Functional properties and skin care effects of sodium trehalose sulfate.

BACKGROUND: It is known that heparinoid, a mucopolysaccharide polysulfate, is effective in improving rough skin and promoting blood circulation as medicines for diseased areas. However, heparinoid has a molecular weight of more than 5000 and cannot penetrate healthy stratum corneum. OBJECTIVE: We tested the efficacy of sulfated oligosaccharides with a molecular weight of less than 2000 on the human skin barrier function and moisturizing function. METHODS: We measured the transepidermal water loss (TEWL) of a three-dimensional human epidermis model cultured for 3 days after topical application of sulfated oligosaccharides, then observed the effects on TEWL suppression. The mRNA levels of proteins involved in intercellular lipid transport and storage in the stratum corneum, and moisture retention were measured using RT-qPCR. RESULTS: An increase in the mRNA levels of the ATP-binding cassette subfamily A member 12 (ABCA12), which transports lipids into stratum granulosum, was confirmed. Increases were also observed in the mRNA levels of filaggrin (FLG), which is involved in the generation of natural moisturizing factors, and of caspase-14, calpain-1 and bleomycin hydrolase, which are involved in the degradation of FLG. Antibody staining confirmed that the application of sodium trehalose sulfate to 3D model skin resulted in more ABCA12, ceramide, transglutaminase1, and FLG than those in controls. In a randomized, placebo-controlled, double-blind study, participants with low stratum corneum water content applied a lotion and emulsion containing sodium trehalose sulfate to their faces for 4 weeks. Sodium trehalose sulfate decreased the TEWL and increased the stratum corneum water content. CONCLUSION: These results suggest that cosmetics containing sodium trehalose sulfate act on the epidermis by increasing barrier factors and moisturizing factors, thereby ameliorating dry skin.

Heparinoid enhances the efficacy of a bactericidal agent by preventing Cutibacterium acnes biofilm formation via quorum sensing inhibition.

Cutibacterium acnes is an opportunistic pathogen in acne vulgaris. C. acnes produces autoinducer-2 (AI-2）, a signaling molecule used for communication known as quorum sensing (QS）. In C. acnes, QS reportedly upregulates biofilm formation leading to resistance against bactericidal agents. In this study, we analyzed how heparinoid affected QS and biofilm formation of the opportunistic pathogen C. acnes. We also verified whether heparinoid would suppress biofilm formation and enhance the efficacy of the bactericidal agent 4-isopropyl-3-methylphenol (IPMP) against C. acnes biofilms. We ran an AI-2 bioassay using Vibrio harveyi ATCC BBA-1121. Heparinoid exhibited inhibitory activity against AI-2 at concentrations of 0.003-0.005%, suggesting an AI-2 analog-derived or C. acnes culture supernatant-derived inhibition of the AI-2 activity. To evaluate how heparinoid suppresses biofilm formation in C. acnes, we completed a biofilm assay in 96-well plates. We also evaluated the bactericidal activity of IPMP against the C. acnes biofilm prepared with or without heparinoid. Heparinoid inhibited C. acnes biofilm formation and IPMP bactericidal efficacy increased upon heparinoid-mediated suppression of biofilm formation. In this study, we clarified that heparinoid inhibits the AI-2-mediated QS of C. acnes, thereby suppressing biofilm formation and increasing IPMP bactericidal efficacy, potentially suppressing acne vulgaris.

Chitosan/Alginate Nanoparticles for the Enhanced Oral Antithrombotic Activity of Clam Heparinoid from the Clam Coelomactra antiquata.

Chitosan/alginate nanoparticles (DG1-NPs and DG1/Cur-NPs) aiming to enhance the oral antithrombotic activity of clam heparinoid DG1 were prepared by ionotropic pre-gelation. The influence of parameters, such as the concentration of sodium alginate (SA), chitosan (CTS), CaCl2, clam heparinoid DG1, and curcumin (Cur), on the characteristics of the nanoparticles, were investigated. Results indicate that chitosan and alginate can be used as polymer matrices to encapsulate DG1, and nanoparticle characteristics depend on the preparation parameters. Nano-particles should be prepared using 0.6 mg/mL SA, 0.33 mg/mL CaCl2, 0.6 mg/mL CTS, 7.2 mg/mL DG1, and 0.24 mg/mL Cur under vigorous stirring to produce DG1-NPS and DG1/Cur-NPS with small size, high encapsulation efficiency, high loading capacity, and negative zeta potential from approximately -20 to 30 mV. Data from scanning electron microscopy, Fourier-transform infrared spectrometry, and differential scanning calorimetry analyses showed no chemical reaction between DG1, Cur, and the polymers; only physical mixing. Moreover, the drug was loaded in the amorphous phase within the nanoparticle matrix. In the acute pulmonary embolism murine model, DG1-NPs enhanced the oral antithrombotic activity of DG1, but DG1/Cur-NPs did not exhibit higher antithrombotic activity than DG1-NPs. Therefore, the chitosan/alginate nanoparticles enhanced the oral antithrombotic activity of DG1, but curcumin did not further enhance this effect.

Antithrombotic Activity of Heparinoid G2 and Its Derivatives from the Clam Coelomactra antiquata.

Clam heparinoid G2 (60.25 kDa) and its depolymerized derivatives DG1 (24.48 kDa) and DG2 (6.75 kDa) prepared from Coelomactra antiquata have been documented to have excellent fibrinolytic and anticoagulant activity. In this study, to further explore the antithrombotic activity of G2, DG1 and DG2, azure A, sheep plasma, and clot lytic rate assays were used to determine their anticoagulant and thrombolytic activity in vitro. The results indicated that the anticoagulant titer of G2 was approximately 70% that of heparin and the thrombolytic activity of DG2 was greater than G2, DG1, and heparin activities. Moreover, in a carrageenan-induced venous thrombosis model, oral administration of G2 and DG1 each at 20 mg/kg and 40 mg/kg for 7 days significantly reduced blacktail thrombus formation, increased tissue-type plasminogen activator, fibrin degradation products, and D-dimer levels, decreased von Willebrand factor and thromboxane B2 levels, and restored phylum and genus abundance changes of intestinal bacteria. DG2 had no antithrombotic effect. At 20 mg/kg, G2, DG1, and heparin had comparable antithrombotic activities, and DG1 at 40 mg/kg had more muscular antithrombotic activity than G2. Thus, DG1 could be an antithrombotic oral agent owing to its more robust antithrombotic activity and lower molecular weight.

Oversulfated dermatan sulfate and heparinoid in the starfish Lysastrosoma anthosticta: Structures and anticoagulant activity.

Crude anionic polysaccharides extracted from the Pacific starfish Lysastrosoma anthosticta were separated by anion-exchange chromatography into fractions LA-F1 and LA-F2. The main fraction LA-F1 was solvolytically desulfated giving rise to preparation LA-F1-DS with a structure of dermatan core [→3)-ß-d-GalNAc-(1→4)-α-l-IdoA-(1→]n. Reduction of LA-F1 afforded preparation LA-F1-RED composed mainly of the repeating disaccharide units →3)-ß-d-GalNAc4R-(1→4)-α-l-Ido2S3S-(1→, where R was SO3- or H. Analysis of the NMR spectra of the parent fraction LA-F1 led to determine the main component as the oversulfated dermatan sulfate LA-Derm bearing sulfate groups at O-2 and O-3 of α-l-iduronic acid, as well as at O-4 of some N-acetyl-d-galactosamine residues. The minor fraction LA-F2 contained a mixture of LA-Derm and heparinoid LA-Hep, the latter being composed of the fragments →4)-α-d-GlcNS3S6S-(1→4)-α-l-IdoA2S3S-(1→ and →4)-α-d-GlcNS3S-(1→4)-α-l-IdoA2S3S-(1→. The presence of 2,3-di-O-sulfated iduronic acid residues is very unusual both for natural dermatan sulfate and heparinoid. Preparations LA-F1, LA-F2 and LA-F1-RED demonstrated significant anticoagulant effect in vitro.

Topical Applications of a Heparinoid-Containing Product Attenuate Glucocorticoid-Induced Alterations in Epidermal Permeability Barrier in Mice.

INTRODUCTION: Either systemic or topical glucocorticoids (GCs) can cause significant adverse effects on cutaneous structure and function. Although some products and ingredients can improve GC-induced abnormalities in epidermal permeability barrier, the efficacy is moderate. Prior studies in normal mice showed that topical applications of a heparinoid-containing product, Hirudoid® cream, improve epidermal barrier function by upregulation of epidermal proliferation, expression of mRNA for epidermal differentiation, and lipid production. OBJECTIVE: The objective of this study was to assess whether topical applications of this product could prevent GC-induced changes in epidermal function in murine skin. MATERIALS AND METHODS: One group of C57BL/6J mice was treated topically with 0.05% clobetasol propionate twice daily for 6 days, while another group was treated topically with Hirudoid® cream 30 min after each application of clobetasol propionate. Untreated mice served as normal controls. Transepidermal water loss (TEWL) rates, stratum corneum hydration, and skin surface pH were measured using respective probes connected to an MPA5 physiology monitor. qPCR was used to measure the expression levels of mRNA for keratinocyte differentiation-related proteins and lipid synthetic enzymes. RESULTS: Co-applications of Hirudoid® cream with GC minimally, but significantly, increased skin thickness in comparison to GC treatment alone (p < 0.05), in parallel with increased expression levels of mRNA for PCNA in both the dermis and the epidermis. Moreover, Hirudoid® cream largely prevented GC-induced elevation in basal TEWL (p < 0.001) and delay in barrier recovery (p < 0.05), accompanied by upregulation in the expression levels of mRNA for epidermal involucrin, HMGCoA, and SPT1. However, both stratum corneum hydration and skin surface pH were comparable in the skin treated with GC alone versus GC + Hirudoid® cream. CONCLUSION: Topical heparinoid-containing product can partially prevent GC-induced alterations in some epidermal functions.

Highly sensitive real-time PCR method to identify species origin in heparinoids.

Heparinoids are the starting material for sulodexide production, a drug used as intravenous anti-coagulant, as an alternative to heparin. The origin determination in the starting material for sulodexide, heparin, and derivatives is crucial for safety (including the impact related to bovine spongiform encephalopathy) and efficacy of the final products. Therefore, European countries have decided to approve the production of heparin only from porcine intestinal mucosa. PCR (polymerase chain reaction) methods are available to evaluate the origin species of crude heparin, during heparin production process, while they lack for the same analysis in heparinoids during sulodexide manufacturing processes. Notably, two main critical issues occur during the origin determination by using PCR for heparinoid analysis: first, heparin has been known to inhibit DNA polymerase activity and, second, the DNA amounts are very low in these samples. To overcome these critical issues, our proposed method is based on two fundamental steps, the DNA concentration by glycogen treatment and DNA purification, which occur before and after DNA extraction, respectively. Finally, by applying real-time PCR, we amplify three specific DNA sequences of ruminant species (bovine, ovine, and caprine), to assess possible contamination, and one from swine, to confirm the origin species. To date, such a method is the only one that determines origin species by PCR for heparinoids that guarantee quality, safety, and traceability of heparin-derived pharmaceutical products. In conclusion, our proposed method is an alternative to nuclear magnetic resonance and ELISA methods, because real-time PCR offers significant advantages in sensitivity, specificity, and robustness. Graphical Abstract.

Prurigo nodularis as a sweat gland/duct-related disorder: resolution associated with restoration of sweating disturbance.

Little attention has been given to the involvement of sweat glands/ducts in the pathogenesis of prurigo nodularis (PN). According to recent studies, PN is likely to develop under conditions characterized by dry skin, such as atopic dermatitis (AD), suggesting a strong impact of skin dryness on PN development. No therapeutic modalities produced complete resolution of PN without exacerbations. We previously reported that increases in skin dryness by sweating disturbance could initiate the development of AD. We investigated whether sweating responses were impaired in refractory PN lesions; and, if so, we asked whether the PN lesions could resolve by restoring sweating disturbance. Using the impression mold technique, which allows an accurate quantification of individual sweat gland/duct activity, we examined basal sweating under quiescent conditions and inducible sweating responses to thermal stimulus in PN lesions and normal-appearing skin in the same patients before and after treatment with a moisturizer or topical corticosteroids. Sweating disturbance, either basal or inducible, was most profoundly detected in the "hub" structure corresponding to the center of PN papule before the treatment. This sweating disturbance was immunohistochemically associated with the leakage of sweat into the dermis. This disturbance was restored by treatment with a moisturizer. Our limitations include a relatively small patient cohort and lack of blinding. Sweating disturbance could be one of the aggravating factors of PN development. Refractory PN with low skin hydration may resolve by restoring sweating disturbance.

A topical heparinoid-containing product improves epidermal permeability barrier homeostasis in mice.

Because of the importance of epidermal functions, including stratum corneum hydration and maintenance of permeability barrier homeostasis, in the pathogenesis of a variety of cutaneous and systemic disorders, a wide range of products has been developed to improve epidermal functions. However, the underlying mechanisms whereby certain products, including heparinoid-containing product, are far little understood. In the present study, we assessed the impact of a heparinoid-containing product, Hirudoid® cream, on epidermal permeability barrier function and expression levels of a panel of epidermal mRNA related to the formation/maintenance of the permeability barrier in mouse skin. Our results showed that while the baseline levels of transepidermal water rates remained unchanged, treatment with Hirudoid® cream twice daily for 7 days significantly accelerated permeability barrier recovery and increased stratum corneum hydration. In parallel, expression levels of epidermal mRNA for certain differentiation marker-related proteins, lipid synthetic enzymes, keratinocyte proliferation and antimicrobial peptides also increased significantly. Together, these results provide the underlying mechanisms by which topical Hirudoid® cream improves epidermal permeability barrier and antimicrobial function. Because of its benefits for epidermal functions, heparinoid-containing product could be more useful in the management of skin conditions, characterized by abnormal permeability barrier and antimicrobial function.

Efficacy of heparinoid PSS in treating cardiovascular diseases and beyond-A review of 31 years clinical experiences in China.

Heparin is a life-saving drug with multiple molecular targets and mostly well known for its anticoagulant and antithrombotic pharmacological effects in treating cardiovascular diseases. All the heparin-like polysaccharides that mimic the biological activities of heparin are called heparinoids. However, heparin has no pharmacological effect if taken orally and has to be used by injection in hospital settings. Thus, heparinoids that can be taken orally are critically needed. Propylene glycol alginate sodium sulfate (PSS) is the world's first oral heparinoid used in treating cardiovascular diseases approved by Chinese Food and Drug Administration in 1987. PSS is produced by modifying partially hydrolyzed alginate, one of the most abundant marine polysaccharides isolated from brown algae, by epoxypropane esterification and by chemical sulfation. It is used for treating and preventing cardiovascular-related diseases. The low cost (US$1.29/100 tablets, ~4 tablets/day), remarkable clinical effects, and convenient oral administration make PSS an ideal long-term cardiovascular disease-prevention drug. PSS is also clinically trialed for treating diabetes and diabetes-associated complications, hepatitis, kidney, skin, and many other diseases in China. PSS is available in most drug stores in China, and millions of patients take PSS routinely during the past 31 years. The 24,089 reported clinical cases as well as the structure, preparation, clinical efficacy, adverse reactions, pharmacokinetics, pharmacodynamics, and future perspectives of PSS based on the results of peer-reviewed publications will be discussed. This review should bring the knowledge of PSS gained in China to the world to stimulate in depth academic and clinical studies of PSS and other heparinoids.

Adjustment of Conditions for Combining Oxybutynin Transdermal Patch with Heparinoid Cream in Mice by Analyzing Blood Concentrations of Oxybutynin Hydrochloride.

The combination of skin external preparation and transdermal patch is influenced by drug absorption through the skin. We investigated the effect of heparinoid cream on the transdermal absorption of oxybutynin hydrochloride using an oxybutynin transdermal patch and determined the combined effect of these medications. Normal skin and dry dorsal skin in hairless mice were treated with heparinoid cream, followed by the application of the oxybutynin transdermal patch. A blood sample was collected from the mouse tail vein and the blood concentration of oxybutynin hydrochloride was analyzed by LC-MS/MS. Transepidermal water loss, the hydration level of the stratum corneum, and the stratum corneum thickness in the dorsal skin were measured. The blood concentration and area under the curve (AUC)0→24 of oxybutynin hydrochloride increased when the 4.0-cm2 oxybutynin transdermal patch was applied 1 h after the application of the moisturizer, compared to the values without moisturizer. Normal skin and dry skin did not affect this result. As the hydration level of the stratum corneum and stratum corneum thickness increased before patch application by pre-treatment with moisturizer, it was suggested that transdermal absorption of oxybutynin hydrochloride was increased by skin hydration. The increased blood concentration of oxybutynin hydrochloride was regulated by changing the effective area of the patch and applying additional moisturizer at intervals. The pharmacokinetics of oxybutynin hydrochloride under the regulation of combination treatment was similar to that of treatment without moisturizer. These findings indicate that the application conditions of the oxybutynin transdermal patch and heparinoid cream influence the proper use of the patch.

Properties of Heparinoids Premixed with Tumor-Derived Extracellular Vesicles.

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.

Heparinoid suppresses Der p-induced IL-1ß production by inhibiting ERK and p38 MAPK pathways in keratinocytes.

Epidermal keratinocytes initiate skin inflammation by activating immune cells. The skin barrier is disrupted in atopic dermatitis (AD) and epidermal keratinocytes can be exposed to environmental stimuli, such as house dust mite (HDM) allergens. We showed previously that HDM allergens activate the NLRP3 inflammasome of keratinocytes, thereby releasing pro-inflammatory cytokines. Heparinoid is an effective moisturizer for atopic dry skin. However, a recent report showed that heparinoid treatment can improve inflammation of lichen planus. Therefore, we hypothesized that it acts on epidermal keratinocytes not only as a moisturizer, but also as a suppressant of the triggers of skin inflammation. We found that HDM allergen-induced interleukin (IL)-1ß release from keratinocytes was inhibited significantly by heparinoid pretreatment without affecting cell viability. However, heparinoid did not affect caspase-1 release, suggesting that heparinoid did not affect HDM allergen-induced inflammasome activation. Heparinoid treatment not only decreased intracellular levels of pro-IL-1ß, but also suppressed IL-1ß messenger RNA (mRNA) expression in keratinocytes. Among the intracellular signalling pathways, the activation of extracellular signal-regulated kinase and p38 pathways, which are required for IL-1ß expression in keratinocytes, was inhibited by heparinoid treatment. The inhibitory effect of heparinoid on IL-1ß mRNA expression was also confirmed with living skin equivalents. Our results demonstrated that heparinoid suppresses the initiation of keratinocyte-mediated skin inflammation.

Efficacy of heparinoid moisturizer as a prophylactic agent for radiation dermatitis following radiotherapy after breast-conserving surgery: a randomized controlled trial.

BACKGROUND: The application of heparinoid moisturizer for 2 weeks following whole-breast radiotherapy (WBRT) was previously reported to significantly increase skin water content (WC) and help improve skin dryness and desquamation. The prospective open-label, randomized trial included an exploratory arm to investigate the preventive efficacy of heparinoid moisturizer for acute radiation dermatitis (ARD). METHODS: Between April 2011 and April 2013, patients receiving WBRT were assigned (1:2:2) to receive either: moisturizer for prophylaxis (group P), moisturizer starting 2 weeks after WBRT for treatment (group M), and no moisturizer (group C). This paper presents the results of comparison between the exploratory arm and no moisturizer group. Skin WC was measured prior to WBRT, on the last day of WBRT, and 2 weeks, 4 weeks and 3 months following WBRT. Signs and symptoms were also assessed. RESULTS: Comparing two groups, WC values were significantly higher in group P until 4 weeks following WBRT. At 2 weeks following WBRT, mean WC values in group P and C were 38.5 ± 6.1 arbitrary units (a.u.) and 30.2 ± 7.8 a.u., respectively (P < 0.001). In group C, dryness was more severe at 2 and 4 weeks following WBRT and desquamation more severe until 3 months following WBRT. However, the erythema score showed no difference between the two groups. Regarding symptoms, group C pain scores on the last day of WBRT were significantly higher than in group P (P < 0.030). CONCLUSIONS: The preventive application of heparinoid moisturizer has the potential of reducing skin desquamation and dryness in patients receiving WBRT.

Evaluation of Moisturizing Effect of Heparinoid Ointment (Hirudoid Soft Ointment) Diluted by White Petrolatum (Propeto).

Steroid ointments are frequently mixed with moisturizer. It was reported that steroid ointments mixed with moisturizer increase permeability. There are only few studies done on the permeability of the moisturizer. We researched moisturizing effect of heparinoid ointment (Hirudoid Soft ointment) diluted with white petrolatum (Propeto) on the dry skin models by measuring water content of stratum. Two to four fold dilution of Hirudoid to white petrolatum resulted in a significant decrease in the moisturizing effect of the active ingredient. There was no significant difference in moisturizing effect between four times diluted mixture and white petrolatum alone. This leads to the conclusion that steroid ointment mixture with moisturizer is frequently used, but we should take more caution regarding the decrease of moisturizing effect.

New insight into the effects of heparinoids on complement inhibition by C1-inhibitor.

Complement activation is of major importance in numerous pathological conditions. Therefore, targeted complement inhibition is a promising therapeutic strategy. C1-esterase inhibitor (C1-INH) controls activation of the classical pathway (CP) and the lectin pathway (LP). However, conflicting data exist on inhibition of the alternative pathway (AP) by C1-INH. The inhibitory capacity of C1-INH for the CP is potentiated by heparin and other glycosaminoglycans, but no data exist for the LP and AP. The current study investigates the effects of C1-INH in the presence or absence of different clinically used heparinoids on the CP, LP and AP. Furthermore, the combined effects of heparinoids and C1-INH on coagulation were investigated. C1-INH, heparinoids or combinations were analysed in a dose-dependent fashion in the presence of pooled serum. Functional complement activities were measured simultaneously using the Wielisa(®) -kit. The activated partial thrombin time was determined using an automated coagulation analyser. The results showed that all three complement pathways were inhibited significantly by C1-INH or heparinoids. Next to their individual effects on complement activation, heparinoids also enhanced the inhibitory capacity of C1-INH significantly on the CP and LP. For the AP, significant potentiation of C1-INH by heparinoids was found; however, this was restricted to certain concentration ranges. At low concentrations the effect on blood coagulation by combining heparinoids with C1-INH was minimal. In conclusion, our study shows significant potentiating effects of heparinoids on the inhibition of all complement pathways by C1-INH. Therefore, their combined use is a promising and a potentially cost-effective treatment option for complement-mediated diseases.

Anticoagulant and FGF/FGFR signal activating activities of the heparinoid propylene glycol alginate sodium sulfate and its oligosaccharides.

Propylene glycol alginate sodium sulfate (PSS), prepared by chemical sulfation of alginate, has been used for treating cardiovascular diseases in China for nearly 30 years. In the current study, the PSS was hydrolyzed partially by an environment-friendly solid phase acid degradation method, and then separated by using a Bio-Gel P6 chromatographic column. Thirteen PSS oligosaccharide fractions were obtained and characterized by ESI-MS. The results of different coagulation assays showed that a high molecular weight and a higher degree of sulfation were essential for the anticoagulant activity of the PSS because the PSS oligosaccharides exhibited no detectable anticoagulant activity. In contrast, not only PSS but also certain oligosaccharides showed significant activities in stimulation of FGF1, 2, 7, 8, 9 or 10 induced cell proliferation in FGFR1c-expressing BaF3 cells. Such properties made the PSS and its oligosaccharides promising compounds in the regulation of FGF-dependent development, treatment of cancer, and wound healing processes.

Pharmacology of Heparin and Related Drugs.

Heparin has been recognized as a valuable anticoagulant and antithrombotic for several decades and is still widely used in clinical practice for a variety of indications. The anticoagulant activity of heparin is mainly attributable to the action of a specific pentasaccharide sequence that acts in concert with antithrombin, a plasma coagulation factor inhibitor. This observation has led to the development of synthetic heparin mimetics for clinical use. However, it is increasingly recognized that heparin has many other pharmacological properties, including but not limited to antiviral, anti-inflammatory, and antimetastatic actions. Many of these activities are independent of its anticoagulant activity, although the mechanisms of these other activities are currently less well defined. Nonetheless, heparin is being exploited for clinical uses beyond anticoagulation and developed for a wide range of clinical disorders. This article provides a "state of the art" review of our current understanding of the pharmacology of heparin and related drugs and an overview of the status of development of such drugs.

Heparinoids activate a protease, secreted by mucosa and tumors, via tethering supplemented by allostery.

Activation by glycosaminoglycans (GAGs) is an emerging trend among extracellular proteases important in disease. ProMMP-7, the zymogen of a matrix metalloproteinase secreted by mucosal epithelial and tumor cells, is activated at their surfaces by sulfated GAGs, but how? ProMMP-7 is activated in trans by representative heparin oligosaccharides in a length-dependent manner, with a large jump in activation at lengths of 16 monosaccharides. Imaging by atomic force microscopy visualized small complexes of proMMP-7 molecules linked by 8-mer lengths of heparinoids and extended assembles formed with 16-mer lengths of heparin. Complexes of proMMP-7 with polydisperse heparin or heparan sulfate were more diverse. Heparinoids evidently accelerate activation by tethering multiple proMMP-7 molecules together for proteolytic attack among neighbors. Removal of either the prodomain or C-terminal peptide sequence of KRSNSRKK from MMP-7 prevents formation of the long arrays induced by heparin 16-mers or heparan sulfate. The role of the C-terminus in activation assays suggests it contributes to remote, allosteric binding of GAGs. Enhancement of proteolytic velocity of MMP-by GAGs indicates them to be effectors of V-type allostery. GAGs from proteoglycans appear to assemble proMMP-7 molecules for activation, an event preceding its tumorigenic or antibacterial proteolytic activities at cell surfaces.

Modulation of protein C activation by histones, platelet factor 4, and heparinoids: new insights into activated protein C formation.

OBJECTIVE: Histones are detrimental in late sepsis. Both activated protein C (aPC) and heparin can reverse their effect. Here, we investigated whether histones can modulate aPC generation in a manner similar to another positively charged molecule, platelet factor 4, and how heparinoids (unfractionated heparin or oxygen-desulfated unfractionated heparin with marked decrease anticoagulant activity) may modulate this effect. APPROACH AND RESULTS: We measured in vitro and in vivo effects of histones, platelet factor 4, and heparinoids on aPC formation, activated partial thromboplastin time, and murine survival. In vitro, histones and platelet factor 4 both affect thrombin/thrombomodulin aPC generation following a bell-shaped curve, with a peak of >5-fold enhancement. Heparinoids shift these curves rightward. Murine aPC generation studies after infusions of histones, platelet factor 4, and heparinoids supported the in vitro data. Importantly, although unfractionated heparin and 2-O, 3-O desulfated heparin both reversed the lethality of high-dose histone infusions, only mice treated with 2-O, 3-O desulfated heparin demonstrated corrected activated partial thromboplastin times and had significant levels of aPC. CONCLUSIONS: Our data provide a new contextual model of how histones affect aPC generation, and how heparinoid therapy may be beneficial in sepsis. These studies provide new insights into the complex interactions controlling aPC formation and suggest a novel therapeutic interventional strategy.