ArticleLiterature Review

Role of heparin and non heparin binding serpins in coagulation and angiogenesis: A complex interplay

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Abstract

Pro-coagulant, anti-coagulant and fibrinolytic pathways are responsible for maintaining haemostatic balance under physiological conditions. Any deviation from these pathways would result in hypercoagulability leading to life threatening diseases like myocardial infarction, stroke, portal vein thrombosis, deep vein thrombosis (DVT) and pulmonary embolism (PE). Angiogenesis is the process of sprouting of new blood vessels from pre-existing ones and plays a critical role in vascular repair, diabetic retinopathy, chronic inflammation and cancer progression. Serpins; a superfamily of protease inhibitors, play a key role in regulating both angiogenesis and coagulation. They are characterized by the presence of highly conserved secondary structure comprising of 3 β-sheets and 8–9 α-helices. Inhibitory role of serpins is modulated by binding to cofactors, specially heparin and heparan sulfate proteoglycans (HSPGs) present on cell surfaces and extracellular matrix. Heparin and HSPGs are the mainstay of anti-coagulant therapy and also have therapeutic potential as anti-angiogenic inhibitors. Many of the heparin binding serpins that regulate coagulation cascade are also potent inhibitors of angiogenesis. Understanding the molecular mechanism of the switch between their specific anti-coagulant and anti-angiogenic role during inflammation, stress and regular haemostasis is important. In this review, we have tried to integrate the role of different serpins, their interaction with cofactors and their interplay in regulating coagulation and angiogenesis.

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... The activity of both tPA and uPA is regulated by specific plasminogen activator inhibitors (PAIs) of which the principal is PAI type 1 (PAI-1), a member of the serine protease inhibitor superfamily (SERPINS) (74). Tight connection of fibrinolysis with coagulation is further provided by thrombin, which enhances fibrinolysis that induces the expression and activity of tPA, and causes inactivation of PAI-1 by forming a complex with it. ...
... Antithrombin (AT, previously also called antithrombin III) belongs to the family of SERPINS and it inhibits several activated coagulation factors: FVIIa in complex with TF, FXa that dissociates from the TF-bearing cell (74) and, as the name suggests, thrombin via the formation of the thrombinantithrombin (TAT) complex. Binding of cofactor heparin and heparin-like molecules are required for achieving inhibitory rates of AT. ...
... Other noteworthy inhibitors from the SERPINS family are heparin cofactor II (HCII), C1 inhibitor (C1INH) and protein C inhibitor (PCI). The inhibitory role of SERPINS is modulated by binding to cofactors, especially glycosaminoglycans like heparin, which present on cell surfaces and on the extracellular matrix (74,106). HCII acts similarly to AT in the negative regulation of thrombin (107). ...
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Significant progress has been made in understanding the complex interactions between the coagulation system and inflammation and autoimmunity. Increased blood-brain-barrier (BBB) permeability, a key event in the pathophysiology of multiple sclerosis (MS), leads to the irruption into the central nervous system of blood components that include virtually all coagulation/hemostasis factors. Besides their cytotoxic deposition and role as a possible trigger of the coagulation cascade, hemostasis components cause inflammatory response and immune activation, sustaining neurodegenerative events in MS. Early studies showing the contribution of altered hemostasis in the complex pathophysiology of MS have been strengthened by recent studies using methodologies that permitted deeper investigation. Fibrin(ogen), an abundant protein in plasma, has been identified as a key contributor to neuroinflammation. Perturbed fibrinolysis was found to be a hallmark of progressive MS with abundant cortical fibrin(ogen) deposition. The immune-modulatory function of the intrinsic coagulation pathway still remains to be elucidated in MS. New molecular details in key hemostasis components participating in MS pathophysiology, and particularly involved in inflammatory and immune responses, could favor the development of novel therapeutic targets to ameliorate the evolution of MS. This review article introduces essential information on coagulation factors, inhibitors, and the fibrinolytic pathway, and highlights key aspects of their involvement in the immune system and inflammatory response. It discusses how hemostasis components are (dys)regulated in MS, and summarizes histopathological post-mortem human brain evidence, as well as cerebrospinal fluid, plasma, and serum studies of hemostasis and fibrinolytic pathways in MS. Studies of disease-modifying treatments as potential modifiers of coagulation factor levels, and case reports of autoimmunity affecting hemostasis in MS are also discussed.
... Especially since MDMs are known to express low levels of CD4 compared to HSPGs, the initial attachment of HIV-1 to HSPGs may represent a crucial rate limiting step in HIV-1 entry [57,58]. SERPIN C1 and SLPI are also known to bind to the syndecan family of HSPGs [61][62][63]. Thus, taken together, it is likely that SERPIN C1 or SLPI pretreatment interferes with the initial attachment of HIV-1 to HSPGs and inhibits entry into MDMs. ...
... Especially since MDMs are known to express low levels of CD4 compared to HSPGs, the initial attachment of HIV-1 to HSPGs may represent a crucial rate limiting step in HIV-1 entry [57,58]. SERPIN C1 and SLPI are also known to bind to the syndecan family of HSPGs [61][62][63]. Thus, taken together, it is likely that SERPIN C1 or SLPI pre-treatment interferes with the initial attachment of HIV-1 to HSPGs and inhibits entry into MDMs. ...
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The impact of steroid hormones estrogen and progesterone on human immunodeficiency virus type 1 (HIV-1) replication is well documented. However, the exact mechanism involved in the regulation of HIV-1 replication by estrogen and progesterone is still unclear. In the present study, we wanted to elucidate the molecular mechanisms underlying the modulation of HIV-1 replication by estrogen and progesterone. To achieve this goal, we used real-time quantitative PCR arrays (PCR arrays) to identify differentially expressed host genes in response to hormone treatments that are involved in antiviral responses. Our in vitro results suggest that treatment with high doses of estrogen and progesterone promotes the expression of host antiviral factors Secretory leukocyte protease inhibitor (SLPI) and Serpin family C member 1 (SERPIN C1) among others produced in response to HIV-1 infection. SLPI is an enzyme that inhibits human leukocyte elastase, human cathepsin G, human trypsin, neutrophil elastase, and mast cell chymase. SERPIN C1 is a plasma protease inhibitor that regulates the blood coagulation cascade by the inhibition of thrombin and other activated serine proteases of the coagulation system. A dose dependent downmodulation of HIV-1 replication was observed in monocyte-derived macrophages (MDMs) pre-treated with the two proteins SLPI and SERPIN C1. Further investigations suggests that the host antiviral factors, SLPI and SERPIN C1 act at the pre-integration stage, inhibiting HIV-1 viral entry and leading to the observed downmodulation of HIV-1 replication. Our studies would help identify molecular mechanisms and pathways involved in HIV-1 pathogenesis.
... Furthermore, Bhakuni et al. (2016) and Rema et al. (2003) reported that molecular weight of heparin plays an important role on its angiogenic activity [18,19]. According to Rema et al. (2003) and Collen et al. (2000), low molecular weight heparin (LMWH) has shown to inhibit angiogenesis compared to high molecular weight heparin (HMWH) on a chorioallantoic membrane assay (CAM assay) [19,20]. ...
... Furthermore, Bhakuni et al. (2016) and Rema et al. (2003) reported that molecular weight of heparin plays an important role on its angiogenic activity [18,19]. According to Rema et al. (2003) and Collen et al. (2000), low molecular weight heparin (LMWH) has shown to inhibit angiogenesis compared to high molecular weight heparin (HMWH) on a chorioallantoic membrane assay (CAM assay) [19,20]. ...
Article
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Formation of blood vessels during bone regeneration represents a major challenge for tissue engineered constructs. Poor revascularization can lead to scaffold failure and consequently, leads to non-healing fracture. Heparin is known to bind with angiogenic growth factors influencing the process of new blood vessels formation. There are several problems associated with the use of growth factors in clinic such as low stability, uncontrolled delivery to the site, and high price. The aim of the present study was to explore the potential of heparin to produce pro-angiogenic bone regeneration materials. Chitosan/hydroxyapatite freeze-gelled scaffolds were prepared and loaded with heparin. Different concentrations of heparin were successfully loaded onto the scaffolds, its release from the scaffold was analysed by toluidine blue assay and their angiogenic effect was evaluated by chorioallantoic membrane (CAM) assay to determine the optimal concentration of heparin to induce a proangiogenic effect. It was noted that low heparin concentrations exhibited a positive effect, with approximately 28 μg per scaffold indicating a significant increment in blood vessels. The synthesized materials showed no cytotoxic effects when evaluated by using U2OS cell line.
... Furthermore, Bhakuni et al. (2016) and Rema et al. (2003) reported that molecular weight of heparin plays an important role on its angiogenic activity 18,19 . According to Rema et al. (2003) and Collen et al. (2000), low molecular weight heparin (LMWH) has shown to inhibit angiogenesis compared to high molecular weight heparin (HMWH) on a chorioallantoic membrane assay (CAM assay) 19,20 . ...
... Furthermore, Bhakuni et al. (2016) and Rema et al. (2003) reported that molecular weight of heparin plays an important role on its angiogenic activity 18,19 . According to Rema et al. (2003) and Collen et al. (2000), low molecular weight heparin (LMWH) has shown to inhibit angiogenesis compared to high molecular weight heparin (HMWH) on a chorioallantoic membrane assay (CAM assay) 19,20 . ...
Preprint
Full-text available
Formation of blood vessels during bone regeneration represents a major challenge for tissue engineered constructs. Poor revascularization can lead to scaffold failure and consequently, leads to non-healing fracture. Heparin is known to bind with angiogenic growth factors influencing the process of new blood vessels formation. There are several problems associated with the use of growth factors in clinic such as low stability, controlled delivery to the site, and price. The aim of the present study was to explore the potential of heparin to produce pro-angiogenic bone regeneration materials. Chitosan/hydroxyapatite freeze-gelled scaffolds were prepared and loaded with heparin. Different concentrations of heparin were successfully loaded onto the scaffolds, its release from the scaffold was analysed by toluidine blue assay and their angiogenic effect was evaluated by chorioallantoic membrane (CAM) assay to determine the optimal concentration of heparin to induce a proangiogenic effect. It was noted that low heparin concentrations exhibited a positive effect, with approximately 28μg per scaffold indicating a significant increment in blood vessels. The synthesized materials showed no cytotoxic effects when evaluated by using U2OS cell line.
... Furthermore, Bhakuni et al. (2016) and Rema et al. (2003) reported that molecular weight of heparin plays an important role on its angiogenic activity 18,19 . According to Rema et al. (2003) and Collen et al. (2000), low molecular weight heparin (LMWH) has shown to inhibit angiogenesis compared to high molecular weight heparin (HMWH) on a chorioallantoic membrane assay (CAM assay) 19,20 . ...
... Furthermore, Bhakuni et al. (2016) and Rema et al. (2003) reported that molecular weight of heparin plays an important role on its angiogenic activity 18,19 . According to Rema et al. (2003) and Collen et al. (2000), low molecular weight heparin (LMWH) has shown to inhibit angiogenesis compared to high molecular weight heparin (HMWH) on a chorioallantoic membrane assay (CAM assay) 19,20 . ...
Preprint
Full-text available
Formation of blood vessels during bone regeneration represents a major challenge for tissue engineered constructs. Poor revascularization can lead to scaffold failure and consequently, leads to non-healing fracture. Heparin is known to bind with angiogenic growth factors influencing the process of new blood vessels formation. There are several problems associated with the use of growth factors in clinic such as low stability, controlled delivery to the site, and price. The aim of the present study was to explore the potential of heparin to produce pro-angiogenic bone regeneration materials. Chitosan/hydroxyapatite freeze-gelled scaffolds were prepared and loaded with heparin. Different concentrations of heparin were successfully loaded onto the scaffolds, its release from the scaffold was analysed by toluidine blue assay and their angiogenic effect was evaluated by chorioallantoic membrane (CAM) assay to determine the optimal concentration of heparin to induce a proangiogenic effect. It was noted that low heparin concentrations exhibited a positive effect, with approximately 28 µg per scaffold indicating a significant increment in blood vessels. The synthesized materials showed no cytotoxic effects when evaluated by using U2OS cell line.
... Heparin is unable to exert coronary arteriogenic properties but potentiates the activity of growth factors released by exercise and ischaemia in CAD patients. 16,17 Fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGR) and adenosine, among others, are recognized to be a mitogen for the three principal vascular cell types (endothelial cells, smooth muscle cells, and fibroblasts). All are released by ischaemia during exercise and are stabilized or potentiated by heparin, as shown by experimental studies for FGF, 18 VEGF, [16][17][18] and adenosine. ...
... 16,17 Fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGR) and adenosine, among others, are recognized to be a mitogen for the three principal vascular cell types (endothelial cells, smooth muscle cells, and fibroblasts). All are released by ischaemia during exercise and are stabilized or potentiated by heparin, as shown by experimental studies for FGF, 18 VEGF, [16][17][18] and adenosine. 20 In particular, circulating levels of HGF are dramatically increased by an intravenous injection of heparin. ...
Article
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Background: Coronary collateral circulation exerts protective effects on myocardial ischaemia due to coronary artery disease and can be promoted by exercise with heparin co-administration. Whether this arteriogenetic effect is accompanied by functional improvement of left ventricle during stress and lessening of angina symptoms remains unknown. Aims: To evaluate the anti-ischaemic efficacy of heparin plus exercise in coronary artery disease. Methods: In a prospective, single-centre, randomized, double-blind study we recruited 32 'no-option' patients (27 males; mean age 61 ± 8 years) with stable angina, exercise-induced ischaemia and coronary artery disease not suitable for revascularization. All underwent a two-week cycle of exercise (two exercise sessions per day, five days per week) and were randomized (n = 16 per group) to intravenous placebo (0.9% saline) versus unfractionated heparin (5.000 IU intravenously), 10 min prior to exercise. We assessed Canadian Cardiovascular Society angina class, stress electrocardiogram and echo parameters (wall motion score index) and computed tomography angiography for collaterals. Results: After two-week cycle, Canadian Cardiovascular Society class statistically decreased in both groups (heparin plus exercise group: 2.6 ± 0.7 to 1.9 ± 0.7, p < 0.001, exercise group: 2.4 ± 0.7 to 2.1 ± 0.9, p = 0.046). Only the heparin plus exercise group improved time-to-ST segment depression (before 270, 228-327 s vs. after 339, 280-360 s, p = 0.012) and wall motion score index (before 1.38 ± 0.25 vs. after 1.28 ± 0.18, p = 0.005). By multi-slice computed tomography angiography, collaterals improved in 12/15 (80%) in the heparin plus exercise group versus 2/16 (12.5%) in the exercise group (p < 0.001). Conclusion: A two-week, 10-test cycle of heparin plus exercise is better than exercise in improving angina class, myocardial ischaemia and collaterals by computed tomography angiography.
... Its activity is markedly potentiated by heparin. Heparin cofactor II is another serpin that inhibits thrombin in the presence of certain glycosaminoglycans, such as heparin [206]. ...
... In the presence of the cofactor protein S, APC inactivates FVa and FVIIIa [207]. Protein C is inhibited by protein C inhibitor whereas APC is inhibited by α1protease inhibitor [206]. ...
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Despite extensive efforts to optimize laser therapy, i.e., the current gold standard treatment, a majority of port wine stain (PWS) patients responds suboptimally to laser therapy. This paper describes the niceties of a novel PWS treatment modality termed site-specific pharmaco-laser therapy (SSPLT). In contrast to the classic approach of enhancing the extent of intravascular photocoagulation (the photothermal response), SSPLT focuses on optimization of post-irradiation thrombus formation (i.e., the hemodynamic response) by combining conventional laser therapy with the administration of thermosensitive drug delivery systems that encapsulate prothrombotic and antifibrinolytic drugs. The aim of SSPLT is to instill complete lumenal occlusion in target vessels, which has been linked to optimal PWS blanching. Relevance for patients: The current treatment options for PWS patients are limited in efficacy. Novel therapeutic modalities are needed to more effectively treat patients with recalcitrant PWSs. SSPLT is an experimental-stage treatment modality that could serve as an adjuvant to pulsed dye laser therapy for a selected group of patients whose PWS is ill-responsive to standard treatment. The expected clinical result of SSPLT is improved lesional blanching.
... Its activity is markedly potentiated by heparin. Heparin cofactor II is another serpin that inhibits thrombin in the presence of certain glycosaminoglycans, such as heparin [207]. ...
... In the presence of the cofactor protein S, APC inactivates FVa and FVIIIa [208]. Protein C is inhibited by protein C inhibitor whereas APC is inhibited by α1-protease inhibitor [207]. ...
... These antithrombotic agents are multi-targeted, i.e. act on a number of coagulation factors like factor Xa (FXa), thrombin (IIa); new antithrombotic drugs selectively target one specific coagulation factor [2][3][4][5][6][7][8][9][10]. Heparin based anticoagulants have numerous limitations like frequent monitoring, dose response, drug-drug and diet-drug interactions, narrow therapeutic range along with purple toe syndrome, eclampsia, risk of haemorrhage and genetic polymorphisms [5,6,8,12]. Thus these major clinical drawbacks and other therapeutic shortcomings of these conventional anticoagulants indicate the need for better anticoagulant agents with better efficacy, improved safety, cost effectiveness and oral administration [2,3,5,9,[11][12][13][14][15][16][17]. ...
... The results showed that EPS prolonged all the three coagulation pathways (extrinsic, intrinsic and common pathways) indicating a non-specific effect or modulation through an enzyme that acts at all three levels. ATIII is the key regulator of the coagulation cascade, it inhibits fXa and other coagulation proteases like factor IXa, factor Xa, factor XIa and factor XIIa using heparin as cofactor to enhance its inhibition efficiency [11,14,44]. We used autodock to do an interaction analysis of E and EPS binding to ATIII and the results indicated an increase in the binding affinity on account of sulfation ( Fig. 7A and B). ...
Article
Currently available anticoagulants for prevention and treatment of thrombosis have several limitations, thus, small organic scaffolds that can dissolve clots in vivo in a dose dependent manner with lesser side effects are highly desirable. Here we report the synthesis of esculin pentasulfate (EPS) and assessment of its in vitro, in vivo and ex vivo anticoagulant and antithrombotic potential. Assessment of in vitro clotting times showed prolonged activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) in the presence of EPS. EPS also showed remarkable reduction in thrombus formation when administered in occlusion induced thrombotic rats at a low dose (2.5 mg/kg). Further, assessment of clot rate with plasma isolated from EPS treated rats confirmed its anticoagulation potential. EPS at varying concentrations showed no significant cytotoxic effect on HEK293 cell line. Further, molecular docking analysis of EPS with known anticoagulant proteins [(antithrombin (ATIII) and heparin cofactor II (HCF II)] that require heparin revealed good binding affinity (-7.9 kcal/mol) with ATIII but not with HCF II. ATIII when incubated with EPS showed increased fluorescence intensity, with no change in secondary structure. Overall, our results clearly show the in vivo modulation of thrombus formation using a modified natural scaffold EPS.
... Once again, it supports the idea that the ascidian polysaccharide fraction does not have the same mech- teinase inhibitor (ZPI), which can inhibit some homeostatic blood proteases of the intrinsic pathway like kallikrein, active protein C and FXIa, respectively. It is also possible that it can act joining the protease with its respectively serpin through a "bridge" made of its glycosidic chain 32,33 . ...
Article
Aims: The anticoagulant effect and cytotoxicity of a high molecular weight polysaccharide fraction (1000RS) obtained from the tunic of the ascidia Microcosmus exasperatus were evaluated. Methods: Anticoagulant properties of 1000RS was evaluated by activated Partial Thromboplastin Time (aPTT), Thrombin Time (TT), Prothrombin Time (PT), anti-factor Xa and lupic anticoagulant (dRVVT) assays. Cytotoxicity was tested on murine hematopoietic cells using MTT assay. Results: This galactose rich fraction showed to be a potential anticoagulant due to its inhibitory effect on the intrinsic coagulation pathway. At the same time, anticoagulant doses of this fraction have no effect on cellular viability, which means that it can be used as a therapeutic agent. Conclusion: In vitro anticoagulant effect of 1000RS occurs at innocuous doses, however, it still need to be tested using in vivo models and its cytotoxicity evaluated in normal human cell lines.
... Heparin is a well-known regulator of the coagulation cascade that is also a potent inhibitor of angiogenesis [708] (see Figure 12). Heparin, therefore, directly modulates the coagulation cascade and is an excellent anticoagulant in diseases where hypercoagulation is prevalent. ...
Article
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Ischaemia-reperfusion (I-R) injury, initiated via bursts of reactive oxygen species produced during the reoxygenation phase following hypoxia, is well known in a variety of acute circumstances. We argue here that I-R injury also underpins elements of the pathology of a variety of chronic, inflammatory diseases, including rheumatoid arthritis, ME/CFS and, our chief focus and most proximally, Long COVID. Ischaemia may be initiated via fibrin amyloid microclot blockage of capillaries, for instance as exercise is started; reperfusion is a necessary corollary when it finishes. We rehearse the mechanistic evidence for these occurrences here, in terms of their manifestation as oxidative stress, hyperinflammation, mast cell activation, the production of marker metabolites and related activities. Such microclot-based phenomena can explain both the breathlessness/fatigue and the post-exertional malaise that may be observed in these conditions, as well as many other observables. The recognition of these processes implies, mechanistically, that therapeutic benefit is potentially to be had from antioxidants, from anti-inflammatories, from iron chelators, and via suitable, safe fibrinolytics, and/or anti-clotting agents. We review the considerable existing evidence that is consistent with this, and with the biochemical mechanisms involved.
... MenSC upregulated the genes involved in angiogenesis and neovascularisation, such as angiopoietin 1 (ANGPT1), PDGFA, PDGFB and matrix metallopeptidase (MMPS), Extracellular Matrix (ECM) components, Elastin (ELN) and MMP10 that during remodelling of tissue helps to regulate collagen degradation 97 . MenSC showed a lower expression of serpin family E member 2 (SERPINE2), which is an inhibitor of serine protease with anti-angiogenic function, and lower expression of the pro-inflammatory Interleukin (IL)-1β 97,99 . Transforming Growth Factor (TGF)B2, a factor involved in keloid formation was downregulated in MenSC 97,100 . ...
Article
Menstrual-derived Stem Cells (MenSC) are a potential novel source of mesenchymal stem cells. There is an increased interest in investigating the therapeutic potential of MenSC due to the various advantages they exhibit, when compared to other types of stem cells. MenSC are obtained non-invasively from menstrual blood. Thus, collection of MenSC is simple, reproducible and can be carried out periodically, with minimal complications. MenSC are present in abundance, are highly proliferative, exhibit a low immunogenicity and lack ethical issues. MenSC have shown the ability to differentiate into several lineages. The therapeutic potential of MenSC in non-gynaecological applications has been investigated in wound healing, neurological, musculo-skeletal, cardiovascular, respiratory, and liver disorders, as well as in diabetes and cancer. Human clinical trials are limited. To date, therapeutic efficacy and safety have been reported in patients with Avian influenza A subtype H7N9, COVID-19, congestive heart failure, multiple sclerosis and Duchene muscular dystrophy. However, further clinical trials in humans should be conducted, to study the long-term therapeutic effects of these stem cells in various diseases and to further explore their mechanism of action. This systematic review focuses on the application of MenSC in non-gynaecological diseases.
... Proteases acting on thrombi directly are plasmin, brinase and trypsin; while, protease dissolving thrombi indirectly is Tissue Plasminogen Activator (t-PA) [20][21][22][23] . The major concerns against using these thrombolytic drugs are excessive bleeding, anaphylactic reactions, heparin-induced thrombocytopenia and toxicity [24][25][26][27][28] . Hence, currently there is lot of interest in the use of natural products as the new thrombolytic drugs with more potency, safety, chemical stability which is devoid of risk of hemorrhage and toxicity from natural sources. ...
... Thus, blocking angiogenesis is a very promising strategy for treating tumors [178]. Consequently, heparin was shown to inhibit angiogenesis as early as 1985 [179,180]. More studies exist on LMWH inhibiting angiogenesis. ...
Article
Heparin is the earliest and most widely used anticoagulant and antithrombotic drug that is still used in a variety of clinical indications. Since it was discovered in 1916, after more than a century of repeated exploration, heparin has not been replaced by other drugs, but a great progress has been made in its basic research and clinical application. Besides anticoagulant and antithrombotic effects, heparin also has antitumor, anti-inflammatory, antiviral, and other pharmacological activities. It is widely used clinically in cardiovascular and cerebrovascular diseases, lung diseases, kidney diseases, cancer, etc., as the first anticoagulant medicine in COVID-19 exerts anticoagulant, anti-inflammatory and antiviral effects. At the same time, however, it also leads to a lot of adverse reactions, such as bleeding, thrombocytopenia, elevated transaminase, allergic reactions, and others. This article comprehensively reviews the modern research progress of heparin compounds; discusses the structure, preparation, and adverse reactions of heparin; emphasizes the pharmacological activity and clinical application of heparin; reveals the possible mechanism of the therapeutic effect of heparin in related clinical applications; provides evidence support for the clinical application of heparin; and hints on the significance of exploring the wider application fields of heparin.
... Currently, increased circulating levels of PEDF is identified as a compensatory response to both macrovascular injury and microvascular injury in obese patients [120]. In addition, in patients with cardio-metabolic disorder diseases, the circulating levels of PEDF has been confirmed to be closely associated with the development of BMI, indicating that PEDF plays an important role in obesity [121]. ...
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Increasing evidence shows that obesity is the critical factor in shaping cardio-metabolic phenotypes. However, the pathogenic mechanisms remain incompletely clarified. According to the published reports, adipose tissue communicates with several diverse organs, such as heart, lungs, and kidneys through the secretion of various cytokines named adipokines. The adipocytes isolated from obese mice or humans are dysfunctional with aberrant production of pro-inflammatory adipokines, which subsequently induce both acute and chronic inflammatory reaction and facilitate the process of cardio-metabolic disorder complications. Furthermore, the microenvironment within adipose tissue under obese status also influence the secretion of adipokines. Recently, given that several important adipokines have been completely researched and causally involved in various diseases, we could make a conclusion that adipokines play an essential role in modulating the development of cardio-metabolic disorder diseases, whereas several novel adipokines continue to be explored and elucidated. In the present review, we summarized the current knowledge of the microenvironment of adipose tissue and the published mechanisms whereby adipocytes affects obesity and cardiovascular diseases. On the other hand, we also provide the evidence to elucidate the functions of adipokines in controlling and regulating the inflammatory reactions which contribute to obesity and cardiovascular disease.
... AT inhibits fibrin formation, which is the final form of coagulant factor. 6 AT activity is measured with plasma containing sodium citrate. Since the blood level of AT depends on the balance between productivity and consumption, the significance of the coagulation system in vivo, production status, and the etiology of thrombosis can be known. ...
Article
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With recent advances in microsurgical instruments and technique, microvascular anastomosis has become a universal surgical technique; however, thrombosis still presents in a number of cases. Tension, twisting, and compression to the anastomotic site are the main causes of thrombus; however, disorder of the coagulation-fibrinolysis system also need to be considered. To date, only few reports exist regarding thrombosis caused by disorder of coagulant system in microvascular anastomosis. Here we report our 3 cases in which multiple thrombus formation occurred intraoperatively caused by decrease of antithrombin (AT) activity. AT activity was measured twice a day after vascular anastomosis: after surgery and up to 3 days after surgery. Thrombosis was not observed in any of the 3 patients intraoperatively after the transfusion, or thrombosis was not observed in any of 3 patients intraoperatively after the transfusion or postoperatively, and no other complications were observed. In these 3 cases, the thrombus was not caused by technical error or other previously described factors. The observed intraoperative decrease in AT activity was thought to be caused by thrombus formation. It is important that microsurgeons are reminded that disorders of the coagulation-fibrinolysis system could cause thrombosis.
... The combination of exercise and heparin co-administration seems a plausible setup since heparin on its own is not able to activate arteriogenesis but acts as a positive modulator of the arteriogenetic process brought on by exercise-induced ischaemia. [10][11][12][13] All randomised patients underwent exercise treadmill stress echocardiography to document the extent of inducible ischaemia (time to development of angina, ST depression on 12-lead ECG and wall motion abnormality on echo) and cardiac CT angiography (CCTA) to assess coronary collateral circulation using the Rentrop classification. The intervention was a two-week-long, 5 days a week, two sessions a day exercise training programme (standard physical rehabilitation program with modified Bruce protocol). ...
... Heparin takes effect rapidly via intravenous infusion, while warfarin acts slowly via oral administration [33]. Both drugs have a narrow therapeutic window and large variation in responses among individuals, and in rare cases they may cause serious complications (heparin-induced thrombocytopenia and coumarin-induced necrosis) [34]. Thus, clinical monitoring and dose adjustment are required during treatment. ...
... VEGF has been identified as a key regulator of both EC differentiation and general vascular development, through activation of key signaling pathways that regulate these processes [35]. VEGF can further synergize with FGFb to promote angiogenesis [36]. ...
Article
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Pancreatic transplantation remains the only cure for diabetes, but the shortage of donors limits its clinical application. Whole organ decellularized scaffolds offer a new opportunity for pancreatic organ regeneration; however inadequate endothelialization and vascularization can prevent sufficient transport of oxygen and nutrient supplies to the transplanted organ, as well as leading unwanted thrombotic events. In the present study, we explored the re-endothelialization of rat pancreatic acellular scaffolds via circulation perfusion using human skin fibroblasts (FBs) and human umbilical vein endothelial cells (HUVECs). Our results revealed that the cell adhesion rate when these cells were co-cultured was higher than under control conditions, and this increase was associated with increased release of growth factors including VEGF, FGFb, EGF, and IGF-1 as measured by ELISA. When these recellularized organs were implanted in vivo for 28 days in rat dorsal subcutaneous pockets, we found that de novo vasculature formation in the co-culture samples was superior to the control samples. Together these results suggest that endothelial cell and FB co-culture enhances the re-endothelialization and vascularization of pancreatic acellular scaffolds.
... However, some studies indicate a pro-angiogenic property of Thrombospondin-1 (42). Similarly, Pentraxin-3 (43), DPPIV (44)(45)(46), TIMP-1 (36,47), and Serpin E1 (Plasminogen Activator Inhibitor) (48,49) have each also been implicated as having both pro-and anti-angiogenesis properties. Additional studies will determine the specific contribution of each of these mast cellderived proteins in tumor development, but the fact that most of these factors were secreted at levels much greater than VEGF suggests that they could potentially have a greater influence on mast cell-mediated angiogenesis and tumor development than VEGF. ...
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Mast cells are classically recognized as cells that cause IgE-mediated allergic reactions. However, their ability to store and secrete vascular endothelial growth factor (VEGF) suggests a role in vascular development and tumorigenesis. The current study sought to determine if other angiogenesis-related factors, in addition to VEGF, were also secreted by human tissue-derived mast cells. Using proteome array analysis and ELISA, we found that human skin-derived mast cells spontaneously secrete CXCL16, DPPIV, Endothelin-1, GM-CSF, IL-8, MCP-1, Pentraxin 3, Serpin E1, Serpin F1, TIMP-1, Thrombospondin-1, and uPA. We identified three groups based on their dependency for stem cell factor (SCF), which is required for mast cell survival: Endothelin-1, GM-CSF, IL-8, MCP-1, and VEGF (dependent); Pentraxin 3, Serpin E1, Serpin F1, TIMP-1, and Thrombospondin-1 (partly dependent); and CXCL16, DPPIV, and uPA (independent). Crosslinking of FcεRI with multivalent antigen enhanced the secretion of GM-CSF, Serpin E1, IL-8, and VEGF, and induced Amphiregulin and MMP-8 expression. Interestingly, FcεRI signals inhibited the spontaneous secretion of CXCL16, Endothelin-1, Serpin F1, Thrombospondin-1, MCP-1 and Pentraxin-3. Furthermore, IL-6, which we previously showed could induce VEGF, significantly enhanced MCP-1 secretion. Overall, this study identified several angiogenesis-related proteins that, in addition to VEGF, are spontaneously secreted at high concentrations from human skin-derived mast cells. These findings provide further evidence supporting an intrinsic role for mast cells in blood vessel formation.
... The anticoagulant function of agents such as Apixaban and Rivaroxaban is derived from the inhibition of coagulation factor Xa, and is therefore similar to that of a number of LMWH. However, these agents do not require the function of antithrombin III, which also possesses independent anti-angiogenic properties 29 . ...
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... Polymerisation and incorrect folding can occur even at a physiological pH and temperature [75]. It is also possible that the inhibitory effect of serpins could be increased by including a cofactor into the reaction; for example, the inhibitory capacity of human antithrombin against thrombin was optimal when the reaction included the glycosaminoglycan heparin [8,33,74,83]. Taking this into consideration, we tried to inhibit factor Xa by rEnSerp1 in the presence of heparin (heparin sodium salt, 20 nM, Zentiva), but no inhibitory effect was observed (data not shown). In order to detect any possible residual contamination with inhibitory effect in the sample of purified rEnSerp1 expressed in bacteria, a control with bacterial lysate instead of rEnSerp1 was tested. ...
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Background: Serpins are a superfamily of serine peptidase inhibitors that participate in the regulation of many physiological and cell peptidase-mediated processes in all organisms (e.g. in blood clotting, complement activation, fibrinolysis, inflammation, and programmed cell death). It was postulated that in the blood-feeding members of the monogenean family Diplozoidae, serpins could play an important role in the prevention of thrombus formation, activation of complement, inflammation in the host, and/or in the endogenous regulation of protein degradation. Results: In silico analysis showed that the DNA and primary protein structures of serpin from Eudiplozoon nipponicum (EnSerp1) are similar to other members of the serpin superfamily. The inhibitory potential of EnSerp1 on four physiologically-relevant serine peptidases (trypsin, factor Xa, kallikrein, and plasmin) was demonstrated and its presence in the worm’s excretory-secretory products (ESPs) was confirmed. Conclusion: EnSerp1 influences the activity of peptidases that play a role in blood coagulation, fibrinolysis, and complement activation. This inhibitory potential, together with the serpin’s presence in ESPs, suggests that it is likely involved in host-parasite interactions and could be one of the molecules involved in the control of feeding and prevention of inflammatory responses.
... In addition to its well-established anticoagulant function, antithrombin has been shown to have antiangiogenic functions by inhibiting endothelial cell proliferation, blood vessel growth in the chick embryo and tumor growth in mice (Zhang et al., 2004), relating this action to a possible AT antitumoral activity. This antiangiogenic/antitumoral activity of AT was only observed when native AT undergoes conformational alterations induced by proteolytic cleavage (Bhakuni et al., 2016), but the role of native AT on the angiogenic process in physiological conditions has not been sufficiently studied. For this reason, to complement the understanding of the beneficial relationship observed between AT activity and clinical outcomes after cardiac surgery, we analyzed the activity of native AT on endothelial function and focus our attention to two processes, migration and angiogenesis, closely related to the recovery of the vasculature after CPB. ...
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Keywords: angiogenesis, antithrombin, cardiac surgery, cardiopulmonary bypass, dexamethasone, endothelial function
... By contrast, a downregulation of TGFB2 (0.47-fold) was observed, factor which higher activity was related to keloids (Xia et al., 2006). Interestingly, MenSCs compared to UC-MSCs expressed lower but significant levels of SERPINE2 (0.19-fold) and IL1β (0.09-fold), the former encoding a protein that inhibit serine proteases and displays anti-angiogenic function (Bhakuni et al., 2016) and the second the pro-inflammatory IL1β ( Figure 4A, Group 1). The pro-inflammatory condition significantly regulated differentially the mRNA expression of 53% of the genes in MenSCs (Figure 4B, Group 3) while only 33% in UC-MSCs ( Figure 4B, Group 4). ...
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Considerable advances have been made toward understanding the cellular and molecular mechanism of wound healing, however, treatments for chronic wounds remain elusive. Emerging concepts utilizing mesenchymal stem cells (MSCs) from umbilical cord, adipose tissue and bone marrow have shown therapeutical advantages for wound healing. Based on this positive outcome, efforts to determine the optimal sources for MSCs are required in order to improve their migratory, angiogenic, immunomodulatory, and reparative abilities. An alternative source suitable for repetitive, non-invasive collection of MSCs is from the menstrual fluid (MenSCs), displaying a major practical advantage over other sources. This study aims to compare the biological functions and the transcriptomic pattern of MenSCs with umbilical cord MSCs in conditions resembling the wound microenvironment. Consequently, we correlate the specific gene expression signature from MenSCs with changes of the wound matrix signals in vivo. The direct comparison revealed a superior clonogenic and migratory potential of MenSCs as well as a beneficial effect of their secretome on human dermal fibroblast migration in vitro. Furthermore, MenSCs showed increased immunomodulatory properties, inhibiting T-cell proliferation in co-culture. We further, investigated the expression of selected genes involved in wound repair (growth factors, cytokines, chemokines, AMPs, MMPs) and found considerably higher expression levels in MenSCs (ANGPT1 1.5-fold; PDGFA 1.8-fold; PDGFB 791-fold; MMP3 21.6-fold; ELN 13.4-fold; and MMP10 9.2-fold). This difference became more pronounced under a pro-inflammatory stimulation, resembling wound bed conditions. Locally applied in a murine excisional wound splinting model, MenSCs showed a significantly improved wound closure after 14 days, as well as enhanced neovascularization, compared to the untreated group. Interestingly, analysis of excised wound tissue revealed a significantly higher expression of VEGF (1.42-fold) among other factors, translating an important conversion of the matrix signals in the wound site. Furthermore, histological analysis of the wound tissue from MenSCs-treated group displayed a more mature robust vascular network and a genuinely higher collagen content confirming the pro-angiogenic and reparative effect of MenSCs treatment. In conclusion, the superior clonogenicity, immunosuppressive and migration potential in combination with specific paracrine signature of MenSCs, resulted in an enhanced wound healing and cutaneous regeneration process.
... The binding of GAG prevents kallistatin from binding and inhibiting kallikrein, thus allowing activation of factor XII and cleavage of HMWK into bradykinin. Both events result in antiangiogenic and 45,106 An important consequence of this is that competition between kallistatin and vascular endothelial growth factor and bFGF for endothelial GAG binding reduces the angiogenic effects associated with these molecules. 106 ...
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The glycosaminoglycans (GAGs) heparan sulfate, dermatan sulfate, and heparin are important anticoagulants that inhibit clot formation through interactions with antithrombin and heparin cofactor II. Unfractionated heparin, low-molecular-weight heparin, and heparin-derived drugs are often the main treatments used clinically to handle coagulatory disorders. A wide range of proteins have been reported to bind and neutralize these GAGs to promote clot formation. Such neutralizing proteins are involved in a variety of other physiological processes, including inflammation, transport, and signaling. It is clear that these interactions are important for the control of normal coagulation and influence the efficacy of heparin and heparin-based therapeutics. In addition to neutralization, the anticoagulant activities of GAGs may also be regulated through reduced synthesis or by degradation. In this review, we describe GAG neutralization, the proteins involved, and the molecular processes that contribute to the regulation of anticoagulant GAG activity.
... The Kd value is inversely proportional to the Ka value; the higher the Ka value for lunathrombase towards thrombin vs. fibrinogen indicates its higher affinity for thrombin, which is consistent with the spectrofluorometric analysis. The anticoagulant mechanism of lunathrombase appears to differ from that of the currently available anticoagulant drugs such as heparin and warfarin that act via the indirect inhibition of thrombin and by vitamin K antagonism, respectively 40,41 . The dual inhibition of thrombin and FXa by lunathrombase may lead to its consideration as an alternative new drug to the traditional cardiovascular drugs 15 . ...
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The harnessing of medicinal plants containing a plethora of bioactive molecules may lead to the discovery of novel, potent and safe therapeutic agents to treat thrombosis-associated cardiovascular diseases. A 35 kDa (m/z 34747.5230) serine protease (lunathrombase) showing fibrin(ogen)olytic activity and devoid of N- and O- linked oligosaccharides was purified from an extract of aqueous leaves from L. indica. The LC-MS/MS analysis, de novo sequencing, secondary structure, and amino acid composition determination suggested the enzyme's novel characteristic. Lunathrombase is an αβ-fibrinogenase, demonstrating anticoagulant activity with its dual inhibition of thrombin and FXa by a non-enzymatic mechanism. Spectrofluorometric and isothermal calorimetric analyses revealed the binding of lunathrombase to fibrinogen, thrombin, and/or FXa with the generation of endothermic heat. It inhibited collagen/ADP/arachidonic acid-induced mammalian platelet aggregation, and demonstrated antiplatelet activity via COX-1 inhibition and the upregulation of the cAMP level. Lunathrombase showed in vitro thrombolytic activity and was not inhibited by endogenous protease inhibitors α2 macroglobulin and antiplasmin. Lunathrombase was non-cytotoxic to mammalian cells, non-hemolytic, and demonstrated dose-dependent (0.125-0.5 mg/kg) in vivo anticoagulant and plasma defibrinogenation activities in a rodent model. Lunathrombase (10 mg/kg) did not show toxicity or adverse pharmacological effects in treated animals.
... Heparin cofactor II (HCII) functions to inactive thrombin and is therefore involved in the modulation of blood coagulation. HCII is responsible for approximately 20-30% of thrombin inactivation during coagulation (Rau et al. 2007, Bhakuni et al. 2016. HCII is unique in that, in the extravascular lumen, it inhibits only thrombin and no other proteins involved in the coagulation process (Parker and Tollefsen 1985). ...
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Objective: To derive a plasma biomarker protein panel from a list of 141 candidate proteins which can differentiate transient ischaemic attack (TIA)/minor stroke from non-cerebrovascular ('mimic') conditions in emergency department (ED) settings. Design: Prospective clinical study (#NCT03050099) with up to three timed blood draws no more than 36 hours following symptom onset. Plasma samples analysed by multiple reaction monitoring-mass spectrometry (MRM-MS). Participants: 545 participants suspected of TIA enrolled in the EDs of two urban medical centres. Outcomes: 90-day, neurologist-adjudicated diagnosis of TIA informed by clinical and radiological investigations. Results: The final protein panel consists of 16 proteins whose patterns show differential abundance between TIA and mimic patients. Nine of the proteins were significant univariate predictors of TIA [odds ratio (95% confidence interval)]: L-selectin [0.726 (0.596-0.883)]; insulin-like growth factor-binding protein 3 [0.727 (0.594-0.889)]; Coagulation factor X [0.740 (0.603-0.908)]; Serum paraoxonase/lactonase 3 [0.763 (0.630-0.924)]; Thrombospondin-1 [1.313 (1.081-1.595)]; Hyaluronan-binding protein 2 [0.776 (0.637-0.945)]; Heparin cofactor 2 [0.775 (0.634-0.947)]; Apolipoprotein B-100 [1.249 (1.037-1.503)]; and von Willebrand factor [1.256 (1.034-1.527)]. The scientific plausibility of the panel proteins is discussed. Conclusions: Our panel has the potential to assist ED physicians in distinguishing TIA from mimic patients.
... Once again, it supports the idea that the ascidian polysaccharide fraction does not have the same mech- teinase inhibitor (ZPI), which can inhibit some homeostatic blood proteases of the intrinsic pathway like kallikrein, active protein C and FXIa, respectively. It is also possible that it can act joining the protease with its respectively serpin through a "bridge" made of its glycosidic chain 32,33 . ...
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Aims: The anticoagulant effect and cytotoxicity of a high molecular weight polysaccharide fraction (1000RS) obtained from the tunic of the ascidia Microcosmus exasperatus were evaluated. Methods: Anticoagulant properties of 1000RS was evaluated by activated Partial Thromboplastin Time (aPTT), Thrombin Time (TT), Prothrombin Time (PT), anti-factor Xa and lupic anticoagulant (dRVVT) assays. Cytotoxicity was tested on murine hematopoietic cells using MTT assay. Results: This galactose rich fraction showed to be a potential anticoagulant due to its inhibitory effect on the intrinsic coagulation pathway. At the same time, anticoagulant doses of this fraction have no effect on cellular viability, which means that it can be used as a therapeutic agent. Conclusion: In vitro anticoagulant effect of 1000RS occurs at innocuous doses, however, it still need to be tested using in vivo models and its cytotoxicity evaluated in normal human cell lines.
... Serpins regulate the activity of serine proteases in the clotting or thrombotic, and in the clot lysis, or thrombolytic, cascades, preventing excess clotting and end-organ ischemia and also preventing excess bleeding or hemorrhage, respectively [1,14,15]. Serpins also control complement and immune responses, hormone transport and even blood pressure, as well as many other processes such as apoptosis, and neurohormone carriers [16][17][18][19][20][21][22][23]. In disease states such as sepsis, where there is dysregulated bleeding and clotting which is termed disseminated intravascular coagulation (DIC), there is a loss of regulation of the thrombotic and the thrombolytic cascades, both due to loss of protease functions as well as loss of serpin functions [24]. ...
... (B) Several factors of the intrinsic coagulation pathway (gray ovals) can be inhibited by either mosquito or tick serpins (yellow boxes, see Section 4.2). The mammalian serpins that target the same pathway are shown in comparison (blue boxes) [156][157][158]. (C) The evolutionary history of tick and mammalian thrombin-inhibiting serpins does not indicate any orthologies between individual tick and human serpin pairs. ...
Article
Objectives Underlying mechanism for the development of chronic thromboembolic pulmonary hypertension and prognostic biomarkers are not clear yet. Thus, our aim is to assess and identify new biomarkers for the expression of 84 key genes linked to angiogenesis. Methods The patients who had levels more than 1000 dynes·sec·cm-5 were included in the test group while the others were assigned to the control group. 12 specimens were taken from the patients. RT2 Profiler PCR Array was performed to quantify the expression of the 84 key genes. Results Eight patients (six male, 2 female, median age 54.4±13.1), who underwent pulmonary endarterectomy, were included. Pulmonary vascular resistance improved significantly from 811 ± 390 dyn/s/cm⁻⁵ to 413.3 ±144.9 dyn/s/cm⁻⁵ (p<0.005). Difference was also detected in median mean pulmonary arterial pressure as decline from 49.8± 9 mmHg to 32.62±2.50 mmHg (p> 0.005) following surgery. Median length of hospital stay was 11.62±2.97 days. The test group hold a distinct pattern of impaired angiogenic and anti-angiogenic genes. The expression levels of TGFA, TGFB1, THBS2, THBS1, TGFBR1, SERPINE1, SERPINF1, TGFB2, TIMP2, VEGFC, IFNA1, TNF, CXCL10, NOS3, IGF1 and MMP14 were downregulated in the specimens from the patients who had higher pulmonary vascular resistance values, while some genes including PDGFA showed upregulation in the same group which were statistically nonsignificant. Conclusion These results can lead to development of new markers which would become predictors for adverse outcomes of CTEPH patients. Identification of new markers that are related with worse outcomes enable screening patients for early diagnosis and treatment.
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Understanding the combined impacts of genetic variances and post-translational modifications requires new approaches. Here, we delineate proteoforms of plasma serine protease inhibitors and relate specific proteoforms to their interactions in complexes through the use of native mass spectrometry (MS). First, we dissect the proteoform repertoire of an acute-phase plasma protein, serine protease inhibitor A1 (SERPINA1), resolving four SERPINA1 variants (M1V, M1A, M2, and M3) with common single-nucleotide polymorphisms (SNPs). Investigating the glycosylation status of these variants and their ability to form complexes with a serine protease, elastase, we find that fucosylation stabilizes the interaction of the SERPINA1 M1V variant through its core fucosylation on Asn271. In contrast, antennary fucosylation on Asn271 destabilizes SERPINA1-elastase interactions. We unveil the same opposing effects of core and antennary fucosylation on SERPINA3 interactions with chymotrypsin. Together, our native MS results highlight the modulating effects of fucosylation with different linkages on glycoprotein interactions.
Chapter
Wide-ranging studies have recently been performed on adipokines and their association with metabolic processes. Vaspin, a serpin-derived adipokine, is an essential linker between metabolic alterations, obesity, and insulin resistance. Vaspin mRNA is expressed in different tissues and has different roles in various tissues. This adipokine has anti-inflammatory and anti-apoptotic effects and is considered an insulin sensitizer whose intracellular and extracellular levels are affected by metabolic changes. Because vaspin was first identified in the visceral adipose tissue of a rat model of type 2 diabetes and obesity, various laboratory and clinical studies have been performed to introduce it as a biomarker of diabetes. The incidence of insulin resistance in type 2 diabetes is associated with the function of vaspin as an insulin sensitizer, which is quite different from the mechanism of type 1 diabetes. This chapter discusses the studies conducted in recent years about the probability of recognizing vaspin as a biomarker for the prognosis and diagnosis of type 2 diabetes. The potential role of vaspin in the body, molecular mechanisms, and signaling pathways associated with vaspin and type 2 diabetes also has been reviewed to achieve the aim of the chapter. It is concluded that circulating vaspin level may be considered as a potential biomarker in assessing type 2 diabetes and the prognosis of its complications.
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Background Fibrotic remodeling is an essential aspect of heart failure. Human kallistatin (KS, mouse Serpina3c homologs) inhibits fibrosis after myocardial infarction (MI) but the specific underlying mechanism is unknown. Methods A total of 40 heart failure patients (HFPs) were enrolled and their plasma KS was measured using ELISA. Serpina3c−/− and C57BL/6 mice were used to construct the MI model. TGF-β1 or a hypoxic condition was established to interfere with the functioning of cardiac fibroblasts (CFs). RNA-seq was performed to assess the effect of Serpina3c on the transcriptome. Findings The levels of KS were used as a predictor of readmission among the HFPs. Serpina3c expression decreased in MI hearts and CFs. Serpina3c−/− led to the aggravation of MI fibrosis, and increased the proliferation of CFs. The overexpression of Serpina3c in CFs had the opposite effect. Glycolysis-related genes were significantly increased in Serpina3c−/− group by RNA-seq. Enolase (ENO1), which is a key enzyme in glycolysis, increased most significantly. Inhibition of ENO1 could antagonize the promotion of Serpina3c−/− on the proliferation of CFs. Co-IP was performed to verify the interaction between Serpina3c and Nr4a1. Serpina3c−/− inhibited the acetylation of Nr4a1 and increased the degradation of Nr4a1. Activation of Nr4a1 could negatively regulate the expression of ENO1 and inhibited the proliferation of Serpina3c−/− CFs in Serpina3c−/− MI mice. Interpretation Serpina3c inhibits the transcriptional activation of ENO1 by regulating the acetylation of Nr4a1, thereby reducing the fibrosis after MI by inhibiting glycolysis. Serpina3c is a potential target for prevention and treatment of heart failure after MI.
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Thromboembolic diseases are a major cause of mortality in human and the currently available anticoagulants are associated with various drawbacks, therefore the search for anticoagulants that have better safety profile is highly desirable. Compounds that are part of the dietary routine can be modified to possibly increase their anticoagulant potential. We show mannose 2,3,4,5,6-O-pentasulfate (MPS) as a synthetically modified form of mannose that has appreciable anticoagulation properties. An in silico study identified that mannose in sulfated form can bind effectively to the heparin-binding site of antithrombin (ATIII) and heparin cofactor II (HCII). Mannose was sulfated using a simple sulfation strategy-involving triethylamine-sulfur trioxide adduct. HCII and ATIII were purified from human plasma and the binding analysis using fluorometer and isothermal calorimetry showed that MPS binds at a unique site. A thrombin inhibition analysis using the chromogenic substrate showed that MPS partially enhances the activity of HCII. Further an assessment of in vitro blood coagulation assays using human plasma showed that the activated partial thromboplastin time (APTT) and prothrombin time (PT) were prolonged in the presence of MPS. A molecular dynamics simulation analysis of the HCII-MPS complex showed fluctuations in a N-terminal loop and the cofactor binding site of HCII. The results indicate that MPS is a promising lead due to its effect on the in vitro coagulation rate. Communicated by Ramaswamy H. Sarma
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Due to the technological advances, it has been well-established that obesity is strongly correlated with various health problems. Among these problems, dyslipidemia is one of the most important concomitant symptoms under obese status which is the main driving force behind the pathological progression of cardio-metabolic disorder diseases. Importantly, the type of dyslipidemia, arising from concerted action of obesity, has been identified as “metabolic related dyslipidemia”, which is characterized by increased circulating levels of Low density lipoprotein cholesterol (LDL-C), Triglycerides (TG) accompanied by lower circulating levels of High density lipoprotein cholesterol (HDL-C). On the other hand, the metabolic related dyslipidemia is being verified as a vital link between obesity and hypertension, diabetes mellitus, and Cardiovascular disease (CVD). In this review, we summarized the current understanding of metabolic related dyslipidemia and the potential mechanisms which lead to the pathogenesis of obesity. Meanwhile, we also summarized the emerging results which focused on several novel lipid bio-markers in metabolic related dyslipidemia, such as pro-protein convertase subtilisin/kexin type 9 (PCSK9) and sphingosine-1-phosphate (S1P), and their potential use as biomarkers of metabolic related dyslipidemia.
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Serpins are evolutionarily conserved serine protease inhibitors found in many organisms. In arthropods, serpins are involved in feeding, development, oviposition, anti-coagulation and innate immune responses. We characterized of 11 serpins in the tick Rhipicephalus haemaphysaloides. These serpins have orthologous genes in other ticks, as indicated by phylogenetic analysis. Analysis of the reactive center loop and hinge regions of the protein sequences indicated that RHS7 encodes proteins that may lack proteinase inhibitor activity. All R. haemaphysaloides serpins had high amino acid sequence identities to Rhipicephalus microplus serpins. Tissue and temporal transcriptional profiling of eight R. haemaphysaloides serpins located in the ovaries demonstrated that they are transcribed during feeding and oviposition. These suggested their participation in the regulation of tick physiology. Immune serum from rabbits repeatedly infested with larvae, nymphs and adults of R. haemaphysaloides can recognize multiple recombinant serpins, respectively. After gene silencing, the blood feeding to repletion time was significantly longer and the 24 h attachment rate was significantly lower in the RHS3 and RHS7 knock down groups. The RHS9 and RHS11 silenced ticks had significant reduction in repletion time and egg-laying rate. Egg hatchability was significantly decreased in RHS4, RHS5 and RHS9 silenced ticks. All groups had significant reductions in engorged body weight. This study increases information on the serpins of R. haemaphysaloides and suggests that some RHSs are potential targets for development of tick vaccines.
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Increased tendency of cancer patient to develop venous thromboembolism (VTE) is associated with high rates of mortality. Elevation of procoagulant proteins and down regulation of naturally occurring coagulation inhibitors appears to form the basis of high risk of VTE in malignancy. A reduced level of anticoagulant protein like antithrombin (AT) will influence both coagulation and angiogenesis, as its cleaved and latent conformations show potent antiangiogenic activity. We show a concentration dependent perturbation in the secondary and tertiary structures of AT conformers exposed to hypochlorous acid (HOCl). Modulated under a very narrow concentration range of HOCl, native AT undergoes oligomerization, aggregation and fragmentation based on spectroscopic, SDS and native-PAGE studies. Factor Xa inhibition assay demonstrated a progressive decrease in inhibition activity of AT on modification by HOCl. Bis-ANS result showed that hydrophobic patches were more exposed in the case of HOCl-modified AT when assessed fluorometrically. Dosage of HOCl-modified AT in experimental animals induced high titer antibodies showing more specificity towards modified forms in comparison to unmodified forms. Auto-antibodies isolated from cancer patients also showed enhanced binding with HOCl-modified AT in comparison to native AT. Compared to normal AT, structurally and functionally altered conformation of HOCl modified AT showed increased immunogenic sensitivity. HOCl modified AT can contribute to prothrombotic and angiogenic environment during cancer progression/development.
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Ethnopharmacological relevance: Cardiovascular diseases are the major cause of mortality and morbidity, causing over 17.9 million deaths a year worldwide. Currently used therapy is often having side effects and expensive, dietary interventions and alternative medicines are required. Clerodendrum colebrookianum has been used to treat cardiac hypertension but anticoagulant potency was not evaluated. Aim of the study: To characterize an active anticoagulant fraction (AAFCC) and a 30 kDa fibrin(ogen)olytic serine protease (clerofibrase) isolated from aqueous leave extract of C. colebrookianum. Materials and methods: AAFCC/clerofibrase was subjected to extensive biochemical and pharmacological characterization including LC-MS/MS, amino acid compositional and GC-MS analyses. Interaction between clerofibrase with fibrinogen was studied by spectrofluorometric analysis. In vitro thrombolytic, antiplatelet and cytotoxicity assay were performed. In vivo toxicity, anticoagulant, defibrinogen and antithrombotic activities were determined on Swiss albino mice. Results: The in vitro anticoagulant activity of AAFCC was found to be superior to heparin and clerofibrase and comparable to Nattokinase and warfarin. The proteomics and amino acid composition analyses suggest that clerofibrase is a previously uncharacterized novel plant protease capable of degrading the -αβ chains of fibrinogen/fibrin. AAFCC/clerofibrase exerts their anticoagulant action via fibrinogenolytic activity and partially by antiplatelet activity albeit they have no effect on thrombin and FXa inhibition. The spectrofluorometric analysis revealed the binding of clerofibrase to fibrinogen but not to thrombin and FXa. The phytochemical constituents and bioactive components of AAFCC were characterized by biochemical, and GC-MS analyses. The AAFCC and clerofibrase inhibited collagen/ADP-induced mammalian platelet aggregation, showed in vitro thrombolytic activity, and non-cytotoxic to mammalian cells. The AAFCC showed and dose-dependent in vivo plasma defibrinogenating and anticoagulant activities and inhibited k-carrageen-induced thrombus formation in the tails of mice. Conclusion: The potent in vivo anticoagulant and antithrombotic effects of AAFCC suggests its pharmacological significance as herbal anticoagulant drug for the prevention and/or treatment of hyperfibrinogenemia- and thrombosis associated cardiovascular disorders.
Chapter
Serpins have a wide range of functions in regulation of serine proteases in the thrombotic cascade and in immune responses, representing up to 2–10% of circulating proteins in the blood. Selected serpins also have cross-class inhibitory actions for cysteine proteases in inflammasome and apoptosis pathways. The arterial and venous systems transport blood throughout the mammalian body representing a central site for interactions between coagulation proteases and circulating blood cells (immune cells) and target tissues, a very extensive and complex interaction. While analysis of serpin functions in vitro in kinetics or gel shift assays or in tissue culture provides very necessary information on molecular mechanisms, the penultimate assessment of biological or physiological functions and efficacy for serpins as therapeutics requires study in vivo in whole animal models (some also consider cell culture to be an in vivo approach).
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Background: Glycosaminoglycans (GAGs) are polysaccharides that are distributed on respiratory epithelial cells, endothelial cells, and submucosal glands. Uniquely positioned, certain GAGs exhibit anti-inflammatory properties in respiratory diseases and serve important roles in repairing mucosal surfaces and modulating mucociliary clearance. We hypothesized that topical administration of a synthetic GAG (GM-0111) would prevent sinonasal inflammation in a mouse model of rhinosinusitis (RS). Methods: To test our hypothesis, C57BL/6 mice were intranasally administered fluorescent GM-0111, and sinonasal tissues were examined for coating and penetration ability. To test therapeutic feasibility, mice (n = 6) were given GM-0111 or hyaluronic acid (HA; 800 μg dose) prior to inducing RS with inflammatory molecule LL-37 (115 μg dose). After 24 hours, sinonasal tissues were harvested for histological and biochemical analysis of inflammatory markers (inflammatory cell infiltration, lamina propria [LP] thickening, and neutrophil enzyme myeloperoxidase [MPO]) and cell death. Results: GM-0111 was observed within sinonasal tissues 1 hour and 24 hours after intranasal administration, indicating rapid and effective coating and penetration. GM-0111 prevented sinonasal tissues from developing inflammatory changes, with significant reductions in mast cell infiltration (p < 0.05), LP thickening (p < 0.001), and MPO levels (p < 0.01) when compared to tissues treated with LL-37 and those pretreated with HA. GM-0111 reduced cell death within sinonasal tissues in contrast to LL-37-treated tissues. Conclusion: We report a new synthetic GAG (GM-0111) that uniformly coats and penetrates into the sinonasal mucosa to prevent sinonasal inflammation and cell death in a mouse model of RS.
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We recently identified protease nexin-1 (PN-1) or serpinE2, as a possibly underestimated player in maintaining angiogenic balance. Here, we used the well-characterized postnatal vascular development of newborn mouse retina to further investigate the role and the mechanism of action of PN-1 in physiological angiogenesis. The development of retinal vasculature was analysed by endothelial cell staining with isolectin B4. PN-1-deficient (PN-1(-/-)) retina displayed increased vascularization in the postnatal period, with elevated capillary thickness and density, compared to their wild-type littermate (WT). Moreover, PN-1(-/-) retina presented more veins/arteries than WT retina. The kinetics of retinal vasculature development, retinal VEGF expression and overall retinal structure were similar in WT and PN-1(-/-) mice, but we observed a hyperproliferation of vascular cells in PN-1(-/-) retina. Expression of PN-1 was analysed by immunoblotting and X-Gal staining of retinas from mice expressing beta-galactosidase under a PN-1 promoter. PN-1 was highly expressed in the first week following birth and then progressively decreased to a low level in adult retina where it localized on the retinal arteries. PCR arrays performed on mouse retinal RNA identified two angiogenesis-related factors, midkine and Smad5, that were overexpressed in PN-1(-/-) newborn mice and this was confirmed by RT-PCR. Both the higher vascularization and the overexpression of midkine and Smad5 mRNA were also observed in gastrocnemius muscle of PN-1(-/-) mice, suggesting that PN-1 interferes with these pathways. Together, our results demonstrate that PN-1 strongly limits physiological angiogenesis and suggest that modulation of PN-1 expression could represent a new way to regulate angiogenesis.
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Angiogenesis, the process of formation of new blood vessel from pre-existing ones, is involved in various intertwined pathological processes including virus infection, inflammation and oncogenesis, making it a promising target for the development of novel strategies for various interventions. To induce angiogenesis, angiogenic growth factors (AGFs) must interact with pro-angiogenic receptors to induce proliferation, protease production and migration of endothelial cells (ECs). The action of AGFs is counteracted by antiangiogenic modulators whose main mechanism of action is to bind (thus sequestering or masking) AGFs or their receptors. Many sugars, either free or associated to proteins, are involved in these interactions, thus exerting a tight regulation of the neovascularization process. Heparin and heparan sulfate proteoglycans undoubtedly play a pivotal role in this context since they bind to almost all the known AGFs, to several pro-angiogenic receptors and even to angiogenic inhibitors, originating an intricate network of interaction, the so called "angiogenesis glycomic interactome". The decoding of the angiogenesis glycomic interactome, achievable by a systematic study of the interactions occurring among angiogenic modulators and sugars, may help to design novel antiangiogenic therapies with implications in the cure of angiogenesis-dependent diseases.
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Antithrombin III (AT) is the main inhibitor of blood coagulation proteases like thrombin and factor Xa. In this study we report the identification and characterization of several variants of AT for the first time in Indian population. We screened 1950 deep vein thrombosis (DVT) patients for AT activity and antigen levels. DNA sequencing was further carried out in patients with low AT activity and/or antigen levels to identify variations in the AT gene. Two families, one with type I and the other with type II AT deficiency were identified. Three members of family I showed an increase in the coagulation rates and recurrent thrombosis in this family was solely attributed to the rs2227589 polymorphism. Four members of family II spanning two generations had normal antigen levels and decreased AT activity. A novel single nucleotide insertion, g.13362_13363insA in this family in addition to g.2603T>C (p.R47C) mutation were identified. AT purified from patient's plasma on hi-trap heparin column showed a marked decrease in heparin affinity and thrombin inhibition rates. Western blot analysis showed the presence of aggregated AT. We also report a novel point mutation at position g.7549 A>G (p.T280A), that is highly conserved in serpin family. Variant protein isolated from patient plasma indicated loss of regulatory function due to in-vivo polymerization. In conclusion this is the first report of AT mutations in SERPINC1 gene in Indo-Aryan population where a novel point mutation p.T280A and a novel single nucleotide insertion g.13362_13363insA are reported in addition to known variants like p.R47C, p.C4-X and polymorphisms of rs2227598, PstI and DdeI.
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Protein Z (PZ) is a vitamin K-dependent coagulation factor without catalytic activity. Evidence points towards PZ as an independent risk factor for the occurrence of human peripheral arterial disease. However, the role of PZ in ischemia-driven angiogenesis and vascular healing processes has not been elucidated so far. Angiogenic potency of PZ was assessed in established in vitro assays using endothelial cells. PZ-deficient (PZ-/-) mice and their wild-type littermates (PZ+/+) were subjected to hindlimb ischemia. Furthermore, PZ-/- mice were exposed to PZ expressing adenovirus (AdV-PZ) or control adenovirus (AdV-GFP). In an additional set of animals, PZ-/- mice were exposed to AdV-PZ and AdV-GFP, each in combination with the CXCR4 antagonist AMD3100. In vitro, PZ stimulated migratory activity and capillary-like tube formation of endothelial cells comparable to SDF-1. PZ-/- mice exhibited diminished hypoxia-driven neovascularization and reperfusion in post-ischemic hindlimbs, which was restored by adenoviral gene transfer up to levels seen in PZ+/+ mice. The stimulatory impact of PZ on endothelial cells in vitro was abolished by siRNA targeting against PZ and PZ was not able to restore reduced migration after knock-down of CXCR4. The increased surface expression of CXCR4 on PZ-stimulated endothelial cells and the abrogated restoration of PZ-/- mice via AdV-PZ after concomitant treatment with the CXCR4 antagonist AMD3100 supports the idea that PZ mediates angiogenesis via a G-protein coupled pathway and involves the SDF-1/CXCR4 axis. This is underlined by the fact that addition of the G-protein inhibitor PTX to PZ-stimulated endothelial cells abolished the effect of PZ on capillary-like tube formation. The results of the current study reveal a role of PZ in ischemia-induced angiogenesis, which involves a G-protein coupled pathway and a raised surface expression of CXCR4. Our findings thereby extend the involvement of PZ from the coagulation cascade to a beneficial modulation of vascular homeostasis.
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Purpose: We evaluated the role of residual vein thrombosis (RVT) to assess the optimal duration of anticoagulants in patients with cancer who have deep vein thrombosis (DVT) of the lower limbs. Patients and methods: Patients with active cancer and a first episode of DVT treated with low molecular weight heparin (LMWH) for 6 months were eligible. Patients were managed according to RVT findings: those with RVT were randomly assigned to continue LMWH for an additional 6 months (group A1) or to discontinue it (group A2), and patients without RVT stopped LMWH (group B). The primary end point was recurrent venous thromboembolism (VTE) during the 1 year after disconinuation of LMWH, and the secondary end point was major bleeding. Analyses are from the time of random assignment. Results: Between October 2005 and April 2010, 347 patients were enrolled. RVT was detected in 242 patients (69.7%); recurrence occurred in 22 of the 119 patients in group A1compared with 27 of 123 patients in group A2. The adjusted hazard ratio (HR) for group A2 versus A1 was 1.37 (95% CI, 0.7 to 2.5; P = .311). Three of the 105 patients in group B developed recurrent VTE; adjusted HR for group A1 versus B was 6.0 (95% CI, 1.7 to 21.2; P = .005). Three major bleeding events occurred in group A1, and two events each occurred in groups A2 and B. The HR for major bleeding in group A1 versus group A2 was 3.78 (95% CI, 0.77 to 18.58; P = .102). Overall, 42 patients (12.1%) died during follow-up as a result of cancer progression. Conclusion: In patients with cancer with a first DVT, treated for 6 months with LMWH, absence of RVT identifies a population at low risk for recurrent thrombotic events. Continuation of LMWH in patients with RVT up to 1 year did not reduce recurrent VTE.
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Although SW-AT-1, a serpin-type trypsin inhibitor from silkworm (Bombyx mori), was identified in previous study, its structure-function relationship has not been studied. In this study, SW-AT-1 was cloned from the body wall of silkworm and expressed in E. coli. rSW-AT-1 inhibited both trypsin and chymotrypsin in a concentration-dependent manner. The association rate constant for rSW-AT-1 and trypsin is 1.31×10-5 M-1s-1, for rSW-AT-1 and chymotrpsin is 2.85×10-6 M-1s-1. Circular dichroism (CD) assay showed 33% α-helices, 16% β-sheets, 17% turns, and 31% random coils in the secondary structure of the protein. Enzymatic and CD analysis indicated that rSW-AT-1 was stable at wide pH range between 4-10, and exhibited the highest activity at weakly acidic or alkaline condition. The predicted three-dimensional structure of SW-AT-1 by PyMOL (v1.4) revealed a deductive reactive centre loop (RCL) near the C-terminus, which was extended from the body of the molecule. In addition to trypsin cleavage site in RCL, matrix-assisted laser desorption ionization time of flight mass spectrometry indicated that the chymotrypsin cleavage site of SW-AT-1 was between F336 and T337 in RCL. Directed mutagenesis indicated that both the N- and C-terminal sides of RCL have effects on the activity, and G327 and E329 played an important role in the proper folding of RCL. The physiological role of SW-AT-1 in the defense responses of silkworm were also discussed.
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Antithrombin, the principal inhibitor of coagulation proteases, requires allosteric activation by its physiological cofactor, heparin or heparin sulfate to achieve physiologically permissible rates. This forms the basis of heparin's use as a clinical anticoagulant. However, heparin therapy is beset with severe complications, giving rise to the need to search new non-heparin activators of antithrombin, devoid of these complications and with favorable safety profiles. We chose some representative organic compounds that have been shown to be involved in coagulation modulation by affecting antithrombin and applied a blind docking protocol to find the binding energy and interactions of the modified (sulfated) versus unmodified organic scaffolds. Increased sulfation plays a key role in shifting the specificity of organic compounds like quercetin, diosmin, rutin, mangiferin, isomangostin, Trapezifolixanthone and benzofuran towards the heparin binding site (HBS). However, in hesperetin and tetrahydroisoquinoline, sulfation shifts the specificity away from HBS. We have further tried to elucidate changes in the binding affinity of quercetin on account of gradual increase in the number of hydroxyl groups being substituted by sulfate groups. The results show gradual increase in binding energy with increase in sulfation. A theoretical screening approach is an ideal mechanism to predict lead molecules as activators of antithrombin and in determining the specificity for antithrombin.
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Maspin (mammary serine protease inhibitor), is a member of the serine protease inhibitor/non-inhibitor superfamily. Its expression is down-regulated in breast, prostate, gastric and melanoma cancers but over-expressed in pancreatic, gallbladder, colorectal, and thyroid cancers suggesting that maspin may play different activities in different cell types. However, maspin expression seems to be correlated with better prognosis in prostate, bladder, lung, gastric, colorectal, head and neck, thyroid and melanoma cancer. In breast and ovarian cancer maspin significance is associated with its subcellular localization: nucleus maspin expression correlates with a good prognosis, whilst in pancreatic cancer it predicts a poor prognosis. Since tumor metastasis requires the detachment and invasion of tumor cells through the basement membrane and stroma, a selectively increased adhesion by the presence of maspin may contribute to the inhibition of tumor metastasis. Furthermore the different position of maspin inside the cell or its epigenetic modifications may explain the different behavior of the expression of maspin between tumors. The expression of maspin might be useful as a prognostic and possibly predictive factor for patients with particular types of cancer and data can guide physicians in selecting therapy. Its expression in circulating tumor cells especially in breast cancer, could be also useful in clinical practice along with other factors, such as age, comorbidities, blood examinations in order to select the best therapy to be carried out. Focusing on the malignancies in which maspin showed a positive prognostic value, therapeutic approaches studied so far aimed to re-activate a dormant tumor suppressor gene by designed transcription factors, to hit the system that inhibits the expression of maspin, to identify natural substances that can determine the activation and the expression of maspin or possible “molecules binds” to introduce maspin in cancer cell and gene therapy capable of up-regulating the maspin in an attempt to reduce primarily the risk of metastasis. Further studies in these directions are necessary to better define the therapeutic implication of maspin.
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Prostate adenocarcinoma (CaP) patients are classified into low-, intermediate-, and high-risk groups that reflect relative survival categories. While there are accepted treatment regimens for low- and high-risk patients, intermediate-risk patients pose a clinical dilemma, as treatment outcomes are highly variable for these individuals. A better understanding of the factors that regulate the progression of CaP is required to delineate risk. For example, aberrant activation of the Hedgehog (Hh) pathway is implicated in CaP progression. Here, we identify the serine protease inhibitor protease nexin 1 (PN1) as a negative regulator of Hh signaling in prostate. Using human CaP cell lines and a mouse xenograft model of CaP, we demonstrate that PN1 regulates Hh signaling by decreasing protein levels of the Hh ligand Sonic (SHH) and its downstream effectors. Furthermore, we show that SHH expression enhanced tumor growth while overexpression of PN1 inhibited tumor growth and angiogenesis in mice. Finally, using comparative genome hybridization, we found that genetic alterations in Hh pathway genes correlated with worse clinical outcomes in intermediate-risk CaP patients, indicating the importance of this pathway in CaP.
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Endogenous angiogenesis inhibitor that specifically decreases tumor cell proliferation can be used to treat cancer since angiogenesis is required at every step of tumor progression and metastasis. Endothelial cells are the main target for the antiangiogenic therapy because they are non-transformed and easily accessible to angiogenic inhibitors. Antithrombin functions as a principal plasma protein inhibitor of blood coagulation proteinases and belongs to the family of serine protease inhibitors (serpins) which have common mechanism of inhibition. Antithrombin acquires a potent antiangiogenic activity upon conversion of the native molecule to cleaved or latent conformation. Cleaved and latent preparations of bovine and human plasma derived antithrombin inhibited capillary endothelial cell proliferation and the growth of human SK-NAS neuroblastoma and Lewis lung carcinoma tumors in mice as compared to the native antithrombin. The native form of antithrombin binds with high affinity to vascular heparan sulfate proteoglycans containing a specific pentasaccharide sequence and it is this cofactor interaction that activated antithrombin to maximal rate of thrombin inhibition. Upon inhibitory complex formation with target proteinases the antithrombin undergoes stressed to relaxed transformation and lose their high affinity for pentasacchride. Low affinity relaxed conformation with reduced heparin binding like cleaved and latent are antiangiogenic but native high affinity heparin binding stressed conformation is not, indicating the critical importance of heparin affinity in antithrombin antiangiogenic function. Based on evidence of interactions of the endothelial cell growth factors bFGF (Basic fibroblast growth factor) and VEGF (vascular endothelial cell growth factor) with heparin like molecule in matrix, the possibility of antiangiogenic antithrombin to interfere with endothelial cell growth and angiogenesis through heparin mediated mechanism deserves serious consideration and investigation. It is also possible that cleaved and latent conformations with reduced affinity for heparins can also induce conformational change in the antithrombin which can open an epitope on the antithrombin surface for appropriate interactions on the endothelial surface for better antiangiogenic activity. This review illustrates the potential of antithrombin and other serpin family members as endogenous antiangiogenic proteins.
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In vector-borne diseases, the complex interplay between pathogen and its vector's immune system determines the outcome of infection and therefore disease transmission. Serpins have been shown in many animals to be key regulators of innate immune reactions. Their control over regulatory proteolytic cascades ultimately decides whether the recognition of a pathogen will lead to an appropriate immune response. In mosquitoes, serpins (SRPNs) regulate the activation of prophenoloxidase and thus melanization, contribute to malaria parasite lysis, and likely Toll pathway activation. Additionally, in culicine mosquitoes, SRPNs are able to regulate hemostasis in the vertebrate host, suggesting a crucial role during bloodfeeding. This review summarizes the annotation, transcriptional regulation, and current knowledge of SRPN function in the three mosquito species for which the complete genome sequence is available. Additionally, we give a brief overview of how SRPNs may be used to prevent transmission of vector-borne diseases.
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We previously clarified that heparin cofactor II (HCII), a serine proteinase inhibitor, exerts various protective actions on cardiovascular diseases in both experimental and clinical studies. In the present study, we aimed to clarify whether HCII participates in the regulation of angiogenesis. Male heterozygous HCII-deficient (HCII+/−) mice and male littermate wild-type (HCII+/+) mice at the age of 12–16 weeks were subjected to unilateral hindlimb ligation surgery. Laser speckle blood flow analysis showed that blood flow recovery in response to hindlimb ischemia was delayed in HCII+/− mice compared with that in HCII+/+ mice. Capillary number, arteriole number, and endothelial nitric-oxide synthase (eNOS), AMP-activated protein kinase (AMPK), and liver kinase B1 (LKB1) phosphorylation in ischemic muscles were decreased in HCII+/− mice. Human purified HCII (h-HCII) administration almost restored blood flow recovery, capillary density, and arteriole number as well as phosphorylation levels of eNOS, AMPK, and LKB1 in ischemic muscles of HCII+/− mice. Although treatment with h-HCII increased phosphorylation levels of eNOS, AMPK, and LKB1 in human aortic endothelial cells (HAECs), the h-HCII-induced eNOS phosphorylation was abolished by compound C, an AMPK inhibitor, and by AMPK siRNA. In a similar fashion, tube formation, proliferation, and migration of HAECs were also promoted by h-HCII treatment and were abrogated by pretreatment with compound C. HCII potentiates the activation of vascular endothelial cells and the promotion of angiogenesis in response to hindlimb ischemia via an AMPK-eNOS signaling pathway. These findings suggest that HCII is a novel therapeutic target for treatment of patients with peripheral circulation insufficiency.
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Thrombotic complications of unknown etiology remain a serious diagnostic and therapeutic problem. Occurrence of the inherited polymorphisms of genes encoding proteins involved in the coagulation cascade is one of the possible causes of these complications. In recent years, protein Z (PZ) and PZ-dependent protease inhibitor (ZPI) have been added to the list of prothrombotic factors. PZ is a glycoprotein serving as a cofactor of ZPI, which is responsible for the inhibition of prothrombinase. Expression of the PZ gene is under the control of many transcriptional factors; several polymorphisms alternate the rate of gene expression. The present article describes the significance of the ZPI-PZ system in venous and arterial thrombosis, adverse pregnancy outcomes and antiphospholipid syndrome complications.
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The serpin protease nexin-1 (PN-1) is expressed by vascular cells and secreted by platelets upon activation, and it is known to interact with several modulators of angiogenesis, such as proteases, matrix proteins, and glycosaminoglycans. We therefore investigated the impact of PN-1 on endothelial cell angiogenic responses in vitro and ex vivo and in vivo in PN-1-deficient mice. We found that PN-1 is antiangiogenic in vitro: it inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell responses, including proliferation, migration, and capillary tube formation, and decreased cell spreading on vitronectin. These effects do not require the antiprotease activity of PN-1 but involve PN-1 binding to glycosaminoglycans. In addition, our results indicated that PN-1 does not act by blocking VEGF binding to its heparan sulfate proteoglycan coreceptors. The results obtained in vitro were supported ex vivo in PN-1-deficient mice, where the microvascular network sprouting from aortic rings was significantly enhanced. Moreover, in vivo, neovessel formation was promoted in the Matrigel plug assay in PN-1-deficient mice compared to wild-type mice, and these effects were reversed by the addition of recombinant PN-1. Taken together, our results demonstrate that PN-1 has direct antiangiogenic properties and is a yet-unrecognized player in the angiogenic balance.
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Serine protease inhibitors, termed serpins, are key regulators in many biologic events. Protease nexin-1 (PN-1) is a serpin that is barely detectable in plasma but found in many organs and produced by most cell types, including monocytes, platelets, and vascular cells. It has a large inhibition spectrum because it is the most efficient tissue inhibitor of thrombin but also a powerful inhibitor of plasminogen activators and plasmin. It has a high affinity for glycosaminoglycans, such as heparan sulfates, which potentiate its activity toward thrombin and target it to the pericellular space. PN-1 has been previously largely described as a crucial regulator of the proteolytic activity in nerves and of central and peripheral nervous system function. In contrast, little was known about its involvement in hemostasis and vascular biology. This article reviews recent data underlining its emerging role as a key factor in the responses of vessels to injury. Indeed, studies of PN-1-deficient mice have established important antithrombotic and antifibrinolytic properties of this serpin that have heretofore gone unrecognized. The roles of PN-1 in the areas of hemostasis and thrombosis summarized here provide insights that may allow the development of drugs and treatment strategies to prevent or limit thrombotic disorders.
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Heparin is a major anticoagulant with activity mediated primarily through its interaction with antithrombin (AT). Heparan sulfate (HS), structurally related to heparin, binds a wide range of proteins of different functionality, taking part in various physiological and pathological processes. The heparin-AT complex, the most well understood facet of anticoagulation, serves as a prototypical example of the important role of heparin/HS in vascular biology. Extensive studies have identified common structural features in heparin/HS-binding sites of proteins. These include the elucidation of consensus sequences in proteins, patterns of clusters of basic and nonbasic residues, and common spatial arrangements of basic amino acids in the heparin-binding sites. Although these studies have provided valuable information, heparin/HS-binding proteins differ widely in structure. The prediction of heparin/HS-binding proteins from sequence information is not currently possible, and elucidation of protein-binding sites requires the individual study of each glycosaminoglycan-protein complex. Thus, x-ray crystallography and site-directed mutagenesis experiments are among the most powerful tools, providing accurate structural information, facilitating the characterization of heparin-protein complexes. Heparin and structurally related heparan sulfate bind a large number of proteins, taking part in a wide range of biological processes, particularly ones involved in vascular biology. Heparin-binding domains share certain common structural features, but there is no absolute dependency on specific sequences or protein folds.
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Over the past 19 years, we have developed a novel myxoma virus-derived anti-inflammatory serine protease inhibitor, termed a serpin, as a new class of immunomodulatory therapeutic. This review will describe the initial identification of viral serpins with anti-inflammatory potential, beginning with preclinical analysis of viral pathogenesis and proceeding to cell and molecular target analyses, and successful clinical trial. The central aim of this review is to describe the development of two serpins, Serp-1 and Serp-2, as a new class of immune modulating drug, from inception to implementation. We begin with an overview of the approaches used for successful mining of the virus for potential serpin immunomodulators in viruses. We then provide a methodological overview of one inflammatory animal model used to test for serpin anti-inflammatory activity followed by methods used to identify cells in the inflammatory response system targeted by these serpins and molecular responses to serpin treatment. Finally, we provide an overview of our findings from a recent, successful clinical trial of the secreted myxomaviral serpin, Serp-1, in patients with unstable inflammatory coronary arterial disease.
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Pigment epithelium-derived factor (PEDF) is a collagen-binding protein that is abundantly distributed in various tissues, including the eye. It exhibits various biological functions, such as anti-angiogenic, neurotrophic, and neuroprotective activities. PEDF also interacts with extracellular matrix components such as collagen, heparan sulfate proteoglycans (HSPGs), and hyaluronan. The collagen-binding property has been elucidated to be important for the anti-angiogenic activity in vivo (Hosomichi, J., Yasui, N., Koide, T., Soma, K., and Morita, I. (2005) Biochem. Biophys. Res. Commun. 335, 756-761). Here, we investigated the collagen recognition mechanism by PEDF. We first narrowed down candidate PEDF-binding sequences by taking advantage of previously reported structural requirements in collagen. Subsequent searches for PEDF-binding sequences employing synthetic collagen-like peptides resulted in the identification of one of the critical binding sites for PEDF, human α1(I)(929-938) (IKGHRGFSGL). Further analysis revealed that the collagen recognition by PEDF is sequence- and conformation-specific, and the high affinity binding motif is KGXRGFXGL in the triple helix. The PEDF-binding motif significantly overlapped with the heparin/HSPG-binding motif, KGHRG(F/Y). The interaction of PEDF with collagen I was specifically competed with by heparin but not by chondroitin sulfate-C or hyaluronan. The binding sequences for PEDF and heparin/HSPG also overlapped with the covalent cross-linking sites between collagen molecules. These findings imply a functional relationship between PEDF and HSPGs during angiogenesis, and the interaction of these molecules is regulated by collagen modifications.
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The Drosophila genome encodes 29 serpins, most of unknown function. We show here that Spn1 is an active protease inhibitor of the serpin superfamily. Spn1 inhibits trypsin in vitro and regulates the Toll-mediated immune response in vivo. Expression of the Toll-dependent transcripts Drosomycin and IM1 is increased in Spn1 null mutants. Overexpression of Spn1 reduces the induction of Drosomycin upon immune challenge with fungi but not Gram-positive bacteria. Similar reductions in Drosomycin levels are observed in the psh, spz, and grass mutants of the Toll signaling pathway. These results support a role of Spn1 as a repressor of Toll activation upon fungal infection. Epistatic analysis places Spn1 upstream of Spätzle processing enzyme and Grass, in the fungal cell wall-activated side branch of the pathway. Overexpression of the pattern recognition receptor GNBP3 activates the β-1,3-glucan-sensitive side branch of the Toll pathway. The resultant increased Drosomycin level is reduced by concomitant overexpression of Spn1, confirming that Spn1 regulates the fungal cell wall side branch. Spn1 null mutants show altered susceptibility to fungal infection compared to the wild type, demonstrating a requirement for Spn1 in the fine regulation of the immune response.
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SERPINA3K is a member of the serine proteinase inhibitor (SERPIN) family. Here we evaluated the therapeutic effects of SERPINA3K on neovascularization and inflammation in a rat cornea alkali burn model that is commonly employed to study corneal wounding. Topical treatment of the injured rat cornea with SERPINA3K (20 µg/eye/day) for 7 days significantly decreased the neovascular area, compared with the groups treated with BSA or PBS. The SERPINA3K treatment also ameliorated the corneal inflammation as evaluated by the inflammatory index. Furthermore, SERPINA3K enhanced the recovery of corneal epithelium after the alkali injury. Toward the mechanism of action, SERPINA3K down-regulated the expression of the pro-angiogenic and pro-inflammatory factors, vascular endothelial growth factor and tumor necrosis factor-α and up-regulated the expression of the anti-angiogenic factor, pigment epithelium-derived factor. SERPINA3K specifically inhibited growth of vascular endothelial cells. Meanwhile, SERPINA3K significantly up-regulated the expression of EGFR in the corneal epithelium. These findings suggest that SERPINA3K has therapeutic potential for corneal inflammation and NV.
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Objectives.—To review of the state of the art relating to congenital heparin cofactor II deficiency as a potential risk factor for thrombosis, as reflected by the medical literature and the consensus opinion of recognized experts in the field, and to make recommendations for the use of laboratory assays for assessing this thrombotic risk in individual patients. Data Sources.—Review of the medical literature, primarily from the last 10 years. Data Extraction and Synthesis.—After an initial assessment of the literature, including review of clinical study design and laboratory methods, a draft manuscript was prepared and circulated to participants in the College of American Pathologists Conference XXXVI: Diagnostic Issues in Thrombophilia. Recommendations were accepted if a consensus of experts attending the conference was reached. The results of the discussion were used to revise the manuscript into its final form. Conclusions.—Consensus was reached that there is insufficient evidence to recommend testing for heparin cofactor II deficiency in patients with thromboembolic disease.
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Protein Z-dependent protease inhibitor (ZPI) is a 72-kd member of the serpin superfamily of proteinase inhibitors that produces rapid inhibition of factor Xa in the presence of protein Z (PZ), procoagulant phospholipids, and Ca++ (t1/2 less than 10 seconds). The rate of factor Xa inhibition by ZPI is reduced more than 1000-fold in the absence of PZ. The factor Xa–ZPI complex is not stable to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, but is detectable by alkaline–polyacrylamide gel electrophoresis. The combination of PZ and ZPI dramatically delays the initiation and reduces the ultimate rate of thrombin generation in mixtures containing prothrombin, factor V, phospholipids, and Ca++. In similar mixtures containing factor Va, however, PZ and ZPI do not inhibit thrombin generation. Thus, the major effect of PZ and ZPI is to dampen the coagulation response prior to the formation of the prothrombinase complex. Besides factor Xa, ZPI also inhibits factor XIa in the absence of PZ, phospholipids, and Ca++. Heparin (0.2 U/mL) enhances the rate (t1/2 = 25 seconds vs 50 seconds) and the extent (99% vs 93% at 30 minutes) of factor XIa inhibition by ZPI. During its inhibitory interaction with factor Xa and factor XIa, ZPI is proteolytically cleaved with the release of a 4.2-kd peptide. The N-terminal amino acid sequence of this peptide (SMPPVIKVDRPF) establishes Y387 as the P1 residue at the reactive center of ZPI. ZPI activity is consumed during the in vitro coagulation of plasma through a proteolytic process that involves the actions of factor Xa with PZ and factor XIa.
Article
This study was undertaken to determine the levels of protein C antigen and activity and protein C inhibitor in sequential plasma samples of disseminated intravascular coagulation (DIC) patients. Our normal range for both protein C antigen and activity is 70 to 130 U/dL, and protein C inhibitor is 65 to 135 U/dL. A decreased level of protein C activity was found in 96% of the plasma samples from individuals with DIC; the protein C antigen was decreased in 73%. The inhibitor of protein C was decreased in all samples. Analysis of serial samples from patients with DIC reveals that protein C activity and antigen and protein C inhibitor decrease progressively during the initial stages of DIC and remain at a low level for 24 to 48 hours before gradually returning toward normal in nonfatal cases. The protein C activity decreases in parallel with protein C inhibitor and is lower than protein C antigen. In a fatal case of DIC, protein C activity and protein C inhibitor rapidly decreased to undetectable levels; however, protein C antigen was gradually decreasing but still detectable at time of death. In DIC, a discrepancy initially occurs between the activity and antigen of protein C, suggesting a complex with the inhibitor or other inactive forms of protein C. Protein C appears to play a major role in the control of DIC.
Article
A deficiency of alpha 2-antiplasmin has been identified in a female patient with severe and frequent bleeding episodes. Routine coagulation and platelet assays of the patient's plasma were within normal limits. However, abnormally rapid whole blood or dilute plasma clot lysis times and an abnormal FXIII test in which clots were lysed in the presence of urea or saline suggested an abnormal fibrinolytic system. Analysis of alpha 2-antiplasmin levels by radioimmunoassay revealed less than 1.0 microgram/ml alpha 2-antiplasmin. Functional assays indicated an alpha 2-antiplasmin level less than or equal to 10% of normal. Addition of purified alpha 2-antiplasmin to the patient's plasma restored its ability to inhibit plasmin in in vitro assays, and mixtures of patient plasma with normal plasma did not interfere with the antiplasmin activity of the normal plasma. Whereas normal platelets contain 68 ng alpha 2-antiplasmin/10(9) platelets, platelets from the patient contained 30% of the normal level of antigen. Analysis of alpha 2- antiplasmin functional and antigenic levels in the plasma of both parents and four siblings of the propositus provided evidence consistent with an autosomal mechanism of inheritance of alpha 2- antiplasmin deficiency. One sibling appeared to be homozygous and three siblings and the parents were heterozygous for the deficiency. Two heterozygotes had positive bleeding histories. The association of a bleeding disorder with a deficiency of alpha 2-antiplasmin emphasizes that lack of regulation of the fibrinolytic system can result in a hemostatic dysfunction.
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Emerging data have enhanced our understanding of cancer-associated thrombosis, a major cause of morbidity and mortality in patients with cancer. This update will focus on recent findings, including the phenomenon of incidental venous thromboembolism (VTE), novel approaches to risk assessment, and the results of randomized clinical trials focusing on prophylaxis of cancer outpatients. Incidental VTE is an important contributor to rates of cancer-associated VTE and, in terms of outcomes, appears to be as consequential for patients as symptomatic VTE. Multiple biomarkers have been studied, with the highest level of evidence for prechemotherapy elevated platelet counts, elevated leukocyte counts, and low hemoglobin. Other candidate biomarkers, including D-dimer and tissue factor, are currently being evaluated. A recently validated risk score for chemotherapy-associated VTE has now been evaluated in more than 10 000 cancer patients in a variety of clinical settings and trials and is ready for clinical use (Level 1 clinical decision rule). Several randomized clinical trials in solid-tumor patients with low-molecular-weight heparins and semuloparin, an ultra-low-molecular-weight heparin, demonstrate clearly that outpatient thromboprophylaxis is feasible, safe, and effective. Selecting the appropriate patients for prophylaxis, however, continues to be a matter of controversy.
Article
Werner, Sabine, and Richard Grose. Regulation of Wound Healing by Growth Factors and Cytokines. Physiol Rev 83: 835–870, 2003; 10.1152/physrev.00032.2002.—Cutaneous wound healing is a complex process involving blood clotting, inflammation, new tissue formation, and finally tissue remodeling. It is well described at the histological level, but the genes that regulate skin repair have only partially been identified. Many experimental and clinical studies have demonstrated varied, but in most cases beneficial, effects of exogenous growth factors on the healing process. However, the roles played by endogenous growth factors have remained largely unclear. Initial approaches at addressing this question focused on the expression analysis of various growth factors, cytokines, and their receptors in different wound models, with first functional data being obtained by applying neutralizing antibodies to wounds. During the past few years, the availability of genetically modified mice has allowed elucidation of the function of various genes in the healing process, and these studies have shed light onto the role of growth factors, cytokines, and their downstream effectors in wound repair. This review summarizes the results of expression studies that have been performed in rodents, pigs, and humans to localize growth factors and their receptors in skin wounds. Most importantly, we also report on genetic studies addressing the functions of endogenous growth factors in the wound repair process.
Article
Endogenous proteins that promote vascular endothelial cell based inhibition of angiogenesis are an attractive option for antitumor therapy. Inactive cleaved and latent conformations of antithrombin (AT) are antiangiogenic, but not its native form which is an inhibitor of proteases involved in blood coagulation. Unlike native, the cleaved and latent conformations are reactive center loop inserted conformations which binds heparin with very low affinity. We use a sulfoxy modified AT to assess the role of reactive center loop insertion and heparin affinity in antiangiogenic function. Chorioallantoic membrane assay (CAM) shows that antiangiogenic activity of latent and oxidized AT are better than thalidomide, a potent antiangiogenic drug. Wound healing experiments suggest that latent and oxidized conformations can influence endothelial cell migration. Latent and cleaved conformations of AT shows an increase in α-helical content in the presence of unfractionated heparin, but not the oxidized AT. Unlike the loop inserted polymer, cleaved and latent conformations, oxidized AT has factor Xa inhibitory activity indicating that loop insertion is not necessary for antiangiogenic role. The results of our study establish that active conformation of AT can become antiangiogenic while maintaining its anticoagulant activity possibly through chelation of low affinity heparin in the vicinity of endothelial cell.