Definition of novel GP6 polymorphisms and major difference in haplotype frequencies between populations by a combination of in-depth exon resequencing and genotyping with tag single nucleotide polymorphisms

BACKGROUND: Common genetic variants of cell surface receptors contribute to differences in functional responses and disease susceptibility. We have previously shown that single nucleotide polymorphisms (SNPs) in platelet glycoprotein VI (GP6) determine the extent of response to agonist. In addition, SNPs in the GP6 gene have been proposed as risk factors for coronary artery disease. METHODS: To completely characterize genetic variation in the GP6 gene we generated a high-resolution SNP map by sequencing the promoter, exons and consensus splice sequences in 94 non-related Caucasoids. In addition, we sequenced DNA encoding the ligand-binding domains of GP6 from non-human primates to determine the level of evolutionary conservation. RESULTS: Eighteen SNPs were identified, six of which encoded amino acid substitutions in the mature form of the protein. The single non-synonymous SNP identified in the exons encoding the ligand-binding domains, encoding for a 103Leu > Val substitution, resulted in reduced ligand binding. Two common protein isoforms were confirmed in Caucasoid with frequencies of 0.82 and 0.15. Variation at the GP6 locus was characterized further by determining SNP frequency in over 2000 individuals from different ethnic backgrounds. CONCLUSIONS: The SNPs were polymorphic in all populations studied although significant differences in allele frequencies were observed. Twelve additional GP6 protein isoforms were identified from the genotyping results and, despite extensive variation in GP6, the sequence of the ligand-binding domains is conserved. Sequences from non-human primates confirmed this observation. These data provide valuable information for the optimal selection of genetic variants for use in future association studies.     

Usefulness of noninvasive methods including assessment of liver stiffness by 2-dimensional shear wave elastography for predicting esophageal varices

BackgroundThe aim of this study was to predict the presence of esophageal varices (EVs) by noninvasive tools combined with 2-dimensional shear wave elastography (2D-SWE), and to compare the diagnostic capabilities of 2D-SWE with those of transient elastography (TE).MethodsBetween January 2015 and December 2017, 289 patients with compensated advanced chronic liver disease (cACLD) who underwent consecutive 2D-SWE and EGD were enrolled. Capabilities for predicting the presence of EVs of 2D-SWE and models combining 2D-SWE with other noninvasive tools (modified LS-spleen-diameter-to-platelet-ratio score [mLSPS], platelet-spleen ratio score) were compared. A subgroup analysis was performed on 177 patients who also underwent simultaneous TE.ResultsThe area under receiver operating characteristics (AUROCs) for detecting EVs for 2D-SWE alone vs. mLSPS, which included 2D-SWE, were 0.757 (95% confidence interval [CI], 0.701–0.810) and 0.813 (95% CI, 0.763–.857), respectively. The AUROCs for predicting varices needing treatment (VNT) for 2D-SWE and mLSPS were 0.712 (95% CI, 0.621–0.738) and 0.834 (95% CI, 0.785–0.875), respectively. For the 195 patients who underwent simultaneous TE and 2D-SWE, no differences in diagnostic performance were observed.ConclusionsThe diagnostic performance of 2D-SWE is similar to that of TE for predicting the presence of EVs. The mLSPS, which includes 2D-SWE, seemed to be useful for predicting EVs.     

Adhesive surface determines raft composition in platelets adhered under flow

Adhesion to von Willebrand factor (VWF) induces platelet spreading, whereas adhesion to collagen induces aggregation. Here we report that cholesterol-rich domains (CRDs) or rafts play a critical role in clustering of receptors that control these responses. Platelets adhered to VWF and collagen show CRDs concentrated in filopodia which contain both the VWF receptor glycoprotein (GP) Ibalpha and the collagen receptor GPVI. Biochemical analysis of CRDs shows a threefold enrichment of GPIbalpha (but not GPVI) in VWF-adhered platelets and a fourfold enrichment of GPVI (but not GPIbalpha) in collagen-adhered platelets. Depletion of cholesterol (i) leaves the initial adhesion unchanged, (ii) inhibits spreading on VWF and aggregate formation on collagen, (iii) leaves filopodia formation intact, and (iv) reduces the localization in filopodia of GPIbalpha but not of GPVI. These data show that the adhesive substrate determines the composition of CRDs, and that cholesterol is crucial for redistribution of GPIbalpha but not of GPVI.     

Genetic analysis of mucopolysaccharidosis type VI in Taiwanese patients

BACKGROUND: Glutathione peroxidase 1 (GPX1), the key antioxidant enzyme in vascular endothelial cells, has been shown to exert a protective effect against the presence of coronary artery disease (CAD). The 198Pro/leu variant, located at codon 198 of GPX1 gene, has recently been linked to cardiovascular disease, but data were inconsistent. We investigated the association between the occurrence of CAD and the 198Pro/leu variant in a Chinese population. METHODS: A total of 265 unrelated CAD patients and 265 age- and sex-matched control subjects were recruited in this study. The GPX1 198Pro/leu genotype was determined using polymerase chain reaction-restriction fragment length polymorphism. RESULTS: Compared to the 198Pro/Pro carriers, subjects with the variant genotypes (198Pro/leu and 198Leu/leu) had a significantly higher risk of CAD (adjusted OR=2.02, 95%CI=1.27-3.22). In stratified analyses, the variant genotypes were significantly associated with increased CAD risk in subjects <64 y (adjusted OR=2.41, 95%CI=1.16-4.98), males (adjusted OR=1.86, 95%CI=1.09-3.18) and non-smokers (adjusted OR=2.40, 95%CI=1.15-5.01). However, no significant association was observed between this variant and the severity of CAD. CONCLUSION: These data provide evidence that GPX1 198Pro/leu variant genotypes are significantly associated with CAD risk in this Chinese population.     

Physical and Functional Association of the Src Family Kinases Fyn and Lyn with the Collagen Receptor Glycoprotein VI-Fc Receptor γ Chain Complex on Human Platelets

We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor γ chain (FcRγ) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRγ, Syk, and phospholipase Cγ2 (PLCγ2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI–FcRγ complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI–FcRγ complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRγ, Syk, and PLCγ2 and recruited tyrosine-phosphorylated Syk to the GPVI–FcRγ complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRγ and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI–FcRγ complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family–specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRγ, Syk, and PLCγ2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI–FcRγ complex.     

Eco-Doppler de ejercicio en pacientes con miocardiopatía hipertrófica. Factores determinantes de la limitación funcional

Introduction and objectives

At-rest echocardiography is a poor predictor of exercise capacity in patients with hypertrophic cardiomyopathy. We aimed to test the performance of treadmill exercise Doppler echocardiography in the prediction of functional limitations in these patients.

Methods

Eighty-seven consecutive patients with hypertrophic cardiomyopathy underwent treadmill exercise echocardiography with direct measurement of oxygen consumption. Both at rest and at peak exercise, the mitral inflow, mitral regurgitation, left ventricular outflow tract obstruction and mitral annulus velocities were assessed.

Results

Forty-three patients developed left ventricular outflow tract obstruction during exercise, which significantly decreased oxygen consumption (21.3 [5.7] mL/kg/min vs 24.6 [6.1] mL/kg/min; P=.012), and had greater left atrial volume (42.1 [14.5] mL/m² vs 31.1 [11.6] mL/m²; P<.001) and a higher degree of mitral regurgitation and E/E’ ratio during exercise. Exercise variables improved the predictive value of functional capacity (adjusted R² rose from 0.38 to 0.49). Independent predictors of oxygen consumption were age, left atrial volume, E/E’ ratio and the presence of left ventricular outflow tract obstruction. In a subset of patients without left ventricular outflow obstruction, only left ventricular and atrial volume indexes were independent predictors of exercise capacity.

Conclusions

In patients with hypertrophic cardiomyopathy, left ventricular outflow tract obstruction and left atrial volume are the main predictors of exercise capacity. Exercise echocardiography is a better predictor of functional performance than at-rest echocardiography, although its predictive power is under 50%. In nonobstructed patients, left atrial and ventricular volumes were the independent factors.Full English text available from:www.revespcardiol.org/en     

GPVI modulation during platelet activation and storage: its expression levels and ectodomain shedding compared to markers of platelet storage lesion

Platelet storage is associated with deleterious changes leading to the loss of platelet reactivity and response. During storage, platelets experience increased expression and shedding of P-selectin and CD40L as specific markers of platelet activation, whereas GPIbα decreases due to ectodomain shedding. As an important adhesive receptor, GPVI contributes significantly to thrombus formation while its expression and shedding levels during storage of platelet products have not been well characterized yet. This study investigated the modulation of GPVI during platelet storage.

For this study, samples obtained from 10 PRP-platelet concentrates (PCs) were subjected to flow-cytometry analysis to examine the expression of platelet activation markers and GPVI on days 1, 3, and 5 post-storage. To examine the levels of etcodomain shedding of these molecules, microparticle (MP)-free supernatants were also analyzed by either ELISA or Western blot methods.

According to results, the expression levels of P-selectin and CD40L as well as the amounts of their soluble forms significantly increased during storage. The expression of GPIbα and GPVI decreased whereas their shedding significantly increased post-storage. The expression and shedding levels of these two receptors were significantly correlated. Negative correlations between the expressions of GPIbα or GPVI and P-selectin have been observed whereas their shedding levels were significantly relevant together. In a control study, the use of biotinylated platelet resuspended in Tyrode’s buffer in the presence of ionophore with/without EDTA, confirmed the role of calcium in receptors shedding. In citrated PRP-PCs, recalcification of platelets also enhanced shedding levels of both GPIbα and GPVI. Intriguingly, the shedding levels of GPVI in stored PRP-PCs were much higher than those of ionophore-treated controls obtained from washed platelets. The ratios of sGPVI in stored platelet to ionophore-treated controls were also at least six times higher than those of GPIbα during storage. In conclusion, here we showed significant decreases of GPVI expression associated with its increasing levels of shedding during storage, suggesting GPVI as a valid marker of platelet storage lesion. Importantly, we found higher levels of GPVI shedding in stored platelets than those of ionophore-treated non-stored control samples. This suggests whereas platelet receptor shedding is mainly modulated by calcium-dependent signals, either platelet–surface interactions with the container walls during storage or induced shear stress under long-term agitation, might be also involved in the excessive shedding of GPVI during the storage of PCs.     

川续断皂苷Ⅵ对小鼠成肌细胞成骨分化的影响

目的 探讨川续断皂苷Ⅵ对小鼠成肌细胞成骨分化的作用及影响.方法 配制浓度为1×10-8～1×10-3 mol/L的川续断皂苷Ⅵ药物溶液,用细胞计数试剂盒8检测其对小鼠成肌细胞的细胞毒性和增殖能力.然后用碱性磷酸酶活性分析确定促进其成骨分化的最佳浓度.最后采用免疫印迹分析、实时定量PCR和茜素红染色观察成骨分化效果.结果...     

A snake venom metalloproteinase, kistomin, cleaves platelet glycoprotein VI and impairs platelet functions

Summary.  Background and objectives:  Injuries to the vessel wall and subsequent exposure of the matrix of the subendothelial layer resulted in thrombus formation. Platelet glycoprotein (GP) Ib and VI play a crucial role in matrix-induced activation and aggregation of platelets. Methods and results:  In the present study, we reported that the GPIb-cleaving snake venom metalloproteinase (SVMP), kistomin, inhibited collagen-induced platelet aggregation. Moreover, kistomin inhibited platelet aggregation induced by convulxin (CVX, a GPVI agonist) and a GPVI-specific antibody in a concentration and time-dependent manner. Kistomin treatment decreased platelet GPVI but not integrin α2β1 and αIIbβ3, accompanied with the formation of GPVI cleavage fragments, as determined by flow cytometric and Western blot analyses. In addition, intact platelet GPVI and recombinant GPVI were digested by kistomin to release 25- and 35-kDa fragments, suggesting that kistomin cleaved GPVI near the mucin-like region. We designed four synthetic peptides ranging from Leu¹⁸⁰ to Asn²⁴⁹ as the substrates for kistomin and found that kistomin cleaved these synthetic peptides at FSE²⁰⁵/A²⁰⁶TA and NKV²¹⁸/F²¹⁹TT, as analyzed by MALDI-TOF-MS. In addition, GPVI-specific antibody-induced tyrosine kinase phosphorylation in platelets was reduced after kistomin pretreatment, and platelet adhesion to collagen but not to fibrinogen was attenuated by kistomin. Conclusions:  We provided here the first evidence that a P-I snake venom metalloproteinase, kistomin, inhibits the interaction between collagen and platelet GPVI through its proteolytic activity on GPVI, thus providing an alternative strategy for developing new anti-thrombotic agents.