Dual Mechanism of Integrin αIIbβ3 Closure in Procoagulant Platelets

Mattheij, Nadine J.A.; Gilio, Karen; Kruchten, Roger van; Jobe, Shawn M.; Wieschhaus, Adam J.; Chishti, Athar H.; Collins, Peter William; Heemskerk, Johan W. M.; Cosemans, Judith M.E.M.

doi:10.1074/jbc.m112.428359

Cited by 104 publications

(178 citation statements)

References 42 publications

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“…The same holds for the Ca 2+ -dependent protease, calpain-1 (51k), which cleaves many cytoskeletal components and controls integrin closure in procoagulant platelets. 164 Another remarkable fact is that 2 of the highly expressed signaling proteins play a role in PKA-dependent inhibition of platelets, that is, the adenylyl cyclase-associated protein CAP1 (42k) and VASP, which is the major substrate of this cAMP-dependent protein kinase. 165 Although earlier data with mice indicated that the prostacyclin receptor (a key cAMPelevating receptor on platelets) is not required for normal hemostasis, 166 the abundance of these proteins supports a quantitatively important role of cAMP signaling in the control of platelet functions (see also above).…”

Section: What Can Quantitative Proteomics Tell Us About Platelet Biolmentioning

confidence: 99%

What Can Proteomics Tell Us About Platelets?

Burkhart

Gambaryan

Watson

et al. 2014

Circulation Research

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105

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More than 130 years ago, it was recognized that platelets are key mediators of hemostasis. Nowadays, it is established that platelets participate in additional physiological processes and contribute to the genesis and progression of cardiovascular diseases. Recent data indicate that the platelet proteome, defined as the complete set of expressed proteins, comprises >5000 proteins and is highly similar between different healthy individuals. Owing to their anucleate nature, platelets have limited protein synthesis. By implication, in patients experiencing platelet disorders, platelet (dys)function is almost completely attributable to alterations in protein expression and dynamic differences in post-translational modifications. Modern platelet proteomics approaches can reveal (1) quantitative changes in the abundance of thousands of proteins, (2) post-translational modifications, (3) protein–protein interactions, and (4) protein localization, while requiring only small blood donations in the range of a few milliliters. Consequently, platelet proteomics will represent an invaluable tool for characterizing the fundamental processes that affect platelet homeostasis and thus determine the roles of platelets in health and disease. In this article we provide a critical overview on the achievements, the current possibilities, and the future perspectives of platelet proteomics to study patients experiencing cardiovascular, inflammatory, and bleeding disorders.

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Section: What Can Quantitative Proteomics Tell Us About Platelet Biolmentioning

confidence: 99%

What Can Proteomics Tell Us About Platelets?

Burkhart

Gambaryan

Watson

et al. 2014

Circulation Research

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105

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“…After staining for 5 min, samples were analyzed with a FACScan flow cytometer (BD Accuri Cytometer). 6 In a separate set of experiments, reconstituted PRP was activated with tissue factor (2 pM) and CaCl 2 (16.7 mM) in the presence of GPRP (2 mM) at 37°C, after which samples were taken for fluorescent labeling. Analysis was by flow cytometry as described above.…”

Section: Flow Cytometric Platelet Analysesmentioning

confidence: 99%

“…1,2 In a growing thrombus, aggregated and procoagulant platelets form two distinct populations, 3,4 which is at least partly explained by the high Ca 2+ response required for PS exposure and coagulation factor binding, and by the calpain-dependent closure of active α IIb b 3 integrins after PS exposure, thus antagonizing inclusion of procoagulant platelets into a platelet aggregate. 5,6 However, another platelet population has also been identified, usually referred to as coated platelets, 7 which may partly overlap with the two other platelet populations described above. 3 In the initial paper, Dale et al describe COAT platelets (later renamed as coated platelets) as a population of platelets arising after combined stimulation with collagen and thrombin, which bind α-granule proteins, including factor V, fibrinogen, von Willebrand factor, thrombospondin, fibronectin and α 2 -antiplasmin, in a transglutaminase-dependent way via the formation of covalent serotonin conjugations.…”

mentioning

confidence: 99%

Coated platelets function in platelet-dependent fibrin formation via integrin α _IIb β ₃ and transglutaminase factor XIII

et al. 2015

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C oated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin α IIb b 3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin α IIb b 3 . Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin α IIb b 3 . Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α IIb b 3 (Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α IIb b 3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin α IIb b 3 .

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“…In agreement with our previous studies,13 almost all resting and T+C‐ stimulated PS− platelets had intact inner mitochondrial membrane potential, while the majority of PS+ platelets had depolarized mitochondria. PAC1 binding was decreased in PS+ platelets compared with T+C (PS−) platelets, confirming that integrin αIIbβ3 is in a resting conformation on procoagulant platelets, and differentiating this procoagulant platelet subpopulation from pro‐aggregatory platelets 18, 23…”

Section: Discussionmentioning

confidence: 75%

“…Flow cytometric analysis of PS‐exposing platelets in suspension using fluorescently‐labeled annexin A5, first described over two decades ago,20 has become widely used in studies of procoagulant platelets and platelet‐derived extracellular vesicles (EVs) 4, 21, 22, 23, 24, 25. A limitation of traditional flow cytometry is the lack of images of individual analyzed cells.…”

Section: Introductionmentioning

confidence: 99%

Analysis of procoagulant phosphatidylserine‐exposing platelets by imaging flow cytometry

Reddy

Wang

Christensen

et al. 2018

Research and Practice in Thrombosis and Haemostasis

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BackgroundUpon platelet activation, a subpopulation of procoagulant platelets is formed, characterized by the exposure of the anionic aminophospholipid phosphatidylserine (PS) on the surface membrane.ObjectiveTo evaluate procoagulant PS‐exposing platelets by imaging flow cytometry.MethodsPlatelet ultrastructure was examined by transmission electron microscopy, and a comprehensive analysis of procoagulant platelets was performed using imaging flow cytometry; platelets were fluorescently labeled for the markers glycoprotein (GP)IX, activated integrin αIIbβ3, CD62P, and PS exposure.ResultsA subpopulation of platelets stimulated in suspension by the physiological agonists thrombin+collagen, and all platelets stimulated by the calcium ionophore A23187, had a distinct round morphology. These platelets were PS‐exposing, larger in size, had an increased circularity index, and had reduced internal complexity compared with non‐PS‐exposing platelets. They expressed CD62P and αIIbβ3 in an inactive conformation on the surface, and demonstrated depolarized inner mitochondrial membranes. For the first time, using imaging flow cytometry, a large proportion of PS‐exposing platelets possessing platelet‐associated extracellular vesicles (EVs) was observed, which demonstrated heterogeneous platelet marker expression that was different from free released EVs.ConclusionsInnovative imaging flow cytometry allowed detailed fluorescence‐based, quantitative morphometric analysis of PS‐exposing platelets; in becoming procoagulant, platelets undergo remarkable morphological changes, transforming into spherical “balloons,” almost devoid of their normal internal architecture. Almost all PS‐exposing platelets have associated EVs that are not detectable by traditional flow cytometry. While their functions have yet to be fully elucidated, the heterogeneity of platelet‐associated and released EVs suggests that they may contribute to different aspects of hemostasis and of thrombosis.

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Dual Mechanism of Integrin αIIbβ3 Closure in Procoagulant Platelets

Cited by 104 publications

References 42 publications

What Can Proteomics Tell Us About Platelets?

What Can Proteomics Tell Us About Platelets?

Coated platelets function in platelet-dependent fibrin formation via integrin α _IIb β ₃ and transglutaminase factor XIII

Analysis of procoagulant phosphatidylserine‐exposing platelets by imaging flow cytometry

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Dual Mechanism of Integrin αIIbβ3 Closure in Procoagulant Platelets

Cited by 104 publications

References 42 publications

What Can Proteomics Tell Us About Platelets?

What Can Proteomics Tell Us About Platelets?

Coated platelets function in platelet-dependent fibrin formation via integrin α IIb β 3 and transglutaminase factor XIII

Analysis of procoagulant phosphatidylserine‐exposing platelets by imaging flow cytometry

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Product

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Coated platelets function in platelet-dependent fibrin formation via integrin α _IIb β ₃ and transglutaminase factor XIII