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Tumor-derived exosomes are bound and internalized to organ-specific cells, affecting metastasis. Heparan sulfate proteoglycans mediate the interaction between cells and exosomes. Exosome transfer to the recipient cell can be competitively blocked by heparinoids, because heparin is structurally similar to heparan sulfate. It is hypothesized that there may be structural requirements of heparinoids to attenuate the cellular uptake and metastatic activity of tumor-derived exosomes. Here, we compared the properties of unfractionated heparin (UFH), glycol-split UFH, low-molecular-weight heparin (LMWH), glycol-split LMWH, and ultra-LMWH premixed with A549-derived exosomes. Uptake of A549-derived exosomes (0.1 mg/mL) into BEAS-2B cells was significantly blocked by 0.4 mg/mL of heparinoids. Heparinoids attenuated migration of BEAS-2B cells stimulated by A549-derived exosomes. Glycol-split LMWH with no antifactor Xa activity exhibited the strongest antimigratory effects than other heparinoids. Thus, heparinoids with proper molecular weight and structure can inhibit tumor-derived exosomes, not proportionally to the anticoagulant activity.

The microenvironment of pancreatic islets gets disrupted during enzyme digestion and causes islets to remain in a vulnerable state, leading to poor outcome in the initial days of transplantation. To avoid immune invasion while allowing the reconstruction of the microenvironment of the transplanted site, we propose immunoisolation polymers, which can nanoencapsulate islets quickly without cytotoxicity. Here, nonhuman primate (NHP) islets were nanoencapsulated with hyperbranched polyethylene glycol (hb-PEG) and heparin by layer-by-layer technology and transplanted into the kidney subcapsular space of diabetic C57BL/6 mice. An immunosuppressive drug protocol was applied to increase the survival time until the animals were sacrificed. The recipients of NHP islets exhibited high nonfasting blood glucose level (BGL) for 2-3 weeks, which was normalized afterward. Immunohistochemical (IHC) analysis revealed an immature vascular basement membrane and cell surface integrins directly associated with poor initial insulin production. The transplanted grafts regained their own microenvironment within a month without any outside stimuli. No lymphocyte infiltration was observed in the grafts at any time. Humoral and cell-mediated immune responses were prominently diminished by the hb-PEG/Heparin nanoencapsulated islets. Immunoisolation accompanied by an immunosuppressive drug protocol protects islets by helping them avoid immunogenesis while at the same time allowing them to reconstruct their microenvironment.

Intraportal pancreatic islet transplantation incurs huge cell losses during its early stages due to instant blood-mediated inflammatory reactions (IBMIRs), which may also drive regulation of the adaptive immune system. Therefore, a method that evades IBMIR will improve clinical islet transplantation. We used a layer-by-layer approach to shield non-human primate (NHP) islets with polyethylene glycol (nano-shielded islets, NSIs) and polyethylene glycol plus heparin (heparin nano-shielded islets; HNSIs). Islets ranging from 10,000 to 20,000 IEQ/kg body weight were transplanted into 19 cynomolgus monkeys (n = 4, control; n = 5, NSI; and n = 10, HNSI). The mean C-peptide positive graft survival times were 68.5, 64 and 108 days for the control, NSI and HNSI groups, respectively (P = 0.012). HNSI also reduced the factors responsible for IBMIR in vitro. Based on these data, HNSIs in conjunction with clinically established immunosuppressive drug regimens will result in superior outcomes compared to those achieved with the current protocol for clinical islet transplantation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Caspase-activated prodrug chemotherapy is introduced and demonstrated using the composite nanoparticles (NPs), which deliver doxorubicin (DOX) and DEVD-S-DOX together to the tumor tissue. DEVD-S-DOX, DOX linked to a peptide moiety (DEVD), is a prodrug that is cleaved into free DOX by caspase-3 upon apoptosis. DEVD-S-DOX has no therapeutic efficacy, but it changes into free DOX with the expression of caspase-3. With the accumulation of the composite NPs in the tumor tissue by the enhanced permeation and retention (EPR) effect, a small exposure of DOX in the tumor cells initiated apoptosis in a localized area of the tumor tissue, which induced caspase-3 activation. Cleavage of DEVD-S-DOX into free DOX by caspase-3 continued with repetitive activation of caspase-3 and cleavage of DEVD-S-DOX at the tumor site. The composite NPs were characterized with transmittance electron microscopy (TEM) and particle size analyzer. We then evaluated the nanoparticle drug release, therapeutic efficacy, and in vivo biodistribution for tumor targeting using a non-invasive live animal imaging technology and the quantification of DOX with high performance liquid chromatography. DOX-induced apoptosis-targeted chemotherapy (DIATC) was verified by in vitro/in vivo DEVD-S-DOX response to free DOX and cellular uptake behavior of the composite NPs with flow cytometry analysis. Significant antitumor efficacy with minimal cardiotoxicity was also observed, which supported DIATC for improved chemotherapy., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Introduction: Anticoagulants have been prescribed to patients to prevent deep vein thrombosis or pulmonary embolism. However, because of several problems in anticoagulant therapy, much attention has been directed at developing an ideal anticoagulant, and numerous attempts have been made to develop new anticoagulant delivery systems in recent years., Areas Covered: This review discusses the challenges associated with the recent development of anticoagulants and their delivery systems. Various delivery methods have been developed to improve the use of anticoagulants. Recent advances in anticoagulant delivery and antidote development are also discussed in the context of their current progression states., Expert Opinion: There have been many different approaches to developing the delivery system of anticoagulants. One approach has been to expand the use of new oral agents and develop their antidotes. Reducing the size of heparins to use smaller heparins for delivery, and developing oral or topical heparins are also some of the approaches used. Various physical formulations or chemical modifications are other ways that have enhanced the therapeutic potential of anticoagulant agents. On the whole, recent advances have contributed to increasing the efficacy and safety of anticoagulant clinically and have benefited the field of anticoagulant delivery.

Heparin is a highly sulfated, long, and linear polysaccharide, which can inhibit tumor growth by interacting with growth factors such as bFGF and VEGF. Several researchers have shown the anti-angiogenic effect of heparin and its conjugates in relation to growth factor inhibition. For drug development and inhibition of growth factors using heparin conjugates, the molecular size of heparin may be crucial considering the size of the heparin binding site of growth factors. In this study, we synthesized heparin fragments and deoxycholic acid conjugated heparin fragments (HFD) to search for the optimal size-controlled conjugate that will inhibit the angiogenic effect of VEGF165. We have also shown that the HFDs could have an enhanced therapeutic effect in vitro and in vivo consequent to the molecular size control. HFDs have significant anti-angiogenic effects by blocking the angiogenic activity of VEGF165 depending on its molecular size. Among them, HFD2 was a promising candidate for oral angiogenesis inhibitor. These results suggest that size-controlled synthesis is necessary for heparin-based drug development.

Angiogenesis, the formation of new blood vessels, plays a pivotal role in tumor progression and for this reason angiogenesis inhibitors are an important class of therapeutics for cancer treatment. Heparin-based angiogenesis inhibitors have been newly developed as one of such classes of therapeutics and possess a great promise in the clinical context. Taurocholate conjugated low molecular weight heparin derivative (LHT7) has been proven to be a potent, multi-targeting angiogenesis inhibitor against broad-spectrum angiogenic tumors. However, major limitations of LHT7 are its poor oral bioavailability, short half-life, and frequent parenteral dosing schedule. Addressing these issues, we have developed an oral formulation of LHT7 by chemically conjugating LHT7 with a tetrameric deoxycholic acid named LHTD4, and then physically complexing it with deoxycholylethylamine (DCK). The resulting LHTD4/DCK complex showed significantly enhanced oral bioavailability (34.3 ± 2.89%) and prolonged the mean residence time (7.5 ± 0.5 h). The LHTD4/DCK complex was mostly absorbed in the intestine by transcellular pathway via its interaction with apical sodium bile acid transporter. In vitro, the VEGF-induced sprouting of endothelial spheroids was significantly blocked by LHTD4. LHTD4/DCK complex significantly regressed the total vessel fractions of tumor (77.2 ± 3.9%), as analyzed by X-ray microCT angiography, thereby inhibiting tumor growth in vivo. Using the oral route of administration, we showed that LHTD4/DCK complex could be effective and chronically administered as angiogenesis inhibitor., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Oral delivery is the preferred route to deliver therapeutics via nanoparticles due to ease of administration and patient acceptance. Here, we report on the findings of the absorption pathway of taurocholic acid (TCA)-linked heparin and docetaxel (DTX) conjugate, which we refer to as HDTA. We studied the oral absorption of HDTA using a Caco-2 cell transport system and an animal model. We have also used other absorption enhancers, such as ethylene glycol tetraacetic acid (EGTA), or inhibitors, such as sodium azide, to compare the relative permeability of HDTA conjugates. In vivo comparative studies were conducted using free TCA as a pre-administration and exhibited the maximum absorption site of the organ after oral administration of HDTA conjugates. HDTA was found to be absorbed mainly in the ileum and Caco-2 cell monolayer through passive diffusion and bile acid transporters. High fluorescence intensity of HDTA in mice came from the ileum, and it was eliminated from the body through colon. This novel formulation could be further investigated by clinical trials to find the prospect of oral anti-cancer drug delivery through anti-angiogenic treatment strategies., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This study proposed a double-layer shielding method of using 8-arm-PEG-catechol (PEG(8)) and N-hydroxysuccinimidyl-linked unfractionated heparin (UFH-NHS) for the prevention of instant blood-mediated inflammatory reaction (IBMIR) and immune reactions against transplanted pancreatic islets. The surface of islet was evenly covered by PEG(8) and UFH-NHS. Both viability and functionality of islets were evaluated in vitro, and the anti-coagulation effect of conjugated heparin on the islet surface was also evaluated. The inhibition effects of PEG(8)/UFH double-layer shielding system on immune reactions and IBMIR induced by transplanted islets were evaluated in an allograft model. When pancreatic islets of Sprague-Dawley (SD) rats were transplanted in the liver of F344 rats, the mean survival time (MST) of PEG(8)/UFH double-layer shielded islets (6.8 ± 1.6 days) was statistically increased, compared to that of unmodified islets (3.6 ± 1.1 days). Furthermore, when 0.5 mg/kg of FK506 was daily administered, the MST of double-layer shielded islet (15.0 ± 2.1 days) was increased by two-fold, compared to that of unmodified islets treated with the same dose of FK506 (8.0 ± 2.4 days). Therefore, a newly developed strategy of combining the PEG(8)/UFH double-layer shielding system with FK506 would certainly be effective for preventing immune activation and IBMIR against allotransplanted islets., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The chemical conjugate of low molecular weight heparin with taurocholate (LHT7) was previously designed to offer anticancer activity while minimizing the anticoagulant activity. In the present study, we found that the systemic administration of LHT7 in nanolipoplex could substantially enhance tumor vasculature targeting and anticancer effects. Moreover, we found that co-delivery of LHT7 with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, in nanolipoplex could provide synergistic antitumor effect. LHT7/SAHA nanolipoplex was formulated by encapsulating SAHA inside cationic liposomes, followed by complexation of negatively charged LHT7 onto the cationic surfaces of SAHA-loaded liposomes (SAHA-L). LHT7/SAHA nanolipoplex was positively charged with a mean diameter of 117.6 nm, and stable in serum. The nanolipoplex form of LHT7 could alter its pharmacokinetics and biodistribution. Compared to the free form of LHT7, LHT7 in the nanolipoplex showed 1.9-fold higher mean residence time, and higher tumor vasculature accumulation after its intravenous administration. LHT7/SAHA nanolipoplex showed highest antitumor efficacy in SCC-bearing mice, compared to LHT7, SAHA-L and sequential co-administration of LHT7 and SAHA-L. Consistent with the enhanced antitumor effect, the reduction of abnormal vessels in the tumor site was also the highest in the LHT7/SAHA nanolipoplex-treated group. These results suggested the potential of LHT7/SAHA nanolipoplex for enhanced tumor vasculature targeting, and the importance of nanolipoplex-mediated co-delivery with a histone deacetylase inhibitor for maximal anticancer effect., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

This study proposes a combination method of using 3,4-dihydorxy-l-phenylalanine (DOPA) conjugated heparin (heparin-DOPA) and a low dose of anti-CD154 monoclonal antibody (MR-1) treatment to improve the survival time of intrahepatic islet xenograft. To inhibit instant blood mediated inflammatory reactions, heparin-DOPA was directly grafted to the pancreatic islet surface. The surface coverage of heparin-DOPA, the viability and functionality of heparin-DOPA grafted islets were evaluated. In addition, the combined effect of grafted heparin-DOPA and a low dose of MR-1 (a T-cell targeting immunosuppressive drug) on the survival of islet was evaluated in a xenograft model. Both unmodified islets and heparin-DOPA grafted islets were completely rejected within 2 weeks after intraportal transplantation. However, when 0.1 mg/mouse of MR-1 was administered (at day 0, 2, 4, 6 of transplantation) to 11 mice that had heparin-DOPA grafted islets transplanted to, seven out of the recipients maintained normoglycemia over 60 days. Therefore, we propose that a developed combinatory immunoprotection protocol of surface modification of pancreatic islets using heparin-DOPA with a low dose of MR-1 can be effective in prolonging the survival rate of transplanted islets in a xenograft model., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Heparin, as therapeutic medications, cannot be administered orally because of its hydrophilic and high molecular weight. Here, we present a new technology to enhance the absorption of heparin in the intestine through its chemical conjugation with deoxycholic acid (DOCA) that can interact with bile acid transporter in the intestine. For the ampiphilic property and complete dissolution, the modified heparin was physically complexed with dimethylsulfoxide (DMSO). The DOCA-conjugated heparin could form nanoparticles in aqueous solution, whereas it was completely dissolved when treated with above 10% DMSO solution. Molecular dynamics computation study and two-dimensional homonulcear (1)H nuclear overhauser effect spectroscopy (NOESY) NMR spectra demonstrated that one heparin molecule was chemically conjugated with two DOCA molecules that were physically interacted with six DMSO molecules within 4 Å via hydrophobic interactions and partly via hydrogen bonding. Its therapeutic efficacy was also pharmaceutically analyzed. When the DMSO-bound DOCA-conjugated heparin was orally administered into mice, its therapeutic efficacy was enhanced according to the amount of bound DMSO. Also, after oral administration of fluorescence-labeled DMSO-bound DOCA-conjugated heparin, it was circulated in the whole body for above 2 h. However, the DOCA-conjugated heparin without DMSO binding was fast eliminated after oral absorption. This study demonstrates that the interaction of structural constraints, DOCA and DMSO, with heparin can serve as a platform technology for potential macromolecule oral delivery.

Heparin immobilization on surfaces has drawn a great deal of attention because of its potential application in enhancing blood compatibility of various biomedical devices such as catheters, grafts, and stents. Existing methods for the heparin immobilization are based on covalent linkage formation and electrostatic interaction between substrates and heparin molecules. However, complicated multistep procedures and uncontrolled desorption of heparin are limitations of these methods. In this work, we report a new heparin derivative that exhibits robust adhesion on surfaces. The derivative, called hepamine, was prepared via conjugation of dopamine, a mussel-inspired adhesive moiety, onto a heparin backbone. Immersion of poly(urethane) substrates into an aqueous solution of hepamine resulted in robust heparin coating of the poly(urethane), the most widely used polymeric material for blood-contacting medical devices. The hepamine-coated poly(urethane) substrate showed significant inhibition of blood coagulation and platelet adhesion. The use of hepamine for surface modification is advantageous for several reasons: for example, no chemical pretreatment of the substrates is necessary, and surface functionalization is a simple, one-step procedure. Thus, the heparin immobilization method described herein is an excellent alternative approach for the introduction of heparin molecules onto surfaces.

The need for an efficacious and safe oral anticoagulant that does not require monitoring has been largely unmet. Many efforts have centered on preparing orally available heparin to improve patient compliance. In this study, novel orally active heparin derivatives (LHD), i.e. low molecular weight heparin (LMWH) conjugated with deoxycholic acid (DOCA), were evaluated in vitro and in vivo for their enhancement effect of oral heparin absorption. After oral administration of 10 mg/kg of water-soluble LHD, Ws-LHD1.5 showed optimum oral efficacy and its bioavailability was about 24% in rats. The oral absorption of LHD1.5 was also enhanced by several solubilizers, among which Poloxamer 407 provided the best results. When 5 mg/kg of LHD1.5 with Poloxamer 407 was orally administered to monkeys, the maximum anti-FXa activity in plasma was 0.26 +/- 0.04 IU/mL and its bioavailability was 17.4%. In a rat thrombosis model, 5 mg/kg of orally administered LHD1.5 formulated with Poloxamer reduced thrombus formation by 63.9 +/- 16.6%, which was higher than the efficacy of clinically used enoxaparin (49.4 +/- 17.8% at 100 IU/kg, sc). Considering the oral absorption efficacy and therapeutic effect, the conjugation ratio was optimized as about 1.5 molecules of DOCA per mole of heparin. Therefore, LHD1.5 with Poloxamer 407 can be further formulated as a solid oral anticoagulant drug.

Although heparin can regulate angiogenesis, tumor growth and metastasis, its clinical application, as well as that of low-molecular heparin (LMWH), for treating cancer are limited because of heparin's anticoagulant activity and risk of hemorrhages. LMWH-taurocholate conjugates (LHT7), which have low anticoagulant activity, were synthesized. The structural property of LHT was evaluated by circular dichroism and the binding affinity of LHT7 to vascular endothelial growth factor 165 (VEGF(165)) was measured by isothermal titration calorimetry. The inhibitory effect of LHT7 on VEGF-mediated KDR (VEGF-receptor 2) phosphorylation in Human umbilical vein endothelial cells was evaluated. The VEGF(165) dependent Matrigel plug assay was performed to verify the antiangiogenic potential of LHT7 on a VEGF(165) inhibitor. Finally, tumor growth inhibition effects of LHT7 on SCC7 and the survival rate of animal models were investigated. Moreover, MDA-MB231 xenograft mouse model was additionally used to confirm the therapeutic effect of LHT7 on human breast cancer cell line. As a result, LHT7 which has 12.7% of anticoagulant activity of the original LMWH showed a peculiar polyproline-type helical structure. LHT7 binds to VEGF strongly and inhibits VEGF dependent KDR phosphorylation. The results of Matrigel plug assay proved LHT7 as a strong antiangiogenic agent inhibiting VEGF(165). Remarkably, LHT7 showed a significant tumor growth inhibition potential on SCC7 with an increased survival rate. LHT7 also delayed tumor growth in MDA-MB231 human breast cancer cell lines., (Copyright 2008 UICC.)

Purpose: We prepared tumor endothelium targeted cRGD-modified heparin derivative (cRGD-HL) by coupling heparin-lithocholic acid (HL) with cRGDyK, and evaluated inhibition effects of cRGD-HL on angiogenesis and tumor growth., Methods: To evaluate antiangiogenic activity of cRGD-HL, we performed tests on endothelial cell adhesion and migration to vitronectin, tube formation, binding affinity to purified alpha(v)beta(3) integrin, and in vivo Matrigel plug assay. The antitumor activity of cRGD-HL was also evaluated by monitoring tumor growth and microvessel formation in squamous cell carcinoma (SCC7) tumor., Results: The cRGD-HL significantly inhibited adhesion and migration of endothelial cells to vitronectin, and tubular structures of endothelial cells. Compared to cRGDyK and HL, cRGD-HL has high binding affinity to purified alpha(v)beta(3) integrin. The enhanced antiangiogenic effect of cRGD-HL was confirmed in Matrigel assay by showing the significant inhibition of bFGF-driven angiogenesis and blood vessel formation. It was thought that potent antiangiogenic effect of cRGD-HL was probably due to the interference of alpha(v)beta(3)-mediated interaction, resulting in the enhanced antitumoral activity against SCC7 tumor., Conclusion: These results demonstrated that cRGD-modified heparin derivative enhanced anti-angiotherapeutic effects against solid tumor, and therefore, it could be applied to treat various cancers and angiogenic diseases as a potent angiogenesis inhibitor.

We describe new DOC (sodium deoxycholate)-heparin nanoparticles for in vivo tumor targeting and inhibition of angiogenesis based on chemical conjugation and the enhanced permeability and retention (EPR) effect. Heparin has been used as a potent anticoagulant agent for 70 years, and has recently been found to inhibit the activity of growth factors which stimulate the smooth muscle cells around tumor. From the results, DOC and heparin were conjugated by bonding carboxyl groups of heparin with amine groups of aminated sodium deoxycholate. Larger antitumor effects of the DOC-heparin VI (8.5 mol of DOC coupled with 1.0 mol heparin) were achieved in animal studies, compared to heparin alone. We confirmed that the conjugated heparin retained its ability to inhibit binding with angiogenic factor, showing a significant decrease in endothelial tubular formation. These results provide new insights into the nontoxic anticancer drug carrier as well as the design of multifunctional bioconjugates for targeted drug delivery.

Purpose: Our previous study confirmed that heparin-deoxycholic acid conjugate (HD) had a potent antiangiogenic effect and safety to use for long-term treatment. Herein, the combined therapeutic effect of HD and doxorubicin (DOX) was evaluated against squamous cell carcinoma (SCC7) and B16F10 melanoma., Methods: The inhibitory effect of cell proliferation and cellular uptake of HD was studied in SCC7 and B16F10. The combination effects of HD and DOX were evaluated by measuring cytotoxicity and apoptosis as well as tumor growth and apoptosis in vivo against SCC7 and B16F10 tumor-bearing mice., Results: HD displayed potent inhibitory effect on SCC7 and B16F10 cell proliferation, but it showed a low cytotoxic effect. Concurrent treatment of HD and DOX displayed enhanced cytotoxic effects and apoptosis on SCC7 and B16F10. The cellular uptake of HD and DOX was affected by the collective cytotoxic effects of these two drugs: each drug suppressed the tumor growth, and their combined treatment enhanced apoptosis and collectively inhibited the tumor growth of SCC7 and B16F10 in vivo., Conclusion: These results demonstrated that HD with cytostatic and antiangiogenetic activities, enhanced the antitumor activity of DOX against SCC7 and B16F10, and the combined treatment of these two drugs might have enhanced therapeutic efficacy.

Heparin, which has been widely used as an anti-coagulant agent, has potential anti-tumor effects; in particular, low molecular weight heparin (LMWH) may inhibit tumor angiogenesis and/or metastasis with reduced toxicity. For decades, it has been known that malignant cancer cells display abnormally enhanced glucose uptake rates and overexpress glucose transporters (GLUTs) compared to normal cells. With these findings in mind, we introduced a glucose moiety to heparin for the purpose of increasing the concentration of heparin at the tumor site by targeting GLUTs. Three glucosylated heparin (GH) derivatives were prepared by conjugation of glucosamine and heparin in different mole ratios. To evaluate the potential of GH derivatives as anti-cancer drugs, their anti-coagulant activities, inhibitory effects on glucose analog uptake, cellular interactions, tumor growth inhibitory effects and sub-acute toxicities were investigated. The anti-coagulant activities of GH derivatives decreased proportionally to the degree of glucosylation. In vitro, GH derivatives inhibited HUVEC proliferation to a greater extent than heparin. GH derivatives mainly existed outside of cells and interacted with GLUTs on the cell surface, thereby inhibiting glucose uptake into cells. In vivo, GH derivatives significantly suppressed tumor growth compared to control, without systemic toxicity. Therefore, GH derivatives are proposed as potent non-toxic anti-cancer drugs.

Oral delivery of heparin will offer great advantages over injectable heparin therapy in the treatment of patients with deep vein thrombosis. Since heparin absorption in the intestine is restricted due to its physicochemical properties, we designed a bile acid derivative, cationic deoxycholylethylamine (DCEA), to be complexed with anionic low molecular weight heparin (LMWH). Complexation between LMWH and DCEA was saturated above 1:10 molar ratio and improved lipophilicity of LMWH. The LMWH/DCEA complex was completely solubilized in 80% propylene glycol solution. The oral absorption of LMWH in rats was proportional to the molar ratio of DCEA and the administered dose of complex. The C(max) values to the complex molar ratios of 1:0, 1:3, 1:5 and 1:10 were about 0.07, 0.27, 0.83, and 0.47 IU/ml, respectively, and the C(max) values to the doses of 10, 25, 50 mg/kg were 0.16, 0.44, and 0.83 IU/ml, respectively. The LMWH/DCEA complex was found to be absorbable through all regions of the small intestine of rats without causing tissue damage. This study demonstrates the feasibility of oral heparin delivery using the cationic DCEA for chronic administration in clinical trials as an effective therapy.

The inhibitory efficacies of new bile acid acylated-heparin derivative (heparin-DOCA) were evaluated on experimental lung metastasis. We evaluated the effect of heparin-DOCA on intercellular interactions including those between B16F10 and thrombin-activated platelets and TNF-alpha-activated HUVECs, and between B16F10 and immobilized mouse P-selectin. In addition, the inhibitory effects of heparin-DOCA on adhesion and invasion of B16F10 to Matrigel were studied. In an animal mouse study, the blood clot formation and the retention of red fluorescence protein (RFP)-B16F10 in lungs were assessed after heparin-DOCA and RFP-B16F10 intravenous administration. Furthermore, we investigated the anti-metastatic effect of heparin-DOCA against lung metastasis induced by B16F10 and SCC7. Heparin-DOCA inhibited intercellular interactions between B16F10 and activated platelets or activated HUVECs by blocking P- and E-selectin-mediated interactions. Moreover, it reduced adhesion and invasion of B16F10 to ECM, thereby affecting the reduction of early retention of B16F10 in the lung. Heparin-DOCA attenuated lung colony formation on the surfaces and in interior of the lung, and attenuated metastasis by B16F10 and SCC7. These results suggest that heparin-DOCA may have potentials as therapeutic agent that prevents tumor metastasis and progression.

Purpose: Anitangiogenic and apoptotic properties of a novel chemically modified heparin derivative with low anticoagulant activity were evaluated on the experimental in vitro and in vivo model., Materials and Methods: Heparin-lithocholate conjugate (HL) was initially synthesized by covalently bonding lithocholate to heparin. Folate-HL conjugate (FHL) was further synthesized by conjugating folate to HL. Antiangiogenic and apoptotic abilities of HL and FHL were characterized in vitro and in vivo experimentations., Results: Compared to unmodified heparin, both HL and FHL represented a low anticoagulant activity (38 and 28%, respectively). HL and FHL maintained antiangiogenic activity even further modification from the results of Matrigel plugs assay. FHL specifically induced apoptosis on KB cells having highly expressed folate receptor after cellular internalization. Both administered HL and FHL had similar antiangiogenic activity and inhibitory effect on tumor growth in vivo although FHL induced higher apoptosis on tumor tissues., Conclusions: In vivo tumor growth inhibition was possibly due to the decrease of vessel density and apoptotic cell death, although antiangiogenic effect of FHL seemed more actively affected on growth inhibition than apoptotic potential in vivo system. Thus, Low anticoagulant FHL having antiangiogenic and apoptotic properties would provide benefits for the development of a new class of anticancer agent.

Although drug-eluting stents (DESs) have become the most effective means of treating coronary artery disease, safety concerns regarding their thrombogenicities remain to be surmounted. Here, we report on a novel type of DES capable of preventing restenosis and thrombosis. The DES was prepared by coating a bare metal stent with echinomycin (an anti-proliferative drug) in polyurethane by a spray drying method. Hydrophobic heparinized polymer was then topcoated onto stent over echinomycin/PU layer by dipping to improve hemocompatibility. The two-layered stent was characterized regarding surface and cross-sectional morphology, drug release pattern, platelet adhesion in vitro, and restenosis in vivo. It was found that the heparin topcoat acts as a diffusion barrier that allows the controlled release of drug in a sustained manner. Also, the heparin coated layer effectively reduced platelet adhesion, indicating excellent hemocompatibility. From the animal test using pigs, it was evident that the developed DESs can minimize neointimal proliferation and thrombus formation. The devised hydrophobic heparinized polymer-coated DES effectively reduced both restenosis and thrombosis, suggesting that they have potential as tools for the treatment of coronary artery diseases.

Purpose: Chemically modified heparin-DOCA was prepared and found to have markedly lower anticoagulant activity than heparin. In the present study, we elucidated the antiangiogenic and antitumoral activities of heparin-DOCA derivative., Methods: To evaluate the antiangiogenic and antitumoral effects of heparin-DOCA, capillary-like tube formation assay, Matrigel plug assay in vivo, western blotting for FGFR phosphorylation, ERK and p38 MAPK activities, tumor growth of SCC in vivo and immunostaining of blood vessels in tumor tissues were performed., Results: Heparin-DOCA inhibited capillary-like tubular structures of endothelial cells and bFGF-induced neovascularizations in Matrigel plug assays. Signaling experiments showed that heparin-DOCA significantly inhibited angiogenesis by suppressing the phosphorylation of FGFR and its downstream signal pathways (ERK and p38 MAPK activities). The antiangiogenic activity of this heparin derivative was found to be closely associated with antitumoral activity in a mouse model. In addition, histological evaluations supported the inhibitory effect of heparin-DOCA on blood vessel formation in tumor tissues., Conclusion: Heparin-DOCA derivative exerted a significant antitumoral effect by inhibiting angiogenesis resulting from the disruption of FGF/FGFR and its downstream signal pathways, and could be applied to treat various angiogenic diseases.

A chemically modified heparin-DOCA (HD) conjugate was developed as a drug carrier for cancer therapy. HD conjugate was found to have markedly low anticoagulant activity and to form self-assembled nanoparticles in aqueous condition. We observed that HD conjugate prevented squamous cell carcinoma (SCC) and human umbilical vascular endothelial cell (HUVEC) proliferation during BrdU incorporation assays. Here, we prepared doxorubicin-loaded heparin nanoparticles by entrapping doxorubicin into the amphiphilic HD conjugate by physical interaction and characterized the properties of these nanoparticles using Dynamic Light Scattering (DLS) and Atomic Force Microscope (AFM). In this study, doxorubicin-loaded heparin nanoparticles were designed to improve the antitumor effects of nano-sized particles (range of 180 to 210 nm) at high drug-loading efficiencies in the range 64% to 96%. These doxorubicin-loaded heparin nanoparticles displayed sustained drug release patterns. It was confirmed in vivo toxicity studies that HD conjugate did not induce unexpected side effects and that DHN 20 was safer than free DOX. An in vivo study showed that HD conjugate, doxorubicin and DHN 20 (one of doxorubicin-loaded heparin nanoparticles) induced tumor volume reductions of 43%, 56% and 74%, respectively, relative to the saline treated control. These results suggest that the drug-entrapped with heparin nanoparticles might provide a novel therapy for SCC.

The use of heparin as the most potent anticoagulant for the prevention of deep vein thrombosis and pulmonary embolism is nevertheless limited, because it is available to patients only by parenteral administration. Toward overcoming this limitation in the use of heparin, we have previously developed an orally active heparin-deoxycholic acid conjugate (LMWH-DOCA) in 10% DMSO formulation. The present study evaluates the anti-thrombogenic effect of this orally active LMWH-DOCA using a venous thrombosis animal model with Sprague-Dawley rats. When 5 mg/kg of LMWH-DOCA was orally administered in rats, the maximum anti-FXa activity in plasma was 0.35 +/- 0.02, and anti-FXa activity in plasma was maintained above 0.1 IU/ml [the minimum effective anti-FXa activity for the prevention of deep venous thrombosis (DVT) and pulmonary embolism (PE)] for five hours. LMWH-DOCA (5 mg/kg, 430 IU/kg) that was orally administered reduced the thrombus formation by 56.3 +/- 19.8%;on the other hand, subcutaneously administered enoxaparin (100 IU/kg) reduced the thrombus formation by 36.4 +/- 14.5%. Also, LMWH-DOCA was effectively neutralized by protamine that was used as an antidote. Therefore, orally active LMWH-DOCA could be proposed as a new drug that is effective for the longterm prevention of DVT and PE.

Introduction: Heparin, one of the most potent anticoagulants widely used for the treatment and prevention of deep vein thrombosis (DVT) and pulmonary embolism (PE), is currently available to patients only by parental administration. In this study, we propose a new oral delivery system of heparin by conjugating it with deoxycholic acid which reformulated by adding dimethyl sulfoxide to increase its bioavailability., Materials and Methods: The chemical conjugates (LMWH-DOCA) of low molecular weight heparin (4.5 kDa) with deoxycholic acid (DOCA) were synthesized by controlling the conjugation ratio. The absorption of LMWH-DOCA after its oral administration was measured by anti-FXa assay according to the conjugation ratio of DOCA, concentration of DMSO solution and dose of LMWH-DOCA, respectively. Furthermore, the incidences of mucosal damage by LMWH-DOCA in 10% DMSO solution were evaluated using H&E staining and SEM., Results: Three kinds of LMWH-DOCA were synthesized according to the DOCA conjugation ratios of LD1, LD2 and LD3, whose anticoagulant activities were 89, 86 and 85 IU/mg, respectively, and the activity of LMWH was 97 IU/mg. LMWH-DOCA was completely dissolved in 10% DMSO solution, and its bioavailability in the oral dose was significantly increased (17.6% for LD2 in 10% DMSO solution) without causing any damages in intestinal tissues., Conclusions: The chemical conjugate of heparin and DOCA in the soluble state could be efficiently absorbed in the intestine. Therefore, we propose this system as a new strategy of oral heparin delivery for the treatment of patients who are at high risk to DVT and PE.

We validated the application of the surface plasmon resonance (SPR) technique to reliably determine adhesion of drugs to the intestinal wall using heparin-DOCA conjugates, developed to enhance the oral absorption of poorly absorbed heparin. In this study, heparin conjugates, or deoxycholyl-heparin (H-DOCA) and bisdeoxycholyl-heparin (H-bis-DOCA), were synthesized by covalently coupling the synthesized succinimido deoxycholate (DOCA-NHS) or succinimido bis-deoxycholyl-L-lysine (DOCA-bis-NHS) to amine groups of heparin, and their physicochemical and biological properties were evaluated. To mimic the duodenal and ileal surfaces, duodenal and ileal brush border membrane (BBM) vesicles isolated from Sprauge-Dawley (SD) rats were immobilized onto a biosensor chip composed of dextran derivatives with modified lipophilic residues. The adhesion of heparin conjugates on the BBM surface was evaluated by measuring the SPR response signal. The adhesion of heparin conjugates was significantly dependent on the conjugated DOCA molecules: that is, they showed higher adhesion signal on the ileal BBM surface than that on the duodenal BBM surface. In particular, the solubilized heparin conjugates in DMSO solution presented significantly increased adhesion affinity on the ileal BBM surface. The adhesion of heparin conjugates on the intestinal surfaces was successfully assayed using the surface plasmon resonance technique with the sensor chip on which BBM vesicles were immobilized.

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.

We evaluated in vivo anti-thrombogenic effects of three different heparin-coated angiographic catheters with different heparin release profiles. Three different types of heparin-coated 5 French angiographic catheters (rapidly-heparin-releasing, slowly-heparin-releasing, and heparin-adherent catheters) were prepared by coating amphiphilic or hydrophobic heparin derivatives in order to regulate heparin elution. After incubation time of 30 minutes in the arteries of dogs, the amount of thrombus deposition, measured as dry weight, was compared in 10 sets of the three heparin-coated catheters and a non-coated catheter used as control. The surface morphology of catheters after their withdrawal was studied by scanning electron microscopy. The amount of thrombus deposition on the non-coated catheter (374.6 +/- 11.6 mg, mean +/- SD) was significantly larger than those on the rapidly-heparin-releasing (52.0 +/- 12.6), slowly-heparin-releasing (70.4 +/- 23.1), and heparin-adherent catheters (103.7 +/- 39.9) ( p < 0.001, each). The thrombus weights in the three heparin-coated catheters were not statistically different. The scanning electron microscopy showed crusts on the catheter surface, which were thickest in the non-coated catheters. We concluded that the thrombus formation inside a catheter is significantly decreased if the catheter is treated with any of the three heparin-coating methods used in this study. The in vivo anti-thrombogenic effects of these coating methods are not significantly different despite their different heparin release profiles.

The heparin and deoxycholic acid conjugate (heparin-DOCA) synthesized in this study has an amphiphilic property and could be completely dissolved in a co-solvent made of 1,4-dioxane (50 v/v%), n-propanol (25 v/v%), and water (25 v/v%). A polycaprolacton (PCL)-polyethylene glycol (PEG) multiblock copolymer was used as the coating matrix; this polymer also dissolved readily in the same co-solvent. Heparin-DOCA and the PCL-PEG multiblock copolymer were mixed well to produce a transparent coated film on an angio-catheter. The bioactivity of the released heparin-DOCA was determined by evaluating platelet adhesion and fibrin formation on the coated surfaces. The release rate could be controlled by the loading amount of the drug and drug release was maintained for 12 h. The released heparin-DOCA significantly prevented platelet adhesion and fibrin formation at the surfaces, as well as in the bulk in the plasma.

Purpose: In this study, conjugates of heparin and deoxycholic acid were synthesized in order to enhance the heparin absorption in the GI tract. Oral delivery of heparin is a preferred therapy in the treatment of patients who are at high risk of deep vein thrombosis and pulmonary embolism., Methods: Several different kinds of heparin derivatives were synthesized, and their absorption in the GI tract was determined by activated partial thromboplastin time (aPTT) and factor Xa (FXa) assay. Any histological changes caused by heparin derivatives were examined by hematoxylin and eosin (H&E) stain and transmission electron microscopy (TEM)., Results: After administering heparin-DOCA orally, the clotting time in aPTT assay was increased with the increase of the coupled DOCA amount. The maximum clotting time of heparin-DOCA was 136+/-33 sec at 200 mg/kg of oral dose. This value was 7 times higher than the baseline. The absorption of heparin-cholesterol, heparin-palmitic acid, and heparin-lauric acid conjugates in the GI tract was lower than that of heparin-DOCA. Histological examination of the GI tract indicated that heparin derivatives did not cause any damage to the microvilli and the cell layer., Conclusions: DOCA coupled with heparin greatly enhanced absorption of heparin in the GI tract, and this enhancing effect was induced without changing the tissue structure of the GI wall.

Heparin is clinically administered mainly by intravenous injection because of its highly hydrophilic property. A slightly hydrophobic heparin derivative which can be dissolved in organic solvent can be widely used in polymeric devices for clinical applications. In this study, hydrophobic heparin derivatives were prepared by coupling heparin with deoxycholic acid, cholesterol, lauric acid, and palmitic acid, respectively. The hydrophobicity of these heparin derivatives depended on the feed mole ratio of heparin to hydrophobic agents, and they showed good solubility in the co-solvent of acetone and water, as well as in water alone. Also, these heparin derivatives showed high anticoagulant activity. This approach for preparing hydrophobic heparin is expected to advance the drug delivery system by further extending the applications of heparin to medical devices such as cardiopulmonary bypass circuits, heart lung oxygenators, and kidney dialyzers.

Cardiovascular diseases that result from thrombosis of critically situated blood vessels remain the leading cause of death in industrialized countries. One primary clinical treatment is dissolution of the thrombus with thrombolytic agents, plasminogen activators (PA). Activation of plasminogen by a PA agent produces plasmin that degrades fibrin. However, plasmin also degrades other circulating clotting factors. Therefore, thrombolytic therapy, which introduces systemic generation of excess plasmin, carries the risk of hemorrhage. We propose a novel approach that could lead to targeted thrombolysis without bleeding risk. The system is comprised of a protein conjugate made of two parts: a fibrin-targeting antibody (Ab) linked with anionic heparin; and a PA derivatized with cationic species. These two parts are linked via an electrostatic interaction. Because the cationic species are relatively small, the derivatized PA would retain its thrombolytic activity, but this activity would be inhibited after binding with the Ab-heparin counterpart because of the blockage of the PA's active site by these appended macromolecules. Because protamine is a clinical heparin antagonist with a much stronger affinity for heparin than the incorporated cations, it can be used safely to dissociate the modified PA from the Ab-heparin counterpart. Therefore, this approach would permit administration of a fibrin-targeting but inactive thrombolytic, and subsequently, a triggered release of the active modified PA drug in close proximity to the fibrin deposit. These features would enhance the potency and the specificity of the thrombolytic agent and alleviate the bleeding risk by avoiding systemic generation of excess plasmin. In this report, we present preliminary results demonstrating the feasibility of the approach. A cationic octapeptide, (Arg)7-Cys, was linked to urokinase (UK) using the N-succinimidyl-3-[2-pyridylidithio]propionate activation method. This UK peptide retained a significant amount of its catalytic activity, as measured by the S-2251 chromogenic assay. However, this activity was almost completely inhibited (approximately 99%) after the addition of heparin, but was fully reversed (100%) after the addition of protamine.

The authors previously reported an approach that could be used to simultaneously control both heparin and protamine induced complications during extracorporeal perfusion. The approach consists of placing a hollow fiber based bio-reactor containing immobilized protamine (defined as the "protamine bio-reactor") at the distal end of the blood perfusion circuit for extracorporeal heparin removal. Preliminary in vitro and in vivo studies have successfully demonstrated the feasibility of the proposed approach. The authors present an in vivo theoretical model for this extracorporeal heparin removal approach. This model, which consists of a two compartment model for the metabolic clearance of heparin and a plug flow reactor model for the protamine bio-reactor, can be used to optimize and assess the required size of the bio-reactor for effective clinical heparin removal. To examine the utility of such a model, 14 female mongrel dogs (six dogs were used as controls and eight dogs were used to test the bio-reactor) were included in the in vivo study. The femoral artery and femoral vein of the dog were cannulated, and the bio-reactor was attached. Heparin was given intravenously at a dose of 150 IU/kg body weight, and its activity was measured using the aPTT assay. Preliminary studies show that the experimental data fits remarkably well with the theoretical model. The model indicates that the protamine bio-reactor can remove heparin efficiently, even in the presence of competitive binding between heparin and plasma proteins. Under clinical situations, such as hemodialysis and open heart operations, a protamine bio-reactor containing approximately 45,000 hollow fibers is necessary to reduce heparin to less than 10% of its initial concentration. The time required to saturate the protamine bio-reactor with heparin is dependent upon the blood flow rate which, as predicted by the model, is 10 min for open heart surgery (flow rate = 2,000 ml/min) and 60 min for hemodialysis (flow rate = 200 ml/min). A detailed description of the in vivo model, as well as future directions envisioned in the design of a clinically useful heparin removing system are discussed.

The ability of heparin to interact with plasma proteins, in particular antithrombin III (ATIII) and thrombin, is its primary mechanism as an anticoagulant drug. Research efforts have focused on the biological activity of heparin under three conditions: in solution as a free molecule, chemically coupled directly onto a polymer surface, and coupled onto a polymer surface using hydrophilic spacer groups. Each of these conditions yields altered biological activity, presumably a result of differing binding interactions with ATIII and thrombin. In this report, insights into binding interaction of direct versus polyethylene oxide space immobilized heparin with ATIII, thrombin, and the generation of the thrombin-antithrombin complex will be presented.

The chemical immobilization of heparin onto polymeric materials through hydrophilic spacer groups was performed to improve the hemocompatibility of blood-contacting devices. Significant data have been gathered attesting to the biological activity of immobilized heparin in static in vitro studies (clotting times) and dynamic in vivo studies (thrombus formation). However, few studies have been performed to investigate the binding kinetics of spacer-immobilized heparin under flow (shear stress) with antithrombin III (ATIII) and thrombin. To help elucidate this binding mechanism, a mathematical model was developed which parallels experiments to measure protein binding and dissociation at the heparin immobilized surface under flow conditions. Heparinized tubing was prepared by chemically immobilizing a high-ATIII-affinity fraction of heparin onto the surface of poly(ethylene)-oxide grafted, poly(styrene-co-p-aminostyrene)-coated polyethylene tubing. ATIII was first bound onto the immobilized heparin, followed by the introduction of thrombin to interact with ATIII. The concentration of thrombin-ATIII complex (TAT) flowing from the tubing was determined, and the dissociation rate constants (kD) of TAT from immobilized heparin were calculated as a function of flow rate. The results indicate that the dissociation rate constant of TAT varied with flow rate, especially low flow rates, high flow rates, and turbulent flow. As the TAT complex dissociates from immobilized heparin, this "recovered" heparin is available for subsequent binding of more ATIII and thrombin. These in vitro mathematical results may help support mechanisms and hypotheses generated for the biological activity of spacer-immobilized heparin observed during long-term in vivo and ex vivo experiments.

An objective of this research is to verify the mechanism of anticoagulant activity of surface-immobilized heparin in the presence of plasma proteins. The competition and binding interaction between immobilized heparin and antithrombin III (ATIII)/thrombin have been described in vitro. However, the strong ionic character of heparin leads to its specific and nonspecific binding with many other plasma proteins. Most notably, fibronectin contains six active binding sites for heparin which may interfere with the subsequent binding of heparin with ATIII or thrombin. Heparin was covalently immobilized through polyethylene oxide (PEO) hydrophilic spacer groups onto a model surface synthesized by random copolymerization of styrene and p-aminostyrene. The binding interaction of immobilized heparin with ATIII was then determined in the presence of different fibronectin concentrations. The binding interaction was studied by first binding immobilized heparin with ATIII, followed by the introduction of fibronectin; heparin binding with fibronectin, followed by incubation with ATIII, and simultaneous incubation of surface immobilized heparin with ATIII and fibronectin. The extent of ATIII binding to heparin in each experiment was assayed using a chromogenic substrate for ATIII, S-2238. The results of this study demonstrate that the displacement of ATIII from immobilized heparin was proportional to the fibronectin concentration, and was reversible. Furthermore, the binding sequence did not play a role in the final concentration of ATIII bound to immobilized heparin.

The authors previously developed a filter device containing immobilized protamine (termed "protamine filter") that could be used to remove heparin during extracorporeal perfusion. In vivo studies involving dogs showed that the protamine filter removed more than 50% of heparin from the animals' blood circuit in less than 20 min. In addition, the use of the protamine filter did not elicit statistically significant protamine induced hemodynamic and thrombocytopenic responses. Biocompatibility of the protamine filter was also evaluated, with the focus on its effect on the coagulation cascade, the complement system, and the blood antithrombin III levels. Results showed that heparin adsorbed to the protamine coated surface retained 20% of its original activated partial thromboplastin time activity, rendering the coated surface antithrombotic. Activation of the coagulation system by the protamine coated membrane and the untreated cellulose membrane, as measured by the elevation of prothrombin fragment F1 + 2 levels, was statistically identical. The CH50 hemolytic assay showed that the protamine coated membrane produced a reduction of 1.2 +/- 0.8% of the total complement levels, as compared to 9.4 +/- 1.6% by the untreated membrane. In addition, the change in C3a des Arginine levels after 30 min of circulation was 1.5 +/- 0.2 mg/ml by the protamine filter, as compared to 2.1 +/- 0.1 mg/ml by the untreated membrane. Unlike native heparin that would bind with antithrombin, heparin adsorbed on the protamine coated surface was devoid of such activity, and produced no depletion of circulating antithrombin. Because of the limited capacity of the protamine filter, the future system is envisioned to consist of two filters; while one filter is removing heparin the other will be regenerated. With a recently developed heparin sensor, it should be possible to design a sensor directed, biofeedback, two filter heparin removal system.

The immobilization of heparin onto polymeric surfaces using a hydrophilic spacer was effective in curtailing surface induced thrombus formation. In this study, the binding kinetics of immobilized heparin with antithrombin III (ATIII) and thrombin were investigated. Low molecular weight heparin (molecular weight, 6,000 daltons) was fractionated on an ATIII affinity column, and it was immobilized onto a styrene/p-amino styrene random co-polymer surface via hydrophilic spacer groups. This polymer substrate was coated onto glass beads (diameter range, 0.088-0.105 mm). PEO (molecular weight 3,400), modified by tolylene diisocyanate, was covalently coupled as a spacer group, followed by heparin. The bioactivity of immobilized heparin was approximately 16.2%, relative to free heparin, and nearly 1:1 binding between heparin and PEO was calculated. The binding constants of immobilized heparin and ATIII, and immobilized heparin and thrombin, were 0.958 x 10(7) M-1 and 1.76 x 10(8) M-1, respectively. The immobilized heparin bound with both ATIII and thrombin, and the binding mechanism was similar to that of free heparin.