doi:10.1152/ajplung.00289.2011 302:L512-L520, 2012. First published 13 January 2012;Am J Physiol Lung Cell Mol Physiol
Cody, Omar A. Minai and Raed A. DweikMetin Aytekin, Kulwant S. Aulak, Sarah Haserodt, Ritu Chakravarti, Josephpulmonary arterial hypertension: role of nitric oxideAbnormal platelet aggregation in idiopathic
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Abnormal platelet aggregation in idiopathic pulmonary arterial hypertension:role of nitric oxide
Metin Aytekin,1 Kulwant S. Aulak,1 Sarah Haserodt,1 Ritu Chakravarti,1 Joseph Cody,1 Omar A. Minai,2
and Raed A. Dweik1,2
1Departments of Pathobiology/Lerner Research Institute, and 2Pulmonary and Critical Care Medicin /Respiratory Institute,Cleveland Clinic, Cleveland, Ohio
Submitted 30 August 2011; accepted in final form 4 January 2012
Aytekin M, Aulak KS, Haserodt S, Chakravarti R, Cody J,Minai OA, Dweik RA. Abnormal platelet aggregation in idiopathicpulmonary arterial hypertension: role of nitric oxide. Am J PhysiolLung Cell Mol Physiol 302: L512–L520, 2012. First published Janu-ary 13, 2012; doi:10.1152/ajplung.00289.2011.—Idiopathic pulmo-nary arterial hypertension (IPAH) is a rare and progressive disease.Several processes are believed to lead to the fatal progressivepulmonary arterial narrowing seen in IPAH including vasocon-striction, cellular proliferation inflammation, vascular remodeling,abnormalities in the lung matrix, and in situ thrombosis. Nitricoxide (NO) produced by NO synthases (NOS) is a potent vasodi-lator and plays important roles in many other processes includingplatelet function. Reduced NO levels in patients with IPAH areknown to contribute to the development of pulmonary hyperten-sion and its complications. Platelet defects have been implied inIPAH, but original research supporting this hypothesis has beenlimited. Normal platelets are known to have NOS activity, but littleis known about NOS expression and NO production by platelets inpatients with IPAH. Here we characterized the phenotype of theplatelets in IPAH and show a defect in their ability to be activatedin vitro by thrombin receptor activating protein but not adenosinediphosphate. We also show that endothelial NOS (eNOS) levels inthese platelets are reduced and demonstrate that NO is an importantregulator of platelet function. Thus reduced levels of eNOS inplatelets could impact their ability to regulate their own functionappropriately.
nitric oxide synthases; L-NAME; TRAP
PULMONARY ARTERIAL HYPERTENSION (PAH) is a rare and progres-sive disease that leads to deterioration in cardiopulmonaryfunction and premature death. It is characterized by increasesin pulmonary vascular tone, pulmonary vascular remodeling(28), and mean pulmonary artery pressure and an imbalance invasoconstrictors and vasodilators, particularly prostacyclin, en-dothelin-1, and nitric oxide (NO; Ref. 13). Idiopathic pulmo-nary arterial hypertension (IPAH), in which PAH occurs in theabsence of underlying disease, is characterized by complex andabnormal vascular responses and pathology. Several processesare believed to lead to the fatal progressive pulmonary arterialnarrowing seen in pulmonary hypertension (PH) includingvasoconstriction, cellular proliferation inflammation, vascularremodeling, abnormalities in the lung matrix, and in situthrombosis (4, 16, 28).
The thrombotic lesions that are often found in pulmonaryarteries in IPAH suggest that platelet dysfunction is a poten-
tially important pathophysiologic process in the disease (36).Thrombosis in pulmonary arteries could be initiated by abnor-malities in the clotting cascade or platelets. von Willebrandfactor, which plays a crucial role in platelet adhesion andaggregation (23), can be increased in patients with IPAH (44)and may be a predictor of long-term prognosis (19). In additionto having a role in clotting, platelets also release many differentbioactive substances, which regulate vasoconstriction andthrombosis (15).
NO has been proposed as a major physiologic regulator ofblood vessel tone (33, 34). NO in the lung is involved inmultiple functions including vascular smooth muscle relax-ation, proliferation, and inflammation (5, 6). Interestingly,NO and the reaction products of NO are reduced in patientswith IPAH compared with healthy individuals (17) and arerelated in a quantitative fashion to the degree of PAH. Oneof the earliest observations of NO action was that uponplatelets. While this was believed to impact platelets fromthe endothelial-derived NO, it is now very clear that NO isalso produced by platelets themselves (18, 19). Both endo-thelial (eNOS) and inducible NO synthase (iNOS) proteinshave been found in platelets (2, 3, 40). While eNOS appearsto be constitutively present, iNOS protein is found onlyunder inflammatory conditions (40). However, these obser-vations are controversial since other studies fail to detectmRNA or proteins for these enzymes in the platelet (25, 26).
Here we have characterized the phenotype of the platelets inIPAH and have identified that eNOS levels in these plateletsare reduced. We also demonstrate that NO is an importantregulator of platelet function and this reduced level of eNOSwould impact the ability of the platelets to regulate themselvescorrectly. The physiological consequence of this dysregulationleads to several known pathological features of IPAH.
MATERIALS AND METHODS
Study population. Platelet counts were evaluated in 154 patientswith IPAH (114 female, age; 50.8 � 13.5 yr) identified based on theWorld Health Organization criteria as shown in Table 1 (13, 39) and105 healthy control volunteers (83 female, age; 44.3 � 13.5 yr; Table1). Healthy controls were individuals with no history of pulmonary orcardiac disease or symptoms. In a subset of these individuals [22IPAH (14 female, age 51.9 � 12.6 yr) and 21 controls (14 female, age36.5 � 10.4 yr)], we performed more detailed evaluation of plateletstructure and function including cyclooxygenase (COX) and NOSexpression (mRNA: n � 13 IPAH, 12 controls; Western blots: n � 9IPAH, 9 controls).
The platelet aggregation studies were performed on freshly isolatedplatelets from blood obtained from three IPAH patients [at the time of adiagnostic right heart catheterization and before initiation of any PH-specific therapies] and three controls (on the same day as the patients).
Address for reprint requests and other correspondence: R. A. Dweik, Dept.of Pulmonary and Critical Care Medicine, Respiratory Institute, ClevelandClinic, Desk A-90, 9500 Euclid Ave., Cleveland, OH 44195 (e-mail:[email protected]).
Am J Physiol Lung Cell Mol Physiol 302: L512–L520, 2012.First published January 13, 2012; doi:10.1152/ajplung.00289.2011.
Isolated purified platelets were also obtained from normal volun-teers (n � 3) undergoing plasmapheresis for research donation ofblood products from the Cleveland Clinic General Clinical ResearchCenter. The pheresis samples (n � 3) were only used for mRNA andprotein expression experiments but not in the functional studies or todetermine the platelet counts.
All participants signed a consent form that was approved by theCleveland Clinic Institutional Review Board before participation inthese studies.
Platelet isolation. Whole blood from healthy volunteers and fromthe patients with IPAH was collected in citrated tubes (1/7 volumeof [acid citrate dextrose (ACD)]) as described previously (21), andplatelet counts were calculated as platelets � 109 per liter. Bloodwas spun for 20 min at 1,000 rpm at room temperature (RT) toobtain platelet rich plasma (PRP). PRP was placed into anothertube in the presence of 100 nM prostaglandin E1 (PGE1; Sigma catno: P-5515) to prevent aggregation then respun for 20 min at 1,500rpm at RT to get a pellet-containing platelets. The pellet waswashed two times with 1� PIPES-saline-glucose buffer containing5 mM PIPES (Sigma), 145 mM NaCl, 50 mM Na2HPO4, 1 mMMgCl2-6 H2O, and 5.5 mM glucose. Washed platelets were spun10 min at 1,500 rpm at RT, and then mRNA extraction or proteinisolation was performed. Platelets obtained from this source werefurther purified by being mixed with PGE1 to a final concentrationof 100 nM and spun at 1,800 rpm for 20 min. The supernatant wasdiscarded, and the upper layer containing the platelets was care-fully removed, without disturbing the red blood cells, and resus-pended in Tyrode buffer lacking calcium. These platelets were thenrespun, and the process was repeated two more times. At this time, no redblood cell contaminates were visible. After the final spin, platelets wereresuspended in Tyrode buffer containing 1 mM calcium and used in
subsequent experiments. Platelet numbers and purity were confirmedusing Abbott Cell-Dyn 3700 hematology analyzer.
Immunostaining. Isolated platelets were cytospun and then fixed in4% paraformaldehyde for 7 min at RT followed by permeabilizationin 0.5% Triton X-100 for 7 min. Slides were washed with PBS andincubated with primary antibodies for eNOS and CD42b [Biosciences,Pharmingen, and Santa Cruz Biotechnology (Santa Cruz, CA), respec-tively] in humidified chambers for 90 min at RT. After three washesin PBS, they were stained with fluorescence-conjugated secondaryantibodies. Slides were rinsed three times with PBS and once withdistilled water followed by mounting on the glass microscope slidesusing Vectashield mounting medium containing DAPI (Vector Lab-oratories, CA). Confocal XY images were taken using �63 objectivelens (zoom 2) of a Leica TCS-SP/SP-AOSB laser confocal micro-scope (Leica-Microsystems, Wetzlar) using Leica confocal softwareversion 2.5. The excitation (Ex)/emission (Em) wavelengths were asfollows: DAPI: Ex 351 nm, Em 370–420 nm; Alexa Fluor 488: Ex488 nm, Em 500–550 nm; and Alexa Fluor 568: Ex 561 nm, Em575–630 nm.
cDNA synthesis and conventional RT-PCR. Total RNA was iso-lated from platelets using Trizol reagent (Invitrogen) according to themanufacturer’s protocol. For cDNA synthesis, 0.5 ug of RNA fromeach sample was digested by DNAseI (Invitrogen) and total RNA wasreverse-transcribed using the Moloney murine leukemia virus enzyme(Invitrogen) and Oligo dT (Invitrogen). cDNAs were amplified using Taqpolymerase (Invitrogen) with the primer sequences listed in Table 2 andconditions in Table 3. PCR products were separated on 2% agarosegels containing ethidium bromide and visualized under a ultravioletlamp.
Western blot for protein determination. Western blot analysis wasutilized to determine eNOS, neuronal NOS (nNOS), and iNOSproteins in isolated platelets from control and IPAH patients.Isolated platelets were lysed with lysis buffer containing 50 mMTris pH 7,4, 100 mM NaCl, 1 mM EDTA, Nonidet P-40, 10%glycerol, and proteinase inhibitors (200 uM NaO, 20 ug/ml apro-tinin, 5 ug/ml leupeptin, 10 ug/ml pepestatin, 1 mM PMSF, and 1mM DTT) and incubated on ice for 30 min. The protein concen-trations in the supernatants were measured after the lysed plateletswere centrifuged for 30 min at 14,000 rpm at 4°C, and thenproteins were subjected to electrophoresis in SDS-PAGE aftersolubilization in SDS-PAGE sample buffer with 10% �-mercapto-ethanol. The proteins were transferred to nitrocellulose membrane,and Western blot analysis was accomplished by using antibodiesfor eNOS (Biosciences, Pharmingen), nNOS (Biosciences,Pharmingen), and iNOS (Millipore, Temecula, CA) and secondaryantibodies (NA931V; Amersham Biosciences). At the end, West-ern blots were developed using enhanced chemiluminescence (Am-ersham).
Table 1. Demographics and clinical characteristics of IPAHstudy subjects
Table 3. RT-PCR conditions for the different primers
Gene Cycle Profile
eNOS 95°C/30 s; 58°C/30 s; 72°C/45 s for 40 cyclesCOX1 95°C/30 s; 58°C/30 s; 72°C/1 min for 35 cyclesCOX2 95°C/30 s; 58°C/30 s; 72°C/1 min for 35 cyclesGAPDH 95°C/30 s; 58°C/30 s; 72°C/1 min for 35 cycles
For detection of CD63, total protein (15 ug) was lysed andsubsequently separated on a gradient 4 –15% polyacrylamide gel(Bio-Rad) under nonreducing conditions. Protein detection wasperformed with a primary antibody against CD63 (1:200; sc-5275;Santa Cruz Biotechnology) and GAPDH (1:1000; 14C10; CellSignaling, Danvers, MA). For detection of protease-activated re-ceptor-1 (PAR1), total protein (15 ug) was lysed and subsequentlyseparated on a gradient 4 –15% polyacrylamide gel (Bio-Rad)under reducing conditions. Protein detection was performed with aprimary antibody against PAR1 (1:100; ab32611; Abcam, Cam-bridge, MA) and GAPDH (1:10,000; AM4300; Ambion, FosterCity, CA). Blots were incubated with differentially labeled spe-cies-specific secondary antibodies after primary antibody incuba-tion [anti-rabbit IRDye 800CW (green) and anti-mouse IRDye 680(red) 926 –32211 and 926 –32220; LI-COR Biosciences, Lincoln,NE]. Membranes were scanned using the ODYSSEY infraredimaging system (LI-COR Biosciences) and quantitated using Im-ageJ (NIH, Bethesda, MD).
Platelet aggregation assay. Platelets in Tyrode buffer with cal-cium were incubated at RT in the presence or absence of NOinhibitors for �30 min before the aggregation assay. NG-nitro-L-arginine methyl ester (L-NAME), which shows selectivity to eNOSand nNOS, was used at 0, 1, 2, and 5 mM. Aminoguanadine, whichshows selective inhibition to iNOS, was used at 2 mM. At the endof the timed incubations, platelets were subjected to aggregometry.Aggregometry was performed using 2 � 108 platelets/ml in a finalreaction volume of 500 �l. Aggregation was stimulated usingthrombin receptor-activating protein (TRAP) peptide at a finalconcentration of 5 �M. After all the samples were run, an assaywas performed on control platelets to confirm that the plateletswere still capable of aggregation with TRAP peptide to the samedegree as at the start of the experiments. All assays were performedusing a Chrono-log whole blood aggregometer and data wasrecorded using AGROLINK software.
Whenever possible, platelets were diluted to 2 � 108 platelets/mlwith their PRP. However, when platelet numbers from IPAH patientswere �2 � 108, the control PRP was diluted to the same numbers asthat from the undiluted IPAH patients. In all studies, control PRP wasdone at the same time as the IPAH PRP. All aggregation assays wereperformed using a Chrono-log whole blood aggregometer, and datawere recorded using AGROLINK software.
Statistical analysis. All statistical analysis was performed usingJump JMP version 5.0.1.2 for Windows. Continuous variables werecompared with the independent two tailed t-test. P � 0.05 wasconsidered as significant.
RESULTS
Low platelet counts in patients with IPAH compared withcontrols. There was a significant difference in platelet countsbetween IPAH patients and controls [(platelet count � 109/l �SE) 212 � 6 for IPAH group and 266 � 8 for control group;P � 0.0001; Fig. 1]. Although, the patients with IPAH beforeand after therapy consistently had significantly lower plateletcounts than controls there was no significant difference be-tween the platelet count for the patients with IPAH before andafter therapy [(plateletscount � 109/l � SE) 231 � 6 for IPAHbefore therapy and 215 � 7 for IPAH after therapy; P � 0.08].The patients on intravenous prostacyclin therapy, however, hadsignificantly lower platelet counts compared with patients onother PH-specific medications [(platelets countx109/l � SE)173 � 11.2 for the patients in the prostacyclin group and215 � 12.2 for control group; P � 0.02; Fig. 1].
Control and IPAH platelets are mature. To investigate if theplatelets in IPAH patients were younger and therefore suggest-ing higher turnover, we measured the expression of COX-2,which is present in less mature platelets (10) and COX-1,which is present in more mature platelets. mRNA expressionlevels of COX-1 and COX-2 were measured by RT-PCR. COX-1was present in both IPAH and control platelets (Fig. 2A), butCOX-2 was not expressed in either group (Fig. 2B). GAPDHwas used for normalization (Fig. 2C).
Low eNOS protein expression in IPAH platelets comparedwith controls. Expressions of eNOS, nNOS, iNOS, andGAPDH in platelets from patients with IPAH and controlswere evaluated by Western blot analysis. eNOS protein waspresent in both but at much lower levels in IPAH comparedwith controls (Fig. 3A). �-Actin was used to normalize theWestern blot (Fig. 3B). Figure 3C shows the ratios of theoptical densities for eNOS/�-actin protein (IPAH 0.48 �0.05 vs. controls 1.23 � 0.2; P � 0.02). However, iNOS and
Fig. 1. Platelet counts (platelets � 109/ml) were determined in 154 patientswith idiopathic pulmonary arterial hypertension (IPAH) and 105 healthycontrols. Gray area shows the normal range of platelets counts in our clinicallaboratory.
Fig. 2. Cyclooxygenase-1 (COX-1) expression (A) is similar in both IPAH (3representative samples from a total n � 13; lanes 1, 2, and 3) and control (3representative samples from a total n � 12; lanes 3, 4, and 5) platelets, andCOX-2 mRNA expression was not detected in either (B). The housekeepinggene GAPDH was used to normalize (C).
nNOS proteins could not be detected in either IPAH orcontrol (Fig. 3, D and E, respectively) platelets. eNOSmRNA expression analysis was done by RT-PCR. The listof the primers and their conditions are shown in Tables 2and 3, respectively. eNOS mRNA expression was not de-tected in platelets from either IPAH or control individuals(Fig. 3F) despite strong mRNA expression of GAPDH (Fig.3G). We also used immunohistochemistry to determine thesource of NOS. Platelets from Controls and patients withIPAH were stained for the platelet-specific marker CD42band eNOS and are shown side by side in Fig. 4, with A–Drepresenting IPAH and E-H representing controls (A and E:CD42b; B and F: eNOS; C and G: colocalization of the 2;and D and H: secondary antibody staining only). eNOS andCD42b were detected both in IPAH (Fig. 4, A and B,respectively) and control (Fig. 4, E and F, respectively).Colocalization of CD42b and eNOS were shown in Fig. 4Gfor control and in Fig. 4C for IPAH platelets. Figure 4, Dand H, shows negative controls of the same sections stainedby the secondary antibody only. A signal for eNOS proteinis present that co-localizes with the platelet-specific markerCD42b (green color in Fig. 4, C and G), clearly demonstrat-ing that platelets contain eNOS protein. Consistent with theresults obtained from the Western blot data, IPAH patientshave much lower levels of eNOS protein (Fig. 4B) comparedwith controls (Fig. 4F). The negative controls, stained withthe secondary antibody only, showed only slight back-ground of autofluorescence (Fig. 4, D and H).
NO plays a role in platelets aggregation. We investigatedthe effect of NO on platelets using NOS inhibitors. Aggregom-etry using NOS-specific inhibitors clearly demonstrates that
NO is important for platelet aggregation. Consistent with ourfinding that eNOS is present in the platelets, L-NAME, aneNOS/nNOS-selective inhibitor, prevented platelet aggrega-tion in a dose-dependent fashion (Fig. 5A) but aminoguanidine,an iNOS-selective inhibitor, had no effect (Fig. 5B).
Defective platelet aggregation in IPAH patients. IPAH pa-tients have increased in situ thrombosis formation andreduced NO levels. We therefore investigated if plateletaggregation was defective in these patients. We carried outaggregation studies using the activators TRAP or adenosinediphosphate (ADP). Titrating different doses of ADP orTRAP, we determined the level required to induce aggre-gation in control and IPAH patients. Interestingly, the doserequired to activate platelets using ADP was not muchdifferent between IPAH and Control platelets (Fig. 5C). Theeffect of TRAP activation, however, was markedly different(Fig. 5D). In some IPAH patients, even 10 times the con-centration of TRAP could not elicit the same response as thecontrol despite similar ADP responses (Fig. 5C). SinceTRAP activation appeared to be defective in IPAH platelets,we investigated the levels of the TRAP receptor (PAR1) inthese platelets. We saw no PAR1 difference in the levels ofthis protein between IPAH and control platelets (Fig. 6A).There was also no difference in CD63 expression, which ismarker for the dense granules that contain ADP (Fig. 6B).
DISCUSSION
Our data demonstrate that platelet aggregation is defective inIPAH, which may, at least in part, be due to abnormalities inNO levels and NOS expression. We have also conclusively
Fig. 3. Western analysis of endothelial nitric oxidesynthase (eNOS) protein from the platelets of thepatients with IPAH (3 representative samples from atotal n � 9; A, lanes 1, 2, and 3) and control (3representative samples from a total n � 9; A, lanes4, 5, and 6). �-actin was used to normalize (B).Quantitative analysis showed the significant differ-ence of eNOS protein between IPAH and control(C). Inducible NOS (iNOS; D) and neuronal NOS(nNOS; E) proteins could not be detected in eitherIPAH or control platelets protein lysates. eNOSmRNA expression was not detected in platelets ineither IPAH or control platelets (F). Three IPAHpatients (lanes 1, 2, and 3) and 3 controls (lanes 4, 5,and 6) are shown. The PCR product size of eNOSmRNA is 468 bp, and human umbilical vein endo-thelial cell (HUVEC) lines were used as positivecontrol (F). The housekeeping gene GAPDH wasused to normalize (G).
demonstrated that all platelets do contain eNOS protein andthat IPAH platelets have lower eNOS protein levels thancontrols. This important finding suggests that the known NOdeficiency state in IPAH encompasses the platelets as well. Ourdata also confirmed prior reports that platelet counts are de-creased in IPAH individuals compared with controls especiallyin patients receiving IV prostacyclin therapy. Interestingly, ournew findings also demonstrate that circulating platelets fromIPAH patients are mature, suggesting that the low plateletlevels in IPAH are not explained by increased platelet con-sumption.
NO produced by NOS is a potent vasodilator that plays amajor role in lung physiology and is known to be low inpatients with IPAH. One of the earliest observations of NOfunction was the effect on platelets. NO can be derived fromthe endothelium or generated by the platelets. A number ofstudies (25, 32, 38) have found NO generated from platelets,but the identity of the platelet NOS isoform remains con-troversial. The majority of the evidence (1, 8) suggests thatplatelets contain eNOS; however, one study (32) suggestedthat platelets contain no eNOS message or eNOS protein.Another possible source of NO could be iNOS since itsmRNA has been identified in megakaryocytes (the plateletprecursor cells) and in porcine platelets (29). This possibil-ity was corroborated by a study (27) that found that plateletsfrom iNOS-knockout mice have significantly decreased NOproduction and aggregation time compared with controls.However, other studies (1, 12) have found no iNOS proteinin platelets. Therefore, we analyzed for the three majorisoform of NOS by RT/PCR, Western blotting, and immu-nochemistry. Western blotting clearly indicates that eNOSprotein is present in the platelet preparations but not nNOSor iNOS. Interestingly, the level of eNOS protein is dimin-ished in platelets obtained from PH patients compared withcontrols. We next looked for eNOS mRNA in platelets. Ourresults show that both PH and control platelets contain nomRNA for eNOS. Platelets do contain mRNA that is presentfrom the platelet precursor megakaryocyte stage, but newmRNA synthesis does not occur as these cells lack anucleus. Since eNOS mRNA half-life is �14 h (20), and thelife span of a platelet is �1 wk (14, 43), it is unlikely thatwe would find eNOS mRNA in circulating platelets. Thisfinding is consistent with the data from COX-1 absence thatindicate that the platelets used in this experiment are matureand thus older than 14 h. This finding also diminishes thepossibility that the eNOS protein detected by Western blot-ting was derived from other cell types since these cell typeswould contribute to the isolated mRNA and so would havegiven a positive mRNA eNOS signal. To further address theissue of location of eNOS protein, we carried out immuno-cytochemistry using an antibody from a different sourcethan that used in the Western blots. It is clear that cells thatare positive for eNOS protein are ones that were small,anuclear, and positive for the platelets marker CD42b. Thuswe have conclusively demonstrated that eNOS is present inplatelets and that platelets from IPAH patients have muchlower levels of eNOS compared with controls platelets.
The biological effects of NO are complex since it can havedirect effects or can react with other species to generate otherreactive agents. The inhibitory pathway of NO involving directactions on soluble guanylyl cyclase (sGC) leading to thegeneration of cyclic GMP is well characterized. This s mes-senger can then mediate inhibition of platelet aggregation byactivating specific protein kinases that modify actin and myo-sin. In the presence of NO and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxal, an inhibitor of sGC, not all affects of NO are lost,suggesting that there must also be sGC-independent effects forNO on platelets (26).
Our novel findings suggest new potential mechanisms ofhow NO regulates platelet function. In low NO states(simulated by Inhibition of NO synthesis), increased plateletactivation was expected, as the inhibitory blockade caused
Fig. 4. Isolated platelets from the patients with IPAH and control were stainedfor eNOS protein (red) and platelet marker, CD42b, (green). eNOS and CD42bwere detected both in IPAH (A and B, respectively) and control (E and F,respectively). Colocalization of CD42b and eNOS is shown in G for controland in C for IPAH platelets. D and H: negative controls of the same sectionsstained by the secondary antibody only.
by cGMP is removed. We, however, saw the reverse andplatelet aggregation was inhibited. This suggests that NO isalso required for platelet activation, which is consistent withan observed burst of NO upon platelet activation (9, 32).Taken together our data demonstrate that levels of NO arecritical for platelet function. Too low levels of NO (pre-sented here and seen in IPAH patients) as well as too highlevels (previously published; Refs. 30, 35, 37, 41, 42) alterplatelet aggregation.
TRAP activation mimics thrombin activation of PAR1.Activation using TRAP is clearly impaired in PH patients,but the ADP response is not. While the initial TRAPresponse seems normal, i.e., the shape change (increasedabsorbance upon TRAP addition) and start of platelet ag-gregation, this aggregation appears to be reversible and notsustained. This triggers several pathways coupled to G-pro-tein subunits. These G proteins cause the release of calcium,the Rho pathway that results in the rearrangement of theactin cytoskeleton, and granule release. This granule releaseis required to amplify the initial platelet activation signal byreleasing ADP and other agonists. From the aggregationtraces of PH patients, the secondary phase seems lacking.The ability of the platelets to aggregate is not dysfunctional,as ADP elicits a response similar to controls. Since Levelsof PAR1 and CD63 are similar to controls, this suggests that
activation and granule formation are normal but granulecontent maybe aberrant.
Reduced levels of eNOS may explain these observationsin IPAH platelets. Since platelets have to very preciselybalance activation and inhibition, levels of NO need to fallwithin a narrow range. If too much NO is present, thenplatelet activation would be difficult as the sGC inhibitorypathway would predominate. However, too little NO (likewe see in patients with IPAH) would lead to reducedthresholds for platelet activation by reducing the sGC in-hibitory pathway but also by reducing the threshold ofgranule release by N-ethylmaleimide-sensitive factor. Aconsequence of reduced NO would be that platelets wouldbe slowly leaching granular contents. This phenomenon mayexplain the observation of increased plasma levels of sero-tonin, a constituent of dense granules, in IPAH patients (24).Also, since these platelets are more easily activated, due tothe reduced GC pathway inhibition, minor activators couldlead to thrombus formation, which can be found in 50% ofIPAH patients (11). The idea that the platelets exist in apreactivated state is further supported by a study (7) of PAHpatients that found elevated levels of fibrinopeptide, amarker of fibrin breakdown and production, in 100% ofIPAH patients. Other studies (22) found elevated von Wil-lebrand factor antigen levels in IPAH and PH associated
Fig. 5. Platelets were incubated with various con-centrations of NG-nitro-L- arginine methyl ester (L-NAME; A) or aminoguanadine (AG; B) at the end ofa 1 h incubation they were tested for aggregationusing 5 �M receptor-activating protein (TRAP) ac-tivation as described in the text. Platelet rich plasmawas isolated from IPAH patients and from controls.Platelet were then counted and diluted to give thesame concentration between patient and control.Dose-response curve using 0–10 mM ADP and0–30 �M TRAP was carried out. Typical responsesto TRAP (C) or ADP (D) are demonstrated on 3patients and same day controls are shown (n � 3IPAH; n � 3 controls).
with congenital heart disease, elevated levels of solubleP-selectin in IPAH patients, and increased von Willebrandfactor levels at baseline and follow-up are associated withworse survival in patients with PAH. Based on our findings,when levels of eNOS are very low, then only partial acti-vation of platelets occurs, with activation impaired since NOis also required for a full response. This concept is alsosupported by the increased presence of p-selectin ineNOS�/� mice in circulating platelets (31) and PH patientshaving circulating and activated platelets.
A model based on our findings and what is known aboutplatelet function is depicted in Fig. 7. Activation of throm-bin leads to binding and activation of the PAR-1 receptor onplatelets. This causes platelet degranulation and release offactors such as ADP, which potentiates the signal causing astrong aggregation response. Under normal conditions, NOcauses activation of sGC, which prevents premature activa-tion of platelets. However, in IPAH the level of NO isreduced, which reduces the threshold of platelet activation.In healthy controls, NO is increased upon platelet activationleading to full activation. This step is dysregulated in IPAHpatients due to the low level of NO in these patients. Thusplatelet dysfunction in IPAH is related to the known globalNO deficiency in this disease that is now recognized toencompass the platelets. The central defect in eNOS inIPAH platelets could also have profound effects on otherpathobiological features of the disease including (but notlimited to) in situ thrombosis.
GRANTS
Support for this study was provided by National Institutes of Health GrantsHL-081064, HL-107147, HL-095181, and RR026231 and a BRCP 08-049
Fig. 6. Expression levels of CD63 protein in platelet. CD63 expression wasmeasured by Western blot in 3 healthy individuals and 3 IPAH patients (A).Expression levels of PAR1 protein in platelet. PAR1 expression was measuredby Western blot in 3 healthy individuals and three IPAH patients (B; repre-sentative sample from a total n � 9 IPAH; n � 9 controls).
Fig. 7. Activation of platelets by thrombin leads to binding and activation of the protease-activated receptor-1 (PAR1) receptor on platelets. This causes plateletdegranulation and release of factors such as ADP which potentiates the signal causing a strong aggregation response. Normally NO causes activation of solubleguanylyl cyclase (sGC), which prevents premature activation of platelets. However, in pulmonary hypertension (PH) the level of NO is reduced thus reducingthe threshold of platelet activation.
Third Frontier Program Grant from the Ohio Department of Development (toR. A. Dweik). This work was also supported in part by the National Institutesof Health, National Center for Research Resources, CTSA 1UL1RR024989,Cleveland, Ohio and by American Heart Association Grant 0826095H (to M.Aytekin).
DISCLOSURES
No conflicts of interest, financial or otherwise are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: M.A., K.S.A., O.A.M., and R.A.D. conceptionand design of research; M.A., K.S.A., S.H., R.C., and J.C. performedexperiments; M.A. and R.A.D. analyzed data; M.A., K.S.A., and R.A.D.interpreted results of experiments; M.A., K.S.A., and R.A.D. preparedfigures; M.A., O.A.M., and R.A.D. drafted manuscript; M.A., K.S.A., andR.A.D. edited and revised manuscript; M.A., K.S.A., and R.A.D. approvedfinal version of manuscript.
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