Flow cytometric detection of activated mouse integrin αIIbβ3 with a novel monoclonal antibody

Bergmeier, Wolfgang; Schulte, Valerie; Brockhoff, Gero; Bier, Ulrich; Zirngibl, Hubert; Nieswandt, Bernhard

doi:10.1002/cyto.10114

Cited by 137 publications

(127 citation statements)

References 18 publications

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“…In parallel, activation of the main platelet integrin, ␣ IIb ␤ 3 , was assessed. 35 Surprisingly, and in contrast to studies in other cell types, we found that P-selectin expression was not decreased but markedly increased in Cdc42 Ϫ/Ϫ compared with wild-type platelets in response to all tested strong agonists ( Figure 4A; bottom panel). In line with this, release of PF4, a protein specifically stored in platelet ␣ granules, was also moderately but significantly increased in Cdc42 Ϫ/Ϫ platelets upon activation compared with the control (Figure 4B).…”

Section: Increased Secretion In Cdc42 ؊/؊ Plateletscontrasting

confidence: 49%

Multiple alterations of platelet functions dominated by increased secretion in mice lacking Cdc42 in platelets

Pleines

Eckly

Elvers

et al. 2010

Blood

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Platelet activation at sites of vascular injury is crucial for hemostasis, but it may also cause myocardial infarction or stroke. Cytoskeletal reorganization is essential for platelet activation and secretion. The small GTPase Cdc42 has been implicated as an important mediator of filopodia formation and exocytosis in various cell types, but its exact function in platelets is not established. Here, we show that the megakaryocyte/platelet-specific loss of Cdc42 leads to mild thrombocytopenia and a small increase in platelet size in mice. Unexpectedly, Cdc42-deficient platelets were able to form normally shaped filopodia and spread fully on fibrinogen upon activation, whereas filopodia formation upon selective induction of GPIb signaling was reduced compared with wild-type platelets. Furthermore, Cdc42-deficient platelets showed enhanced secretion of ␣ granules, a higher adenosine diphosphate (ADP)/adenosine triphosphate (ATP) content, increased aggregation at low agonist concentrations, and enhanced aggregate formation on collagen under flow. In vivo, lack of Cdc42 resulted in faster occlusion of ferric chloride-injured arterioles. The life span of Cdc42-deficient platelets was markedly reduced, suggesting increased clearing of the cells under physiologic conditions. These data point to novel multiple functions of Cdc42 in the regulation of platelet activation, granule organization, degranulation, and a specific role in GPIb signaling. (Blood. 2010;115(16): 3364-3373) IntroductionAt sites of tissue trauma, platelets become activated and rapidly aggregate to form a plug that seals the wound and limits blood loss. On the other hand, platelet activation in pathologic situations can lead to thrombosis, causing myocardial infarction or stroke. Platelet activation by multiple signaling pathways leads to shape change, release of intracellularly stored granules, and spreading on immobilized ligands. Small GTPases of the Rho family, namely RhoA, Cdc42, and Rac1, are thought to play important roles in the cytoskeletal rearrangements occurring during platelet activation by facilitating the formation of stress fibers, filopodia and lamellipodia, respectively. 1 In platelets, signaling from G protein-coupled receptors, such as the thromboxane or thrombin receptors, as well as immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors (GPVI, CLEC-2) was shown to induce activation of Rho GTPases. 2,3 Cdc42 is a small (ϳ 23 kDa) protein that cycles between a GDP-bound inactive and a GTP-bound active state. 4 Cdc42 is an important mediator of filopodia formation in various cell types. According to the "convergent elongation model," active Cdc42 induces activation of Wiskott-Aldrich Syndrome protein (WASP). WASP subsequently activates the ARP2/3 complex, thereby increasing actin turnover and initiating the formation of parallel actin bundles. [5][6][7] Furthermore, Cdc42 can also bind to and activate IRSp53, which recruits the Ena/vasodilator-stimulated phosphoprotein (VASP) family protein Mena, thus promoting filopodi...

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Section: Increased Secretion In Cdc42 ؊/؊ Plateletscontrasting

confidence: 49%

Multiple alterations of platelet functions dominated by increased secretion in mice lacking Cdc42 in platelets

Pleines

Eckly

Elvers

et al. 2010

Blood

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116

126

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“…We used the JON/A antibody to study the agonist-induced signaling. This antibody reacts selectively with the activated conformer of αIIbβ3 (Bergmeier et al, 2002). We did not observe any significant differences between wild-type and Fyn -/-mice with respect to JON/A binding that was induced by thrombin or two different doses of ADP (Table 1).…”

Section: Characterization Of Fyn -/-Micementioning

confidence: 68%

Analysis of Fyn function in hemostasis and αIIbβ3-integrin signaling

Reddy

Smith

Plow

2008

Journal of Cell Science

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Recent studies have shown that Src-family kinases (SFKs) play an important role in mediating integrin signalling, and the β3 subunit of αIIbβ3 integrin has been shown to interact with multiple SFK members. Here, we analyzed the interactions and functional consequences of Fyn and Src binding to αIIbβ3. Fyn associated with the β3 subunit in resting and thrombin-aggregated platelets, whereas interaction between Src and αIIbβ3 was seen predominantly in resting but not in thrombin-aggregated platelets. We have also observed that Fyn but not Src localized to focal adhesions in CHO cells adherent to fibrinogen through αIIbβ3. On the basis of these differences, we wanted to determine the sequence requirements for the interaction of Fyn and Src within the β3-cytoplasmic domain. Whereas Src association required the C-terminal region of β3, Fyn continued to interact with mutants that could no longer associate with Src and that contained as few as 13 membrane-proximal amino acids of the β3-cytoplasmic tail. Using deletion mutants of β3-cytoplasmic tails expressed as GST-fusion proteins, we narrowed down the Fyn-binding site even further to the amino acid residues 721-725 (IHDRK) of the β3-cytoplasmic domain. On the basis of these observations, we explored whether Fyn–/– mice exhibited any abnormalities in hemostasis and platelet function. We found that Fyn–/– mice significantly differed in their second bleeding times compared with wild-type mice, and platelets from Fyn–/– mice exhibited delayed spreading on fibrinogen-coated surfaces. Using mutant forms of Fyn, it appears that its kinase activity is required for its localization to focal adhesions and to mediate αIIbβ3-dependent cell spreading. Our results suggest that Fyn and Src have distinct requirements for interaction with αIIbβ3; and, consequently, the two SFK can mediate different functional responses.

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“…Moreover, a strong reduction in platelet-mediated adhesion of B16 melanoma cells was achieved after preincubation of murine platelets with a blocking anti-CD61 (␤3 integrin) mAb or an anti-GPIIb/ IIIa Ab (JON/A) (Fig. 3, B and C) (37). To exclude that reduced melanoma cell adhesion was caused by a reduction in the number of present platelets after incubation with RGD or antiGPIIb/IIIa Ab, the number of platelets in the well was analyzed and showed no difference between control and inhibitory protein (supplemental Fig.…”

Section: Resultsmentioning

confidence: 98%

Engagement of αIIbβ3 (GPIIb/IIIa) with ανβ3 Integrin Mediates Interaction of Melanoma Cells with Platelets

Lonsdorf

Kramer

Fahrleitner

et al. 2012

Journal of Biological Chemistry

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102

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A mutual relationship exists between metastasizing tumor cells and components of the coagulation cascade. The exact mechanisms as to how platelets influence blood-borne metastasis, however, remain poorly understood. Here, we used murine B16 melanoma cells to observe functional aspects of how platelets contribute to the process of hematogenous metastasis. We found that platelets interfere with a distinct step of the metastasis cascade, as they promote adhesion of melanoma cells to the endothelium in vitro under shear conditions. Constitutively active platelet receptor GPIIb/IIIa (integrin ␣IIb␤3) expressed on Chinese hamster ovary cells promoted melanoma cell adhesion in the presence of fibrinogen, whereas blocking antibodies to a␤3 integrin on melanoma cells or to GPIIb/IIIa significantly reduced melanoma cell adhesion to platelets. Furthermore, using intravital microscopy, we observed functional platelet-melanoma cell interactions, as platelet depletion resulted in significantly reduced melanoma cell adhesion to the injured vascular wall in vivo. Using a mouse model of hematogenous metastasis to the lung, we observed decreased metastasis of B16 melanoma cells to the lung by treatment with a mAb blocking the a subunit of a␤3 integrin. This effect was significantly reduced when platelets were depleted in vivo. Thus, the engagement of GPIIb/IIIa with a␤3 integrin interaction mediates tumor cell-platelet interactions and highlights how this interaction is involved in hematogenous tumor metastasis.

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Flow cytometric detection of activated mouse integrin αIIbβ3 with a novel monoclonal antibody

Cited by 137 publications

References 18 publications

Multiple alterations of platelet functions dominated by increased secretion in mice lacking Cdc42 in platelets

Multiple alterations of platelet functions dominated by increased secretion in mice lacking Cdc42 in platelets

Analysis of Fyn function in hemostasis and αIIbβ3-integrin signaling

Engagement of αIIbβ3 (GPIIb/IIIa) with ανβ3 Integrin Mediates Interaction of Melanoma Cells with Platelets

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