Abstract 391: Platelet CD40L Affects Thrombus Formation via Phosphatidylinositol 3-kinase ß, But Not Via CD40 or IKKα

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Marijke J Kuijpers ◽  
Nadine J Mattheij ◽  
Lina Cipolla ◽  
Johanna P van Geffen ◽  
Toby Lawrence ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Aggregate Formation ◽  
Phosphatidylinositol 3 Kinase ◽  
Akt Phosphorylation ◽  
Soluble Cd40l ◽  
Granule Secretion ◽  
Collagen Receptor

Objective: To investigate the roles and signaling pathways of CD40L and CD40 in platelet activation and thrombus formation under atherothrombotic conditions. Approach and Results: Mouse platelets lacking CD40L (Cd40lg -/- Apoe -/- ) showed diminished αIIbβ3 activation and α-granule secretion in response to collagen receptor (GPVI) stimulation, while CD40 deficient platelets (Cd40 -/- Apoe -/- ) showed increased responses. ADP- or thrombin-evoked activation was unaffected. In both Cd40lg -/- Apoe -/- and Cd40 -/- Apoe -/- mice, formation of multi-layered thrombi was decreased on both atherosclerotic plaque material and collagen, in comparison to controls. Addition of CD40L prior to perfusion over collagen or plaque material enhanced dense aggregate formation in Apoe -/- , Cd40lg -/- Apoe -/- and Cd40 -/- Apoe -/- blood. CD40L or low GPVI stimulation separately did not cause platelet aggregation. But when combined, aggregation was potentiated, even in the absence of CD40. This potentiation was antagonized by inhibiting PI3Kβ, as well as in platelets from Pik3cb R/R mice. CD40L enhanced Akt phosphorylation at low GPVI stimulation, which was again antagonized by PI3Kβ inhibition and absent in platelets from Pik3cb R/R mice. Finally, Chuk1 A/A Apoe -/- mice, deficient in IKKα, displayed no differences in platelet aggregation - with or without CD40L - nor in thrombus formation in whole blood, indicating that these effects are not mediated via IKKα/NFkB. Conclusions: Under atherothrombotic conditions, CD40L enforces collagen-dependent platelet activation, by supporting integrin αIIbβ3 activation, secretion and dense thrombus formation via PI3Kβ, but not IKKα. Since shedding of CD40L starts minutes after activation, these results point to a joint role of both platelet-bound and soluble CD40L in controlling the size of rapidly formed thrombi.

FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

2018 ◽  
Vol 115 (11) ◽  
pp. 1672-1679 ◽  
Author(s):  
Qi Ma ◽  
Weilin Zhang ◽  
Chongzhuo Zhu ◽  
Junling Liu ◽  
Quan Chen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Mitochondrial Protein ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Akt Phosphorylation ◽  
Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

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.

Role of Caspase in a Subset of Human Platelet Activation Responses

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4222-4231 ◽  
Author(s):  
Anna Shcherbina ◽  
Eileen Remold-O’Donnell
Keyword(s):  
Platelet Activation ◽  
Caspase 3 ◽  
Human Platelet ◽  
Thrombus Formation ◽  
Shape Change ◽  
Vascular Wall ◽  
Human Platelets ◽  
Granule Secretion ◽  
Platelets Function

Abstract Platelets function to protect the integrity of the vascular wall. A subset of platelet activation responses that are especially important for thrombus formation include exposure of phosphatidylserine and release of microparticles, which generate procoagulant surfaces. The resemblance of these platelet activation processes to events occurring in nucleated cells undergoing apoptosis suggests a possible role for caspases, which are major effector enzymes of nucleated cell apoptosis. We demonstrate here the presence of caspase-3 in human platelets and its activation by physiological platelet agonists. Using cell-permeable specific inhibitors, we demonstrate a role for a caspase-3–like protease in the agonist-induced (collagen plus thrombin or Ca2+ ionophore) platelet activation events of phosphatidylserine exposure, microparticle release, and cleavage of moesin, a cytoskeletal-membrane linker protein. The role of caspase-3 in platelet activation is restricted rather than global, because other activation responses,  granule secretion, shape change, and aggregation were unaffected by caspase-3 inhibitors. Experiments with two classes of protease inhibitors show that caspase-3 function is distinct from that of calpain, which is also involved in late platelet activation events. These findings show novel functions of caspase and provide new insights for understanding of platelet activation.

scholarly journals Opposing Roles of GSK3α and GSK3β Phosphorylation in Platelet Function and Thrombosis

2021 ◽  
Vol 22 (19) ◽  
pp. 10656
Author(s):  
Samantha F. Moore ◽  
Ejaife O. Agbani ◽  
Andreas Wersäll ◽  
Alastair W. Poole ◽  
Chris M. Williams ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Glycogen Synthase ◽  
Thrombus Formation ◽  
Resistant Mouse ◽  
Function Expression ◽  
Substrate Phosphorylation ◽  
Granule Secretion

One of the mechanisms by which PI3 kinase can regulate platelet function is through phosphorylation of downstream substrates, including glycogen synthase kinase-3 (GSK3)α and GSK3β. Platelet activation results in the phosphorylation of an N-terminal serine residue in GSK3α (Ser21) and GSK3β(Ser9), which competitively inhibits substrate phosphorylation. However, the role of phosphorylation of these paralogs is still largely unknown. Here, we employed GSK3α/β phosphorylation-resistant mouse models to explore the role of this inhibitory phosphorylation in regulating platelet activation. Expression of phosphorylation-resistant GSK3α/β reduced thrombin-mediated platelet aggregation, integrin αIIbβ3 activation, and α-granule secretion, whereas platelet responses to the GPVI agonist collagen-related peptide (CRP-XL) were significantly enhanced. GSK3 single knock-in lines revealed that this divergence is due to differential roles of GSK3α and GSK3β phosphorylation in regulating platelet function. Expression of phosphorylation-resistant GSK3α resulted in enhanced GPVI-mediated platelet activation, whereas expression of phosphorylation-resistant GSK3β resulted in a reduction in PAR-mediated platelet activation and impaired in vitro thrombus formation under flow. Interestingly, the latter was normalised in double GSK3α/β KI mice, indicating that GSK3α KI can compensate for the impairment in thrombosis caused by GSK3β KI. In conclusion, our data indicate that GSK3α and GSK3β have differential roles in regulating platelet function.

scholarly journals Tannic Acid Inhibits Protein Disulfide Isomerase, Platelet Activation and Thrombus Formation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2418-2418
Author(s):  
Li Zhu
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Tannic Acid ◽  
Protein Disulfide Isomerase ◽  
Thrombus Formation ◽  
Platelet Surface ◽  
Disulfide Isomerase ◽  
Protein Disulfide

Abstract Tannic acid (TA) was a polyphenol that harbors anti-oxidant capacity. A recent report implied that surface coating with TA might blunt thrombosis via altering the structure of fibrinogen. However, the effect of TA on platelet function and in vivo thrombus formation has not been reported. In this study, we showed that TA inhibits PDI activity and attenuates platelet activation. To explore the effects of TA on platelet aggregation, gel-filtered human platelets from healthy human donors were pretreated with TA (10/30/50 μM) or vehicle (0.9% sodium chloride) before being stimulated by various agonists. Turbidity analyses on a Chronolog aggregometer showed that TA dose-dependently inhibited platelet aggregation induced by thrombin, SFLLRN, GYQGQV, collagen, CRP, U46619, and ristocetin. Next, we employed flow cytometry (FACS) to determine the role of TA in platelet activation, including α-granule secretion and integrin activation. Pretreatment of platelets with TA led to significant reductions in surface P-selectin expression and soluble fibrinogen binding, supporting the inhibition of diverse platelet activation pathways. Supportively, platelet spreading on immobilized fibrinogen was significantly suppressed by TA treatment. In addition, cell viability assay with Almar blue agent showed no detrimental impact of TA on the survival of platelets. To ask whether the antiplatelet role of TA might be translated into an antithrombotic efficacy, we tested the effect of TA in both ex vivo and in vivo thrombosis models. Calcein-labeled human whole blood was perfused through microfluidic channels coated with collagen, and adherent platelets were visualized under a fluorescent microscopy. However, treatment with TA suppressed the number of adherent platelets under flow conditions. Moreover, in laser-induced mouse cremaster muscle arteries, administration of TA (5mg/kg) significantly reduced the size of forming thrombi compared with the vehicle. Verification of bleeding risk using tail truncation assay indicated no prolongation of bleeding time in mice receiving TA. Thus, TA shows an antiplatelet effect and may also attenuate thrombus formation. To gain a mechanistic insight to the role of TA in platelet function, we performed a molecular docking screen of the structure of TA and platelet surface proteins using the Autodock Vina software, which displayed the binding of TA with protein disulfide isomerase at the enzymatic active center. We then measured the impact of TA on PDI reductase activity with the dieosin glutathione disulfide assay in vitro (di-GSSG), showing that TA significantly inhibited PDI activity in a concentration-dependent manner. The results were verified in platelets using the 3-(N-Maleimidylpropionyl) biocytin (MPB) labeling, which showed that TA abrogated thrombin-stimulated free thiol formation on platelet surface. Supportively, FACS demonstrated that TA significantly suppressed the binding of fluorescent-labeled PDI to Mn2+-activated platelet integrin β3. Taken together, our findings demonstrated that TA inhibits PDI activity and may become a novel antithrombotic agent. Disclosures No relevant conflicts of interest to declare.

Platelet 12-Lipoxygenase Is Required for Dense Granule Secretion and Platelet Aggregation: Role of 12-hLO In Platelet Hemostasis and Thrombosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3203-3203
Author(s):  
Patrick Apopa ◽  
Megha Patel ◽  
Olivier Boutaud ◽  
Michael Holinstat
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Reactivity ◽  
Dense Granule ◽  
Clot Formation ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
12 Lipoxygenase

Abstract Abstract 3203 Platelet activation plays a central role in regulating hemostasis. Uncontrolled activation of circulating platelets can result in the formation of occlusive thrombi and stroke. Following activation, metabolism of arachidonic acid by 12-lipoxygenase (12-hLO) may play a significant role in regulating the degree and stability of platelet reactivity. Using specific inhibitors for 12-hLO which do not interact with other lipoxygenases or enzymes in the COX-1 pathway, we were able for the first time to asses the involvement of 12-hLO in platelet reactivity. In order to assess the role of 12-hLO in platelet activation and thrombosis, dense granule secretion, platelet aggregation, alpha granule secretion, and platelet adhesion and clot formation under flow were measured. Inhibiting 12-hLO results in a complete inhibition of dense granule secretion with only a partial attenuation of alpha granule secretion indicating a novel regulatory scheme for modulating positive autocrine reinforcement of platelet reactivity and clot formation. Addition of the 12-hLO metabolite, 12-HETE (as low as 250 nM), resulted in a significant (25%) increase in PAR1-mediated dense granule secretion compare to agonist alone indicating that 12-HETE may be the crucial metabolite formed by 12-hLO metabolism of arachidonic acid. Importantly, platelet aggregation and adhesion are also significantly attenuated in the absence of 12-hLO. In fact, collagen-mediated platelet aggregation was shifted over 25 fold to the right in the absence of 12-hLO. These studies support the role of 12-hLO in hemostasis and may be a good target for anti-platelet therapy. Disclosures: No relevant conflicts of interest to declare.

The Role of ABC Transporter Abcc4 in Platelets Physiologic Function and Its Impact On Collagen Meditated Platelet Aggregation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1063-1063
Author(s):  
Satish B. Cheepala ◽  
Kazumasa Takenaka ◽  
Tamara I. Pestina ◽  
Carl W. Jackson ◽  
John D. Schuetz
Keyword(s):  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Platelet Activation ◽  
Cyclic Nucleotide ◽  
Dense Granules ◽  
Collagen Receptor ◽  
Kinase A

Abstract Abstract 1063 Platelet activation is a highly regulated process, and cyclic nucleotide mediated signaling pathways are crucial to effective platelet activation. Vascular injury produces, exposed collagen which binds circulating platelets through the platelet's “collagen” receptor, GPVI, resulting in the activation of guanyly/adenlyl cyclases. These interactions result in the rapid alterations in the cyclic nucleotide concentration inside the platelets leading to activation of protein kinase A and G signaling pathways to modulate platelet function. While, ABCC4 functions as a plasma membrane transporter for cyclic nucleotides its contribution to platelet activation has been obscured because it was reportedly as primarily intracellular in the platelets dense granules. This original report (Jedlitschky, Tirschmann et al. 2004) evaluated ABCC4 localization by immune-fluorescence of platelets attached to collagen coated coverslips. However, attachment via collagen produces platelet activation leading to mobilization and fusion of alpha and dense granules to the plasma membrane, thus under these conditions distinguishing between plasma membrane and dense granules is not possible. We resolved this problem by labeling quiescent platelets with a cell impermeable biotinylating agent (EZ-Link Sulfo-NHS-LC-LC Biotin). Isolation of membrane and internal fraction demonstrated that of over ninety percent of Abcc4 localizes to the plasma membrane. Furthermore, confocal microscopy of platelets stained with specific antibodies against Abcc4 confirmed Abcc4 localization to the plasma membrane. We extended these studies to the Abcc4- knockout (KO) mouse model. The Abcc4- KO mouse does not have any change in the number of platelet or dense granules 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 leading to activation of signaling cascade through protein kinase A or G. Expose of Abcc4-KO platelets to collagen and revealed impaired activation in response to collagen. However, Abcc4-KO platelets activated by either thrombin or ADP (which activate either G-coupled PAR receptors or P2Y12 receptor respectively) shows an aggregation profile almost identical to wildtype platelets, thus indicating the defect in Abcc4 -KO platelet aggregation is specific to the collagen pathway. To understand the basis for the impaired collagen aggregation of Abcc4-KO platelets, we investigated the collagen receptor (GPVI) signaling pathway in Abcc4-KO platelets. Interestingly, in the Abcc4-KO platelets after the platelet activation with collagen, cyclic nucleotide dependent phosphorylation of VASP through protein kinase A or G at Ser-157 or Ser-239 respectively is reduced compared to the wildtype. Notably, Abcc4-KO platelets had reduced GPVI surface expression that correlated with the reduced phosphorylation of VASP after collagen stimulation. The similar, protein levels of Syk and Plcg2, (downstream signaling molecules of GPVI signaling pathway), in the Abcc4 wildtype and KO platelets implies that GPVI expression is the primary defect in Abcc4 deficiency. These results suggest that Abcc4 plays a crucial role in regulating cyclic nucleotides in response to GPVI activation by collagen. These findings suggest ABCC4/Mrp4 loss of function or inhibition (by drugs) may disrupt platelet aggregation under conditions of vascular injury. As, many antiplatelet drugs are potent inhibitors of Abcc4 (e.g., Dipyridamole and Sildenafil) these conclusions have strong implications for not just the development of antiplatelet drugs, but also for further exploring the role of Abcc4 in regulating intracellular nucleotide levels and platelet biology. Disclosures: No relevant conflicts of interest to declare.

scholarly journals β-arrestin-1 participates in thrombosis and regulates integrin αIIbβ3 signalling without affecting P2Y receptors desensitisation and function

2012 ◽  
Vol 107 (04) ◽  
pp. 735-748 ◽  
Author(s):  
Mathieu Schaff ◽  
Nicolas Receveur ◽  
Catherine Bourdon ◽  
Philippe Ohlmann ◽  
François Lanza ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
P2y Receptors ◽  
Integrin Αiibβ3 ◽  
Akt Phosphorylation ◽  
Fibrinogen Binding ◽  
And Function ◽  
G Protein Coupled ◽  
Receptor Desensitisation

Summaryβ-arrestin-1 (β-arr1) and β-arrestin-2 (β-arr2) are cytosolic proteins well-known to participate in G protein-coupled receptor desensitisation and signalling. We used genetically-inactivated mice to evaluate the role of β-arr1 or β-arr2 in platelet function, P2Y receptor desensitisation, haemostasis and thrombosis. Platelet aggregation, soluble fibrinogen binding and P-selectin exposure induced by various agonists were near normal in β-arr1−/− and β-arr2−/− platelets. In addition, deficiency in β-arr1 or β-arr2 was not critical for P2Y receptors desensitisation. A functional redundancy between β-arr1 and β-arr2 may explain these unchanged platelet responses. Interestingly, β-arr1−/− but not β-arr2−/− mice were protected against laser- and FeCl3-induced thrombosis. The tail bleeding times, number of rebleeds and volume of blood loss were unchanged in β-arr1−/− and β-arr2−/− mice, suggesting no defect in haemostasis. β-arr1−/− platelet activation upon adhesion to immobilised fibrinogen was inhibited, as attested by a 37 ± 5% (n = 3, p<0.0001) decrease in filopodia extension, suggesting defective signalling through integrin αIIbβ3. β-arr1 appeared to be located downstream of Src family kinases and to regulate αIIbβ3 signalling by increasing Akt phosphorylation. Overall, this study supports a role for β-arr1 in promoting thrombus formation, in part through its participation in αIIbβ3 signalling, and no role of β-arr1 and β-arr2 in agonist-induced platelet activation and P2Y receptors desensitisation.

Role of Caspase in a Subset of Human Platelet Activation Responses

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4222-4231 ◽  
Author(s):  
Anna Shcherbina ◽  
Eileen Remold-O’Donnell
Keyword(s):  
Platelet Activation ◽  
Caspase 3 ◽  
Human Platelet ◽  
Thrombus Formation ◽  
Shape Change ◽  
Vascular Wall ◽  
Human Platelets ◽  
Granule Secretion ◽  
Platelets Function

Platelets function to protect the integrity of the vascular wall. A subset of platelet activation responses that are especially important for thrombus formation include exposure of phosphatidylserine and release of microparticles, which generate procoagulant surfaces. The resemblance of these platelet activation processes to events occurring in nucleated cells undergoing apoptosis suggests a possible role for caspases, which are major effector enzymes of nucleated cell apoptosis. We demonstrate here the presence of caspase-3 in human platelets and its activation by physiological platelet agonists. Using cell-permeable specific inhibitors, we demonstrate a role for a caspase-3–like protease in the agonist-induced (collagen plus thrombin or Ca2+ ionophore) platelet activation events of phosphatidylserine exposure, microparticle release, and cleavage of moesin, a cytoskeletal-membrane linker protein. The role of caspase-3 in platelet activation is restricted rather than global, because other activation responses,  granule secretion, shape change, and aggregation were unaffected by caspase-3 inhibitors. Experiments with two classes of protease inhibitors show that caspase-3 function is distinct from that of calpain, which is also involved in late platelet activation events. These findings show novel functions of caspase and provide new insights for understanding of platelet activation.

Novel pharmacological inhibitors demonstrate the role of the tyrosine kinase Pyk2 in adhesion and aggregation of human platelets

2016 ◽  
Vol 116 (11) ◽  
pp. 904-917 ◽  
Author(s):  
Marta Zarà ◽  
Ilaria Canobbio ◽  
Caterina Visconte ◽  
Giada Di Nunzio ◽  
Mauro Torti ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Blood Perfusion ◽  
Human Platelets ◽  
Limited Information ◽  
Akt Phosphorylation ◽  
Haematopoietic Cells ◽  
Relevant Role ◽  
Induced Aggregation

SummaryPyk2 is a Ca2+-regulated kinase predominantly expressed in neuronal and in haematopoietic cells. Previous studies on Pyk2-null mice have demonstrated that Pyk2 plays a crucial role in platelet activation and thrombus formation, thus representing a possible target for antithrombotic therapy. Very limited information is available about the role of Pyk2 in human platelets, mainly because of the lack of specific pharmacological inhibitors. In this work, we have tested two novel Pyk2 inhibitors, PF-4594755 and PF-4520440, to validate their specificity and to investigate their ability to modulate platelet activation. Both molecules were able to efficiently block Pyk2 activity in human and mouse platelets stimulated with thrombin or with the Ca2+-ionophore. In wild-type murine platelets, PF-4594755 and PF-4520440 reduced thrombin-induced aggregation to the level observed in Pyk2 knockout platelets, but did not affect aggregation induced by GPVI stimulation. Importantly, neither compounds affected the residual thrombin-induced aggregation of Pyk2-null platelets, thus excluding possible off-target effects. In human platelets, PF-4594755 and PF-4520440 significantly reduced aggregation stimulated by thrombin, but not by the GPVI agonist convulxin. Both inhibitors reduced platelet adhesion on fibrinogen and prevented Akt phosphorylation in adherent cells, indicating that Pyk2 regulates PI3K and cell spreading downstream of integrins in human platelets. Finally, the Pyk2 inhibitors significantly inhibited thrombus formation upon blood perfusion on immobilized collagen under arterial flow rate. These results demonstrate that PF-4594755 and PF-4520440 are specific inhibitors of Pyk2 in intact platelets and allowed to reliably document that this kinase plays a relevant role in human platelet activation.Supplementary Material to this article is available online at www.thrombosis-online.com.

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