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Intercellular calcium communication regulates platelet aggregation and thrombus growth.

Nesbitt WS, Giuliano S, Kulkarni S, Dopheide SM, Harper IS, Jackson SP - J. Cell Biol. (2003)

Bottom Line: In this study, we have examined the mechanisms regulating cytosolic calcium flux during the development of platelet-platelet adhesion contacts under the influence of flow.We demonstrate that ICC is primarily mediated by a signaling mechanism operating between integrin alpha IIb beta 3 and the recently cloned ADP purinergic receptor P2Y12.Furthermore, we demonstrate that the efficiency by which calcium signals are propagated within platelet aggregates plays an important role in dictating the rate and extent of thrombus growth.

View Article: PubMed Central - PubMed

Affiliation: Australian Centre for Blood Diseases, Department of Medicine, Monash University, Box Hill Hospital, Victoria 3128, Australia.

ABSTRACT
The ability of platelets to form stable adhesion contacts with other activated platelets (platelet cohesion or aggregation) at sites of vascular injury is essential for hemostasis and thrombosis. In this study, we have examined the mechanisms regulating cytosolic calcium flux during the development of platelet-platelet adhesion contacts under the influence of flow. An examination of platelet calcium flux during platelet aggregate formation in vitro demonstrated a key role for intercellular calcium communication (ICC) in regulating the recruitment of translocating platelets into developing aggregates. We demonstrate that ICC is primarily mediated by a signaling mechanism operating between integrin alpha IIb beta 3 and the recently cloned ADP purinergic receptor P2Y12. Furthermore, we demonstrate that the efficiency by which calcium signals are propagated within platelet aggregates plays an important role in dictating the rate and extent of thrombus growth.

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Sustained calcium flux is dependent on cooperative signaling between integrin αIIbβ3 and ADP secretion. Isolated platelets reconstituted in Tyrode's buffer with RBCs (50% hematocrit) were perfused through vWf-coated (100 μg/ml) microslides at a shear rate of 1,800 s−1 and subsequently chased by a bolus of 12.5 μM ADP. (A) The percentage (mean ± SEM of three experiments) of the total adherent platelet population forming irreversible stationary adhesion in the presence or absence of 10 μM ADP and/or c7E3. (B) Representative single-platelet calcium flux recordings and concomitant displacement versus time graphs showing platelet behavior at the surface of immobilized vWf. Note that the platelets arrest almost immediately upon ADP addition, concomitant with the onset of oscillatory calcium flux. (C) Representative single-platelet calcium flux recordings and concomitant displacement versus time graphs showing platelet behavior upon ADP addition after pretreatment with the anti-αIIbβ3 blocking c7E3 Fab (20 μg/ml). The shaded boxes indicate the platelet behavior before ADP stimulation. The arrow indicates the point of ADP addition. Note that ADP-dependent oscillatory calcium flux is completely inhibited by c7E3 Fab treatment.
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fig4: Sustained calcium flux is dependent on cooperative signaling between integrin αIIbβ3 and ADP secretion. Isolated platelets reconstituted in Tyrode's buffer with RBCs (50% hematocrit) were perfused through vWf-coated (100 μg/ml) microslides at a shear rate of 1,800 s−1 and subsequently chased by a bolus of 12.5 μM ADP. (A) The percentage (mean ± SEM of three experiments) of the total adherent platelet population forming irreversible stationary adhesion in the presence or absence of 10 μM ADP and/or c7E3. (B) Representative single-platelet calcium flux recordings and concomitant displacement versus time graphs showing platelet behavior at the surface of immobilized vWf. Note that the platelets arrest almost immediately upon ADP addition, concomitant with the onset of oscillatory calcium flux. (C) Representative single-platelet calcium flux recordings and concomitant displacement versus time graphs showing platelet behavior upon ADP addition after pretreatment with the anti-αIIbβ3 blocking c7E3 Fab (20 μg/ml). The shaded boxes indicate the platelet behavior before ADP stimulation. The arrow indicates the point of ADP addition. Note that ADP-dependent oscillatory calcium flux is completely inhibited by c7E3 Fab treatment.

Mentions: To gain further insight into the mechanism by which ADP and integrin αIIbβ3 cooperate to drive cytosolic calcium flux in aggregating platelets, we examined the effect of exogenous ADP addition on cytosolic calcium flux during platelet adhesion to vWf. As demonstrated in Fig. 4 A, in the absence of added ADP, <6% of tethered platelets formed stationary adhesion contacts and elicited a sustained (>30 s) calcium response on a vWf matrix (1,800 s−1). We have previously demonstrated that this oscillatory calcium flux is dependent on integrin αIIbβ3 outside-in signaling, as it is completely inhibited by c7E3 Fab, but not by ADP and TXA2 antagonists (Nesbitt et al., 2002). Exposure of platelets to soluble ADP during surface translocation on vWf resulted in a dramatic increase in the proportion of platelets forming stable adhesion contacts with the vWf surface (>90% of platelets), with all stably adherent platelets exhibiting sustained calcium oscillations (Fig. 4 B). The potentiating effect of ADP on the calcium response of translocating platelets was dependent on integrin αIIbβ3, as it was completely prevented by blocking ligand binding with aggrastat (Fig. 4 C). These studies demonstrate an important cooperative signaling role for ADP and integrin αIIbβ3, in which the latter receptor is essential for sustained cytosolic calcium flux.


Intercellular calcium communication regulates platelet aggregation and thrombus growth.

Nesbitt WS, Giuliano S, Kulkarni S, Dopheide SM, Harper IS, Jackson SP - J. Cell Biol. (2003)

Sustained calcium flux is dependent on cooperative signaling between integrin αIIbβ3 and ADP secretion. Isolated platelets reconstituted in Tyrode's buffer with RBCs (50% hematocrit) were perfused through vWf-coated (100 μg/ml) microslides at a shear rate of 1,800 s−1 and subsequently chased by a bolus of 12.5 μM ADP. (A) The percentage (mean ± SEM of three experiments) of the total adherent platelet population forming irreversible stationary adhesion in the presence or absence of 10 μM ADP and/or c7E3. (B) Representative single-platelet calcium flux recordings and concomitant displacement versus time graphs showing platelet behavior at the surface of immobilized vWf. Note that the platelets arrest almost immediately upon ADP addition, concomitant with the onset of oscillatory calcium flux. (C) Representative single-platelet calcium flux recordings and concomitant displacement versus time graphs showing platelet behavior upon ADP addition after pretreatment with the anti-αIIbβ3 blocking c7E3 Fab (20 μg/ml). The shaded boxes indicate the platelet behavior before ADP stimulation. The arrow indicates the point of ADP addition. Note that ADP-dependent oscillatory calcium flux is completely inhibited by c7E3 Fab treatment.
© Copyright Policy
Related In: Results  -  Collection

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fig4: Sustained calcium flux is dependent on cooperative signaling between integrin αIIbβ3 and ADP secretion. Isolated platelets reconstituted in Tyrode's buffer with RBCs (50% hematocrit) were perfused through vWf-coated (100 μg/ml) microslides at a shear rate of 1,800 s−1 and subsequently chased by a bolus of 12.5 μM ADP. (A) The percentage (mean ± SEM of three experiments) of the total adherent platelet population forming irreversible stationary adhesion in the presence or absence of 10 μM ADP and/or c7E3. (B) Representative single-platelet calcium flux recordings and concomitant displacement versus time graphs showing platelet behavior at the surface of immobilized vWf. Note that the platelets arrest almost immediately upon ADP addition, concomitant with the onset of oscillatory calcium flux. (C) Representative single-platelet calcium flux recordings and concomitant displacement versus time graphs showing platelet behavior upon ADP addition after pretreatment with the anti-αIIbβ3 blocking c7E3 Fab (20 μg/ml). The shaded boxes indicate the platelet behavior before ADP stimulation. The arrow indicates the point of ADP addition. Note that ADP-dependent oscillatory calcium flux is completely inhibited by c7E3 Fab treatment.
Mentions: To gain further insight into the mechanism by which ADP and integrin αIIbβ3 cooperate to drive cytosolic calcium flux in aggregating platelets, we examined the effect of exogenous ADP addition on cytosolic calcium flux during platelet adhesion to vWf. As demonstrated in Fig. 4 A, in the absence of added ADP, <6% of tethered platelets formed stationary adhesion contacts and elicited a sustained (>30 s) calcium response on a vWf matrix (1,800 s−1). We have previously demonstrated that this oscillatory calcium flux is dependent on integrin αIIbβ3 outside-in signaling, as it is completely inhibited by c7E3 Fab, but not by ADP and TXA2 antagonists (Nesbitt et al., 2002). Exposure of platelets to soluble ADP during surface translocation on vWf resulted in a dramatic increase in the proportion of platelets forming stable adhesion contacts with the vWf surface (>90% of platelets), with all stably adherent platelets exhibiting sustained calcium oscillations (Fig. 4 B). The potentiating effect of ADP on the calcium response of translocating platelets was dependent on integrin αIIbβ3, as it was completely prevented by blocking ligand binding with aggrastat (Fig. 4 C). These studies demonstrate an important cooperative signaling role for ADP and integrin αIIbβ3, in which the latter receptor is essential for sustained cytosolic calcium flux.

Bottom Line: In this study, we have examined the mechanisms regulating cytosolic calcium flux during the development of platelet-platelet adhesion contacts under the influence of flow.We demonstrate that ICC is primarily mediated by a signaling mechanism operating between integrin alpha IIb beta 3 and the recently cloned ADP purinergic receptor P2Y12.Furthermore, we demonstrate that the efficiency by which calcium signals are propagated within platelet aggregates plays an important role in dictating the rate and extent of thrombus growth.

View Article: PubMed Central - PubMed

Affiliation: Australian Centre for Blood Diseases, Department of Medicine, Monash University, Box Hill Hospital, Victoria 3128, Australia.

ABSTRACT
The ability of platelets to form stable adhesion contacts with other activated platelets (platelet cohesion or aggregation) at sites of vascular injury is essential for hemostasis and thrombosis. In this study, we have examined the mechanisms regulating cytosolic calcium flux during the development of platelet-platelet adhesion contacts under the influence of flow. An examination of platelet calcium flux during platelet aggregate formation in vitro demonstrated a key role for intercellular calcium communication (ICC) in regulating the recruitment of translocating platelets into developing aggregates. We demonstrate that ICC is primarily mediated by a signaling mechanism operating between integrin alpha IIb beta 3 and the recently cloned ADP purinergic receptor P2Y12. Furthermore, we demonstrate that the efficiency by which calcium signals are propagated within platelet aggregates plays an important role in dictating the rate and extent of thrombus growth.

Show MeSH
Related in: MedlinePlus