Misshapen/NIK-related kinase (MINK1) is involved in platelet function, hemostasis, and thrombus formation

Abstract PI3-kinase (phosphoinositide 3-kinase) is an important signaling molecule that is activated downstream of various receptors upon platelet activation. PI3-kinase activation leads to the generation of PIP3 (Phosphatidylinositol (3,4,5)-trisphosphate) subsequently leading to the recruitment of PH (pleckstrin homology) domain containing proteins to the plasma membrane. Our laboratory screened for proteins that interacted with PIP3 (Phosphatidylinositol (3,4,5)-trisphosphate) using PIP3 beads. One of the proteins that interacted with PIP3 was ELMO1 (Engulfment and cell motility-1). ELMO1 is a scaffold protein with no catalytic activity and is well known to regulate actin cytoskeletal rearrangement via Rac1 in other cells. However, it is not known whether ELMO1 is expressed in platelets and if so, does it regulate platelet functional responses. Here, we show that ELMO1 is present in both human and murine platelets. We used ELMO1-deficient (ELMO1-/-) mice to study its role in platelets. ELMO1-/- murine platelets showed enhanced platelet aggregation and dense granule secretion in response to the GPVI agonist, CRP (Figure 1 A & B), compared to the wildtype controls although there was no difference in GPVI expression levels between the two. There was no difference observed in response to AYPGKF- or 2-MeSADP. These data suggest that ELMO1 plays a specific role downstream of GPVI pathway but GPCRs. Moreover, ELMO1-/- platelets exhibited enhanced clot retraction and spreading indicating its role in Glycoprotein IIb/IIa (GPIIb/IIIa) mediated outside-in signaling. Furthermore, whole blood from ELMO1-/- mice perfused over collagen under arterial shear conditions exhibited enhanced thrombus formation. In an in vivo pulmonary thromboembolism model, ELMO1-/- mice showed reduced survival compared to the wildtype control. ELMO1-/- mice also showed shorter time to occlusion and increased thrombus stability using the ferric-chloride injury model indicating the role of ELMO1 in thrombus formation in vivo. At the molecular level, Rac1 activity was enhanced in ELMO1-/- murine platelets compared to the wildtype control in response to CRP (Figure 1C). Together, these data suggest that ELMO1 regulates Rac1 activity upon GPVI-mediated thrombus formation and it may play a negative regulator role in both inside-out and outside-in signaling, which might involve Rac1. Figure 1 Representative figure of (A) platelet aggregation and (B) dense granule secretion. (C) Washed platelets were stimulated with CRP 1.25 μg/mL for the indicated times. GST-PAK-RBD was used to pull-down active Rac1 from platelet lysates and was detected using specific antibody to Rac1 by Western blot. WT = Wildtype mice. ELMO1-/- = ELMO1-deficient mice. CRP = collagen related protein. Figure 1. Representative figure of (A) platelet aggregation and (B) dense granule secretion. (C) Washed platelets were stimulated with CRP 1.25 μg/mL for the indicated times. GST-PAK-RBD was used to pull-down active Rac1 from platelet lysates and was detected using specific antibody to Rac1 by Western blot. WT = Wildtype mice. ELMO1-/- = ELMO1-deficient mice. CRP = collagen related protein. Disclosures No relevant conflicts of interest to declare.

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Granules and thrombus formation

Blood ◽

10.1182/blood-2009-05-220665 ◽

2009 ◽

Vol 114 (5) ◽

pp. 932-933 ◽

Cited By ~ 6

Author(s):

Walter H. A. Kahr

Keyword(s):

Thrombus Formation ◽

Dense Granule ◽

Snare Protein ◽

Laser Injury ◽

Platelet Accumulation ◽

Dense Granule Secretion ◽

Granule Secretion

In this issue of Blood, Graham and colleagues demonstrate the importance of platelet dense granule secretion for in vivo platelet accumulation following laser injury, which is mediated by the SNARE protein Endobrevin/VAMP-8.

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Analysis of microvascular thrombus mechanobiology with a novel particle-based model

10.1101/2021.06.07.447380 ◽

2021 ◽

Author(s):

Anastasia A Masalceva ◽

Valeriia N Kaneva ◽

Mikhail A Panteleev ◽

Fazoil Ataullahanov ◽

Vitaly Volpert ◽

...

Keyword(s):

Thrombus Formation ◽

Dense Granule ◽

Aggregate Formation ◽

Mechanical Stabilization ◽

Dense Granules ◽

Virtual Particles ◽

Platelet Accumulation ◽

Dense Granule Secretion ◽

Granule Secretion

Platelet accumulation at the site of vascular injury is regulated by soluble platelet agonists, which induce various types of platelet responses, including integrin activation and granule secretion. The interplay between local biochemical cues, mechanical interactions between platelets and macroscopic thrombus dynamics is poorly understood. Here we describe a novel computational model of microvascular thrombus formation for detailed analysis of thrombus mechanics. Adopting a previously developed two-dimensional particle-based model focused on the thrombus shell formation, we revise it to introduce platelet agonists. Blood flow is simulated via computational fluid dynamics approach. In order to model soluble platelet activators, we apply Langevin dynamics to a large number of non-dimensional virtual particles. Taking advantage of the available data on platelet dense granule secretion kinetics, we model platelet degranulation as a stochastic agonist-dependent process. The new model qualitatively reproduces enhanced thrombus formation due to granule secretion in line with in vivo findings and provides a mechanism for thrombin confinement at the early stages of aggregate formation. Our calculations also predict that release of dense granules results in additional mechanical stabilization of the inner layers of the thrombus. Distribution of the inter-platelet forces throughout the aggregate reveals multiple weak spots in the outer regions of thrombus, which are expected to result in mechanical disruptions at the later stages of thrombus formation.

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The Predominant Role of Arrestin3 in General GPCR Desensitization in Platelets

Journal of Clinical Medicine ◽

10.3390/jcm10204743 ◽

2021 ◽

Vol 10 (20) ◽

pp. 4743

Author(s):

Preeti Kumari Chaudhary ◽

Sanggu Kim ◽

Soochong Kim

Keyword(s):

Platelet Aggregation ◽

Thrombus Formation ◽

Dense Granule ◽

Functional Responses ◽

Erk Phosphorylation ◽

Dense Granule Secretion ◽

Granule Secretion ◽

Gpcr Desensitization

Arrestins in concert with GPCR kinases (GRKs) function in G protein-coupled receptor (GPCR) desensitization in various cells. Therefore, we characterized the functional differences of arrestin3 versus arrestin2 in the regulation of GPCR signaling and its desensitization in platelets using mice lacking arrestin3 and arrestin2. In contrast to arrestin2, platelet aggregation and dense granule secretion induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in arrestin3-deficient platelets compared to wild-type (WT) platelets, while non-GPCR agonist CRP-induced platelet aggregation and secretion were not affected. Surprisingly, in contrast to GRK6, platelet aggregation induced by the co-stimulation of serotonin and epinephrine was significantly potentiated in arrestin3-deficient platelets, suggesting the central role of arrestin3 in general GPCR desensitization in platelets. In addition, the second challenge of ADP and AYPGKF restored platelet aggregation in arrestin3-deficient platelets but failed to do so in WT and arrestin2-deficient platelets, confirming that arrestin3 contributes to GPCR desensitization. Furthermore, ADP- and AYPGKF-induced Akt and ERK phosphorylation were significantly increased in arrestin3-deficient platelets. Finally, we found that arrestin3 is critical for thrombus formation in vivo. In conclusion, arrestin3, not arrestin2, plays a central role in the regulation of platelet functional responses and thrombus formation through general GPCR desensitization in platelets.

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Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2

Blood ◽

10.1182/blood-2010-10-312199 ◽

2011 ◽

Vol 118 (2) ◽

pp. 416-424 ◽

Cited By ~ 35

Author(s):

Olga Konopatskaya ◽

Sharon A. Matthews ◽

Matthew T. Harper ◽

Karen Gilio ◽

Judith M. E. M. Cosemans ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Molecular Mechanisms ◽

Thrombus Formation ◽

Dense Granule ◽

Molecular Pathway ◽

Kinase C ◽

Pkc Isoforms ◽

Dense Granule Secretion ◽

Granule Secretion

Abstract Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

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A dual role for integrin-linked kinase in platelets: regulating integrin function and α-granule secretion

Blood ◽

10.1182/blood-2008-03-148502 ◽

2008 ◽

Vol 112 (12) ◽

pp. 4523-4531 ◽

Cited By ~ 43

Author(s):

Katherine L. Tucker ◽

Tanya Sage ◽

Joanne M. Stevens ◽

Peter A. Jordan ◽

Sarah Jones ◽

...

Keyword(s):

Thrombus Formation ◽

Dense Granule ◽

Conditional Knockout ◽

Knockout Mouse Model ◽

Conditional Knockout Mouse ◽

Fibrinogen Binding ◽

Integrin Linked Kinase ◽

Dense Granule Secretion ◽

Granule Secretion

Abstract Integrin-linked kinase (ILK) has been implicated in the regulation of a range of fundamental biological processes such as cell survival, growth, differentiation, and adhesion. In platelets ILK associates with β1- and β3-containing integrins, which are of paramount importance for the function of platelets. Upon stimulation of platelets this association with the integrins is increased and ILK kinase activity is up-regulated, suggesting that ILK may be important for the coordination of platelet responses. In this study a conditional knockout mouse model was developed to examine the role of ILK in platelets. The ILK-deficient mice showed an increased bleeding time and volume, and despite normal ultrastructure the function of ILK-deficient platelets was decreased significantly. This included reduced aggregation, fibrinogen binding, and thrombus formation under arterial flow conditions. Furthermore, although early collagen stimulated signaling such as PLCγ2 phosphorylation and calcium mobilization were unaffected in ILK-deficient platelets, a selective defect in α-granule, but not dense-granule, secretion was observed. These results indicate that as well as involvement in the control of integrin affinity, ILK is required for α-granule secretion and therefore may play a central role in the regulation of platelet function.

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Impaired Platelet Responses to Thromboxane a2 and Thrombin in Mice Lacking Receptor-Interacting Protein Kinase 3

Blood ◽

10.1182/blood.v124.21.2762.2762 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2762-2762

Author(s):

Yiwen Zhang ◽

Jian Zhang ◽

Rong Yan ◽

Jie Zhang ◽

Mengxing Chen ◽

...

Keyword(s):

Platelet Aggregation ◽

Protein Kinase ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Thromboxane A2 ◽

Dense Granule ◽

In Vivo Model ◽

Interacting Protein ◽

Granule Secretion

Abstract Objective: Receptor-interacting protein 3 (RIP3) is a member of RIP family with a Ser/Thr protein kinase domain in its amino-terminus which is essential for kinase activity and autophosphorylation. The roles of RIP3 in embryonic development and different disease pathologies, such as inflammation and infections, have been reported in recent years. However, the role of RIP3 in thrombosis and hemostasis remains unknown. Methods: Hematologic analysis was performed and tail bleeding time was monitored. Mouse platelets were isolated from anti-coagulated whole blood. Platelet aggregation and secretion were recorded at real time. Platelet P-selectin exposure and specific fibrinogen binding were detected by flow cytometry. TXA2 generation was measured with enzyme immunoassay (EIA) kit. Protein phosphorylations were detected by western blotting. Result: RIP3-/- mice had tail-bleeding times that were significantly prolonged compared with their wild type littermates. In an in vivo model of mesenteric arteriole thrombosis, mice lacking RIP3 exhibited delayed thrombus formation, fewer accumulated platelets, smaller thrombi, and prolonged occlusion times. RIP3 was expressed in both human and mouse platelets. Deletion of RIP3 in mouse platelets caused a marked defect in aggregation and attenuated dense granule secretion in response to low doses of thrombin or a thromboxane A2 (TXA2) analogue, U46619. The defect in ADP secretion appears responsible for the impaired platelet aggregation, because addition of exogenous ADP rescued the reduced platelet aggregation. Although TXA2 generation and α-granule secretion were not impaired, integrin αIIbβ3 activation was attenuated in RIP3-/- platelets. Moreover, phosphorylation of Akt induced by U46619 or thrombin was markedly reduced in the absence of RIP3. Activation of Akt signaling restored the impaired aggregation of RIP3-/- platelets. ERK and p38 phosphorylation elicited by either U46619 or thrombin was attenuated in RIP3-/- platelets. In contrast, U46619- and thrombin-induced activation of PTEN, PDK1, or Src was not impaired in RIP3-/- platelets. Conclusion: Our data demonstrate a novel role for RIP3 in amplifying U46619- and thrombin-induced platelet activation by mediating Akt-dependent ADP secretion, and in supporting hemostasis and thrombus formation in vivo. RIP3 may represent a novel target to modulate PARs and TP signaling and a potential new target for antithrombotic strategy. Disclosures No relevant conflicts of interest to declare.

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Critical role of peroxisome proliferator-activated receptor α in promoting platelet hyperreactivity and thrombosis under hyperlipidemia

Haematologica ◽

10.3324/haematol.2021.279770 ◽

2021 ◽

Author(s):

Li Li ◽

Jiawei Zhou ◽

Shuai Wang ◽

Lei Jiang ◽

Xiaoyan Chen ◽

...

Keyword(s):

Platelet Activation ◽

Thrombus Formation ◽

Critical Role ◽

Dense Granule ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Important Regulator ◽

Dense Granule Secretion ◽

Granule Secretion ◽

Underlying Mechanisms

Platelet hyperreactivity and increased atherothrombotic risk are specifically associated with dyslipidemia. Peroxisome proliferator-activated receptor alpha (PPARα) is an important regulator of lipid metabolism. It was suggested to affect both thrombosis and hemostasis, yet the underlying mechanisms are not well understood. In this study, the role and mechanism of PPARα in platelet activation and thrombosis related to dyslipidemia were examined. Employing mice with deletion of PPARα (Pparα -/-), we demonstrated that PPARα is required for platelet activation and thrombus formation. The effect of PPARα is critically dependent on platelet dense granule secretion, and is contributed by p38MAPK/Akt, fatty acid β- oxidation, and NAD(P)H oxidase (NOX) pathways. Importantly, PPARα and the associated pathways mediated a prothrombotic state induced by high-fat diet (HFD) and platelet hyperactivity provoked by oxidized low density lipoproteins (oxLDL). Platelet reactivities were positively correlated with the expression levels of PPARα, as revealed by data from wild-type (WT), chimeric (Pparα +/-), and Pparα -/- mice. This positive correlation was recapitulated in platelets from hyperlipidemic patients. In a lipid-treated megakaryocytic cell line, lipid-induced reactive oxygen species (ROS)-NF-κB pathway was revealed to upregulate platelet PPARα in hyperlipidemia. These data suggested platelet PPARα critically mediates platelet activation and contributes to prothrombotic status under hyperlipidemia.

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Non-genomic effects of the Pregnane X Receptor negatively regulate platelet functions, thrombosis and haemostasis

Scientific Reports ◽

10.1038/s41598-019-53218-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Gagan D. Flora ◽

Khaled A. Sahli ◽

Parvathy Sasikumar ◽

Lisa-Marie Holbrook ◽

Alexander R. Stainer ◽

...

Keyword(s):

Platelet Function ◽

Thrombus Formation ◽

Pregnane X Receptor ◽

Human Platelets ◽

Regulatory Effects ◽

Granule Secretion ◽

Species Specific ◽

Platelet Functions

AbstractThe pregnane X receptor (PXR) is a nuclear receptor (NR), involved in the detoxification of xenobiotic compounds. Recently, its presence was reported in the human vasculature and its ligands were proposed to exhibit anti-atherosclerotic effects. Since platelets contribute towards the development of atherosclerosis and possess numerous NRs, we investigated the expression of PXR in platelets along with the ability of its ligands to modulate platelet activation. The expression of PXR in human platelets was confirmed using immunoprecipitation analysis. Treatment with PXR ligands was found to inhibit platelet functions stimulated by a range of agonists, with platelet aggregation, granule secretion, adhesion and spreading on fibrinogen all attenuated along with a reduction in thrombus formation (both in vitro and in vivo). The effects of PXR ligands were observed in a species-specific manner, and the human-specific ligand, SR12813, was observed to attenuate thrombus formation in vivo in humanised PXR transgenic mice. PXR ligand-mediated inhibition of platelet function was found to be associated with the inhibition of Src-family kinases (SFKs). This study identifies acute, non-genomic regulatory effects of PXR ligands on platelet function and thrombus formation. In combination with the emerging anti-atherosclerotic properties of PXR ligands, these anti-thrombotic effects may provide additional cardio-protective benefits.

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Enrichment of FLI1 and RUNX1 mutations in families with excessive bleeding and platelet dense granule secretion defects

Blood ◽

10.1182/blood-2013-06-506873 ◽

2013 ◽

Vol 122 (25) ◽

pp. 4090-4093 ◽

Cited By ~ 70

Author(s):

Jacqueline Stockley ◽

Neil V. Morgan ◽

Danai Bem ◽

Gillian C. Lowe ◽

Marie Lordkipanidzé ◽

...

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Transcriptional Activity ◽

Dense Granule ◽

Amino Acid Substitutions ◽

Dna Binding Domain ◽

Excessive Bleeding ◽

Key Points ◽

Dense Granule Secretion ◽

Granule Secretion

Key Points Novel FLI1 and RUNX1 alterations were identified in 6 of 13 patients with excessive bleeding and platelet granule secretion defects. Two FLI1 alterations predicting amino acid substitutions in the DNA-binding domain of FLI1 abolished transcriptional activity of FLI1.

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