Acetylsalicylic acid loaded poly(vinyl alcohol) hemodialysis membranes: effect of drug release on blood compatibility and permeability

Membranes developed from poly(vinyl alcohol) (PVA) have superior permeability because of the highly hydrophilic character of PVA. However, its blood compatibility needs to be further improved. For this we have developed acetylsalicylic acid (ASA, aspirin) loaded PVA membranes. It seems that the slow release of aspirin from the membrane provides a surface concentration of aspirin sufficient for partially inhibiting platelet adhesion. PVA membrane with 531 micrograms cm-2 of ASA loaded, may be selected for hemodialysis applications. This may help to reduce the amount of heparin infused during hemodialysis, thereby reducing the side-effects associated with the systemic administration of heparin.     

Development of a membrane oxygenator for ECMO using a novel fine silicone hollow fiber

One of the limitations of conventional silicone hollow fiber oxygenators compared with microporous membrane oxygenators is poor gas permeability. However, the silicone hollow fiber is free from plasma leakage, which is the major life limiting factor of the microporous membrane oxygenator. It has been difficult to fabricate a fine, thin hollow fiber for reduction of resistance to gas permeability because of the poor mechanical strength of conventional silicone materials. The authors developed a novel silicone material with sufficient mechanical strength, and a fine silicone hollow fiber with a diameter of 30 microns and wall thickness of 50 microns, which is approximately half that of a conventional silicone hollow fiber. Using this newly developed silicone hollow fiber, the authors developed a compact extracapillary flow membrane oxygenator. The oxygenator consists of fine silicone hollow fibers inserted in a housing made of polycarbonate. The housing is a cylindrical case, 20 cm long and 55 mm in inside diameter. The hollow fibers are cross-wound. The surface area of the membrane is 2.0 m2, and priming volume is 230 ml. Gas transfer performance of the newly developed oxygenator was evaluated by in vitro experiments. Oxygen and carbon dioxide transfer rates were 195 ml/min and 165 ml/min, at a blood flow rate 3 L/min. The novel silicone membrane oxygenator developed in this study can be used for extended duration in such applications as extracorporeal membrane oxygenation.     

Nitric oxide prevents human platelet adhesion to fiber membranes in whole blood

During cardiopulmonary bypass or long-term extracorporeal life support, foreign surface induced platelet deposition in the oxygenator causes deterioration of gas exchange. In this study, the authors evaluated the effectiveness of nitric oxide (NO) in reducing the adhesion of platelets in whole blood to the surface of hollow fiber membranes. For this purpose, a test chamber was designed consisting of a gas exchanger with ten mitsubishi multi-layered composite hollow fibers (MHF: 257 mm OD; 203 mm ID; 70 mm length) and a polypropylene tube (16 mm OD; 100 mm length). Pure N2 (control) or nitric oxide (NO) (100 ppm, 200 ppm in N2) were delivered into the test chamber previously filled with 13 ml human whole blood. Platelet counts and platelet factor 4 (PF4), as a measure of platelet activation, were measured before and after either 1 or 2 hr of testing, and fibers were observed under scanning electron microscopic study (SEM) after each experiment. In the control and 100 ppm NO groups, platelet counts decreased and the level of PF4 increased during the 1 hr period. In the 200 ppm NO group, almost no platelet deposition could be observed on the surface of fibers under SEM. In conclusion, NO flow through hollow fiber membranes can markedly reduce platelet adhesion. Additional quantitative studies should define the optimal concentration for this effect and determine if this finding could improve oxygenator function, especially under conditions of long-term support.     

Antioxidant status and alpha1-antiproteinase activity in subarachnoid hemorrhage patients

Grafting of polyethylene glycol chains onto cellulosic membrane can be expected to reduce the interaction between blood (plasma protein and cells) and the membrane surface. Alkylether carboxylic acid (PEG acid) grafted high flux cellulosic membranes for hemodialysis, in which the polyethylene glycol chain bears an alkyl group at one side and a carboxyl group at the other side, have been developed and evaluated. PEG acid-grafted high flux cellulosic membranes with various grafting amounts have been compared with respect to platelet adhesion, the contact phase of blood coagulation, and complement activation in vitro. A new method of quantitating platelet adhesion on hollow-fiber membrane surfaces has been developed, which is based on the determination of lactate dehydrogenase (LDH) activity after lysis of the adhered platelets. PEG acid-grafted high flux cellulosic membranes showed reduced platelet adhesion and complement activation effects in grafting amounts of 200 ppm or higher without detecting adverse effects up to grafting amounts of 850 ppm. The platelet adhesion of a PEG acid-grafted cellulosic membrane depends on both the flux and grafting amounts of the membrane. It is concluded that the grafting of PEG acid onto a cellulosic membrane improves its biocompatibility as evaluated in terms of platelet adhesion, complement activation, and thrombogenicity.     

An adiabatic multiple spin-echo pulse sequence: removal of systematic errors due to pulse imperfections and off-resonance effects

Poly(2-methacryloyloxyethyl phosphorylcholine) (pMPC) was grafted onto the surface of a silicon rubber (SR) membrane (pMPC-SR) by plasma induced grafted copolymerization (PIP). Argon plasma was used to activate the SR surfaces. Determination was also made of the influences of grafted copolymerization reaction time, reaction temperature, and monomer concentration on polymerization yield. The surface properties of SR were characterized by ATR-FTIR, ESCA, and SEM. In those analyses the ATR-FTIR spectra indicated that the pMPC grafted onto the SR surface at 1720 and 3300 cm(-1). The elemental composition and different carbon bindings on the surface of the SR were examined by ESCA. An increasing P1s/C1s value g was obtained in the grafted polymerization yield with a concentration of 0.05-0.5M of MPC in the isolated ethanol solution. The surface morphologies of pMPC-SR differed more than those of control and Ar plasma treated surfaces. The difference could have been caused by the homogeneous graft polymerization of pMPC onto the SR membrane. In the biological analyses, protein adsorption on pMPC-SR surfaces was reduced. The reduced level increased with an increase in the pMPC grafted amount. The epithelial cell attachment and growth onto these samples were suppressed. The blood compatibility for a series of pMPC-SR surfaces was examined by platelet adhesion. Blood platelet morphologies in contact with the high ratio of pMPC-SR surfaces were maintained, meaning that in this case the release reaction for platelets never occurred. Consequently, the high amount of pMPC-SR surface had excellent blood compatibility, further suggesting that prevention of adhesion, activation of platelets, and adsorption of blood protein could be achieved.     

Development of a silicone hollow fiber membrane oxygenator for ECMO application

A new silicone hollow fiber membrane oxygenator for extracorporeal membrane oxygenation (ECMO) was developed using an ultrathin silicone hollow fiber, with a 300 microm outer diameter and a wall thickness of 50 microm. The hollow fibers were mechanically cross-wound on the flow distributor to achieve equal distribution of blood flow without changing the fiber shape. The housing, made of silicone coated acryl, was 236 mm long with an inner diameter of 60 mm. The surface area was 1.0 m2 for prototype 211, and 1.1 m2 for prototype 209. The silicone fiber length was 150 mm, and the silicone membrane packing density was 43% for prototype 211 and 36% for prototype 209. Prototype 211 has a priming volume of 208 ml, and prototype 209 has a priming volume of 228 ml. The prototype 211 oxygenator demonstrates a gas transfer rate of 120 +/- 5 ml/min (mean +/- SD) for O2 and 67 +/- 12 ml/min for CO2 under 2 L of blood flow and 4 L of O2 gas flow. Prototype 209 produced the same values. The blood side pressure drop was low compared with the silicone sheet oxygenator (Avecor, 1500ECMO). These results showed that this new oxygenator for ECMO had efficiency similar to the silicone sheet oxygenator that has a 50% larger surface area. These results suggest that the new generation oxygenator using an ultrathin silicone hollow fiber possesses sufficient gas transfer performance for long-term extracorporeal lung support.     

Membrane versus bubble oxygenator in hyperthermic regional perfusion: a prospective randomized clinical study

In a prospective randomized clinical study a routinely used bubble oxygenator (Bentley-5) was compared with a hollow fiber membrane oxygenator (D 701 Masterflo 34) during hyperthermic isolated extremity perfusion. This was done to find out whether there were differences between the two oxygenators in hemolysis, cellular damage, oxygenation and temperature achieved during extremity perfusion. In 30 perfusions blood samples were obtained at defined times: plasma hemoglobin (Hb), serum lactate dehydrogenase (s-LDH), number of erythrocytes, mean corpuscular volume (MCV), hemoglobin and bilirubin were determined for hemolysis, leukocyte count (neutrophils, lymphocytes, monocytes) and platelets as a check for cellular damage, and PO2, PCO2, O2 saturation and pH to define blood oxygenation and CO2 elimination. Maximal increase in temperature after 30 min and perfusion time until maximum tissue temperature were also recorded. The membrane oxygenator yielded better results from the aspect of hemolysis: s-LDH and plasma Hb were significantly different (p < 0.001). Cellular damage was less with the membrane oxygenator: platelet differences were significant (p < 0.01). Oxygenation and hyperthermia were obtained more quickly and were better controllable in membrane oxygenator. Further advantages for the patient were the smaller volume of blood needed for priming in a membrane oxygenator (750 vs. 1,200 ml) and improved safety resulting from a 'closed' perfusion system. On the basis of the clinical prospective randomized trial conducted, we conclude that membrane oxygenators must be adopted as the new standard in isolated hyperthermic extremity perfusion.     

Contribution of platelet microparticle formation and granule secretion to the transmembrane migration of phosphatidylserine

Activation of human platelets by complement proteins, C5b-9, thrombin plus collagen, or a Ca2+ ionophore results in surface exposure of phosphatidylserine (PS), accompanied by the expression of membrane catalytic activity for the tenase (VIIaIXa) and prothrombinase (VaXa) coagulation enzyme complexes. The mechanism underlying this surface exposure of PS upon platelet activation remains unresolved. Using fluorescent derivatives of PS (NBD-PS), we have investigated how the transmembrane migration of PS is related to microvesiculation of the platelet plasma membrane and to fusion of storage granules with the plasma membrane. Gel-filtered platelets were incubated with NBD-PS, allowing 90 +/- 10% of the incorporated NBD-PS to accumulate into the inner leaflet of the plasma membrane. Migration of NBD-PS from the inner leaflet to the plasma membrane surface was monitored by time-based flow cytometry, and correlated with the appearance of platelet microparticles and alpha-granule secretion. Platelet activation by C5b-9 or the Ca2+ ionophore, A23187, increased surface exposure of NBD-PS, due to acceleration of the apparent rate of migration from inner to outer plasma membrane leaflets. The onset of this accelerated migration of NBD-PS to the surface coincided with the onset of plasma membrane vesiculation, and the NBD-PS that partitioned into the membrane of the shed microparticle was also rapidly exposed to the surface (t1/2 < 2 min). In addition to a temporal correlation, microparticle formation and the surface exposure of inner leaflet NBD-PS showed a similar requirement for Ca2+. These results demonstrate that agonist-induced microvesiculation of the platelet plasma membrane is accompanied by accelerated migration of a PS analogue from the inner leaflet to the surface of the shed microparticle membrane, suggesting the mechanism by which induction of platelet microparticle formation exposes catalytic surface for tenase and prothrombinase assembly.     

Anterior, posterior, or laparoscopic approach for the management of adrenal diseases?

An important role in the formation of hemostasis defects in uremic patients is attributed to platelet dysfunction. An essential role in platelet structure and function is played by membrane phospholipids (PL). They are asymmetrically distributed within the platelet membrane: outer surface is composed mainly of sphingomyelin (Sph) and phosphatidylcholine (PC). During platelet activation a translocation of phosphatidylserine (PS) and phosphatidylethanolamine (PE) from inner to outer membrane surface is observed. Phosphatidylinositol (PI) is not translocated. Lipid abnormalities are common in uremic patients. According to some authors erythropoietin (EPO) has been reported to alter lipid metabolism. In our recent works a positive influence of EPO on platelet PL composition in uremic patients has been indicated. The aim of this study was the assessment of the EPO influence (applied 4000 U per week) on platelet membrane PL distribution in chronically hemodialyzed patients. The PL distribution was determined using nonpenetrating tracer (TNBS) by Vale method, and using high purified phospholipases hydrolysis according to Chap method. Our results indicate that during EPO therapy the PS, PE, Sph and PC exposition at the outer surface of platelet membrane (in patients hemodialyzed without EPO widely disturbed compared with healthy controls) approaches to normal values. These results confirm our recent observations that EPO profoundly interferes with lipid metabolism. The smaller PS exposition at the outer platelet surface during EPO treatment suggests less platelet activation, and might partially explains the positive EPO influence on platelet hemostasis.     

High flux hemofiltration

Experiments were performed using a new hollow fiber hemofilter. Ultrafiltration rates and whole blood urea clearances were measured in post-dilution hemofiltration. High ultrafiltration rates were obtained with the new hemofilter. No adverse effects were detected despite filtration fractions above 45%. These experiments suggest that it is possible in post-dilution hemofiltration to obtain small solute clearances comparable to those of hemodialysis without apparent deleterious effects.     

The changes of Mac-1 and L-selectin expression on granulocytes and soluble L-selectin level during hemodialysis

L-selectin and Mac-1 expressed on leukocytes are critical for leukocyte adhesion to inflamed endothelium. L-selectin is known to be rapidly shed from the cell surface of granulocytes after activation. In the present study the change of expressions of these adhesion molecules on granulocytes were analyzed by flow cytometry, and the serum concentration of shed L-selectin (soluble L-selectin; sL-selectin) was measured by enzyme-linked immunosorbent assay (ELISA) during hemodialysis in patients treated with regenerated cellulose membranes (RC group) versus polysulfone membranes (PS group). In the RC group, Mac-1 expression on granulocytes increased significantly at 30 min after the initiation of hemodialysis (p < 0.05) compared with predialysis values, coinciding with the nadir of dialysis-induced granulocytopenia. Granulocyte L-selectin expression decreased significantly at 15 min after the initiation of hemodialysis (p < 0.05) and remained decreased through the course of dialysis session, compared with predialysis values. Serum sL-selectin level significantly increased at 15 min after the initiation of hemodialysis (p < 0.05), compared with predialysis values. In the PS group, no significant variation in Mac-1 and L-selectin expression on granulocytes and serum sL-selectin level were detected. This reciprocal change of Mac-1 and L-selectin on granulocyte cell surface was attributed to development of granulocytopenia and subsequent reversal during dialysis with cellulose membranes. In this study, we confirmed the shedding of L-selectin during cellulosic dialysis by ELISA. The increase in sL-selectin, which has potential activity of inhibiting L-selectin-dependent adhesion of granulocyte to endothelium, might be involved in rebound granulocytosis during dialysis with cellulose membranes and impairment of the granulocyte function in patients on chronic hemodialysis.     

High dose rate endobronchial brachytherapy: results and complications in 189 patients

No quantitative, simple and non-radioactive method has been described for measuring the platelet content of experimental thrombi. The aim of the present study was to develop a simple method for quantifying platelets in thrombi formed on thrombogenic surfaces in flowing native human blood. To test the relevance of this new method, the effect of unfractionated heparin on arterial thrombus formation was investigated. Tissue factor (TF)- and collagen-coated coverslips were exposed to non-anticoagulated blood at an arterial wall shear rate (2,600 s(-1)) for 1 to 4 min. Platelet deposition was quantified by measuring the P-selectin (PS) and beta-thromboglobulin (betaTG) content of dissolved plasmin-digested thrombi using immunoenzymoassays; fibrin deposition was determined by measuring the D-dimer levels. These results were compared to those established by morphometrical analysis. Morphometric evaluation showed that fibrin deposition was maximum on TF by 1 min perfusion time. Platelets deposited subsequently and reached a maximum at 3 min. On collagen, platelets deposited directly on the collagen fibrils without detectable fibrin deposit. Platelet deposition increased from 1 to 4 min. Platelet deposition quantified by PS was correlated to the values obtained by morphometry (r = 0.72, r = 0.67, p <0.001, on TF and collagen, respectively). As compared to PS, betaTG measurements gave an underestimation of the size of the thrombus platelet number. Unfractionated heparin infused through a mixing device proximal to the perfusion chamber to obtain plasma concentrations of 0.5, 1 and 3 IU/ml, reduced fibrin deposition on TF-coated coverslips in a dose-dependent manner (77% reduction at 3 IU/ml, p <0.01), but had no significant effect on platelet deposition (33% at 3 IU/ml, p >0.05). In contrast, heparin had no effect on fibrin or platelet deposition on collagen-coated coverslips. Thus, a new quantitative and simple method for measuring platelet deposition in flowing blood has been developed and characterized. Utilizing this system, we have demonstrated that unfractionated heparin did not inhibit arterial thrombus formation either on procoagulant or on proaggregant surface.     

Social support, need for support, and anxiety among women graduate students

The Bernard-Soulier syndrome (BSS) is characterized by thrombocytopenia with giant platelets, a prolonged bleeding time with defective platelet adhesion to the subendothelium related to a defect in platelet membrane glycoprotein Ib (GPIb) and a decreased prothrombin consumption. The mechanism of the latter abnormality remains unknown. In this study, we showed that this defect was corrected by the addition of purified human factor VIII (FVIII) to blood from four patients with BSS. The correction of prothrombin consumption was almost complete at concentrations between 1.5 and 3 IU/ml of FVIII procoagulant activity (VIII:C) and partially abolished by a monoclonal antibody which neutralizes VIII:C. This correction was specific for FVIII and was not observed after addition of purified human FIX. It was obtained, in the same magnitude range, with FVIII complexed to von Willebrand factor (vWF) but not with free vWF. These data provide a new insight into the knowledge of the physiological interaction between the platelet membrane and the vWF-FVIII complex facilitating plasma coagulation activation and may lead to helpful therapeutic advances.     

Encapsulation of OKT3 cells in hollow fibers

Encapsulation of an OKT3 cell line in hollow fibers was evaluated in vitro and in vivo. The cell line is a mouse hybridoma producing immunoglobulin G2a (IgG2a) against CD3 human T lymphocytes and thus may function as a nonspecific activation system of a subpopulation of human T lymphocytes. For encapsulation purpose, hollow fibers of polypropylene K600 PP Accurel (Akzo, Germany) were selected. Hollow fibers were siliconized to improve membrane biocompatibility for in vivo experiments. The siliconized hollow fibers exhibited acceptable diffusive permeability (P) [ml/min/m2] for small solutes (for creatinine, p = 63.9 +/- 2.0, n = 3) and larger solutes (for albumin, p = 16.9 +/- 1.9, n = 3; for IgG, p = 1.0 +/- 0.2, n = 3). The 12 cm long hollow fibers were filled with a suspension of OKT3 cells of an average density of 10(6) cells/ ml, and both ends were sealed. The encapsulated cells were cultivated in RPMI 1640/10% CS medium at 37 degrees C, 5% CO2 for a period of 3 to 4 days. After the culture period, the medium was tested on human peripheral blood lymphocytes for the presence of anti-CD3 antibody and read in a flow FACS-trac cytometer (Becton Dickinson Immunocytochemistry Systems, San Diego, CA). The tightness of hollow fiber sealing was tested with a bubble point method. The number of cells increased after cultivation by four- to nine-fold on average (n = 11). Ten experiments were performed in vivo with OKT3 cells encapsulated in hollow fibers and implanted subcutaneously into mice for 3 days. In 50% of the experiments, some anti-CD3 antigens on human lymphocytes were found; however, the difference, in comparison with control, in percent of CD3+ was insignificant. In conclusion, the hollow fiber method for cultivation of hybridoma cells in vitro allows for separation of cells from the medium containing secreted anti-CD3 antibodies and is effective in maintaining cell viability. In vivo application needs additional study.     

An N-substituted polyurea coating with high affinity for heparin

A new N-substituted polyurea with tertiary amino groups in the polycarbamidic chain (NPUTA) has been synthesized. The polymer is soluble in C1-C4 alcohols, has high adhesion to polar molds, and has high H2O uptake (130-150%). The material can be coated on many biomaterials (polyurethanes, charcoal hemosorbents, cellulosic hemodialysis membranes), and high amounts of heparin can be adsorbed onto treated surfaces. NPUTA cast from 0.5-3.5% ethanol solutions can absorb large amounts of heparin from anti-coagulant solution (40-60 micrograms/cm2) and heparinized plasma. Heparin release into phosphate buffered saline (PBS) solution or plasma is minimal. The influence of NPUTA solution concentration and pre-absorbed heparin on the protein adsorption, platelet adhesion, surface induced hemolysis, and complement activation of these films has been investigated. Radiolabeled protein assays, radiolabeled platelet assays, and other methods were used. It was shown that modified surfaces for the listed materials, with heparinization, demonstrate improved in vitro blood compatibility without any changes in functional properties. For example, treatment with NPUTA/heparin does not reduce sorption of middle molecules by activated charcoal hemosorbent, while markedly and significantly decreasing platelet adhesion and complement activation. NPUTA/heparin modified, glutaraldehyde treated bovine pericardium exhibited significantly reduced calcification in a rat subcutaneous implant model. Other ex vivo circulation experiments also confirm the blood compatibility of different NPUTA treated surfaces.     

Preparation and performance of CeO_2 hollow spheres and nanoparticles

CeO_2 hollow spheres were synthesized by polystryrene sphere(PS) templates and CeO_2 nanoparticles were prepared by a facile method. The as-obtained products were characterized by scanning electron microscopy(SEM), N_2 adsorption-desorption, X-ray diffraction(XRD), Fourier transform infrared spectroscopy(FTIR) and UV-vis diffuse reflectance spectra. The results showed that the structure of the obtained CeO_2 hollow spheres was hollow microsphere with a diameter of 380 nm and the average particle size of CeO_2 nanoparticles was about 1700 nm. The two samples' Brunauer-Emmett-Teller(BET) surface area was 67.1 and 37.2 m~2/g. The CeO_2 hollow spheres had a better performance than nanoparticles at UV-shielding because of higher surface area and the structure of hollow sphere.     

Solute washout experiments for characterizing mass transport in hollow fiber immunoisolation membranes

The transport characteristics of immunoisolation membranes can have a critical effect on the design of hybrid artificial organs and cell therapies. However, it has been difficult to quantitatively evaluate the desired transport properties of different hollow fiber membranes due to bulk mass transfer limitations in the fiber lumen and annular space. An attractive alternative to existing methodologies is to use the rate of solute removal or "washout" from the annular space during constant flow perfusion through the fiber lumen. Experimental washout curves were obtained for glucose and a 10 kD dextran in two different hollow fiber devices. Data were analyzed using a theoretical model which accounts for convective and diffusive transport in the lumen, membrane, and annular space. The model was in good agreement with the experimental results and provided an accurate measure of the effective membrane diffusion coefficient for both small and large solutes. This approach should prove useful in theoretical analyses of solute transport and performance of hollow fiber artificial organs.     

A fifty percent reduction of platelet surface glycoprotein Ib does not affect platelet adhesion under flow conditions

Glycoprotein (GP) Ib is an adhesion receptor on the platelet surface that binds to von Willebrand Factor (vWF). vWF becomes attached to collagens and other adhesive proteins that become exposed when the vessel wall is damaged. Several investigators have shown that during cardiopulmonary bypass (CPB) surgery and also during platelet activation in vitro by thrombin or thrombin receptor activating peptide (TRAP) GPIb disappears from the platelet surface. Such a disappearance is presumed to lead to a decreased adhesive capacity. In the present study, we show that a 65% decrease in platelet surface expression of GPIb, due to stimulation of platelets in Orgaran anticoagulated whole blood with 15 micromol/L TRAP, had no effect on platelet adhesion to both collagen type III and the extracellular matrix (ECM) of human umbilical vein endothelial cells under flow conditions in a single-pass perfusion system. In contrast to adhesion, ristocetin-induced platelet agglutination was highly dependent on the presence of GPIb. Immunoelectron microscopic studies showed that GPIb almost immediately returned to the platelet surface once platelets had attached to collagen. In a subsequent series of experiments, we showed that when less than 50% of GPIb was blocked by an inhibitory monoclonal antibody against GPIb (6D1), platelet adhesion under flow conditions remained unaffected.     

Miniaturized supported liquid membrane device for selective on-line enrichment of basic drugs in plasma combined with capillary zone electrophoresis

A hollow fiber miniaturized supported liquid membrane (SLM) device for sample preparation is connected on-line with capillary electrophoresis and used for determination of a basic drug, bambuterol, in human plasma. The analyte is extracted from the outside of the hollow fiber (donor) through the liquid membrane (pores of the fiber impregnated with organic solvent) into the acceptor solution in the fiber lumen. The process is driven by differences in pH between the donor and acceptor solution. The whole volume of the acceptor solution can then be injected into the CZE capillary by using the double-stacking procedure for large volume-injection. Very clean extracts of low ionic strength are obtained from the SLM treatment, making this sample pretreatment method compatible with the CZE double-stacking procedure, which in turn makes it possible to inject large volumes of sample onto the separation capillary. Good performance of the whole procedure is demonstrated, and detection limits in the low nanomolar range were obtained in spite of the relatively weak UV absorbance of bambuterol. Extractions through the miniaturized SLM unit can be performed for 5-6 h without regenerating the fiber. The regeneration procedure was tested, and no relevant changes in the performance of the extraction could be found after seven regenerations, allowing the same fiber to be used for a week.     

Flow behavior of erythrocytes in microvessels and glass capillaries: effects of erythrocyte deformation and erythrocyte aggregation

The pharmacodynamic effects of different intravenous and subcutaneous doses of a new recombinant hirudin (IK-HIR02) on platelet adhesion, platelet-induced thrombin formation and on platelet count have been studied in 18 healthy volunteers in a bicenter study. Single intravenous bolus injections of 0.1, 0.2 and 0.3 mg/kg IK-HIR02 in 6 volunteers caused a significant dose-dependent prolongation of platelet-induced thrombin generation time (PITT) and a significant inhibition of platelet adhesion to glass. Single subcutaneous doses of 0.1, 0.25 and 0.5 mg/kg IK-HIR02 slightly prolonged PITT and inhibited platelet adhesion to glass for up to 8 h. Repeat subcutaneous injections of 0.3 mg/kg IK-HIR02 b.i.d. in 6 healthy volunteers led to a prolongation of PITT and also to a reduction of platelet adhesion. In platelet-rich plasma (PRP) from blood samples which had been collected using hirudin as anticoagulant (0.7 micrograms/ml), the platelet count was constantly higher than in citrate PRP which had been sampled at the same time. The recombinant hirudin IK-HIR02 inhibits platelet adhesion to glass and also PITT. Both effects which have not been described before are most likely due to a direct inhibition of thrombin-induced platelet activation. These effects may contribute to the antithrombotic action of hirudin and probably have to be considered when hirudin is used in higher doses as an antithrombotic agent together with platelet function inhibitors to avoid excessive bleeding.