HEMA-Lysine-Based Cryogels for Highly Selective Heparin Neutralization.

Unfractionated heparin (UFH) and its low-molecular-weight fragments (LMWH) are widely used as anticoagulants for surgical procedures and extracorporeal blood purification therapies such as cardiovascular surgery and dialysis. The anticoagulant effect of heparin is essential for the optimal execution of extracorporeal blood circulation. However, at the end of these procedures, to avoid the risk of bleeding, it is necessary to neutralize it. Currently, the only antidote for heparin neutralization is protamine sulphate, a highly basic protein which constitutes a further source of serious side events and is ineffective in neutralizing LMWH. Furthermore, dialysis patients, due to the routine administration of heparin, often experience serious adverse effects, among which HIT (heparin-induced thrombocytopenia) is one of the most severe. For this reason, the finding of new heparin antagonists or alternative methods for heparin removal from blood is of great interest. Here, we describe the synthesis and characterization of a set of biocompatible macroporous cryogels based on poly(2-hydroxyethyl methacrylate) (pHEMA) and L-lysine with strong filtering capability and remarkable neutralization performance with regard to UFH and LMWH. These properties could enable the design and creation of a filtering device to rapidly reverse heparin, protecting patients from the harmful consequences of the anticoagulant.

In vitro and in vivo safety studies indicate that R15, a synthetic polyarginine peptide, could safely reverse the effects of unfractionated heparin.

Unfractionated heparin (UFH) is an anionic glycosaminoglycan that is widely used to prevent blood clotting. However, in certain cases, unwanted side effects can require it to be neutralized. Protamine sulfate (PS), a basic peptide rich in arginine, is the only approved antagonist for UFH neutralization. Many adverse reactions occur with the clinical application of PS, including systemic hypotension, pulmonary hypertension, and anaphylaxis. We previously described R15, a linear peptide composed of 15 arginine molecules, as a potential UFH antagonist. In this study, the in-depth safety of R15 was explored to reveal its merits and associated risks in comparison with PS. In vitro safety studies investigated the interactions of R15 with erythrocytes, fibrin, complement, and rat plasma. In vivo safety studies explored potential toxicity and immunogenicity of R15 and the UFH-R15 complex. Results showed that both PS and R15 can induce erythrocyte aggregation, thicken fibrin fibers, activate complement, and cause anticoagulation in a concentration-dependent manner. However, those influences weakened in whole blood or in live animals and were avoided when R15 was in a complex with UFH. We found dramatically enhanced complement activation when there was excess UFH in analyses involving UFH-PS complexes, and a slight increase in those involving UFH-R15 complexes. Within 2 h, R15 was degraded in rat plasma in vitro, whereas PS was not. Enhanced creatinine was found after a single intravenous injection of PS or R15 (900 U·kg-1 , body weight), suggesting possible abnormal renal function. The UFH-PS complex, but not the UFH-R15 complex, exhibited obvious immunogenicity. In conclusion, R15 is nonimmunogenic and potentially safe at a therapeutic dose to reverse the effects of UFH.

Heparin Binding to an Engineered Virus-like Nanoparticle Antagonist.

The anticoagulant activity of heparin administered during medical interventions must be reversed to restore normal clotting, typically by titrating with protamine. Given the acute toxicity associated with protamine, we endeavored to generate safer heparin antagonists by engineering bacteriophage Qß virus-like particles (VLPs) to display motifs that bind heparin. A particle bearing a single amino acid change from wild-type (T18R) was identified as a promising candidate for heparin antagonism. Surface potential maps generated through molecular modeling reveal that the T18R mutation adds synergistically to adjacent positive charges on the particle surface, resulting in a large solvent-accessible cationic region that is replicated 180 times over the capsid. Chromatography using a heparin-sepharose column confirmed a strong interaction between heparin and the T18R particle. Binding studies using fluorescein-labeled heparin (HepFL) resulted in a concentration-dependent change in fluorescence intensity, which could be perturbed by the addition of unlabeled heparin. Analysis of the fluorescence data yielded a dissociation constant of approximately 1 nM and a 1:1 binding stoichiometry for HepFL:VLP. Dynamic light scattering (DLS) experiments suggested that T18R forms discrete complexes with heparin when the VLP:heparin molar ratios are equivalent, and in vitro clotting assays confirmed the 1:1 binding stoichiometry as full antagonism of heparin is achieved. Biolayer interferometry and backscattering interferometry corroborated the strong interaction of T18R with heparin, yielding Kd ∼ 1-10 nM. These biophysical measurements further validated T18R, and VLPs in general, for potential clinical use as effective, nontoxic heparin antagonists.

[Neutralization of anticoagulant activity of heparin by N-[(2-hydroxy-3-trimethylammonium) propyl] chloride derivatives of chitosan].

Alkylated derivatives of low molecular weight chitosan with different substitution degrees of 98, 40, and 9% (I, II, and III respectively) have been synthesized. The structure of the obtained derivatives was defined by spectral assays (IR-spectroscopy and proton magnetic resonance). Chitosan derivatives were characterized with positive zeta-potential (33­51 mV) and solubility from 2 to 100 mg/mL in pH 7.4 and 25°C. It was shown that, at a concentration of 0.0014­0.0029 mg/mL, derivative I, as well as protamine sulfate, could be used to neutralize the anticoagulant activity of unfractionated or low molecular weight heparin. At a concentration of 0.0029­0.58 mg/mL, derivative I enhanced platelet aggregation, which would be necessary when hemostatic compounds or materials were used. Derivatives II and III enhanced platelet aggregation to a lesser extent.

Novel HIT antibody detection method using Sonoclot® coagulation analyzer.

INTRODUCTION: Since heparin-induced thrombocytopenia (HIT), caused by the generation of antibodies against platelet factor 4 (PF4)/heparin complexes (HIT antibodies), may induce serious complications due to thrombosis, a prompt diagnosis is desirable. Functional tests with platelet activation to detect HIT antibodies are useful for diagnosis of HIT, in particular (14)C-selotonin release assay (SRA). However, they are complicated and so can be performed only in limited laboratories. We tested if a blood coagulation test using Sonoclot® analyzer can serve for the detection of HIT antibodies. MATERIALS AND METHODS: A murine monoclonal antibody (HIT-MoAb) against PF4/heparin complexes was used as an alternative to human HIT antibodies. To the mixture of HIT-MoAb and heparin (0.5 U/mL, final), whole blood obtained from a healthy volunteer was added, and then the activated clotting time (ACT), clot rate (CR), and area under the curve (AUC) were measured with Sonoclot® analyzer for 30minutes. RESULTS: The HIT-MoAb (30 to 100µg/mL, final) concentration dependently suppressed the anticoagulation activity (prolongation of ACT and decrease of CR and AUC) of heparin. CONCLUSIONS: The suppression of anticoagulation effect of heparin by HIT-MoAb was demonstrated by measurements using Sonoclot® analyzer. This method may provide a new tool for screening of HIT antibodies.

Low molecular weight protamine (LMWP): a nontoxic protamine substitute and an effective cell-penetrating peptide.

Low molecular weight protamine (LMWP) is a peptide fragment produced in our laboratory from enzymatic digestion of native protamine. More than 30 papers studying the properties and applications of LMWP have been published by our group in various journals since its initial discovery in 1999. Results have shown that LMWP could completely neutralize the anticoagulant functions of both heparin and low molecular weight heparin (LMWH), with reduced antigenicity and cross-reactivity toward the mice-derived anti-protamine antibodies. Aside from its potential as a heparin/LMWH antagonist, LMWP also shows the ability to retard insulin adsorption by the formation of an insoluble complex, making it a less toxic long-lasting insulin product than the conventional neutral protamine Hagedorn (NPH) insulin for diabetic control. Importantly, LMWP (Sequence: VSRRRRRRGGRRRR), with 10 arginine residues in its structure, could function as a cell-penetrating peptide (CPP), also termed protein transduction domain (PTD), to achieve effective intracellular protein or gene delivery in clinical practice. In this paper, we present a thorough review of our work related to LMWP, with the aim of providing readers an insight into its potential to be a clinical protamine substitute as well as a non-toxic cell penetrating peptide applicable to achieve intracellular protein and gene delivery.

A chemically-modified inactive antithrombin as a potent antagonist of fondaparinux and heparin anticoagulant activity.

BACKGROUND: Heparin and its analogs, mediating their anticoagulant activity through antithrombin (AT) activation, remain largely used for the preventive and curative treatment of thrombosis. The major adverse reaction of these drugs is the bleeding risk associated with overdose. Unfractionnated heparin (UFH) can be efficiently and rapidly neutralized by protamine sulfate, but this reversal partially neutralizes low-molecular-weight heparin (LMWH) and is inefficient in reversing fondaparinux. To secure administration of AT-mediated anticoagulants and counteract bleeding disorders, we previously designed a recombinant inactive AT as an antidote to heparin derivatives. OBJECTIVES: To get around the limited production level of recombinant AT, we propose in this study an alternative strategy to produce a chemically modified inactive AT, exhibiting increased heparin affinity, as an antagonist of heparin analogs. METHODS: Plasma-derived AT was chemically modified with 2,3 butanedione, a diketone known to specifically react with the arginine side chain. The chemical reaction was conducted in the presence of heparin to preserve basic residues within the heparin binding site from modifications. RESULTS: AT treated by butanedione and selected for its high heparin affinity (AT-BD) was indeed modified on reactive Arg393 and thus exhibited decreased anticoagulant activity and increased heparin affinity. AT-BD was able to neutralize anticoagulant activity of heparin derivatives in vitro and in vivo and was devoid of intrinsic anticoagulant activity, as assessed by activated partial thromboplastin time assay. CONCLUSIONS: AT-BD appears to be as efficient as protamine to neutralize UFH in vivo but could be more largely used because it also reverses fondaparinux and LMWH.

Neutralizing the anticoagulant activity of ultra-low-molecular-weight heparins using N-acetylglucosamine 6-sulfatase.

Heparin has been the most commonly used anticoagulant drug for nearly a century. The drug heparin is generally categorized into three forms according to its molecular weight: unfractionated (UF, average molecular weight 13 000), low molecular weight (average molecular weight 5000) and ultra-low-molecular-weight heparin (ULMWH, average molecular weight 2000). An overdose of heparin may lead to very dangerous bleeding in patients. Protamine sulfate may be administered as an antidote to reverse heparin's anticoagulant effect. However, there is no effective antidote for ULMWH. In the current study, we examine the use of human N-acetylglucosamine 6-sulfatase (NG6S), expressed in Chinese hamster ovary cells, as a reversal agent for ULMWH. NG6S removes a single 6-O-sulfo group at the non-reducing end of the ULMWH Arixtra(®) (fondaparinux), effectively removing its ability to bind to antithrombin and preventing its inhibition of coagulation factor Xa. These results pave the way to developing human NG6S as an antidote for neutralizing the anticoagulant activity of ULMWHs.

The C-terminal fragment of axon guidance molecule Slit3 binds heparin and neutralizes heparin's anticoagulant activity.

Slit3 is a large molecule with multiple domains and belongs to axon guidance families. To date, the biological functions of Slit3 are still largely unknown. Our recent study demonstrated that the N-terminal fragment of Slit3 is a novel angiogenic factor. In this study, we examined the biological function of the C-terminal fragment of human Slit3 (HSCF). The HSCF showed a high-affinity binding to heparin. The binding appeared to be heparin/heparan sulfate-specific and depends on the size, the degree of sulfation, the presence of N- and 6-O-sulfates and carboxyl moiety of the polysaccharide. Functional studies observed that HSCF inhibited antithrombin binding to heparin and neutralized the antifactor IIa and Xa activities of heparin and the antifactor IIa activity of low-molecular-weight heparin (LMWH). Thromboelastography analysis observed that HSCF reversed heparin's anticoagulation in global plasma coagulation. Taken together, these observations demonstrate that HSCF is a novel heparin-binding protein that potently neutralizes heparin's anticoagulation activity. This study reveals a potential for HSCF to be developed as a new antidote to treat overdosing of both heparin and LMWH in clinical applications.

High-throughput turbidimetric screening for heparin-neutralizing agents and low-molecular-weight heparin mimetics.

Safer heparin-neutralizing agents are currently required to replace protamine, the use of which causes adverse effects such as anaphylaxia. Low-molecular-weight (LMW) heparin mimetics that potentiate antithrombin III (AT) action are also valuable as anti-thrombotics. This paper describes a high-throughput assay for both heparin-neutralizing agents and LMW heparin mimetics without the use of blood preparations. The assay is based on turbidimetric measurement of a solution of collagen, heparin, and a test compound. Native collagen molecules spontaneously form insoluble fibrils when transferred to a physiological buffer, and this process is inhibited by heparin. In the presence of a heparin-neutralizing agent or an LMW heparin mimetic, the inhibitory effect of heparin is canceled and turbidity increase is retrieved. We demonstrated that this assay is effective in detecting potential agents with high reliability (Z' factor=0.9). The screening of a chemical library (34400 compounds) was further performed in a 384-well format, and led to the identification of a novel heparin-neutralizing agent. Since this assay protocol is feasible for an automated high-throughput screening (HTS) system, it could enhance the lead seeking process for drugs related to heparin/heparan sulfate (HS) functions.

Cationic derivatives of dextran and hydroxypropylcellulose as novel potential heparin antagonists.

Cationic derivatives of dextran (Dex) and hydroxypropylcellulose (HPC) were studied as potential alternatives of protamine sulfate (PS) used in the reversal of anticoagulant activity of heparin. The modification was performed by the attachment of cationic groups to the Dex main chain or by grafting short side chains of a polycation onto HPC. The cationic derivatives of these polysaccharides were found to bind heparin with the efficiency increasing with growing degree of cationic modification. The degree of cationic modification and consequently the ζ potential of the polymers do not have to be high to achieve effective heparin binding. The size of the complexes of cationic Dex with unfractionated heparin (UFH) is a few micrometers. For complexes of cationic HPC and UFH the size is much below 1 µm, both below and above the lower critical solution temperature of HPC. None of the cationic polysaccharides studied caused hemolysis. The concentrations of the polymers inducing the aggregation of human erythrocytes in vitro were determined.

A magnetofluorescent nanoparticle for ex-vivo cell labeling by covalently linking the drugs protamine and Feraheme.

We synthesized a nanoparticle (NP) for ex-vivo cell labeling and MRI tracking by covalently coupling the C-terminus of a rhodamine-labeled protamine (ProRho) to Feraheme (FH) in order to yield the nanoparticle denoted ProRho-FH. Since protamine can adsorb to certain charged surfaces, we confirmed a covalent interaction between ProRho and FH by heparin affinity chromatography. ProRho-FH lacks a net charge (zeta potential approximately 0) due to the combination of negative FH and positive ProRho charges. ProRho-FH was readily internalized by U87 cells and mouse mesenchymal stem cells as determined by FACS and MR relaxometry. Finally, some 4,000 stem cells were implanted in a mouse brain and imaged by MRI. Due to its lack of net surface charge, ProRho-FH relies on the internalizing properties of the surface guanidinium groups present in the arginine-rich protamine to induce NP uptake. ProRho-FH is a unique cell-labeling agent due to its synthesis using two approved drugs, magnetofluorescence, site-specific covalent attachment chemistry, and lack of surface charge.

Efficacy of fragmin/protamine microparticles containing fibroblast growth factor-2 (F/P MPs/FGF-2) to induce collateral vessels in a rabbit model of hindlimb ischemia.

OBJECTIVES: The localized delivery of exogenous, angiogenic growth factors such as fibroblast growth factor (FGF)-2 has become a promising alternative treatment of peripheral artery disease (PAD) and critical limb ischemia (CLI). The present study describes the efficacy of fragmin/protamine microparticles containing FGF-2 (F/P-MPs/FGF-2) to promote vessel growth in a rabbit model of hindlimb ischemia. METHODS: A total of 24 rabbits were used to construct a model of hindlimb ischemia by resection of the left femoral artery. The rabbits were randomly divided into four groups 10 days after surgery (day 0); group A: control (non-treated; 1 mL of phosphate-buffered saline [PBS]); group B: FGF-2 (100 µg FGF-2 in 1 mL PBS)-treated; group C: F/P-MPs (12 mg dried F/P MPs in 1 mL PBS)-treated; and group D; F/P MPs/FGF-2 (100 µg FGF-2 and 12 mg dried F/P MPs in 1 mL PBS)-treated (n = 6 each). The drugs were administered intramuscularly to each group. Blood flow and blood pressure were measured in each group on days 0, 14, and 28. Angiography was performed to assess arteriogenesis on day 28. The number of capillaries on day 28 was determined by direct counting CD31(-) and α-smooth muscle antibody (α-SMA)-positive vessels. RESULTS: Neither death nor wound infection was observed throughout the experiment. The F/P MPs/FGF-2-treated group showed marked improvement in the blood flow ratio, blood pressure ratio, and capillary number in comparison to the control group, FGF-2-treated group, and F/P MPs-treated group. The F/P MPs-treated group showed intermediate improvement in blood flow ratio and capillary number in comparison to the control group and FGF-2-treated group. CONCLUSIONS: The F/P MPs/FGF-2-treated group strongly induced functional collateral vessels in the rabbit model of hindlimb ischemia, indicating a possible therapy for PAD.

Supramolecular protamine/Gd-loaded liposomes adducts as relaxometric protease responsive probes.

A new approach to enzyme-responsive MRI agents based on the use of liposomes loaded with a high number of paramagnetic metal complexes (Gd-HPDO3A) is presented. It relies on the disruption of low relaxivity aggregates formed by liposomes and a macromolecular substrate that is selectively cleaved by the enzyme of interest. The interaction of anionic liposomes composed of POPC:CHOL:DPGS and the cationic protein protamine yields a poorly soluble supramolecular assembly endowed with a low relaxivity. The action of the serine protease trypsin causes the digestion of protamine and the consequent de-assembly of the supramolecular adduct. The process is accompanied by an overall relaxation enhancement of solvent water protons as consequence of the dissolution of the aggregated liposomes. The observed increase of relaxivity is linearly dependent on the enzyme concentration. An illustrative example of the possible use of the herein presented responsive agent has been reported. It consists of the entrapment of the supramolecular assembly in alginate microcapsules that have often been used as envelopes for in vivo applications of stem cells and pancreatic islets. The change in the observed longitudinal relaxation rate R(1) (leading to an hyperintense signal in the corresponding MR images) may act as a sensor of the protease activity in the biological environment in which the capsules is located.

Isolation and characterization of two new Lys49 PLA2s with heparin neutralizing properties from Bothrops moojeni snake venom.

Among the proteins and peptides already characterized in Bothrops moojeni venom, two novel phospholipases A(2) (PLA(2)) have been purified and fully sequenced by ESI-MS/MS techniques. Both of them belong to the enzymatically non-active Lys49 variants of PLA(2). They consist of 122 amino acids and share a characteristic sequence in their C-terminal region composed of clusters of basic amino acids known to interact with heparin. Thus, as already established, heparin can be used as an antidote to antagonize some myotoxic PLA(2)s from venoms of Bothrops genus. The two PLA(2) variants were shown to interact in vitro with unfractionated heparin (UFH) and low molecular weight heparin (LMWH), neutralizing their anticoagulant properties. Although the influences of PLA(2)s from snake venoms on the blood coagulation system are known, their use to antagonize the anticoagulant effect of heparin in vitro or in vivo has never been proposed. These finding recommend diagnostic and therapeutic applications, which are currently investigated.

From heparin to EP217609: the long way to a new pentasaccharide-based neutralisable anticoagulant with an unprecedented pharmacological profile.

The elucidation of the structure of the antithrombin binding sequence in heparin has given a large impulse to the rational design of heparin related drugs. De novo chemical synthesis of the corresponding pentasaccharide as well as simplified analogues has provided very specific, antithrombin-mediated inhibitors of factor Xa with various pharmacokinetic profiles. Fondaparinux and idraparinux are examples of such compounds that have found clinical application as antithrombotics. Because of the very specific binding to antithrombin the pharmacokinetics of pentasaccharides can be predicted and transferred to other molecules covalently bound to them. The new chemical entities thus obtained display a wide array of antithrombotic activities, giving improved heparin molecules as well as new anticoagulants, devoid of the undesired side effects of heparin and with unprecedented pharmacological profiles. In this context, a direct thrombin inhibitor was covalently coupled to a pentasaccharide by an inert spacer. This compound, EP42675 exerts antithrombin mediated anti-factor Xa activity together with direct thrombin inhibiting capacity. It displays favourable pharmacokinetics as imposed by the pentasaccharide. EP42675 was further modified by the introduction of a biotin moiety in its structure. The new entity obtained, EP217609 exerts the same pharmacological profile as EP42675 and it can be instantaneously neutralised by injection of avidin. Due to this unprecedented mechanism of anticoagulant activity and its ability to be neutralised, EP217609 deserves to be investigated in clinical settings where direct thrombin inhibition is required.

Development of idraparinux and idrabiotaparinux for anticoagulant therapy.

Idraparinux is an analogue of fondaparinux binding with high affinity to antithrombin. It was designed for weekly, rather than daily, administration, with an exceptionally long half-life. One potential problem with small heparin-like fragments of this type is the difficulty of neutralising excessive activity in the case of side-effects or overdose. The efficacy of idraparinux was was proven in clinical studies with patients suffering from venous thromboembolism (VTE) or atrial fibrillation. Due to major bleeding events during treatment for more than six months the development of idraparinux was stopped. Idrabiotaparinux has an attached biotin moiety at the non-reducing end unit, which allows its neutralisation with avidin, an egg-derived protein with low antigenicity. This compound is currently investigated in clinical trials for prevention of recurrent VTE in patients with acute pulmonary embolism. The future of idrabiotaparinux depends also on the safety and efficacy of avidin.

Parviz Lalezari: an autobiography.

Doctor Parviz Lalezari, currently a clinical professor of Medicine and Pathology at Albert Einstein College of Medicine in New York, describes highlights of his research career since 1958. He became the director of the blood bank at Montefiore Hospital in New York City in 1961, director of the Division of Immunohematology until 1996, and then until 2001, was President and chief executive officer of the Bergen Community Regional Blood Center in New Jersey. Doctor Lalezari was born in Iran in 1931, and after graduation from Medical School, he came to the United States in 1956. His initial research was on leukocyte antibodies. After modifying the available antibody detection techniques, he discovered that like hemolytic disease of the newborn and neonatal immune thrombocytopenia, fetal-maternal neutrophil incompatibility can cause neonatal neutropenia. He identified the targets of these antibodies and showed that they were expressed only on peripheral blood neutrophils. Doctor Lalezari also discovered that a common form of neutropenia in early childhood was caused by development of autoantibodies, which surprisingly were directed against the same neutrophil-specific antigens involved in fetal-maternal incompatibility. In 1959, a heparin-neutralizing drug (Polybrene) was introduced to be used after open-heart surgery. Lalezari discovered that Polybrene, a quaternary ammonium polymer, reacted with sialic acid molecules on the red blood cell (RBC) surface, causing the RBCs to aggregate. Later, realizing that the repelling forces generated by the RBC surface membrane charges were responsible for failure of the small IgG antibody molecules to agglutinate the RBCs, he used Polybrene to neutralize the RBC surface negative charge to allow the IgG antibody molecules to induce hemagglutination. This became The Polybrene test, which is to be used in RBC antibody detection.

Strategies for improving the functionality of an affinity bioreactor.

Heparin employed in extracorporeal blood circulation (ECBC) procedures (e.g. open heart operations) often leads to a high incidence of bleeding complications. Protamine employed in heparin neutralization, on the other hand, can cause severe adverse reactions. We previously developed an approach that could prevent both heparin- and protamine-induced toxic side effects concomitantly. This approach consisted of placing a hollow fiber-based bioreactor device containing immobilized protamine (termed a "protamine bioreactor") at the distal end of the ECBC procedure. This protamine bioreactor would remove heparin after heparin served its anticoagulant purpose in the ECBC device, thereby eliminating heparin-induced bleeding risks. In addition, this protamine bioreactor would prevent protamine from entering the patients, thereby aborting any protamine-induced toxic effects. Both in vitro and in vivo studies have successfully demonstrated the feasibility of this approach. Despite promises, early findings also revealed two shortcomings that must be overcome for the protamine bioreactor to be applied clinically. The first drawback was that the cyanate ester linkages, involved in conjugating protamine to the bioreactor device, were unstable and prone to hydrolysis, resulting in the leakage of a significant amount of protamine into circulation during application of the protamine bioreactor. The second deficiency was that the capacity of the protamine bioreactor in heparin removal was rather low, owing to the limited surface area of the hollow fibers for protamine immobilization and subsequently heparin adsorption. In this paper, we present novel strategies to overcome these two limitations. A new conjugation method based on the use of 4-(oxyacetyl)phenoxyacetic acid (OAPA) as the activating reagent was employed to yield stable linkages, via the abundant arginine residues of protamine, onto the hollow fibers. Results showed that while the amount of protamine immobilized on each gram of fibers was relatively comparable between the OAPA and the previous CNBr activation methods (7.45 mg/g versus 7.69 mg/g fibers), there was virtually no detectable leaching of immobilized protamine from the bioreactor by the OAPA method, comparing to 35% leaching of protamine by the previous CNBr method following 72 h of storage of the bioreactor in PBS buffer at 37 degrees C. To improve the capacity and functionality of the protamine bioreactor, two novel approaches were adopted. Long chain and high molecular weight poly-lysine was linked to the hollow fibers, prior to protamine coupling, to create multiple layers of immobilized protamine for subsequent heparin adsorption. In addition, a poly(ethylene glycol) (PEG) chain was inserted between protamine and the hollow fibers to yield a three-dimensional, free dynamic motion for immobilized protamine. Preliminary observations indicated that a four- to five-fold enhancement in heparin adsorption was attained by utilizing each of these new approaches. Aside from their current use, these new strategies can also be employed generically to improve the functionality of any affinity-type bioreactor. Indeed, efforts have been made recently in utilizing these approaches to develop a clinically usable GPIIb/IIIa bioreactor for the treatment of immune thrombocytopenic purpura (ITP)-an autoimmune disease.