Decreased annexin A5 mRNA placental expression in pregnancies complicated by fetal growth restriction.

BACKGROUND: The placental anticoagulant protein Annexin A5 (ANXA5) is highly expressed on the apical surfaces of syncytiotrophoblasts and plays an important role in maintaining blood fluidity in the placental circulation. We investigated the mRNA and protein expression of ANXA5 in placentas from pregnancies complicated by fetal growth restriction (FGR) compared with uncomplicated pregnancies. MATERIALS AND METHODS: Placental tissue was collected from 18 pregnancies complicated by FGR and 16 pregnancies with a normal outcome. ANXA5 mRNA expression was quantified by Real-Time PCR (RT-PCR), and protein concentrations were measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: A decreased ANXA5 mRNA expression was observed in placenta samples from FGR-affected pregnancies compared to those from uncomplicated pregnancies. However, similar ANXA5 protein levels were measured in both specimen groups. No correlation was observed between ANXA5 mRNA and protein levels. CONCLUSIONS: Transcriptional ANXA5 down-regulation was demonstrated in FGR-affected pregnancies, although protein levels were similar in FGR-related placentas and controls. We can speculate that either recruitment of the protein from the bloodstream or increased apoptosis or post-transcriptional modifications occur, which affect ANXA5 protein levels in FGR-related placentas. Further studies are required to reveal the role of ANXA5 in FGR pathology.

Development of a real-time PCR detection method for a FCGR2A polymorphism in the LightCycler and application in the heparin-induced thrombocytopenia syndrome.

OBJECTIVE: The FcgammaRIIa receptor is responsible for the activation of platelets by antibodies in heparin-induced thrombocytopenia (HIT). The c.497G>A polymorphism in the corresponding FCG2RA gene (H131R) has been implicated in the HIT syndrome and we aimed at its rapid and reliable determination. DESIGN AND METHODS: We designed a novel asymmetric real-time PCR method in the LightCycler that uses two hybridization probes and is followed by melting curve analysis. Seventy-one post-cardiac-surgery HIT Greek patients well ascertained by clinical data, immunological and functional tests (PAT, CD62P-selectin and microparticle flow cytometric detection) were studied, along with a clinically relevant group of 49 thrombocytopenic control patients and 119 healthy subjects. RESULTS: The developed method has excellent analytical characteristics (linear and efficient amplification, precision), has wide DeltaT(m) between the two alleles H and R (11.53 degrees C), and is in 100% concordance with validated controls and another commonly used screening method. The RR percentage increased from 10% in the control populations to 24% in the HIT patient group. CONCLUSION: The described method is technically simple, robust, fast, and accurate. A statistically significant difference was found in the comparison between the groups of HIT patients and healthy subjects [RR vs. RH+ HH, chi(2) test, p=0.01, OR (95% C.I.) 2.81 (1.21-4.68)]. The RR frequency in the Greek population was found to be the lowest among Caucasians.

Effect of exogenous nitric oxide during cardiopulmonary bypass on lung postperfusion histology.

We tested the hypothesis that nitric oxide (NO) administered during cardiopulmonary bypass (CPB) would preserve platelets and prevent postperfusion lung changes. Ten anesthetized Yorkshire pigs were put on normothermic CPB (right atrium to aorta) with a roller pump and membrane oxygenator for 1 hour. In the study group (n = 5), NO was delivered in the oxygenator's gas inflow line with a MiniNO system at 5-10 ppm throughout CPB. In controls (n = 5), NO was not used. Crystalloid solution and norepinephrine were used to maintain blood pressure > or = 60 mm Hg. Fifteen minutes after CPB termination, all pigs were killed with intravenous potassium chloride and exsanguinated via the right atrium. Organ samples were put in formalin solution, processed in paraffin blocks, and stained with hematoxylin and eosin. We did not observe any thrombi in any perfusion system. There were no differences observed in platelet counts and aggregation ability to ADP and collagen, or in neutrophil counts between groups. Bleeding times were similar between groups before and after CPB. Also, there was no significant difference in factor XIIa and fibrinopeptide A levels between groups. Methemoglobin did not exceed normal levels. Lungs were devoid of neutrophils after perfusion in NO-treated pigs, whereas many neutrophils were present in the respiratory membrane of controls. Low-dose exogenous NO in the oxygenator's gas intake has no demonstrable effect on platelet number or function, but prevents neutrophil adhesion to lungs with a possible beneficial effect on postperfusion pulmonary morbidity.

Preoperative detection and management of immune heparin-induced thrombocytopenia in patients undergoing heart surgery with iloprost.

OBJECTIVE: The objective of this study was to evaluate our protocol for the identification and management of patients with immune heparin-induced thrombocytopenia undergoing cardiac surgery. METHODS: Among 1518 patients who underwent cardiac surgery between June 1998 and May 2001, 32 (2.1%) presented with platelet counts less than 150,000/mm3 preoperatively or a history of prolonged (>3 days) intravenous exposure to heparin or both. These 32 patients were evaluated with an enzyme-linked immunosorbent assay for antibodies against heparin-platelet factor 4 complex. Platelets of patients with detected antibodies were tested with the prostacyclin analog iloprost for inhibition of heparin aggregation and determination of the inhibiting concentration and corresponding intravenous infusion rate of iloprost. Patients with antibodies received heparin after complete platelet inhibition with iloprost infusion. Hypotension was prevented or treated with intravenous noradrenaline. Ten randomly selected patients with similar preoperative characteristics, no previous extended exposure to heparin, and normal platelet counts served as controls. RESULTS: Ten of the 32 patients (group A, 31.3%) and none of the controls had antibodies against heparin-platelet factor 4 complex. Patients in group A underwent surgery with iloprost (6-24 ng.kg(-1).min(-1)) and had their blood pressure maintained at greater than 95 mm Hg with norepinephrine infusion (1-4 microg.kg(-1).min(-1)). Operative mortality was zero. There were no thrombotic complications or bleeding requiring exploration. One patient in group A bled 1310 mL/6 hours but did not need exploration. There was no difference in postoperative blood loss and morbidity between groups. Platelet counts were reduced by 12.5% +/- 8.7% (group A) and 38.1% +/- 15.2% (control) (P <.001) 1 hour postoperatively and reached preoperative values by the fifth postoperative day. CONCLUSIONS: Immune heparin-induced thrombocytopenia can be detected preoperatively among patients with a low platelet count or a history of prolonged heparin exposure or both. Cardiac surgery can be safely undertaken using iloprost-induced platelet inhibition during heparinization.

A prospective, double-blind study on the efficacy of the bioline surface-heparinized extracorporeal perfusion circuit.

BACKGROUND: We evaluated the newly introduced Bioline heparin coating and tested the hypothesis that surface heparinization limited to the oxygenator and the arterial filter will ameliorate systemic inflammation and preserve platelets during cardiopulmonary bypass (CPB). METHODS: In a prospective double-blind study, 159 patients underwent coronary revascularization using closed-system CPB with systemic heparinization, mild hypothermia (33 degrees C), a hollow-fiber oxygenator, and an arterial filter. The patients were randomly divided in three groups. In group A (controls, n = 51), surface heparinization was not used. In group B (n = 52), the extracorporeal circuits were totally surface-heparinized with Bioline coating. In group C (n = 56), surface heparinization was limited to oxygenator and arterial filter. RESULTS: No significant difference was noted in patient characteristics and operative data between groups. Operative (30-day) mortality was zero. Platelet counts dropped by 12.3% of pre-CPB value among controls at 15 minutes of CPB, but were preserved in groups B and C throughout perfusion (p = 0.0127). Platelet factor 4, plasmin-antiplasmin levels, and tumor necrosis factor-alpha increased more in controls during CPB than in groups B or C (p = 0.0443, p = 0.0238 and p = 0.0154 respectively). Beta-thromboglobulin, fibrinopeptide-A, prothrombin fragments 1 + 2, factor XIIa levels, bleeding times, blood loss, and transfusion requirements were similar between groups. Intensive care unit stay was shorter in groups B and C than in controls (p = 0.037). CONCLUSIONS: Surface heparinization with Bioline coating preserves platelets, ameliorates the inflammatory response and is associated with a reduced fibrinolytic activity during CPB. Surface heparinization limited to the oxygenator and the arterial filter had similar results as totally surface-heparinized circuits.