Anna-Karin Wihlborg

ADP Receptor P2Y12 Is Expressed in Vascular Smooth Muscle Cells and Stimulates Contraction in Human Blood Vessels

Objective—ADP plays an important role in platelet aggregation by activating P2Y12 receptors. We assessed the hypothesis that P2Y12 receptors are expressed in vascular smooth muscle cells (VSMC). Methods and Results—P2Y12 receptor mRNA was found to have a high expression among the P2 receptors in human VSMC, significantly higher than the other 2 ADP receptors (P2Y1 and P2Y13, real-time polymerase chain reaction). Western blots gave a band of 50 kD, similar to that in platelets. To unmask a P2Y12 receptor-mediated vasoconstriction by simulating the in vivo situation, vessels were precontracted to a submaximal level. 2-MeSADP stimulated contractions in vessel segments from internal mammary artery (IM), IM branches and small veins (Emax=15±6% of 60mmol/L K+ contraction, pEC50=5.6±0.6, Emax=21±1%, pEC50=6.8±0.1, and Emax=48±9%, pEC50=6.6±0.4). The selective P2Y12 antagonist AR-C67085 blocked 2-MeSADP contractions. The contraction was not reduced in patients using clopidogrel, a drug inhibiting ADP-induced platelet aggregation by blocking the P2Y12 receptor. This may be explained by the high instability of the active clopidogrel metabolite that never reaches the systemic circulation. Conclusion—ADP acting on P2Y12 receptors not only is important for platelet activation but also stimulates vasoconstriction. Stable drugs with antagonistic effects on P2Y12 receptors, affecting both platelets and VSMC, could be of double therapeutic benefit in their prevention of both thrombosis and vasospasm.

Positive Inotropic Effects by Uridine Triphosphate (UTP) and Uridine Diphosphate (UDP) via P2Y2 and P2Y6 Receptors on Cardiomyocytes and Release of UTP in Man During Myocardial Infarction

The aim of this study was to examine a possible role for extracellular pyrimidines as inotropic factors for the heart. First, nucleotide plasma levels were measured to evaluate whether UTP is released in patients with coronary heart disease. Then, inotropic effects of pyrimidines were examined in isolated mouse cardiomyocytes. Finally, expression of pyrimidine-selective receptors (a subgroup of the P2 receptors) was studied in human and mouse heart, using real time polymerase chain reaction, Western blot, and immunohistochemistry. Venous plasma levels of UTP were increased (57%) in patients with myocardial infarction. In electrically stimulated cardiomyocytes the stable P2Y2/4 agonist UTPγS increased contraction by 52%, similar to β1-adrenergic stimulation with isoproterenol (65%). The P2Y6-agonist UDPβS also increased cardiomyocyte contraction (35%), an effect abolished by the P2Y6-blocker MRS2578. The phospholipase C inhibitor U73122 inhibited both the UDPβS and the UTPγS-induced inotropic effect, indicating an IP3-mediated effect via P2Y6 receptors. The P2Y14 agonist UDP-glucose was without effect. Quantification of mRNA with real time polymerase chain reaction revealed P2Y2 as the most abundant pyrimidine receptor expressed in cardiomyocytes from man. Presence of P2Y6 receptor mRNA was detected in both species and confirmed at protein level with Western blot and immunohistochemistry in man. In conclusion, UTP levels are increased in humans during myocardial infarction, giving the first evidence for UTP release in man. UTP is a cardiac inotropic factor most likely by activation of P2Y2 receptors in man. For the first time we demonstrate inotropic effects of UDP, mediated by P2Y6 receptors via an IP3-dependent pathway. Thus, the extracellular pyrimidines (UTP and UDP) could be important inotropic factors involved in the development of cardiac disease.

Extracellular nucleotides induce vasodilatation in human arteries via prostaglandins, nitric oxide and endothelium-derived hyperpolarising factor.

The present study was aimed at examining P2 receptor‐mediated vasodilatation in human vessels. The isometric tension was recorded in isolated segments of the human left internal mammary artery branches precontracted with 1 μM noradrenaline. Endothelial denudation abolished the dilator responses. The selective P2Y1 agonist, 2‐MeSADP, induced a potent vasodilatation (pEC50=6.9±0.1). The P2Y1 antagonist of 10 μM, MRS 2216, shifted the 2‐MeSADP concentration‐response curve 1.1 log units to the right. The combined P2Y1 and P2X agonist, 2‐MeSATP, stimulated a dilatation with a potency similar to that of 2‐MeSADP. Furthermore, MRS 2216 had a similar antagonistic effect on both 2‐MeSATP and 2‐MeSADP indicating that P2X receptors do not mediate vasodilatation. Both the P2Y2/4 agonist, UTPγS and the P2Y6 agonist, UDPβS, stimulated potent dilatations (pEC50=7.8±0.4 for UTPγS and 8.4±0.2 for UDPβS). The 2‐MeSADP‐induced nitric oxide (NO)‐mediated dilatation was studied in the presence of 10 μM indomethacin, 50 nM charybdotoxin and 1 μM apamin. The involvement of the endothelium‐derived hyperpolarising factor (EDHF) was investigated in the presence of 0.1 mM L‐NOARG and indomethacin. The involvement of prostaglandins was investigated in the presence of L‐NOARG, charybdotoxin and apamin. Both NO, EDHF and prostaglandins mediated 2‐MeSADP dilatation with similar efficacy (Emax=25±5% for NO, 25±6% for EDHF and 27±5% for prostaglandins). In conclusion, extracellular nucleotides induce endothelium‐derived vasodilatation in human vessels by stimulating P2Y1, P2Y2/4 and P2Y6 receptors, while P2X receptors are not involved. Endothelial P2Y receptors mediate dilatation by release of EDHF, NO and prostaglandins

Quantification of ADP and ATP receptor expression in human platelets.

Summary.  The mechanism of ADP‐mediated platelet activation has been difficult to unravel due to the large number of receptors for extracellular nucleotides (P2 receptors). mRNA levels in circulating platelets are very low, but have been shown to be translationally active. By optimizing mRNA extraction and using real time (RT)‐PCR we were able to establish a protocol for highly sensitive platelet mRNA quantification in human regular blood samples. In platelets from healthy volunteers, only P2X1, P2Y1 and P2Y12 were found in significant levels, with the following order of expression: P2Y12 >> P2X1 > P2Y1. Other P2 receptors (P2Y2, P2Y4, P2Y6, P2Y11, P2Y13, P2X4, P2X7) had very low expression. As a control measurement to exclude contamination, P2 receptors in buffy coat were quantified but had a completely different profile. Incubation in vitro revealed a more rapid degradation rate for P2X1 receptor mRNA than for P2Y1 and P2Y12, indicating that the level of P2X1 may be relatively higher in newly released platelets and in megacaryocytes. In conclusion, we have developed the first protocol for quantifying mRNA expression in human platelets limiting the P2 receptor drug development targets to P2Y12, P2Y1 and P2X1. Furthermore, the method could be used to study platelet expression for any gene in human materials.

Genome-wide association study identifies multiple susceptibility loci for multiple myeloma

Multiple myeloma (MM) is a plasma cell malignancy with a significant heritable basis. Genome-wide association studies have transformed our understanding of MM predisposition, but individual studies have had limited power to discover risk loci. Here we perform a meta-analysis of these GWAS, add a new GWAS and perform replication analyses resulting in 9,866 cases and 239,188 controls. We confirm all nine known risk loci and discover eight new loci at 6p22.3 (rs34229995, P=1.31 × 10−8), 6q21 (rs9372120, P=9.09 × 10−15), 7q36.1 (rs7781265, P=9.71 × 10−9), 8q24.21 (rs1948915, P=4.20 × 10−11), 9p21.3 (rs2811710, P=1.72 × 10−13), 10p12.1 (rs2790457, P=1.77 × 10−8), 16q23.1 (rs7193541, P=5.00 × 10−12) and 20q13.13 (rs6066835, P=1.36 × 10−13), which localize in or near to JARID2, ATG5, SMARCD3, CCAT1, CDKN2A, WAC, RFWD3 and PREX1. These findings provide additional support for a polygenic model of MM and insight into the biological basis of tumour development.

Residual platelet ADP reactivity after clopidogrel treatment is dependent on activation of both the unblocked P2Y(1) and the P2Y (12) receptor and is correlated with protein expression of P2Y (12).

Two ADP receptors have been identified on human platelets: P2Y1 and P2Y12. The P2Y12 receptor blocker clopidogrel is widely used to reduce the risks in acute coronary syndromes, but, currently, there is no P2Y1 blocker in clinical use. Evidence for variable responses to clopidogrel has been described in several reports. The mechanistic explanation for this phenomenon is not fully understood. The aim of this study was to examine mechanisms responsible for variability of 2MeS-ADP, a stable ADP analogue, induced platelet reactivity in clopidogrel-treated patients. Platelet reactivity was assessed by flow cytometry measurements of P-selectin (CD62P) and activated GpIIb/IIIa complex (PAC-1). Residual 2MeS-ADP activation via the P2Y12 and P2Y1 receptors was determined by co-incubation with the selective antagonists AR-C69931 and MRS2179 in vitro. P2Y1 and P2Y12 receptor expression on both RNA and protein level were determined, as well as the P2Y12 H1 or H2 haplotypes. Our data suggest that the residual platelet activation of 2MeS-ADP after clopidogrel treatment is partly due to an inadequate antagonistic effect of clopidogrel on the P2Y12 receptor and partly due to activation of the P2Y1 receptor, which is unaffected by clopidogrel. Moreover, a correlation between increased P2Y12 protein expression on platelets and decreased response to clopidogrel was noticed, r2=0.43 (P<0.05). No correlation was found between P2Y12 mRNA levels and clopidogrel resistance, indicating post-transcriptional mechanisms. To achieve additional ADP inhibition in platelets, antagonists directed at the P2Y1 receptor could be more promising than the development of more potent P2Y12 receptor antagonists.

Identification of sequence variants influencing immunoglobulin levels

Immunoglobulins are the effector molecules of the adaptive humoral immune system. In a genome-wide association study of 19,219 individuals, we found 38 new variants and replicated 5 known variants associating with IgA, IgG or IgM levels or with composite immunoglobulin traits, accounted for by 32 loci. Variants at these loci also affect the risk of autoimmune diseases and blood malignancies and influence blood cell development. Notable associations include a rare variant at RUNX3 decreasing IgA levels by shifting isoform proportions (rs188468174[C>T]: P = 8.3 × 10−55, β = −0.90 s.d.), a rare in-frame deletion in FCGR2B abolishing IgG binding to the encoded receptor (p.Asn106del: P = 4.2 × 10−8, β = 1.03 s.d.), four IGH locus variants influencing class switching, and ten new associations with the HLA region. Our results provide new insight into the regulation of humoral immunity.

Direct evidence for a polygenic etiology in familial multiple myeloma

Although common risk alleles for multiple myeloma (MM) were recently identified, their contribution to familial MM is unknown. Analyzing 38 familial cases identified primarily by linking Swedish nationwide registries, we demonstrate an enrichment of common MM risk alleles in familial compared with 1530 sporadic cases (P = 4.8 × 10-2 and 6.0 × 10-2, respectively, for 2 different polygenic risk scores) and 10 171 population-based controls (P = 1.5 × 10-4 and 1.3 × 10-4, respectively). Using mixture modeling, we estimate that about one-third of familial cases result from such enrichments. Our results provide the first direct evidence for a polygenic etiology in a familial hematologic malignancy.

The multiple myeloma risk allele at 5q15 lowers ELL2 expression and increases ribosomal gene expression

Recently, we identified ELL2 as a susceptibility gene for multiple myeloma (MM). To understand its mechanism of action, we performed expression quantitative trait locus analysis in CD138+ plasma cells from 1630 MM patients from four populations. We show that the MM risk allele lowers ELL2 expression in these cells (Pcombined = 2.5 × 10−27; βcombined = −0.24 SD), but not in peripheral blood or other tissues. Consistent with this, several variants representing the MM risk allele map to regulatory genomic regions, and three yield reduced transcriptional activity in plasmocytoma cell lines. One of these (rs3777189-C) co-locates with the best-supported lead variants for ELL2 expression and MM risk, and reduces binding of MAFF/G/K family transcription factors. Moreover, further analysis reveals that the MM risk allele associates with upregulation of gene sets related to ribosome biogenesis, and knockout/knockdown and rescue experiments in plasmocytoma cell lines support a cause–effect relationship. Our results provide mechanistic insight into MM predisposition.ELL2 was recently discovered as a susceptibility gene for multiple myeloma (MM). Here, they show that the MM risk allele lowers ELL2 expression in plasma cells, that it also upregulates gene sets related to ribosome biogenesis, and that one of the linked variants reduces binding of MAFF/G/K family transcription factors.