Heparin-binding Site Research Articles

Efficient drug delivery by novel cell-penetrating peptide derived from Midkine, with two heparin binding sites braced by a length-specific helix

Cell-penetrating peptides (CPPs) have been regarded as potential drug carriers for cancer therapy. However, most well-studied CPPs fail to deliver exogenous drugs efficiently and selectively. In this study, a tumour-targeted CPP with high efficiency derived from heparin-binding domain (HBD) of Midkine (named HMD) was discovered. HMD exhibited higher delivery efficiency than classic CPPs (TAT and R9) and manifested selectivity in tumour cells. Normally, the positive charge is the key factor for the transmembrane activity of CPPs such as TAT and R9. Here, the length of α-helix inside CPP was found also important for in the recognition of heparan sulphate proteoglycans (HSPGs). Subsequently, the introduction of HMD enhanced the inhibitory effect of Momordica antiviral protein of 30 kDa (MAP30) on tumour cells, resulting in a 6.07-fold and 5.42-fold increase in HeLa cells and MGC80-3 cells respectively without enhanced cytotoxicity in normal cells. These results show that HMD possesses high efficiency and good tumour specificity and can be utilised as a promising agent for the tumour-targeted delivery of drug. This study is also a supplement to the existing theories about the biological activities of the α-helix in CPPs.

High Risk of Venous Thrombosis Recurrence in Fully Anticoagulated Patient with Antithrombin Deficiency during COVID-19: A Case Report

AbstractCoagulation dysfunction is a serious issue in patients with Coronavirus disease-19 (COVID-19). With regard to recently published studies, a high number of patients with acute respiratory distress syndrome (ARDS) secondary to COVID-19 developed life-threatening thrombotic complications despite anticoagulation. We report a case of young woman with the type-II heparin-binding site (HBS) antithrombin (AT) deficiency (Budapest 3-homozygous), who developed acute deep vein thrombosis on two occasions due to COVID-19 infection in the course of stable anticoagulation with vitamin K antagonist. The first thrombotic event was observed during mild COVID-19 infection, while the second thrombotic event she developed 2 months after she was negative for severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2). Our case highlights the complexity of the treatment in this particular type of thrombophilia and the need for precaution even in mild forms of viral infection. In the treatment of acute thrombosis, AT-deficient patients may benefit from the use of AT concentrate along with low-molecular weight heparin (LMWH), while in cases of type II-HBS, AT supplementation is mandatory.

Molecular Mechanism of Inhibition of Polysialyltransferase Domain (PSTD) by Heparin.

The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. It has been proposed that NCAM polysialylation could be inhibited by two types of heparin inhibitors, low molecular heparin (LMWH) and heparin tetrasaccharide (DP4). This review summarizes how the interactions between Polysialyltransferase Domain (PSTD) in ST8SiaIV and CMP-Sia, and between the PSTD and polySia take place, and how these interactions are inhibited by LMWH and DP4. Our NMR studies indicate that LMWH is a more effective inhibitor than DP4 for inhibition of NCAM polysialylation. The NMR identification of heparin-binding sites in the PSTD may provide insight into the design of specific inhibitors of polysialylation.

Ulvan mediated VE cadherin antibody and REDV peptide co-modification to improve endothelialization potential of bioprosthetic heart valves

An aging population and a rapid increase in the incidence of degenerative valve diseases have led to greater use of bioprosthetic heart valves (BHVs). The durability of glutaraldehyde cross-linked bioprostheses currently available for clinical use is poor due to calcification, coagulation, and degradation. Decellularization can partially reduce calcification by removal of xenogenic cells, but can also lead to thrombosis, which can be addressed by further surface modification. The natural sulfated polysaccharide ulvan possesses antithrombotic and anti-inflammatory properties, and can behave as a heparinoid to immobilize proteins through their heparin binding sites. VE-cadherin antibody and the Arg-Glu-Asp-Val (REDV) peptide can facilitate selective endothelial cell attachment, adhesion and proliferation. In this study, we functionalized decellularized porcine pericardium (DPP) with ulvan, REDV, and VE-cadherin antibody (U-R-VE). Ulvan was covalently modified to act as a protective coating and spacer for VE-cadherin antibody, and to immobilize REDV. In in vitro tests, we found that functionalization significantly and selectively promoted adhesion and growth of endothelial cells while reducing platelet adhesion, inflammation, and in vitro calcification of DPPs. In an in vivo subdermal implantation model, U-R-VE modified DPP exhibited greater endothelialization potential and biocompatibility compared with unmodified pericardium. Thus, U-R-VE modification provides a promising solution to the problem of preparing BHVs with enhanced endothelialization potential.

Antibody epitopes in vaccine-induced immune thrombotic thrombocytopaenia.

Vaccine-induced immune thrombotic thrombocytopaenia (VITT) is a rare adverse effect of COVID-19 adenoviral vector vaccines1-3. VITT resembles heparin-induced thrombocytopaenia (HIT) in that it is associated with platelet-activating antibodies against platelet factor4 (PF4)4; however, patients with VITT develop thrombocytopaenia and thrombosis without exposure to heparin. Here we sought to determine the binding site on PF4 of antibodies from patients with VITT. Using alanine-scanning mutagenesis5, we found that the binding of anti-PF4 antibodies from patients with VITT (n=5) was restricted to eight surface amino acids on PF4, all of which were located within the heparin-binding site, and that the binding was inhibited by heparin. By contrast, antibodies from patients with HIT (n=10) bound to amino acids that corresponded to two different sites on PF4. Biolayer interferometry experiments also revealed that VITT anti-PF4 antibodies had a stronger binding response to PF4 and PF4-heparin complexes than did HIT anti-PF4 antibodies, albeit with similar dissociation rates. Our data indicate that VITT antibodies can mimic the effect of heparin by binding to a similar site on PF4; this allows PF4 tetramers to cluster and form immune complexes, which in turn causes Fcγ receptorIIa (FcγRIIa; also known as CD32a)-dependent platelet activation. These results provide an explanation for VITT-antibody-induced platelet activation that could contribute to thrombosis.

ACE2-Independent Interaction of SARS-CoV-2 Spike Protein with Human Epithelial Cells Is Inhibited by Unfractionated Heparin.

Coronaviruses such as SARS-CoV-2, which is responsible for COVID-19, depend on virus spike protein binding to host cell receptors to cause infection. The SARS-CoV-2 spike protein binds primarily to ACE2 on target cells and is then processed by membrane proteases, including TMPRSS2, leading to viral internalisation or fusion with the plasma membrane. It has been suggested, however, that receptors other than ACE2 may be involved in virus binding. We have investigated the interactions of recombinant versions of the spike protein with human epithelial cell lines that express low/very low levels of ACE2 and TMPRSS2 in a proxy assay for interaction with host cells. A tagged form of the spike protein containing the S1 and S2 regions bound in a temperature-dependent manner to all cell lines, whereas the S1 region alone and the receptor-binding domain (RBD) interacted only weakly. Spike protein associated with cells independently of ACE2 and TMPRSS2, while RBD required the presence of high levels of ACE2 for interaction. As the spike protein has previously been shown to bind heparin, a soluble glycosaminoglycan, we tested the effects of various heparins on ACE2-independent spike protein interaction with cells. Unfractionated heparin inhibited spike protein interaction with an IC50 value of <0.05 U/mL, whereas two low-molecular-weight heparins were less effective. A mutant form of the spike protein, lacking the arginine-rich putative furin cleavage site, interacted only weakly with cells and had a lower affinity for unfractionated and low-molecular-weight heparin than the wild-type spike protein. This suggests that the furin cleavage site might also be a heparin-binding site and potentially important for interactions with host cells. The glycosaminoglycans heparan sulphate and dermatan sulphate, but not chondroitin sulphate, also inhibited the binding of spike protein, indicating that it might bind to one or both of these glycosaminoglycans on the surface of target cells.

Activating Adenosine Monophosphate-Activated Protein Kinase Mediates Fibroblast Growth Factor 1 Protection From Nonalcoholic Fatty Liver Disease in Mice.

Fibroblast growth factor (FGF) 1 demonstrated protection against nonalcoholic fatty liver disease (NAFLD) in type 2 diabetic and obese mice by an uncertain mechanism. This study investigated the therapeutic activity and mechanism of a nonmitogenic FGF1 variant carrying 3 substitutions of heparin-binding sites (FGF1△HBS ) against NAFLD. FGF1△HBS administration was effective in 9-month-old diabetic mice carrying a homozygous mutation in the leptin receptor gene (db/db) with NAFLD; liver weight, lipid deposition, and inflammation declined and liver injury decreased. FGF1△HBS reduced oxidative stress by stimulating nuclear translocation of nuclear erythroid 2 p45-related factor 2 (Nrf2) and elevation of antioxidant protein expression. FGF1△HBS also inhibited activity and/or expression of lipogenic genes, coincident with phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and its substrates. Mechanistic studies on palmitate exposed hepatic cells demonstrated that NAFLD-like oxidative damage and lipid accumulation could be reversed by FGF1△HBS . In palmitate-treated hepatic cells, small interfering RNA (siRNA) knockdown of Nrf2 abolished only FGF1△HBS antioxidative actions but not improvement of lipid metabolism. In contrast, AMPK inhibition by pharmacological agent or siRNA abolished FGF1△HBS benefits on both oxidative stress and lipid metabolism that were FGF receptor (FGFR) 4 dependent. Further support of these in vitro findings is that liver-specific AMPK knockout abolished therapeutic effects of FGF1△HBS against high-fat/high-sucrose diet-induced hepatic steatosis. Moreover, FGF1△HBS improved high-fat/high-cholesterol diet-induced steatohepatitis and fibrosis in apolipoprotein E knockout mice. These findings indicate that FGF1△HBS is effective for preventing and reversing liver steatosis and steatohepatitis and acts by activation of AMPK through hepatocyte FGFR4.

Rapid evolution of mammalian APLP1 as a synaptic adhesion molecule

Amyloid precursor protein (APP) family members are involved in essential neuronal development including neurite outgrowth, neuronal migration and maturation of synapse and neuromuscular junction. Among the APP gene family members, amyloid precursor-like protein 1 (APLP1) is selectively expressed in neurons and has specialized functions during synaptogenesis. Although a potential role for APLP1 in neuronal evolution has been indicated, its precise evolutionary and functional contributions are unknown. This study shows the molecular evolution of the vertebrate APP family based on phylogenetic analysis, while contrasting the evolutionary differences within the APP family. Phylogenetic analysis showed 15 times higher substitution rate that is driven by positive selection at the stem branch of the mammalian APLP1, resulting in dissimilar protein sequences compared to APP/APLP2. Docking simulation identified one positively selected site in APLP1 that alters the heparin-binding site, which could affect its function, and dimerization rate. Furthermore, the evolutionary rate covariation between the mammalian APP family and synaptic adhesion molecules (SAMs) was confirmed, indicating that only APLP1 has evolved to gain synaptic adhesion property. Overall, our results suggest that the enhanced synaptogenesis property of APLP1 as one of the SAMs may have played a role in mammalian brain evolution.

Optimized expression and purification of a soluble BMP2 variant based on in-silico design

Bone morphogenetic protein 2 (BMP21) is a highly interesting therapeutic growth factor due to its strong osteogenic/osteoinductive potential. However, its pronounced aggregation tendency renders recombinant and soluble production troublesome and complex. While prokaryotic expression systems can provide BMP2 in large amounts, the typically insoluble protein requires complex denaturation-renaturation procedures with medically hazardous reagents to obtain natively folded homodimeric BMP2. Based on a detailed aggregation analysis of wildtype BMP2, we designed a hydrophilic variant of BMP2 additionally containing an improved heparin binding site (BMP2-2Hep-7M). Consecutive optimization of BMP2-2Hep-7M expression and purification enabled production of soluble dimeric BMP2-2Hep-7M in high yield in E. coli. This was achieved by a) increasing protein hydrophilicity via introducing seven point mutations within aggregation hot spots of wildtype BMP2 and a longer N-terminus resulting in higher affinity for heparin, b) by employing E. coli strain SHuffle® T7, which enables the structurally essential disulfide-bond formation in BMP2 in the cytoplasm, c) by using BMP2 variant characteristic soluble expression conditions and application of l-arginine as solubility enhancer. The BMP2 variant BMP2-2Hep-7M shows strongly attenuated although not completely eliminated aggregation tendency.

Competitive coordination of the dual roles of the Hedgehog co-receptor in homophilic adhesion and signal reception.

Hedgehog (Hh) signaling patterns embryonic tissues and contributes to homeostasis in adults. In Drosophila, Hh transport and signaling are thought to occur along a specialized class of actin-rich filopodia, termed cytonemes. Here, we report that Interference hedgehog (Ihog) not only forms a Hh receptor complex with Patched to mediate intracellular signaling, but Ihog also engages in trans-homophilic binding leading to cytoneme stabilization in a manner independent of its role as the Hh receptor. Both functions of Ihog (trans-homophilic binding for cytoneme stabilization and Hh binding for ligand sensing) involve a heparin-binding site on the first fibronectin repeat of the extracellular domain. Thus, the Ihog-Ihog interaction and the Hh-Ihog interaction cannot occur simultaneously for a single Ihog molecule. By combining experimental data and mathematical modeling, we determined that Hh-Ihog heterophilic interaction dominates and Hh can disrupt and displace Ihog molecules involved in trans-homophilic binding. Consequently, we proposed that the weaker Ihog-Ihog trans interaction promotes and stabilizes direct membrane contacts along cytonemes and that, as the cytoneme encounters secreted Hh ligands, the ligands trigger release of Ihog from trans Ihog-Ihog complex enabling transport or internalization of the Hh ligand-Ihog-Patched -receptor complex. Thus, the seemingly incompatible functions of Ihog in homophilic adhesion and ligand binding cooperate to assist Hh transport and reception along the cytonemes.

Investigation of the Differences in Antithrombin to Heparin Binding among Antithrombin Budapest 3, Basel, and Padua Mutations by Biochemical and In Silico Methods.

Antithrombin (AT) is a serine protease inhibitor, its activity is highly accelerated by heparin. Mutations at the heparin-binding region lead to functional defect, type II heparin-binding site (IIHBS) AT deficiency. The aim of this study was to investigate and compare the molecular background of AT Budapest 3 (p.Leu131Phe, ATBp3), AT Basel (p.Pro73Leu), and AT Padua (p.Arg79His) mutations. Advanced in silico methods and heparin-binding studies of recombinant AT proteins using surface plasmon resonance method were used. Crossed immunoelectrophoresis and Differential Scanning Fluorimetry (NanoDSF) were performed in plasma samples. Heparin affinity of AT Padua was the lowest (KD = 1.08 × 10−6 M) and had the most severe consequences affecting the allosteric pathways of activation, moreover significant destabilizing effects on AT were also observed. KD values for AT Basel, ATBp3 and wild-type AT were 7.64 × 10−7 M, 2.15 × 10−8 M and 6.4 × 10−10 M, respectively. Heparin-binding of AT Basel was slower, however once the complex was formed the mutation had only minor effect on the secondary and tertiary structures. Allosteric activation of ATBp3 was altered, moreover decreased thermostability in ATBp3 homozygous plasma and increased fluctuations in multiple regions of ATBp3 were observed by in silico methods suggesting the presence of a quantitative component in the pathogenicity of this mutation due to molecular instability.

SARS-CoV-2 spike protein interactions with amyloidogenic proteins: Potential clues to neurodegeneration

The post-infection of COVID-19 includes a myriad of neurologic symptoms including neurodegeneration. Protein aggregation in brain can be considered as one of the important reasons behind the neurodegeneration. SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) binds to heparin and heparin binding proteins. Moreover, heparin binding accelerates the aggregation of the pathological amyloid proteins present in the brain. In this paper, we have shown that the SARS-CoV-2 S1 RBD binds to a number of aggregation-prone, heparin binding proteins including Aβ, α-synuclein, tau, prion, and TDP-43 RRM. These interactions suggests that the heparin-binding site on the S1 protein might assist the binding of amyloid proteins to the viral surface and thus could initiate aggregation of these proteins and finally leads to neurodegeneration in brain. The results will help us to prevent future outcomes of neurodegeneration by targeting this binding and aggregation process.

Towards better understanding of the heparin role in NETosis: Feasibility of using native mass spectrometry to monitor interactions of neutrophil elastase with heparin oligomers

Neutrophil elastase is a serine protease released by neutrophils, and its dysregulation has been associated with a variety of debilitating pathologies, most notably cystic fibrosis. This protein is also a prominent component of the so-called neutrophil extracellular traps (NETs), whose formation is a part of the innate immunity response to invading pathogens, but also contributes to a variety of pathologies ranging from autoimmune disorders and inflammation to cancer to thrombotic complications in COVID-19. Retention of neutrophil elastase within NETs is provided by ejected DNA chains, although this protein is also capable of interacting with a range of other endogenous polyanions, such as heparin and heparan sulfate. In this work, we evaluate the feasibility of using native mass spectrometry (MS) as a means of studying interactions of neutrophil elastase with heparin oligomers ranging from structurally homogeneous synthetic pentasaccharide fondaparinux to relatively long (up to twenty saccharide units) and structurally heterogeneous chains produced by partial depolymerization of heparin. The presence of heterogeneous N-glycan chains within neutrophil elastase and the structural heterogeneity of heparin oligomers render the use of standard MS to study their complexes impractical. However, supplementing MS with limited charge reduction in the gas phase allows meaningful data to be extracted from MS measurements. In contrast to earlier molecular modeling studies where a single heparin-binding site was identified, our work reveals the existence of multiple binding sites, with a single protein molecule being able to accommodate up to three decasaccharides. The measurements also reveal the ability of even relatively short heparin oligomers to bridge two protein molecules, suggesting that characterization of these complexes using native MS can shed light on the structural properties of NETs. Lastly, the use of MS allows the binding preferences of heparin oligomers to neutrophil elastase to be studied with respect to specific structural properties of heparin, such as the level of sulfation (i.e., charge density). All experimental measurements are carried out in parallel with molecular dynamics simulations of the protein/heparin oligomer systems, which are in remarkable agreement with the experimental data and highlight the role of electrostatic interactions as dominant forces governing the formation of these complexes.

Binding of the human antioxidation protein α1-microglobulin (A1M) to heparin and heparan sulfate. Mapping of binding site, molecular and functional characterization, and co-localization in vivo and in vitro

Heparin and heparan sulfate (HS) are linear sulfated disaccharide polymers. Heparin is found mainly in mast cells, while heparan sulfate is found in connective tissue, extracellular matrix and on cell membranes in most tissues. α1-microglobulin (A1M) is a ubiquitous protein with thiol-dependent antioxidant properties, protecting cells and matrix against oxidative damage due to its reductase activities and radical- and heme-binding properties. In this work, it was shown that A1M binds to heparin and HS and can be purified from human plasma by heparin affinity chromatography and size exclusion chromatography. The binding strength is inversely dependent of salt concentration and proportional to the degree of sulfation of heparin and HS. Potential heparin binding sites, located on the outside of the barrel-shaped A1M molecule, were determined using hydrogen deuterium exchange mass spectrometry (HDX-MS). Immunostaining of endothelial cells revealed pericellular co-localization of A1M and HS and the staining of A1M was almost completely abolished after treatment with heparinase. A1M and HS were also found to be co-localized in vivo in the lungs, aorta, kidneys and skin of mice. The redox-active thiol group of A1M was unaffected by the binding to HS, and the cell protection and heme-binding abilities of A1M were slightly affected. The discovery of the binding of A1M to heparin and HS provides new insights into the biological role of A1M and represents the basis for a novel method for purification of A1M from plasma.

Age and Origin of the Founder Antithrombin Budapest 3 (p.Leu131Phe) Mutation; Its High Prevalence in the Roma Population and Its Association With Cardiovascular Diseases.

Background: Antithrombin (AT) is one of the most important regulator of hemostasis. AT Budapest 3 (ATBp3) is a prevalent type II heparin-binding site (IIHBS) deficiency due to founder effect. Thrombosis is a complex disease including arterial (ATE) and venous thrombotic events (VTE) and the Roma population, the largest ethnic minority in Europe has increased susceptibility to these diseases partly due to their unfavorable genetic load. We aimed to calculate the age and origin of ATBp3 and to explore whether the frequency of it is higher in the Roma population as compared with the general population from the corresponding geographical area. We investigated the association of ATBp3 with thrombotic events in well-defined patients' populations in order to refine the recommendation when testing for ATBp3 is useful.Methods and Results: Prevalence of ATBp3, investigated in large samples (n = 1,000 and 1,185 for general Hungarian and Roma populations, respectively) was considerably high, almost 3%, among Roma and the founder effect was confirmed in their samples, while it was absent in the Hungarian general population. Age of ATBp3—as calculated by analysis of 8 short tandem repeat sequences surrounding SERPINC1—was dated back to XVII Century, when Roma migration in Central and Eastern Europe occurred. In our IIHBS cohort (n = 230), VTE was registered in almost all ATBp3 homozygotes (93%) and in 44% of heterozygotes. ATE occurred with lower frequency in ATBp3 (around 6%); it was rather associated with AT Basel (44%). All patients with ATE were young at the time of diagnosis. Upon investigating consecutive young (<40 years) patients with ATE (n = 92) and VTE (n = 110), the presence of ATBp3 was remarkable.Conclusions: ATBp3, a 400-year-old founder mutation is prevalent in Roma population and its Roma origin can reasonably be assumed. By the demonstration of the presence of ATBp3 in ATE patients, we draw the attention to consider type IIHBS AT deficiency in the background of not only VTE but also ATE, especially in selected populations as young patients without advanced atherosclerosis. We recommend including the investigation of ATBp3 as part of thrombosis risk assessment and stratification in Roma individuals.

Evidence of a putative glycosaminoglycan binding site on the glycosylated SARS-CoV-2 spike protein N-terminal domain

SARS-CoV-2 has rapidly spread throughout the world’s population since its initial discovery in 2019. The virus infects cells via a glycosylated spike protein located on its surface. The protein primarily binds to the angiotensin-converting enzyme-2 (ACE2) receptor, using glycosaminoglycans (GAGs) as co-receptors. Here, we performed bioinformatics and molecular dynamics simulations of the spike protein to investigate the existence of additional GAG binding sites on the receptor-binding domain (RBD), separate from previously reported heparin-binding sites. A putative GAG binding site in the N-terminal domain (NTD) of the protein was identified, encompassing residues 245–246. We hypothesized that GAGs of a sufficient length might bridge the gap between this site and the PRRARS furin cleavage site, including the mutation S247R. Docking studies using GlycoTorch Vina and subsequent MD simulations of the spike trimer in the presence of dodecasaccharides of the GAGs heparin and heparan sulfate supported this possibility. The heparan sulfate chain bridged the gap, binding the furin cleavage site and S247R. In contrast, the heparin chain bound the furin cleavage site and surrounding glycosylation structures, but not S247R. These findings identify a site in the spike protein that favors heparan sulfate binding that may be particularly pertinent for a better understanding of the recent UK and South African strains. This will also assist in future targeted therapy programs that could include repurposing clinical heparan sulfate mimetics.

Molecular Targeting of VEGF with a Suramin Fragment-DOCA Conjugate by Mimicking the Action of Low Molecular Weight Heparins.

Molecular targeting of growth factors has shown great therapeutic potential in pharmaceutical research due to their roles in pathological conditions. In the present study, we developed a novel suramin fragment and deoxycholic acid conjugate (SFD) that exhibited the potential to bind to the heparin-binding site (HBD) of vascular endothelial growth factor (VEGF) and to inhibit its pathogenic action for the first time. Notably, SFD was optimally designed for binding to the HBD of VEGF using the naphthalenetrisulfonate group, allowing to observe its excellent binding efficacy in a surface plasmon resonance (SPR) study, showing remarkable binding affinity (KD = 3.8 nM) as a small molecule inhibitor. In the tubular formation assay, it was observed that SFD could bind to HBD and exhibit antiangiogenic efficacy by inhibiting VEGF, such as heparins. The cellular treatment of SFD resulted in VEGF-inhibitory effects in human umbilical vein endothelial cells (HUVECs). Therefore, we propose that SFD can be employed as a novel drug candidate to inhibit the pathophysiological action of VEGF in diseases. Consequently, SFD, which has a molecular structure optimized for binding to HBD, is put forward as a new chemical VEGF inhibitor.

Heparin Binding to Resting and Activated Platelets

AbstractHeparin inhibits platelet function and can cause thrombocytopenia. In an effort to understand these phenomena, we have measured the binding of (3H)-heparin to resting and stimulated platelets. In platelet-rich plasma, a single class of saturable heparin binding sites was observed (apparent dissociation constant [kd] ~ 0.55μg/mL, R ~ 0.059 μg/109 cells). In gel-filtered platelets, a similar class of sites was present but with a greater binding capacity (apparent kd1 ~ 0.56 μg/mL, R ~ 0.44 μg/109 cells). Gel-filtered platelets that had been stimulated with thrombin displayed two classes of binding sites: a high-affinity class (apparent kd1 ~1.1 μg/mL, R1 ~ 0.39 μg/109 cells) corresponding to that of the unstimulated cells, and a low-affinity class (apparent kd2 ~13 μg/mL, R2 ~ 2.2 μg/109 cells). Heparin binding was also increased in platelet-rich plasma when the cells had been stimulated by adenosine diphosphate (ADP) to release, but not when ADP caused primary aggregation without release. Binding was not dependent on extracellular calcium, nor was it reduced by monoclonal antibodies to platelet membrane glycoproteins la/Ila, lb, llb/llla, or IV. Because the apparent dissociation constant of the high-affinity sites (~0.55 μg/mL) falls in the range of heparin concentrations achieved clinically, these binding sites may be involved in the platelet dysfunction and immune-mediated thrombocytopenia associated with therapeutic heparin. The low-affinity, high-capacity class of sites, which appears after cell stimulation, may participate in the process of platelet adhesion.

The Tat Protein of Human Immunodeficiency Virus Type-1 Promotes Vascular Cell Growth and Locomotion by Engaging the α5β1 and αvβ3 Integrins and by Mobilizing Sequestered Basic Fibroblast Growth Factor

AbstractThe Tat protein of human immunodeficiency virus type-1 (HIV-1) has been shown to be released during acute infection of T cells by HIV-1 and to promote angiogenesis and Kaposi's sarcoma (KS) development in infected individuals. In this study, we investigated the molecular mechanisms responsible for the angiogenic effects of Tat. The results shown herein indicate that two different Tat domains cooperate to induce these effects by different pathways. The arginine-glycine-aspartic acid (RGD) sequence present at the carboxyterminal of Tat mediates vascular cell migration and invasion by binding to the α5β1 and αvβ3 integrins. This interaction also provides endothelial cells with the adhesion signal they require to grow in response to mitogens. At the same time, the Tat basic sequence retrieves into a soluble form extracellular basic fibroblast growth factor (bFGF) bound to heparan sulfate proteoglycans by competing for heparin-binding sites. This soluble bFGF mediates Tat-induced vascular cell growth. These effects resemble those of extracellular matrix proteins, suggesting that Tat enhances angiogenesis and promotes KS progression by a molecular mimicry of these molecules.

High resolution structure of human apolipoprotein (a) kringle IV type 2: beyond the lysine binding site

Lipoprotein (a) [Lp(a)] is characterized by an LDL-like composition in terms of lipids and apoB100, and by one copy of a unique glycoprotein, apo(a). The apo(a) structure is mainly based on the repetition of tandem kringle domains with high homology to plasminogen kringles 4 and 5. Among them, kringle IV type 2 (KIV-2) is present in a highly variable number of genetically encoded repeats, whose length is inversely related to Lp(a) plasma concentration and cardiovascular risk. Despite it being the major component of apo(a), the actual function of KIV-2 is still unclear. Here, we describe the first high-resolution crystallographic structure of this domain. It shows a general fold very similar to other KIV domains with high and intermediate affinity for the lysine analog, ε-aminocaproic acid. Interestingly, KIV-2 presents a lysine binding site (LBS) with a unique shape and charge distribution. KIV-2 affinity for predicted small molecule binders was found to be negligible in surface plasmon resonance experiments; and with the LBS being nonfunctional, we propose to rename it "pseudo-LBS". Further investigation of the protein by computational small-molecule docking allowed us to identify a possible heparin-binding site away from the LBS, which was confirmed by specific reverse charge mutations abolishing heparin binding. This study opens new possibilities to define the pathogenesis of Lp(a)-related diseases and to facilitate the design of specific therapeutic drugs.