Chandler Loop Method Research Articles

Hemocompatibility of styrenic block copolymers for use in prosthetic heart valves.

Certain styrenic thermoplastic block copolymer elastomers can be processed to exhibit anisotropic mechanical properties which may be desirable for imitating biological tissues. The ex-vivo hemocompatibility of four triblock (hard–soft–hard) copolymers with polystyrene hard blocks and polyethylene, polypropylene, polyisoprene, polybutadiene or polyisobutylene soft blocks are tested using the modified Chandler loop method using fresh human blood and direct contact cell proliferation of fibroblasts upon the materials. The hemocompatibility and durability performance of a heparin coating is also evaluated. Measures of platelet and coagulation cascade activation indicate that the test materials are superior to polyester but inferior to expanded polytetrafluoroethylene and bovine pericardium reference materials. Against inflammatory measures the test materials are superior to polyester and bovine pericardium. The addition of a heparin coating results in reduced protein adsorption and ex-vivo hemocompatibility performance superior to all reference materials, in all measures. The tested styrenic thermoplastic block copolymers demonstrate adequate performance for blood contacting applications.

Antithrombogenic Properties of Amphiphilic Block Copolymer Coatings: Evaluation of Hemocompatibility Using Whole Blood.

Antithrombogenicity is one of the most critical properties required for materials used in biomedical devices, particularly in devices that contact blood. The antithrombogenicity of surfaces coated with amphiphilic block copolymers composed of hydrophobic poly(2-methoxyethyl acrylate) (M) and hydrophilic poly(N,N-dimethylacrylamide) (D) segments was investigated using plasma protein and whole blood with regard to protein adsorption, thrombus formation, platelet activation, and clotting kinetics. Three types of block copolymers and a random copolymer were synthesized using one-pot reversible addition-fragmentation chain-transfer (RAFT) polymerization under conditions of high yield and high molecular weight. Triblock and 4-arm block copolymers with MDM and (MD)4 architecture, respectively, showed good adhesion to both organic and inorganic substrates, including polyvinyl chloride (PVC) tubes, and the resulting coated surfaces showed superior protein repellency and hemocompatibility compared to the diblock or random copolymer coatings and noncoated control. In a Chandler-loop method with whole blood, PVC tubes coated with MDM and (MD)4 showed improved thromboresistance and adsorption resistance to blood-derived proteins. This high hemocompatibility was also confirmed with human whole blood by thrombelastography (suppression of blood-clotting behavior in both intrinsic and extrinsic coagulation pathways) and platelet function analyses (significant reductions in the aggregation activity of platelets under two types of stimulation). The antithrombogenicity has been discussed based on the structural analyses of the MDM-coated surface. The results of this study will enable the development of more effective biomedical and analytical devices with excellent antithrombogenic characteristics by using a simple and environmentally friendly approach.

Hyaluronan and dextran modified tubes resist cellular activation with blood contact

This study was undertaken to evaluate the effects of thin film hyaluronic acid and dextran surface coatings to blunt cellular activation in a laboratory model of extracorporeal blood circulation. The inner lumen surface of polyurethane (PU) and poly(vinyl) chloride (PVC) tubing was grafted with hyaluronic acid and dextran. Surfaces were characterized for the presence of the grafted layer using ellipsometry, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Persistence of the surface layer was maintained for up to 5 days of continuous exposure to shear flow using a Chandler loop apparatus. The Chandler loop method was used to study human whole blood activation activity. Whole blood aggregometry and flow cytometry measures of CD18, CD62L, CD62P, Annexin V and myeloperoxidase performed on blood samples exposed to the tubing for up to three hours were complemented by scanning electron microscopy (SEM) analysis of adherent cells and state of activation. In these studies commercial hospital products and uncoated PVC and PU tubes were used as controls. We found that hyaluranized PU and PVC conferred the greatest resistance to blood activation and that dextranization of the PU and PU tubing also provided significant diminution of the bioresponses measured. Based on our findings, we suggest that surface coating with hyaluronic acid or dextran acts as a potent shield from blood cellular activation during forms of extracorporeal circulation.

The use of the Chandler loop to examine the interaction potential of NXY‐059 on the thrombolytic properties of rtPA on human thrombi in vitro

Recombinant tissue-type plasminogen activator (rtPA) is the only globally approved treatment for acute ischaemic stroke. Other potential treatments might be administered with rtPA, making it important to discover whether compounds interfere with rtPA-induced lysis. We evaluated methods for examining the effect of the neuroprotectant NXY-059 on the lytic property of rtPA. Plasma clot formation and lysis in the presence of rtPA and NXY-059 was measured as the change in plasma turbidity. The effect of NXY-059 on rtPA-induced lysis was similarly assessed on preformed clots. Lysis of the thrombus formed in a Chandler loop measured release of fluorescent-tagged fibrinogen that had been incorporated during thrombus formation. Thrombi were exposed to both rtPA and NXY-059 throughout lysis in the presence of 80% autologous plasma and the release of label during lysis was measured. Data interpretation is limited in the clot lysis experiments because either the rtPA was present during clot formation or the drug was added to a clot formed in static conditions. In contrast, thrombi were formed in dynamic flow conditions in the Chandler loop and the time course of lysis in plasma was examined. rtPA increased thrombolysis and the antifibrinolytic trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCA) inhibited lysis. Lysis induced by rtPA was unaltered by NXY-059. The Chandler loop method provides a reliable technique for examining the effect of compounds on rtPA-induced lysis in vitro and demonstrated that NXY-059 does not alter rtPA-induced lysis at clinically relevant concentrations of either drug.

Prototype therapeutic ultrasound emitting catheter for accelerating thrombolysis.

The aim of this study was to evaluate the performance of a new therapeutic ultrasound device for accelerating thrombolysis by urokinase. Artificial thrombi were produced in vitro by Chandler's loop method. Urokinase (240 IU) was added to the artificial thrombi (n = 5) before exposure to ultrasound with the new catheter-delivered device. Ultrasound (1.3 MHz) was applied with an intensity of 0.3 W/cm2 for 60 s. The degree of fibrinolysis was obtained by measurement of thrombus weight and calculated as percentages. Fibrinolysis was compared between control thrombus groups (n = 5) treated with urokinase alone or with ultrasound alone (analysis of variance, P < 0.05). Ultrasound plus urokinase (33.1 +/- 6.1% [mean +/- standard deviation]) was significantly different urokinase alone (12.3 +/- 2.4%) after 15 min of incubation. Ultrasound alone resulted in no fibrinolysis (0.9 +/- 2.5%). Ultrasound energy from the catheter-based device was sufficient to induce acceleration of fibrinolysis with urokinase.

Enhancement of Fibrinolysis with Ultrasound Energy

The effect of ultrasound energy on fibrinolysis of artificial thrombus in vitro was investigated. Thrombi produced by the Chandler loop method were exposed to low-energy ultrasound (5,000-6,000 Pa) in an ultrasound bath (48 kHz) for 60 seconds. Fibrinolysis with urokinase was enhanced from 40.6% +/- 1.8% to 59.2% +/- 2.6% (mean +/- standard deviation) with ultrasound exposure after a 60-minute incubation. Ultrasound alone without urokinase resulted in no fibrinolysis. In a second experiment, a newly developed miniature ultrasound-emitting ceramic element (2 x 1 x 5 mm) was attached to the tip of a catheter. Ultrasound exposure (225 kHz) from this device markedly enhanced fibrinolysis with urokinase from 8.9% +/- 1.5% to 37.3% +/- 0.8% (total ultrasound exposure 60 seconds, intensity 30 mW/cm2) after a 30-minute incubation. After a 120-minute incubation, fibrinolysis with ultrasound exposure was 61.1% +/- 1.8% versus 46.7% +/- 0.5% for the unexposed group. Ultrasound enhancement of fibrinolysis was less pronounced with longer incubation time (60 or 120 minutes). Ultrasound energy enhanced fibrinolysis with urokinase, especially in the early phase of lysis. This new device may shorten the time needed to complete fibrinolysis and reduce total drug dosage needed for treatment of thromboembolic diseases.

Identification of a low molecular weight polypeptide of pregnant bovine uterine origin (LMW-UDF): influence on coagulation system [formula omitted]. part I

Acidified extracts of pregnant bovine uterus were found to contain a heparin-affinity polypeptide(s). The eluted peak was assayed in the Chandler loop method for whole blood and fibrin thrombus generation. Significant differences in the mean weight and size of whole blood thrombi (243.5 ± 50 mg and 3.5 cm ± 0.6 cm) occurred with 1 μg of purified polypeptide when compared to control samples containing albumin (47.87 ± .30 g and 0.8 cm ± 0.3 cm). This activity was detected by the Chandler loop method with 100 ng to 10 μg of protein when analyzed in a dose-response fashion. When tested for fibrin thrombus formation this activity persisted. Experiments with 125I fibrinogen revealed no net increase or decrease of uptake into whole blood thrombi when purified polypeptide was added. The heparin-affinity polypeptide(s) possessed a molecular weight of approx- imately 6-4 kDa when examined by SDS-PAGE. We have therefore designated this polypeptide as low molecular weight-uterine derived factor (LMW-UDF).

The effect of hyperlipoproteinemia on thrombolysis: In vitro studies using purified lipoproteins from normolipemic donors

To determine whether or not lipoproteins affect thrombolysis in vitro using Chandler's loop method, VLDL, LDL, and HDL fractions from healthy male donors were obtained by ultracentrifugation. The lipoproteins were used to enrich whole blood or fibrin thrombi radiolabeled with 125-I-fibrinogen. Lipoprotein-enriched or unenriched thrombi were perfused in rotating Chandler loops with lipoprotein-enriched or unenriched plasma, respectively. Lysis was initiated by adding high molecular weight urokinase or single chain pro-urokinase to the perfusion medium. In some experiments, plasminogen was also added. Variation in the amounts of these activators and plasminogen permitted study of the effect of lipoproteins over a range of thrombolysis. No consistent statistically significant difference in the degree or time course of lysis of lipoprotein-enriched vs. unenriched thrombi or perfusion media was found. These studies, using normal lipoproteins, do not preclude the possibility that lipoproteins from patients with type IIa, IIb, or IV hyperlipoproteinemia may be genetically abnormal or may function pathologically, resulting in an effect on thrombolysis.

ヒトメラノーマ由来培養細胞株の産生するｔｉｓｓｕｅ‐ｔｙｐｅ ｐｌａｓｍｉｎｏｇｅｎ ａｃｔｉｖａｔｏｒ（ｔ‐ＰＡ）について

We have purified the tissue-type plasminogen activator (t-PA) produced by human melanoma cell line and have investigated its protein-chemical characteristics and fibrinolytic activity. Either one-chain or two-chain t-PA was obtained in the presence or absence of aprotinin during cell culture in purification steps. The N-terminal amino acid sequence of the one-chain t-PA was shown to start with Ser-Tyr-Gln-Val-. The amidolysis of S-2288 by t-PA was found to be greatly altered when the one-chain form was changed to a two-chain form by plasmin or trypsin treatment. When the fibrinolytic activities of t-PA and urokinase (UK) were compared using clot lysis time assay, t-PA and UK having the same unit base were found to have the nearly similar lysis time. On the other hand when fibrin plate assay was used, UK, having a unit of less than 1/10th of that of t-PA, was found to have the same fibrinolytic activity as t-PA.Furthermore, when the modified Chandler's loop method was used, t-PA was found to be several times more effective than UK in thrombolysis. Whereas UK drastically lowered the amount of fibrinogen and plasminogen in blood, t-PA hardly showed any effect on them.

Stability of Urokinase in Sodium Cefalotin Preparations

Stability of urokinase (UM) in sodium cefalotin preparations (CET) was investigated in terms of the agent's appearance, change of pH, and activity as determined by fibrin-plate Method and Chandler's Loop method. Electrophoresis was also used in the same study. The results are summarized as follows:1) UM activity was significantly decreased after mixing. However, residual potency of CET remained above 96% even 24 hours after mixing. 2) UM was unstable in CET in various concentrations or IV infusions. Same tendency was observed with UM in standard CET. 3) From the results of electrophoresis, it was found that CET contained some foreign matters.It was also found in the above study that CET or foreign matters in CET caused the decrease in UM activity. It is, therefore, concluded that UM is incompatible, with CET.

適切なＵＫ投与量の決定ならびに血せん溶解に影響する諸因子について

The direct clotiysis by urokinase (UK) may be due to exogenous clotlysis. On the other hand, the clot formed in the presence of UK may undergo endogenous clotlysis. To elucidate the optimal dosage of UK in the exogenous and endogenous clotlysis, the Chandler's loop method and thromboelastography (TEG) were used.Fibrinogen, plasminogen, α2-macroglobulin, α1-antitrypsin, antithrombin III, α2-plasmin inhibitor, C1-inactivator, hematocrit and platelet count were measured. The correlation between these factors and clotlysis time on TEG were analysed.In the loop method, UK agents from six companies (A-F) were used. previously formed Chandler's artificial clots were placed into loops containing 200, 120 and 60IU/ml of UK in saline solution, and these loops were rotated for 4-24 hours. Percentage reduction of clot weight was measured (Fig. 1).In TEG, clotlysis time on TEG was measured using 5, 10, 20, 25, 30, 40, 50 and 100IU/ml of UK.The artificial clots in 200IU/ml of UK solution showed mean clotlysis 55.8% in 4 hours by the Chandler's technique. By TEG, the clot formed in the presence of 10-20IU/ml of UK revealed clotlysis in 46-120min after clotting.C1-INA and α2-PI showed correlation between clotlysis time on TEG (Fig. 2).

Studies on Stability of Urokinase Preparation

In order to evaluate the fibrinolytic activities of 3 kinds of urokinase preparation, the fibrinplate, fibrin-tube and modified Chandler's loop method developed by us were compared.1) The modified Chandler's loop method was found to be most sensitive and physiological of the 3 assay methods.2) Preparations A and B, consisting mainly of high molecular weight of 54, 000 daltons showed satisfactorily high value by the 3 assays, but preparation C with low molecular weight of 33, 000 daltons showed relatively lower activity by the modified Chandler's loop method. These results support the validity of Lormeau's theory (1978).3) Three kinds of urokinase preparation were stable when they were dissolved in buffer solution at pH 7.2 at room temperature for several hours.

Clinical application of Chandler's loop method for evaluation of thrombic and thrombolytic states

The Chandler's loop method has been widely used for producing thrombus in vitro and evaluating thrombolytic agents. In the present study, the Chandler's loop method was used for evaluation of thrombic and thrombolytic states in 50 normal subjects and 20 patients with cerebral thrombosis. In normal subjects, the experimental clot showed increase in weight with progression of age, while thrombolytic effect showed little correlation with the age. The clot weight from stroke patients immediately after the onset of accident varied from twice of, to less than, the weight from normal subjects, and thrombolysis in vitro by Urokinase tended to occur more easily in larger clots. Correlation between decrease in thrombus weight and increase in thrombolytic rate were observed in convalescent stage of the disease. Within 2 weeks after discontinuation of Urokinase treatment, an acute rebound rise in thrombus weight was frequently seen. Experimental thrombus formation and thrombolytic rate in the present study might be correlated to the homeostatic balance in the normal and the diseased and reveal some aspects of pathophysiological mechanisms in thrombosis.

Inhibitory effect of tiaramide on ADP-induced aggregation in rabbit platelets.

Tiaramide in 10(-4) or 10(-5) M depressed the ADP-induced aggregation of rabbit platelets using the turbidimetric method. In modified Chandler's loop method, tiaramide in the same concentration accelerated the restoration of the time course of disaggregation.

Prolongation of thrombus-formation time in rabbits given chondroitin sulfate A

Summary A single intravenous injection of 80 mg chondroitin sulfate A/kg of body weight resulted in a significant increase in thrombus-formation time over pre-injection levels in rabbits as determined by the Chandler loop method. This effect did not occur immediately after injection but thrombus-formation times began to increase 3–6 h after injection and reached maximal levels (approximately 4 times pre-injection levels) 12 h after injection. 24 and 48 h after injection thrombus-formation times averaged 3 times pre-injection levels. Thereafter they dropped although they were still elevated over pre-injection levels 120 h after injection. Rabbits injected with 40 mg or 20 mg chondroitin sulfate A/kg of body weight also exhibited an increase in thrombus-formation time although to a lesser extent. No significant increase in thrombus-formation time over pre-injection levels occurred following the intravenous injection of saline solution alone. A single intravenous injection of heparin sodium (2000 U. heparin/kg of body weight) resulted in a striking prolongation of thrombus-formation time ( i.e. , in excess of 4 times the maximal prolongation obtained with chondroitin sulfate A) in blood taken 1.5 h after injection but thrombus-formation times averaged only slightly longer than pre-injection levels in blood taken 12 h after injection. Findings indicate that the prolongation of thrombus-formation time following the intravenous administration of chondroitin sulfate A was not due to a reduction in blood platelet count or plasma fibrinogen levels.