The influence of different heparin surface concentrations and antithrombin-binding capacity on inflammation and coagulation

The corline heparin surface (CHS) used in the extracorporeal circuit during coronary artery bypass grafting is shown to decrease the activation of inflammation and coagulation. Synchrotron radiation studies have shown that a single layer of the CHS may not completely cover the substrate surface. However, a double layer of CHS results in a uniform surface. We investigated the effect of surfaces with different surface concentrations of heparin on cell activation and coagulation compared to an uncoated surface. The CHS is prepared by a conditioning layer of polymeric amine onto which a macromolecular heparin conjugate is attached. We used PVC tubing, uncoated or modified with a single or double layer of the CHS, and circulated fresh whole blood from healthy volunteers in a loop model system at 37 degrees C up to 4 h. Blood was drawn from the loops at different times and activation of inflammation and coagulation was studied by real-time PCR, flow cytometry and ELISA. The activation of leukocytes and platelets and formation of leukocyte-platelet aggregates were reduced by use of the single-layered CHS compared to the uncoated surface. Use of double-layered CHS resulted in significantly reduced cell activation and thrombin generation. Development of the CHS obtained by the double layer of the coating has improved the biocompatibility of the surface.     

Effect of hirudin versus heparin on hemocompatibility of blood contacting biomaterials: an in vitro study

Hirudin serves as an alternative anticoagulant for extracorporeal blood circulation. Comparing anticoagulation with hirudin (2.5 or 5.0 microg/mL) and heparin (2.0 or 4.0 IU/mL) human blood was circulated in a modified 'Chandler System' using PVC-tubes for 2 hours at 37 degrees C. Activation of coagulation (thrombin-antithrombin III-complex, prothrombin fragment 1+2 and D-Dimer), platelet (platelet factor 4 - PF4) and complement systems was analyzed. Both heparin concentrations and 5.0 microg/dL hirudin led to as significantly less activated plasmatic coagulation as 2.5 microg/dL hirudin. Decreased levels of PF4 and anaphylatoxin C5a (p<0.05) as well as terminal complement complex demonstrated improved hemocompatibility after anticoagulation with heparin in contrast to hirudin. Because initial coagulation cascade, platelet activation and complement activation is less influenced by hirudin than by heparin, hemocompatibility is more dependent on the characteristics of the biomaterials used. This predestines hirudin as anticoagulant for in vitro studies analyzing hemocompatibility of biomaterials or surface modifications.     

In vitro hemocompatibility of albumin-heparin multilayer coatings on polyethersulfone prepared by the layer-by-layer technique

Polyethersulfone foils (PES)--a unique material for blood purification membranes--were coated with a multilayer assembly of heparin (unfractionated or high anticoagulant activity fraction heparin) and albumin (albumin-heparin coatings), or with a multilayer of albumin (albumin coating), using the layer-by-layer technique. The coatings combine advantages of albumin (reduction of nonspecific interactions) and heparin (specific interactions with blood coagulation proteins). The differences between the two heparins, while significant for their biological activity, had only a minor effect on the multilayer assembly with albumin monitored in situ by reflection infrared spectroscopy (FTIR MIRS). Uncoated as well as modified PES surfaces were evaluated using an in vitro assay with freshly drawn, slightly heparinized (1.5 IU heparin/mL) human whole blood. The blood was circulated with a roller pump over the sample surfaces in shear flow across rectangular slit channels ( app. 6 mL/min and 120 s(-1)) for 1.5 h at 37 degrees C. All coatings effectively reduced platelet adhesion and activation according to the PF4 release. The activation of coagulation evaluated as TAT generation was significantly lowered for the coating composed of albumin and high activity heparin. A further beneficial effect of the heparin containing coatings was reduced complement activation as determined by different complement fragments.     

Quality assessment of heparin coatings by their binding capacities of coagulation and complement enzymes

In vitro testing of blood contacting materials before clinical application is generally advisable. Four heparin coatings from different manufacturers were tested for adsorbed proteins and soluble activation markers. The surface with the highest antithrombin, thrombin, high-molecular-weight-kininogen (HMWK) and the lowest fibrinogen binding capacity (Carmeda, Medtronic) showed significantly lower levels of granulocytes and platelet activation (beta-TG, PMN-elastase release). No statistically significant differences in soluble markers of the coagulation system could be detected (F1 + 2, TAT). Interestingly, complement activation (TCC) was significantly reduced within the group of the lowest adsorption of the complement factor C3. Our data demonstrate that there is a relation between the binding affinity of proteins (C1-inhibitor, C3-complement) and the consecutive changes in complement activation (TCC). Therefore, measuring adsorbed proteins on artificial surfaces is a suitable, sensitive and very reproducible method for assessing the thrombogenicity of biomaterials.     

Hemocompatibility of heparin-coated surfaces and the role of selective plasma protein adsorption.

Although several studies have shown that heparin-coated surfaces reduce the activation of both the complement system and the coagulation system, there is still inadequate understanding of the factors initiating and controlling blood activation at these surfaces. We investigated the adsorption profile of 12 common plasma proteins (and the platelet receptor CD41) to a heparin coating (Carmeda BioActive surface (CBAS)) compared to uncoated controls (PVC) by using an in vitro whole blood Chandler-Loop model. Surface bound proteins were studied kinetically by a direct ELISA technique. Western blots were performed on the SDS eluates in order to detect adsorbed cleavage products and denatured proteins. Changes in plasma levels of neutrophil activation markers, platelet activation, coagulation activation, complement activation and the inflammatory response were measured by conventional ELISAs. This study showed significant differences in adsorption patterns among the heparin-coated and the uncoated surfaces, notably for fibronectin, fibrinogen, C3 and high molecular weight kininogen (HMWK). The kinetic studies confirmed the results obtained from Western blots and indicated specific adsorption profiles of plasma proteins. We assume that at least some of the improved blood compatibility of the heparin-coated surfaces may be ascribed to the selective uptake and cleavage of plasma proteins.     

Surface-attached PEO in the form of activated Pluronic with immobilized factor H reduces both coagulation and complement activation in a whole-blood model

In the present work we have bound Pluronic, a class of triblock copolymers consisting of a block of polypropylene oxide (PPO) surrounded on each side by polyethylene oxide (PEO) blocks, to polystyrene surfaces and investigated the thrombogenicity and complement activation of this construct upon exposure to whole blood. The surface was highly inert towards coagulation, unfortunately at the expense of increased complement activation. We, therefore, as an alternative approach, used End-Group Activated Pluronic to conjugate factor H, a regulator of complement activation (RCA), to the surface. The bound factor H did not detach from the surface upon incubation with human serum. Furthermore, factor H bound in a physiological conformation could to a significant degree attenuate complement activation at the Pluronic surface. Thus, we have created a hybrid surface in which the coagulation-inert properties of the original Pluronic are supplemented with a specific complement-inhibitory effect. Medical device technology includes numerous potential applications for crosslinkers that are capable of specifically binding biomolecules to surfaces with retained activity. These applications include coupling of functional biomolecules to biomedical devices such as stents and grafts. The biomolecule may be an RCA, antibody, or other beneficial ligand.     

In vitro hemocompatibility of self-assembled monolayers displaying various functional groups

Self-assembled monolayers (SAMs) of alkanethiols with various terminating groups (-OH, -CH3, -COOH) and binary mixtures of these alkanethiols were studied with respect to their hemocompatibility in vitro by means of freshly taken human whole blood. The set of smooth monomolecular films with graded surface characteristics was applied to scrutinize hypotheses on the impact of surface chemical-physical properties on distinct blood activation cascades, i.e. to analyze -OH surface groups vs. complement activation, acidic surface sites vs. contact activation/coagulation and surface hydrophobicity vs. thrombogenicity. Blood and model surfaces were analyzed after incubation for the related hemocompatibility parameters. Our results show that the adhesion of leukocytes is abolished on a -CH3 surface and greatly enhanced on surfaces with -OH groups. The opposite was detected for the adhesion of platelets. A strong correlation between the activation of the complement system and the adhesion of leukocytes with the content of -OH groups could be observed. The contact activation for hydrophilic surfaces was found to scale with the amount of acidic surface sites. However, the coagulation and platelet activation did not simply correlate with any surface property and were therefore concluded to be determined by a superposition of contact activation and platelet adhesion.     

Mechanism of complement activation during extracorporeal blood-biomaterial interaction: effects of heparin coated and uncoated surfaces

In vitro studies with miniaturized rotating circuits and heparinized human blood, as well as long-term extracorporeal membrane oxygenation with either heparin coated (HBS) or uncoated surfaces connected to adult sheep, were performed comparing the impact on complement activation in blood and on surfaces. Analysis of surface bound complement proteins revealed significantly reduced binding of activated C3 and C5b-9 to HBS in vitro, compared with uncoated surfaces, which was probably due to more HBS bound complement inhibitors (C1-Inhibitor, factor H) being present. This was reflected by significantly reduced activation of the alternative pathway (C3bBbP) and terminal complex (SC5b-9) by HBS but slightly increased levels of classic pathway complex (C1rs-C1-inhibitor). These results were confirmed during in vivo study by analysis of hemolytic complement function, activation specific C3 derived split products, and surface bound complement proteins. Increased binding of complement regulators to HBS appears to effectively reduce complement activation by biomaterials, leading to improved long-term biocompatibility.     

Tubing loops as a model for cardiopulmonary bypass circuits: Both the biomaterial and the blood-gas phase interfaces induce complement activation in anin vitro model

We describe here a model for the study of blood/surface and blood/air interaction as encountered in cardiopulmonary bypass (CPB) circuits. Polyethylene tubing was filled with serum or blood and closed end to end into loops whereby the volume of the remaining air bubble was inversely varied with respect to that of the fluid. The loops were rotated vertically in a water bath at 37°C. The profiles of C3a, iC3, and TCC generation were similar to those observed at surgery, involving CPB. Soluble heparin and heparan sulfate inhibited both C3a and TCC formation, but surface-conjugated heparin had only a minor effect. Binding of C3 and/or C3 fragments to the heparin surface was much reduced compared to the amine matrix to which heparin was linked, but compared with the polyethylene surface the effect was less pronounced. These data suggest that, in addition to the biomaterial surface, the blood-gas interface seems to play an important role in the activation of complement and that this activation is inhibitable by high concentrations of soluble glucose aminoglycans.     

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.     

Importance of a biofouling-resistant phospholipid polymer to create a heparinized blood-compatible surface

Heparinization is believed to be one of the methods to suppress thrombus formation on blood-contacting surfaces. However, this study hypothesizes that heparinization alone might not be sufficient to provide a blood-compatible surface; that is, a surface property that resists biofouling is necessary to obtain an effective heparin-modified surface. 2-Methacryloyloxyethyl phosphorylcholine (MPC) polymers with 2-aminoethyl methacrylate (AEMA) were synthesized to immobilize heparin through ionic bonding. The primary amino groups of AEMA were considered to be the polymer surface because the zeta-potential of the surface was positive when the mole fraction of the AEMA units was above 0.2. The antithrombogenic character of the polymer surface modified with heparin was evaluated by both Lee-White and microsphere column methods. The coagulation period of human whole blood in the absence of anticoagulant in glass tubing coated with the MPC polymer was longer than that in the original glass tube. Cell adhesion was completely inhibited on the MPC polymer surface after contact with human whole blood without anticoagulant. However, many adherent blood cells were observed on poly(2-ethylhexyl methacrylate-co-AEMA) (no MPC unit) even after heparinization. These results strongly indicate that the MPC polymer is a useful substrate where the heparin works well and that the heparin-immobilized MPC polymer has superior blood compatibility to the simple MPC polymer.     

Importance of a biofouling-resistant phospholipid polymer to create a heparinized blood-compatible surface

Heparinization is believed to be one of the methods to suppress thrombus formation on blood-contacting surfaces. However, this study hypothesizes that heparinization alone might not be sufficient to provide a blood-compatible surface; that is, a surface property that resists biofouling is necessary to obtain an effective heparin-modified surface. 2-Methacryloyloxyethyl phosphorylcholine (MPC) polymers with 2-aminoethyl methacrylate (AEMA) were synthesized to immobilize heparin through ionic bonding. The primary amino groups of AEMA were considered to be the polymer surface because the ζ-potential of the surface was positive when the mole fraction of the AEMA units was above 0.2. The antithrombogenic character of the polymer surface modified with heparin was evaluated by both Lee-White and microsphere column methods. The coagulation period of human whole blood in the absence of anticoagulant in glass tubing coated with the MPC polymer was longer than that in the original glass tube. Cell adhesion was completely inhibited on the MPC polymer surface after contact with human whole blood without anticoagulant. However, many adherent blood cells were observed on poly(2-ethylhexyl methacrylate-co-AEMA) (no MPC unit) even after heparinization. These results strongly indicate that the MPC polymer is a useful substrate where the heparin works well and that the heparin-immobilized MPC polymer has superior blood compatibility to the simple MPC polymer.     

Experimental Interactions of Components of Hemodialysis Units with Human Blood

An in vitro model test system for estimation of the blood compatibility of hemodialysis membranes and tubing is described. The model test system consists of a modified hemodialysis unit and blood pump through which fresh citrated human blood is circulated. The effects of the use of different pump and tubing types upon hematologic and blood coagulation parameters are described. Preexposure of test surfaces to albumin appeared to enhance blood compatibility characteristics of the model test system, whereas preexposure to a high density lipoprotein preparation or a proteinpolysaccharide preparation was without appreciable benefit. Use of blood from subjects receiving aspirin resulted in enhanced blood compatibility in the test system as did use of heparin. Use of Warfarin or dextran did not appear to enhance blood compatibility of test surfaces under the conditions of this test system. Dialysis membranes and tubing which formed parts of the test system were examined by scanning and transmission electron microscopy in control tests and in tests for effects of proteins and antithrombotic agents.     

In vitro comparative evaluation under static conditions of the hemocompatibility of four types of tubing for cardiopulmonary bypass.

A comparative in vitro assessment of 4 types of tubing representative of the materials currently used in cardiopulmonary bypass (CPB) procedures was conducted under static conditions using liquid extracts of the materials or direct contact with fresh human blood or serum. The parameters monitored were biomarkers of coagulation and fibrinolytic cascades, the complement system and cell activation. Silicone and PVC tubing were shown to be non-cytotoxic and non-hemolytic. Heparin-coated PVC tubing did present a certain degree of cytotoxicity especially when in direct contact. Thrombosis was found to be significantly lower with the same heparin-coated material. To a lesser extent, platinum-cured silicone also showed a reduced thrombotic tendency. None of the materials activated platelets or the complement system. With platinum-cured silicone tubing, constant and lower leukocyte adhesion was evidenced at the different experimental time points. This could reflect reduced cell activation.     

Thrombin, kallikrein and complement C5b-9 adsorption on hydrophilic and hydrophobic titanium and glass after short time exposure to whole blood

Hydrophilic and hydrophobic titanium and glass were exposed to capillary whole blood between 5s and 24h. The time-sequence for adsorption of thrombin, kallikrein and complement C5b-9, and their relationship with adherent platelets and polymorphonuclear granulocyte (PMN) activation were investigated. Adsorbed thrombin and kallikrein were measured by cleavage of specific chromogenic substances, S-2238 and S-2303, respectively. Complement C5b-9 and expression of CD11b, CD66b, CD62P and Pan-platelets were measured by immunofluorescence. Thrombin and kallikrein were present on the surfaces during the whole investigated periods. Platelet adhesion and PMN cell adhesion and activation on all surfaces and activation of platelets on hydrophobic surfaces showed a similar pattern to thrombin adsorption. Kallikrein adsorption had a different pattern on each surface. C5b-9 was detected between 32min and 24h of blood exposure and a varying pattern of C5b-9 coverage was observed on each surface. In conclusion, our results indicate that the interaction between material and blood coagulation and kinin-activating proteins regulate the adhesion and activation of blood cells, whereas after longer time the coagulation and kallikrein-kinin system play minor roles and the complement system is decisive for mediating and elongating the inflammatory process.     

Layer-by-layer co-immobilization of soluble complement receptor 1 and heparin on islets

Early graft loss due to instant blood-mediated inflammatory reactions (IBMIRs) is a major obstacle of clinical islet transplantation; inhibition of blood coagulation and complement activation is necessary to inhibit IBMIRs. Here, human soluble form complement receptor 1 (sCR1) and heparin were co-immobilized onto the surfaces of islet cells. sCR1 molecules carrying thiol groups were immobilized through maleimide-poly(ethylene glycol)-phospholipids anchored in the lipid bilayers of islet cells. Heparin was immobilized on the sCR1 layer via the affinity between sCR1 and heparin, and additional layers of sCR1 and heparin were formed layer-by-layer. The sCR1 and heparin molecules in these layers maintained anti-complement activation and anti-coagulation activities, respectively. This promising method could be employed to reduce the number of islet cells required to reverse hyperglycemia and prolong graft survival in both allo- and xeno-islet transplantation.     

The effect of heparin rinse on the biocompatibility of continuous veno-venous hemodiafiltration

The aims of our cross-over randomized study were (1) to assess hemostasis in patients with acute renal failure (ARF) and (2) to determine whether or not the generally recommended heparin rinse of the extracorporeal circuit (ECC) prior to the procedure affects thrombogenicity, complement activation, and leukocyte count in blood during continuous venovenous hemodiafiltration (CVVHDF). Eleven critically ill ARF patients were treated, in random order, using CVVHDF in postdilution setup following ECC rinse with saline (A) with heparin at a concentration of 2,000 IU/L (10 procedures), (B) with heparin at a concentration of 10,000 IU/L (7 procedures), and (C) without heparin (9 procedures). Except for the rinse, anticoagulation therapy did not differ in individual patients during the procedures. Blood was withdrawn before, and at minutes 15, 60, and 360 invariably at diafilter inlet and outlet. Compared with healthy individuals, patients showed lower blood thrombocyte counts (153 vs 233*10(9)/L, p<0.01, arithmetic means, Student's t test), longer aPTT (44 vs 36 s, p<0.05), higher plasma levels of heparin (0.1 vs 0.0 U/mL, p<0.05), D-dimer (1129 vs 36 ng/mL, p<0.001) and beta-thromboglobulin (BTG) (159 vs 37 U/mL, p<0.001) prior to CVVHDF. The comparison of procedures with different rinsing technique did not reveal any significant difference in their effects on blood thrombocyte and leukocyte counts, aPTT, plasma levels of heparin, BTG, thrombin-antithrombin III complexes, D-dimer, or the C5a complement component. CONCLUSIONS: (1) Patients indicated for CVVHDF show impaired hemostasis involving thrombocytes, coagulation, and fibrinolysis, (2) no beneficial effect of heparin rinse on CVVHDF ECC thrombogenicity, complement activation or blood leukocyte counts was demonstrated.     

Blood plasma coagulation studied by surface plasmon resonance

A surface plasmon resonance (SPR) apparatus was used to investigate blood plasma coagulation in real time as a function of thromboplastin and heparin concentrations. The response curves were analyzed by curve fitting to a sigmoid curve equation, followed by extraction of the time constant. Clotting activation by thromboplastin resulted in increased time constant, as compared to spontaneously clotted plasma, in a dose dependent way. Addition of heparin to the thromboplastin-activated plasma counteracted this effect. Atomic force microscopy (AFM) pictures of sensor surfaces dried after completed clotting, revealed differences in fibrin network structures as a function of thromboplastin concentration, and the fiber thickness increased with decreased thromboplastin concentration. The physical reason for the SPR signal observed is ambiguous and is therefore discussed. However, the results summarized in the plots and the fibrin network properties observed by AFM correlate well with present common methods used to analyze blood coagulation.     

Surface modification of chitosan membranes by complexation-interpenetration of anionic polysaccharides for improved blood compatibility in hemodialysis

Chitosan membrane surface was modified by cornplexation and interpenetration of anionic polysaccharides - heparin and dextran sulfate - for improved blood compatibility in hemodialysis. Electron spectroscopy for chemical analysis results showed a characteristic sulfur (S) and sodium (Na) peaks after modification with dextran sulfate. The sulfur/carbon (S/C) atomic composition ratio increased from 0.03 to 0.08 when the bulk dextran sulfate concentration used for modification was increased from 2.5 to 10 mg ml^-1 . The permeability of urea and creatinine did not change significantly upon modification with heparin or dextran sulfate. Surface modification, however, did decrease the permeability coefficients of glucose, vitamin B-2, and vitamin B-12. Unlike Cuprophan, chitosan and surface-modified chitosan membranes did not significantly activate the complement system as measured by the serum iC3b concentration. Compared to forty and sixty fully-activated platelets present on control surfaces, surface modification with heparin and dextran sulfate significantly reduced the number of adherent platelets per 25 000 μm² area and the extent of platelet activation. Surface modification with anionic polysaccharides, however, did significantly shorten the plasma recalcification time leading to fibrin clot formation. The results of this study show that chitosan membrane surface can be modified by complexation-interpenetration of anionic modifying agents. The modified membranes do resist complement activation and platelet adhesion and activation.     

Moderation of prekallkrein–factor XII interactions in surface activation of coagulation by protein-adsorption competition

Traditional biochemistry of contact activation of blood coagulation suggesting that anionic hydrophilic surfaces are specific activators of the cascade is inconsistent with known trends in protein adsorption. To investigate contact activation reactions, a chromogenic assay was used to measure prekallkrein (PK) hydrolysis to kallikrein (Kal) by activated factor XII (FXIIa) at test hydrophilic (clean glass) and hydrophobic (silanized glass) surfaces in the presence of bovine serum albumin (BSA). Hydrolysis of PK by FXIIa is detected after contact of the zymogen FXII with a test hydrophobic surface only if putatively-adsorbed FXIIa is competitively displaced by BSA. By contrast, FXIIa activity is detected spontaneously following FXII activation by a hydrophilic surface and requires no adsorption displacement. These results (i) show that an anionic hydrophilic surface is not a necessary cofactor for FXIIa-mediated hydrolysis of PK, (ii) indicate that PK hydrolysis does not need to occur by an activation complex assembled directly on an anionic, activating surface, (iii) confirms that contact activation of FXII (autoactivation) is not specific to anionic hydrophilic surfaces, and (iv) demonstrates that protein-adsorption competition is an essential feature that must be included in any comprehensive mechanism of surface-induced blood coagulation.