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Long-term antithrombotic protection by in vivo depletion of platelet glycoprotein VI in mice.

Nieswandt B, Schulte V, Bergmeier W, Mokhtari-Nejad R, Rackebrandt K, Cazenave JP, Ohlmann P, Gachet C, Zirngibl H - J. Exp. Med. (2001)

Bottom Line: JAQ1-treated mice were completely protected for at least 2 wk against lethal thromboembolism induced by infusion of a mixture of collagen (0.8 mg/kg) and epinephrine (60 microg/ml).The tail bleeding times in JAQ1-treated mice were only moderately increased compared with control mice probably because the treatment did not affect platelet activation by other agonists such as adenosine diphosphate or phorbol myristate acetate.These results suggest that GPVI might become a target for long-term prophylaxis of ischemic cardiovascular diseases and provide the first evidence that it is possible to specifically deplete an activating glycoprotein receptor from circulating platelets in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, General Surgery, Witten/Herdecke University, 42117 Wuppertal, Germany. nieswand@klinikum-wuppertal.de

ABSTRACT
Coronary artery thrombosis is often initiated by abrupt disruption of the atherosclerotic plaque and activation of platelets on the subendothelial layers in the disrupted plaque. The extracellular matrix protein collagen is the most thrombogenic constituent of the subendothelial layer; therefore, a selective inhibition of the collagen activation pathway in platelets may provide strong antithrombotic protection while preserving other platelet functions. Here we demonstrate that treatment of mice with a monoclonal antibody against the activating platelet collagen receptor glycoprotein VI (GPVI; JAQ1) results in specific depletion of the receptor from circulating platelets and abolished responses of these cells to collagen and collagen-related peptides (CRPs). JAQ1-treated mice were completely protected for at least 2 wk against lethal thromboembolism induced by infusion of a mixture of collagen (0.8 mg/kg) and epinephrine (60 microg/ml). The tail bleeding times in JAQ1-treated mice were only moderately increased compared with control mice probably because the treatment did not affect platelet activation by other agonists such as adenosine diphosphate or phorbol myristate acetate. These results suggest that GPVI might become a target for long-term prophylaxis of ischemic cardiovascular diseases and provide the first evidence that it is possible to specifically deplete an activating glycoprotein receptor from circulating platelets in vivo.

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JAQ1 induces internalization of GPVI in vivo Fc independently. Mice were injected with 100 μg biotinylated JAQ1 or DOM1 (anti-GPV) and platelets were analyzed at the indicated time points. (a) Flow cytometric analysis of platelets from JAQ1-treated or control mice. Diluted control blood was incubated with biotinylated rat IgG2a (10 μg/ml; control [ctrl]) or JAQ1 (10 μg/ml; in vitro) for 6 h at 37°C. Subsequently, these samples and samples from JAQ1-treated mice (ex vivo) were incubated with FITC-labeled JAQ1 (5 μg/ml), streptavidin (Strep; 5 μg/ml), or convulxin (Cvx; 5 μg/ml) for 15 min at RT and analyzed directly. Platelets were gated by FSC/SSC characteristics and Fl2 intensity (anti–mouse GPIIb/IIIaPE). (b) Detection of surface-bound biotinylated DOM1 (anti-GPV) ex vivo. The staining was performed as described for biotinylated JAQ1. Results in panels a and b are expressed as mean log Fl1 ± SD (n = 6). (c) Top: whole platelet proteins were separated by SDS-PAGE under reducing conditions and biotinylated JAQ1 was detected with HRP-labeled streptavidin/ECL. For detection of GPVI and GPIIIa, the proteins were separated under nonreducing conditions and immunoblotted with FITC-labeled JAQ1 or EDL1 followed by HRP-labeled rabbit anti-FITC/ECL. (d) Mice were injected with 100 μg Fab fragments of JAQ1 and platelets were analyzed in a Western blot for the presence of GPVI and GPIIIa after 48 h. These platelets did not aggregate in response to collagen (50 μg/ml), CRP (30 μg/ml), or Cvx (10 μg/ml), whereas ADP (10 μM) induced normal aggregation.
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Figure 5: JAQ1 induces internalization of GPVI in vivo Fc independently. Mice were injected with 100 μg biotinylated JAQ1 or DOM1 (anti-GPV) and platelets were analyzed at the indicated time points. (a) Flow cytometric analysis of platelets from JAQ1-treated or control mice. Diluted control blood was incubated with biotinylated rat IgG2a (10 μg/ml; control [ctrl]) or JAQ1 (10 μg/ml; in vitro) for 6 h at 37°C. Subsequently, these samples and samples from JAQ1-treated mice (ex vivo) were incubated with FITC-labeled JAQ1 (5 μg/ml), streptavidin (Strep; 5 μg/ml), or convulxin (Cvx; 5 μg/ml) for 15 min at RT and analyzed directly. Platelets were gated by FSC/SSC characteristics and Fl2 intensity (anti–mouse GPIIb/IIIaPE). (b) Detection of surface-bound biotinylated DOM1 (anti-GPV) ex vivo. The staining was performed as described for biotinylated JAQ1. Results in panels a and b are expressed as mean log Fl1 ± SD (n = 6). (c) Top: whole platelet proteins were separated by SDS-PAGE under reducing conditions and biotinylated JAQ1 was detected with HRP-labeled streptavidin/ECL. For detection of GPVI and GPIIIa, the proteins were separated under nonreducing conditions and immunoblotted with FITC-labeled JAQ1 or EDL1 followed by HRP-labeled rabbit anti-FITC/ECL. (d) Mice were injected with 100 μg Fab fragments of JAQ1 and platelets were analyzed in a Western blot for the presence of GPVI and GPIIIa after 48 h. These platelets did not aggregate in response to collagen (50 μg/ml), CRP (30 μg/ml), or Cvx (10 μg/ml), whereas ADP (10 μM) induced normal aggregation.

Mentions: To examine the mechanisms underlying the loss of GPVI, mice were injected with biotinylated JAQ1 and the amount of surface-bound mAb was determined flow cytometrically ex vivo at early time points after injection. Interestingly, as soon as 6 h after injection only very low levels of surface-bound JAQ1 were detectable and the signals further decreased to control after 24 and 48 h (Fig. 5 a), whereas JAQ1FITC and CvxFITC bound to the platelets at no time point. These data suggested that the JAQ1–GPVI complex had been cleared from the surface of those platelets within 6 h. In contrast, platelets from mice injected with a biotinylated mAb against GPV 24 constantly yielded positive staining with FITC-labeled streptavidin (Fig. 5 b). In the next step, we tested whole cell lysates from platelets of JAQ1-treated mice for the presence of GPVI and the biotinylated mAb. As shown in Fig. 5 c, JAQ1 was strongly detectable in platelets 6 h after injection, whereas signals markedly decreased at 24 h and even more at 48 h. A similar picture was found for GPVI, strongly suggesting that the JAQ1–GPVI complex had become internalized and was degraded within 2 d. In contrast to its in vivo effects, JAQ1 did not induce any detectable downregulation of surface GPVI within 6 h incubation at 37°C on washed platelets or in whole blood (heparinized or citrated), indicating that a second signal may be required to induce this effect and that this signal is absent in vitro (Fig. 5 a).


Long-term antithrombotic protection by in vivo depletion of platelet glycoprotein VI in mice.

Nieswandt B, Schulte V, Bergmeier W, Mokhtari-Nejad R, Rackebrandt K, Cazenave JP, Ohlmann P, Gachet C, Zirngibl H - J. Exp. Med. (2001)

JAQ1 induces internalization of GPVI in vivo Fc independently. Mice were injected with 100 μg biotinylated JAQ1 or DOM1 (anti-GPV) and platelets were analyzed at the indicated time points. (a) Flow cytometric analysis of platelets from JAQ1-treated or control mice. Diluted control blood was incubated with biotinylated rat IgG2a (10 μg/ml; control [ctrl]) or JAQ1 (10 μg/ml; in vitro) for 6 h at 37°C. Subsequently, these samples and samples from JAQ1-treated mice (ex vivo) were incubated with FITC-labeled JAQ1 (5 μg/ml), streptavidin (Strep; 5 μg/ml), or convulxin (Cvx; 5 μg/ml) for 15 min at RT and analyzed directly. Platelets were gated by FSC/SSC characteristics and Fl2 intensity (anti–mouse GPIIb/IIIaPE). (b) Detection of surface-bound biotinylated DOM1 (anti-GPV) ex vivo. The staining was performed as described for biotinylated JAQ1. Results in panels a and b are expressed as mean log Fl1 ± SD (n = 6). (c) Top: whole platelet proteins were separated by SDS-PAGE under reducing conditions and biotinylated JAQ1 was detected with HRP-labeled streptavidin/ECL. For detection of GPVI and GPIIIa, the proteins were separated under nonreducing conditions and immunoblotted with FITC-labeled JAQ1 or EDL1 followed by HRP-labeled rabbit anti-FITC/ECL. (d) Mice were injected with 100 μg Fab fragments of JAQ1 and platelets were analyzed in a Western blot for the presence of GPVI and GPIIIa after 48 h. These platelets did not aggregate in response to collagen (50 μg/ml), CRP (30 μg/ml), or Cvx (10 μg/ml), whereas ADP (10 μM) induced normal aggregation.
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Related In: Results  -  Collection

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Figure 5: JAQ1 induces internalization of GPVI in vivo Fc independently. Mice were injected with 100 μg biotinylated JAQ1 or DOM1 (anti-GPV) and platelets were analyzed at the indicated time points. (a) Flow cytometric analysis of platelets from JAQ1-treated or control mice. Diluted control blood was incubated with biotinylated rat IgG2a (10 μg/ml; control [ctrl]) or JAQ1 (10 μg/ml; in vitro) for 6 h at 37°C. Subsequently, these samples and samples from JAQ1-treated mice (ex vivo) were incubated with FITC-labeled JAQ1 (5 μg/ml), streptavidin (Strep; 5 μg/ml), or convulxin (Cvx; 5 μg/ml) for 15 min at RT and analyzed directly. Platelets were gated by FSC/SSC characteristics and Fl2 intensity (anti–mouse GPIIb/IIIaPE). (b) Detection of surface-bound biotinylated DOM1 (anti-GPV) ex vivo. The staining was performed as described for biotinylated JAQ1. Results in panels a and b are expressed as mean log Fl1 ± SD (n = 6). (c) Top: whole platelet proteins were separated by SDS-PAGE under reducing conditions and biotinylated JAQ1 was detected with HRP-labeled streptavidin/ECL. For detection of GPVI and GPIIIa, the proteins were separated under nonreducing conditions and immunoblotted with FITC-labeled JAQ1 or EDL1 followed by HRP-labeled rabbit anti-FITC/ECL. (d) Mice were injected with 100 μg Fab fragments of JAQ1 and platelets were analyzed in a Western blot for the presence of GPVI and GPIIIa after 48 h. These platelets did not aggregate in response to collagen (50 μg/ml), CRP (30 μg/ml), or Cvx (10 μg/ml), whereas ADP (10 μM) induced normal aggregation.
Mentions: To examine the mechanisms underlying the loss of GPVI, mice were injected with biotinylated JAQ1 and the amount of surface-bound mAb was determined flow cytometrically ex vivo at early time points after injection. Interestingly, as soon as 6 h after injection only very low levels of surface-bound JAQ1 were detectable and the signals further decreased to control after 24 and 48 h (Fig. 5 a), whereas JAQ1FITC and CvxFITC bound to the platelets at no time point. These data suggested that the JAQ1–GPVI complex had been cleared from the surface of those platelets within 6 h. In contrast, platelets from mice injected with a biotinylated mAb against GPV 24 constantly yielded positive staining with FITC-labeled streptavidin (Fig. 5 b). In the next step, we tested whole cell lysates from platelets of JAQ1-treated mice for the presence of GPVI and the biotinylated mAb. As shown in Fig. 5 c, JAQ1 was strongly detectable in platelets 6 h after injection, whereas signals markedly decreased at 24 h and even more at 48 h. A similar picture was found for GPVI, strongly suggesting that the JAQ1–GPVI complex had become internalized and was degraded within 2 d. In contrast to its in vivo effects, JAQ1 did not induce any detectable downregulation of surface GPVI within 6 h incubation at 37°C on washed platelets or in whole blood (heparinized or citrated), indicating that a second signal may be required to induce this effect and that this signal is absent in vitro (Fig. 5 a).

Bottom Line: JAQ1-treated mice were completely protected for at least 2 wk against lethal thromboembolism induced by infusion of a mixture of collagen (0.8 mg/kg) and epinephrine (60 microg/ml).The tail bleeding times in JAQ1-treated mice were only moderately increased compared with control mice probably because the treatment did not affect platelet activation by other agonists such as adenosine diphosphate or phorbol myristate acetate.These results suggest that GPVI might become a target for long-term prophylaxis of ischemic cardiovascular diseases and provide the first evidence that it is possible to specifically deplete an activating glycoprotein receptor from circulating platelets in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, General Surgery, Witten/Herdecke University, 42117 Wuppertal, Germany. nieswand@klinikum-wuppertal.de

ABSTRACT
Coronary artery thrombosis is often initiated by abrupt disruption of the atherosclerotic plaque and activation of platelets on the subendothelial layers in the disrupted plaque. The extracellular matrix protein collagen is the most thrombogenic constituent of the subendothelial layer; therefore, a selective inhibition of the collagen activation pathway in platelets may provide strong antithrombotic protection while preserving other platelet functions. Here we demonstrate that treatment of mice with a monoclonal antibody against the activating platelet collagen receptor glycoprotein VI (GPVI; JAQ1) results in specific depletion of the receptor from circulating platelets and abolished responses of these cells to collagen and collagen-related peptides (CRPs). JAQ1-treated mice were completely protected for at least 2 wk against lethal thromboembolism induced by infusion of a mixture of collagen (0.8 mg/kg) and epinephrine (60 microg/ml). The tail bleeding times in JAQ1-treated mice were only moderately increased compared with control mice probably because the treatment did not affect platelet activation by other agonists such as adenosine diphosphate or phorbol myristate acetate. These results suggest that GPVI might become a target for long-term prophylaxis of ischemic cardiovascular diseases and provide the first evidence that it is possible to specifically deplete an activating glycoprotein receptor from circulating platelets in vivo.

Show MeSH
Related in: MedlinePlus