Important Role In Platelet Activation Research Articles

High-Speed Confocal Imaging Reveals Complex Calcium Transients in Plateles

Intracellular calcium transients have long been known to play an important role in platelet activation. The main processes involved are the IP3 mediated release of calcium from the endoplasmic reticulum and the consequent activation of the store-operated calcium entry (SOCE) mechanism, consisting of the STIM-Orai system and TRPC channels. Traditionally the following methods have been used to measure the calcium level in platelets: flow cytometry, spectrofluorimetry and fluorescent microscopy, with Fura-2-AM used as a calcium indicator. These methods, however, are either incapable of visualising the time course of the calcium transient for the same cell during the activation process or show only the composite calcium level for all the cells.Combining co-loading with Fluo-4-AM and Fura-Red-AM and high-speed confocal imaging (Zeiss LSM Live) we have been able to visualize the calcium transients in case of platelet activation. We investigated the effects of thrombin receptor activating peptide (TRAP), acting on the receptor involved in the physiological platelet activation.Our results indicate that cellular calcium response to TRAP was highly heterogeneous, with only 22.2% (95% CI: 15.1-30.8%) of the platelets exhibiting a calcium transient after activation by TRAP. The non-responding cells still showed an increase of intracellular calcium level when calcium ionophore A23187 was applied. By first using a calcium-free buffer and re-adding calcium after TRAP activation we were also able to distinctly measure SOCE on these cells. Also, by directly activating IP3 receptors with thimerosal, we have been able to measure the effects of inhibitors of the phospholipase C pathway on SOCE.We believe that the proposed imaging method is useful for studying the calcium homeostasis of platelets and possibly other blood cells. The observed heterogeneity of platelet responses may have important clinical and pharmacological consequences.

Platelet activation after endovascular repair of abdominal aortic aneurysm.

To investigate the effect of endovascular aneurysm repair (EVAR) on platelet (PLT) function and reveal possible associated factors. Fifty consecutive patients were included. PLT count and activation (CD62P-CD36), white blood cell (WBC) count, and high sensitivity C-reactive protein (hs-CRP) were measured preoperatively, at the first and third postoperative day. EVAR elicited a significant reduction in PLT count from baseline to first day after EVAR (p < 0.001), while no significant difference was noted between the first and third day. Furthermore, CD62P expression was markedly elevated at the first day after EVAR (median % positive PLTs from 13.7 at baseline to 22.1, p = 0.05), but returned to baseline levels by the third day. Maximum abdominal aortic aneurysm diameter was the only factor that significantly affected the CD62P values (p = 0.005). Postoperative CD36 values were significantly correlated with total aneurysm volume (p = 0.05) and were higher in endografts made from polyester (p = 0.01). There were no correlation between PLT activation and hs-CRP, WBC, maximum temperature, and 30-day morbidity. EVAR has elucidated a significant reduction in PLT count and increase in PLT activation at the immediate postoperative period. The type of the endograft material and the aneurysm maximum diameter and volume appear to play an important role in PLT activation.

Characterization of UBO-QIC as a Gαq inhibitor in platelets

Gαq plays an important role in platelet activation by agonists such as thrombin, adenosine diphosphate (ADP) and thromboxane. The significance of Gαq signaling in platelets was established using YM254890, a Gαq/11-specific inhibitor and Gαq knockout murine platelets. However, YM-254890 is no longer available for investigators and there is a need to characterize other Gαq inhibitors. The aim of this study is to characterize the specificity of a compound, {L-threonine,(3R)-N-acetyl-3-hydroxy-L-leucyl-(aR)-a-hydroxybenzenepropanoyl-2,3-idehydro-N-methylalanyl-L-alanyl-N-methyl-L-alanyl-(3R)-3-[[(2S,3R)-3-hydroxy-4-methyl-1-oxo-2-[(1-oxopropyl)amino]pentyl]oxy]-L-leucyl-N,O-dimethyl-,(7→1)-lactone (9CI)} (UBO-QIC), as a Gαq inhibitor in platelets. Human platelets treated with UBO-QIC showed a concentration-dependent inhibition of platelet aggregation and secretion by protease-activated receptors (PAR) agonists, U46619 and ADP. UBO-QIC also abolished Gαq pathway signaling events such as calcium mobilization and pleckstrin phosphorylation. UBO-QIC had no nonspecific effects on the Gα12/13 pathway since platelet shape change was intact in Gαq knockout murine platelets stimulated with PAR agonists in the presence of the inhibitor. In addition, UBO-QIC-treated platelets did not affect collagen-related peptide-induced platelet activation suggesting that this inhibitor had no non-specific effects on the GPVI pathway. Furthermore, Akt phosphorylation downstream of the Gαi and Gαz pathways, and vasodilator-stimulated phosphoprotein phosphorylation downstream of the Gαs pathway were not inhibited in UBO-QIC-treated platelets. UBO-QIC is a specific inhibitor for Gαq, which can be a useful tool for investigating Gαq-coupled receptor signaling pathways in platelets.

Characterization of a novel function-blocking antibody targeted against the platelet P2Y1 receptor.

Platelet hyperactivity is associated with vascular disease and contributes to the genesis of thrombotic disorders. ADP plays an important role in platelet activation and activates platelets through 2 G-protein-coupled receptors, the Gq-coupled P2Y1 receptor (P2Y1R), and the Gi-coupled P2Y12 receptor. Although the involvement of the P2Y1R in thrombogenesis is well established, there are no antagonists that are currently available for clinical use. Our goal is to determine whether a novel antibody targeting the ligand-binding domain, ie, second extracellular loop (EL2) of the P2Y1R (EL2Ab) could inhibit platelet function and protect against thrombogenesis. Our results revealed that the EL2Ab does indeed inhibit ADP-induced platelet aggregation, in a dose-dependent manner. Furthermore, EL2Ab was found to inhibit integrin GPIIb-IIIa activation, dense and α granule secretion, and phosphatidylserine exposure. These inhibitory effects translated into protection against thrombus formation, as evident by a prolonged time for occlusion in a FeCl3-induced thrombosis model, but this was accompanied by a prolonged tail bleeding time. We also observed a dose-dependent displacement of the radiolabeled P2Y1R antagonist [(3)H]MRS2500 from its ligand-binding site by EL2Ab. Collectively, our findings demonstrate that EL2Ab binds to and exhibits P2Y1R-dependent function-blocking activity in the context of platelets. These results add further evidence for a role of the P2Y1R in thrombosis and validate the concept that targeting it is a relevant alternative or complement to current antiplatelet strategies.

Ca2+Influx through P2X1 Receptors Amplifies P2Y1 Receptor-Evoked Ca2+Signaling and ADP-Evoked Platelet Aggregation

Many cells express both P2X cation channels and P2Y G-protein-coupled receptors that are costimulated by nucleotides released during physiologic or pathophysiologic responses. For example, during hemostasis and thrombosis, ATP-gated P2X1 channels and ADP-stimulated P2Y1 and P2Y12 G-protein coupled receptors play important roles in platelet activation. It has previously been reported that P2X1 receptors amplify P2Y1-evoked Ca(2+) responses in platelets, but the underlying mechanism and influence on function is unknown. In human platelets, we show that maximally activated P2X1 receptors failed to stimulate significant aggregation but could amplify the aggregation response to a submaximal concentration of ADP. Costimulation of P2X1 and P2Y1 receptors generated a superadditive Ca(2+) increase in both human platelets and human embryonic kidney 293 (HEK293) cells via a mechanism dependent on Ca(2+) influx rather than Na(+) influx or membrane depolarization. The potentiation, due to an enhanced P2Y1 response, was observed if ADP was added up to 60 seconds after P2X1 activation. P2X1 receptors also enhanced Ca(2+) responses when costimulated with type 1 protease activated and M1 muscarinic acetylcholine receptors. The P2X1-dependent amplification of Gq-coupled [Ca(2+)]i increase was mimicked by ionomycin and was not affected by inhibition of protein kinase C, Rho-kinase, or extracellular signal-regulated protein kinase 1/2, which suggests that it results from potentiation of inositol 1,4,5-trisphosphate receptors and/or phospholipase C. We conclude that Ca(2+) influx through P2X1 receptors amplifies Ca(2+) signaling through P2Y1 and other Gq-coupled receptors. This represents a general form of co-incidence detection of ATP and coreleased agonists, such as ADP at sites of vascular injury or synaptic transmitters acting at metabotropic Gq-coupled receptors.

Abstract 196: NADPH Oxidase Isoforms NOX1 and NOX2 Differentially Regulate GPVI- and Non-GPVI-Dependent ROS Generation and Platelet Activation

Reactive oxygen species (ROS) play an important role in platelet activation. Although diverse biochemical reactions contribute to ROS generation, NADPH oxidases (NOX) have emerged as critical sources of agonist induced ROS generation in platelets. Platelets express two NOX isoforms, NOX1 and NOX2. In this study we investigated the effects of ML171 (0.1-1 μM), a selective NOX1 inhibitor (ACS Chem Biol. 5, 981-993, 2010), and Phox-I (1-10 μM), a rationally designed small molecule inhibitor of NOX2 (Chem. Biol. 19, 699-710, 2012), to define the roles of NOX1 and NOX2 in ROS-mediated platelet activation by diverse agonists. A two-minute pre-incubation of washed human platelets with ML171 or Phox-I inhibited CRP, a GPVI agonist, induced ROS generation, ATP secretion and platelet aggregation. In contrast, only platelets treated with Phox-I, but not ML171, inhibited CRP induced binding of PAC-1 to platelets and expression of P-selectin. Treatment of platelets with Phox-I, but not ML-171, inhibited thrombin or U46619, a stable analog of TXA 2 , induced ROS generation, P-selectin expression, ATP secretion and aggregation. Both Phox-I and ML171 inhibited CRP induced phosphorylation of myosin light chain (MLC) and Akt. However, only Phox-I inhibited thrombin induced phosphorylation of MLC or Akt. Treatment of platelets with Phox-I or ML171 only minimally or partially inhibited CRP induced phosphorylation of p38-MAPK or ERK. In other experiments, incubation of platelets with varying concentrations of both ML171 and Phox-I did not exhibit additive effect on CRP induced ROS generation. These results imply that ROS generation by these NOX isoforms may be sequential. These data suggest that NOX1 is specifically involved in GPVI mediated, whereas NOX2 is involved in GPVI- and non-GPVI-dependent, signals in ROS generation and platelet activation.

Platelet size and density affect shear-induced thrombus formation in tortuous arterioles

Thrombosis accounts for 80% of deaths in patients with diabetes mellitus. Diabetic patients demonstrate tortuous microvessels and larger than normal platelets. Large platelets are associated with increased platelet activation and thrombosis, but the physical effects of large platelets in the microscale processes of thrombus formation are not clear. Therefore, the objective of this study was to determine the physical effects of mean platelet volume (MPV), mean platelet density (MPD) and vessel tortuosity on platelet activation and thrombus formation in tortuous arterioles. A computational model of the transport, shear-induced activation, collision, adhesion and aggregation of individual platelets was used to simulate platelet interactions and thrombus formation in tortuous arterioles. Our results showed that an increase in MPV resulted in a larger number of activated platelets, though MPD and level of tortuosity made little difference on platelet activation. Platelets with normal MPD yielded the lowest amount of mural thrombus. With platelets of normal MPD, the amount of mural thrombus decreased with increasing level of tortuosity but did not have a simple monotonic relationship with MPV. The physical mechanisms associated with MPV, MPD and arteriole tortuosity play important roles in platelet activation and thrombus formation.

Staphylococcal Extracellular Adherence Protein Induces Platelet Activation by Stimulation of Thiol Isomerases

Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation. The staphylococcal extracellular adherence protein (Eap) functions as an adhesin for host plasma proteins. Therefore we tested the effect of Eap on platelets. We found a strong stimulation of the platelet-surface thiol isomerases protein disulfide isomerase and endoplasmic reticulum stress proteins 57 and 72 by Eap. Eap induced thiol isomerase-dependent glycoprotein IIb/IIIa activation, granule secretion, and platelet aggregation. Treatment of platelets with thiol blockers, bacitracin, and anti-protein disulfide isomerase antibody inhibited Eap-induced platelet activation. The effect of Eap on platelets and protein disulfide isomerase activity was completely blocked by glycosaminoglycans. Inhibition by the hydrophobic probe bis(1-anilinonaphthalene 8-sulfonate) suggested the involvement of hydrophobic sites in protein disulfide isomerase and platelet activation by Eap. In the present study, we found an additional and yet unknown mechanism of platelet activation by a bacterial adhesin, involving stimulation of thiol isomerases. The thiol isomerase stimulatory and prothrombotic features of a microbial secreted protein are probably not restricted to S aureus and Eap. Because many microorganisms are coated with amyloidogenic proteins, it is likely that the observed mechanism is a more general one.

Effects of physiologically relevant dynamic shear stress on platelet complement activation

Disturbed shear stress, commonly found in cardiovascular diseases, plays important roles in platelet activation and functions. It has been reported that when activated by elevated shear stress, platelets were able to support complement activation to completion. In this study, through a dynamic cone and plate shearing device, three physiologically relevant shear stresses were applied to platelets, mimicking the shear conditions when platelets pass through a normal left coronary artery (0.05–1 Pa), a 60% stenosis (elevated shear stress at 6.5 Pa for less than 0.1 s), and when platelets are trapped in a recirculation zone past a stenosis (<0.5 Pa). After shear exposure, platelet-surface complement activation (C1q, C4d, iC3b, and SC5b-9 depositions) was measured using a solid-phase ELISA approach and flow cytometry. Production of complement regulatory proteins – C1-inhibitor (C1-INH) and complement receptor 1 (CR1), was also measured. Results demonstrated that low-pulsatile shear stress (recirculation) was able to initiate platelet complement activation, by increasing C1q deposition significantly. Both pathological shear stresses triggered significant increases in C1 inhibitor generation and noticeable changes in CR1 production, effectively preventing complement activation from completion. These results suggested that for platelets, low-pulsatile shear stress may be more pro-atherogenic, compared to elevated shear stress, especially when the shear stress exposure time is short.

The Second Extracellular Loop of the Thromboxane Receptor Plays an Important Role in Platelet Activation

In efforts to more effectively target pathways for antithrombotic activity, there is interest in utilizing antibodies targeting ligand binding domains for various platelet receptors. To this end, we hypothesize that an antibody (designated C-EL2Ab), targeting the second extracellular loop (C-EL2) of the thromboxane receptor (TPR), which contains the ligand binding domain, exhibits antagonistic activity against platelet TPRs. Our results show that C-EL2Ab inhibited TPR-mediated aggregation (triggered by the agonists U46619 and arachidonic acid) in vitro in both human and murine platelets, in a dose-dependent fashion. On the other hand, control experiments revealed that C-EL2Ab lacks the ability to inhibit aggregation stimulated by ADP. Consistent with the notion of receptor antagonism, ligand binding studies demonstrated that C-EL2Ab has the capacity to completely displace the radiolabeled TPR antagonist H3-SQ29,548. In addition, separate control studies showed that normal rabbit IgG or a TPR-specific Ab that targets the first EL, were devoid of effects on aggregation regardless of the agonist used. Taken together, these findings indicate that: the C-EL2 of TPR plays a crucial role in platelet activation, and hence may serve as a target for therapeutic interventions; and the C-EL2Ab has the potential to be used as an antiplatelet agent.

Candida albicans and its metabolite gliotoxin inhibit platelet function via interaction with thiols

Platelets bind to Candida albicans, the major cause of candidiasis. But in contrast to other microorganisms the fungus does not aggregate platelets. Gliotoxin (GT), which possesses immunosuppressive properties, is produced by various fungi, including the opportunistic pathogens Aspergillus fumigatus and C. albicans . Its mode of action involves the formation of mixed disulfides with host proteins. Disulfide exchanges play an important role in platelet activation. Therefore, the effect of C. albicans and GT on platelet function was tested. C. albicans yeast cells (5,000-10,000 cells/microl) and GT, in pathophysiologically relevant concentrations (0.05-0.5 microM), inhibited platelet fibrinogen binding, anti gp IIb/IIIa antibody PAC-1 binding, aggregation and procoagulant activity in a dose-dependent manner. Alpha granule release, measured via CD62P surface expression, was not affected. Addition of reduced glutathione partially counteracted the effect of C. albicans and GT on platelet fibrinogen binding and platelet aggregation. The C. albicans metabolite GT features antithrombotic properties in addition to its immunosuppressive functions. Since treatment with reduced glutathione partially counteracted the inhibitory effect of C. albicans yeast cells and GT on platelet fibrinogen binding, the antithrombotic activity is likely to depend on the disulfide bridge of this mycotoxin. GT production by C. albicans could contribute to its survival in the blood stream during vascular infections. The knowledge of the underlying mechanisms of the antithrombotic properties might help to treat fungal infections as well as thrombosis.

Transforming growth factor-β–induced protein (TGFBIp/β ig-h3) activates platelets and promotes thrombogenesis

AbstractTransforming growth factor-β–induced protein (TGFBIp)/βig-h3 is a 68-kDa extracellular matrix protein that is functionally associated with the adhesion, migration, proliferation, and differentiation of various cells. The presence of TGFBIp in platelets led us to study the role of this protein in the regulation of platelet functions. Upon activation, platelet TGFBIp was released and associated with the platelets. TGFBIp mediates not only the adhesion and spread of platelets but also activates them, resulting in phosphatidylserine exposure, α-granule secretion, and increased integrin affinity. The fasciclin 1 domains of TGFBIp are mainly responsible for the activation of platelets. TGFBIp promotes thrombus formation on type I fibrillar collagen under flow conditions in vitro and induces pulmonary embolism in mice. Moreover, transgenic mice, which have approximately a 1.7-fold greater blood TGFBIp concentration, are significantly more susceptible to collagen- and epinephrine-induced pulmonary embolism than wild-type mice. These results suggest that TGFBIp, a human platelet protein, plays important roles in platelet activation and thrombus formation. Our findings will increase our understanding of the novel mechanism of platelet activation, contributing to a better understanding of thrombotic pathways and the development of new antithrombotic therapies.

RACK1 associates with CLEC-2 and promotes its ubiquitin–proteasome degradation

CLEC-2 is a C-type lectin-like receptor and plays an important role in platelet activation. Snake venom toxin rhodocytin and the endogenous sialoglycoprotein podoplanin are identified as ligands for CLEC-2 and function as stimulators in platelet activation. We also previously indentified two splice variants of murine CLEC-2 as well as a soluble fragment cleaved from the full-length form. However, little is known about the interacting partners with the cytoplasmic region of CLEC-2. In this study, we reported that RACK1, the receptor for activated C-kinase 1, associated with the cytoplasmic tail of CLEC-2. Moreover, overexpression of RACK1 decreased the stability of CLEC-2 through promoting its ubiquitin–proteasome degradation, without impairing surface expression and downstream signaling of CLEC-2. Taken together, these results suggest RACK1 as a novel modulator of CLEC-2 expression.

Novel mutations in GP IIb gene in Glanzmann's thrombasthenia from India

The integrin GPIIb-IIIa plays an important role in platelet activation and plug formation. Quantitative and qualitative dysfunction in this receptor causes Glanzmann's thrombasthenia (GT) and leads to a bleeding tendency. Mutations in the GPIIb or GPIIIa gene are known to be responsible for the inherited form of this disease, which is characterized by mucocutaneous bleeding and other clinically severe bleeding manifestations. GT is an autosomal inherited platelet function. Mutations in the GPIIIa gene but not GPIIb gene in GT patients from Western India have been studied. Hence, this study was designed and included for the first time two mutation detection strategies: single strand conformation polymorphism and conformation sensitive gel electrophoresis followed by sequencing. Patients diagnosed as GT in our laboratory were screened for mutations. We report 12 mutations in 13 patients, of which 11 are novel mutations. Six mutations were missense, four were deletions and two were splice junction mutations. The study thus identifies novel mutations in the GPIIb gene in patients from Western India, implicating the importance of certain amino acids in structure–function correlations as well as enabling prenatal diagnosis in these families.

The Role of Akt3 in Platelet Activation

Akt is a family of serine/threonine kinases that are activated downstream of Phosphoinositide 3 Kinases (PI3K). There are three known Akt isoforms: Akt1, Akt2, and Akt3. Both Akt1 and Akt2 have been shown to be expressed in platelets and play important roles in PI3K-dependent platelet activation signaling. Previous studies have concluded that Akt3 is not expressed in platelets; however, we show that Akt3 is in fact a major Akt isoform present in platelets, and demonstrate that Akt3 plays an important role in platelet activation. In order to determine if Akt3 mRNA is present in platelets, RNA was isolated from platelets and RT-PCR experiments were performed using Akt3 specific primers. Akt3 mRNA was detected in both human and mouse platelets, and the possibility that Akt3 mRNA came from leukocyte contamination was excluded because Akt3 was not detected with RT-PCR using RNA purified from the same number of leukocytes as that in platelet preparation. To assess if Akt3 protein is present in platelets, western blot analysis was performed using an Akt3 specific antibody. Results revealed that Akt3 is highly expressed in human and mouse platelets. In order to assess the relative amount of Akt3 present in platelets, total Akt levels and phosphorylated Akt levels were measured in Akt3 knockout mouse platelets in comparison with wild type mouse platelets. Akt3 knockout mouse platelets showed a significant decrease in total Akt, and also a significant reduction in phosphorylated Akt, as indicated by Western blot analysis of phospho-Thr308-Akt and phospho-Ser473-Akt antibodies. Thus, our data indicates Akt3 is a major Akt isoform expressed in platelets. To determine the role of Akt3 in platelet activation, platelets from wild type mice and Akt3 knockout mice were stimulated with low concentrations of platelet agonists. Akt3 knockout mouse platelets exhibited impaired aggregation and ATP secretion in response to low dose thrombin and collagen compared with wild type controls, indicating that Akt3 plays an important role in promoting platelet activation. In order to assess the signaling molecules downstream from Akt3, Akt3 knockout and wild type mouse platelets were stimulated with low dose thrombin and phosphorylation of GSK-3β, an enzyme recently reported to negatively regulate platelet function downstream from Akt1 or Akt2 in platelets, was assessed. Western blot for phospho-Ser9 of GSK-3β in Akt3 knockout mouse platelets stimulated with thrombin showed less phosphorylation compared to Akt1 or Akt2 knockout mouse platelets, indicating that Akt3 is the major Akt isoform responsible for GSK-3β phosphorylation. Thus, our data shows that Akt3 is a major Akt isoform in platelets and plays an important role in platelet activation. Since phosphorylation of GSK-3β at Ser9 has been reported to reverse the inhibitory effect of GSK-3β on platelet function, our data also suggest that the stimulatory role of Akt3 in platelet activation may be mediated by negative regulation of GSK-3β function.

The Role of LIM Kinase-1 in the Glycoprotein Ib-IX Complex-Mediated Platelet Activation and Thrombus Formation

LIM Kinase (LIMK)-1 is a member of the LIMK family of serine-threonine protein kinases that phosphorylates actin-binding protein cofilin and regulates actin cytoskeleton organization. LIMK1 is expressed in many cell types including platelets but the exact role of LIMK1 in platelet function remains unclear. To determine the role of LIMK1 in platelet activation, wild type or LIMK1 knockout mouse platelets were stimulated with platelet agonists. Platelet aggregation and granule secretion were analyzed. Integrin-dependent second wave of platelet aggregation induced by von Willebrand factor (VWF) in the presence of VWF activator botrocetin was abolished in LIMK1 knockout platelets. In contrast, platelet aggregation in response to the agonist peptide of protease-activated receptor-4 (PAR4, thrombin receptor), ADP and collagen was either not affected or enhanced in LIMK1 knockout platelets in comparison with wild type mouse platelets. Thus, LIMK appears to play an important role in platelet activation stimulated by VWF binding to its platelet receptor, glycoprotein Ib-IX complex (GPIb-IX) but had no stimulatory effect on or negatively regulate the GPIb-IX-independent platelet activation pathways mediated by PAR-4, ADP receptors and collagen receptors. To determine whether ligand binding to GPIb-IX stimulates LIMK activation and function, platelets were stimulated with VWF in the presence of either ristocetin or botrocetin, and immunoblotted with antibodies specifically recognizing phosphorylated LIMK1 (Serine 505) or cofilin (Serine 3). VWF induced phosphorylation of LIMK1 and LIMK substrate cofilin. Thus, VWF indeed stimulates LIMK1 activation and function. An important physiological role of GPIb-IX in platelets is to mediate platelet adhesion to subendothelial-bound VWF under shear stress at sites of vascular injury. To determine whether LIMK1 is important in platelet adhesion, we investigated whether LIMK1 knockout affected platelet adhesion to VWF-coated surfaces. LIMK1 knockout platelets are defective in mediating stable platelet adhesion to vWF under shear stress, suggesting that LIMK1 plays an important role in GPIb signaling and GPIb-IX-mediated integrin activation that is required for stable platelet adhesion under shear stress. Importantly, LIMK1 knockout mice showed significant delay in the formation of occlusive thrombus following FeCl3-induced carotid artery injury in comparison with wild type mice, indicating that the role of LIMK1 in GPIb-IX-mediated platelet activation is important in in vivo thrombosis. Together, our study reveals that LIMK1 plays an important role in GPIb-IX-mediated platelet activation and arterial thrombosis in vitro and in vivo.

Signaling-mediated Functional Activation of Inducible Nitric-oxide Synthase and Its Role in Stimulating Platelet Activation

Nitric oxide (NO) is a short lived secondary messenger, synthesized by nitric-oxide synthases (NOS). It is believed that the activity of inducible NOS (iNOS) is regulated primarily at the transcription level by inducing expression of iNOS mRNA and protein, which then continuously produces NO, until its degradation. Platelets do not have the nuclear transcriptional regulatory mechanisms of the iNOS gene and are believed to generate NO in response to agonist stimulation via endothelial NOS (eNOS). However, here we show that agonist-induced NO production is only partially eNOS-dependent and is also mediated by iNOS. Platelet agonist-induced NO production is significantly reduced in iNOS-knockout platelets. Platelet NO production occurs within seconds after agonist addition and is not accompanied by changes in iNOS protein levels, indicating a signaling-mediated functional activation mechanism of iNOS. Importantly, iNOS knock-out and iNOS inhibitors reduce agonist-induced platelet secretion and aggregation and cGMP levels, indicating that iNOS activation is important in stimulating platelets via the newly identified NO-cGMP-dependent platelet secretion pathway. Furthermore, iNOS knock-out mice have prolonged bleeding time, suggesting that this novel mode of regulation of iNOS activity plays a physiologically relevant role in hemostasis.

A Murine Monoclonal Antibody That Binds N-Terminal Extracellular Segment of Human Protease-Activated Receptor-4

Abstract A monoclonal antibody that recognizes native G protein coupled receptors (GPCR) is generally difficult to obtain. Protease-activated receptor-4 (PAR4) is a GPCR that plays an important role in platelet activation as a low-affinity thrombin receptor. By immunizing peptide corresponding to the N-terminal segment of human PAR4, we obtained a monoclonal antibody that recognizes cell surface expressed PAR4. Epitope mapping using a series of artificial fusion proteins that carry PAR4-derived peptide revealed that the recognition motif is fully contained within the 6-residue portion adjacent to the thrombin cleavage site. The antibody blocked PAR4 peptide cleavage by thrombin, suggesting its utility in the functional study of PAR4 signaling.

Proteomic analysis of differential protein expression in rat platelets treated with notoginsengnosides

Sanqi, the root of Panax notoginseng, is a popularly used traditional Chinese medicine with cardiovascular effects. Notoginsengnosides (NG) isolated from Sanqi could inhibit ADP-induced platelet aggregation of rat washed platelets. To identify the possible target proteins of NG in platelets, two-dimensional gel electrophoresis (2-DE)-based comparative proteomics was performed and proteins altered in expressional level after NG treatment were identified by MALDI-TOF MS/MS. Treatment of 200 microg/ml NG caused regulation of the levels of 12 proteins, which play important roles in platelet activation, oxidative stress and cytoskeleton. In the NG-treated platelets, there were increase in the levels of growth factor receptor-bound protein 2 (Grb2), thrombospondin 1, tubulin alpha 6 and decrease in the levels of thioredoxin, Cu-Zn superoxide dismutase, DJ-1 protein, peroxiredoxin 3, thioredoxin-like protein 2, ribonuclease inhibitor, potassium channel subfamily V member 2, myosin regulatory light chain 9 and laminin receptor 1. The change in the levels of these proteins caused by NG treatment might contribute to the inhibitive effect of NG on platelet aggregation. Furthermore, analysis of the reactive oxygen species (ROS) level indicated that NG could decrease the ROS level in platelets. The regulation of ROS level might play important role in the effect of NG on platelets.

Disodium Disuccinate Astaxanthin Prevents Carotid Artery Rethrombosis and ex vivo Platelet Activation

Background/Aims:The disodium disuccinate derivative of astaxanthin (DDA) is a carotenoid antioxidant under development for the treatment of ischemic cardiovascular events. Recent evidence suggests that reactive oxygen species (ROS) play an important role in platelet activation. This study seeks to investigate the effects of a reactive oxygen species quencher, DDA, in a canine model of carotid artery thrombosis. Methods:After formation of an occlusive carotid thrombus, dogs were administered recombinant tissue plasminogen activator intra-arterially to achieve thrombolysis in the presence of either 0.9% NaCl solution or DDA (10–50 mg/kg i.v. infusion). Ex vivo platelet aggregation and tongue bleeding times were measured before and after drug administration. Residual thrombus mass was analyzed at the end of each experiment. Results:The data indicated a dose- dependent reduction in the incidence of carotid artery rethrombosis. In addition, platelet aggregation and thrombus weights were dose-dependently inhibited by DDA. No change was recorded in tongue bleeding time among the treatment groups. Conclusions:The data demonstrate that at the doses used in this study, DDA significantly reduced the incidence of secondary thrombosis while maintaining normal hemostasis. The results suggest that upon further study, DDA may one day find utility in revascularization procedures.