Introduction
Platelet activation plays an important role in the processes of hemostasis and pathophysiology. Thrombin, a key enzyme in the blood coagulation cascade and a potent platelet activator [
1], can activate human platelets through proteolytic activation of two protease-activated receptors (PARs), PAR1 and PAR4 [
2]. Research in the field has indicated that PAR1 is a high affinity thrombin receptor that signals through at least 3 classes of G proteins (Gα
12, Gα
q, and Gα
i/o), whereas PAR4 has been shown to signal through at least 2 G protein signaling pathways (Gα
12 and Gα
q but not Gα
i/o) [
3-
5]. PAR1 may be activated in the presence of low levels of circulating thrombin, whereas PAR4 may become active following the initial clot formation when the local thrombin concentration is significantly increased.
The purpose of this study was to determine the expression of PARs on platelets in healthy individuals. To our knowledge, the present study is the first to report the expression of PARs mRNA on platelets in healthy individuals in China.
Materials and methods
Experiment reagents
Trizol reagent was bought from Gibco BRL Co., USA. One-step RNA PCR kit was provided by TaKaRa Co., Japan. Agarose gels were from Sigma Co., USA. Ethidium bromide was procured from Sigma Co., USA.
Laboratory apparatus
PTC150-PCR thermo-cycler was bought from MJ Research Co., USA. Electrophoresis apparati were from Pharmacia Biotech Co., Sweden. The JS-380 automatic gel image analysis instrument was procured from Shanghai Peiqing Technology Co. Ltd., China.
Preparation of platelet-rich plasma (PRP)
Thirty healthy volunteers, with ages ranging from 21 to 29 (24.9 ± 2.1) years, including 15 males and 15 females, were recruited from February to April 2008 from Union Hospital, Wuhan, China. The volunteers were nonsmokers, had no remarkable personal and family medical histories, and did not take any medication for at least 10 days before blood collection. A physical examination and routine laboratory tests, including white blood cell and platelet counts, mean platelet volume, plasma fibrinogen, C-reactive protein (CRP), and von Willebrand factor plasma levels, were performed. The study was approved by the local ethics committee. Informed consent was obtained from all individuals prior to platelet donation. Venous blood was collected by using a 19-gauge needle between 8:00 and 10:00 a.m. after an overnight fast, in tubes containing 0.105 mol/L sodium citrate. The first 2 mL of blood was discarded. PRP was obtained by centrifugation at 150 g for 10 min at 4°C.
Platelet RNA extraction and reverse transcription
Total RNA was extracted by using Trizol reagent according to the manufacturer’s instructions. The concentration of RNA was determined by measuring the absorbance (A) at 260 nm. Total RNA was stored at -80°C. For the reverse transcription-polymerase chain reaction (RT-PCR), 1 µg of total RNA from each sample was re-suspended in a 25 µL final volume of reaction buffer, which contained 10× one-step RNA PCR buffer, 25 mmol/L MgCl2, 10 mmol/L dNTP mixture, 20 U RNase inhibitor, 2.5 U AMV RTase XL, 2.5 U AMV-Optimized Taq, 20 µmol/L forward primer, 20 µmol/L reverse primer, and RNase-free dH2O. Primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services (Shanghai, China) (Table 1). The number of cycles and annealing temperature were optimized for each primer pair. For PAR1, amplification was initiated by 5 min of denaturation at 94°C for 1 cycle, followed by 35 cycles at 94°C for 30 s, 51°C for 30 s, and 72°C for 1 min. For PAR4, amplification was initiated by 5 min of denaturation at 94°C for 1 cycle, followed by 35 cycles at 94°C for 30 s, 57°C for 30 s, and 72°C for 1 min. For β-actin, amplification was initiated by 5 min of denaturation at 94°C for 1 cycle, followed by 35 cycles at 94°C for 30s, 55°C for 30 s, and 72°C for 1 min. After the last cycle of amplification, samples were incubated for 10 min at 72°C. The amplified RT-PCR products were subjected to electrophoresis at 75 V through 1.2% agarose gels for 45 min. A 2000 bp DNA ladder marker was used as a molecular marker. Agarose gels were stained with 0.5 mg/mL ethidium bromide TAE buffer. The gel bands were examined by using a JS-380 gel imaging system.
Statistical analysis
The data were expressed as and analyzed by using SPSS 11.0 software package.
Results
In the present study we found that the average levels of PAR1 mRNA and PAR4 mRNA on platelets in healthy individuals were 0.1601 ± 0.0269 and 0.1073 ± 0.0194 respectively (Fig. 1).
Discussion
Thrombin, a key enzyme in blood coagulation, activates human platelets
via two 7-transmembrane G-protein-coupled protease-activated receptors (PAR1 and PAR4) and activation of either is sufficient to trigger platelet secretion and aggregation [
7,
8].
PARs are G-protein-coupled receptors activated by particular serine proteases
via a unique mechanism, as follows: site-specific proteolysis of the amino-terminal exodomain of the receptor makes a neo-N-terminal exposed, which acts as a self-activating “tethered” ligand [
9,
10]. PAR activation of platelets results in the release of a number of small molecules and protein modulators related to platelet function, including ATP and ADP [
11]. Once they are released, ADP further stimulates the platelet in an autocrine fashion through purinergic receptors on the platelet surface [
12].
Cook
et al has reported that pharmacological blockade of PAR1 or genetic deficiency of PAR4 could inhibit arterial thrombosis in animal models [
13]. Sambrano
et al have observed that platelets from PAR4-deficient mice failed to change shape, mobilize calcium, secrete ATP or aggregate in response to thrombin. This result demonstrates that PAR signaling is necessary for mouse platelet activation by thrombin [
14].
Because of the importance of thrombin signaling in platelets among the multiple pathways and cell types that govern hemostasis and thrombosis, we propose that a selective PAR-1 antagonist has the potential for significant utility in clinical practice.
The therapeutic potential of a PAR-1 antagonist as an antithrombotic agent was first reported in a study with an antibody directed to the extracellular PAR-1 domain that binds thrombin’s exo- site region with high affinity [
13]. In this model of mechanical injury in African Green monkeys, the disruption of thrombin/PAR-1 binding effectively limited experimental thrombosis. More recently, two small peptides were reported to exert antithrombotic actions
via PAR-1 antagonism. The heptapeptide AFLARAA inhibited arterial thrombosis in a rabbit model of electrolytic injury [
15], and the peptide RPPGF delayed coronary occlusion in a canine model using electrolytic injury [
16]. In both cases, however, the mechanism of action of these peptides is unclear because PAR-1 is not expressed in either rabbit or canine platelets [
17,
18]. The development of a selective, small-molecule PAR-1 antagonist, such as RWJ-58259, which directly blocks the tethered ligand of PAR-1, provides a distinct interventional approach to thrombin-induced PAR-1 activation [
19]. Moreover, because PAR antagonists do not affect the ability of thrombin to cleave fibrinogen, these probably cause fewer bleeding complications than thrombin inhibitors. Therefore, PAR1 and PAR4 represent promising targets for the development of new antithrombotic drugs. A number of PAR1 antagonists have been developed, including peptide-mimetic or non-peptide PAR1 antagonists [
20-
22]. For instance, RWJ-56110, a potent synthetic PAR1 antagonist, inhibited platelet aggregation caused by a low concentration (0.05 U/mL) of thrombin. YD-3, a non-peptide PAR4 antagonist, alone had little or no effect on thrombin-induced platelet aggregation, but significantly enhanced the antiaggregatory activity of the PAR1 antagonist.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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