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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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Platelets from JAQ1-treated mice do not respond to CRP and collagen. (a) Two-color flow cytometric analysis of platelets from JAQ1-treated or control mice 3 d after Ab injection. Diluted whole blood was stimulated with 10 μM ADP or 10 μg/ml CRP for 2 min and subsequently incubated with antifibrinogenFITC and anti–P-selectinPE Abs for 10 min at RT and analyzed directly. Platelets were gated by FSC/SSC characteristics and Fl3 intensity (anti–mouse GPIb αPE/Cy5). The data shown are representative of six mice per group. Similar results were obtained on days 7 and 14 after Ab injection. (b) Heparinized prp from the indicated mice was stimulated with collagen (50 μg/ml), ADP (10 μM), or PMA (50 ng/ml). Light transmission was recorded on a Fibrintimer 4 channel aggregometer. (c) Heparinized prp from control mice was incubated with stirring in the presence of irrelevant rat IgG2a (20 μg/ml; ○) or JAQ1 (20 μg/ml; ▿) for 5 min before the addition of the indicated concentrations of collagen. In parallel, prp from JAQ1-treated mice was tested (□). Results are expressed as the maximum (max.) platelet aggregation ± SD for groups of each six mice.
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Figure 2: Platelets from JAQ1-treated mice do not respond to CRP and collagen. (a) Two-color flow cytometric analysis of platelets from JAQ1-treated or control mice 3 d after Ab injection. Diluted whole blood was stimulated with 10 μM ADP or 10 μg/ml CRP for 2 min and subsequently incubated with antifibrinogenFITC and anti–P-selectinPE Abs for 10 min at RT and analyzed directly. Platelets were gated by FSC/SSC characteristics and Fl3 intensity (anti–mouse GPIb αPE/Cy5). The data shown are representative of six mice per group. Similar results were obtained on days 7 and 14 after Ab injection. (b) Heparinized prp from the indicated mice was stimulated with collagen (50 μg/ml), ADP (10 μM), or PMA (50 ng/ml). Light transmission was recorded on a Fibrintimer 4 channel aggregometer. (c) Heparinized prp from control mice was incubated with stirring in the presence of irrelevant rat IgG2a (20 μg/ml; ○) or JAQ1 (20 μg/ml; ▿) for 5 min before the addition of the indicated concentrations of collagen. In parallel, prp from JAQ1-treated mice was tested (□). Results are expressed as the maximum (max.) platelet aggregation ± SD for groups of each six mice.

Mentions: The effect of JAQ1 on circulating platelets was studied ex vivo at different time points after Ab injection. The basal surface expression of the major GP receptors GPIIb/IIIa and GPIb-IX-V, CD9 and integrin α2β1 was unchanged compared with control platelets at 3, 7, and 14 d after Ab injection (Table ). At no time after Ab injection did circulating platelets show any signs of activation, as demonstrated by the lack of surface bound fibrinogen and surface-expressed P-selectin (Table ). On days 3, 7, and 14, platelets from JAQ1-treated mice were resistant towards activation with the CRP (up to 30 μg/ml), which is known to be a strong GPVI-specific platelet agonist (31; Fig. 2 a). In contrast, ADP induced normal activation (fibrinogen binding) of these platelets. Furthermore, platelets from JAQ1-treated mice were completely resistant to activation with collagen at concentrations of up to 50 μg/ml ex vivo, and this profound inhibitory effect also lasted for at least 14 d upon a single injection of 100 μg JAQ1 (Fig. 2 b). In contrast to collagen, ADP and PMA induced normal aggregation of these platelets, indicating that JAQ1 specifically blocked GPVI-dependent platelet activation pathways whereas other functions were not affected. In vitro, saturating concentrations of JAQ1 (20 μg/ml) only displayed a limited inhibitory effect on collagen-induced platelet aggregation which could be overcome when collagen concentrations higher than ∼7 μg/ml were used (Fig. 2 c), confirming earlier results 29.


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)

Platelets from JAQ1-treated mice do not respond to CRP and collagen. (a) Two-color flow cytometric analysis of platelets from JAQ1-treated or control mice 3 d after Ab injection. Diluted whole blood was stimulated with 10 μM ADP or 10 μg/ml CRP for 2 min and subsequently incubated with antifibrinogenFITC and anti–P-selectinPE Abs for 10 min at RT and analyzed directly. Platelets were gated by FSC/SSC characteristics and Fl3 intensity (anti–mouse GPIb αPE/Cy5). The data shown are representative of six mice per group. Similar results were obtained on days 7 and 14 after Ab injection. (b) Heparinized prp from the indicated mice was stimulated with collagen (50 μg/ml), ADP (10 μM), or PMA (50 ng/ml). Light transmission was recorded on a Fibrintimer 4 channel aggregometer. (c) Heparinized prp from control mice was incubated with stirring in the presence of irrelevant rat IgG2a (20 μg/ml; ○) or JAQ1 (20 μg/ml; ▿) for 5 min before the addition of the indicated concentrations of collagen. In parallel, prp from JAQ1-treated mice was tested (□). Results are expressed as the maximum (max.) platelet aggregation ± SD for groups of each six mice.
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Related In: Results  -  Collection

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Figure 2: Platelets from JAQ1-treated mice do not respond to CRP and collagen. (a) Two-color flow cytometric analysis of platelets from JAQ1-treated or control mice 3 d after Ab injection. Diluted whole blood was stimulated with 10 μM ADP or 10 μg/ml CRP for 2 min and subsequently incubated with antifibrinogenFITC and anti–P-selectinPE Abs for 10 min at RT and analyzed directly. Platelets were gated by FSC/SSC characteristics and Fl3 intensity (anti–mouse GPIb αPE/Cy5). The data shown are representative of six mice per group. Similar results were obtained on days 7 and 14 after Ab injection. (b) Heparinized prp from the indicated mice was stimulated with collagen (50 μg/ml), ADP (10 μM), or PMA (50 ng/ml). Light transmission was recorded on a Fibrintimer 4 channel aggregometer. (c) Heparinized prp from control mice was incubated with stirring in the presence of irrelevant rat IgG2a (20 μg/ml; ○) or JAQ1 (20 μg/ml; ▿) for 5 min before the addition of the indicated concentrations of collagen. In parallel, prp from JAQ1-treated mice was tested (□). Results are expressed as the maximum (max.) platelet aggregation ± SD for groups of each six mice.
Mentions: The effect of JAQ1 on circulating platelets was studied ex vivo at different time points after Ab injection. The basal surface expression of the major GP receptors GPIIb/IIIa and GPIb-IX-V, CD9 and integrin α2β1 was unchanged compared with control platelets at 3, 7, and 14 d after Ab injection (Table ). At no time after Ab injection did circulating platelets show any signs of activation, as demonstrated by the lack of surface bound fibrinogen and surface-expressed P-selectin (Table ). On days 3, 7, and 14, platelets from JAQ1-treated mice were resistant towards activation with the CRP (up to 30 μg/ml), which is known to be a strong GPVI-specific platelet agonist (31; Fig. 2 a). In contrast, ADP induced normal activation (fibrinogen binding) of these platelets. Furthermore, platelets from JAQ1-treated mice were completely resistant to activation with collagen at concentrations of up to 50 μg/ml ex vivo, and this profound inhibitory effect also lasted for at least 14 d upon a single injection of 100 μg JAQ1 (Fig. 2 b). In contrast to collagen, ADP and PMA induced normal aggregation of these platelets, indicating that JAQ1 specifically blocked GPVI-dependent platelet activation pathways whereas other functions were not affected. In vitro, saturating concentrations of JAQ1 (20 μg/ml) only displayed a limited inhibitory effect on collagen-induced platelet aggregation which could be overcome when collagen concentrations higher than ∼7 μg/ml were used (Fig. 2 c), confirming earlier results 29.

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