Structural Basis of Regulation of von Willebrand Factor Binding to Glycoprotein Ib

Activation by elongational flow of von Willebrand factor (VWF) is critical for primary hemostasis. Mutations causing type 2B von Willebrand disease (VWD), platelet-type VWD (PT-VWD), and tensile force each increase affinity of the VWF A1 domain and platelet glycoprotein Ibα (GPIbα) for one another; however, the structural basis for these observations remains elusive. Directed evolution was used to discover a further gain-of-function mutation in A1 that shifts the long range disulfide bond by one residue. We solved multiple crystal structures of this mutant A1 and A1 containing two VWD mutations complexed with GPIbα containing two PT-VWD mutations. We observed a gained interaction between A1 and the central leucine-rich repeats (LRRs) of GPIbα, previously shown to be important at high shear stress, and verified its importance mutationally. These findings suggest that structural changes, including central GPIbα LRR-A1 contact, contribute to VWF affinity regulation. Among the mutant complexes, variation in contacts and poor complementarity between the GPIbα β-finger and the region of A1 harboring VWD mutations lead us to hypothesize that the structures are on a pathway to, but have not yet reached, a force-induced super high affinity state.     

中国鄂温克族和达斡尔族人群中血小板膜糖蛋白Ib(GPIb)的多态性

研究旨在进一步探索同一人种的不同民族之间GPIb的多态性是否存在差异。血小板从84名健康鄂温克族,85名达斡尔族青年志愿采血分离而获得,血小板样品经SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）后,再转移至硝酸纤维素膜上（Western blotting),用生物素化的素胚凝集素（WGA/Bio)偶联辣根过氧化酶复合物试剂盒（ABCkit)分步孵育,再以4-氯-1-萘酚显影,膜上呈现的深灰色带即是GPIbα链,其分子量分别为141、136、132、128kD,此即A、B、C、D四型,此为基因型,其中每两型构成一个表型,本研究证明,在我国鄂温克族人群中,等位基因A、B、C、D的频率分别为0.113,0.375,0.381,0.131;在达斡尔族中,A型为0.124,B型为0.406,C型为0.359。D型为0.112。经统计学分析。在这两个民族的人群之间,其GPIb各基因频率无显性差异。两个民族人群的GPIb表型经卡方检验也无显性差异。由此结果说明,鄂温克,达斡尔两个民族的人群之间,GPIb的多态性特征是相似的。     

Involvement of platelet glycoprotein Ib in platelet microparticle mediated neutrophil activation

Summary Platelet microparticles (MPs) are membrane vesicles shed by platelets after activation, and carry antigens characteristic of intact platelets, such as glycoprotein (GP) IIb/IIIa, GPIb and P-selectin. Elevated platelet MPs have been observed in many disorders in which platelet activation is documented. Recently, platelet GPIb has been implicated in the mediation of platelet–leukocyte interaction via binding to its ligand Mac-1 on leukocyte. The role of GPIb for mediating adhesion-activation interactions between platelet MPs and leukocytes has not been clarified. In this study we investigate the role of GPIb in the interplay between platelet MPs and neutrophils. Platelet MPs were obtained from collagen-stimulated platelet-rich plasma (PRP). In a study model of neutrophil aggregation, platelet MPs can serve a bridge to support neutrophil aggregation under venous level shear stress, suggesting that platelet MPs may enhance leukocyte aggregation, which would bear clinical relevance in diseases where the platelet MPs are elevated. The level of aggregation can be reduced by GPIb blocking antibodies, AP1 and SZ2, but not by anti-CD18 mAb. The GPIb blocking antibodies also decreased platelet MP-mediated neutrophil activation, including β2 integrin expression, adherence-dependent superoxide release and platelet MP-mediated neutrophil adherence to immobilized fibrinogen. Our data provide the evidence for the involvement of GPIb–Mac-1 interaction in the cross-talk between platelet MPs and neutrophils.     

Hydrogen peroxide release from human blood platelets

The release of hydrogen peroxide from human blood platelets after stimulation with particulate membrane-perturbing agents has been determined by fluorescence using scopoletin as the detecting agent. Platelet suspensions containing less than 1 polymorphonuclear leukocyte/10⁸ platelets showed a significant release of hydrogen peroxide (6.11 nmol/10⁹ platelets per 20 min, S.D., 0.26, n=9) after addition of zymosan or latex particles, compared to unstimulated platelets. The release of hydrogen peroxide was only observed when the scopoletin was added to the platelet suspensions during the stimulation. Any attempt to determine hydrogen peroxide release in the supernatant at the end of the incubation with zymosan or latex failed. A NADH-dependent production of hydrogen peroxide was observed by measuring the difference of oxygen uptake in the presence and absence of catalase (500 units), which was not inhibited by potassium cyanide (1 mM). By this method the NADH-dependent cyanide-insensitive peroxide production and release was 6.0 nmol/10⁹ platelets per 20 min from resting platelets (S.D., 2, n=6) vs. 15 nmol/10⁹ platelets per 20 min from stimulated platelets (S.D., 2, n=6).     

Depression is associated with an increase in the expression of the platelet adhesion receptor glycoprotein Ib

There is a significant association between cardiovascular disease and depression. Previous studies have documented changes in platelets in depression. It is unknown if depression causes functional changes in platelet surface receptors. Therefore, we analyzed (1) the surface expression of glycoprotein (GP)Ib and the integrin receptor _IIbβ_IIIa, receptors involved in platelet adhesion and aggregation, (2) CD62 (P-selectin) and CD63, integral granule proteins translocated during platelet activation, (3) platelet aggregation in response to ADP and (4) plasma levels of glycocalicin and von Willebrand factor (vWF), in depressed patients compared to healthy volunteers. Fifteen depressed patients with a Hamilton depression score of at least 22 and fifteen control subjects were studied. Platelets were assessed for surface expression levels of GPIb, _IIbβ_IIIa, CD62 and CD63 by flow cytometry. Genomic DNA was isolated to investigate a recently described polymorphism in the 5’ untranslated region of the GPIb gene. The number of GPIb receptors was significantly increased on the surface of platelets from patients with depression compared to control subjects. Surface expression of CD62 was also significantly increased in the depressed patients versus control subjects. There was no significant difference between depressed patients and healthy volunteers in the surface expression of _IIbβ_IIIa or CD63, or in glycocalicin or vWF plasma concentration, or ADP-induced aggregation. There was no difference in allele frequency of the Kozak region polymorphism of the GPIb gene, which can affect GPIb expression. The results of this study demonstrate that the number of GPIb receptors on platelets are increased in depression and suggest a novel risk factor for thrombosis in patients with depression.     

Cyclic AMP-binding proteins in human blood platelets detected by photoaffinity labelling

Cyclic AMP inhibits platelet aggregation induced by physiological agents. 8 Azido [³²P]cyclic AMP (N₃ cyclic AMP) has been utilized as a photoaffinity probe to define the cyclic AMP-binding proteins present in unperturbed human platelets and their subcellular fractions. Specificity of cyclic AMP binding was determined by contrasting binding in the presence and absence of excess unlabelled cyclic AMP, cyclic GMP and 5′-AMP. Binding was unaffected by 5′-AMP and obliterated by cyclic AMP. Four major species of binding proteins, 49 000, 42 000, 39 000, 37 000, were obtained in all platelet fractions (crude homeogenate, cytosol, membranes and granules). Two-dimensional gel electrophoresis of platelet cytosol resolved the major molecular weight species into 15 specific cyclic AMP binding proteins of four molecular weight classes differing by charge density. These studies suggest that platelets contain an array of specific cyclic AMP-binding proteins which may function in hemostatic regulation.     

Crosslinking of platelet glycoprotein Ib by N-succinimidyl(4-azidophenyldithio)propionate and 3,3′-dithiobis(sulfosuccinimidyl propionate)

To examine the relationship between glycoprotein Ib and other proteins in the platelet membrane and the interaction of this protein with thrombin, platelets were crosslinked by two cleavable reagents, SADP (N-succinimidyl(4-azidophenyldithio)propionate) and DTSSP (3,3′-dithiobis(sulfosuccinimidyl propionate)). Two-dimensional, unreduced-reduced sodium dodecyl sulphate (SDS)-polyacrylamide electrophoresis and staining by silver or wheat germ agglutinin-conjugated peroxidase, after protein transfer to nitrocellulose, demonstrated that SADP intramolecularly crosslinked glycoprotein Ib and formed intermolecular complexes of glycoprotein IIb and some high molecular weight proteins. DTSSP intermolecularly crosslinked glycoprotein Ib, glycoprotein IIb, and other high molecular weight proteins. With a low concentration of ¹²⁵I-labeled TLCK-thrombin (6 nM), crosslinking with SADP yielded a 200 000 Da complex containing radioactive-labeled thrombin, and high TLCK-thrombin concentration (0.1 μM) gave the complex and a 167 000 band. α- and TLCK-thrombin crosslinking with DTSSP also yielded the 200 000 complex, with the remaining radioactivity in a band corresponding to a highly crosslinked complex. The 200 000 complex formed by reaction with SADP or DTSSP was markedly reduced by preincubation of platelets with excess unlabeled TLCK-thrombin and had a pI similar to glycoprotein Il. These results suggest that glycoprotein Il is one of the proteins composing the high affinity receptor for thrombin.     

Evidence for the presence of tropomyosin in the cytoskeletons of ADP- and thrombin-stimulated blood platelets

Stimulation of porcine platelets with ADP or thrombin and subsequent analyses of their cytoskeletons by SDS-polyacrylamide gel electrophoresis have shown the presence of a 30.5-kDa polypeptide in the cytoskeletons of activated as well as aggregated platelets. This polypeptide comigrates with pure porcine platelet tropomyosin in SDS gels, their mobilities being similarly and markedly decreased in the presence of 6 M urea. One-dimensional peptide mapping after limited proteolysis by Staphylococcus aureus protease gives the same pattern for pure tropomyosin and the 30.5-kDa polypeptide. This latter may thus be identified as the porcine platelet tropomyosin subunit, the role of which may not be solely structural.     

Metallokinetic characteristics of antidiabetic bis(allixinato)oxovanadium(IV)-related complexes in the blood of rat

The antidiabetic effect of vanadium is a widely accepted phenomenon; some oxovanadium(IV) complexes have been found to normalize high blood glucose levels in both type 1 and type 2 diabetic animals. In light of the future clinical use of these complexes, the relationship among their chemical structures, physicochemical properties, metallokinetics, and antidiabetic activities must be closely investigated. Recently, we found that among bis(3-hydroxypyronato)oxovanadium(IV) [VO(3hp)₂] related complexes, bis(allixinato)oxovanadium(IV) [VO(alx)₂] exhibits a relatively strong hypoglycemic effect in diabetic animals. Next, we examined its metallokinetics in the blood of rats that received five VO(3hp)₂-related complexes by the blood circulation monitoring–electron paramagnetic resonance method. The metallokinetic parameters were obtained from the blood clearance curves based on a two-compartment model; most parameters, such as area under the concentration curve and mean residence time, correlated significantly with the in vitro insulinomimetic activity in terms of 1/IC₅₀ (IC₅₀ is the 50% inhibitory concentration of the complex required for the release of free fatty acids in adipocytes) and the lipophilicity of the complex (log P _com). The oxovanadium(IV) concentration was significantly higher and the species resided longer in the blood of rats that received VO(alx)₂ than in the blood of rats that received VO(3hp)₂ or bis(kojato)oxovanadium(IV); VO(alx)₂ also exhibited higher log P _com and 1/IC₅₀ values. On the basis of these results, we propose that the introduction of lipophilic groups at the C2 and C6 positions of the 3hp ligand is an effective method to enhance the hypoglycemic effect of the complexes, as supported by the observed in vivo exposure and residence in the blood.     

Adenosine and blood platelets

Adenosine is an important regulatory metabolite and an inhibitor of platelet activation. Adenosine released from different cells or generated through the activity of cell-surface ectoenzymes exerts its effects through the binding of four different G-protein-coupled adenosine receptors. In platelets, binding of A₂ subtypes (A_2A or A_2B) leads to consequent elevation of intracellular cyclic adenosine monophosphate, an inhibitor of platelet activation. The significance of this ligand and its receptors for platelet activation is addressed in this review, including how adenosine metabolism and its A₂ subtype receptors impact the expression and activity of adenosine diphosphate receptors. The expression of A₂ adenosine receptors is induced by conditions such as oxidative stress, a hallmark of aging. The effect of adenosine receptors on platelet activation during aging is also discussed, as well as potential therapeutic applications.     

O-Linked Glycosylation of the Mucin Domain of the Herpes Simplex Virus Type 1-specific Glycoprotein gC-1 Is Temporally Regulated in a Seed-and-spread Manner

The herpes simplex virus type 1 (HSV-1) glycoprotein gC-1, participating in viral receptor interactions and immunity interference, harbors a mucin-like domain with multiple clustered O-linked glycans. Using HSV-1-infected diploid human fibroblasts, an authentic target for HSV-1 infection, and a protein immunoaffinity procedure, we enriched fully glycosylated gC-1 and a series of its biosynthetic intermediates. This fraction was subjected to trypsin digestion and a LC-MS/MS glycoproteomics approach. In parallel, we characterized the expression patterns of the 20 isoforms of human GalNAc transferases responsible for initiation of O-linked glycosylation. The gC-1 O-glycosylation was regulated in an orderly manner initiated by synchronous addition of one GalNAc unit each to Thr-87 and Thr-91 and one GalNAc unit to either Thr-99 or Thr-101, forming a core glycopeptide for subsequent additions of in all 11 GalNAc residues to selected Ser and Thr residues of the Thr-76–Lys-107 stretch of the mucin domain. The expression patterns of GalNAc transferases in the infected cells suggested that initial additions of GalNAc were carried out by initiating GalNAc transferases, in particular GalNAc-T2, whereas subsequent GalNAc additions were carried out by followup transferases, in particular GalNAc-T10. Essentially all of the susceptible Ser or Thr residues had to acquire their GalNAc units before any elongation to longer O-linked glycans of the gC-1-associated GalNAc units was permitted. Because the GalNAc occupancy pattern is of relevance for receptor binding of gC-1, the data provide a model to delineate biosynthetic steps of O-linked glycosylation of the gC-1 mucin domain in HSV-1-infected target cells.     

CHO细胞表达糖蛋白的研究进展*

中国仓鼠卵巢细胞(Chinese hamster ovary cells,CHO)表达系统因具有较高密度培养、高表达和相对完整的蛋白质糖基化修饰系统等特点,成为生产糖蛋白广泛应用的宿主表达细胞之一。目前已产生不同的CHO细胞系和各种功能细胞株以满足对糖蛋白的大量生产和其他实验需求。近年来,随着基因工程、蛋白质工程、细胞工程和发酵调控等技术的发展应用,由CHO细胞生产糖蛋白的产量和糖基化修饰程度取得了突破。然而,随着生物制品市场对于糖蛋白的需求增加,如何获得大量、均质的糖蛋白也成为急需解决的问题。综述了不同工程CHO表达系统的研究、应用、糖基化修饰系统,以及影响外源糖蛋白在CHO系统表达和糖基化修饰的理化因素,结合文献总结并预测了未来CHO细胞表达系统研究的四个具有重大意义的研究方向,以期在未来可以改善由CHO细胞表达糖蛋白的产量和质量。     

Glycoprotein Ib distribution on the surface of platelets in resting and activation states: An electron microscope study

Summary Using a monoclonal antibody (TM60) against glycoprotein (GP) Ib, we determined immunocytochemically how GPIb is distributed on the platelet surface. When glutaraldehyde-fixed platelets were incubated with TM60, a uniform distribution of ferritin particles which represent the localization of GPIb was observed on the surface membrane of platelets. The particles were distributed at intervals of about 100 nm. The number of ferritin particles on the surface of one side were 2070–4150 (2940 ± 790; mean ±s.d.,n = 10) under the scanning electron microscope. The distribution of ferritin particles was somewhat disarranged on the surface of unfixed platelets incubated with TM60 compared to that in the fixed platelets. Cluster-like structures of ferritin particles were observed in several places. When platelets were activated with ristocetin or thrombin, the distribution of ferritin particles was disturbed and cluster formation was observed in several places on the surface. These findings suggest that GPIb is uniformly distributed on the surface of platelets in the resting state, and that cluster formation occurs during activation of platelets.     

Ⅰ型单纯疱疹病毒 gD基因片段的高效表达及抗原性分析

目的 获得高表达的Ⅰ型单纯疱疹病毒(HSV)被膜糖蛋白gD（简称gD1）基因的工程菌。方法 通过计算机分析,筛选出疱疹病毒gD1中优势抗原决定簇的基因片段。将克隆的基因片段插入表达载体pTrxA内,转化大肠杆菌Rosetta,以异丙基-β-D-硫代半乳糖苷诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物。　结果　PCR扩增出约930bp的gD1编码基因目的片段,与预期片段大小相符,经测序鉴定无基因突变;所构建pTrxA-gD1重组表达质粒阳性克隆经PCR与双酶切鉴定,与预期结果一致;含有pTrxA-gD1重组质粒的大肠杆菌Rosetta诱导后得到了高效达,SDS-PAGE显示表达产物约Mr48000（Dalton）。免疫印迹结果表明表达产物具有较好的抗原性。结论　成功构建了pTrxA-gD1表达质粒,实现了成熟gD1蛋白在大肠杆菌中的高效表达,表达产物具有好的抗原性。     

Characterization of α2-adrenergic receptors in human platelets by binding of a radioactive ligand [3H]yohimbine

[³H]Yohimbine, a potent α₂-adrenergic antagonist, was used to label the α₂-adrenergic receptors in membranes isolated from human platelets. Binding of [³H]yohimbine to platelet membranes appears to have all the characteristics of binding to α₂-adrenergic receptors. Binding reached a steady state in 2–3 min at 37°C and was completely reversible upon the addition of excess phentolamine or yohimbine (both at 10^?5 M;$\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 2.37}\text{min}$). [³H]Yohimbine bound to a single class of noncooperative sites with a dissociation constant of 1.74 nM. At saturation, the total number of binding sites was calculated to be 191 fmol/mg protein. [³H]Yohimbine binding was stereo-specifically inhibited by epinephrine: the (?) isomer was 11-times more potent than the (+) isomer. Cathecholamine agonists competed for the occupancy of the [³H]yohimbine-binding sites with an order of potency: clonidine > (?)-epinephrine > (?)-norepinephrine >> (?)-isoproterenol. The potent α₂-adrenergic antagonist, phentolamine, competed for the sites whereas the β-antagonist, (±)-propanolol, was a very weak inhibitor. 0.1 mM GTP reduced the bindng affinity of the agonists, while producing no change in antagonist-binding affinity. Dopamine and serotonine competed only at very high concentrations. Similarly, muscarinic cholinergic ligands were also poor inhibitors of [³H]yohimbine binding. These results suggest tht [³H]yohimbine binding to human platelet membranes is specific, rapid, saturable, reversible and, therefore, can be successfully used to label α₂-adrenergic receptors.     

The effect of oral contraceptives on rat platelet membrane glycoproteins

Female rats were administered oral contraceptives and the levels of sialic acid on platelet membrane and granule glycoproteins were compared to controls using a sialic acid assay and a fluorescein-conjugated wheat germ agglutinin binding assay and also by measuring the binding of ¹²⁵I-labelled wheat germ agglutinin to glycoprotein bands from platelets separated by polyacrylamide electrophoresis. The contraceptive-treated rats showed increased levels of glycoprotein sialylation which may partly explain the altered physiological function of the platelets.     

单纯疱疹病毒Ⅰ型糖蛋白D在酵母中的表达

从提取的HSV-1基因组中扩增得到编码gD蛋白胞外区1～314aa的基因gDt,将其插入毕赤酵母表达质粒pPIC9K的醇氧化酶(AOX1)启动子下游,构建携带gDt的重组载体,经电转化GS115菌株和G418筛选,得到了高效分泌表达gD蛋白的毕赤酵母菌株,表达量达到250mg/L,该目的蛋白可被gD单抗(1-I-9)特异性识别.表达产物经离子交换、金属螯合、分子筛柱层析纯化后得到纯度较高的重组蛋白.重组gD蛋白免疫BALB/c小鼠可诱生一定水平的特异性抗体,表明该蛋白具有较好的免疫原性,能够诱导小鼠产生体液免疫应答.     

Spinnokinetic analyses of blood disposition and biliary excretion of nitric oxide (NO)-Fe(II)-N-(dithiocarboxy)sarcosine complex in rats: BCM-ESR and BEM-ESR studies

Nitric oxide (NO) is well known to have a wide variety of biological and physiological functions in animals. On the basis of the fact that Fe(II)-dithiocarbamates react with NO, a Fe(II)-N-(dithiocarboxy)sarcosine complex (Fe(II)-DTCS) was proposed as a trapping agent for endogenous NO. However, quantitative pharmacokinetic investigation for NO-Fe(II)-dithiocarbamate complexes in experimental animals has been quite limited. This paper describes the results on the quantitative pharmacokinetic features of a NO-Fe(II)-N-DTCS in both the blood and bile of rats following intravenous (i.v.) administration of the complex. For this purpose, we applied two in vivo methods, i.e. (1) in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) which previously developed, and (2) in vivo biliary excretion monitoring-electron spin resonance (BEM-ESR). We monitored real-time ESR signals due to nitrosyl-iron species in the circulating blood and bile flow. The ESR signal due to NO-Fe(II)-DTCS was stable in biological systems such as the fresh blood and bile. In in vivo BCM- and BEM-ESR, the pharmacokinetic parameters were calculated on the basis of the two-compartment and hepatobiliary transport models. The studies also revealed that the compound is widely distributed in the peripheral organs and partially excreted into the bile. We named a kinetic method to follow spin concentrations as spinnokinetics and this method will be useful for detecting and quantifying the endogenously generated NO in Fe(II)-DTCS administered animals.     

Glycoprotein secretion in the mouse submandibular gland as revealed by radioautography after L-3H-fucose injection

Summary Glycoprotein secretion in the mouse submandibular gland was investigated by light microscope radioautography of semi-thin sections after the administration of L-³H-fucose. The incorporation of the precursor in the acini was negligible. ³H-fucose was taken up in the paranuclear region of the cells lining the intercalated, secretory, striated and excretory ducts. This labeling pattern was interpreted as addition of the precursor to glycoproteins within the Golgi apparatus. Incorporation in the intercalated duct was restricted to the cells with fine cytoplasmic granules. The glycoproteins synthesized by the intercalated and secretory ducts were transported to the saliva by the secretion granules. It is assumed that the glycoproteins synthesized in the striated and excretory ducts are plasma membrane glycoproteins which seem to renew continuously. Quantitation of the radioautographs supplied data concerning the incorporation of ³H-fucose into newly synthesized glycoproteins as well as the renewal of the labeled macromolecules in each duct.     

The role of blood platelets in tumor angiogenesis

Coagulation abnormalities occur frequently in cancer patients. It is becoming evident that blood platelets have an important function in this process. However, understanding of the underlying mechanisms is still very modest. In this review, we discuss the role of platelets in tumor angiogenesis and growth and suggest their potential significance in malignancies. Platelets contain various pro-and antiangiogenic molecules, which seem to be endocytosed and sequestered in different populations of α-granules. Furthermore, tumor endothelial cells are phenotypically and functionally different from endothelial cells in healthy tissue, stimulating local platelet adhesion and subsequent activation. As a consequence, platelets are able to secrete their angiogenic and angiostatic content, most likely in a regulated manner. The overall effect of these platelet–endothelium interactions appears to be proangiogenic, stimulating tumor angiogenesis. We favor the view that local adhesion and activation of blood platelets and dysregulation of coagulation represent underestimated pathways in the progression of cancer.