In-vacuum Faraday isolation remote tuning.

In-vacuum Faraday isolators (FIs) are used in gravitational wave interferometers to prevent the disturbance caused by light reflected back to the input port from the interferometer itself. The efficiency of the optical isolation is becoming more critical with the increase of laser input power. An in-vacuum FI, used in a gravitational wave experiment (Virgo), has a 20 mm clear aperture and is illuminated by an almost 20 W incoming beam, having a diameter of about 5 mm. When going in vacuum at 10(-6) mbar, a degradation of the isolation exceeding 10 dB was observed. A remotely controlled system using a motorized lambda=2 waveplate inserted between the first polarizer and the Faraday rotator has proven its capability to restore the optical isolation to a value close to the one set up in air.

Endothelial dysfunction in haemophilia patients.

Haemophilia patients may develop cardiovascular diseases, suggesting that their clotting defect does not protect them completely from atherosclerosis and its complications. We aimed to evaluate cardiovascular risk factors and, for the first time, the presence of endothelial dysfunction in middle-aged haemophilia patients. We studied 40 patients with haemophilia A and B (24 with moderate-severe disease and 16 with mild disease), and 40 healthy controls. Flow-mediated dilation (FMD), carotid ultrasound (US) intima media thickness (IMT), arterial blood pressure, body mass index (BMI), cholesterol, triglycerides, glucose, insulin, lipoprotein(a) and homocysteine levels were measured, and PAI-1 and t-PA levels before and after venous occlusion (VO), and antibodies to HIV, HBV and HCV were assayed. At least one cardiovascular risk factor was detected in 87.5% of patients, and 2 or more in 47.5% of cases. At US exam, none of the patients had significant carotid stenosis or significant differences in IMT compared to controls. In contrast, all the patients had a significant FMD impairment, associated with a reduced t-PA release after VO in 70% of cases. PAI-1 levels significantly correlated with BMI, triglycerides and insulin values. Fifteen haemophilia patients with chronic viral hepatitis and/or HIV infection showed a significantly lower FMD than patients without active infection. We found an endothelial dysfunction with impaired FMD and t-PA release in our haemophilia patients, usually associated with cardiovascular risk factors. Other pathogenic mechanisms, such as chronic viral infections, are likely to be involved in this endothelial damage, however.

In-vacuum optical isolation changes by heating in a Faraday isolator.

We describe a model evaluating changes in the optical isolation of a Faraday isolator when passing from air to vacuum in terms of different thermal effects in the crystal. The changes are particularly significant in the crystal thermal lensing (refraction index and thermal expansion) and in its Verdet constant and can be ascribed to the less efficient convection cooling of the magneto-optic crystal of the Faraday isolator. An isolation decrease by a factor of 10 is experimentally observed in a Faraday isolator that is used in a gravitational wave experiment (Virgo) with a 10 W input laser when going from air to vacuum. A finite element model simulation reproduces with a great accuracy the experimental data measured on Virgo and on a test bench. A first set of measurements of the thermal lensing has been used to characterize the losses of the crystal, which depend on the sample. The isolation factor measured on Virgo confirms the simulation model and the absorption losses of 0.0016 +/- 0.0002/cm for the TGG magneto-optic crystal used in the Faraday isolator.

Blood oxidative status and selectins plasma levels in healthy donors receiving granulocyte-colony stimulating factor.

Recombinant human G-CSF (rHuG-CSF) is used for hematopoietic progenitor cells (HPC) mobilization and collection. Activation of polymorphonuclear leukocytes (PMN) is present during rHuG-CSF treatment and is associated with endothelial cell dysfunction and hypercoagulation. We evaluated whether PMN activation by rHuG-CSF may alter the blood oxidative status and subsequently affect the vascular cell function. Fourteen healthy individuals received rHuG-CSF for HPC harvesting. Blood was drawn before starting rHuG-CSF (T0), on the last day of rHuG-CSF (T1) and 1 week after stopping rHuG-CSF (T2). Levels of CD11b, myeloperoxidase (MPO), hydroperoxides, nitric oxide (NO), and soluble endothelium (sES), leukocyte (sLS), and platelet (sPS) selectins were measured. During rHuG-CSF, CD11b, MPO and hydroperoxides significantly increased, while NO levels significantly decreased, compared with T0. At T2 all these markers returned to baseline values. Significant increments of all selectins were observed during rHuG-CSF. At T2 sES and sEP significantly decreased back to pre-treatment values, whereas sLS remained significantly high. These data show that rHuG-CSF induces a transient inflammatory status characterized by circulating activated PMN, which release reactive oxygen species and intracellular proteases, promoting the onset of an abnormal oxidative status. This process may modify the hemostatic balance towards a pro-thrombotic state.

Clinical use of thrombolytic agents in venous thromboembolism.

The use of thrombolytic agents in venous thromboembolism has been shown to be highly effective. Patients treated with lytic agents show more rapid clot resolution and lung reperfusion and more rapid and greater reversal of the abnormal hemodynamic responses to pulmonary embolism than patients receiving heparin. Moreover, lytic therapy removes thromboemboli more completely from the pulmonary microcirculation, whereas residual thromboemboli tend to accumulate with heparin therapy. In addition, lytic therapy tends to preserve the venous valves, whereas distortion and destruction occur with heparin therapy. Hence, lytic therapy confers a number of short- and long-term benefits not observed with heparin therapy.

Heparin neutralizing activity (HNA) and antithrombin III in coronary artery disease.

Plasma from 14 patients with severe and diffuse coronary atherosclerosis has been compared with that obtained from a normal control group. While a decreased heparin-thrombin clotting time was demonstrated in the patient group, suggesting an increased level of circulating platelet factor 4, there was no significant alteration in plasma antithrombin III level. These results do not support a recent suggestion of a mild chronic intravascular coagulation in atherosclerosis.

Serial determinations of PF4 and beta TG: comparisons between multiple venipunctures vs a catheter infusion system.

Platelet factor 4 (PF4) and beta thromboglobulin (beta TG) are platelet-specific proteins which are released upon platelet aggregation and which can be accurately measured by radioimmunoassay. We devised a catheter-infusion system that enables serial determinations of these proteins. In 20 subjects (10 healthy volunteers and 10 patients with stable coronary artery disease), we compared samples collected by individual venipunctures with those simultaneously obtained by means of a simple catheter-infusion system. At least 5 samples were obtained over a period of time which was as long as 60 min, and at least 30 min. Subjects with stable coronary artery disease were selected so that they would be expected to have stable and normal PF4 and beta TG levels. Thus, elevations of either PF4 or beta TG would represent artifacts secondary to sampling technique. Analysis of the results demonstrated that the catheter-infusion system was equivalent to individual venipunctures for determination of PF4 and beta TG. 16.8% of samples obtained via the catheter and 17.2% of those obtained by individual venipunctures were spuriously elevated. A second series of studies were performed to refine the technique further by examining the impact of infusion rate and the addition of citrate phosphate dextrose (CPD) to the infusate. Ten additional subjects had catheter systems utilized in both arms simultaneously. The addition of CPD resulted in significantly less abnormal values at slower infusion rates (1 and 2.5 cc/min). At 5 cc/min D5/w or saline alone are suitable. These investigations confirm that this simple catheter system is equivalent to individual venipunctures for determination of PF4 and beta TG while avoiding patient discomfort.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutralization of the antiheparin activity of platelet factor 4 by a monoclonal antibody.

We have produced a panel of monoclonal antibodies (mAbs) against rabbit platelet factor 4 (PF4). Two of these mAbs have been characterized in this study. In particular the antibody called 10B2, which also recognizes the human molecule, is able to block PF4's ability to neutralize heparin in a modified Heparin-Factor Xa chromogenic assay. The inhibition appears to be more than 95% at 1:1 mAb/PF4 molar ratio both for purified rabbit and human PF4. Similar results were obtained using supernatants from stimulated human platelets (90% of inhibition at 1:1 mAb/PF4 molar ratio) or using Fab fragments from 10B2. Studies to determine the antigenic determinant against which 10B2 is directed, show that this is an assembled epitope which involves disulfide bonds of the PF4.

Clearance and in vivo release by heparin of human platelet factor 4 (PF4) in the rabbit.

13 male New Zealand rabbits were injected with two different doses (25 micrograms/Kg and 100 micrograms/Kg) of human platelet factor 4 antigen (PF4). The disappearance of the protein was extremely fast with an half-life for the fast component of 1.07 +/- 0.16 and 1.76 +/- 0.11 min respectively. The half-life for the slow component, detectable only with the highest dosage, was 18.8 min. The administration of 2500 I.U. of heparin 30 min after PF4 administration induced a partial release of the injected protein and its clearance from plasma was slow, with half-life of 23.3 +/- 5.9 min and 30.9 +/- 2.19 min respectively.

Plasma markers of endothelial dysfunction in pulmonary hypertension.

STUDY OBJECTIVES: To evaluate and to correlate endothelial cell dysfunction, using recently available plasma markers, with the magnitude of pulmonary artery pressure in patients with severe pulmonary hypertension (PH). DESIGN: Selected plasma markers of endothelial cell dysfunction were studied: nitric oxide (NO), thrombomodulin, tissue factor pathway inhibitor, and soluble endothelium, leukocyte, and platelet selectins (sE-, sL-, sP-selectins, respectively). SETTING: Padova University Hospital and Department of Pathology and Pharmacology, Loyola University of Chicago, Chicago, IL. PATIENTS: Fifteen patients had severe PH (four men and 11 women; mean age, 49.7 +/- 2.9 years: seven patients had primary pulmonary hypertension [PPH] and eight patients had secondary pulmonary hypertension [SPH]), and 20 patients were healthy control subjects. MEASUREMENT AND RESULTS: In patients with PH, sP- and sE-selectins were elevated, whereas sL-selectin was lower in comparison with the selectin levels in control subjects. However, the differences between patients with PH and control subjects were significant only for sL-selectin (p < 0.0001) and sE-selectin (p < 0.03). The NO level was significantly lower in patients with PH compared with the NO level in control subjects (p < 0.01). No difference in tissue factor pathway inhibitor level was noted between control subjects and patients with PH. Only a weak correlation was found between thrombomodulin plasma levels and magnitude of systolic pulmonary artery pressure (r = -0.528, p < 0.05). CONCLUSIONS: Our data are in keeping with the evidence for significant endothelial cell dysfunction in patients with PH and the need for chronic anticoagulation believed to increase survival in these patients. In addition, these data seem to suggest a need for newer agents that are able to increase the antithrombotic endothelial function.

Prospective study of a standardized questionnaire to improve clinical estimate of pulmonary embolism.

A standardized questionnaire was administered to 100 patients suspected of having pulmonary embolism to investigate whether clinical data may be reliably used in the decision-making process of such patients. Questionnaire data were compared with lung scintigraphy outcome and 16 variables were selected as significantly associated with the scintigraphic diagnosis. Based on these variables, patients with abnormal scintigraphy compatible with pulmonary embolism and patients with scintigraphy not compatible with embolism were classified in accordance with the scintigraphic diagnosis. When these 16 variables were tested in an additional validation group, clinical and scintigraphic diagnosis matched in most cases. These results show that clinical data can be used to predict the outcome of lung perfusion scintigraphy in patients suspected of pulmonary embolism. Use of a standardized questionnaire can improve the accuracy of pretest assessment of such patients and positively influence the decisions to start heparin coverage or obtain pulmonary angiography.

Preparation of a new nerve guide from a poly(L-lactide-co-6-caprolactone).

A copolymer of L-lactide and 6-caprolactone (50:50, w/w) was synthesized and characterized. The thermal behaviour of this material did not show any crystallinity for several months; only after more than 1 yr of aging at room temperature and, particularly, in the in vitro degradation tests did it partially crystallize. The values of tensile strength, percent elongation at break and elastic modulus were, respectively, 25 MPa, 490% and 3 MPa. Transparent, elastic nerve guides having inner diameter of 1.3 mm and wall thickness of 175 microns were prepared.

Effectiveness of a bioabsorbable conduit in the repair of peripheral nerves.

A new conduit made with a bioabsorbable copolymer, poly (L-lactide-co-6-caprolactone), was evaluated in an animal model as a guide for nerve regeneration. The conduit had an inner diameter of 1.3 mm and a wall thickness of 175 microns. Segments of length 1.2 cm were interposed between the proximal and distal stumps of transected ischiatic nerves in Wistar rats, bridging a nerve gap of 1 cm. All of the procedure was performed under general anaesthesia using microsurgical techniques. Controls were performed at 1, 3 and 6 months and it was demonstrated that the conduit was still undamaged after 30 d. Progressive signs of degradation appeared at 90 and 180 d. Nerve regeneration in the lumen was effective as confirmed by histological and electron microscopical investigations. These preliminary results emphasize the interesting properties of the conduit with regard to the achievement of a neural prosthesis.

Improved method for purification of human platelet factor 4 by affinity and ion-exchange chromatography.

A simple method for the reproducible purification of human platelet factor 4 (PF4) is described. PF4 is obtained in a highly purified form from platelet concentrate by utilizing a combination of affinity and FPLC ion-exchange chromatography. In every instance, after elution from heparin affinity and cation exchange chromatography, SDS gel electrophoresis reveals a single band attributable to PF4. Moreover, the amino acid composition of PF4 isolated by this method is compatible with that described for a PF4 cDNA clone and with other reported PF4 analysis. The purified protein is used to study in vitro the affinity of PF4 for several glycosaminoglycans (GAGs), by measuring the fluorescence of each PF4-GAG complex bound to fluorescamine. PF4 affinity for GAGs is as follows: heparin greater than heparan sulphate much greater than dermatan sulphate.

Platelet factor 4 (PF4) and heparin-released platelet factor 4 (HR-PF4) in patients with cardiovascular disorders.

The recent introduction of the determinations of platelet factor 4 (PF4) and beta-thromboglobulin (beta TG) by radioimmunoassay provided a new tool to obtain knowledge of in vivo platelet activation. We evaluated the plasma level of PF4 and beta TG in 14 normal subjects (mean PF4 7.7 ng/ml; beta TG 28.8 ng/ml), in 29 patients with chronic stable cardiovascular disorders (mean PF4 9.8 ng/ml; beta TG 32.6 ng/ml) and in 15 diabetics with vascular disease (mean PF4 14.5 ng/ml; beta TG 41.8 ng/ml). The great majority had normal values and no statistical differences were noted among the three groups (p greater than 0.05). Fifteen days of treatment with 150 mg daily of dipyridamole produced a significant reduction in the levels of both proteins (p less than 0.01), in contrast of the daily administration of 650 mg of aspirin, which failed to produce any significant change (p greater than 0.5). The patients and the normal subjects were also administered 3,000 USP units intravenously of porcine heparin. The values of the heparin released-platelet factor 4 (HR-PF4), evaluated 5 minutes after the injection, showed a good correlation between platelet concentration and HR-PF4 levels (z = 2.37, p less than 0.02) in the patients. The determination of standard residual following linear regression analysis of HR-PF4 indicated the presence of two distinct patient populations. One group, including the vast majority of patients, did not differ from the control (patients mean HR-PF4 111.1 ng/ml; controls: mean HF-PF4 136 ng/ml). The other group, with severe cardiovascular disease, but with normal levels of PF4 and beta TG in almost all patients and similar platelet concentrations, showed a significantly higher HR-PF4 (219 ng/ml). Neither aspirin nor dipyridamole had any effect on the level of HR-PF4. This HR-PF4 could represent a possible marker of the interaction of platelets with a seriously damaged atherosclerotic vessel wall.

Tissue factor pathway inhibitor levels in patients with homocystinuria.

Thrombotic events are a well-recognized complication of homocystinuria. However, the mechanisms involved in the atherogenic and thrombotic effects of homocyst(e)ine remain incompletely understood. The objective of this study was to determine the role of endothelial cell activation/damage as indicated by levels of thrombomodulin, tissue factor and tissue factor pathway inhibitor, and factor VII activity in patients with homocystinuria. Six patients with homocystinuria, nonresponsive to pyridoxine, treated only with trimethylglycine (betaine) were injected with a bolus of 20 IU/kg body weight of unfractionated commercial heparin to induce the release of tissue factor pathway inhibitor from the vascular endothelium. Tissue factor, thrombomodulin, and factor VII activity were measured by enzyme-linked immunosorbent assay and clotting assay before heparin administration. Tissue factor pathway inhibitor antigen and activity were measured before and 5 minutes after the bolus of heparin. Levels of homocyst(e)ine were elevated (patients: 144.2+/-19.2 micromol/L; controls: 10.2+/-0.9 micromol/L); however, levels of thrombomodulin, tissue factor, and tissue factor pathway inhibitor antigen were not statistically different from the control group. In contrast, tissue factor pathway inhibitor activity showed a significantly increased level (patients: 2.09+/-0.34 U/L; controls: 1.14+/-0.20 U/L; p<0.05) that was correlated with homocyst(e)ine. Factor VII activity was significantly decreased (patients: 64.7+/-5.1%; controls: 91.4+/-4.7%; p<0.05) and inversely correlated with homocyst(e)ine. After heparin the patients released higher amounts of tissue factor pathway inhibitor antigen and activity compared with the control group; however, the difference was not statistically significant. Although not treated with antithrombotic drugs, none of the patients had any thromboembolic complications after starting betaine. In addition to betaine treatment, the enhanced factor pathway inhibitor antigen activity observed in this small series of patients suggests that factor pathway inhibitor antigen may play an additional, as yet unexplained, role in this genetic disorder.

Platelet count, anti-heparin/platelet factor 4 antibodies and tissue factor pathway inhibitor plasma antigen level in chronic dialysis.

We studied 50 chronic dialysis patients with end-stage renal disease. Mean platelet count was within normal limits. An inverse linear correlation was observed between pre-dialysis platelet count and serum creatinine (r=0.304, p=0.038). Dialysis caused a decrease in platelet count (216+/-80x10(9)/L, pre; 198+/-68, post; p=0.0001), and the higher the pre-dialysis platelet count, the greater the decrease (r=0.623, p=0.0001). Post-dialysis triglyceride decreased (1.67+/-1.27 mmol/L, pre; 1.23+/-0.96, post; p=0.0001). Tissue factor pathway inhibitor (TFPI) antigen plasma level was higher in uremic patients than in controls (114+/-42 ng/ml vs. 64+/-12, p=0.0001). TFPI increased 2.3 times following dialysis and such an increase was directly correlated with post-dialysis plasma heparin concentration (r=0.571, p=0.0002) and inversely correlated with post-dialysis triglyceride variation (r=0.407, p=0.005). Six of fifty patients (12%) had anti-heparin/platelet factor 4 antibodies (Hab), 3 IgG, and 3 IgM. Female sex and the use of cuprophane membranes were more frequent among Hab-positive patients (p=0.0001), while a lower percentage of them were on anti-aggregating drugs as compared to Hab-negative patients (p=0.002). Only one Hab-positive patient was slightly thrombocytopenic and none showed bleeding or thrombotic manifestations. Serum albumin and y globulin decreased following dialysis in Hab-positive patients, while the opposite was seen in those Hab-negative (-2.47+/-1.72 g/L, vs. 0.21+/-1.77, p=0.001 and -0.48+/-0.60 g/L vs. 0.64+/-0.97, p=0.007, respectively). In vivo factors other than Hab are involved in the development of heparin-induced thrombocytopenia. Besides a blunted immunological response, increased levels of TFPI, the use of anti-aggregating drugs, and the observed behavior of serum proteins might play a role in this regard.

Disappearance of human platelet factor 4 (PF4) in rabbits: does an immediate component exist?

Twelve male New Zealand rabbits were injected with 21 micrograms/kg of human platelet factor 4 antigen (PF4). The decay of the protein followed a monoexponential curve for the first 5 mins, with a half-life (t 1/2) of 1.94 mins and a calculated concentration at 0 time (CO) of 79.4 ng/ml. Five rabbits were pre-treated with heparin (2.500 I.U. i.v.) and 3 mins later were injected with the same amount of PF4. PF4 decay followed a monoexponential curve with a t 1/2 of 25.3 mins, and with CO of 380.8 ng/ml. This value is not greatly different from the one calculated assuming an immediate and uniform distribution in plasma (482.7 ng/ml for a plasma volume of 43.5 ml/kg). The 12 rabbits injected with PF4 were divided in 3 groups, in which heparin was given at 10', 30' or 60' after PF4, respectively. After heparin the peak levels of PF4 were 139.9 ng/ml, 65.3 ng/ml and 52.7 ng/ml, respectively. The following monoexponential PF4 decay had t 1/2 of 20.7, 25.6 and 26.0 mins, respectively. In a separate group of 4 animals, we studied heparin decay after an intravenous bolus of 2.500 I.U. Heparin decay could not be described by a monoexponential equation and was different from the decay of PF4 injected after heparin. On the basis of the present data we suggest the presence of an immediate component of PF4 decay, most likely due to uptake by the tissues. Heparin pretreatment may avoid this uptake process.

Tissue factor pathway inhibitor (TFPI) activity in uremic patients during hemodialysis.

We studied tissue factor pathway inhibitor (TFPI) activity during hemodialysis in 10 uremic patients who were not receiving anticoagulant for at least 120 minutes. TFPI activity before dialysis was normal (patients 107 +/- 5.8%, controls 104 +/- 4.5%). During extracorporeal circuit it rose progressively with a statistically significant difference, reaching a plateau between 60 and 120 minutes. Since thrombin induces a marked redistribution and release of TFPI from stimulated endothelial cells and platelets contain about 10% of TFPI activity that is secreted following activation it is possible that thrombin-induced release of TFPI by endothelium and platelets could account for the increased TFPI we found during hemodialysis. To investigate this possibility we measured during dialysis beta-thromboglobulin (beta-TG), thrombin-antithrombin complex (TAT) and prothrombin fragment 1.2 (F 1.2). The increased levels of beta-TG, TAT and F1.2 we noted during extracorporeal circuit are in keeping with this concept. One hundred eighty minutes after initiation of dialysis, by which time all patients were receiving heparin there was a further increase in TFPI (to more than 200% of baseline), due to the presence of the glycosaminoglycan. This was due the previously reported displacement by heparin of the major intravascular pool of TFPI, from endothelial cell surfaces.