Arterial thrombosis: relevance of a model with two levels of severity assessed by histologic, ultrastructural and functional characterization

Hechler, Béatrice; Nonne, Christelle; Eckly, Anita; Magnenat, Stéphanie; Rinckel, Jean‐Yves; Denis, Cécile V.; Freund, Monique; Cazenave, Jean‐Pierre; Lanza, François; Gachet, Christian

doi:10.1111/j.1538-7836.2009.03666.x

Cited by 77 publications

(91 citation statements)

References 36 publications

(70 reference statements)

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“…Therefore, this treatment is considered a reasonable model of thrombosis (43,44). Although other models often damage endothelial cells but do not cause them to detach from the vessel wall to expose collagen, recent studies, published after these studies were completed, from Hechler et al (45) have demonstrated that it is possible to denude the endothelium by laser injury if properly applied. In WT mice, we found that the time to occlusion time is 6.1 Ϯ 2.2 min, which is reasonably consistent with other data (46).…”

Section: Discussionmentioning

confidence: 99%

Cbl-b Is a Novel Physiologic Regulator of Glycoprotein VI-dependent Platelet Activation

Daniel

Dangelmaier

Mada

et al. 2010

Journal of Biological Chemistry

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Platelet activation by newly exposed basement membrane collagen is a key initial step in both hemostasis and thrombosis (1). The complex of glycoprotein VI (GPVI) 2 and the Fc receptor ␥-chain is primarily responsible for activation of platelets by collagen (2). This receptor acts via a PLC␥2-dependent pathway, leading to generation of autocoids that recruit additional platelets to the site of injury. Signaling downstream of the GPVI/Fc receptor ␥ chain is similar to signaling initiated by activation of immune receptors on T-and B-lymphocytes. The Fc receptor ␥-chain contains an immunoreceptor tyrosine activation motif. Binding of collagen to the receptor is thought to cluster the receptors, which initiates the phosphorylation of the immunoreceptor tyrosine activation motif tyrosines by an Src family kinase. Similar to signaling through other immune receptors, Syk tyrosine kinase binds to the phosphorylated immunoreceptor tyrosine activation motif to start a cascade of phosphorylations and protein interactions resulting in the activation of PLC␥2.Hematopoietic cells express two members of the Cbl family of E3 ligases namely, c-Cbl and Cbl-b (3-5). The Cbl family proteins have been shown to be key regulators of intracellular signaling in immune cells (6, 7). As E3 ligases, Cbl proteins can catalyze the transfer of ubiquitin molecules to their substrates. A primary role for ubiquitylation is targeting the substrates of E3 ligases to the proteasome, where the substrates are proteolytically degraded. Because of this activity, Cbl family members have come to be considered as negative regulators of signaling. In platelets, c-Cbl promotes ubiquitylation of Syk and appears to be a negative regulator of platelet activation. Murine platelets deficient in c-Cbl show enhanced platelet aggregation in response to convulxin, a GPVI agonist (8, 9). Furthermore, we have shown that Syk is not ubiquitylated in c-Cbl Ϫ/Ϫ platelets, in contrast to normal platelets (9).The presence of Cbl-b in platelets and any role it might have in signaling have not been investigated. Because Cbl-b is also an E3 ligase, one might expect that it would also be a negative regulator of cell function. However, several reports have indicated that Cbl-b may play a positive role in both T cell and B cell signaling. Cbl-b interacts with Zap-70, leading to activation of T-cell-specific transcription factor NF-AT (10). Cbl-b Ϫ/Ϫ DT40 B cells display reduced PLC␥2 activation and Ca 2ϩ mobilization upon B-cell receptor stimulation, and the overexpression of Cbl-b results in an enhanced agonist-dependent Ca 2ϩ mobilization (11). These results may be explained by another function of the Cbl molecules, namely that they have several domains in addition to those responsible for the E3 ligase activity, which, acting as a scaffold, can bind to a variety of signaling molecules, bringing them into close proximity (6,12,13).Given the importance of Cbl-b to regulation of signaling in lymphocytes, we have investigated whether Cbl-b regulates platelet activation downstream ...

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Section: Discussionmentioning

confidence: 99%

Cbl-b Is a Novel Physiologic Regulator of Glycoprotein VI-dependent Platelet Activation

Daniel

Dangelmaier

Mada

et al. 2010

Journal of Biological Chemistry

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“…10 To be active fully, Akt requires phosphorylation on Thr 308 by PDK1 21 and Ser 9 in GSK3a and b, respectively) by Akt following platelet stimulation. GSK3b, the most abundant isoform, 40 has been shown to regulate negatively platelet functions 22 possibly by downregulating a IIb b 3 outside-in signaling, at least in vitro under ADP stimulation. 33 Here we show that pharmacologic inhibition of GSK3 rescues thrombus stabilization of PI3Kb-null platelet or AZD6482-treated human platelets under high shear rate.…”

Section: Discussionmentioning

confidence: 99%

“…To analyze the role of platelet PI3Kb in arterial thrombus stability in vivo, we performed a laser beam-induced local deep lesion of mesenteric arterioles 22 and monitored platelet thrombus formation by intravital microscopy. WT platelets formed a thrombus at the site of injury (supplemental Video 3; Figure 4A) that was either massive (thrombus size .28 000 mm 2 ) in 51.5% of the cases, strongly reducing the lumen of the vessel, or smaller (thrombus size ,28 000 mm 2 ) in 48.5% of the cases.…”

Section: Detachment Of Platelet Emboli From the Thrombus Formed On Dementioning

confidence: 99%

Platelet PI3Kβ and GSK3 regulate thrombus stability at a high shear rate

Laurent

Séverin

Hechler

et al. 2015

Blood

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Key Points• A key role for platelet PI3Kb, but not a, and for GSK3 in thrombus stability at a high shear rate.• Risk of platelet emboli formation on PI3Kb inhibition in vivo.Class IA phosphoinositide 3-kinase b (PI3Kb) is considered a potential drug target in arterial thrombosis, which is a major cause of death worldwide. Here we show that a striking phenotype of mice with selective p110b deletion in the megakaryocyte lineage is thrombus instability at a high shear rate, which is an effect that is not detected in the absence of p110a in platelets. The high shear rate-dependent thrombus instability in the absence of p110b is observed both ex vivo and in vivo with the formation of platelet emboli. Moreover, PI3Kb is required for the recruitment of new platelets to a growing thrombus when a pathological high shear is applied. Treatment of human blood with AZD6482, a selective PI3Kb inhibitor, phenocopies p110b deletion in mouse platelets, which highlights the role of the kinase activity of p110b. Within the growing platelet thrombus, p110b inactivation impairs the activating phosphorylations of Akt and the inhibitory phosphorylation of GSK3. In accord with these data, pharmacologic inhibition of GSK3 restores thrombus stability. Thus, platelet PI3Kb is not essential for thrombus growth and stability at normal arterial shear but has a specific and critical role in maintaining the integrity of the formed thrombus on elevation of shear rate, suggesting a potential risk of embolization on treatment with PI3Kb inhibitors. (Blood. 2015;125(5):881-888)

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“…As previously shown, laser-induced thrombosis in the cremaster microcirculation represents a model of localized, thrombin-driven thrombosis. 27,28 When subjected to this model, CalDAG-GEFI Ϫ/Ϫ mice showed decreased platelet accumulation compared with WT mice in both arterioles (Figure 2A-B) and to a lesser extent in venules ( Figure 2D-E). Thrombi in venules of CalDAG-GEFI Ϫ/Ϫ mice grew slowly but remained stably anchored to the vessel wall.…”

Section: Thrombotic Profile Of Caldag-gefi ؊/؊ and Clopidogrel-treatementioning

confidence: 99%

The kinetics of αIIbβ3 activation determines the size and stability of thrombi in mice: implications for antiplatelet therapy

Stolla

Stefanini

Roden

et al. 2011

Blood

115

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Two major pathways contribute to Rasproximate-1-mediated integrin activation in stimulated platelets. Calcium and diacyglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI, Ras-GRP2) mediates the rapid but reversible activation of integrin ␣IIb␤3, while the adenosine diphosphate receptor P2Y12, the target for antiplatelet drugs like clopidogrel, facilitates delayed but sustained integrin activation. To establish CalDAG-GEFI as a target for antiplatelet therapy, we compared how each pathway contributes to thrombosis and hemostasis in mice. Ex vivo, thrombus formation at arterial or venous shear rates was markedly reduced in CalDAG-GEFI ؊/؊ blood, even in the presence of exogenous adenosine diphosphate and thromboxane A 2 . In vivo, thrombosis was virtually abolished in arterioles and arteries of CalDAG-GEFI ؊/؊ mice, while small, hemostatically active thrombi formed in venules. Specific deletion of the C1-like domain of CalDAG-GEFI in circulating platelets also led to protection from thrombus formation at arterial flow conditions, while it only marginally increased blood loss in mice. In comparison, thrombi in the micro-and macrovasculature of clopidogrel-treated wild-type mice grew rapidly and frequently embolized but were hemostatically inactive. Together, these data suggest that inhibition of the catalytic or the C1 regulatory domain in CalDAG-GEFI will provide strong protection from atherothrombotic complications while maintaining a better safety profile than P2Y12 inhibitors like clopidogrel. (Blood. 2011; 117(3):1005-1013) IntroductionArterial thrombosis in the coronary or cerebrovascular circulation is the principal pathological process underlying acute coronary syndrome and ischemic stroke, which together represent the leading cause of morbidity and mortality in industrialized countries. 1 Platelet activation is a central event in the pathogenesis of arterial thrombosis. Currently, the most powerful antiplatelet agents used in the clinic are inhibitors of cyclooxygenase-1 (acetylsalicylic acid, aspirin), the platelet adenosine diphosphate (ADP) receptor P2Y12 (eg, clopiodgrel or Plavix), and integrin ␣IIb␤3 (eg, abciximab or Reopro). 2,3 These agents have all been shown to improve clinical outcomes in large-scale randomized controlled trials. However, all therapies have limitations that include uncertainty about optimal dosing, questions about resistance, and issues regarding the lack of reversibility in situations where bleeding risks are high.␣IIb␤3, the platelet fibrinogen receptor, is the best-studied member of the integrin family. 4,5 Like most integrins, especially those regulating adhesion and trafficking of blood cells, it is expressed in a low-affinity state on resting platelets. Engagement of agonist receptors on the platelet surface triggers intracellular signaling events, which lead to inside-out activation of ␣IIb␤3. Deficiency in ␣IIb␤3 completely inhibits the ability of platelets to aggregate and adhere to sites of injury. 6,7 Consequently, inhibitors to integrin ␣IIb␤3 show...

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Arterial thrombosis: relevance of a model with two levels of severity assessed by histologic, ultrastructural and functional characterization

Abstract: thrombosis: relevance of a model with two levels of severity assessed by histologic, ultrastructural and functional characterization. J Thromb Haemost 2010; 8: 173-84.

Cited by 77 publications

References 36 publications

Cbl-b Is a Novel Physiologic Regulator of Glycoprotein VI-dependent Platelet Activation

Cbl-b Is a Novel Physiologic Regulator of Glycoprotein VI-dependent Platelet Activation

Platelet PI3Kβ and GSK3 regulate thrombus stability at a high shear rate

The kinetics of αIIbβ3 activation determines the size and stability of thrombi in mice: implications for antiplatelet therapy

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