Abstract

Platelets play a crucial role in the physiology of primary hemostasis and pathophysiologic processes such as arterial thrombosis. Accumulating evidence suggests a role of reactive oxygen species (ROSs) in platelet activation. Here we show that platelets activated with different agonists produced intracellular ROSs, which were reduced by reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase inhibitors and superoxide scavengers. In addition, we demonstrate that ROSs produced in platelets significantly affected αIIbβ3 integrin activation but not alpha and dense granule secretion and platelet shape change. Thrombin-induced integrin αIIbβ3 activation was significantly decreased after pretreatment of platelets with NAD(P)H oxidase inhibitors (diphenylene iodonium [DPI] [45% ± 9%] and apocynin [43% ± 11%]) and superoxide scavengers (tiron [60% ± 9%] and Mn(III)tetrakis (1-methyl-4-pyridyl)porphyrin [MnTMPyP] [70% ± 6%]). These inhibitors also reduced platelet aggregation and thrombus formation on collagen under high shear and achieved their effects independent of the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway.

Introduction

Platelet activation is a complex process that involves different cellular signaling pathways. Recently it has been suggested that reactive oxygen species (ROSs) take part in platelet activation.1  ROSs are mostly generated by reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase, an enzyme complex that was primarily described in phagocytes, and has bactericidal function in these cells.2  Within the vessel wall, endothelial cells, vascular smooth muscle cells, and fibroblasts express nonphagocytic NAD(P)H oxidase isoforms that produce mostly intracellular ROSs involved in cellular signaling.3  Here, generated ROSs act as second messengers in control of different physiologic responses such as gene expression, apoptosis, and proliferation.2 

In platelets the presence of NAD(P)H oxidase subunits has been shown by several groups.4-6  ROSs may regulate platelet function by decreasing NO bioavailability because ROSs scavenge platelet or endothelium-derived nitric oxide (NO).7-9  However, it is still not clear whether the only possible mechanism for the regulation of platelet activation by ROSs is due to decreased NO bioavailability or perhaps due to a direct role of ROSs in the control of platelet functions.1  Because the source of intracellular ROS production as well as the potential role of ROSs in platelets are not well defined, it was our goal (1) to determine which platelet agonists cause ROS production; (2) to ascertain possible sources of ROSs; (3) to test if platelet ROSs affect platelet function by altering integrin activation or platelet secretion; and (4) to test the hypothesis that inhibition of ROSs in platelets increases platelet NO/cyclic guanosine monophosphate (NO/cGMP) levels and vasodilator-stimulated phosphoprotein (VASP) phosphorylation and thus inhibits platelets. Here, we report that NAD(P)H oxidase activation in platelets generates intracellular ROSs, which are involved, at least partially, in the regulation of αIIbβ3 activation without affecting the NO/cGMP pathway, granule secretion, and platelet shape change.

Study design

Measurement of ROS production

Platelets and neutrophils were isolated from whole blood obtained from healthy volunteers as described previously10,11  according to our institutional guidelines and the Declaration of Helsinki. Approval for this study was obtained from the local ethics committee of the University of Wuerzburg, and informed consent was provided according to the Declaration of Helsinki. For detection of intracellular ROSs, platelets and neutrophils were preloaded with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Molecular Probes, Göttingen, Germany) and measured by flow cytometry (FACSCalibur; Becton Dickinson, Heidelberg, Germany). Extracellular ROS production was measured by L-012 (100 μM; Wako, Neuss, Germany) in chemiluminescence assay (Wallac Victor 1420, Perkin Elmer Wallac Life and Analytical Sciences, Boston, MA).

Analysis of αIIbβ3 activation, P-selectin expression, aggregation, and flow chamber experiments

For the determination of P-selectin expression, washed platelets were stained with R-phycoerythrin (RPE)–conjugated anti-CD62P antibody (DAKO, Glostrup, Denmark) and, for αIIbβ3 activation, with fluorescein isothiocyanate (FITC)–PAC-1 antibody (Becton Dickinson). Aggregation was carried out using a Bio/Data PAP-4 aggregometer (Bio/Data, Horsham, PA) with platelet-rich plasma as previously described.10 

Flow chamber experiments were carried out as described previously,12  with coverslips coated with Horm-type collagen under high shear conditions (1000 s–1, 4 minutes) with the anticoagulated whole blood.

Serotonin secretion, thromboxane synthase activity, and cGMP and cAMP levels

Analysis of serotonin secretion and thromboxane synthase activity was determined as described previously.13  Levels of cGMPand cyclic adenosine monophosphate (cAMP) were measured in washed platelets according to the manufacturer's protocol (R&D Systems, Wiesbaden-Nordenstadt, Germany).

Data analysis

All experiments were performed at least in triplicate, and data shown are means ± SD. Data were analyzed using analysis of variance (ANOVA) followed by the Bonferroni test or Student t test. Differences were considered significant when the significance was P < .05.

Results and discussion

In previous studies, the production of ROSs was mainly observed with collagen-stimulated5,14-16  and thrombin-stimulated7,11  platelets. Here we show intracellular ROS production also with thrombin receptor activating peptide 6 (Trap6) and the stable thromboxane A2 analog U46619, but not with adenosine diphosphate (ADP) stimulation (Figure 1A), while no extracellular signal for ROSs was observed (not shown). Control experiments were done in neutrophils, which produced both intracellular and extracellular ROSs after phorbol myristate acetate (PMA) stimulation (Figure 1A).

To investigate the possible source and type of intracellular ROS production, we used different inhibitors and superoxide scavengers. Inhibitors of mitochondrial respiration, xanthine oxidase, and nitric oxide synthase (NOS) had no effect on ROS production, while NAD(P)H oxidase inhibitors, diphenylene iodonium (DPI) and apocynin, and the cyclooxygenase (COX) inhibitor acetylsalicylic acid (ASA) significantly inhibited thrombin-induced ROS production (Figure 1B), indicating that NAD(P)H oxidase and COX are the likely sources of ROSs in platelets. However, COX is a part of prostaglandin endoperoxide H synthase (PGHS), which produces peroxy compounds like 12(R)-hydroxyperoxy-eicosatetraenoic acid (15(R)-HPETE) and could in turn oxidize H2DCF-DA. Furthermore, non-ROS–related enzymatic oxidation of H2DCF-DA could occur, as shown by Larsen et al.17  The lack of effect of NAD(P)H oxidase inhibitors and superoxide scavengers on thromboxane synthase activity (not shown) and the additive inhibition of ROSs by DPI and ASA or tiron and ASA (not shown) indicate that NAD(P)H oxidase–generated ROSs are independent from COX. That the intracellular superoxide scavengers tiron and superoxide dismutase (SOD) mimetic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), but not the hydroxyl scavenger mannitol, inhibited ROS production also indicates superoxide as the major radical in platelets.

To address the role of ROSs in platelet regulation, we assessed the major markers of platelet activation, namely P-selectin expression and dense granule secretion (serotonin), shape change, platelet aggregation, thrombus formation, and integrin activation (Figure 2). Platelet aggregation induced by Trap6 was significantly reduced in the presence of DPI and apocynin (Figure 2A). Aggregation traces showed no loss of shape change even when high concentrations of DPI (300 μM) and apocynin (4.8 mM) were used (not shown). This was further confirmed by unaltered myosin light chain (MLC) phosphorylation and Rho family small G protein RhoA activation (not shown). Integrin αIIbβ3 activation plays a major role in the regulation of platelet adhesion and aggregation. Integrin αIIbβ3 activation was inhibited by NAD(P)H oxidase inhibitors and superoxide scavengers. ASA, as previously shown,18,19  had no effect on integrin activation, indicating that peroxy compounds do not play a significant role in integrin activation. At high shear, activated integrins are a key step in platelet-platelet interactions and thrombus formation. Flow chamber experiments over a collagen-coated surface showed significantly reduced thrombus formation under high shear flow conditions (1000 s1) in the presence of DPI or apocynin (Figure 2D). Furthermore, platelet stimulation by thrombin, Trap6, U46619, convulxin, and ADP showed a similar pattern of αIIbβ3 activation and ROS production (Figure 2C). Integrin inhibition by ROSs was independent of Rap1 (small guanosine triphosphatase [GTPase] activated by many different platelets agonists) stimulation because no changes in Rap1b activation were observed (not shown). NAD(P)H oxidase inhibitors and superoxide scavengers had no significant effect on alpha and dense granule secretion (not shown).

Figure 1.

Intracellular ROS production in platelets and platelet sources of ROSs. (A) Washed human platelets (300 × 109/L) were preincubated with H2DCF-DA (50 μM) for 30 minutes, 37°C in phosphate-buffered saline (PBS)/5.5 mM glucose/1 mM EDTA (ethylenediaminetetraacetic acid), excessive dye was washed out, and platelets were resuspended in HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer. Platelets were stimulated with thrombin (0.2 U/mL), Trap6 (30 μM), U46619 (1 μM), convulxin (100 ng/mL), or ADP (50 μM) for 1 minute. Samples were then analyzed for intracellular ROS production by flow cytometry. In control experiments, ROS production was measured in neutrophils after PMA (0.1 μM) stimulation. Neutrophils produced high amounts of extracellular ROSs (420-fold increase versus control after 20 minutes of PMA stimulation) as measured by L-012. Intracellular production (H2DCF-DA, 10 μM) was much lower (PMA stimulation 6-fold increase versus control). In both cases, ROS production was inhibited by DPI and tiron. No signal for extracellular ROSs was observed in platelets in the same experiment (not shown). Data are represented as arbitrary units, mean ± SD from 5 experiments, control taken as 1 (*significantly different at P < .05 compared with control; #significantly different at P < .05 compared with PMA). (B) Washed human platelets (300 × 109/L) were preincubated with inhibitors of NAD(P)H oxidase (DPI [10 μM] or apocynin [600 μM]), mitochondrial metabolism (rotenone [100 μM]), xanthine oxidase (oxypurinol [100 μM]), COX (ASA 100 μM]), or NOS (nitro-l-arginine methyl ester [l-NAME]) for 5 minutes. Then, after activation by thrombin (0.2 U/mL) for 1 minute, ROS production was measured by flow cytometry. Platelet ROS production was also inhibited with intracellular superoxide scavengers tiron (3 mM) and MnTMPyP (100 μM) after thrombin stimulation. Data are shown as arbitrary units, mean ± SD, thrombin-induced ROS production taken as 1 (*significantly different at P < .05 compared with thrombin; n = 6). Error bars indicate 50 (from mean).

Figure 1.

Intracellular ROS production in platelets and platelet sources of ROSs. (A) Washed human platelets (300 × 109/L) were preincubated with H2DCF-DA (50 μM) for 30 minutes, 37°C in phosphate-buffered saline (PBS)/5.5 mM glucose/1 mM EDTA (ethylenediaminetetraacetic acid), excessive dye was washed out, and platelets were resuspended in HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer. Platelets were stimulated with thrombin (0.2 U/mL), Trap6 (30 μM), U46619 (1 μM), convulxin (100 ng/mL), or ADP (50 μM) for 1 minute. Samples were then analyzed for intracellular ROS production by flow cytometry. In control experiments, ROS production was measured in neutrophils after PMA (0.1 μM) stimulation. Neutrophils produced high amounts of extracellular ROSs (420-fold increase versus control after 20 minutes of PMA stimulation) as measured by L-012. Intracellular production (H2DCF-DA, 10 μM) was much lower (PMA stimulation 6-fold increase versus control). In both cases, ROS production was inhibited by DPI and tiron. No signal for extracellular ROSs was observed in platelets in the same experiment (not shown). Data are represented as arbitrary units, mean ± SD from 5 experiments, control taken as 1 (*significantly different at P < .05 compared with control; #significantly different at P < .05 compared with PMA). (B) Washed human platelets (300 × 109/L) were preincubated with inhibitors of NAD(P)H oxidase (DPI [10 μM] or apocynin [600 μM]), mitochondrial metabolism (rotenone [100 μM]), xanthine oxidase (oxypurinol [100 μM]), COX (ASA 100 μM]), or NOS (nitro-l-arginine methyl ester [l-NAME]) for 5 minutes. Then, after activation by thrombin (0.2 U/mL) for 1 minute, ROS production was measured by flow cytometry. Platelet ROS production was also inhibited with intracellular superoxide scavengers tiron (3 mM) and MnTMPyP (100 μM) after thrombin stimulation. Data are shown as arbitrary units, mean ± SD, thrombin-induced ROS production taken as 1 (*significantly different at P < .05 compared with thrombin; n = 6). Error bars indicate 50 (from mean).

Figure 2.

NAD(P)H oxidase inhibitors and superoxide scavengers reduce platelet aggregation, αIIbβ3 activation, and thrombus formation under high shear flow conditions. (A) Platelet-rich plasma was preincubated with DPI (50 μM) and apocynin (1.2 mM) and then stimulated with Trap6 (30 μM) and allowed to aggregate for 5 minutes, with stirring at 1000 rpm. Platelet aggregation was demonstrated by the change in light transmission. A representative aggregation tracing from 3 independent experiments is shown. (B) Washed human platelets (300 × 109/L) were preincubated with DPI (10 μM), apocynin (600 μM), tiron (3 mM), MnTMPyP (100 μM), or ASA (100 μM) and stimulated with thrombin (0.2 U/mL) for 1 minute. Integrin αIIbβ3 activation was measured with FITC–PAC-1 monoclonal antibody (mAb). DPI, apocynin, tiron, and MnTMPyP, but not ASA, inhibited αIIbβ3 activation. Data are shown as arbitrary units, mean ± SD, thrombin-induced PAC-1 binding taken as 1 (*significantly different at P < .05 compared with thrombin; n = 5). In the same experimental conditions, platelets were analyzed for VASP phosphorylation23  by Western blot and for cGMP levels. NAD(P)H oxidase inhibitors and superoxide scavengers did not significantly change either VASP phosphorylation or cGMP levels in platelets. Sodium nitroprusside (SNP) (1 μM) was used as a positive control for phosphorylated VASP (P-VASP) and cGMP. Data for cGMP are shown as fold increase, mean ± SD, thrombin taken as 1 (*significantly different at P < .05 compared with thrombin; n = 3). (C) ROS production and αIIbβ3 activation measured by FITC–PAC-1 mAb after stimulation with different platelet agonists, thrombin (0.2 U/mL), Trap6 (30 μM), U46619 (1 μM), convulxin (100 ng/mL), and ADP (50 μM). (D) Inhibition of thrombus formation after treatment of platelets with DPI and apocynin. Whole blood from different individuals was treated with DMSO (control), DPI ([ii] 100 μM), or apocynin ([iii] 1.2 mM) and was perfused over a collagen-coated surface for 4 minutes under high shear conditions (1000 s–1). The chamber was rinsed, and phase-contrast images were taken with a Zeiss Axiovert 200 inverted microscope, using a 63 ×/0.75 numeric aperture objective (both from Carl Zeiss, Göttingen, Germany), and a CV-M300 CCD camera (Visitron, Munich, Germany) connected to an S-VHS video recorder (AG-7355; Panasonic, Matsushita Electric, Japan). Videotaped images were evaluated using Meta Morph (Visitron), a computer-assisted image analysis program. The results of the experiments are summarized as bar graphs showing percentage surface area coverage (iv). DPI and apocynin caused 18% ± 5% and 20% ± 6% inhibition of platelet adhesion, respectively (*significantly different at P < .05 compared with control; n = 3). The images shown are representative of 3 individual experiments.

Figure 2.

NAD(P)H oxidase inhibitors and superoxide scavengers reduce platelet aggregation, αIIbβ3 activation, and thrombus formation under high shear flow conditions. (A) Platelet-rich plasma was preincubated with DPI (50 μM) and apocynin (1.2 mM) and then stimulated with Trap6 (30 μM) and allowed to aggregate for 5 minutes, with stirring at 1000 rpm. Platelet aggregation was demonstrated by the change in light transmission. A representative aggregation tracing from 3 independent experiments is shown. (B) Washed human platelets (300 × 109/L) were preincubated with DPI (10 μM), apocynin (600 μM), tiron (3 mM), MnTMPyP (100 μM), or ASA (100 μM) and stimulated with thrombin (0.2 U/mL) for 1 minute. Integrin αIIbβ3 activation was measured with FITC–PAC-1 monoclonal antibody (mAb). DPI, apocynin, tiron, and MnTMPyP, but not ASA, inhibited αIIbβ3 activation. Data are shown as arbitrary units, mean ± SD, thrombin-induced PAC-1 binding taken as 1 (*significantly different at P < .05 compared with thrombin; n = 5). In the same experimental conditions, platelets were analyzed for VASP phosphorylation23  by Western blot and for cGMP levels. NAD(P)H oxidase inhibitors and superoxide scavengers did not significantly change either VASP phosphorylation or cGMP levels in platelets. Sodium nitroprusside (SNP) (1 μM) was used as a positive control for phosphorylated VASP (P-VASP) and cGMP. Data for cGMP are shown as fold increase, mean ± SD, thrombin taken as 1 (*significantly different at P < .05 compared with thrombin; n = 3). (C) ROS production and αIIbβ3 activation measured by FITC–PAC-1 mAb after stimulation with different platelet agonists, thrombin (0.2 U/mL), Trap6 (30 μM), U46619 (1 μM), convulxin (100 ng/mL), and ADP (50 μM). (D) Inhibition of thrombus formation after treatment of platelets with DPI and apocynin. Whole blood from different individuals was treated with DMSO (control), DPI ([ii] 100 μM), or apocynin ([iii] 1.2 mM) and was perfused over a collagen-coated surface for 4 minutes under high shear conditions (1000 s–1). The chamber was rinsed, and phase-contrast images were taken with a Zeiss Axiovert 200 inverted microscope, using a 63 ×/0.75 numeric aperture objective (both from Carl Zeiss, Göttingen, Germany), and a CV-M300 CCD camera (Visitron, Munich, Germany) connected to an S-VHS video recorder (AG-7355; Panasonic, Matsushita Electric, Japan). Videotaped images were evaluated using Meta Morph (Visitron), a computer-assisted image analysis program. The results of the experiments are summarized as bar graphs showing percentage surface area coverage (iv). DPI and apocynin caused 18% ± 5% and 20% ± 6% inhibition of platelet adhesion, respectively (*significantly different at P < .05 compared with control; n = 3). The images shown are representative of 3 individual experiments.

Several studies suggested that intracellularly produced ROSs scavenge endothelial or platelet-derived NO in a fast reaction forming peroxynitrite (ONOO) as an end product.7,9,20  The NO/cGMP pathway is a well-established mechanism of platelet inhibition and can be monitored by cGMP-dependent protein kinase (cGK)– and cAMP-dependent protein kinase (cAK)–mediated VASP phosphorylation.21  Therefore, we hypothesized that inhibition of ROS production in platelets might lead to an increase of cGMP levels and VASP phosphorylation, because more NO should be present. VASP phosphorylation closely correlates with the inhibition of the integrin αIIbβ3 activation on human platelets.22  However, changes in VASP phosphorylation or GMP levels (Figure 2B) or cAMP (not shown) levels were not observed. Collectively, our data suggest that NAD(P)H oxidase–generated ROSs are involved in integrin activation and act by other mechanisms than scavenging NO. Although the molecular mechanism of integrin activation following cellular activation and ROS production is presently not clear, our results strongly suggest an involvement of intracellular ROSs in integrin regulation.

Prepublished online as Blood First Edition Paper, June 23, 2005; DOI 10.1182/blood-2005-03-1047.

Supported in part by the Deutsche Forschungsgemeinschaft and by a grant from the Bayerische Forschungsstiftung.

A.J.B. was responsible for platelet experiments, analysis, and writing initial drafts of the paper (as part of her PhD thesis work); S.G. was PhD advisor for A.J.B., analyzed data, and helped write the paper; J.G. advised in the design and interpretation of platelet reactive oxygen species (ROS) experiments; B.A. and M.P. provided advice, help, and analysis of the blood-flow experiments; B.N. provided advice on the blood-flow experiments and helped write parts of the paper; and U.W. was responsible for the initial plan of the work, advising A.J.B. in her PhD work, analyzing data, and writing and finalizing the paper.

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