Percent inhibition of the endogenous thrombin potential (ETP) as a parameter of APC sensitivity was determined in plasmas using a thrombin generation-based APC resistance test probed with rAPC. All results were normalized using pooled normal plasma (PNP) as a reference. RESULTS: Normalized percent inhibition of ETP by APC was lower in patients with LA [61.4%, 95% confidence interval (CI) 45.8-74.5%] compared to controls (107.8%, 95% CI: 107.1-109.3%). In patients with LA and APS, median inhibition was lower than in patients with LA without APS (44.6%, 95% CI: 30.1-55.7% vs. 78.8%, 95% CI: 73.9-95.8%). This difference also persisted when patients on warfarin therapy were excluded from the APS subgroup. CONCLUSIONS: APC resistance can be demonstrated with a thrombin generation-based test in a majority of patients with the LA laboratory phenotype. A history of thrombotic events in patients with LA is associated with a stronger resistance to the anticoagulant effect of APC.

The performance of STA-Liatest D-dimer assay in out-patients with suspected pulmonary embolism.

Several studies have shown that D-dimer can reliably rule out pulmonary embolism (PE) in out-patients. However, various assays have different sensitivities and specificities to detect thrombosis. Our aim was to evaluate the performance of STA-Liatest D-Di in out-patients referred for suspected PE in a prospective outcome study. 495 consecutive patients referred to Østfold Hospital Trust-Fredrikstad, Norway for suspected PE between February 2002 and December 2003, were recruited in a study evaluating a decision-based algorithm combining clinical probability (CP), D-dimer, and multi-slice computer tomography (MSCT). D-dimer was performed as a first step test. No further testing was carried out in patients with D-dimer < or =0.4 mg/l and low/intermediate CP. The remaining patients proceeded to MSCT. All patients were followed up for 3 months to assess the 3-month thromboembolic risk. The final cohort consisted of 432 patients. PE was diagnosed in 102 (23%) patients. At a D-dimer cut-off point of 0.4 mg/l the tests had the highest sensitivity (100%) and specificity (36%). It safely ruled out PE in 120 (28%) patients. Kappa-coefficients for comparisons versus VIDAS and Asserachrom showed good concordance. STA-Liatest is a reliable and effective assay that can safely rule out PE in out-patients with a performance comparable with that of enzyme-linked immunosorbent assay-based d-dimer levels.

Hormone replacement therapy and acquired resistance to activated protein C: results of a randomized, double-blind, placebo-controlled trial.

Recent studies suggest that low-dose oral contraceptives may cause acquired resistance to activated protein C (APC). The aims of this study were to determine whether hormone replacement therapy (HRT) may also induce acquired APC resistance and to study the effects of APC resistance on the risk of recurrent thrombosis. The patients comprised 140 females with at least one previous venous thromboembolism (VTE), who were randomized to receive continuous treatment with 2 mg 17-beta-oestradiol and 1 mg norethisterone acetate (n = 71) or placebo (n = 69). Normalized APC sensitivity ratios (nAPCsr) were calculated by measurement of the effect of APC on thrombin generation in plasma collected at baseline and after 3 months of treatment. Of the 140 women, 121 had plasma samples collected both at baseline and after 3 months. The nAPCsr increased significantly (P < 0.001) on HRT (n = 62), both in females not carrying the factor V(Leiden) mutation [mean change 0.57 (95% CI 0.45-0.70), n = 50] and in females heterozygous for the factor V(Leiden) mutation [mean change 1.10 (0.71-1.49), n = 12], but remained unchanged on placebo (n = 59). The baseline nAPCsr as well as the increase in nAPCsr associated with HRT use was not higher in the five women who subsequently developed recurrent VTE. Free protein S and free TFPI were both important parameters for the acquired APC resistant phenotype. We conclude that HRT diminishes the efficacy by which APC downregulates in-vitro thrombin formation in a similar fashion to that observed with low-dose oral contraceptives, but the increase in nAPCsr alone is not sufficient to explain the increased risk of VTE associated with use of HRT.

The effects of hormone replacement therapy (HRT) on hemostatic variables in women with previous venous thromboembolism--results from a randomized, double-blind, clinical trial.

In a recent randomized, double-blind, placebo-controlled trial of women with a history of venous thromboembolism (VTE), we found that hormone replacement therapy (HRT) was associated with an early excess risk of recurrent thrombosis. The aims of the present study were to characterize the effects of HRT on coagulation in these women to elucidate the mechanism(s) by which HRT increases the risk of thrombosis. The study comprised 140 women who were randomized to receive continuous treatment for 24 months with once daily 2 mg 17-beta-estradiol plus 1 mg norethisterone acetate (n = 71) or placebo (n = 69). HRT caused significant increases in prothrombin fragments 1+2, thrombin-antithrombin complex, and D-Dimer after 3 months, but these changes were less pronounced on prolonged treatment. The increases in markers of activated coagulation was higher in those women who subsequently developed recurrent thrombosis, but was similar in carriers and non-carriers of the factor V Leiden mutation. HRT had no effects on fibrinogen and factor VIII. Activated factor VII, but not factor VII antigen, decreased significantly on HRT as compared with placebo. The coagulation inhibitors antithrombin, protein C, and TFPI, but not protein S, all showed significant sustained decreases in the HRT group as compared with placebo. Antithrombin and protein C decreased by 8-12% on HRT, whereas TFPI activity decreased by 12-17% and TFPI free antigen by 29-30%. In multivariate analysis, only TFPI activity was a significant predictor for the increased activation of coagulation. We conclude that HRT was associated with early activation of coagulation, which corroborates the finding of an early risk of recurrent VTE. This activation may in part be explained by reduction in circulating anticoagulants.

Structure and anticoagulant activity of a fucosylated chondroitin sulfate from echinoderm. Sulfated fucose branches on the polysaccharide account for its high anticoagulant action.

A polysaccharide isolated from the body wall of the sea cucumber Ludwigothurea grisea has a backbone like that of mammalian chondroitin sulfate: [4-beta-D-GlcA-1-->3-beta-D-GalNAc-1]n but substituted at the 3-position of the beta--glucuronic acid residues with sulfated alpha--fucopyranosyl branches (Vieira, R. P., Mulloy, B., and Mourão, P. A. S. (1991) J. Biol. Chem. 266, 13530-13536). Mild acid hydrolysis removes the sulfated alpha--fucose branches, and cleaved residues have been characterized by 1H NMR spectroscopy; the most abundant species is fucose 4-O-monosulfate, but 2,4- and 3, 4-di-O-sulfated residues are also present. Degradation of the remaining polysaccharide with chondroitin ABC lyase shows that the sulfated alpha-L-fucose residues released by mild acid hydrolysis are concentrated toward the non-reducing end of the polysaccharide chains; enzyme-resistant polysaccharide material includes the reducing terminal and carries acid-resistant -fucose substitution. The sulfated alpha-L-fucose branches confer anticoagulant activity on the polysaccharide. The specific activity of fucosylated chondroitin sulfate in the activated partial thromboplastin time assay is greater than that of a linear homopolymeric alpha-L-fucan with about the same level of sulfation; this activity is lost on defucosylation or desulfation but not on carboxyl-reduction of the polymer. Assays with purified reagents show that the fucosylated chondroitin sulfate can potentiate the thrombin inhibition activity of both antithrombin and heparin cofactor II.

Phase I study of intraventricular recombinant tissue plasminogen activator for treatment of posthaemorrhagic hydrocephalus.

AIM: Phase I study to evaluate intraventricular fibrinolytic treatment with recombinant tissue plasminogen activator (tPA) as a method of clearing blood from the cerebrospinal fluid, and thus preventing permanent hydrocephalus. METHODS: Twenty two preterm infants, aged 7 to 26 days, with progressive posthaemorrhagic ventricular dilatation (ventricular width > 4 mm over 97th centile) received one to five intraventricular bolus injections of 1.0 mg or 0.5 mg tPA at intervals of one to seven days. RESULTS: The mean cerebrospinal fluid concentration of tPA 24 hours after 1 mg was 1860 micrograms/ml. The half life of tPA in cerebrospinal fluid was about 24 hours. Twenty one (95%) infants survived, 12 (55%) without shunt surgery. One infant had secondary intraventricular haemorrhage. CONCLUSION: Intraventricular tPA resulted in survival without a shunt for most of the infants, but with some risk. Failure may have been due to plasminogen deficiency, an inhibitor, or late intervention.

Thrombin-inhibitor complexes in the blood during and after delivery.

Activation of coagulation leads to generation of thrombin which in turn is inactivated by the formation of thrombin-antithrombin (TAT) complexes, and thrombin-heparin cofactor complexes (T-HCII). These complexes were measured in plasma by ELISA methods. During normal delivery, the median TAT level in ten women increased from 4.1 to 7.8 times the median normal reference level. There was great individual variation, and levels 42 and 56 times normal median were found in two women shortly after normal delivery. The median T-HCII levels increased only moderately from 2.3 to 3.1 times median normal reference. D-dimer values were elevated in 28 out of the 30 samples. In blood sampled 1-2 days after delivery, the median TAT level was 2.5 times the median normal reference. The median T-HCII level was now 5.6 times the median normal reference value. The values were stable during the first 4 days post partum, and there was little difference between those delivered vaginally or by Caesarean section (C-section). D-dimer values were above normal reference in all women, and higher in women delivered by C-section. In conclusion, increasing TAT levels during labour and delivery indicated generation of thrombin which was mainly inactivated by antithrombin. The T-HCII levels increased less during delivery. In the early post partum period, the T-HCII levels were relatively more increased than the TAT levels. These results suggest that intravascularly generated thrombin is preferably inactivated by antithrombin, even in parturient women. In the post partum period, formation of T-HCII complexes was more evident, possibly reflecting extravascular inactivation of thrombin.

Endogenous tissue plasminogen activator in neonatal cerebrospinal fluid.

Tissue type plasminogen activator (tPA) plays a role in differentiation of neurones and activity-dependent structural changes in neurones. We hypothesised that tPA would also be present in CSF during fibrinolysis after intraventricular haemorrhage. We measured tPA antigen in CSF from 13 normal newborn infants and 14 infants with post-haemorrhagic ventricular dilatation (PHVD). tPA was undetectable or at the limit of detection (1 microgram/l) in normal CSF. The CSF tPA concentration ranged from 1.3 to 3.5 micrograms/l in the infants with PHVD. Serial tapping in one infant showed persistence of tPA in the CSF from 3 to 8 weeks of age. We conclude that endogenous tPA may be part of the physiological response to intraventricular haemorrhage or may be present as a result of passive diffusion into the CSF.

Low levels of plasminogen in cerebrospinal fluid after intraventricular haemorrhage: a limiting factor for clot lysis?

The aim of this study was to measure plasminogen in the cerebrospinal fluid (CSF) of control neonates with no infection or haemorrhage and in infants who had suffered intraventricular haemorrhage (IVH). A chromogenic substrate method was used. The 16 reference infants had a median CSF plasminogen level of 0.74% of that of normal adult plasma (range 0.17-1.1%). The 11 infants with IVH had a median CSF plasminogen level of 0.55% of normal adult plasma (range 0-4.4%). Six of the IVH infants went on to develop permanent hydrocephalus despite the use of intraventricular plasminogen activators. Endogenous fibrinolysis and the potential for fibrinolytic treatment in the CSF may be limited by low concentrations of plasminogen, and administration of recombinant plasminogen may assist attempts to clear intraventricular blood clots.

Intraventricular streptokinase increases cerebrospinal fluid D dimer in preterm infants with posthaemorrhagic ventricular dilatation.

Failure to lyse multiple small blood clots in the cerebrospinal fluid (CSF) reabsorption pathways may be one of the mechanisms leading to posthaemorrhagic ventricular dilatation (PHVD). It has been suggested that intraventricular administration of streptokinase may resolve PHVD but it is not known whether such treatment produces an increase in fibrin degradation products in the CSF. Ventricular CSF was collected from six infants with PHVD before and during intraventricular treatment with streptokinase 1000 units/h. In all six infants, CSF D dimer increased during streptokinase treatment. Median D dimer before treatment was 1642 micrograms/l and during treatment 5440 micrograms/l (p < 0.05). Undetectable D dimer levels in plasma during streptokinase treatment ruled out the possibility that D dimer had merely diffused into the CSF. This augmentation of local fibrinolysis may have therapeutic potential. There was no evidence of systemic fibrinolysis.