scholarly journals Platelet–Neutrophil Crosstalk in Atherothrombosis

2019 ◽  
Vol 119 (08) ◽  
pp. 1274-1282 ◽  
Author(s):  
Joachim Pircher ◽  
Bernd Engelmann ◽  
Steffen Massberg ◽  
Christian Schulz
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Intracellular Signaling ◽  
Arterial Thrombosis ◽  
Plaque Rupture ◽  
Bleeding Risk ◽  
Potential Therapeutic Target ◽  
Extracellular Traps ◽  
Subendothelial Matrix

AbstractAtherothrombosis is a frequent cause of cardiovascular mortality. It is mostly triggered by plaque rupture and exposure of the thrombogenic subendothelial matrix, which initiates platelet aggregation and clot formation. Current antithrombotic strategies, however, target both thrombosis and physiological hemostasis and thereby increase bleeding risk. Thus, there is an unmet clinical need for optimized therapies. Neutrophil activation and consecutive interactions of neutrophils and platelets contribute mechanistically to thromboinflammation and arterial thrombosis, and thus present a potential therapeutic target. Platelet–neutrophil interactions are mediated through adhesion molecules such as P-selectin and P-selectin glycoprotein ligand 1 as well as glycoprotein Ib and macrophage-1 antigen, which mediate physical cell interactions and intracellular signaling. Release of soluble mediators as well as direct signaling between platelets and neutrophils lead to their reciprocal activation and neutrophil release of extracellular traps, scaffolds of condensed chromatin that play a prothrombotic role in atherothrombosis. In this article, we review the role of neutrophils and neutrophil-derived prothrombotic molecules in platelet activation and atherothrombosis, and highlight potential therapeutic targets.

On The Mechanism Of Heparin-Induced Potentiation Of Platelet Aggregation

1981 ◽  
Author(s):  
M Yamamoto ◽  
K Watanabe ◽  
Y Ando ◽  
H Iri ◽  
N Fujiyama ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Coagulation Factors ◽  
Marked Enhancement ◽  
Matrix Bound ◽  
Induced Aggregation ◽  
Aggregation Of Platelets ◽  
Plasma Heparin

It has been suggested that heparin caused potentiation of aggregation induced by ADP or epinephrine. The exact mechanism of heparin-induced platelet activation, however, remained unknown. In this paper, we have investigated the role of anti-thrombin III ( AT ) in heparin-induced platelet activation using purified AT and AT depleted plasma. When ADP or epinephrine was added to citrated PRP one minute after addition of heparin ( 1 u/ml, porcine intestinal mucosal heparin, Sigma Co. USA ), marked enhancement of platelet aggregation was observed, compared with the degree of aggregation in the absence of heparin. However, in platelet suspensions prepared in modified Tyrode’s solution, heparin exhibited no potentiating effect on platelet aggregation induced by epinephrine or ADP. Potentiation of epinephrine- or ADP-induced platelet aggregation by heparin was demonstrated when purified AT was added to platelet suspensions at a concentration of 20 μg/ml. AT depleted plasma, which was prepared by immunosorption using matrix-bound antibodies to AT, retained no AT, while determination of α1-antitrypsinα2- macroglobulin and fibrinogen in AT depleted plasma produced values which corresponded to those of the original plasma when dilution factor was taken into account. The activities of coagulation factors were also comparable to those of the original plasma. Heparin exhibited potentiating effect on ADP- or epinephrine-induced aggregation of platelets in original plasma, but no effect in AT depleted plasma. When purified AT was added back to AT depleted plasma at a concentration of 20 μg/ml, potentiation of platelet aggregation by heparin was clearly demonstrated.Our results suggest that effect of heparin on platelet aggregation is also mediated by anti-thrombin III.

scholarly journals ROLE OF NITRIC OXIDE (NO) IN CAPSAICIN MEDIATED ANTI-PLATELET ACTIVITY IN IN VITRO, IN VIVO, EX-VIVO MODEL OF PLATELET AGGREGATION ASSAY AND ARTERIAL THROMBOSIS IN RAT: POTENTIAL THERAPEUTIC TARGET?

2018 ◽  
Vol 10 (4) ◽  
pp. 44
Author(s):  
Mihir K Patel ◽  
Kiranj K. Chaudagar ◽  
Anita A. Mehta
Keyword(s):  
Platelet Aggregation ◽  
Arterial Thrombosis ◽  
Ex Vivo ◽  
Platelet Activity ◽  
Aggregation Assay ◽  
Thrombosis Model ◽  
Platelet Aggregation Assay

Objective: Although recent advances in the treatment of congestive heart disease, mortality among patients’ remains a questionable remark. Therefore, we evaluated the role of capsaicin on in vitro and ex vivo platelet aggregation induced by Adenosine Di-Phosphate (ADP) as well as in in vivo thrombosis models and role of NO, KATP was also identified in the capsaicin-induced anti-platelet animal model as well as in vivo model of arterial thrombosis.Methods: According to body weight wistar rats were divided into five groups. Group I and Group II was treated with saline and capsaicin (3 mg/kg, i. v), while animals from Group III were treated with N(ω)-nitro-L-arginine methyl ester (L-NAME) (30 mg/kg, i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group IV animals were treated with glibenclamide (10 mg/kg,i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group V was considered as a positive control and administered clopidogrel (30 mg/kg, p. o). Animals were subjected for in vitro, ex-vivo platelet aggregation assay. ADP (30µM) was utilized as an aggregating agent in these experiments. After these assays; animals of each group were subjected for subaqueous tail bleeding time in a rat model and FeCl3-induced arterial thrombosis model in rats.Results: In ADP-induced in vitro platelet aggregation, a significant reduction in % platelet aggregation was observed at 50µM (64.35±4.641) and 100µM (52.72±4.192) concentration of capsaicin as compared to vehicle control (85.82±3.716). Capsaicin (3 mg/kg, i. v) also showed a significant reduction (49.53±4.075) in ex-vivo ADP-induced platelet aggregation as compared to vehicle control (89.38±2.057). In FeCl3 induced arterial thrombosis model, Capsaicin (3 mg/kg, i. v) exhibited an increase in time to occlusion in this rodent model and presence of the L-NAME and glibenclamide had inhibited the activity of capsaicin.Conclusion: In our study, capsaicin (50 µM, 100µM) exhibited potent anti-platelet activity in ADP-induced platelet aggregation, similarly capsaicin exhibited significant anti-platelet action in the ex-vivo study. Moreover, the presence of L-NAME and glibenclamide inhibited the anti-thrombotic and anti-platelet action of capsaicin. Therefore, it was concluded that NO and KATP may be involved in the anti-thrombotic action of capsaicin.

scholarly journals Cysteine sulfenylation by CD36 signaling promotes arterial thrombosis in dyslipidemia

2020 ◽  
Vol 4 (18) ◽  
pp. 4494-4507 ◽  
Author(s):  
Moua Yang ◽  
Wei Li ◽  
Calvin Harberg ◽  
Wenjing Chen ◽  
Hong Yue ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Hydrogen Peroxide ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Arterial Thrombosis ◽  
Reactive Oxygen ◽  
Src Family Kinases ◽  
Oxygen Species ◽  
Carbon Nucleophiles ◽  
Platelet Proteins

Abstract Arterial thrombosis in the setting of dyslipidemia promotes clinically significant events, including myocardial infarction and stroke. Oxidized lipids in low-density lipoproteins (oxLDL) are a risk factor for athero-thrombosis and are recognized by platelet scavenger receptor CD36. oxLDL binding to CD36 promotes platelet activation and thrombosis by promoting generation of reactive oxygen species. The downstream signaling events initiated by reactive oxygen species in this setting are poorly understood. In this study, we report that CD36 signaling promotes hydrogen peroxide flux in platelets. Using carbon nucleophiles that selectively and covalently modify cysteine sulfenic acids, we found that hydrogen peroxide generated through CD36 signaling promotes cysteine sulfenylation of platelet proteins. Specifically, cysteines were sulfenylated on Src family kinases, which are signaling transducers that are recruited to CD36 upon recognition of its ligands. Cysteine sulfenylation promoted activation of Src family kinases and was prevented by using a blocking antibody to CD36 or by enzymatic degradation of hydrogen peroxide. CD36-mediated platelet aggregation and procoagulant phosphatidylserine externalization were inhibited in a concentration-dependent manner by a panel of sulfenic acid–selective carbon nucleophiles. At the same concentrations, these probes did not inhibit platelet aggregation induced by the purinergic receptor agonist adenosine diphosphate or the collagen receptor glycoprotein VI agonist collagen-related peptide. Selective modification of cysteine sulfenylation in vivo with a benzothiazine-based nucleophile rescued the enhanced arterial thrombosis seen in dyslipidemic mice back to control levels. These findings suggest that CD36 signaling generates hydrogen peroxide to oxidize cysteines within platelet proteins, including Src family kinases, and lowers the threshold for platelet activation in dyslipidemia.

scholarly journals Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization

2020 ◽  
Vol 21 (11) ◽  
pp. 3932 ◽  
Author(s):  
Preeti Kumari Chaudhary ◽  
Sanggu Kim ◽  
Youngheun Jee ◽  
Seung-Hun Lee ◽  
Kyung-Mee Park ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
G Protein ◽  
Platelet Activation ◽  
Functional Role ◽  
Thrombus Formation ◽  
Receptor Activation ◽  
G Protein Coupled ◽  
Gpcr Desensitization ◽  
Stimulation Of

Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and α-granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6−/− platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6−/− platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled α2A adrenergic receptors, respectively, was not affected in GRK6−/− platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6−/− platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKCδ) phosphorylation were significantly potentiated in GRK6−/− platelets. Finally, GRK6−/− mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6−/− mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.

scholarly journals Targeting myeloid-cell specific integrin α9β1 inhibits arterial thrombosis in mice

Blood ◽  
2020 ◽  
Vol 135 (11) ◽  
pp. 857-861 ◽  
Author(s):  
Nirav Dhanesha ◽  
Manasa K. Nayak ◽  
Prakash Doddapattar ◽  
Manish Jain ◽  
Gagan D. Flora ◽  
...  
Keyword(s):  
Arterial Thrombosis ◽  
Potential Role ◽  
Myeloid Cell ◽  
Neutrophil Extracellular Traps ◽  
Cathepsin G ◽  
Wild Type ◽  
Extracellular Traps ◽  
Laser Injury ◽  
Activated Platelets

Abstract Evidence suggests that neutrophils contribute to thrombosis via several mechanisms, including neutrophil extracellular traps (NETs) formation. Integrin α9β1 is highly expressed on neutrophils when compared with monocytes. It undergoes affinity upregulation on neutrophil activation, and stabilizes adhesion to the activated endothelium. The role of integrin α9 in arterial thrombosis remains unexplored. We generated novel myeloid cell-specific integrin α9−/− mice (α9fl/flLysMCre+) to study the role of integrin α9 in arterial thrombosis. α9fl/fl littermates were used as controls. We report that α9fl/flLysMCre+ mice were less susceptible to arterial thrombosis in ferric chloride (FeCl3) and laser injury-induced thrombosis models with unaltered hemostasis. Neutrophil elastase-positive cells were significantly reduced in α9fl/flLysMCre+ mice concomitant with reduction in neutrophil count, myeloperoxidase levels, and red blood cells in the FeCl3 injury-induced carotid thrombus. The percentage of cells releasing NETs was significantly reduced in α9fl/flLysMCre+ mouse neutrophils stimulated with thrombin-activated platelets. Furthermore, we found a significant decrease in neutrophil-mediated platelet aggregation and cathepsin-G secretion in α9fl/flLysMCre+ mice. Transfusion of α9fl/fl neutrophils in α9fl/flLysMCre+ mice restored thrombosis similar to α9fl/fl mice. Treatment of wild-type mice with anti-integrin α9 antibody inhibited arterial thrombosis. This study identifies the potential role of integrin α9 in modulating arterial thrombosis.

scholarly journals Interplay between Nox2 Activity and Platelet Activation in Patients with Sepsis and Septic Shock: A Prospective Study

2020 ◽  
Vol 2020 ◽  
pp. 1-6
Author(s):  
Giusy Tiseo ◽  
Elena Cavarretta ◽  
Arianna Forniti ◽  
Cristina Nocella ◽  
Sebastiano Sciarretta ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Septic Shock ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Healthy Controls ◽  
Sample Collection ◽  
Oxidative Stress Status ◽  
Sepsis And Septic Shock ◽  
A Prospective Study

Background. Although preclinical studies highlighted the potential role of NADPH oxidase (NOX) in sepsis, only few studies evaluated the oxidative stress in patients with sepsis and septic shock. The objective of the study is to appraise the oxidative stress status and platelet function in patients with sepsis and septic shock compared to healthy controls. Methods and Results. Patients with sepsis or septic shock admitted to the hospital Policlinico Umberto I (Sapienza University, Rome) underwent a blood sample collection within 1 hour from admission. Platelet aggregation, serum thromboxane B2 (TxB2), soluble NOX2-derived peptides (sNox2-dp), and hydrogen peroxide breakdown activity (HBA) were measured and compared to those of healthy volunteers. Overall, 33 patients were enrolled; of these, 20 (60.6%) had sepsis and 13 (39.4%) septic shock. Compared to healthy controls ( n = 10 , age 67.8 ± 3.2 , male 50%), patients with sepsis and septic shock had higher platelet aggregation (49% (IQR 45-55), 60% (55.75-67.25), and 73% (IQR 69-80), respectively, p < 0.001 ), higher serum TxB2 (77.5 (56.5-86.25), 122.5 (114-131.5), and 210 (195-230) pmol/L, respectively, p < 0.001 ), higher sNox2-dp (10 (7.75-12), 19.5 (17.25-21), and 33 (29.5-39) pg/mL, respectively, p < 0.001 ), and lower HBA (75% (67.25-81.5), 50% (45-54.75), and 27% (21.5-32.5), respectively, p < 0.001 ). Although not statistically significant, a trend in higher levels of serum TxB2 and sNox2-dp in patients who died was observed. Conclusions. Patients with septic shock exhibit higher Nox2 activity and platelet activation than patients with sepsis. These insights joined to better knowledge of these mechanisms could guide the identification of future prognostic biomarkers and new therapeutic strategies in the scenario of septic shock.

A Dual Role for the α-Subunit KVGFFKR Motif of αIIbβ3: Integrin Activation and Intracellular Signaling.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1545-1545
Author(s):  
Kelly Aylward ◽  
Marc Devocelle ◽  
Niamh Moran
Keyword(s):  
Platelet Aggregation ◽  
Ligand Binding ◽  
Tyrosine Phosphorylation ◽  
Cho Cells ◽  
Intracellular Signaling ◽  
Dual Role ◽  
Cytoskeletal Reorganization ◽  
Integrin Activation ◽  
Α Subunit

Abstract The platelet-specific integrin αIIbβ3 plays a critical role in platelet aggregation and pathological thrombosis. Integrin affinity and ligand binding are regulated by the highly conserved αIIb membrane-proximal motif 989KVGFFKR995. We have recently shown that this motif is dependent on the presence of two phenylalanines (FF) for its activity. In order to investigate the role of KVGFFKR on integrin transmembrane signaling we used two parallel systems: (1) stable Chinese Hamster Ovary (CHO) cells expressing mutant αIIbβ3 integrins and (2) platelets treated with synthetic palmitylated (pal) peptides corresponding to the seven amino acid motif. In CHO cells, we chose cytoskeletal reorganization as a means to explore outside-in signaling. Alanine substitutions were introduced to the α-subunit KVGFFKR domain and co-expressed with wildtype β3. Cells were stably transfected with wildtype αIIb(992FF993)/β3, αIIb(992AA993)/β3 and αIIb (992AF993)/β3 to produce the FF, AA and AF cells respectively. Their ability to reorganize their cytoskeleton upon adhesion to fibrinogen was then determined. Even though double alanine substitution produced a constitutively activated integrin, the AA cells were unable to give rise to cytoskeletal reassembly as seen in the FF and AF cells. Using phalloidin as a marker, the AA cells displayed polymerized F-actin but failed to show the elaborate elongated stress fibers formed in the FF and AF cells. To further investigate the role of the KVGFFKR motif on downstream signaling events, we focused on using pal-peptides in platelets. We have shown that in addition to stimulating platelet aggregation presumably by facilitating the spatial separation of the integrin cytoplasmic tails, pal-KVGFFKR (pal-FF) induced tyrosine phosphorylation even in the absence of ligand (EDTA:5mM) or (ReoPro:10μg/ml). The tyrosine phosphoproteome associated with alanine-substituted peptides pal-KVGAFKR (pal-AF) and pal-KVGFAKR (pal-FA) was similar to that of pal-FF. However there is a remarkable absence of a specific 100kDa band (probably α-actinin) in the phosphoprotein profile in response to pal-KVGAAKR (pal-AA) both with peptide treatment alone and in the presence of TRAP. A closer look at ppFAK125 revealed that its tyrosine phosphorylation is also inhibited by pal-AA. Since α-actinin and ppFAK125 phosphorylation are closely linked events it supports α-actinin as the 100kDa missing phosphoprotein. However, pal-AA did not inhibit ppSyk72or ppSrc60 activation. Moreover pal-AA was identified as a potent antagonist, inhibiting platelet aggregation, PAC-1 binding and tyrosine phosphorylation. In summary, a double alanine substitution of the α-subunit membrane proximal domain disturbs cytoskeletal reorganization downstream, even though this substitution produces a constitutively activated integrin. This suggests a signaling role for the conserved α-integrin motif in addition to regulating integrin affinity. Furthermore in platelets, pal-FF peptide, by mimicking the endogenous αIIb KVGFFKR sequence can both activate the integrin and contribute to an intracellular signaling response even when ligand binding is absent. Taken together, both the stable cell system and pal-peptides in platelets support a role for the KVGFFKR domain in outside-in signaling. Also since pal-AA is an antagonist of integrin function it highlights the complexity of the proximal regulation of αIIbβ3 activation and suggests a dual role for this motif in integrin activation and intracellular signaling.

The Endocannabinoid 2-Arachidonoylglycerol Regulates Platelet Function.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3904-3904
Author(s):  
Samantha Baldassarri ◽  
Alessandra Bertoni ◽  
Paolo Lova ◽  
Stefania Reineri ◽  
Chiara Sarasso ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Cannabinoid Receptors ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Dependent Manner ◽  
Cb2 Receptor ◽  
Thromboxane A2 Receptor

Abstract 2-Arachidonoylglycerol (2-AG) is a naturally occurring monoglyceride that activates cannabinoid receptors and meets several key requisites of an endogenous cannabinoid substance. It is present in the brain and hematopoietic cells, including macrophages, lymphocytes and platelets. 2-AG is released from cells in a stimulus-dependent manner and is rapidly eliminated by uptake into cells and enzymatic hydrolysis in arachidonic acid and glycerol. 2-AG might exert a very fine control on platelet function either through mechanisms intertwining with the signal transduction pathways used by platelet agonists or through mechanisms modulating specific receptors. The aim of this study was to define the role of 2-AG in human platelets and characterize the mechanisms by which it performs its action. Platelets from healthy donors were isolated from plasma by differential centrifugations and gel-filtration on Sepharose 2B. The samples were incubated with 2-AG (10–100 μM) under constant stirring in the presence or absence of various inhibitors. Platelet aggregation was measured by Born technique. We have found that stimulation of human platelets with 2-AG induced irreversible aggregation, which was significantly enhanced by co-stimulation with ADP (1–10 μM). Furthermore, 2-AG-dependent platelet aggregation was completely inhibited by ADP scavengers, aspirin, and Rho kinase inhibitor, as well as by antagonists of the 2-AG receptor (CB2), of the ADP P2Y12 receptor, and of the thromboxane A2 receptor. We further investigated the role of endocannabinoids on calcium mobilization. Intracellular [Ca2+] was measured using FURA-2-loaded platelets prewarmed at 37°C under gentle stirring in a spectrofluorimeter. 2-AG induced rapid increase of cytosolic [Ca2+] in a dose-dependent manner. This effect was partially blocked by ADP scavengers and CB2 receptor antagonists. Furthermore, 2-AG-induced [Ca2+] mobilization was totally suppressed by aspirin or the thromboxane A2 receptor antagonist. These results suggest that 2-AG is able to trigger platelet activation, and that this action is partially mediated by CB2 receptor and ADP. Furthmore, 2-AG-dependent platelet activation is totally dependent on thromboxane A2 generation.

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

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 112-112
Author(s):  
Aleksandra Stojanovic ◽  
Matvey Gorovoy ◽  
Tatyana Voyno-Yasenetskaya ◽  
Xiaoping Du
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Adhesion ◽  
Wild Type ◽  
Glycoprotein Ib ◽  
Lim Kinase ◽  
And Function

Abstract 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.

The Abcc4 Knockout Reveals An Important Role for Abcc4 in Platelet Aggregation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1141-1141
Author(s):  
Satish Babu Cheepala ◽  
Kazumasa Takenaka ◽  
Tamara I. Pestina ◽  
Carl W. Jackson ◽  
Schuetz John
Keyword(s):  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Cyclic Nucleotides ◽  
Cyclic Nucleotide ◽  
Critical Role ◽  
Antiplatelet Drugs ◽  
Null Mouse ◽  
Dense Granules

Abstract Abstract 1141 Cyclic nucleotides have an important role in platelet aggregation and the role of phosphodiesterases in regulating their concentration is well known. Currently it is unknown if plasma membrane cyclic nucleotide export proteins regulate cyclic nucleotide concentrations in platelets. The ATP-binding cassette transporter, ABCC4 functions as a cyclic nucleotide exporter that is highly expressed in platelets. However, its role as a cyclic nucleotide transporter in platelets is unknown, because it was reportedly localized intracellularly in the platelet dense granules. This original report (Jedlitschky, Tirschmann et al. 2004) evaluated ABCC4 localization by immune-fluorescence of platelets after attachment to collagen coated coverslips. However, collagen attachment activates platelets causing mobilization and fusion of alpha and dense granules to the plasma membrane, thus rendering conditions that distinguish between plasma membrane and dense granules almost impossible. To resolve this problem we isolated the platelets under conditions that minimize activation during isolation. Subsequently, these platelets membranes were labeled with the cell impermeable biotinylating agent (EZ-Link Sulfo-NHS-LC-LC Biotin). Analysis of total platelet lysate detected the dense granule marker, P-selectin and Abcc4. However, after precipitation of the plasma membrane with streptavidin-beads, we detected only Abcc4. This indicates Mrp4 is at the plasma membrane. We confirmed Abcc4 localization by confocal microscopy on platelets that were treated with a monoclonal antibody specific to Abcc4. Evidence that Abcc4 regulates cyclic nucleotide levels under basal conditions was then provided by the findings that Abcc4-null platelets have elevated cyclic nucleotides. We further used the Abcc4-null mouse model to explore the role of Abcc4 in platelet biology. The Abcc4-null mouse does not have any change in the platelet or dense granules number compared to the wild type mouse. Platelet activation in vivo can be initiated by interaction with collagen through the GPVI receptor that is expressed at the plasma membrane of the platelets. At the molecular level, the initiation of platelet activation by collagen results in an increase in the cyclic nucleotide concentration and phosphorylation of vasodilator-stimulated phosphoprotein (VASP) which can attenuate aggregation. To determine the Abcc4 role in this process we exposed Abcc4-null platelets to collagen and discovered that these platelets have impaired activation in response to collagen. However, Abcc4-null platelets activated by thrombin or ADP, which activate either G-coupled PAR receptors or P2Y12 receptor respectively, show an aggregation profile almost identical to wildtype platelets, thus indicating the defect in Abcc4-null platelet aggregation is specific to the collagen initiated pathway. To understand the basis for the impaired aggregation of Abcc4-null platelets, we examined VASP phosphorylation after collagen treatment, and discovered that the cyclic nucleotide dependent phosphorylation of VASP (Ser 157) is elevated in the Abcc4-null platelets. These results strongly suggest that Abcc4-null platelets have impaired GPVI activation by collagen due to elevated cyclic nucleotide concentrations. Based on these studies we conclude that Abcc4 plays a critical role in regulating platelet cyclic nucleotide concentrations and its absence or perhaps inhibition (by drugs) impairs the aggregation response to collagen. Because many antiplatelet drugs are potent inhibitors of Abcc4 (e.g., Dipyridamole and Sildenafil) these findings have strong implications for not just the development of antiplatelet drugs, but also for understanding the role of Abcc4 in regulating intracellular nucleotide levels. Jedlitschky, G., K. Tirschmann, et al. (2004). “The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage.” Blood 104(12): 3603–10. This work was supported by NIH and by the American Lebanese Syrian Associated Charities (ALSAC). Disclosures: No relevant conflicts of interest to declare.

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