TREM-like transcript 1: a more sensitive marker of platelet activation than P-selectin in humans and mice

Key Points Platelet activation in vitro results in a more rapid and greater upregulation of TLT-1 surface expression compared with P-selectin. TLT-1 is more rapidly translocated to the surface of activated platelets than P-selectin during thrombus formation in vivo.

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Impaired platelet activation and cAMP homeostasis in MRP4-deficient mice

Blood ◽

10.1182/blood-2015-02-631044 ◽

2015 ◽

Vol 126 (15) ◽

pp. 1823-1830 ◽

Cited By ~ 35

Author(s):

Benoit Decouture ◽

Elise Dreano ◽

Tiphaine Belleville-Rolland ◽

Orjeta Kuci ◽

Blandine Dizier ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Platelet Activation ◽

Thrombus Formation ◽

Key Points ◽

Platelet Signaling ◽

Deficient Mice ◽

Kinase A

Key PointsIn vivo and in vitro thrombus formation is altered in MRP4-deficient mice. MRP4 modulates the cAMP–protein kinase A platelet signaling pathway.

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Soluble Platelet Release Factors as Biomarkers for Cardiovascular Disease

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.684920 ◽

2021 ◽

Vol 8 ◽

Author(s):

Gaukhar Baidildinova ◽

Magdolna Nagy ◽

Kerstin Jurk ◽

Philipp S. Wild ◽

Hugo ten Cate ◽

...

Keyword(s):

Platelet Activation ◽

Thrombus Formation ◽

Coagulation System ◽

Thrombotic Risk ◽

Activated Platelets ◽

Platelet Release ◽

Thrombotic Diseases ◽

Arterial Vascular Disease

Platelets are the main players in thrombotic diseases, where activated platelets not only mediate thrombus formation but also are involved in multiple interactions with vascular cells, inflammatory components, and the coagulation system. Although in vitro reactivity of platelets provides information on the function of circulating platelets, it is not a full reflection of the in vivo activation state, which may be relevant for thrombotic risk assessment in various disease conditions. Therefore, studying release markers of activated platelets in plasma is of interest. While this type of study has been done for decades, there are several new discoveries that highlight the need for a critical assessment of the available tests and indications for platelet release products. First, new insights have shown that platelets are not only prominent players in arterial vascular disease, but also in venous thromboembolism and atrial fibrillation. Second, knowledge of the platelet proteome has dramatically expanded over the past years, which contributed to an increasing array of tests for proteins released and shed from platelets upon activation. Identification of changes in the level of plasma biomarkers associated with upcoming thromboembolic events allows timely and individualized adjustment of the treatment strategy to prevent disease aggravation. Therefore, biomarkers of platelet activation may become a valuable instrument for acute event prognosis. In this narrative review based on a systematic search of the literature, we summarize the process of platelet activation and release products, discuss the clinical context in which platelet release products have been measured as well as the potential clinical relevance.

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IκB kinase 2 is not essential for platelet activation

Blood Advances ◽

10.1182/bloodadvances.2019001044 ◽

2020 ◽

Vol 4 (4) ◽

pp. 638-643

Author(s):

Manuel Salzmann ◽

Sonja Bleichert ◽

Bernhard Moser ◽

Marion Mussbacher ◽

Mildred Haase ◽

...

Keyword(s):

Platelet Activation ◽

Active Site ◽

Bleeding Time ◽

Thrombus Formation ◽

Human Platelets ◽

Iκb Kinase ◽

Specific Deletion

Abstract Platelets are small anucleate cells that release a plethora of molecules to ensure functional hemostasis. It has been reported that IκB kinase 2 (IKK2), the central enzyme of the inflammatory NF-κB pathway, is involved in platelet activation, because megakaryocyte/platelet-specific deletion of exons 6 and 7 of IKK2 resulted in platelet degranulation defects and prolonged bleeding. We aimed to investigate the role of IKK2 in platelet physiology in more detail, using a platelet-specific IKK2 knockout via excision of exon 3, which makes up the active site of the enzyme. We verified the deletion on genomic and transcriptional levels in megakaryocytes and were not able to detect any residual IKK2 protein; however, platelets from these mice did not show any functional impairment in vivo or in vitro. Bleeding time and thrombus formation were not affected in platelet-specific IKK2-knockout mice. Moreover, platelet aggregation, glycoprotein GPIIb/IIIa activation, and degranulation were unaltered. These observations were confirmed by pharmacological inhibition of IKK2 with TPCA-1 and BMS-345541, which did not affect activation of murine or human platelets over a wide concentration range. Altogether, our results imply that IKK2 is not essential for platelet function.

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Inhibitory effects of lycopene on in vitro platelet activation and in vivo prevention of thrombus formation

Journal of Laboratory and Clinical Medicine ◽

10.1016/j.lab.2005.03.018 ◽

2005 ◽

Vol 146 (4) ◽

pp. 216-226 ◽

Cited By ~ 27

Author(s):

George Hsiao ◽

Ying Wang ◽

Nien-Hsuan Tzu ◽

Tsorng-Hang Fong ◽

Ming-Yi Shen ◽

...

Keyword(s):

Platelet Activation ◽

Thrombus Formation ◽

Inhibitory Effects ◽

In Vitro Platelet Activation

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A Role for Bile Salt-Dependent Lipase in Platelet Activation and in Thrombus Formation in Vivo.

Blood ◽

10.1182/blood.v104.11.3526.3526 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3526-3526 ◽

Cited By ~ 1

Author(s):

Laurence Panicot-Dubois ◽

Christophe Dubois ◽

Barbara C. Furie ◽

Bruce Furie ◽

Dominique Lombardo

Keyword(s):

Bile Salt ◽

Platelet Activation ◽

Thrombus Formation ◽

Pancreatic Acinar Cells ◽

Cremaster Muscle ◽

Terminal Domain ◽

Laser Injury ◽

Phosphatidylserine Exposure

Abstract Bile Salt Dependent Lipase (BSDL) is an enzyme secreted by pancreatic acinar cells. BSDL, in the presence of primary bile salts, participates in the hydrolysis of dietary lipid esters in the duodenum lumen. This 105 kDa N and O-glycosylated protein has been detected in the plasma of normal subjects. Recent in vitro and in vivo studies demonstrated that pancreatic BSDL reaches the blood via transcytosis through enterocytes. Other studies showed that pancreatic human BSDL is captured by human umbilical vein endothelial cells and induces the proliferation of smooth muscle cells in vitro at BSDL concentrations found in blood, suggesting that this enzyme may play a role in hemostasis and thrombosis. However the specific role of circulating BSDL is unknown. The goal of this study was to determine the possible involvement of circulating BSDL in thrombus formation. We investigated the participation of circulating mouse BSDL in thrombus formation using widefield intravital microscopy in the cremaster muscle of living mice. Thrombi were formed following laser injury of the vessel wall of an arteriole in the cremaster muscle. Pancreatic mouse BSDL, a 74 kDa glycoprotein, was detected using several antibodies directed against either the whole human BSDL (pAbL64, pAbL32) or a peptide based on a sequence in the N-terminal domain of BSDL (Ser326-Thr350; pAbAntipeptide). Mouse and human BSDL share about 80% sequence homology, the main difference localized in the C-terminal domain, which is truncated to the mouse BSDL compared with the human enzyme. All the antibodies are able to specifically recognize the mouse pancreatic BSDL. Using antibodies pAbL64, pAbL32, or pAbAntipeptide we observed specific accumulation of circulating mouse BSDL into the growing thrombus. The circulating BSDL co-localized with platelets present in the thrombus. These results suggest that circulating BSDL is involved in thrombus formation in vivo. In order to determine if BSDL plays a role in platelet activation and aggregation, we performed in vitro studies on human washed platelets. BSDL increased both the amount of phosphatidylserine exposure on the surface of platelets and the activation of αIIbβ3 induced by thrombin. These results indicate that this enzyme can amplify the activation of platelets in vitro. While BSDL alone cannot induce the aggregation of platelets, this enzyme significantly increases the amount of platelet aggregation induced by SFLLRN peptide or thrombin. Altogether, these data suggeste that circulating BSDL participates in the thrombus formation after laser injury of the arterial wall and can amplify both the activation of platelets and the phosphatidylserine exposure, increasing the thrombotic response after vessel injury. This mechanism may be operative in the development of venous thromboembolic disease in pancreatic cancer.

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A Significant Fraction of ADAMTS13 Activity Is Associated with Activated Platelets and Their Microparticles (PMP): Implication for Regulating ADAMTS13 Activity.

Blood ◽

10.1182/blood.v108.11.1066.1066 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1066-1066

Author(s):

Wenche Jy ◽

Andrew Lin ◽

Loreta Bidot ◽

Jaehoon Bang ◽

Eugene Ahn ◽

...

Keyword(s):

Platelet Activation ◽

Control Level ◽

In Vitro Study ◽

Ionophore A23187 ◽

Adamts13 Activity ◽

Activated Platelets ◽

Membrane Bound ◽

Low Levels

Abstract BACKGROUND: Deficiency of ADAMTS13, vWF cleaving protease, is known to be associated with TTP and some other microangiopathies, but low levels were reported in other diseases such as ITP, DIC, lupus and other thromboses. Although inhibitory autoantibodies were demonstrated in TTP, the mechanisms underlying reduced levels of ADAMTS13 in other disorders remains unclear. We tested the hypothesis that ADAMTS13 is associated with cell membranes and derived microparticles, especially from activated platelet and their microparticles (PMP), which could modulate the enzyme activities of ADAMTS13. METHODS: PRP was prepared by centrifuging citrated normal blood for 10 min at 160×g, and PPP by further centrifuging for 10 min at 3,000×g, and particle-free plasma (PFP) by further centrifuging for 15 min at 20,000×g. ADAMTS13 activity was assayed by the FRETS-VWF73 method of Kokame et al [Br J Haematol 129:93, 2005] using the Fluoroskan Ascent plate reader. Platelets were activated by ADP (10 μM) or ionophore A23187 (2 μM). RESULTS: The ADAMTS13 activity (A) of pooled PPP of 10 controls was defined as 100%. (1) In vitro study: (1a) ADAMTS13 activity was not significantly different between PPP and resting PRP. However, if the platelets in PRP were first activated by ADP for 1hr, a significant reduction of activity was observed (A = 85 ±7%, p<0.05). If the activated platelets were removed, the activity of the supernatant fell to 79 ±10% p<0.05) of the control level, and was further reduced by higher centrifugation to remove PMP (A = 66 ±12%, p<0.01). (1b) Activation by A23187, a stronger agonist producing 2–3 fold more PMP than ADP (confirmed by flow cytometry), induced a more dramatic reduction in PRP (A = 78±8%, p<0.01), and after removal of platelets (A = 71 ±11%, p<0.01), and after removal of PMP (48 ±11%, p<0.01). (1c) Interestingly, resuspending the activated platelets did not restore ADAMTS13 activity, although resuspending the PMP did partially restore the activity. (2) In vivo study: PPP from 13 patients (6 ITP, 4 APS, 3 lupus) were analyzed. The majority (11/13) of PPP samples lost activity after removal of PMP (A = 79 ±12% in PPP vs. 64 ±11% in PFP; p <0.02). CONCLUSION: These data show that a significant but variable fraction of ADAMTS13 activity is associated with activated platelets and PMP. This has several implications. First, distinguishing soluble from membrane-bound ADAMTS13 may lead to better correlation of activities with clinical findings, and may help explain low levels of ADAMTS13 in some disorders associated with platelet activation and high PMP. Second, this interaction may play a role in regulating ADAMTS13 activity. Third, membrane-bound ADAMTS13 may clear more readily from circulation, therefore inhibiting platelet activation or MP formation may have benefits for the management of microangiopathies.

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STIM1 Deficiency Results In Impaired Platelet Procoagulant Activity and Protection From Arterial Thrombosis

Blood ◽

10.1182/blood.v116.21.485.485 ◽

2010 ◽

Vol 116 (21) ◽

pp. 485-485

Author(s):

Firdos Ahmad ◽

Lucia Stefanini ◽

Timothy Daniel Ouellette ◽

Teshell K Greene ◽

Stefan Feske ◽

...

Keyword(s):

Platelet Activation ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Critical Role ◽

Phosphatidyl Serine ◽

Flow Chamber ◽

Procoagulant Activity ◽

Static Conditions

Abstract Abstract 485 Platelet activation is a central event in thrombosis and hemostasis. We recently demonstrated that most aspects of platelet activation depend on synergistic signaling by two signaling modules: 1) Ca2+/CalDAG-GEFI/Rap1 and 2) PKC/P2Y12/Rap1. The intracellular Ca2+ concentration of platelets is regulated by Ca2+ release from the endoplasmic reticulum (ER) and store-operated calcium entry (SOCE) through the plasma membrane. Stromal interaction molecule 1 (STIM1) was recently identified as the ER Ca2+ sensor that couples Ca2+ store release to SOCE. In this study, we compared the activation response of platelets lacking STIM1−/− or CalDAG-GEFI−/−, both in vitro and in vivo. To specifically investigate Ca2+-dependent platelet activation, some of the experiments were performed in the presence of inhibitors to P2Y12. The murine Stim1 gene was deleted in the megakaryocyte/platelet lineage by breeding Stim flox/flox mice with PF4-Cre mice (STIM1fl/fl). STIM1fl/fl platelets showed markedly reduced SOCE in response to agonist stimulation. aIIbβ3 activation in STIM1fl/fl platelets was significantly reduced in the presence but not in the absence of the P2Y12 inhibitor, 2-MesAMP. In contrast, aIIbb3 activation was completely inhibited in 2-MesAMP-treated CalDAG-GEFI−/− platelets. Deficiency in STIM1, and to a lesser extent in CalDAG-GEFI, reduced phosphatidyl serine (PS) exposure in platelets stimulated under static conditions. PS exposure was completely abolished in both STIM1fl/fl and CalDAG-GEFI−/− platelets stimulated in the presence of 2-MesAMP. To test the ability of platelets to form thrombi under conditions of arterial shear stress, we performed flow chamber experiments with anticoagulated blood perfused over a collagen surface. Thrombus formation was abolished in CalDAG-GEFI−/− blood and WT blood treated with 2-MesAMP. In contrast, STIM1fl/fl platelets were indistinguishable from WT platelets in their ability to form thrombi. STIM1fl/fl platelets, however, were impaired in their ability to express PS when adhering to collagen under flow. Consistently, when subjected to a laser injury thrombosis model, STIM1fl/fl mice showed delayed and reduced fibrin generation, resulting in the formation of unstable thrombi. In conclusion, our studies indicate a critical role of STIM1 in SOCE and platelet procoagulant activity, but not in CalDAG-GEFI mediated activation of aIIbb3 integrin. Disclosures: No relevant conflicts of interest to declare.

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CLEC-2 is an essential platelet-activating receptor in hemostasis and thrombosis

Blood ◽

10.1182/blood-2009-05-222273 ◽

2009 ◽

Vol 114 (16) ◽

pp. 3464-3472 ◽

Cited By ~ 130

Author(s):

Frauke May ◽

Ina Hagedorn ◽

Irina Pleines ◽

Markus Bender ◽

Timo Vögtle ◽

...

Keyword(s):

Platelet Activation ◽

Thrombus Formation ◽

Antibody Treatment ◽

Cellular Activation ◽

Primary Hemostasis ◽

Arterial Thrombus ◽

Normal Adhesion

Abstract Damage to the integrity of the vessel wall leads to exposure of the subendothelial extracellular matrix (ECM), triggering platelet activation and aggregation. This process is essential for primary hemostasis but it may also lead to arterial thrombosis. Although the mechanisms underlying platelet activation on the ECM are well explored, it is less clear which receptors mediate cellular activation in a growing thrombus. Here we studied the role of the recently identified C-type lectin-like receptor 2 (CLEC-2) in this process. We show that anti–CLEC-2 antibody treatment of mice leads to complete and highly specific loss of CLEC-2 in circulating platelets for several days. CLEC-2–deficient platelets displayed normal adhesion under flow, but subsequent aggregate formation was severely defective in vitro and in vivo. As a consequence, CLEC-2 deficiency was associated with increased bleeding times and profound protection from occlusive arterial thrombus formation. These results reveal an essential function of CLEC-2 in hemostasis and thrombosis.

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The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation

10.1101/2021.01.24.427281 ◽

2021 ◽

Author(s):

Leila Revollo ◽

Glenn Merrill-Skoloff ◽

Karen De Ceunynck ◽

James R. Dilks ◽

Mattia Bordoli ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Platelet Activation ◽

Platelet Function ◽

Secretory Pathway ◽

Thrombus Formation ◽

Extracellular Proteins ◽

Normal Platelet

AbstractTyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate Lonesome Kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet ɑ-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology, but have dramatic changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets demonstrate a significant decrease of several tyrosine phosphobands. Functional testing of VLK-deficient platelets shows decreased PAR4- and collagen-mediated platelet aggregation, but normal responses to ADP. Dense granule and α-granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased PAR4-mediated Akt (S473) and Erk1/2(T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets demonstrate strongly reduced platelet accumulation and fibrin formation following laser-injury of cremaster arterioles compared to controls. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function.

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The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation

Blood ◽

10.1182/blood.2020010342 ◽

2021 ◽

Author(s):

Leila Denise Revollo ◽

Glenn Merrill-Skoloff ◽

Karen De Ceunynck ◽

James R Dilks ◽

Shihui Guo ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Platelet Activation ◽

Platelet Function ◽

Secretory Pathway ◽

Thrombus Formation ◽

Extracellular Proteins ◽

Normal Platelet

Tyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate Lonesome Kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet ɑ-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology, but have significant changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets demonstrate a significant decrease of several tyrosine phosphobands. Functional testing of VLK-deficient platelets shows decreased PAR4- and collagen-mediated platelet aggregation, but normal responses to ADP. Dense granule and a-granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased PAR4-mediated Akt (S473) and Erk1/2 (T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets demonstrate strongly reduced platelet accumulation and fibrin formation following laser-injury of cremaster arterioles compared to controls, but normal bleeding times. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function.

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