Actions of thrombin in the interstitium.

Thrombin is a pleiotropic enzyme best known for its contribution to fibrin formation and platelet aggregation during vascular hemostasis. There is increasing evidence to suggest a role for thrombin in the development of interstitial fibrosis, but interstitial thrombin has not been demonstrated by the direct determination of activity. Rather its presence is inferred by products of thrombin action such as fibrin and activated fibroblasts. This review will focus on possible mechanisms of thrombin formation in the interstitial space, the possible actions of thrombin, processes regulating thrombin activity in the interstitial space, and evidence supporting a role for thrombin in fibrosis.

Continuous cardiac output during off-pump coronary artery bypass surgery: pulse-contour analyses vs pulmonary artery thermodilution.

BACKGROUND: No gold standard method exists for monitoring continuous cardiac output (CO). In this study, the agreement between the two most frequently used methods, PiCCO pulse-contour analysis (PCCO) and STAT pulmonary artery thermodilution (STAT-CO), was assessed during multiple-vessel off-pump coronary artery bypass (OPCAB) surgery. METHODS: Thirty patients were enrolled in the study. Two time periods were defined during surgery; Period 1 included positioning of the heart and stabilizer device and Period 2 included the coronary occlusion. Measurements were obtained every minute during both periods. The agreement for the continuous CO and the change in CO (DeltaCO) was estimated using the Bland-Altman method. RESULTS: Significant changes in mean arterial pressure (DeltaMAP), central venous saturation, PCCO and STAT-CO were seen only during Period 1. DeltaMAP correlated only with changes in PCCO, (P < 0.001, r = 0.60). The mean difference (2sd) between PCCO and STAT-CO ranged from - 0.29 (1.82) to - 0.71 (2.57) litre min(-1), and the percentage error varied from 32 to 50%. For the CO measurements, the limits of agreements did not differ between Period 1 and Period 2. In contrast, for the DeltaCO measurements, the limits of agreements were wider in Period 1 than in the more haemodynamically stable Period 2. CONCLUSIONS: CCO and STAT-CO show large discrepancies in CO during OPCAB surgery. Clinically acceptable agreement was seen only for trends in CO during haemodynamically stable periods.

Antibody therapy (IVIG): evaluation of the use of genomics and proteomics for the study of immunomodulation therapeutics.

BACKGROUND AND OBJECTIVES: Intravenous immunoglobulin (IVIG) is used for an increasingly diverse number of therapeutic applications as an immunomodulation drug. Although it has demonstrated therapeutic effectiveness, the mechanism of action of IVIG in these disorders is poorly understood; this lack of understanding complicates rational clinical application and reimbursement for 'off-label' use. MATERIALS AND METHODS: Selected literature on the clinical use of IVIG as an immunomodulation drug is reviewed. We present a brief description of DNA microarray and protein microarray technology and the application of such technologies to the study of immune system cells. The several studies on the application of DNA microarray technology to study gene expression in response to IVIG are presented. RESULTS: There is increasing data on the use of DNA microarray and protein microarray technology to study gene expression in immune system cells including T cells, B cells, macrophages, and leucocytes. There is less information on the effect of IVIG on gene expression in immune system cells. However, there is sufficient information available to suggest that this is a practical approach with the caveat that such work will require careful experimental design and clear definition of the normal population. CONCLUSIONS: DNA and protein microarray assays can be used to (i) provide rational indications for the clinical use of IVIG, (ii) provide for specific analysis of raw material and end product IVIG in screening for content related to immunomodulation, and (iii) accelerate the development of next generation products which would be more focused and/or targeted therapeutics.

Agreement between PiCCO pulse-contour analysis, pulmonal artery thermodilution and transthoracic thermodilution during off-pump coronary artery by-pass surgery.

BACKGROUND: Haemodynamic instability during off-pump coronary artery bypass surgery (OPCAB) may appear rapidly, and continuous monitoring of the cardiac index (CI) during the procedure is advisable. With the PiCCO monitor, CI can be measured continuously and almost real time with pulse-contour analysis and intermittently with transthoracic thermodilution. The agreement between pulmonal artery thermodilution CI (Tpa), transthoracic thermodilution CI (Tpc) and pulse-contour CI (PCCI) during OPCAB surgery has not been evaluated sufficiently. METHODS: In 30 patients scheduled for OPCAB surgery, a pulmonary artery catheter and a PiCCO catheter were inserted. At different time points during surgery, Tpa, Tpc and PCCI were compared. Measurements were performed after induction of anesthesia (T1), after pericardiothomy (T2), after grafting on the anterior (T3), posterior (T4) and lateral (T5) walls and after chest closure (T6). The PCCI was recalibrated at time point T2-T6. RESULTS: Mean difference and the limits of agreements (percentage error) between Tpa and Tpc were: -0.14 +/- 0.60 (22.0%) l/min/m2, between Tpa and PCCI: -0.07 +/- 0.92 (33.5%) l/min/m2 and between Tpc and PCCI: 0.10 +/- 1.00 (35.5%) l/min/m2. For changes in CI from one time point to the next (DeltaCI), the limits of agreements between DeltaCI Tpa and DeltaCI Tpc were 0.04 +/- 0.90 l/min/m2, between DeltaCI Tpa and DeltaCI PCCI: -0.02 +/- 1.22 l/min/m2 and between DeltaCI Tpc and DeltaCI PCCI: -0.08 +/- 1.32 l/min/m2. CONCLUSION: In OPCAB surgery, limits of agreement comparing thermodilution methods were smaller than comparing PCCI with thermodilution. Recalibration of PCCI is therefore advisable.

Sternal wound infections in patients undergoing open heart surgery: randomized study comparing intracutaneous and transcutaneous suture techniques.

BACKGROUND: Intracutaneous suture technique has been our standard method for closing sternal wounds in cardiac surgery, mainly for cosmetic reasons. However, an increased rate of postoperative infections has been reported in cosmetic surgery with this method compared with the percutanous or transcutaneous closure technique. A comparison of these two techniques in cardiac surgery is presented. METHODS: In a randomized study, 300 patients were selected to intracutaneous suture (n = 150) or percutanous suture (n = 150). The endpoints were superficial and deep sternal wound infections within 6 weeks postoperatively. RESULTS: The total infection rate was lower in the percutanous group compared with the intracutaneous group (3% versus 8%) (p = 0.007). The superficial infection rate was lower in the percutaneous group (2.3% versus 6.7%) (p = 0.01), whereas there was no statistically significant difference in the deep infection rate between the groups. CONCLUSIONS: The percutaneous suture technique reduces the incidence of superficial wound infections, but not the deep infection rate in open heart surgery. There was no difference in the cosmetic results on a visual scale, assessed by the patients.

Graft control by transit time flow measurement and intraoperative angiography in coronary artery bypass surgery.

BACKGROUND: The aim of this study was to compare the relationship between intraoperative transit time flow measurements and angiographic findings with long-term graft patency in 72 patients who underwent coronary artery bypass surgery. METHODS: Transit time flow measurements with recording of mean flow and pulsatility indexes were performed after completion of the anastomoses. Coronary angiography was performed on-table while the patients were still in general anesthesia, and then at follow-up three months and 12 months after surgery. Based on angiography, the grafts were graded as type A (fully patent), type B (having more than 50% diameter reduction), or type O (occluded). RESULTS: Of the 67 left internal mammary artery (LIMA) grafts, 51 (76%) were type A on-table, 14 (21%) were type B, and two (3%) were type O. Of the 57 saphenous vein grafts, 49 (86%) were type A, 7 (12%) were type B, and one (2%) was type O. For both LIMA and vein grafts, there were no differences in flow (p = 0.69 and 0.47, respectively) or pulsatility index (p = 0.79 and 0.83) between type A and B. There were also no differences in flow (p = 0.37 and 0.7) or pulsatility index (p = 0.37 and 0.24) between type B on-table that either normalized or persisted occluded at the follow-up. Transit time flow measurement failed to detect an occluded LIMA graft as shown by intraoperative angiography. CONCLUSIONS: Blood flow measurements performed intraoperatively could not identify significant lesions in arterial or vein grafts, and could not predict graft patency. We have become cautious in interpreting flow measurements alone and combine blood flow recordings with intraoperative angiography in the assessment of graft quality.

Ventricular fibrillation during off-pump coronary artery bypass grafting: transcranial Doppler and clinical findings.

A 73-year-old male developed ventricular fibrillation which lasted for 2 min and 24 s, during off-pump coronary artery bypass grafting. Cerebral hemodynamics were assessed by continuous transcranial Doppler monitoring. Ventricular fibrillation resulted in an immediate fall in cerebral blood flow velocities to almost zero with only slight fluctuations. This was then followed by a pronounced reactive hyperperfusion. Cerebral magnetic resonance imaging examinations and detailed neurological and neuropsychological evaluations were performed before and at 3 and 12 months after surgery. No evidence of cerebral damage was found.

The CarboMedics supraannular top hat valve improves prosthesis size in the aortic root.

BACKGROUND AND AIM OF THE STUDY: Aortic valve replacement can result in patient-valve mismatch and attenuated left ventricular remodeling. Using CarboMedics mechanical valves, we examined if the supraannular Top Hat prosthesis provided a size advantage over the intraannular valve. METHODS: Seventeen patients with aortic stenosis and aortic root < or = 23 mm were randomized to receive a CarboMedics supraannular Top Hat valve (n = 7) or an intraannular valve (n = 10). Doppler echocardiography was performed preoperatively, and after three months. RESULTS: There was no difference in aortic annulus size, but mean prosthesis size was significantly larger in the Top Hat group than in the intraannular group (25.00 mm versus 21.60 mm); the mean size improvement for Top Hat patients was 3.14 mm. After three months, all patients had excellent functional improvement and low transvalvular pressure gradients, with slightly higher effective valve opening area in the Top Hat group. CONCLUSION: The supraannular Top Hat valve provides an advantage of one to two sizes over the intraannular valve, and improves the effective valve opening area. Both valves offer favorable hemodynamic performance and functional improvement.

Issues with the assay of factor VIII activity in plasma and factor VIII concentrates.

A review of the literature suggests that assays accurate for the determination of factor VIII in plasma samples may not necessarily retain this accuracy when used for the determination of factor VIII in high-purity factor VII concentrates such as Hemofil M. Review of assay data suggests that it is imperative to obtain maximal activation of the factor VIII in the sample with thrombin when using an assay system of isolated coagulation factors such as the two-stage assay or the various chromogenic substrate assays. Based on a combination of ease and reproducibility of performance and correlation of in vivo and in vitro measurements. it is recommended that the one-stage activated partial thromboplastin time performed with plasma from an individual with severe hemophilia A be used for the measurement of factor VIII potency. Chromogenic substrate assays can be used if care is taken to assure optimal activation of factor VIII by thrombin in the assay and the presence of sufficient factor IXa, phospholipid and calcium ions to stabilize factor VIIIa during the assay process.

Colorimetric protein assay techniques.

There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

Measurement of active coagulation factors in Autoplex-T with colorimetric active site-specific assay technology.

Autoplex-T is a partially activated prothrombin complex concentrate used primarily for the treatment of patients expressing factor VIII inhibitors. While Autoplex-T has a demonstrated record of clinical effectiveness, the procoagulant composition of this material has not been reported. This absence of composition data is a reflection of the lack of techniques appropriate for accurately measuring an individual protease such as factor IXa in complex mixtures of similar proteases. The development of Colorimetric Active Site-Specific ImmunoAssay technology (CASSIA) has permitted the accurate analysis of the coagulant enzymes present in Autoplex-T. Ten lots of Autoplex-T were reacted with both biotinylated phenylalanylprolylarginine chloromethylketone and biotinylated glutamylglycylarginine chloromethylketone. Only activated forms of the clotting factors present in Autoplex-T react with the peptide chloromethylketones and were thus separated from the other proteins present in Autoplex-T by adsorption onto streptavidin. The individual proteins bound to streptavidin were then detected with specific antibodies. Mean results from the analysis of ten lots of Autoplex-T (mean values) are as follows: factor Xla, 5.9 nM or 0.8 microg/ml; factor Xa, 46.5 nM or 2.1 microg/ml; factor IXa, 177.8 nM or 11.7 microg/ml; factor VIIa, 68.6 nM or 3.3 microg/ml and factor IIa, 5.3 nM or 0.2 microg/ml. These results are discussed with respect to the mechanism of action of Autoplex-T in the treatment of factor VIII inhibitor patients.

Endothelin-1 and neutrophil activation during heparin-coated cardiopulmonary bypass.

BACKGROUND: Heparin-coated circuits attenuate the systemic inflammatory response to cardiopulmonary bypass. The present study compares two different heparin coatings in terms of the release of endothelin-1 and neutrophil glycoproteins. METHODS: Forty low-risk patients undergoing coronary artery bypass grafting were investigated, having cardiopulmonary bypass with a Duraflo II heparin-coated circuit (n = 10), an identical but uncoated circuit (n = 10), a Carmeda BioActive Surface heparin-coated circuit (n = 10), or an identical but uncoated circuit (n = 10). A standard systemic heparin dosage was used in all patients. Endothelin-1 and the neutrophil glycoproteins lactoferrin and myeloperoxidase were quantified throughout the operation and 3 hours postoperatively. RESULTS: Enhanced plasma levels of endothelin-1, lactoferrin, and myeloperoxidase were observed during and after uncoated cardiopulmonary bypass, but this was not associated with clinical side effects. Compared with the respective uncoated controls, Duraflo II attenuated only the lactoferrin levels, whereas Carmeda BioActive Surface was associated with lower levels of both endothelin-1, lactoferrin, and myeloperoxidase. Of the two heparin coatings, Carmeda BioActive Surface proved more effective than Duraflo II in attenuating the levels of these substances. CONCLUSIONS: The plasma levels of endothelin-1, lactoferrin, and myeloperoxidase increase during cardiopulmonary bypass in coronary artery bypass grafting, but this has no clinical side effects in low-risk patients. The increase is attenuated using heparin-coated extracorporeal circuits, and then more effectively by Carmeda BioActive Surface than by Duraflo II.

Applications of site-specific chemical modification in the manufacture of biopharmaceuticals: I. An overview.

The chemical modification of proteins has long been a useful approach to elucidating structure-function relationships. Recently, these same chemistries have been finding application in the preparation of proteins for both diagnostic and therapeutic use. These applications include alterations to introduce new properties, such as improved stability, or new traits, such as drug binding and transport, that take advantage of both broad and narrow ranges of selectivity. Site-specific chemical modification of proteins, when combined with the powerful advantages of site-specific mutagenesis, can yield protein agents superior to those generated by either approach alone.

Elucidation of N-linked oligosaccharide structures of recombinant human factor VIII using fluorophore-assisted carbohydrate electrophoresis.

Characterization of the carbohydrate moiety is a critical measure of manufacturing process consistency of recombinant human Factor VIII (rFVIII) in Chinese-hamster ovary (CHO) cells. FVIII, a large (300 kDa) glycoprotein, is employed therapeutically for the correction of haemophilia A. While N-linked and O-linked oligosaccharides are found in this protein, the current study focuses on the N-linked oligosaccharides. The N-linked oligosaccharides from rFVIII were released using either peptide N-glycosidase F or endoglycosidase H, derivatized with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonate, and analysed by fluorophore-assisted carbohydrate electrophoresis (FACE). The electrophoretically resolved oligosaccharide bands were isolated and individual bands subjected to digestion with defined pools of exoglycosidases and re-electrophoresed on FACE sequencing gels. The resulting gel patterns were interpreted, based on band mobility shifts, to obtain the sequence structure of the oligosaccharides. A total of eight acidic and 12 neutral structures were identified, and the majority of the oligosaccharides (approximately 92%) were found to be sialylated. All of the major oligosaccharide structures found in CHO-cell-derived rFVIII have also been reported to be present in plasma-derived FVIII. Among the most abundant are disialylated, biantennary, core-fucosylated (approximately 40%), followed by trisialylated, triantennary, core-fucosylated and monosialo, biantennary, core-fucosylated structures (each approximately 18%). The Gal alpha 1-3Gal structures reported to be present in baby-hamster-kidney-cell-derived rFVIII were not found in the CHO-cell-derived protein. The glycosylation patterns were consistent in six random lots of rFVIII [coefficient of variation (%) 3-14] based on percentage lane luminance data of bands that represent approximately 98% of all asparagine-linked oligosaccharides.