Interaction of antithrombin III with surface-immobilized albumin-heparin conjugates

The interaction between antithrombin III (ATIII) and albumin-heparin conjugates covalently coupled onto carboxylated polystyrene beads either in buffer containing albumin or in plasma was studied using 14C-labeled ATIII. Binding isotherms of ATIII were modeled using a summation of two Langmuir equations. These equations describe the binding of ATIII to two different sets of binding sites, one with a high, the other with a low affinity for ATIII. The average binding constants for the binding of ATIII to these sites are 9 × 106 L/mol and 0.3 × 106 L/mol, respectively. The binding of ATIII to surface binding sites with a high affinity for ATIII was correlated with the presence of specific ATIII binding sites in the immobilized heparin. Binding of ATIII from albumin solutions to binding sites with a low affinity for ATIII was dominated by nonspecific binding of ATIII to the immobilized heparin. A third small fraction of the surface bound ATIII is probably adsorbed to sites on the surface not covered with heparin. In the case of the binding of ATIII to the heparinized surface from plasma solutions, a fraction of initially adsorbed ATIII was desorbed by other plasma proteins. This desorption in combination with direct competition between ATIII and other plasma proteins resulted in lower ATIII surface concentrations using plasma as compared to the ATIII surface concentrations obtained using albumin solutions. The binding of ATIII to nonspecific binding sites was almost completely inhibited in the presence of plasma proteins. The amount of ATIII bound to immobilized heparin via specific ATIII binding sites was 30% lower in plasma solutions as compared to the specific binding of ATIII using albumin solutions. It is concluded that the accessibility of immobilized heparin for ATIII in plasma decreases by binding of heparin-binding proteins onto the immobilized heparin and/or by adsorption of other plasma proteins on the heparinized surface

Sterilization of heparinized Cuprophan hemodialysis membranes

The effects of sterilization of dry heparinized Cuprophan hemodialysis membranes by means of ethylene oxide (EtO) exposure, gamma irradiation, or steam on the anticoagulant activity and chemical characteristics of immobilized heparin and the permeability of the membrane were investigated. Sterilization did not result in a release of heparin or heparin fragments from heparinized Cuprophan. Sterilization of heparinized Cuprophan by means of EtO exposure and gamma irradiation induced a slight, insignificant decrease of the anticoagulant activity. In contrast, steam-sterilized heparinized Cuprophan showed a higher anticoagulant activity than unsterilized heparinized Cuprophan, which was most likely caused by cleavage of some of the covalent bonds between heparin and Cupropha. The effects of sterilization on the permeability of unmodified Cuprophan and heparinized Cuprophan were compared. The permeability of unmodified Cuprophan for vitamin B12 (Vit B12) and sulfobromophthalein (SBP) was reduced by 20–35% after EtO exposure and gamma irradiation and was reduced by 90–95% after steam sterilization. The water permeability of unmodified Cuprophan remained the same after EtO exposure and gamma irradiation but also dramatically reduced after steam sterilization. These reductions were ascribed to the collapse of pores of the membrane. The permeability of heparinized Cuprophan was not affected by EtO exposure and gamma irradiation but dramatically reduced after steam sterilization, although to a lesser extent than in the case of unmodified Cuprophan. Apparently, the presence of immobilized heparin (partially) prevented the collapse of pores during sterilization. Gamma irradiation was recommended as the preferred method of sterilization for heparinized Cuprophan.\u

Design of a new type of coating for the controlled release of heparin

Thrombus formation at the surface of blood contacting devices can be prevented by local release of heparin. Preferably, the release rate should be constant for prolonged periods of time. The minimum heparin release rate to achieve thromboresistance will be different for various applications and should therefore be adjustable. In this study a new type of heparin release system is presented which may be applied as a coating for blood contacting devices. The system is based on the covalent immobilization of heparin onto porous structures via hydrolysable bonds. This approach was evaluated by the immobilization of heparin onto a porous cellulosic substrate via ester bonds. Cuprophan was used as a model substrate and N,N¿-carbonyldiimidazole as a coupling agent. Heparinized Cuprophan incubated in phosphate buffered saline showed a release of heparin due to the hydrolysis of the ester bonds between heparin and Cuprophan. The release rate could be easily adjusted by varying the amount of coupling agent used during immobilization. Cuprophan with a rather stable heparin coating (release rate: 6.1 mU/cm2·h) and Cuprophan which shows a substantial release of heparin (release rate up to 23.0 mU/cm2·h) could be prepared. Except when the release was relatively high, release rates were constant for at least 1 week. Storage of the release system at ambient conditions up to 6 months or sterilization by means of steam, ethylene oxide exposure, or gamma irradiation did not affect the release properties. It was concluded that this concept for a heparin release system is highly promising to prepare thromboresistant surfaces for various blood contacting devices

An in vitro study of the adhesion of blood platelets onto vascular catheters. Part I

The adhesion of human blood platelets onto vascular catheters was studied using a specially designed perfusion chamber. Polyurethane catheters were exposed to citrated human blood for different periods (up to 20 min) and at different wall shear rates (190, 260, 330 sec-1). The rate of platelet adhesion was determined using 111In-labeled platelets, while the morphology of adhering platelets was investigated using scanning electron microscopy. A linear increase in platelet adhesion was found within the first 10 min of perfusion, after which a plateau value was reached. The number of adhering platelets did not vary significantly with the shear rates applied, which may indicate that within the range of shear rates studied, the adhesion of platelets onto the catheter surface is mainly determined by the rate of the reaction between the platelets and the material surface. Catheters coated with a conjugate of heparin and albumin showed a four- to five-fold reduction in platelet adhesion as compared to uncoated catheters. This reduction in platelet adhesion was not only due to the presence of albumin moieties at the surface but also to the presence of heparin residues in the adsorbed albumin-heparin conjugate

Signal amplification on planar and gel-type sensor surfaces in surface plasmon resonance-based detection of prostate-specific antigen

This article describes surface plasmon resonance (SPR)-based detection of prostate-specific antigen (PSA), comparing amplification with colloidal gold (10 nm diameter) and latex microspheres (120 nm diameter) on planar- and gel-type sensor surfaces. As matrix, 3% BSA in PBS was used. Experimental data were compared with model calculations that predict the SPR signal that results from covering of the different sensor surfaces with each of the particles used. Amplification with latex particles gave a higher signal than did that with colloidal gold. However, the limit of detection (LOD) attained by latex amplification was not as good as that obtained after gold amplification, and this was unexpected. LOD and sensitivity of the amplified PSA assays when performed with the planar-type sensor disc were equally good or better compared with those when performed with the gel-type sensor disc. Indirect evidence indicates a restricted accessibility of the gel layer on the gel-type sensor toward the colloidal gold. Application of colloidal gold led to a sensitivity increase of approximately three orders of magnitude compared with nonamplified detection. The corresponding LOD was approximately 0.15 ng PSA/ml, which is sufficient for measuring enhanced, clinically relevant PSA levels (>4 ng/ml)

Clinical characteristics of women captured by extending the definition of severe postpartum haemorrhage with 'refractoriness to treatment': a cohort study

Background: The absence of a uniform and clinically relevant definition of severe postpartum haemorrhage hampers comparative studies and optimization of clinical management. The concept of persistent postpartum haemorrhage, based on refractoriness to initial first-line treatment, was proposed as an alternative to common definitions that are either based on estimations of blood loss or transfused units of packed red blood cells (RBC). We compared characteristics and outcomes of women with severe postpartum haemorrhage captured by these three types of definitions. Methods: In this large retrospective cohort study in 61 hospitals in the Netherlands we included 1391 consecutive women with postpartum haemorrhage who received either ≥4 units of RBC or a multicomponent transfusion. Clinical characteristics and outcomes of women with severe postpartum haemorrhage defined as persistent postpartum haemorrhage were compared to definitions based on estimated blood loss or transfused units of RBC within 24 h following birth. Adverse maternal outcome was a composite of maternal mortality, hysterectomy, arterial embolisation and intensive care unit admission. Results: One thousand two hundred sixty out of 1391 women (90.6%) with postpartum haemorrhage fulfilled the definition of persistent postpartum haemorrhage. The majority, 820/1260 (65.1%), fulfilled this definition within 1 h following birth, compared to 819/1391 (58.7%) applying the definition of ≥1 L blood loss and 37/845 (4.4%) applying the definition of ≥4 units of RBC. The definition persistent postpartum haemorrhage captured 430/471 adverse maternal outcomes (91.3%), compared to 471/471 (100%) for ≥1 L blood loss and 383/471 (81.3%) for ≥4 units of RBC. Persistent postpartum haemorrhage did not capture all adverse outcomes because of missing data on timing of initial, first-line treatment. Conclusion: The definition persistent postpartum haemo