Heparin, a member of the glycosaminoglycan family, is renowned as the most negatively charged biomolecule discovered within the realm of human biology. This polysaccharide serves a vital role as a regulator for various proteins, cells, and tissues within the human body, positioning itself as a pivotal macromolecule of significance. The domain of biology has witnessed substantial interest in the intricate design of heparin and its derivatives, particularly focusing on heparin-based polymers and hydrogels. This intrigue spans a wide spectrum of applications, encompassing diverse areas such as protein adsorption, anticoagulant properties, controlled drug release, development of implants, stent innovation, enhancement of blood compatibility, acceleration of wound healing, and pioneering strides in tissue engineering. This comprehensive overview delves into a multitude of developed heparin conjugates, employing various methods, and explores their functions in both the biomedicine and electronics fields. The efficacy of materials derived from heparin is also thoroughly investigated, encompassing considerations such as thrombogenicity, drug release kinetics, affinity for growth factors (GFs), biocompatibility, and electrochemical analyses. We firmly believe that by redirecting focus towards research and advancements in heparin-related polymers/hydrogels, this study will ignite further research and accelerate potential breakthroughs in this promising and evolving field of discovery.

Biomaterial science has contributed tremendously to developing nanoscale materials for delivering biologically active compounds, enhancing protein stability, and enabling its therapeutic use. This paper presents a process of formation of polyelectrolyte multilayer (PEM) prepared by sequential adsorption of positively charged polydiallyldimethylammonium chloride (PDADMAC) and negatively charged heparin sodium salt (HP), from low polyelectrolyte concentration, on a solid substrate. PEM was further applied as a platform for the adsorption of a brain-derived growth factor (BDNF), which is a protein capable of regulating neuronal cell development. The multilayers containing BDNF were thoroughly characterized by electrokinetic (streaming potential measurements, SPM) and optical (optical waveguide lightmode spectroscopy, OWLS) techniques. It was found that BDNF was significantly adsorbed onto polyelectrolyte multilayers terminated by HP under physiological conditions. We further explore the effect of established PEMs in vitro on the neuroblastoma SH-SY5Y cell line. An enzyme-linked immunosorbent assay (ELISA) confirmed that BDNF was released from multilayers, and the use of the PEMs intensified its cellular uptake. Compared to the control, PEMs with adsorbed BDNF significantly reduced cell viability and mitochondrial membrane polarization to as low as 72% and 58%, respectively. HPLC analysis showed that both PDADMAC-terminated and HP-terminated multilayers have antioxidative properties as they almost by half decreased lipid peroxidation in SH-SY5Y cells. Finally, enhanced formation of spheroid-like, 3D structures was observed by light microscopy. We offer a well-characterized PEM with antioxidant properties acting as a BDNF carrier, stabilizing BDNF and making it more accessible to cells in an inhomogeneous, dynamic, and transient in vitro environment. Described multilayers can be utilized in future biomedical applications, such as boosting the effect of treatment by selective anticancer as adjuvant therapy, and in biomedical research for future development of more precise neurodegenerative disease models, as they enhance cellular 3D structure formation.

Dendritic polyglycerol sulfate (dPGS) has originally been investigated as an anticoagulant to potentially substitute for the natural glycosaminoglycan heparin. Compared to unfractionated heparin, dPGS possesses lower anticoagulant activity but a much higher anticomplementary effect. Since coagulation, complement activation, and inflammation are often present in the pathophysiology of numerous diseases, dPGS polymers with both anticoagulant and anticomplementary activities represent promising candidates for the development of polymeric drugs of nanosized architecture. In this review, we describe the nanomedical applications of dPGS based on its anti-inflammatory activity. Furthermore, the application of dPGS as a carrier molecule for diagnostic molecules and therapeutic drugs is reviewed, based on the ability to target tumors and localize in tumor cells. Finally, the application of dPGS for inhibition of virus infections is described.

Interactions between chemokines and glycosaminoglycans (GAGs) are crucial for the physiological and pathophysiological activities of chemokines. GAGs are therefore commonly designated as chemokine coreceptors which are deeply involved in the chemokine-signaling network. Studying the interaction of chemokines with GAGs is therefore a major prerequisite to fully understand the biological function of chemokines. GAGs are, however, a very complex class of biomacromolecules which cannot be produced by conventional recombinant methods and which, if purchased from commercial suppliers, are often not subjected to rigorous quality control and therefore frequently differ in batch characteristics. This naturally impacts chemokine-GAG interaction studies. In order to standardize the quality of our GAG ligands, we have therefore established protocols for the preparation and characterization of GAGs from various cells and tissues, for which we give practical examples relating to the major GAG classes heparin, heparan sulfate, and chondroitin sulfate. We will also outline robust and sensitive protocols for chemokine-GAG interaction studies. By this means, a better and more common understanding of the involvement of GAGs in chemokine-signaling networks can be envisaged.

Mesoporous silica MCM-41 and SBA-15 were chosen to study the adsorption and release of bulky biomolecule heparin, in order to develop new heparin controlled delivery system and expand the application of mesoporous materials in life science. To explore how the structure of support such as pore size and surface state affects the accommodation and release of heparin, we used decane as swelling agent to enlarge pores of MCM-41, introduced amino groups for improving the biocompatibility of support, and controllably retained templates in the as-synthesized sample. The influence of modification on the structure of samples was investigated by XRD and N(2) adsorption-desorption, whereas their performance of adsorbing and releasing heparin was assessed with that of toluidine blue method. Both enlarged pore and organic modification significantly promoted the adsorption and prolonged the release of heparin in MCM-41, and the release was characterized with a three-stage release model. The mechanism of heparin release from mesoporous material was studied by fitting the release profiles to the theoretical equation. As expected, some mesoporous composites could release heparin in the long term with tuned dosage.

The anionic polysaccharides heparin and heparan sulphate play essential roles in the regulation of many physiological processes. Heparin is often used as an analogue for heparan sulphate. Despite knowledge of an NMR solution structure and 19 crystal structures of heparin-protein complexes for short heparin fragments, no structures for larger heparin fragments have been reported up to now. Here, we show that solution structures for six purified heparin fragments dp6-dp36 (where dp stands for degree of polymerisation) can be determined by a combination of analytical ultracentrifugation, synchrotron X-ray scattering, and constrained modelling. Analytical ultracentrifugation velocity data for dp6-dp36 showed sedimentation coefficients that increased linearly from 1.09 S to 1.84 S with size. X-ray scattering of dp6-dp36 gave radii of gyration R(G) that ranged from 1.33 nm to 3.12 nm and maximum lengths that ranged from 3.0 nm to 12.3 nm. The higher resolution of X-ray scattering revealed an increased bending of heparin with increased size. Constrained molecular modelling of 5000 randomised heparin conformers resulted in 9-15 best-fit structures for each of dp18, dp24, dp30, and dp36 that indicated flexibility and the presence of short linear segments in mildly bent structures. Comparisons of these solution structures with crystal structures of heparin-protein complexes revealed similar ranges of phi (phi) and psi (psi) angles between iduronate and glucosamine rings. We conclude that heparin in solution has a semi-rigid and extended conformation that is preformed for its optimal binding to protein targets without major conformational changes.

We previously reported that heparin post-transcriptionally stimulates the production of hepatocyte growth factor (HGF). In this study, we addressed the size-dependency of heparin fragments on the HGF-inducing activity aiming to obtain fragments without antiblood coagulant activity. Heparin fragments, produced by digestion with heparinase, were size-fractionated and tested for HGF-inducing activity in cultured human fibroblasts. The HGF-inducing activity deceased with the reduction in oligosaccharide size. Decasaccharides exerted an activity comparable with undigested heparin, while smaller oligosaccharides showed lesser activities. The anticoagulant activity of heparin fragments also decreased with size and anticoagulant activity of decasaccharides was <13% that of undigested heparin. Further fractionation of decasaccharides by anion-exchange chromatography revealed that most of the decasaccharides had HGF-inducing activity and the extent of sulfation was roughly related to the activity. The lack of N-sulfation in heparin markedly reduced HGF-inducing activity, whereas 2-O-desulfation or 6-O-desulation had a lesser influence. Moreover, an N-sulfated disaccharide showed significant HGF-inducing activity, suggesting the involvement of N-sulfation in HGF-inducing activity. Because of the much reduced anticoagulant activity, potential applications of heparin-derived oligosaccharides such as decasaccharides is considerable as a therapeutic agent for many diseases.

Intimatan (dermatan 4,6-O-disulfate), a heparin cofactor II agonist, is a highly sulfated negatively charged semisynthetic polysaccharide. The present study examined the hypothesis that Intimatan reduces complement-mediated myocardial injury. The rabbit isolated heart was perfused with 4% normal human plasma (NHP) as a source of complement in the absence or presence of Intimatan (5 microM). Heat-inactivated human plasma (HIHP) was used as a negative control. Previous studies demonstrated that contact of rabbit tissue with human plasma results in activation of the alternative pathway of the human complement system, leading to irreversible myocardial injury. In the presence of NHP, left ventricular end-diastolic pressure (LVEDP) was increased significantly to 61.8+/-11.7 mm Hg compared to a value of 17.2+/-6.1 mm Hg in hearts perfused in the presence of HIHP. Left ventricular developed pressure (LVDP) was reduced significantly upon exposure to NHP, 19.3+/-10.2 (NHP) vs. 54.0+/-8.0 mm Hg (HIHP). Functional impairment in the presence of NHP was accompanied by a significant release of cardiac troponin I (cTnI; 131.8+/-20.3 ng/ml) as compared to hearts exposed to HIHP (0.8+/-0.8). Intimatan treatment improved cardiac function and maintained viability of cardiac myocytes (LVEDP 14.6+/-5.6, LVDP 58.0+/-8.1 mm Hg and cTnI 6.7+/-5.2 ng/ml). Immunohistochemical staining demonstrated that Intimatan pretreatment prevented deposition of the human membrane attack complex (MAC) in hearts exposed to NHP. The results indicate that Intimatan, a glycosaminoglycan (GAG), can reduce tissue injury and preserve organ function that otherwise would be compromised during activation of the human complement cascade.

Various deoxycholic acid (DOCA) bearing heparin (HD) amphiphilic conjugates with different degrees of substitution (DS) with DOCA were synthesized using heparin as a hydrophilic segment and DOCA as a hydrophobic segment. Structural characteristics of these HD conjugates were investigated using 1H NMR, dynamic light scattering, zeta potential, transmission electron microscopy (TEM), and fluorescence spectroscopy. HD conjugates provided monodispersed self-aggregates in water, with mean diameters decreasing with increasing DOCA DS in the range of 120-200 nm. HD aggregates were covered with negatively charged heparin shells, exhibiting xi potentials near -56 mV. The critical aggregation concentration (cac) of the HD (0.02-0.003 mg/mL) depended upon DOCA DS. TEM images demonstrated that the shape of the self-aggregates was spherical. Partition equilibrium constants, Kv, for pyrene in the self-aggregates in water indicated that increasing DS enhanced the hydrophobicity of the self-aggregate inner core. The mean aggregation number of DOCA per hydrophobic microdomain, estimated by the fluorescence quenching methods using cetylpyridinium chloride, indicated that five to nine of HD chains comprised a hydrophobic domain in the conjugates.

Hereditary angioedema (HAE) is a rare disorder characterized by episodes of angioedema of the skin, mucous membranes, and gastrointestinal tract resulting from a defect in the gene that produces C1 esterase inhibitor. Although in vitro laboratory data and past reports suggested that heparin might be efficacious in preventing HAE attacks, no controlled study has been reported to examine heparin's efficacy in this regard.

We sought to determine the safety and efficacy of inhaled and subcutaneous heparin versus that of placebo in the prevention of HAE attacks.

We performed a double-blind, double-dummy, saline placebo-controlled, randomized, 3-way crossover study with 11 visits.

The study was designed to enroll 24 patients. Twenty-two patients were randomized and received the study drug. Patients did not have a significant decrease in average flare intensity after they received injected or inhaled heparin compared with that seen after placebo, the primary endpoint. However, when patients received injected heparin, they had a statistically significant decrease in average flare intensity compared with that seen with inhaled heparin after a normalizing transformation was applied. When the means are back transformed, this translates into median flare intensities of 9.2, 8.0, and 5.1 in the patients treated with inhaled heparin, placebo, and injected heparin, respectively. There were no significant differences when individual symptoms were examined, when total numbers of flares over a 6-week observation period were examined, or when global evaluations by the patients and investigators were evaluated. Adverse event severity was fairly uniform across treatments, with the majority of events classified as moderate and the remainder split between mild and severe. Injected heparin treatment was associated with higher rates of relatedness than other treatments, which was partially explained by 17 adverse events specifically related to the injection process itself (tenderness, bruising, redness, pain, and itching at the injection site). The injection treatment was also associated with a larger overall number of reported adverse events (70 vs 48 in the placebo treatment). Tenderness and bruising at the injection site were entirely confined to the injected heparin treatment.

Injected and inhaled heparin failed to attenuate average flare intensity, the primary endpoint, compared with placebo. Interestingly, after patients injected heparin, they had a significant decrease in average flare intensity compared with that seen after inhalation of heparin. There were no differences among groups in other efficacy parameters. Taken together, these data indicate that commercial heparin was ineffective in preventing exacerbations of HAE.

Specific sequences in heparin are responsible for its modulation of the biological activity of proteins. As part of a program to characterize heparin-peptide and heparin-protein binding, we are studying the interaction of chemically discrete heparin-derived oligosaccharides with peptides and proteins. We report here the isolation and characterization, by one- and two-dimensional 1H NMR spectroscopies, of ten hexasaccharides, one pentasaccharide, and one octasaccharide serine that were isolated from depolymerized porcine intestinal mucosal heparin. Hexasaccharides were chosen for study because they fall within the size range, typically tetra- to decasaccharide in length, of heparin sequences that modulate the activity of proteins. The depolymerization reaction was catalyzed by heparinase I (EC 4.2.2.7) in the presence of histamine, which binds site specifically to heparin. Histamine increases both the rate and extent of heparinase I-catalyzed depolymerization of heparin. It is proposed that oligosaccharides produced by heparinase I-catalyzed depolymerization can inhibit the enzyme by binding to the imidazolium group of histidine-203, which together with cysteine-135 forms the catalytic domain of heparinase I. The increased rate and extent of depolymerization are attributed to competitive binding of the oligosaccharides by histamine.

The complement system is an innate, cytotoxic host defence system that normally functions to eliminate foreign pathogens. However, considerable evidence suggests that complement plays a key role in the pathophysiology of ischaemic heart disease (IHD). Experimental models of acute myocardial infarction (MI) and autopsy specimens taken from acute MI patients demonstrate that complement is selectively deposited in areas of infarction. Furthermore, inhibition of complement activation or depletion of complement components prior to myocardial reperfusion has been shown to reduce complement-mediated tissue injury in numerous animal models. IHD remains a leading cause of patient morbidity and mortality. Considerable effort in recent years has therefore been directed by biotechnology and pharmaceutical industries towards the development of novel, human complement inhibitors. Proposed anticomplement therapeutic strategies include the administration of naturally occurring or recombinant complement regulators, anticomplement monoclonal antibodies, and anticomplement receptor antagonists. Although data regarding the effectiveness of anticomplement therapy in humans is limited at present, a number of novel anticomplement therapeutic strategies are currently in clinical trials. The role of complement in IHD and potential for pharmacological intervention is reviewed.

In this chapter we have described anti-inflammatory functions of heparin distinct from its traditional anticoagulant activity. We have presented in vivo data showing heparin's beneficial effects in various preclinical models of inflammatory disease as well as discussed some clinical studies showing that the anti-inflammatory activities of heparin may translate into therapeutic uses. In vivo models that use low-anticoagulant heparins indicate that the anticoagulant activity can be distinguished from heparin's anti-inflammatory properties. In certain cases such as hypovolemic shock, the efficacy of a low-anticoagulant heparin derivative (GM1892) exceeds heparin. Data also suggest that nonconventional delivery of heparin, specifically via inhalation, has therapeutic potential in improving drug pharmacokinetics (as determined by measuring blood coagulation parameters) and in reducing the persistent concerns of systemic hemorrhagic complications. Results from larger clinical trials with heparin and LMW heparins are eagerly anticipated and will allow us to assess our predictions on the effectiveness of this drug class to treat a variety of human inflammatory diseases.

The ability of the heparin derivative, N-acetylheparin (NHEP) to protect the heart from regional ischemia/reperfusion injury was examined in vivo. NHEP (2 mg/kg i.v.) or vehicle was administered 2 h before occlusion of the left circumflex coronary (LCX) artery. Open-chest, anesthetized rabbits were subjected to 30 min of regional myocardial ischemia followed by 5 h of reperfusion. Myocardial myeloperoxidase activity, membrane attack complex (MAC) deposition and IL-8 generation were assessed in supernatant samples from the area at risk. Infarct size in rabbits pretreated with NHEP (32.5 +/- 3.8%, n = 10) decreased by 41% compared to infarct size in rabbits that received vehicle (55.3 +/- 4.9%, n = 10; p = 0.002). Accumulation of neutrophils within the ischemic region, as assessed by myeloperoxidase activity, declined by 45% (p < 0.05) in AAR from NHEP-treated animals compared to AAR from vehicle-treated animals. Levels of MAC and IL-8 obtained from AAR were less in NHEP-pretreated animals compared to controls. These results suggest that NHEP may protect the myocardium by inhibiting complement activation and subsequent neutrophil infiltration.

Myocardial ischemia/reperfusion injury is accompanied by an inflammatory response contributing to reversible and irreversible changes in tissue viability and organ function. Endothelial and leukocyte responses are involved in tissue injury, orchestrated primarily by the complement cascade. Anaphylatoxins, and assembly of the membrane attack complex contribute directly and indirectly to further tissue damage. Tissue salvage can be achieved by depletion of complement components, thus making evident a contributory role for the complement cascade in ischemia/reperfusion injury. The complexity of the complement cascade provides numerous sites as potential targets for therapeutic interventions designed to modulate the complement response to injury. The latter is exemplified by the ability of a soluble form of complement receptor 1 (sCR1) to decrease infarct size in in vivo models of ischemia/reperfusion injury as well as prevent myocyte and vascular injury and organ dysfunction by interdicting assembly of the membrane attack complex. Effective inhibitors of complement are not limited to newly developed compounds or solubilized forms of endogenous regulators of complement activation. Therapeutic agents in common use, such as heparin and related non-anticoagulant glycosaminoglycans, are known to inhibit the complement activation in vitro as well as in vivo and may prove useful as cytoprotective agents.

The cytoprotective action of reviparin-sodium (LU-47311: Clivarin), a low-molecular-weight heparin, was examined in an ex vivo model of complement-mediated myocardial injury. The effective concentration of reviparin was determined by using an in vitro rabbit erythrocyte-lysis assay using 4% normal human plasma. Reviparin (0.01-2.73 mg/ml) reduced erythrocyte lysis in a concentration-dependent manner. Subsequently, 0.91 mg/ml of reviparin was evaluated in an ex vivo rabbit isolated-heart model of human complement-mediated injury. Hearts perfused in the presence of 0.91 mg/ml of reviparin (n = 10) demonstrated significant preservation of ventricular function compared with vehicle-treated hearts (n = 10), as evidenced by coronary artery perfusion pressure, left ventricular developed pressure, and left ventricular end-diastolic pressure. A reduction in myocyte creatine kinase release was observed in reviparin-treated hearts compared with controls. Myocardial injury in vehicle-treated hearts was associated with an increased assembly of the membrane-attack complex, as determined by immunohistochemical localization of C5b-9 neoantigen. Reviparin decreased fluid-phase Bb formation detected in the lymphatic drainage of plasma-perfused hearts. The results of this study demonstrate that reviparin inhibits complement-mediated myocardial injury as assessed in an ex vivo experimental model of complement activation.

Surfaces of medical devices made of polymeric materials may promote thrombosis and inflammation. Therefore, in an attempt to produce surfaces which might diminish biomaterial-mediated thrombosis and inflammation, surface derivatization with 2-acrylamido-2-methylpropanesulphonic acid (AMPS) was carried out. The derivatization procedure generates free radicals which initiate the copolymerization of AMPS monomers directly to a polyurethane surface. In an in vitro blood loop study using non-anticoagulated human blood, the resulting AMPS-derivatized material completely abrogates the generation of fibrinopeptide A, decreases the production of beta-thromboglobulin and C3a, and decreases the adherence of platelets. The derivatized material also attracts fewer adherent neutrophils when implanted in mice. However, AMPS derivatization unexpectedly increases the recruitment of macrophages to implanted material and promotes the formation of adherent sleeve thrombi on central venous catheters indwelling in non-anticoagulated canine femoral veins. Thus, AMPS derivatization has highly variable effects on inflammatory and thrombotic systems. Further investigation is clearly required to determine the mechanisms underlying both desired and adverse effects.

We have shown previously that a low-molecular-weight fucan extracted from the brown seaweed Ascophylum nodosum strongly inhibited human complement activation in vitro and its mechanism of action was largely elucidated. We further investigated the influence of molecular weight and chemical composition of fucan on its anticomplementary activity. The capacity of 12 fragments of fucan (ranging from a molecular weight of 4100 to 214,000) to prevent complement-mediated haemolysis of sheep erythrocytes (classical pathway) and of rabbit erythrocytes (alternative pathway) increased with increasing molecular weight, and reached a plateau for 40,000 and 13,500, respectively. The most potent fucan fractions were 40-fold more active than heparin in inhibiting the classical pathway. They were, however, as active as heparin in inhibiting the alternative pathway. In addition, we have developed a haemolytic test based on the CH50 protocol, which allows discrimination between activators and inhibitors of complement proteins. Although the mannose content within the different fucan fragments did not vary, the galactose and glucuronic acid contents increased with increasing activity, suggesting that these residues should be essential for full anticomplementary activity. Meanwhile, sulphate groups appeared to be necessary, but were clearly not a sufficient requirement for anticomplementary activity of fucans. Taken together, these data illustrate the prospects for the use of fucans as potential anti-inflammatory agents.

Four hexasaccharides representing major structural sequences of heparin were isolated and characterized after degradation of heparin by heparinase. The structures were determined from two-dimensional 1H NMR spectroscopy including TOCSY (total correlated spectroscopy), COSY (correlated spectroscopy), and ROESY (rotating frame nuclear Overhauser enhancement spectroscopy) methods, providing new data on hexasaccharides. One of the hexasaccharides, the last eluting component from anion exchange chromatography, was derived from the tri-sulfated repeating disaccharide, alpha-L-idopyranosyluronic acid 2-sulfate-(1-->4)-2-amino-2-deoxy-D-glucopyranose 6,N-disulfate, and having the structure delta UAp2S-(1)-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S. The second hexasaccharide contained a nonsulfated D-glucuronic acid unit instead of the L-iduronic acid adjacent to the reducing end, and having the structure delta UAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-beta-D- GlcAp-(1-->4)-alpha-D-GlcNp2S6S. The last two hexasaccharides were obtained in lower yield and they have not been isolated and characterized before. The structure of the third saccharide corresponded to a trimer of the repeating disaccharide except for the lack of a 6-O-sulfate group at the reducing end glucosamine residue; deltaUAp2S-(1-->4)-alpha-D-Glcnp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L-IdoAp2S -(1-->4)-alpha- D-GlcNp2S. The fourth and last hexasaccharide were less sulfated and the following structure was established delta UAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- Idop2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp-(1-->4)-alpha-D-GlcNpAc6S. Analysis of the ROESY spectra revealed conformational difference of the glucosidic linkage alpha-L-IdoAp-(1-->4)-alpha-D-GlcNp between the hexasaccharides and longer heparin chains.

Evidence is presented that heparin pretreatment produces protective effects on myocardial tissue distinct from its anticoagulant activity. The present study examines the ability of heparin sulfate and N-acetyl heparin (a derivative of heparin devoid of anticoagulant effects) to protect the heart from injury associated with global ischemia and reperfusion. Male New Zealand White rabbits were administered either heparin sulfate (n = 7, 300 U/kg i.v.), N-acetyl heparin (n = 6, 1.73 mg/kg i.v.), or vehicle (n = 6). Two hours after treatment, the hearts were removed, perfused on a Langendorff apparatus, and subjected to 30 minutes of global ischemia, followed by 45 minutes of reperfusion. During reperfusion, creatine kinase concentrations in the coronary sinus effluent were greater in hearts from vehicle-treated rabbits compared with hearts from N-acetyl heparin-treated and heparin-treated rabbits. Left ventricular end-diastolic pressure after 45 minutes of reperfusion in the vehicle-treated group was 64 +/- 15 mm Hg compared with 17 +/- 4 and 10 +/- 3 mm Hg in the heparin-pretreated and N-acetyl heparin-pretreated groups, respectively. Heparin, but not N-acetyl heparin, increased the activated partial thromboplastin time, consistent with its known anticoagulant action. Heparin and N-acetyl heparin inhibited complement-mediated erythrocyte lysis in a concentration-dependent manner. The glycosaminoglycans, in contrast to r-hirudin, reduced complement activation-induced injury in the rabbit isolated heart. The results demonstrate that heparin or N-acetyl heparin, administered to the intact rabbit, protects the isolated heart from subsequent myocardial dysfunction secondary to ischemia/reperfusion. The cardioprotective effects of heparin and N-acetyl heparin are independent of an antithrombin mechanism.

Heparin has been shown to inhibit activity of the alternative, classical and terminal pathways of complement by regulating C1, C1 inhibitor, C4 binding protein, C3b, factor H and S-protein. In vivo, heparin inhibits cobra venom factor activation of complement in a dose-related manner in guinea pigs. However, the ability of heparin and of modified heparin to inhibit complement activation in serum has not been examined systematically. The present study compared commercial heparin with a modified heparin that has reduced anticoagulant activity (N-desulfated, N-acetylated heparin) for ability to inhibit cobra venom factor and zymosan-induced complement activation in guinea pig and human serum. Both heparins inhibited cobra venom factor and zymosan-induced consumption of C3 activity in both human and guinea pig serum. In both serum types, commercial heparin was about twice as active as modified heparin on a weight basis for ability to inhibit cobra venom factor-induced complement activation. Both heparins also inhibited zymosan-induced complement activation in human serum. About four times more heparin was required to inhibit cobra venom factor-induced complement activation in guinea pig serum than in human serum while heparin was more than ten times more active in human serum than in guinea pig serum when zymosan was used as the activator of complement. This study suggests that heparin is considerably more effective in regulating complement activity in humans than in guinea pigs, an animal model in which heparin clearly has in vivo capacity to regulate complement activity. These observations represent an important step in the development of new clinically relevant oligosaccharide-derived pharmacologic agents to regulate complement activity.

Many diverse effects of heparin on the complement system have been reported. In only a few cases have the sites or the mechanisms of these effects been identified. In order to understand these results we sought to comprehensively analyze which complement proteins interact with heparin and which do not. Purified components of the classical, alternative and terminal pathways of complement were radiolabeled and their affinity for heparin determined. Affinity chromatography of normal human serum on heparin-agarose allowed a complete analysis of complement proteins and confirmed the results obtained with radiolabeled purified components. Of the 22 complement proteins examined, 13 bound heparin (C1q, C2, C4, C4bp, C1INH, B, D, H, P, C6, C8, C9, and vitronectin) while 9 did not bind heparin (C1r, C1s, C3, Factor I, C5, C7, C3b, Ba and Bb). These observations help explain the many effects heparin has on the complement system and they identify the proteins which need to be examined in order to explain these effects.

1. Six different monoclonal IgG mouse antibodies to heparin lyase I from Flavobacterium heparinum were prepared. 2. The monoclonal antibodies were used to detect heparin lyases I, II and III by dot-blotting immunoassay and by Western blotting. 3. Individual antibodies showed different reactivity toward the three heparin lyases. 4. The reactivity of two of the monoclonal antibodies was destroyed by exposing heparin lyases to sodium dodecyl sulfate. 5. The antibodies can be used to rapidly distinguish between the three heparin lyases.

Earlier work from our laboratory demonstrated that heparin inhibits type I collagen and DNA synthesis in fetal rat calvariae in vitro. In this paper we have analyzed the structural features of heparin that determine its inhibitory effect on collagen synthesis. These experiments were performed using unmodified heparins and low-molecular-weight heparins from different manufacturers, nonheparin glycosaminoglycans, desulfated heparins, anticoagulant and nonanticoagulant heparin, and chemically defined heparin oligosaccharides. Low-molecular-weight heparin (Mr 3700-5100) inhibited collagen synthesis, but oligosaccharides (disaccharides to decasaccharide, Mr 665-3000) did not. The glycosaminoglycans chondroitin sulfate B, heparan sulfate, and hyaluronic acid did not alter collagen synthesis but dextran sulfate was as inhibitory as unmodified heparin. Nonanticoagulant as well as anticoagulant low-molecular-weight heparin fractions inhibited collagen synthesis. Modification of heparin by total desulfation, O-desulfation, or N-desulfation and re-N-acetylation resulted in the loss of inhibitory property, suggesting that the degree of sulfation contributed to heparin's inhibitory effect. Low-molecular-weight heparins from different manufacturers were just as inhibitory as native heparin on collagen synthesis. We therefore conclude that low-molecular-weight heparin compounds offer no protection against heparin-induced osteoporosis. Our findings also suggest that the size and sulfation of a heparin-derived oligosaccharide contribute to its ability to inhibit collagen synthesis in bone.

The rapid preparation of multimilligram quantities of five heparin-derived oligosaccharides (1-5) is described. These oligosaccharides are the final products obtained from the action of heparin lyase (heparinase, E.C. 4.2.2.7) at its primary sites in the heparin polymer. Five oligosaccharides comprise from 75-85 wt% of commercial porcine mucosal heparins and are recovered in good yield and high purity. Four of these five oligosaccharides were further acted upon at much lower rates by prolonged treatment with heparin lyase or heparan monosulfate lyase (heparitinase, E.C. 4.2.2.8), revealing the subspecificities of these enzymes. These oligosaccharides were used as defined substrates for heparin lyase and heparan monosulfate lyase and their kinetic constants were obtained. Potential applications for these oligosaccharides include their use as defined substrates for purification of heparin monosulfate lyases, and for establishing the catalytic purity of enzyme preparations.

Eighteen patients undergoing aortobifemoral graft surgery for severe aortoiliac atherosclerotic disease received a bolus injection of 10,000 anti-Xa units of either unfractionated heparin (UFH) or low molecular weight heparin (LMWH) into the distal aorta as prophylaxis against thromboembolic complications related to clamping. Heparin activity was measured by factor Xa inhibition and by prolongation of the APTT. In both groups there was a delay before peak levels of heparin were observed. In the LMWH group, this amounted to 30 min. In the UFH group, APTT was prolonged by 46 s, 7 min after injection but only by 5 s at the end of the operation. In contrast, in the LMWH group, the prolongation in APTT 7 min after injection was less (34 s) but more sustained since a 12.5 s prolongation was still present at the end of the operation. During surgery, heparin activity exceeded 0.7 U/ml in the LMWH group, compared to significantly lower levels in the UFH group (less than or equal to 0.20 U/ml). By the end of the operation no heparin activity was detectable in the UFH group. Protein C antigen decreased after heparin injection and this fall was more pronounced in the UFH group. The level of C1q (a subcomponent of the first component of the complement system) was decreased in the UFH group (P less than 0.04), whereas in the LMWH group C1q levels increased. Platelet aggregation with collagen was inhibited to a significantly greater degree in the LMWH group than the UFH group (54% compared with 23%) (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Polyanions and polycations inhibit activity of the alternative and classical pathways of complement. We compared polyanions (commercial porcine heparin, chondroitin sulfate A, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C and heparatin sulfate) with polycations (salmon sperm protamine sulfate, poly-L-lysine, poly-L-arginine, polybrene and a synthetically prepared portion of platelet factor 4) for ability to inhibit alternative and classical pathway activity. The polyanions had considerably more activity on the alternative than on the classical pathway, whereas the polycations more profoundly inhibited classical than alternative pathway activity. For example, heparin, a polyanion, at 1.0 micrograms (7.7 x 10(-7) M based upon an Mr average of 13000)/10(7) cellular intermediates, inhibited alternative pathway activity and classical pathway activity by 77 and 14%, respectively, whereas protamine sulfate, a polycation, at 0.25 micrograms/10(7) cellular intermediates, inhibited these two pathways by 34 and 98%, respectively. These studies suggest that the capacity to inhibit complement activity is a common feature of highly charged substances and the polyanions preferentially inhibit the alternative pathway while polycations preferentially inhibit the classical pathway. In vivo these highly charged substances could play an important role in the tissues in regulating the activity of both pathways of complement.

Heparins from different species and tissues show similar levels of ATIII and HCII mediated anti-IIa activities. On fractionation, chains containing predominantly ATIII or HCII activities could not be separated. Oligosaccharide mapping demonstrates that the concentration of an oligosaccharide comprising a portion of heparin's ATIII binding site in a particular heparin fraction correlates with ATIII mediated anti-IIa activity, but does not correlate with HCII mediated anti-IIa activity. These results suggest that ATIII and HCII do not share a common binding site. Partial enzymatic depolymerization of heparin resulted in large oligosaccharides which could be purified and partially characterized. Although oligosaccharides of degree of polymerization (dp) 18 and 20 showed significant ATIII and HCII mediated anti-IIa activities no separation of these activities resulted. These data suggest however that a minimum chain length of dp18 was required for HCII mediated anti-IIa activity.

Fluid phase heparin inhibits formation of the classical and alternative pathway C3 convertase of complement in assays performed either with purified complement proteins or in whole serum. Experiments using oligosaccharides of homogeneous mol. wt obtained by mild nitrous hydrolysis of heparin, demonstrated that the inhibitory activity of heparin increased exponentially with mol. wt for fragments containing between 4 and 14 saccharidic units and that fragments of mol. wt above 4700 (greater than 14 saccharidic units) had a similar anti-complementary activity to that of native heparin. Fragments of homogeneous mol. wt (octasaccharides) separated by ion exchange chromatography on the basis of negative charges, exhibited increasing inhibitory activity with increasing sulfate content. Over-sulfation of fragments of defined mol. wt resulted in a constant enhancement of the relative capacity of each fragment species to inhibit formation of the classical and alternative pathway C3 convertases. A synthetic pentasaccharide representing the minimal critical sequence responsible for the binding of heparin to anti-thrombin III exhibited a similar inhibitory capacity on formation of the C3 convertases as another synthetic pentasaccharide that was devoid of anti-Xa activity. These studies contribute to define a minimal structure of the heparin molecule with C3b- and C4b-binding capacity and definitively establish the independency of the anti-coagulant and anti-complementary sites on the heparin molecule.

Using size- uniform mixtures of di-, tetra-, octa- and decasaccharides obtained from the depolymerization of heparin with heparinase, we have studied the activity that these low molecular weight heparin fragments may have on the acrosome reaction and sperm nuclei decondensation processess. Swelling of human spermatozoa nuclei was stimulated by heparin and their fragments and was dependent on the incubation time and directly correlated with the size of the fragment tested. Disaccharides were unable to increase the number of swollen nuclei. At short times (2-8 hrs) decasaccharides were the most active substances tested, including heparin. Only heparin and the tetra- and decasaccharides showed a significant increase in the number of acrosome-reacted spermatozoa, both fragments were more active than heparin at 2 hour incubation. Hexa- and octasaccharides induced a slight increase in the number of acrosome reacted spermatozoa and disaccharides were ineffective. The presence in animal systems of oligosaccharides derived from macromolecules and having specific biochemical properties, remembers the recent discovery of to those mediated by oligosaccharins in plants may exist in animals.

Extracorporeal medical devices such as the hemodialyzer rely on systemic heparinization to prevent thrombus formation. Heparin, however, can lead to serious hemorrhagic complications. A blood filter containing immobilized heparinase, a heparin specific enzyme, was used to degrade heparin into small fragments which have significantly less anticoagulant activity than the parent compound. The heparinase filter was tested in the extracorporeal circuit during the hemodialysis of adult sheep. At a blood flow of 200 ml/min, the clearance of heparin varied from 50 to 70 ml/min (N = 16) depending on the amount of immobilized heparinase in the filter. Hemolysis was insignificant as measured by the animals' red cell counts, hematocrit, total hemoglobin and a plasma-free hemoglobin value of 89 +/- 33 mg/dl (N = 16) (less than 1% of the total hemoglobin). The white cell counts dropped to 47 +/- 7% (N = 16) of the initial value at 20 minutes and rebounded to 72 +/- 10% (N = 16) after one hour. The platelet counts decreased to 55 +/- 8% (N = 16) of the initial value after one hour. No change in heparin clearance was observed when reactors were used repeatedly in adult sheep over a 10 week period. The red cell counts, white cell counts, platelet counts, total hemoglobin and hematocrit did not change after 10 weeks of exposure to the device. These results suggest that with further study, heparinase may be useful in removing heparin used to anticoagulate blood in extracorporeal circuits.

Heparin-derived oligosaccharides, prepared by using flavobacterial heparinase, having a high degree of heterogeneity (sequence variability) were resolved into sharp well-defined bands by using polyacrylamide gel electrophoresis (PAGE). The use of a stacking gel and a high-density-pore-gradient resolving gel was primarily responsible for the success of this separation. Low-Mr standards of known structure and having a degree of polymerization (dp) 2-6 were used to establish that the separation on gradient PAGE was primarily dependent on molecular size. High-Mr oligosaccharides (dp 8-20) were prepared using strong-anion-exchange h.p.l.c. and were used to help characterize the gradient PAGE separation. Kinetic profiles were obtained for the depolymerization of heparin and heparan sulphate with heparinase and heparitinase respectively. The utility of this approach in sequencing oligosaccharides derived from glycosaminoglycans is discussed.

The need to fully heparinize patients undergoing extracorporeal therapy often leads to hemorrhagic complications. Two approaches have been used to solve this problem. The first involves full heparinization of blood entering the extracorporeal device followed by the elimination of heparin from the blood returned to the patient using an immobilized heparinase reactor system. Animal studies have demonstrated the successful elimination of heparin's anticoagulant activity using this reactor. The second approach uses very low molecular weight (VLMW) heparins with improved properties. Although low molecular weight heparins and heparinoids have been successfully used in hemodialysis, these preparations are polydisperse mixtures. New VLMW heparins are described which are pure, monodisperse, structurally defined drugs and show improved pharmacokinetics and greater specificity than heparin. The separation of ATIII and HCII mediated activity against factors IIa and Xa may permit extracorporeal therapy with only partial anticoagulation resulting in increased antithrombotic activity with decreased hemorrhagic side-effects. Finally, these VLMW heparins suggest certain desirable structural characteristics in the design blood compatible non-thrombotic synthetic polymers for use in extracorporeal devices.

The molecular weight and degree of sulfation has been obtained for di-, tetra- and hexasaccharide fragments of heparin obtained by enzymatic depolymerization of porcine mucosal heparin. The sodium salt form of the sulfated oligosaccharide is adsorbed onto an immobilized cationic surfactant film which is inserted directly into the mass spectrometer. Analyses are routinely obtained on 25-50 microgram samples in less than an hour. This approach provides rapid confirmatory structural information that is complementary to existing methodologies.

The first use of computer-simulation studies to examine heparin's structure has been reported. The product distributions obtained when porcine mucosal heparins were depolymerized with heparinase have been compared to computer-simulated distributions. The modeled distribution was relatively unaffected by the polydispersity and molecular weight of heparin. However, the percent of heparinase-cleavable glycosidic linkages and their distribution throughout the polymer resulted in a marked change in the simulated product distribution. The similarity between experimentally observed and computer-simulated product distributions is consistent with the random distribution of heparinase-cleavable sites in porcine mucosal heparin. Finally, a random distribution of N-acetyl residues with respect to heparinase-cleavable sites was experimentally observed.

Heparin has been enzymatically depolymerized with heparinase (heparin lyase (EC 4.2.2.7)) and then separated into di-, tetra-, hexa-, octa-, and decasaccharide mixtures by low-pressure gel-permeation chromatography (GPC). These sized mixtures were resolved by strong anion-exchange (SAX) HPLC into multiple components. The fractions from the SAX-HPLC were collected and characterized for size by GPC-HPLC and sulfate content by ion chromatography. This study provides detailed methodology for the separation of larger and more highly sulfated oligosaccharides than previously reported. It describes the first use of ion chromatography for the accurate determination of the sulfate content of heparin oligosaccharides, a method which can also be applied to heparin and other glycosaminoglycans.

The structure of heparin was examined by characterizing a disaccharide and five of the more than a dozen tetrasaccharide components obtained by its depolymerization with flavobacterial heparinase. Enzymic depolymerization of porcine mucosal heparin results in a mixture of di-, tetra-, hexa- and higher oligo-saccharides. The di- and tetra-saccharide components represent 75mol/100mol of these heparin fragments. Ion-exchange chromatography indicates the presence of only one disaccharide, deltaIdu2S(1----4)-alpha-D-GlcNS6S (where Idu is iduronic acid, deltaIdu is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is glucosamine, GlcA is glucuronic acid and S is sulphate), but results in the isolation of five major and at least seven minor tetrasaccharide components. The structures of the disaccharide and five major tetrasaccharides were determined by chemical, enzymic, electrophoretic and spectroscopic methods, including 13C, 1H n.m.r. and fast atom bombardment-m.s. The structure of these five tetrasaccharides are: delta Idu2S(1----4)-alpha-D-GlcNS6S(1---4)-alpha-L-Idu2S(1-- --4)-alpha -D-GlcNS6S; delta Idu2S(1----4)-alpha-D-GlcNS6S(1----4)-beta-D-GlcA(1--- -4)- alpha-D-GlcNS6S; delta Idu2S(1----4)-alpha-D-GlcNS(1----4)-beta-D-GlcA delta Idu2S(1----4)-alpha-D-GlcNAc(1----4)-beta-D-GlcA(1----4)- alpha-D-GlcNS6S; and delta Idu2S(1----4)-alpha- D-GlcNAc(1----4)-alpha-L-Idu(1----4)-alpha-D-GlcNS6S. Biological activity for the disaccharide and the five major tetrasaccharides was examined, and none of them were found to possess significant anticoagulant activity.

Polyclonal IgG rabbit antibodies were prepared against a purified heparinase from Flavobacterium heparinum. Immuno-affinity purification of crude and partially purified heparinase is described. The resulting enzyme was of comparable purity to that prepared using the standard multistep purification scheme. The antibodies prepared were found to increase the activity of bound heparinase.