Reactivities of the S2 and S3 subsite residues of thrombin with the native and heparin-induced conformers of antithrombin.

A pentasaccharide (PS) fragment of heparin capable of activating antithrombin (AT) markedly accelerates the inhibition of factor Xa by AT, but has insignificant effect on inhibition of thrombin. For inhibition of thrombin, the bridging function of a longer polysaccharide chain is required to accelerate the reaction. To study the basis for the similar reactivity of thrombin with the native or heparin-activated conformers of AT, several residues surrounding the active site pocket of thrombin were targeted for mutagenesis study. Leu99 and Glu192, the variant residues influencing the S2 and S3 subsite specificity of thrombin were replaced with Tyr and Gln. The Tyr60a, Pro60b, Pro60c, and Trp60d residues forming part of the S2 specificity pocket were deleted from the B-insertion loop of the wild-type and Leu99/Glu192 --> Tyr/Gln thrombins. Kinetic studies indicated that the reactivities of all mutants with AT were moderately or severely impaired. Although heparin largely corrected the defect in reactivities, it also markedly elevated the stoichiometries of inhibition with the mutants. Interestingly, PS also accelerated AT inhibition of the mutants 5-68-fold, suggesting that the mutants are able to discriminate between the native and activated conformers of AT. Based on these results and the recent crystal structure determination of AT in complex with PS, a model for thrombin-AT interaction is proposed in which the S2 and S3 subsite residues of thrombin are critical for recognition of the P2 and P3 residues of AT in the native conformation. In the activated conformation, other residues are made accessible for interaction with the protease, and the similar reactivity of thrombin with the native and heparin-activated conformers of AT may be coincidental. The results further suggest that the S2 and S3 subsite residues are crucial in controlling the partitioning of the thrombin-AT intermediate into the alternative inhibitory or substrate pathways of the reaction.     

Ligand-dependent and -independent interactions with the transforming growth factor type II and I receptor subunits reside in the aminoterminal portion of the ectodomain of the type III subunit

Summary The type III receptor for transforming growth factor beta (TGFβ), which exhibits no kinase activity, binds TGFβ1 and TGFβ2 and is involved in assembly and activity of the multi-subunit TGFβ signal transduction complex. Recently we showed that TGFβ receptor type III (TβRIII) can participate in a complex composed of the dimeric TGFβ ligand and a type III, II, and I receptor subunit. The interaction of the TβRIII subunit with TβRII is TGFβ-dependent, whereas interaction with TβRI is TGFβ-independent. Here we use coexpression of the three types of TGFβ receptors in baculoviral-infected insect cells to determine which parts of the unglycosylated TβRIII receptor participate in the binding of TGFβ, the TGFβ-dependent interaction with TβRII and the TGFβ-independent interaction with TβRI. The results suggest that the first 500 amino acid residues in the aminoterminal portion of TβRIII exhibit all three properties.     

低分子肝素联合丹参注射液治疗急性心肌梗死的临床研究

目的:观察低分子肝素联合丹参注射液治疗急性ST段抬高型心肌梗死的临床疗效及安全性。方法:按照随机原则将78例急性心肌梗死患者分成两组,在常规溶栓治疗的基础上,其中对照组39人采用低分子肝素治疗,治疗组患者在对照组治疗的基础上给予丹参注射液治疗,对两组临床费用、住院时间和冠脉再通进行评价。结果:治疗组的临床费用、住院时间和冠脉再通与对照组相比,有统计学差异(P0.05)。结论:低分子肝素联合丹参注射液治疗急性心肌梗死的临床疗效确切,值得临床推广。     

A purification process for heparin and precursor polysaccharides using the pH responsive behavior of chitosan

The contamination crisis of 2008 has brought to light several risks associated with use of animal tissue derived heparin. Because the total chemical synthesis of heparin is not feasible, a bioengineered approach has been proposed, relying on recombinant enzymes derived from the heparin/HS biosynthetic pathway and Escherichia coli K5 capsular polysaccharide. Intensive process engineering efforts are required to achieve a cost‐competitive process for bioengineered heparin compared to commercially available porcine heparins. Towards this goal, we have used 96‐well plate based screening for development of a chitosan‐based purification process for heparin and precursor polysaccharides. The unique pH responsive behavior of chitosan enables simplified capture of target heparin or related polysaccharides, under low pH and complex solution conditions, followed by elution under mildly basic conditions. The use of mild, basic recovery conditions are compatible with the chemical N‐deacetylation/N‐sulfonation step used in the bioengineered heparin process. Selective precipitation of glycosaminoglycans (GAGs) leads to significant removal of process related impurities such as proteins, DNA and endotoxins. Use of highly sensitive liquid chromatography‐mass spectrometry and nuclear magnetic resonance analytical techniques reveal a minimum impact of chitosan‐based purification on heparin product composition. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1348–1359, 2015     

Heparin promotes fibrillation of most phenol-soluble modulin virulence peptides from Staphylococcus aureus

Phenol-soluble modulins (PSMs), such as α-PSMs, β-PSMs, and δ-toxin, are virulence peptides secreted by different Staphylococcus aureus strains. PSMs are able to form amyloid fibrils, which may strengthen the biofilm matrix that promotes bacterial colonization of and extended growth on surfaces (e.g., cell tissue) and increases antibiotic resistance. Many components contribute to biofilm formation, including the human-produced highly sulfated glycosaminoglycan heparin. Although heparin promotes S. aureus infection, the molecular basis for this is unclear. Given that heparin is known to induce fibrillation of a wide range of proteins, we hypothesized that heparin aids bacterial colonization by promoting PSM fibrillation. Here, we address this hypothesis using a combination of thioflavin T-fluorescence kinetic studies, CD, FTIR, electron microscopy, and peptide microarrays to investigate the mechanism of aggregation, the structure of the fibrils, and identify possible binding regions. We found that heparin accelerates fibrillation of all α-PSMs (except PSMα2) and δ-toxin but inhibits β-PSM fibrillation by blocking nucleation or reducing fibrillation levels. Given that S. aureus secretes higher levels of α-PSM than β-PSM peptides, heparin is therefore likely to promote fibrillation overall. Heparin binding is driven by multiple positively charged lysine residues in α-PSMs and δ-toxins, the removal of which strongly reduced binding affinity. Binding of heparin did not affect the structure of the resulting fibrils, that is, the outcome of the aggregation process. Rather, heparin provided a scaffold to catalyze or inhibit fibrillation. Based on our findings, we speculate that heparin may strengthen the bacterial biofilm and therefore enhance colonization via increased PSM fibrillation.     

Parathyroid hormone is a heparin/polyanion binding protein: binding energetics and structure modification

The interaction of four representative polyanions with parathyroid hormone (PTH) residues 1-84 has been investigated utilizing a variety of spectroscopic and calorimetric techniques. Each of the polyanions employed demonstrate enthalpically driven binding to PTH (1-84) with significant affinity. The polyanions heparin, dextran sulfate, phytic acid, and sucrose octasulfate induce alpha-helical structure in PTH to varying extents depending on the ratio of polyanion to protein employed. Intrinsic and extrinsic fluorescence spectroscopy suggests significant protein tertiary structure alteration upon polyanion binding. Although structural modification occurred upon polyanion binding, PTH colloidal stability was increased depending on the ratio of polyanion to protein used. Nevertheless, the bioactivity of PTH in the presence of various ratios of heparin was not altered. The potential biological significance of PTH/polyanion interactions is discussed.     

Heparin/polypyrrole (PPy) composite on gold-coated matrix for the neurite outgrowth of PC12 cells by electrical stimulation

A heparin/polypyrrole (PPy) composite, an electrical conducting polymer, was designed to enhance the interactions between a gold-coated matrix and nerve cells, with the cell (PC12 cells) interactions investigated under different conditions, both with and without electrical stimulation. The heparin concentration in the composites increased with increasing current density under the preparation condition, indicating that the heparin concentration in the composite could be controlled by managing the current density. Optical imaging showed that PC12 cells well attached to the PPy surfaces covered with heparin, but were poorly interacted to PPy surfaces without the heparin and gold coated matrix. The neurite length of the PC12 cells on the surfaces with an electrical stimulation (100 mV for 1h) significantly increased, with a median length of 77.5 μm; whereas, that without electrical stimulation was 10∼20 μm. Therefore, the heparin/polypyrrole (PPy) composite may provide insight for the development of an ideal nerve guidance channel.     

Saturation Mutagenesis of the Antithrombin Reactive Center Loop P14 Residue Supports a Three-step Mechanism of Heparin Allosteric Activation Involving Intermediate and Fully Activated States

Past studies have suggested that a key feature of the mechanism of heparin allosteric activation of the anticoagulant serpin, antithrombin, is the release of the reactive center loop P14 residue from a native state stabilizing interaction with the hydrophobic core. However, more recent studies have indicated that this structural change plays a secondary role in the activation mechanism. To clarify this role, we expressed and characterized 15 antithrombin P14 variants. The variants exhibited basal reactivities with factors Xa and IXa, heparin affinities and thermal stabilities that were dramatically altered from wild type, consistent with the P14 mutations perturbing native state stability and shifting an allosteric equilibrium between native and activated states. Rapid kinetic studies confirmed that limiting rate constants for heparin allosteric activation of the mutants were altered in conjunction with the observed shifts of the allosteric equilibrium. However, correlations of the P14 mutations'' effects on parameters reflecting the allosteric activation state of the serpin were inconsistent with a two-state model of allosteric activation and suggested multiple activated states. Together, these findings support a minimal three-state model of allosteric activation in which the P14 mutations perturb equilibria involving distinct native, intermediate, and fully activated states wherein the P14 residue retains an interaction with the hydrophobic core in the intermediate state but is released from the core in the fully activated state, and the bulk of allosteric activation has occurred in the intermediate.     

Interaction of angio-associated migratory cell protein with the TPα and TPβ isoforms of the human thromboxane A₂ receptor

In humans, thromboxane (TX) A₂ signals through the TPα and TPβ isoforms of its G-protein coupled TXA₂ receptor (TP) to mediate a host of (patho)physiologic responses. Herein, angio-associated migratory cell protein (AAMP) was identified as a novel interacting partner of both TPα and TPβ through an interaction dependent on common (residues 312-328) and unique (residues 366-392 of TPβ) sequences within their carboxyl-terminal (C)-tail domains. While the interaction was constitutive in mammalian cells, agonist-stimulation of TPα/TPβ led to a transient dissociation of AAMP from immune complexes which coincided with a transient redistribution of AAMP from its localization in an intracellular fibrous network. Although the GTPase RhoA is a downstream effector of both AAMP and the TPs, AAMP did not influence TP-mediated RhoA or vice versa. Small interfering RNA (siRNA)-mediated disruption of AAMP expression decreased migration of primary human coronary artery smooth muscle cells (1° hCoASMCs). Moreover, siRNA-disruption of AAMP significantly impaired 1° hCoASMC migration in the presence of the TXA₂ mimetic U46619 but did not affect VEGF-mediated cell migration. Given their roles within the vasculature, the identification of a specific interaction between TPα/TPβ and AAMP is likely to have substantial functional implications for vascular pathologies in which they are both implicated.