| Diabetes mellitus (DM) is a chronic disease with many risk factors, complex pathogenesis and the course of lifelong.Atherothrombotic cardiovascular and thrombosis events, including dyslipidemia, obesity, hypertension, hypercoagulable state,, are critically risk factors and its huge variety of hazards leads to serious complications in diabetic patients. Cardiovascular and cerebrovascular events are the most serious complications of diabetes and the main reason affecting the quality of diabetes patients life, what’s more, it threatens the lives of diabetes patients.Recent studies have found that platelet hyperactivity, including platelet adhesion, aggregation and release reaction, a critical factor in the pathogenesis of intravascular coagulation, thrombosis and atherosclerosis (2), plays a major role in this phenomenon in type2diabetes.Arachidonic acid (AA) is contained Platelet membrane phospholipids and phospholipase A2is stored platelet intracellular. When the platelet surface is activated, phospholipase A2is also activated.As a result, AA is separated from the plasma membrane phospholipids and produce prostaglandin G2and prostaglandin H2in the role of cyclooxygenase-1, which catalyze thromboxane synthase and form a large number of thromboxane A2in platelet. Thus COX-1and thromboxane synthase is a key enzyme in the process of generating TXA2and PGH2.TXA2is one of the most powerful vasoconstrictor substances in vivo, while TXA2inhibits adenylate cyclase in platelet membrane to make the production of cyclic AMP which inhibits platelet activation, aggregation, and thrombosis be decreased. TXA2which have very short half-life is rapidly inactivated and degraded to TXB2. by a variety of ways,TXA2may be determined by measuring its stable metabolite, such as serum or plasma thromboxane B2or urinary11-dehydro thromboxane B2. Thromboxane B2levels is largely dependent on platelet COX-1,so it can be used to measure inhibitory effect of low-dose aspirin on platelets.Many experiments have proved that there is L-arginine/nitric oxide system in platelets.L-Arg generates NO and L-citrulline in the role of oxygen molecules and nitric oxide synthase in platelets.NO activates guanylate cyclase which elevates the levels of cyclic guanosine monophosphate, and then inhibits platelet activation and aggregation. NOS activity is a key step in the course of NO synthesis. NO can inhibit platelet aggregation and platelet activation and reduce degranulation and the release of the active substance.Some studies have shown that the more thromboxane A2releases, the stronger platelet sensitivity is.Increased platelet activity is not only due to substance-induced activation of coagulation,but also the functional status of platelets caused by the loss of anticoagulant mechanism of inhibitory effect. Platelet endothelial nitric oxide synthase activity is significantly lower than normal, indicating that eNOS activity may play an important role in the development of cardiovascular complications of diabetes.In type2diabetes, aspirin can make an effect on anti-platelet activation therapy for the prevention of diabetic vascular disease occurrence and development of a positive clinical significance.Aspirin can acetylate platelet COX-1and prevent arachidonic acid transforming to thromboxane A2(TXA2), thereby inhibiting the synthesis of TXA2, blocking the TXA2-induced platelet aggregation and thrombosis, playing important role in anti-platelet. Since platelets are non-nuclear cells and can not create new COX-1, so that COX-1activity of the aspirin inhibition is irreversible. Studies have shown that aspirin can increase the synthesis of NO activity by activating normal platelets eNOS, inhibit the platelet aggregation and play a role on protecting vascular endothelium.In addition to diect anti-platelet aggregation effect, aspirin can also make an anti-platelet indirect effect on anti-inflammatory and anti-oxidation by inhibiting inflammatory cytokines, peroxidase.In type2diabetes, although aspirin can be effective on anti-platelet activation and prevent the cardiovascular complications, the events of aspirin resistance has often occurred. Insulin resistance occupies a central position in the pathogenesis of type2diabetes. IR causes an increase of oxidative stress which elevates levels of ox-LDL, nuclear factor κB (NF-κB) activation, endothelial NOS uncoupling, and thereby promote platelet adhesion, aggregation and thrombosis. IR increases levels of free radicals and reactive oxygen species, which in turn further damage to the insulin signal transduction pathway metabolism and vascular factors that promote the formation of thrombus. Therefore, we speculate whether high insulin affects the synthesis of TXA2by aspirin-induced COX pathway and NO production by NOS and L-arginine transport process.This study explored whether insulin affects aspirin-inhibited platelet aggregation TXA2and NO synthesis production in vitro.In this study, insulin increases the synthesis of aspirin-induced TXA2, but does not affect the production of NO with platelet-rich plasma by platelet aggregation instrument, ELISA and other methods to detect in vitro. MATERIALS AND METHODS1. Experimental subjectsTo choose healthy human volunteers from the305th hospital of Chinese People’s Liberation Army using fresh blood to prepare platelet. Inclusion criteria were:(1). free from hemostatic disorders, diabetes, and infectious, hepatic, renal, cardiovascular, and malignant diseases, hypertension;(2). non-smoking human volunteers;(3). did not take any medicine during the preceding two weeks;(4). did not ingest alcohol in the previous24hours;(5). Elbow vein exposure significantly and is thicker;(6). The blood samples were collected after overnight fasting (12hours at least).2. Experimental methods2.1Preparation of platelet plasma2.1.1Blood collectionThe blood samples were collected after overnight fasting via forearm vein punctures performed by a trained nurse using a21G needle without a tourniquet at22℃-24℃room temperature. Blood was first injected gently into two blood tubes and then into PT tubes containing1volume of acid-citrate-dextrose for9volumes of blood,and the tubes were mixed by gentle inversion.2.1.2Preparation of platelet-rich plasma(PRP)Anti-coagulated blood was centrifuged at100x g for15minutes at21℃without braking. The supernatants were collected by foul disposable straw into a clean centrifugal tube.2.1.3Preparation of platelet-poor plasma(PPP)A part of the platelet-rich plasma was centrifuged at2000x g for10minutes at21℃without braking. The supernatants were collected by foul disposable straw into other clean centrifugal tube.2.1.3Preparation of insulin-aspirin PRP, The equivalent volume of platelet-rich plasma (PRP) prepared by hand was preincubated with insulin(30,100,300μU/mL) in37℃water for3minutes, which was labeled A, B, C group and then stimulated with different concentrations of aspirin (0,75,150,300μmol/L) for30minutes in water of37℃. The platelet maximal aggregation each sample were analyzed by platelet aggregation system.2.2Platelet aggregationBlank parameters are set with containing PPP cup in platelet aggregation system. PRP prepared as described above will be transferred to the inspection cup, added25μL of ADP or AA, observed and recorded the process of platelet aggregation within5min.2.3Thromboxane B2synthesisIn PRP, TXB2levels were measured at the end of the aggregation tests (i.e.,5min after the addition of agonists-ADP/AA) after blocking platelet responses with indomethacin and citrate-dextrose. Each sample was then spun at8,000g for2min,and supernatants were stored and frozen at-80℃. TXB2was measured by using the EIA kit.2.4cGMP productionPlatelet samples (PRP) have been repeated with20-min preincubations with the NOS inhibitor L-NMMA (100μmol/L), or the guanylate cyclase inhibitor MB (50μmol/L).cGMP was measured in PRP samples incubated at37℃for3min with30and300μU/mL insulin and then exposed to aspirin (30μmol/L for30min). Platelet reactions were stopped with30%trichloroacetic acid. Precipitated proteins were removed by20-min centrifugation at2,000g at4℃. After the addition of lmol/L of hydrochloric acid,the supernatant was submitted to10extractions with ethylic ether to remove trichloroacetic acid. Samples were then lyophilized and kept at-80℃until determination. cGMP measurement was carried out using a radio immune assay kit. 3. Statistical analysisAll data were presented as means±SD. Results concerning platelet aggregation responses and agonist-induced thromboxane B2synthesis have been evaluated with a parpmetric generalized linear model two-factor within subject ANVOA for repeated measures. When only two measures had to be compared, we used Student t test for paired data when appropriated. The statistics were analyzed using SPSS for Windows13.0. A value of P<0.05was considered statistically significant.Results1. Platelet aggregation1.1In response to ADPThe inhibitory effects of0,75,150, and300μmol/L aspirin differed in the presence of300μU/mL vs. in the presence of30μU/mL (P<0.05)(87.62±6.23%vs84.64±8.09%,60.85±16.24%vs48.13±19.27%,55.28±16.48%vs44.30±20.50%,49.36±16.77%vs35.30±16.81%),whereas in the presence of300μU/mL vs. in the presence of100μU/mL and in the presence of30μU/mL vs. in the presence of100μU/mL were not significant.In the absence of L-ASA, high insulin did not modify platelet aggregation; the main effect (i.e., on the whole sample) of aspirin was significant (P<0.05for all the concentrations vs. baseline); whereas the inhibitory effects of300and of150μmol/L L-ASA did not differ (P>0.05).1.2In response to AAThe inhibitory effects ofO,5,30, and100μmol/L aspirin differed in the presence of300μU/mL vs. in the presence of100μU/mL (89.62±4.23%vs89.43±4.88%,88.03±6.93%vs86.30±4.43%,,17.33±2.69%vs10.10±2.34%,13.77±2.76%vs8.15±2.55%) and300μU/mL vs. in the presence of30μU/mL (P<0.05)(89.62±4.23%vs88.45±4.32%,88.03±6.93%vs86.56±5.18%,,17.33±2.69% vs7.60±2.20%,13.77±2.76%vs4.56±2.15%), whereas in the presence of30μU/mL vs. in the presence of100μU/mL was not significant.In the absence of L-ASA, high insulin did not modify platelet aggregation; the main effect (i.e., on the whole sample) of aspirin was significant (P<0.05for all the concentrations vs. baseline); whereas the inhibitory effects of30and of100μmol/L L-ASA did not differ (P>0.05)2Thromboxane B2synthesisThe inhibitory effects of0,75,150, and300μmol/L aspirin differed in the presence of300μU/mL vs. in the presence of30μU/mL (3.65±0.07ng/mL vs3.43±0.59ng/mL,1.68±0.68ng/mL vs1.00±0.51ng/mL,1.37±0.69ng/mL vs0.86±0.44ng/mL,1.08±0.55ng/mL vs0.85±0.40ng/mL), P<0.05.3cGMP productionIn the presence of30μU/mL insulin,300μmol/L aspirin increased intraplatelet cGMP, this effect is attributable to the NO-induced activation of guanylate cylase, but it was not completely prevented by both L-NMMA and MB.ConclusionsThe findings demonstrate that high insulin (300μU/mL) environment, will impair the effect of aspirin on platelet aggregation In vitro,and increase the content of TXA2, but does not affect the synthesis of NO/cGMP production.These findings provide new insights into the relationship between insulin therapy and process of thrombosis in the type2diabetes mellitus. |
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