Quantitative isolation of RNA from human platelets

Djaffar, Isabelle; Vilette, Didier; Bray, Paul F.; Rosa, Jean‐Philippe

doi:10.1016/0049-3848(91)90187-2

Cited by 26 publications

(19 citation statements)

References 15 publications

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“…There are some previous studies in which regular RT-PCR has been used to detect mRNA in platelets [19][20][21], but no-one has been able to set up a quantification method, probably due to the large amount of total RNA needed for previous RNA quantification methods. Realtime PCR is a recent quantitative method with the advantage that extremely low amounts of sample RNA are needed [25].…”

Section: Discussionmentioning

confidence: 99%

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Quantification of ADP and ATP receptor expression in human platelets

Wang

Östberg

Wihlborg

et al. 2003

Journal of Thrombosis and Haemostasis

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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 P2X 1 , P2Y 1 and P2Y 12 were found in significant levels, with the following order of expression: P2Y 12 >> P2X 1 > P2Y 1 . Other P2 receptors (P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 13 , P2X 4 , P2X 7 ) 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 P2X 1 receptor mRNA than for P2Y 1 and P2Y 12 , indicating that the level of P2X 1 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 P2Y 12 , P2Y 1 and P2X 1 . Furthermore, the method could be used to study platelet expression for any gene in human materials.Keywords: P2X receptor, P2Y receptor, platelets, real time-PCR, Western blot.Platelets play a crucial role in the maintenance of normal hemostasis and are involved in the development of pathological thrombus formation leading to vascular occlusion which is an important mechanism in myocardial infarction and stroke [1,2]. A large number of endogenous mediators can activate platelets, such as thromboxane A2, adenosine diphosphate (ADP), collagen, von Willebrand factor, thrombin, epinephrine and 5-hydroxitryptamine (5-HT). To get full activation of the platelets, these agonists are dependent on two positive feedback loops: the formation of thromboxane A2 by cyclooxygenase in the platelets and the release of ADP from dense platelet granules. Thromboxane A2 and ADP then activates specific receptors on the extracellular side of the platelet membrane.Therapeutic intervention aimed at the first positive feedback loop by inhibiting cyclooxygenase with aspirin is highly efficient in reducing death and cardiovascular events. However, ADP may be even more important as evidenced by the CAPRIE study, in which the ADP receptor inhibition was more beneficial than aspirin in reducing cardiovascular events [3,4].Recently, progress has been made in the understanding of the mechanisms of ADP mediated platelet aggregation, where at least three receptors are considered to be involved; the P2Y 12 , P2Y 1 , and P2X 1 receptors [5]. The importance of the P2Y 12 receptor is proven by the effects of clopidogrel, which after metabolization in the liver, acts as an irreversible antagonist at P2Y 12 receptors. Knockout of the P2Y 1 receptor in mice has demonstrated its importance for thrombus formation, blee...

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Section: Discussionmentioning

confidence: 99%

“…Even though mRNA in platelets has been detected with PCR [19][20][21], no previous methods for quantification of platelet mRNA has been reported. The mRNA of a gene could reflect two aspects.…”

mentioning

confidence: 99%

Quantification of ADP and ATP receptor expression in human platelets

Wang

Östberg

Wihlborg

et al. 2003

Journal of Thrombosis and Haemostasis

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“…Polymerase Chain Reaction (PCR) Amplification of Platelet cDNA-Fresh human platelets were isolated and platelet total RNA prepared (48). Approximately 600 ng of RNA were used to synthesize cDNA, using random hexamers and the Geneamp RNA PCR kit (Perkin-Elmer).…”

Section: Methodsmentioning

confidence: 99%

Dystrophin-related Protein in the Platelet Membrane Skeleton

Earnest¹,

Santos²,

Zuerbig³

et al. 1995

Journal of Biological Chemistry

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The platelet membrane is lined with a membrane skeleton that associates with transmembrane adhesion receptors and is thought to play a role in regulating the stability of the membrane, distribution and function of adhesive receptors, and adhesive receptor-induced transmembrane signaling. When platelets are lysed with Triton X-100, cytoplasmic actin filaments can be sedimented by centrifugation at low g-forces (15,600 ؋ g) but the membrane skeleton requires 100,000 ؋ g. The present study shows that DRP (dystrophin-related protein) sediments from lysed platelets along with membrane skeleton proteins. Sedimentation results from association with the membrane skeleton because DRP was released into the detergent-soluble fraction when actin filaments were depolymerized. Interaction of fibrinogen with the integrin ␣ IIb ␤ 3 induces platelet aggregation, transmembrane signaling, and the formation of integrin-rich cytoskeletal complexes that can be sedimented from detergent lysates at low g-forces. Like other membrane skeleton proteins, DRP redistributed from the high-speed pellet to the integrin-rich low-speed pellet of aggregating platelets. One of the signaling enzymes that is activated following ␣ IIb ␤ 3 -ligand interactions in a platelet aggregate is calpain; DRP was cleaved by calpain to generate a ϳ140-kDa fragment that remained associated with the low-speed detergent-insoluble fraction. These studies show that DRP is part of the platelet membrane skeleton and indicate that DRP participates in the cytoskeletal reorganizations resulting from signal transmission between extracellular adhesive ligand and the interior of the cell.Duchenne muscular dystrophy is one of the most common inherited human diseases. It is caused by a defective gene that codes for a 427-kDa protein, dystrophin (1-5). The deduced amino acid sequence of dystrophin shows that it consists of four domains and suggests that it is a cytoskeletal protein (6). The major rod-shaped domain contains 24 spectrin-like repeats. This domain is flanked on the amino terminus by a domain that has a high degree of homology to the actin-binding domains of spectrin and ␣-actinin, and on the carboxyl terminus by a cysteine-rich domain that shows some homology to a Ca 2ϩ -binding region in ␣-actinin. The most carboxyl-terminal end of dystrophin consists of a short domain that has no homology to any known protein and appears to play a role in linking the molecule to the plasma membrane (7,8). Recent studies using purified protein or recombinant fragments containing the putative actin-binding domain (9 -11) have shown that the protein can bind to actin filaments in vitro, supporting the idea that this molecule functions as a cytoskeletal protein. The finding that dystrophin exists in a submembranous location (8,12,13) and that the carboxyl-terminal end of the molecule associates tightly with a complex of membrane glycoproteins (termed dystroglycan) (14 -17) suggests that dystrophin is a component of a submembranous cytoskeleton.Although there is now considerable inform...

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“…Few previous studies have focused on platelet mRNA quantification. 50,51 Qualitative and semiquantitative studies have shown that platelets contain undegraded ribosomal RNA as well as intact mRNAs. The absence or quantitative alteration of ␣ IIb ␤ 3…”

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confidence: 99%

An intronic polymorphism in the PAR-1 gene is associated with platelet receptor density and the response to SFLLRN

Dupont

Fontana

Bachelot‐Loza

et al. 2003

Blood

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Protease-activated receptor 1 (PAR-1), the main thrombin receptor on vascular cells, plays a key role in platelet activation. We examined the range of PAR-1 expression on platelets, obtained twice, 1 week apart, from 100 healthy subjects and found a 2-fold interindividual variation in receptor numbers (95% CI 858-1700). Because PAR-1 density was stable with time (r 2 76%, P < .001), we sought a genetic explanation for the observed variability. To validate this approach, we also analyzed the 2 1 genotype according to receptor density and platelet mRNA expression data. We found that the number of PAR-1 receptors on the platelet surface is associated with the intervening sequence IVSn14 A/T intronic variation. The number of receptors was also found to govern the platelet response to the SFLLRN agonist, in terms of aggregation and P-selectin expression. The T allele (allelic frequency, 0.14) can be considered as an allele with decreased expression , because it was associated with lower PAR-1 expression on the platelet surface and with a lower response to SFLLRN. The IVSn14 A/T intronic variation may therefore be clinically relevant. (Blood.

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Quantitative isolation of RNA from human platelets

Cited by 26 publications

References 15 publications

Quantification of ADP and ATP receptor expression in human platelets

Quantification of ADP and ATP receptor expression in human platelets

Dystrophin-related Protein in the Platelet Membrane Skeleton

An intronic polymorphism in the PAR-1 gene is associated with platelet receptor density and the response to SFLLRN

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