Fibrinogen-like protein 1 (FGL1): the next immune checkpoint target.

Immune checkpoint therapy has achieved significant efficacy by blocking inhibitory pathways to release the function of T lymphocytes. In the clinic, anti-programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) monoclonal antibodies (mAbs) have progressed to first-line monotherapies in certain tumor types. However, the efficacy of anti-PD-1/PD-L1 mAbs is still limited due to toxic side effects and de novo or adaptive resistance. Moreover, other immune checkpoint target and biomarkers for therapeutic response prediction are still lacking; as a biomarker, the PD-L1 (CD274, B7-H1) expression level is not as accurate as required. Hence, it is necessary to seek more representative predictive molecules and potential target molecules for immune checkpoint therapy. Fibrinogen-like protein 1 (FGL1) is a proliferation- and metabolism-related protein secreted by the liver. Multiple studies have confirmed that FGL1 is a newly emerging checkpoint ligand of lymphocyte activation gene 3 (LAG3), emphasizing the potential of targeting FGL1/LAG3 as the next generation of immune checkpoint therapy. In this review, we summarize the substantial regulation mechanisms of FGL1 in physiological and pathological conditions, especially tumor epithelial to mesenchymal transition, immune escape and immune checkpoint blockade resistance, to provide insights for targeting FGL1 in cancer treatment.

Hypochlorite-induced oxidation of fibrinogen: Effects on its thermal denaturation and fibrin structure.

BACKGROUND: Human fibrinogen, which plays a key role in plasma haemostasis, is a highly vulnerable target for oxidants. Fibrinogen undergoes posttranslational modifications that can potentially disrupt protein structure and function. METHODS: For the first time, by differential scanning calorimetry, dynamic and elastic light scattering and confocal laser scanning microscopy, the consequences of HOCl/-OCl-induced oxidation of fibrinogen on its thermal denaturation, molecular size distribution and fibrin clot network have been explored. RESULTS: Within a wide range of HOCl/-OCl concentrations (50-300 µM), the molecular size distribution remained unimodal; however, the average size of the hydrated molecules decreased. HOCl/-OCl-induced oxidation of fibrinogen resulted in the diminished thermal stability of regions D and E. As evidenced by elastic light scattering and confocal laser scanning microscopy, HOCl/-OCl caused the formation of abnormal fibrin with a decreased diameter of individual fibres. CONCLUSIONS: The current results along with data from previous studies enable one to conclude that the effect of HOCl/-OCl-mediated oxidation on the thermal stability of region D is influenced directly by oxidative damage to the D region structure. Since the E region is not subjected to oxidative modification, its structural damage is likely to be mediated by the oxidation of other protein structures, in particular α-helical coiled-coils. GENERAL SIGNIFICANCE: The experimental findings acquired in the current study could help to elucidate the consequences of oxidative stress in vivo on damage to the structure of fibrinogen/fibrin under the action of different ROS species.

The hemostatic and thrombotic complications of liver disease.

Hepatic cirrhosis leads to numerous hematologic derangements resulting in a complex and tenuously rebalanced hemostatic milieu. The utility of common hematologic tests including the INR and aPTT in assessing hemostatic and thrombotic risk in patients with cirrhosis is limited, and consensus on transfusion thresholds and proper management of thrombotic complications continues to evolve. This review summarizes the pathophysiology of key derangements of hemostasis including those of platelets, von Willebrand factor, pro- and anticoagulation factors, and fibrin. Additionally, the pathogenesis, consequences, optimal management, and prevention of major thrombotic and bleeding complications in cirrhosis arte discussed.

Fibrinogen/AKT/Microfilament Axis Promotes Colitis by Enhancing Vascular Permeability.

BACKGROUND & AIMS: Increased vascular permeability (VP) has been indicated to play an important role in the pathogenesis of inflammatory bowel disease (IBD). However, the pathological causes of increased intestinal VP in IBD remain largely unknown. METHOD: Fibrinogen level was measured in dextran sulphate sodium (DSS)-induced colitis and patients with ulcerative colitis. Gly-Pro-Arg-Pro acetate (GPRP), an Fg inhibitor, was used to detect the effect of Fg inhibition on the pathogenesis of DSS-induced colitis, as indicated by tissue damage, cytokine release and inflammatory cell infiltration. Miles assay was used to detect vascular permeability. RESULTS: Through tandem mass tag-based quantitative proteomics, fibrinogen (Fg) was found to be upregulated in the colon of DSS-treated mice, which was consistent with increased Fg level in colon sample of patients with ulcerative colitis. Gly-Pro-Arg-Pro acetate (GPRP), an Fg inhibitor, significantly alleviated DSS-induced colitis as indicated by improvement of body weight loss and mortality. GPRP decreased colonic inflammation and VP in DSS-treated mice. In vivo, Fg enhanced VP as indicated by Miles assay, which was significantly inhibited by GRPR, AKT (serine/threonine kinase 1) inhibitors and low doses of Jasplakinolide which induced actin polymerization, while was dramatically enhanced by Cytochalasin D (an actin polymerization inhibitor). Moreover, activation of AKT was found in vessels of DSS-treated mice. In vitro, Fg induced activation of AKT and depolymerization of microfilament and promoted cell-to-cell disaggregation. Furthermore, inhibition of AKT decreased Fg-induced microfilament depolymerization. CONCLUSIONS: Our findings highlight the importance of Fg in regulating colitis by modulation of VP via activating AKT and subsequent depolymerization of microfilament and suggest Fg as an attractive target for anti-colitis treatment.

Blood, Sweat, and Fears: A Novel Mutation Associated With Anaphylaxis and Nonresponse in a Patient With Afibrinogenemia.

Congenital afibrinogenemia is a rare disorder characterized by a lack of detectable fibrinogen. The mainstay of treatment for acute bleeding episodes or perioperative management is replacement with fibrinogen concentrate or fibrinogen-containing blood products. The development of neutralizing antibodies and severe allergic reactions to fibrinogen replacement is rarely reported in afibrinogenemia patients. Here the treatment regimen is described for a 6-year-old girl with a severe allergic reaction to multiple fibrinogen-containing products who became refractory to treatment because of a presumed inhibitor to fibrinogen.

Effects of fibrinogen synthesis inhibition on vascular cognitive impairment during traumatic brain injury in mice.

Traumatic brain injury (TBI) is associated with increased blood content of fibrinogen (Fg), called hyperfibrinogenemia (HFg), which results in enhanced cerebrovascular permeability and leads to short-term memory (STM) reduction. Previously, we showed that extravasated Fg was deposited in the vasculo-astrocyte interface and was co-localized with cellular prion protein (PrPC) during mild-to-moderate TBI in mice. These effects were accompanied by neurodegeneration and STM reduction. However, there was no evidence presented that the described effects were the direct result of the HFg during TBI. We now present data indicating that inhibition of Fg synthesis can ameliorate TBI-induced cerebrovascular permeability and STM reduction. Cortical contusion injury (CCI) was induced in C57BL/6J mice. Then mice were treated with either Fg antisense oligonucleotide (Fg-ASO) or with control-ASO for two weeks. Cerebrovascular permeability to fluorescently labeled bovine serum albumin was assessed in cortical venules following evaluation of STM with memory assessement tests. Separately, brain samples were collected in order to define the expression of PrPC via Western blotting while deposition and co-localization of Fg and PrPC, as well as gene expression of inflammatory marker activating transcription factor 3 (ATF3), were characterized with real-time PCR. Results showed that inhibition of Fg synthesis with Fg-ASO reduced overexpression of AFT3, ameliorated enhanced cerebrovascular permeability, decreased expression of PrPC and Fg deposition, decreased formation of Fg-PrPC complexes in brain, and improved STM. These data provide direct evidence that a CCI-induced inflammation-mediated HFg could be a triggering mechanism involved in vascular cognitive impairment seen previously in our studies during mild-to-moderate TBI.

FGL1 regulates acquired resistance to Gefitinib by inhibiting apoptosis in non-small cell lung cancer.

BACKGROUND: This study investigated the role of fibrinogen-like protein 1 (FGL1) in regulating gefitinib resistance of PC9/GR non-small cell lung cancer (NSCLC). METHODS: The effect of different concentrations of gefitinib on cell proliferation were evaluated using the CCK-8 assay. FGL1 expression in the normal human bronchial epithelial cell line Beas-2B, as well as four lung tumor cell lines, H1975, A549, PC9, and PC9/GR, was investigated by using western blotting and qRT-PCR. FGL1 was knocked down using small interfering RNA to evaluate the effects of FGL1 on PC9 and PC9/GR. The correlation between FGL1 expression and gefitinib resistance was determined in vitro via CCK-8 and colony formation assays, and flow cytometry and in vivo via flow cytometry and immunohistochemistry. RESULTS: FGL1 expression was significantly upregulated in non-small cell lung cancer cells with EGFR mutation and higher in the gefitinib-resistant NSCLC cell line PC9/GR than in the gefitinib-sensitive NSCLC cell line PC9. Further, FGL1 expression in PC9 and PC9/GR cells increased in response to gefitinib treatment in a dose-dependent manner. Knockdown of FGL1 suppressed cell viability, reduced the gefitinib IC50 value, and enhanced apoptosis in PC9 and PC9/GR cells upon gefitinib treatment. Mouse xenograft experiments showed that FGL1 knockdown in PC9/GR tumor cells enhanced the inhibitory and apoptosis-inducing actions of gefitinib. The potential mechanism of gefitinib in inducing apoptosis of PC9/GR cells involves inhibition of PARP1 and caspase 3 expression via suppression of FGL1. CONCLUSIONS: FGL1 confers gefitinib resistance in the NSCLC cell line PC9/GR by regulating the PARP1/caspase 3 pathway. Hence, FGL1 is a potential therapeutic target to improve the treatment response of NSCLC patients with acquired resistance to gefitinib.

Targeting fibrinogen-like protein 1 is a novel therapeutic strategy to combat obesity.

Fibrinogen-like-protein 1 (FGL1) is a novel hepatokine that plays an important role in hepatic steatosis and insulin resistance. Although FGL1 expression can be detected in adipose tissues, the functions of FGL1 in adipose tissues are still unknown. In this study, 356 participants with (body mass index (BMI) ≥25 kg/m2 ; n = 134) or without obesity (BMI <25 kg/m2 ; n = 222) were recruited, and we found that the plasma FGL1 concentrations were significantly higher in obese group than those of in the normal weight group, and were positively correlated with age, BMI, waist circumference, fat content, plasma glucose at 2 hours during an oral glucose tolerance test, and the insulin sensitivity index. In univariate analyses, BMI, waist circumference, total fat, visceral fat, and subcutaneous fat areas were positively correlated with FGL1 levels. After adjusting for age and gender, obesity indices, including the BMI and different fat areas, remained significantly associated with FGL1 levels. In order to investigate the causal relationship between FGL1 and obesity, animal and cell models were used. Overexpression of FGL1 in epididymal adipose tissue by lentiviral vector encoding FGL1 increased the fat pad size, whereas FGL1-knockdown by lentiviral vector encoding short-hairpin RNA targeted to FGL1 decreased high-fat diet-induced adiposity. In addition, 3T3-L1 adipocytes were used to clarify the possible mechanism of FGL1-induced adipogenesis. FGL1 induced adipogenesis through an ERK1/2-C/EBPß-dependent pathway in 3T3-L1 adipocytes. These findings highlight the pathophysiological role of FGL1 in obesity, and FGL1 might be a novel therapeutic target to combat obesity.

Soluble fibrinogen-like protein 2 promotes the growth of hepatocellular carcinoma via attenuating dendritic cell-mediated cytotoxic T cell activity.

BACKGROUND: Soluble fibrinogen-like protein 2 (sFGL2), a secretory protein expressed by regulatory T cells (Tregs) with immunosuppressive activity, is highly expressed in both the peripheral blood and tumor tissue of patients with hepatocellular carcinoma (HCC); however, sFGL2 function in HCC remains largely unknown. Here, we elucidated the potential role of sFGL2 in HCC progression. METHODS: T cells, dendritic cells (DCs), and related cytokines in the tumor microenvironment were comparatively analyzed in BALB/c and C57BL/6 mice bearing transplanted hepatomas harboring Fgl2-knockout or receiving sFGL2-antibody treatment. Additionally, the effects of sFGL2 on DCs and T cells were evaluated in vivo and ex vivo. RESULTS: The growth of both subcutaneously and orthotopically transplanted hepatomas was inhibited in Fgl2-knockout mice and those treated with the sFGL2 antibody, respectively, as compared with controls. Moreover, sFGL2 depletion enhanced the proportion and cytotoxicity of cytotoxic T cells, promoted DC maturation, and improved DC activity to proliferate T cells in the tumor microenvironment. Furthermore, we detected lower levels of interleukin (IL)-35 in both types of transplanted hepatomas and higher level of IL-6 in orthotopically transplanted hepatomas following sFGL2 depletion. Mechanistically, we found that sFGL2 impaired bone-marrow-derived DC (BMDCs) function by inhibiting phosphorylation of Akt, nuclear factor-kappaB, cAMP response element binding protein, and p38 and downregulating the expression of major histocompatibility complex II, CD40, CD80, CD86, and CD83 on BMDCs in vitro. CONCLUSIONS: Our data suggest that sFGL2 promotes hepatoma growth by attenuating DC activity and subsequent CD8+ T cell cytotoxicity, suggesting sFGL2 as a novel potential therapeutic target for HCC treatment.

An anticoagulant peptide from beta-casein: identification, structure and molecular mechanism.

Various bioactive peptides are identified from casein hydrolysates. YQEPVLGPVR (PICA), a novel antithrombotic peptide derived from beta-casein (fragment 193-202), was identified by high-performance liquid chromatography - liquid chromatography-mass spectrometry/mass spectrometry. The anticoagulation activity assay showed that this peptide has a strong anticoagulant activity. It was proved that the peptide did not interact with the active site of thrombin to inhibit thrombin, and that it inhibited thrombin activity by binding the exosite-1 of thrombin, which was also confirmed by the fibrinogen clotting time assay. It was shown that PICA prolonged fibrinogen clotting time in a dose-dependent manner. Secondary structures of the thrombin-PICA complex were also measured by circular dichroism to prove that PICA can combine with thrombin. Moreover, Discovery Studio 2017 R2 software was used for molecular docking to provide the potential mechanism for the antithrombotic activity of the peptide. These results suggested that PICA probably can be used as an antithrombotic ingredient in the functional food industry.

Anticoagulant activity of two novel polysaccharides from flowers of Apocynum venetum L.

Two novel polysaccharides were obtained from flowers of Apocynum venetum L., and named as Vp2a-II and Vp3. Their average molecular weights were 7 kDa and 9 kDa, respectively. Methods of monosaccharide analysis, acid hydrolysis and methylation analysis, Fourier transform-infrared spectroscopy (FT-IR), gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy were used to identify the structure of Vp2a-II and Vp3. Vp2a-II was composed of →6)-ß-d-Glcp-(1 → 6)-α-d-Galp-(1→ residues. Vp3 was composed of α-d-GlcpA-(3 → α-d-GalpA residues. The anticoagulant activity was evaluated by activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), and fibrinogen (FIB) assays in vitro. Results indicated that Vp3 exhibited the anticoagulant activity.

Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration.

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer's disease (AD). However, the mechanisms that link disruption of the blood-brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377-395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.

Fibrinogen Activates BMP Signaling in Oligodendrocyte Progenitor Cells and Inhibits Remyelination after Vascular Damage.

Blood-brain barrier (BBB) disruption alters the composition of the brain microenvironment by allowing blood proteins into the CNS. However, whether blood-derived molecules serve as extrinsic inhibitors of remyelination is unknown. Here we show that the coagulation factor fibrinogen activates the bone morphogenetic protein (BMP) signaling pathway in oligodendrocyte progenitor cells (OPCs) and suppresses remyelination. Fibrinogen induces phosphorylation of Smad 1/5/8 and inhibits OPC differentiation into myelinating oligodendrocytes (OLs) while promoting an astrocytic fate in vitro. Fibrinogen effects are rescued by BMP type I receptor inhibition using dorsomorphin homolog 1 (DMH1) or CRISPR/Cas9 activin A receptor type I (ACVR1) knockout in OPCs. Fibrinogen and the BMP target Id2 are increased in demyelinated multiple sclerosis (MS) lesions. Therapeutic depletion of fibrinogen decreases BMP signaling and enhances remyelination in vivo. Targeting fibrinogen may be an upstream therapeutic strategy to promote the regenerative potential of CNS progenitors in diseases with remyelination failure.

Spatholobus suberectus Dunn. constituents inhibit sortase A and Staphylococcus aureus cell clumping to fibrinogen.

Sortases are a family of Gram-positive transpeptidases responsible for anchoring surface protein virulence factors to the peptidoglycan cell wall layer. In Staphylococcus aureus (S. aureus), deletion of sortase isoform results in a significant reduction in virulence and infection potential. Twenty flavonoids were isolated from the stem of the folk medicinal plant Spatholobus suberectus Dunn. These compounds were tested against S. aureus-derived sortase A (SrtA), a key transpeptidase for bacterial virulence. Among these active flavonoids, 7-hydroxy-6-methoxy-flavanone (3) and formononetin (10) were identified as compounds with promising SrtA inhibitory activity. These compounds also exhibited inhibitory activity against S. aureus cell clumping to fibrinogen. The suppression of cell-clumping activity indicates the potential of these compounds in the treatment of S. aureus infections via the inhibition of SrtA.

Characterisation of a novel, high affinity and selective αvß6 integrin RGD-mimetic radioligand.

The alpha-v beta-6 (αvß6) integrin has been identified as playing a key role in the activation of transforming growth factor-ß (TGFß) that is hypothesised to be pivotal in the development of cancer and fibrotic diseases. Therefore, the αvß6 integrin is an attractive therapeutic target for these debilitating diseases and a drug discovery programme to identify small molecule αvß6 selective arginyl-glycinyl-aspartic acid (RGD)-mimetics was initiated within GlaxoSmithKline. The primary aim of this study was to pharmacologically characterise the binding to αvß6 of a novel clinical candidate, compound 1, using a radiolabelled form. Radioligand binding studies were completed with [(3)H]compound 1 against the human and mouse soluble protein forms of αvß6 to determine accurate affinity estimates and binding kinetics. The selectivity of compound 1 for the RGD integrin family was also determined using saturation binding studies (αvß1, αvß3, αvß5, αvß8, α5ß1 and α8ß1 integrins) and fibrinogen-induced platelet aggregation (αIIbß3 integrin). In addition, the relationship between divalent metal cation type and concentration and αvß6 RGD site binding was also investigated. Compound 1 has been demonstrated to bind with extremely high affinity and selectivity for the αvß6 integrin and has the potential as a clinical tool and therapeutic for investigating the role of αvß6 in a range of disease states both pre-clinically and clinically. In addition, this is the first study that has successfully applied radioligand binding to the RGD integrin field to accurately determine the affinity and selectivity profile of a small molecule RGD-mimetic.

Neprilysin Inhibits Coagulation through Proteolytic Inactivation of Fibrinogen.

Neprilysin (NEP) is an endogenous protease that degrades a wide range of peptides including amyloid beta (Aß), the main pathological component of Alzheimer's disease (AD). We have engineered NEP as a potential therapeutic for AD but found in pre-clinical safety testing that this variant increased prothrombin time (PT) and activated partial thromboplastin time (APTT). The objective of the current study was to investigate the effect of wild type NEP and the engineered variant on coagulation and define the mechanism by which this effect is mediated. PT and APTT were measured in cynomolgus monkeys and rats dosed with a human serum albumin fusion with an engineered variant of NEP (HSA-NEPv) as well as in control plasma spiked with wild type or variant enzyme. The coagulation factor targeted by NEP was determined using in vitro prothrombinase, calibrated automated thrombogram (CAT) and fibrin formation assays as well as N-terminal sequencing of fibrinogen treated with the enzyme. We demonstrate that HSA-NEP wild type and HSA-NEPv unexpectedly impaired coagulation, increasing PT and APTT in plasma samples and abolishing fibrin formation from fibrinogen. This effect was mediated through cleavage of the N-termini of the Aα- and Bß-chains of fibrinogen thereby significantly impairing initiation of fibrin formation by thrombin. Fibrinogen has therefore been identified for the first time as a substrate for NEP wild type suggesting that the enzyme may have a role in regulating fibrin formation. Reductions in NEP levels observed in AD and cerebral amyloid angiopathy may contribute to neurovascular degeneration observed in these conditions.

Nanoscale Plasma Coating Inhibits Formation of Staphylococcus aureus Biofilm.

Staphylococcus aureus commonly infects medical implants or devices, with devastating consequences for the patient. The infection begins with bacterial attachment to the device, followed by bacterial multiplication over the surface of the device, generating an adherent sheet of bacteria known as a biofilm. Biofilms resist antimicrobial therapy and promote persistent infection, making management difficult to futile. Infections might be prevented by engineering the surface of the device to discourage bacterial attachment and multiplication; however, progress in this area has been limited. We have developed a novel nanoscale plasma coating technology to inhibit the formation of Staphylococcus aureus biofilms. We used monomeric trimethylsilane (TMS) and oxygen to coat the surfaces of silicone rubber, a material often used in the fabrication of implantable medical devices. By quantitative and qualitative analysis, the TMS/O2 coating significantly decreased the in vitro formation of S. aureus biofilms; it also significantly decreased in vivo biofilm formation in a mouse model of foreign-body infection. Further analysis demonstrated TMS/O2 coating significantly changed the protein adsorption, which could lead to reduced bacterial adhesion and biofilm formation. These results suggest that TMS/O2 coating can be used to effectively prevent medical implant-related infections.

Targeted disruption of fibrinogen like protein-1 accelerates hepatocellular carcinoma development.

Fibrinogen like protein-1 (Fgl1) is a predominantly liver expressed protein that has been implicated as both a hepatoprotectant and a hepatocyte mitogen. Fgl1 expression is decreased in hepatocellular carcinoma (HCC) and its loss correlates with a poorly differentiated phenotype. To better elucidate the role of Fgl1 in hepatocarcinogenesis, we treated mice wild type or null for Fgl1 with diethyl nitrosamine and monitored for incidence of hepatocellular cancer. We find that mice lacking Fgl1 develop HCC at more than twice the rate of wild type mice. We show that hepatocellular cancers from Fgl1 null mice are molecularly distinct from those of the wild type mice. In tumors from Fgl1 null mice there is enhanced activation of Akt and downstream targets of the mammalian target of rapamycin (mTOR). In addition, there is paradoxical up regulation of putative hepatocellular cancer tumor suppressors; tripartite motif-containing protein 35 (Trim35) and tumor necrosis factor super family 10b (Tnfrsf10b). Taken together, these findings suggest that Fgl1 acts as a tumor suppressor in hepatocellular cancer through an Akt dependent mechanism and supports its role as a potential therapeutic target in HCC.

Analysis of the binding of bovine and human fibrinogen to ferritin: evidence that fibrinogen is a common ferritin-binding protein in mammals.

Both human and horse fibrinogen are heme-binding proteins, and horse fibrinogen also exhibits heme-mediated ferritin binding. This study found that bovine and human fibrinogen are heme-mediated ferritin-binding proteins and demonstrated direct binding of bovine ferritin to protoporphyrin (PPIX) and its derivatives or to Zn ions. Binding of bovine and human fibrinogen to bovine spleen ferritin coated on microtiter plate wells was detected using an anti-human fibrinogen antibody, and this binding was inhibited in a dose-dependent manner by hemin (iron-PPIX) and also inhibited by Zn-PPIX. PPIX showed less of an inhibitory effect on the binding of bovine and human fibrinogen to bovine ferritin. The inhibitory effect of Sn-PPIX was similar to that of PPIX, but with respect to human fibrinogen, PPIX did not inhibit the binding of human fibrinogen to ferritin. Bovine fibrinogen immobilized on CNBr-activated Sepharose 4B beads showed affinity for hemin, Sn-PPIX, Zn-PPIX, and iron-free PPIX in the order Sn-PPIX < iron-free PPIX < hemin < Zn-PPIX. The fibrinogen beads also directly bound to zinc ions. These results suggest that bovine fibrinogen is a heme- and zinc-binding protein and that binding of circulating mammalian fibrinogen to ferritin is heme mediated.

Spirulan from blue-green algae inhibits fibrin and blood clots: its potent antithrombotic effects.

We investigated in vitro and in vivo fibrinolytic and antithrombotic activity of spirulan and analyzed its partial biochemical properties. Spirulan, a sulfated polysaccharide from the blue-green alga Arthrospira platensis, exhibits antithrombotic potency. Spirulan showed a strong fibrin zymogram lysis band corresponding to its molecular mass. It specifically cleaved Aα and Bß, the major chains of fibrinogen. Spirulan directly decreased the activity of thrombin and factor X activated (FXa), procoagulant proteins. In vitro assays using human fibrin and mouse blood clots showed fibrinolytic and hemolytic activities of spirulan. Spirulan (2 mg/kg) showed antithrombotic effects in the ferric chloride (FeCl3 )-induced carotid arterial thrombus model and collagen and epinephrine-induced pulmonary thromboembolism mouse model. These results may be attributable to the prevention of thrombus formation and partial lysis of thrombus. Therefore, we suggest that spirulan may be a potential antithrombotic agent for thrombosis-related diseases.