Patency of heparin-PVA coated tubes at low flow rates

The patency of heparin-PVA coated 1.14 mm i.d. polyethylene tubes was greater than that of control tubes without heparin in a novel test section that enabled ex vivo evaluation in dogs at low flow and shear rates (2-10 ml/min). Low shear rates were achieved by diverting a small portion of a chronic A-V shunt flow through Y-connectors into the small diameter test tubing. For shear rates in the range of 200-650 s-1 half the heparin-PVA tubes remained patent for longer than 72 min whereas half the control tubes (PVA only) only remained patent for longer than 30 min. At higher shear rates (500-1000 s-1), the 50% patency times for heparin-PVA and PVA were 225 and 123 min respectively. The increased patency at the higher shear rates was attributed more to the effect of the connector than just shear rate. The higher shear rates were achieved by changing from a Y-connector in which the small diameter side-branch was cut flush to the larger diameter main line to one in which the small diameter test tubing protruded into the lumen of the main line; the latter 'protruding' connector was presumed to result in less platelet pre-activation than the other. This method has been found useful for assessing the thrombogenicity of heparinized tubes at low flow rates.     

Water content and compression modulus of some heparin-PVA hydrogels

The effects of changes in the heparin-PVA formulation on the water content, compression modulus and molecular weight between cross-links of the resulting gel was determined. Molecular weight between cross-links was calculated directly from the compression modulus and the swelling ratio. Glutaraldehyde and MgCl2 displayed the effects expected of them as cross-linking agent and catalyst respectively. On the other hand, formaldehyde appeared to be a non-essential component since its absence had little effect on gel properties and its presence did not affect the water content as originally predicted. Understanding the effect of each formulation component on gel properties enables alteration of gel water content and hence permeability for special applications.     

Effect of heparin-PVA hydrogel on platelets in a chronic canine arterio-venous shunt

Polyvinyl alcohol (PVA) hydrogel, with or without heparin, was reactive towards canine platelets in a chronic arteriovenous shunt as demonstrated by an increase in platelet regeneration time, a systemic decrease in platelet count and transient decrease in platelet serotonin content. Immobilized heparin (heparin-PVA) had no effect whereas unmodified polyethylene was found to be unreactive despite similar levels of platelet deposition as measured by SEM and a higher in vitro reactivity (J. Biomed. Mater. Res., this issue). Twenty-centimeter lengths of hydrogel coated polyethylene tubing were inserted between the arterial and venous portions of the shunt and left in place for 4-6 days, without the complicating artifacts of anticoagulation, anesthesia, or surgical intervention. Regeneration time was measured as the return to normal platelet cyclooxygenase (co) activity after a single 240-mg dose of aspirin, with co activity measured in vitro as malondialdehyde production. Although measuring new platelet production, regeneration time is an indirect measure of platelet consumption, so that the reduced regeneration time seen here was presumed to reflect enhanced material associated consumption and thromboembolism. Like other hydrogels, PVA does not appear to be "thromboadherent" but it does appear thrombogenic. Immobilized heparin had no additional effect, presumably because the platelet response was dominated by the reactivity of the underlying substrate.     

A model of thrombin inactivation in heparinized and nonheparinized tubes with consequences for thrombus formation

The role of flow and mass transport in determining procoagulant concentration at the wall of synthetic and natural cylindrical blood vessels is analyzed theoretically. The model assumes steady laminar flow and considers, in addition to the fluid dynamic parameters, three rate-determining steps: production of procoagulant (thrombin) and its inactivation at the wall, as well as inactivation in the fluid bulk. The ratio of thrombin wall concentration to production rate Cw/N emerges as a critical parameter in characterizing the behavior of the tube wall. With a wall-inactivation rate typical of heparinized materials, Cw/N = 11.1 s/cm, independent of flow (shear rate) and axial position. This is significantly less than the range of Cw/N (50-500 s/cm) for which the thrombin concentration is high enough to result in significant fibrin formation and thrombosis. Hence little fibrin formation and a high degree of thromboresistance is expected for heparinized materials. Nonheparinized materials have Cw/N values above this range, which are only weakly dependent on shear rate and diameter, suggesting that flow-induced dispersion of thrombin (or other procoagulants) has limited impact on the thrombin wall concentration. These latter results appear to refute the conventional wisdom that attributes the relative patency of large-diameter vessels and differences between venous and arterial thrombi to such flow effects. It is likely that additional factors such as flow pulsatility and wall geometry must be considered to account for these observations.     

The modulation of gene expression in osteoblasts by thrombin coated on biphasic calcium phosphate ceramic

For many years, fibrin sealants were associated with bone substitutes to promote bone healing. However, the osteoblastic response to fibrin sealant components remains poorly documented. In this study, MC3T3-E1 osteoblastic cells were cultured on biphasic calcium phosphate ceramic (MBCP) coated with Tissucol components (thrombin and fibrinogen). Analysis of osteoblastic differentiation markers by RT-PCR revealed that MBCP coated with Tissucol stimulated mRNA levels for osteocalcin and alkaline phosphatase (ALP). Of all the components of Tissucol, thrombin has been reported to affect osteoblastic behavior. Our results demonstrated that low thrombin concentrations (0.5-5 U/ml) stimulated mRNA levels for ALP, whereas high thrombin concentrations (50-100 U/ml) decreased mRNA levels for ALP and PTH/PTHrP receptor and also increased mRNA level for the osteoclastogenesis inhibitor OPG. As thrombin stimulated angiogenesis, we then wondered whether thrombin could influence the expression of angiogenic factors. Low thrombin concentrations were shown to up-regulate mRNA levels for VEGF-B and VEGF-R1, suggesting an autocrine/paracrine role for VEGF-B. Higher thrombin concentrations also up-regulated mRNA for VEGF-A and neuropilin-1. In conclusion, the association of MBCP with thrombin and fibrinogen appears to be a convenient scaffold for bone cell differentiation. Thrombin could also acts at the cellular level by increasing the angiogenic potential of osteoblasts as well as their responsiveness to thrombin and VEGF.     

Development of coated tubes RIA for serum T3 (tri-iodothyronine) for production scale

A coating procedure that could provide immobilization of antibodies, with increased binding capacity, that is cost effective, simple, robust, and appropriate for production scale application, is described. This coating approach of T3 antibodies to the polystyrene tubes has been systematically investigated to determine its utility for the development of coated tube Radioimmunoassay (RIA) for T3 in human serum. Further, the results obtained by the developed coating procedure are found to be comparable with those obtained by the "gold standard," the liquid phase RIA for T3. The coating procedure is completed in three major steps, each step involving an overnight incubation. The normal rabbit gamma-globulins are physically adsorbed onto the polystyrene tubes and incubated. After washing, a second antibody (goat anti-rabbit antiserum) is added and incubated. To this antigen specific antibody is added (T3 antibody produced in rabbit) and further incubated. Finally, the non-specific sites on the tubes are saturated by the blocking solution. The concentration of normal rabbit globulin, titers of second antibody and T3 antibody, and time required for coating are optimized to arrive at a suitable coating protocol. The coated tubes were evaluated for precision, reproducibility, and stability. Various parameters such as total reaction volume, incubation time and temperature, total number and volume of washings, concentration of 8-anilino-1-naphthalene sulfonic acid (ANS), and quantity of tracer per tube are optimized to arrive at a suitable standard curve. The optimized assay is validated for the quality control parameters such as intra- and inter-assay variations, recovery, and parallelism. The developed coated tubes assay had an assay range of 0.3-4.8 ng/mL with a sensitivity of 0.3 ng/mL at 90% B/B0. Batch to batch variation in coating was < 10%. The coated tubes were stable up to 1 year, which is adequate for production scale.     

Evaluation in an emergency department of rapid separator tubes containing thrombin for serum preparation prior to hs-cTnT and CK-MB analyses

Background

In our emergency department, we collect blood in Rapid Serum Tubes (RSTs; Becton Dickinson, Franklin Lakes, NJ), in which clotting times are reduced. We investigated the influence of RST use on cardiac troponin T (hs-cTnT) and creatine kinase-MB (CK-MB) test results, in comparison with the use of tubes featuring a separator gel containing a clotting activator (SSTs; Green-vac, Yongin, Korea).

Methods

Samples from 60 patients were divided into equal aliquots and placed into RSTs and SSTs; hs-cTnT and CK-MB concentrations were determined using an autoanalyzer (Elecsys 2010) running commercial assays (Roche Diagnostics, Penzberg, Germany). Between-tube differences in CK-MB and hs-cTnT values were compared using the paired t-test, and correlations among variables were evaluated by calculation of Spearman correlation coefficients (r values). Deming regression analysis was performed and Bland-Altman plots were constructed.

Results

The hs-cTnT and CK-MB test results obtained from samples placed into RSTs and SSTs did not differ (p?>?0.1). The correlations between the concentrations of hs-cTnT and CK-MB in samples placed into RSTs and SSTs were good; both r values were unity (p?<?0.001). Deming regression analysis yielded the equation: RST [hs-cTnT]?=?0.98 SST [hs-cTnT]?+?0.69 pg/ml; and RST [CK-MB]?=?0.95 SST [CK-MB]–0.09 ng/ml. The biases of 1.4 pg/ml (95% CI: minus 8.1–10.7 pg/ml) for hs-cTnT levels and 0.249 ng/ml (95% CI: minus 0.682–1.681 ng/ml) for CK-MB levels assayed using either tube was acceptable.

Conclusion

The hs-cTnT and CK-MB test results did not significantly differ when either tube was used. RST tube use was associated with a short clotting time; this was an advantage in an emergency laboratory setting.

     

Analysis of cellular morphology, adhesion, and proliferation on uncoated and differently coated PVC tubes used in extracorporeal circulation (ECC)

The biggest challenge to improve extracorporeal circulation (ECC) circuits lays on avoiding platelet adhesion to their surfaces, because this contributes to thrombus formation, resulting in the activation of blood coagulation. One approach to minimize this effect is to improve the biocompatibility of ECC circuits by modifying their surfaces. This can be achieved by coating them with heparin or phospholipids. The present study investigated the adhesion and morphology characteristics of fibroblastic and blood cells cultured on uncoated poly (vinyl) chloride PVC tubes as well as on heparin, phosphatidylcholine (DMPC), and phosphatidylethanolamine (DMPE) -coated tubing. The results showed the importance of uniform coating regardless of the substance used, because the coatings cover the grooves on PVC surfaces, which favor cell adhesion. The comparison among the three different coatings showed the best biocompatibility results for the PVC tubes coated with heparin, followed by the coating with DMPE and with DMPC. For all coated tubes, cells did not spread on the PVC surfaces and, consequently, did not adhere to their surfaces, increasing the overall biocompatibility of PVC tubes. However, possible DMPE's alkylation, caused by sterilization, resulted in increased material hydrophobicity, which explains the decrease in fibroblastic adhesion. Furthermore, sterilization of DMPC-PVC improves its hydrophilic character, also decreasing adhesion. Based on these results, coating PVC with the phospholipids DMPC and DMPE seems to be a promising technique to improve the biocompatibility of PVC tubes, and is worthy of further investigation.     

Peptidomimetic thrombin inhibitors

The central position of thrombin in the coagulation cascade has made it a popular target for discovery of novel antithrombotic agents. Starting with hirudin, a natural peptide isolated from the medicinal leech, its shorter synthetic analogue hirulog, and argatroban,the first therapeutically used synthetic small-molecule thrombin active site inhibitor, hundreds of direct thrombin inhibitors have been discovered over the last 20 years. Most of them are peptidomimetic compounds,based on the amino acid sequence of fibrinogen which binds into the thrombin active site. Since elucidation of the crystal structure of human thrombin in 1989, the structure-based design of low-molecular-weight peptidomimetic thrombin inhibitors has been greatly aided by the use of x-ray crystallographic analysis of thrombin-inhibitor complexes. The ultimate goal of most research programmes and drug optimization strategies is to develop an orally bioavailable, small-molecule,direct thrombin inhibitor that would be suitable for once or twice daily dosing. An overview of the most advanced peptidomimetic direct thrombin inhibitors bivalirudin, argatroban, ximelagatran and dabigatran is presented.     

Skewed X-Chromosome Inactivation in Scleroderma

Scleroderma is a female-prevalent autoimmune disease of unclear etiology. Two fundamental gender differences, skewed X-chromosome inactivation (XCI) and pregnancy-related microchimerism, have been implicated in scleroderma. We investigated the XCI patterns of female scleroderma patients and the parental origin of the inactive X chromosome in those patients having skewed XCI patterns (>80%). In addition, we investigated whether a correlation exists between XCI patterns and microchimerism in a well-characterized cohort. About 195 female scleroderma patients and 160 female controls were analyzed for the androgen receptor locus to assess XCI patterns in the DNA extracted from peripheral blood cells. Skewed XCI was observed in 67 (44.9%) of 149 informative patients and in 10 of 124 healthy controls (8.0%) [odds ratio (OR) = 9.3 (95% confidence interval (CI) 4.3-20.6, P < 0.0001)]. Extremely skewed XCI (>90%) was present in 44 of 149 patients (29.5%) but only in 3 of 124 controls (2.4%; OR = 16.9; 95% CI 4.8-70.4, P < 0.0001). Parental origin of the inactive X chromosome was investigated for ten patients for whom maternal DNA was informative, and the inactive X chromosome was of maternal origin in eight patients and of paternal origin in two patients. Skewed XCI mosaicism could be considered as an important risk factor in scleroderma.     

Isolation of bound thrombin consisting of thrombin and fibrin N-terminal fragment from clot lysate

It has been reported that thrombin is liberated from fibrin clots by the action of fibrinolytic enzymes. It has also been reported that the liberated thrombin complexes with fibrin fragment E or (DD)E, which are denoted as bound thrombin. However, bound thrombin has not been isolated from clot lysate, and the structural characteristics of isolated bound thrombin have not been specified. In this study, we attempted to isolate the bound thrombin from clot lysate and to clarify its structural features. Rabbit fibrinogen was clotted with bovine thrombin, and clot lysate was prepared with urokinase. The bound thrombin was isolated from clot lysate by serial chromatography using a Sepharose 4B column immobilizing an anti-bovine thrombin antibody and a Sepharose 4B column immobilizing an anti-rabbit fibrinogen antibody. SDS-PAGE under unreduced conditions demonstrated that there were two different protein bands in the isolated bound thrombin. On a C4 reverse-phase HPLC, the bound thrombin from clot lysate was resolved by 4 M urea into alpha-thrombin and a fibrin fragment, the N-terminal regions of which were identified as alpha-, beta- and gamma-chains. Thus, in the bound thrombin, thrombin molecule would bind to rabbit fibrin fragment consisting of N-terminal central domain.     

Effects of the oral direct thrombin inhibitor ximelagatran on p-selectin expression and thrombin generation in atrial fibrillation

This study investigated the pharmacodynamic effects of the oral direct thrombin inhibitor ximelagatran on platelet activation and thrombin generation in patients with nonvalvular atrial fibrillation. Using an open, group-matched study design, the effects of ximelagatran (36 mg twice daily for 5 days) were studied in 12 patients with permanent nonvalvular atrial fibrillation and in 12 healthy controls. After ximelagatran for 5 days, elevated platelet P-selectin expression in atrial fibrillation patients was lowered to that during coumarin treatment or in controls but had no effect in control subjects. Using the endogenous thrombin potential as a marker, ximelagatran decreased and delayed thrombin generation in both groups. In conclusion, direct thrombin inhibition with ximelagatran reduced elevated platelet P-selectin expression and inhibited thrombin generation.     

Multifunctional roles of thrombin.

Thrombin is an unique molecule that functions both as a procoagulant and anticoagulant. In its procoagulant role it activates platelets through its receptor on the platelets. It regulates its own generation by activating coagulation factors V, VIII and even XI resulting in a burst of thrombin formation. It activates factor XI, thus preventing fibrin clots from undergoing fibrinolysis. Thrombin not only cleaves fibrinogen to fibrin, but also through the activation of factor XIII effects the cross-linking of fibrin monomers to produce a firm fibrin clot. Thrombin's role as an anticoagulant is mediated through binding to thrombomodulin, a receptor protein on the endothelial membrane of the blood vessel, initiating a series of reactions that leads to fibrinolysis. Thrombin has chemotactic properties enabling it to exert its effects during inflammation and vascular injury. It has a mitogenic effect stimulating growth of mammalian cells, fibroblasts and macrophage-like tumor cell lines. It has also been implicated in brain development. A molecule with multifunctional roles such as thrombin has its activity in vivo modulated by only a few endogenous inhibitors.