End-point Attachment Research Articles

Effect of heparinization on promoting angiogenesis of decellularized kidney scaffolds.

Native decellularized extracellular matrix provides an adequate platform for tissues and organs and promotes the development of organogenesis and tissue remodeling. However, thrombosis poses a great challenge that hinders the transplantation for a substantial organ in vivo. Therefore, anticoagulation and re-reendothelialization of organ biological scaffolds are the primary concerns to be addressed before orthotopic transplantation. Herein, a heparinized decellularized kidney scaffold (HEP-DKSs) was prepared using end-point attachment technology, followed by binding the vascular endothelial growth factor (VEGF) to greatly improve the hemocompatibility and angiogenesis of DKSs. Based on the anticoagulant, co-culture of human umbilical vein endothelial cells, and subcapsular transplantation of kidney experiments, HEP-VEGF-DKSs are shown to reduce platelet adhesion, which is crucial for subsequent vascularization and slow release of heparin and VEGF, suggesting its ability of improve neovascularization. Taken together, these data indicated an optimal anticoagulation function of HEP-VEGF-DKSs and the potential of vascularization for regeneration of whole decellularized kidney.

Effect of Heparinization on Promoting Angiogenesis of Decellularized Kidney Scaffolds

In recent years, the native decellularized extracellular matrix (ECM) provides a scaffold for tissues and organs, but also supports organogenesis and tissue remodeling. However, the anti-coagulation properties have always been a challenge in the creation of the ideal three dimensional bioscaffold materials for tissue engineering organ construction. This hurdle is also an inevitable issue that researchers have to face in the revascularization of the decellularized kidney scaffolds (DKSs). This manuscript describes the strategies to accelerate the endothelialization of blood vessels of DKSs based on anticoagulation, compared to previous reports in our research. In this study, we prepared a heparinized decellularized kidney scaffold (Hep-DKSs) by end-point attachment (EPA) technology, then binding the vascular endothelial growth factor (VEGF) with the Hep-DKSs in order to rapidly promote the formation of endothelialization of DKSs. Following, the anticoagulant properties and cytocompatibility of the modified scaffolds were investigated in vitro by means of Toluidine blue staining, Platelet adhesion assay and Co-culture of human umbilical vein endothelial cells (HUVECs). Subsequently, the modified scaffolds were transplantated to intrarenal subcapsular of the recipient rat. Then the transplanted specimens were observed on 1, 3, 7, 14, 21 and 28 days after transplantation. The results in vivo showed that the numbers of neovascularization were higher in the heparin cross-linking VEGF decellularized kidney scaffolds (HEP-VEGF-DKSs) than that of other decellularized renal scaffolds. Taken together, these data suggest that HEP-VEGF-DKSs have a good anticoagulation property and biocompatibility, and can slowly release heparin and VEGF, suggesting its ability of improving neovascularization better and faster and potential use for regeneration of whole decellularized kidney.

Heterotopic vascularization and functionalization of implantable bio engineered hepatic tissue alleviates liver injury in rats.

The challenge of using bioengineered liver lies in sustaining the quantity of high-quality hepatocytes and the vasculature for blood perfusion. We characterized the heparinization of a porcine decellularized liver scaffold (DLS) as a carrier to support hepatocyte angiogenesis, thereby developing functional and vascularized hepatic tissue useful to treat liver injury. The porcine DLS was obtained by the removal of cellular components and then subjected to heparinization by the end-point attachment technique. The heparinized DLSs were recellularized with rat hepatocyte spheroids to construct engineered hepatic tissue. The hepatic tissue was heterotopically implanted in the omentum majus of a rat model with liver injury induced by carbon tetrachloride (CCl4 ). Hepatocyte spheroids in the heparinized DLS remained viable for at least 10weeks in vivo. The entire scaffold was populated with hepatocytes and arranged well. The volume of the heparinized DLS group was expanded over 400-fold. Liver-specific functions such as albumin synthesis, glycogen storage and cytochrome P 3A4 activity were highly expressed in the hepatic tissue. In addition, endothelial cells were recruited, as shown by CD31 staining, and new blood vessels formed, as visualized by fluorescein isothiocyanate-labelled dextran intravital confocal microscopy. The heparinized bioengineered hepatic tissue alleviated CCl4 -induced liver injury by regulating the deposition and degradation of the extracellular matrix. Primary hepatocyte spheroids survived for an extended time on the heparinized DLS and expanded to generate vascularized and functional bioengineered hepatic tissue that can alleviate liver injury in rats.

The effect of heparinized decellularized scaffolds on angiogenic capability

The immobilization of heparin, a new and versatile approach to the surface modification of decellularized tissues, has the potential to greatly improve the hemocompatibility of engineered tissue constructs derived from decellularized organs. We report on porcine decellularized liver scaffolds (DLSs) heparinized by the end-point attachment (EPA) technique. The heparinized DLSs (HEP-DLSs) have the ability to bind and slowly release heparin-binding growth factors. We hypothesized that DLS-immobilized heparin acts as an antithrombotic coating reagent and binds vascular endothelial growth factor (VEGF) to induce angiogenesis in the DLSs. Human umbilical vein endothelial cells (HUVECs) seeded on HEP-VEGF-DLSs attached and remained bioactive. Using the chicken chorioallantoic membrane (CAM) assay, we found that the HEP-VEGF-DLSs induced a significant and rapid enhancement of angiogenesis compared with native DLSs. Scaffolds were implanted in the greater omentum of rats and evaluated after 7, 14, 21, and 28 days. There were significant increases in the numbers of blood vessels in the HEP-VEGF-DLSs compared with native DLSs at all time-points. The modified method introduced in this article could overcome obstacles faced by conventional matrices that lack the ability to induce rapid and sufficient vascularization. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3021-3030, 2016.

Hemocompatibility improvement of perfusion-decellularized clinical-scale liver scaffold through heparin immobilization.

Whole-liver perfusion-decellularization is an attractive scaffold–preparation technique for producing clinical transplantable liver tissue. However, the scaffold’s poor hemocompatibility poses a major obstacle. This study was intended to improve the hemocompatibility of perfusion-decellularized porcine liver scaffold via immobilization of heparin. Heparin was immobilized on decellularized liver scaffolds (DLSs) by electrostatic binding using a layer-by-layer self-assembly technique (/h-LBL scaffold), covalent binding via multi-point attachment (/h-MPA scaffold), or end-point attachment (/h-EPA scaffold). The effect of heparinization on anticoagulant ability and cytocompatibility were investigated. The result of heparin content and release tests revealed EPA technique performed higher efficiency of heparin immobilization than other two methods. Then, systematic in vitro investigation of prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT), platelet adhesion and human platelet factor 4 (PF4, indicates platelet activation) confirmed the heparinized scaffolds, especially the /h-EPA counterparts, exhibited ultralow blood component activations and excellent hemocompatibility. Furthermore, heparin treatments prevented thrombosis successfully in DLSs with blood perfusion after implanted in vivo. Meanwhile, after heparin processes, both primary hepatocyte and endothelial cell viability were also well-maintained, which indicated that heparin treatments with improved biocompatibility might extend to various hemoperfusable whole-organ scaffolds’ preparation.

Preparation and characterization of heparinized multi-walled carbon nanotubes

The characteristics of heparinized multiwalled carbon nanotubes (MWNTs) were investigated in terms of the activated partial thromboplastin time (APTT) to verify the heparin activity, a carbazole assay was done to measure the content of the immobilized heparin, and the octanol–water partition coefficient was assessed to determine the lipophilicity. Two heparin-immobilized MWNTs were prepared to evaluate their differences. The first preparation method involved polymer-coated MWNTs with heparin indirectly center-point-attached. In the second approach, heparin was directly end-point-attached through its reducing end onto acid-treated MWNTs. The blood compatibility of MWNTs to which heparin was end-point-attached through its reducing end was greatly enhanced compared to that of the MWNTs onto which heparin was center-point-attached. The APTT and carbazole assay results demonstrated that heparinized MWNTs prepared through end-point attachment result in prolonged plasma-based anticoagulant activity. The blood compatibility of MWNTs heparinized by end-point attachment was not decreased up to the fourth pasteurization. Heparinized MWNTs were also studied using octanol–water partition, which should be useful for exploring heparinized MWNTs as drug carriers including delivery systems. The results of octanol/water partition on the design of heparinized MWNTs prepared by end-point attachment with a specific binding can facilitate the design of drug delivery carriers with high blood compatibility.

Lower Limb Revascularization with a New Bioactive Prosthetic Graft: Early and Late Results

The aim of the study was to evaluate the immediate and long-term results of femoropopliteal bypasses performed with a new bioactive heparin-treated expanded polytetrafluoroethylene (ePTFE) graft in a single-center experience. From March 2002 to April 2006, 51 patients underwent lower limb revascularization with a new bioactive ePTFE prosthetic graft with covalent end-point attachment of heparin to the graft surface. Data concerning preoperative assessment, intraoperative strategy, drug administration, and follow-up surveillance program were prospectively collected in a dedicated database; early results were analyzed in terms of graft patency, amputation rate, and deaths. Follow-up consisted of clinical and duplex scan examination at 1, 6, and 12 months and yearly thereafter. Midterm results in terms of primary and secondary patency, limb salvage, and survival were analyzed. Patients were predominantly male (35 patients, 71%), with a mean age of 71 years (SD = 9.05). Indications for surgical revascularization were critical limb ischemia in 36 patients and severe intermittent claudication in 15 patients. Interventions were performed for occlusion of a native vessel in 35 cases, whereas 12 patients had late thrombosis of a femoropopliteal bypass; the remaining four patients were operated on for an occluded popliteal artery aneurysm. Intervention consisted of below-knee bypass in 34 patients, while the other 17 had an above-knee revascularization. No perioperative deaths occurred. Cumulative 30-day graft patency was 88%, with an amputation rate of 4% (two cases). Results were similar in above- and below-knee revascularizatons. Mean duration of follow-up was 18 months (SD = 7). Cumulative estimated 24-month survival and primary patency rates were 97% and 80.2%, respectively; the corresponding limb salvage rate was 85.7%. Long-term results did not significantly differ in above- and below-knee revascularizatons. In our experience, the use of a modified ePTFE graft with covalent end-point linkage of heparin molecules on the graft surface provides good early and midterm results, with low rates of graft thrombosis and amputation.

ATTEMPTS: a heparin/protamine-based delivery system for enzyme drugs

A prodrug delivery system termed “Antibody Targeted, Triggered, Electrically Modified Prodrug-Type Strategy (ATTEMPTS)” has been developed to permit the antibody-directed administration of inactive enzyme drug including tissue-type plasminogen activator (tPA), and allow a subsequent triggered release of the active tPA at the target site. Cation-modified tPA (mtPA) was attached to a heparin–antifibrin complex via ionic interaction, and the active tPA can subsequently be released by the addition of protamine, a competitive heparin inhibitor. Anti-fibrin IgG was conjugated to heparin via an end-point attachment to form the heparin–antifibrin complex which provides the targeting efficiency of the final heparin/mtPA complex. Cation modification was performed by either chemical conjugation by linking (Arg) 7Cys to tPA with N-succinimidy-3-(2-pyridyldithio) propionate or by recombinant DNA methods. Results show that the modification process did not significantly alter the specific activity of tPA with regard to plasminogen activation, fibrin-binding ability, and response toward fibrinogen. The complexes of both modified tPA–heparin did not yield any intrinsic catalytic activity owing to the blockage of the active site of tPA by the attached heparin. On the other hand, heparin-induced inhibition of modified tPA activity was reversed by adding protamine, which is similar to that of a prodrug delivery system. These results suggest that heparin/protamine-based enzyme delivery systems may be a useful tool to improve current enzyme therapeutic status, as well as thrombolytic therapy, by both regulating the release of active enzyme and aborting the associated systemic toxic effect. Currently, modification of enzyme drugs has been optimized by recombinant DNA technology assisted by computer simulation. In addition, the original strategy has been revised to obtain enhanced therapeutic efficacy.

ATTEMPTS: A heparin/protamine-based prodrug approach for delivery of thrombolytic drugs

The aim of this study is to develop a heparin/protamine-based prodrug system for the controlled delivery of enzyme such as tissue-type plasminogen activator (tPA). This approach, termed antibody targeted, triggered, electrically modified prodrug-type strategy (ATTEMPTS), would permit antibody-directed administration of inactive tPA, and allow a subsequent triggered release of the active tPA at the target site. Cation-modified tPA (mtPA) was attached to a heparin–antifibrin complex via ionic interaction. The active tPA can be subsequently released by the addition of protamine, a competitive heparin inhibitor. Anti-fibrin IgG was conjugated to heparin via an end-point attachment to form the heparin–antifibrin–complex which provides the targeting efficiency of the final heparin–mtPA complex. Cation-modification was performed either by chemical conjugation by linking (Arg) 7Cys to tPA with N-succinimidy-3-(2-pyridyldithio) propionate or by recombinant DNA method. Results show that the chemical modification process did not significantly alter specific activity of tPA with regard to plasminogen activation, fibrin-binding ability, and response toward fibrinogen. Expressed modified tPA (EmtPA) produced by recombinant DNA methods retained the same catalytic activity of the parent tPA, as well as a dynamic catalytic behavior depending upon the presence of heparin and protamine. Both types of modified tPA, especially the mtPA demonstrated a significantly higher affinity toward heparin or heparin–antifibrin complex than native tPA. In addition, the complexes of mtPA–heparin did not yield any intrinsic clot lysis activity owing to the blockage of the active site of tPA by attached heparin. On the other hand, heparin-induced inhibition of both mtPA and EmtPA activity was reversed by adding protamine, as confirmed by chromogenic and in vitro clot lysis assays. These results suggested that a heparin/protamine-based tPA delivery system may be a useful tool to improve current thrombolytic therapeutic status, by both precisely regulating the release of active tPA and aborting the associated bleeding risk. Alternatively, this ATTEMPTS approach could also be used to deliver enzyme drugs while diminishing their associated toxic effects.

Effects of heparin immobilization on the surface characteristics of a biological tissue fixed with a naturally occurring crosslinking agent (genipin): an in vitro study

Heparinized biomaterials have been used to manufacture blood-contacting prostheses. The present study was intended to characterize the surface properties of a genipin-fixed biological tissue immobilized with heparin using the methods of ionic binding (the /h-i tissue) or covalent binding via multi-point attachment (the /h-m tissue) or end-point attachment (the /h-e tissue). The surface characteristics of test tissues evaluated were water contact angle, surface tension, protein adsorption, platelet adhesion, and cellular compatibility. Nonheparinized and the glutaraldehyde-fixed counterparts were used as controls. It was found that immobilization of heparin on the glutaraldehyde- and genipin-fixed tissues increased their hydrophilicity and surface tension and suppressed their mole ratio of adsorbed fibrinogen to adsorbed albumin and the amount of platelets adhered. Among the heparinized tissues, the /h-m tissue was more hydrophobic and had a higher mole ratio of adsorbed fibrinogen to adsorbed albumin and a greater amount of platelets adhered than the /h-i and /h-e tissues. In general, the surface characteristics of the /h-i tissue were comparable to the /h-e tissue. However, it is known that the ionically immobilized heparin may be displaced from the surface by an ion-exchange mechanism when exposed to blood. There were no significant differences in hydrophilicity, surface tension, the mole ratio of adsorbed fibrinogen to adsorbed albumin, and the amount of platelet adhesion between the glutaraldehyde- and genipin-fixed tissues in comparison with their respective counterparts. However, the cellular compatibility of the genipin-fixed tissues with or without heparinization was significantly superior to its glutaraldehyde-fixed counterparts.

Synthesis and characterization of positively charged tPA as a prodrug using heparin/protamine-based drug delivery system.

Positively charged peptides [(Arg)7 Cys] were successfully linked to tissue-specific plasminogen activator (tPA) using cross-linking agent N-succinimidyl 3-(2-pyridyldithio) propionate. Specific amidolytic activity of this tPA/(Arg)7 Cys (termed modified tPA, mtPA) was 3900 IU/microg as compared to 5800 IU/microg of the parent tPA. Both activation of plasminogen with mtPA (Km= 2.7 mM(-1)) and tPA (Km= 1.1 mM(-1)) in a purified system followed Michaelis-Menten kinetics. In addition, (Arg)7 Cys modification did not result in significant changes in the fibrin-binding ability of tPA, and mtPA still retained a response to fibrinogen similar to that of the parent tPA. Compared with tPA, mtPA showed much stronger heparin affinity, and the heparin/mtPA complex was stable in human plasma. The activity of mtPA in such a complex was inhibited by heparin, and, unlike tPA, the heparin/mtPA complex did not cause statistically meaningful depletion of plasminogen, fibrinogen, and alpha2-antiplasmin in plasma. Using the chromogenic and the in vitro clot lysis assay, it was demonstrated that the heparin-induced inhibition of the mtPA activity was easily reversed following the addition of an adequate amount of protamine. To enhance the clot-targeting efficiency of the heparin/mtPA complex further, anti-fibrin immunoglobulin (IgG) was conjugated to heparin via an end-point attachment of heparin to the sugar moieties in the Fc region of the IgG. Results show that the activity of mtPA could also be blocked by the heparin/anti-fibrin IgG conjugate. These findings suggest the applicability of the heparin/protamine delivery system to abort the potential bleeding risks associated with clinical use of tPA.

Biocompatible surfaces by immobilization of heparin on diamond-like carbon films deposited on various substrates

Although graphitic carbon has been known and used as a biomaterial for a very long time, the excellent biocompatibility of diamond-like carbon (DLC) films has been addressed only in a few cases. We anticipate the combination of bioinert DLC films and surface-immobilized bioactive biomolecules with antithrombogenic properties, such as the polysaccharide heparin, as a straightforward concept to optimize the haemocompatibility of a wide variety of materials. Therefore, to assess this property in view of an application as vascular grafts, surface analytical studies on DLC-coated and heparinized polymer and silicon substrates were performed. The DLC films were deposited on polytetrafluoroethylene (PTFE), PTFE vascular prostheses, polystyrene and silicon wafers by an energetic acetylene plasma beam and subsequently exposed to an ammonia plasma before heparin was covalently coupled to such functionalized surfaces by an end-point attachment method. The biochemical characterization by thrombin tests revealed, in accordance with XPS measurements, a high amount of heparin on the surface and an extended blood coagulation time. Thus, the efficiency of the plasma treatment for functionalization and successful improvement of the haemocompatibility of DLC films could be shown. Copyright © 2000 John Wiley & Sons, Ltd.

Monoclonal antibodies against heparin and heparinoids.

The antigenicity of glycosaminoglycans and galactosaminoglycans is very weak. Accordingly, only a few reports on the successful production of monoclonal antibodies against glycosaminoglycans and galactosaminoglycans have been published. Antibodies have been raised against the heparinlike compounds heparan sulfate, chondroitin sulfate, and keratan sulfate. The production of heparin antibodies was reported. But further analysis showed that the antibodies were not directed against native heparin but against heparin conjugates or chemically modified heparins. After immunization with a heparin-bovine serum albumin conjugate, prepared by reductive amination, a monoclonal antibody against heparin and heparin fractions was obtained (Huhle et al: Semin Thromb Hemostas 20:193-204, 1995). For further analysis, tyramine, which was covalently bound to low-molecular-mass heparin (LMMH) by endpoint attachment (Malsch et al: Anal Biochem 217:255-264, 1994), was labeled with iodine-125 at the aryl residue. The tracer antibody complex was immunoprecipitated by goat anti-mouse immunoglobulin IgG. H1.18 recognized specifically intact heparin and heparin fractions. The lower detection limit for heparin preparations was 100 ng/mL. The H1.18 antibody was purified by ammonium sulfate precipitation. H1.18 remained biologically and functionally active after purification. The smallest disaccharide that was detected by H1.18 was found to be iduronic acid-anhydromannose, obtained by nitrous acid degradation of LMMH. Endpoint attachment of tyramine to anhydromannose did not modify the binding of the disaccharide to H1.18, indicating the importance of iduronic acid for binding of glycosaminoglycans to the antibody.

Pharmacokinetic properties of LMW-heparin-tyramine fractions with high or low affinity to antithrombin III in the rat.

We have recently presented evidence that a macrophage scavenger receptor-mediated pathway is responsible for the hepatic uptake of unfractionated heparins and low-molecular-weight heparins (LMWHs) in the rat. The same receptor-mediated pathway was partially responsible for the removal of oxidized low-density lipoprotein. Unfractionated and fractionated LMWHs exert their anticoagulatory effects predominately by reversibly binding to the plasma glycoprotein antithrombin III. In this study LMWHs modified by endpoint attachment of tyramine were radiolabeled and their fractions with low or high affinity to AT-III studied in vivo in rats. The high-affinity fraction was predominately cleared from the circulation by a hepatic uptake mechanism. About 25% of the injected high-affinity tracer material was recovered, whereas only about 8% of the low-affinity material was found in the liver after 180 minutes. Blocking the scavenger receptor-mediated liver RES uptake mechanism by maleylated bovine serum albumin led to a considerable decline in liver uptake (9 versus 25%). The low-affinity material was rapidly eliminated into the urine after 180 minutes. About 45% of the low-affinity material was excreted versus 23% of the high-affinity material. Tight binding to AT-III prevented LMWH-tyramine from being rapidly cleared from the circulation via the kidneys into the urine; instead, the scavenger receptor-mediated hepatic uptake mechanism seemed to be more dominant in removing material with high affinity to AT-III from blood.

Characterisation of silica-based heparin-affinity adsorbents through column chromatography of plasma fractions containing thrombin

Different heparin-silicas, synthesised in this laboratory via directional end-point attachment of heparin (H) onto various amino-derivatised silicas, have been evaluated in packed-bed and expanded-bed column chromatographic experiments using crude preparations of the therapeutic protein, thrombin (T). Adsorbent capacities, determined through batch adsorption experiments, were verified by employing frontal analysis in packed-bed systems. The performance of these adsorbents was also investigated in terms of thrombin purification factors and recoveries. The potential of the heparin-silicas was further examined in the expanded-bed column chromatographic mode using a scaled-up procedure. With heparin-Fractosil 1000 adsorbents, capacities of around 100 000 U thrombin/ml adsorbent could be achieved. Heparin-Fractosil 1000 adsorbents of intermediate heparin content (around 4 mg heparin/ml sorbent) displayed binding stoichiometries similar to that of the commercial heparin-Sepharose (2.6–2.7 mol T/mol H). Furthermore, binding stoichiometries were largely unaffected by increasing the heparin content on the heparin-Fractosil 1000 adsorbents from 0.8 to 4.6 mg of heparin/ml of sorbent. This result suggests that optimal binding site accessibilities for the thrombin-heparin interaction occurs at lower ligand density values. The binding capacity values determined from frontal analysis were confirmed by the recovery data, thus indicating minimal irreversible adsorption. Specific activities of ca. 2100 U/mg were obtained for thrombin when affinity-purified on these heparin-LiChroprep Si60 or heparin-Fractosil 1000 adsorbents. These values were higher than the maximum achievable purity obtained through alternative, multi-step chromatographic purification procedures reported by other investigators. These results indicated that the packed-bed performances with these silica-based adsorbents were superior to currently available commercial soft gel adsorbents, with the more dense heparin-silicas exhibiting very good potential for use in expanded-bed applications.

Perfusion Techniques for Heparin-Bonded Circuits

Recent advances in cardiopulmonary bypass techniques include the development of durable, covalently bonded heparin coated surfaces using end-point attachment techniques. Advantages of heparin-coated cardiopulmonary bypass include reduced systemic heparin, avoidance of protamine sulfate at the completion of the procedure, enhanced biocompatibility of the circuit, successful application of cardiopulmonary bypass in cases where bleeding may become a problem, and in application of assist devices. Our experience with this technology has led to the development of guidelines and management strategies. We generally consider application fortwo categories: 1) pharmacotoxicity, i.e., allergic reaction to heparin or protamine; and 2) progression of problems, such as prolonged bleeding tendencies (aspirin) or hemorrhage from cerebral vascular accident. Applications of this technology require tip to tip heparin-bonding, "streamlining" the circuit, meticulous attention to fluid mechanics, crystalloid cardioplegia, precise heparin management, and reversed Trendelenburg position for post-cardiopulmonary bypass volume.

Binding of fluorescent-labeled low molecular mass heparin to latex microspheres and to rat and human leukocytes.

Fluorescent-labeled LMMH may be used to investigate the pharmacokinetics of heparins. FITC has been specifically bound by endpoint attachment to LMMH-tyramine. In the present article two in vitro methods are described with the intention of developing new ex vivo test systems. To determine the concentration of heparin, protamine has been bound to the surface of magnetic latex particles. LMMH-tyramine-FITC bound dose dependently in a linear range from 0.1 to 30 micrograms/mL to the protamine-coated latex particles. The fluorescence intensity of the latex particles was analyzed using flow cytometry. No differences in the dilution curves were observed between buffer system, rat and human plasma. This indicated that no interference of heparin binding took place with plasma proteins with respect to the binding to protamine. In the second method the binding of LMMH-tyramine-FITC on rat and human leukocytes was analyzed using flow cytometry. LMMH-tryamine-FITC bound dose dependently to rat and human granulocyes, monocytes, and lymphocytes. A linear relationship of binding was obtained for all three cell lines; the sensitivity was 0.1 microgram/mL for monocytes and lymphocytes and 0.03 microgram/mL for granulocytes without differences between rat and human cells. The data demonstrate a dose-dependent binding of LMMH-tyramine-FITC to protamine coated latex beads and to rat and human leukocytes.

Complement activation by polymethyl methacrylate (PMMA) is minimized by end-point heparin attachment

Complement activation by polymethyl methacrylate (PMMA) is minimized by end-point heparin attachment

Modification of the Complement Binding Properties of Polystyrene: Effects of End‐Point Heparin Attachment

In recent years, conjugation of heparin to biomaterials has been shown to improve its biocompatibility. The purpose of the present work was to compare complement activation and binding of C3 to unmodified and heparin-treated polystyrene surfaces of microtitre plates. When polystyrene was incubated with human serum, C3 was deposited on the surface by both adsorption and binding dependent on activation of the classical (CPW) and alternative (APW) pathways. After end-point attachment of heparin, significant C3 deposition, although at reduced levels, occurred only by CPW-mediated mechanisms, while adsorption and APW-mediated binding were strongly reduced. Generally, the modified surface bound lower amounts of protein, e.g. serum albumin and IgG, than the unmodified. By contrast, it had increased affinity for C1q which leads to binding of C1 and activation of complement via the CPW. Nevertheless, the net effect of the surface modification on the complement reaction was an overall reduction of C3 binding due to obliteration of APW. This can be related to an enhanced factor H/I-dependent down-regulation of C3b and to the lowered protein-adsorbing property of the surface, both of which have inhibitory effects on APW and on the C3 shunt-dependent activation of the complement system.

Enhanced patency of small-diameter tubings after surface immobilization of heparin fragments. A study in the dog.

The luminal surfaces of polyethylene and polyurethane tubings of 1 mm inner diameter and 1 m in length were coated with heparin by end point attachment and thereafter sterilized in ethylene oxide. The tubings were inserted as arterio-venous shunts from the aorta to the superior caval vein via a carotid artery and a jugular vein. Patency was assessed with an ultrasound doppler flow meter and flow measurements. All nonheparinized shunts occluded within 10 days with a mean elapsed time from the insertion of the tubing is the last positive doppler registration being 3.5 days. The corresponding patency time for heparinized shunts was 25.9 days. The occlusion of the heparinized tubings was due to the incorporation of the ends of the tubings in the walls of aorta and caval vein, whereas the nonheparinized tubings occluded as a result of clot formation. It is concluded that surface immobilization of heparin with sequences capable of activating antithrombin may be beneficial in the development of small-diameter vascular protheses.