Limits...
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.

Show MeSH

Related in: MedlinePlus

Reduced adhesion to collagen and abolished procoagulant response of GPVI-depleted platelets. (a) Platelets from JAQ1-treated mice (day 7) bind normal amounts of plasma vWF in the presence of botrocetin (2 μg/ml; solid line). Bound vWF was detected by FITC-labeled anti-vWF Abs (10 μg/ml). No binding was detected in the absence of botrocetin (shaded area). Normal activation of β1-integrins on platelets from JAQ1-treated mice in response to thrombin (0.1 U/ml). Resting (shaded area) or thrombin activated (solid line) platelets were incubated with FITC-labeled 9EG7 (5 μg/ml) for 15 min at RT and analyzed directly. (b) Washed platelets from control or JAQ1-treated mice (day 7) were incubated in collagen-coated microtiter plates in the presence or absence of MgCl2 (1 mM)/CaCl2 (1 mM) for the indicated times and adherent platelets were quantitated fluorimetrically. The data shown are from a single experiment, representative of five identical experiments and expressed as the mean of triplicate readings ± SD. (c) Flow cytometric analysis of annexin V-FITC binding to platelets from control and JAQ1-treated (day 7) mice activated with a combination of collagen (50 μg/ml) and thrombin (0.01 U/ml).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195902&req=5

Figure 6: Reduced adhesion to collagen and abolished procoagulant response of GPVI-depleted platelets. (a) Platelets from JAQ1-treated mice (day 7) bind normal amounts of plasma vWF in the presence of botrocetin (2 μg/ml; solid line). Bound vWF was detected by FITC-labeled anti-vWF Abs (10 μg/ml). No binding was detected in the absence of botrocetin (shaded area). Normal activation of β1-integrins on platelets from JAQ1-treated mice in response to thrombin (0.1 U/ml). Resting (shaded area) or thrombin activated (solid line) platelets were incubated with FITC-labeled 9EG7 (5 μg/ml) for 15 min at RT and analyzed directly. (b) Washed platelets from control or JAQ1-treated mice (day 7) were incubated in collagen-coated microtiter plates in the presence or absence of MgCl2 (1 mM)/CaCl2 (1 mM) for the indicated times and adherent platelets were quantitated fluorimetrically. The data shown are from a single experiment, representative of five identical experiments and expressed as the mean of triplicate readings ± SD. (c) Flow cytometric analysis of annexin V-FITC binding to platelets from control and JAQ1-treated (day 7) mice activated with a combination of collagen (50 μg/ml) and thrombin (0.01 U/ml).

Mentions: It is currently thought that GPVI is the platelet collagen receptor for activation, whereas integrin α2β1 and GPIb-V-IX (via vWF) mediate adhesion. As shown before (Table ), the basal surface expression of both receptors was not influenced by the JAQ1 treatment. Further experiments demonstrated that platelets from JAQ1-treated mice bound normal levels of vWF in the presence of botrocetin, and thrombin induced normal activation of β1-integrins, as assessed with the mAb 9EG7, which specifically recognizes the activated form of the β1 subunit (33; Fig. 6 a). In the next step, the adhesion of platelets from JAQ1-treated mice to collagen was tested in a static assay. As shown in Fig. 6 b, the adhesion of platelets from JAQ1-treated mice was strongly reduced as compared with control platelets and was abolished in the absence of extracellular free magnesium/calcium, strongly suggesting it to be mediated predominantly by integrin α2β1 34. It is well known that GPVI is critically involved in the procoagulant response of platelets where stimulated platelets expose negatively charged phosphatidylserine (PS) at the plasma membrane which facilitates thrombin generation 35. Indeed, platelets from JAQ1-treated mice did not expose PS in response to a combination of collagen and thrombin on day 3, 7, and 14 after Ab injection, as demonstrated by the lack of annexin V binding (Fig. 6 c).


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)

Reduced adhesion to collagen and abolished procoagulant response of GPVI-depleted platelets. (a) Platelets from JAQ1-treated mice (day 7) bind normal amounts of plasma vWF in the presence of botrocetin (2 μg/ml; solid line). Bound vWF was detected by FITC-labeled anti-vWF Abs (10 μg/ml). No binding was detected in the absence of botrocetin (shaded area). Normal activation of β1-integrins on platelets from JAQ1-treated mice in response to thrombin (0.1 U/ml). Resting (shaded area) or thrombin activated (solid line) platelets were incubated with FITC-labeled 9EG7 (5 μg/ml) for 15 min at RT and analyzed directly. (b) Washed platelets from control or JAQ1-treated mice (day 7) were incubated in collagen-coated microtiter plates in the presence or absence of MgCl2 (1 mM)/CaCl2 (1 mM) for the indicated times and adherent platelets were quantitated fluorimetrically. The data shown are from a single experiment, representative of five identical experiments and expressed as the mean of triplicate readings ± SD. (c) Flow cytometric analysis of annexin V-FITC binding to platelets from control and JAQ1-treated (day 7) mice activated with a combination of collagen (50 μg/ml) and thrombin (0.01 U/ml).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195902&req=5

Figure 6: Reduced adhesion to collagen and abolished procoagulant response of GPVI-depleted platelets. (a) Platelets from JAQ1-treated mice (day 7) bind normal amounts of plasma vWF in the presence of botrocetin (2 μg/ml; solid line). Bound vWF was detected by FITC-labeled anti-vWF Abs (10 μg/ml). No binding was detected in the absence of botrocetin (shaded area). Normal activation of β1-integrins on platelets from JAQ1-treated mice in response to thrombin (0.1 U/ml). Resting (shaded area) or thrombin activated (solid line) platelets were incubated with FITC-labeled 9EG7 (5 μg/ml) for 15 min at RT and analyzed directly. (b) Washed platelets from control or JAQ1-treated mice (day 7) were incubated in collagen-coated microtiter plates in the presence or absence of MgCl2 (1 mM)/CaCl2 (1 mM) for the indicated times and adherent platelets were quantitated fluorimetrically. The data shown are from a single experiment, representative of five identical experiments and expressed as the mean of triplicate readings ± SD. (c) Flow cytometric analysis of annexin V-FITC binding to platelets from control and JAQ1-treated (day 7) mice activated with a combination of collagen (50 μg/ml) and thrombin (0.01 U/ml).
Mentions: It is currently thought that GPVI is the platelet collagen receptor for activation, whereas integrin α2β1 and GPIb-V-IX (via vWF) mediate adhesion. As shown before (Table ), the basal surface expression of both receptors was not influenced by the JAQ1 treatment. Further experiments demonstrated that platelets from JAQ1-treated mice bound normal levels of vWF in the presence of botrocetin, and thrombin induced normal activation of β1-integrins, as assessed with the mAb 9EG7, which specifically recognizes the activated form of the β1 subunit (33; Fig. 6 a). In the next step, the adhesion of platelets from JAQ1-treated mice to collagen was tested in a static assay. As shown in Fig. 6 b, the adhesion of platelets from JAQ1-treated mice was strongly reduced as compared with control platelets and was abolished in the absence of extracellular free magnesium/calcium, strongly suggesting it to be mediated predominantly by integrin α2β1 34. It is well known that GPVI is critically involved in the procoagulant response of platelets where stimulated platelets expose negatively charged phosphatidylserine (PS) at the plasma membrane which facilitates thrombin generation 35. Indeed, platelets from JAQ1-treated mice did not expose PS in response to a combination of collagen and thrombin on day 3, 7, and 14 after Ab injection, as demonstrated by the lack of annexin V binding (Fig. 6 c).

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