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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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Related in: MedlinePlus

ICC requires integrin αIIbβ3 engagement. ICC is dependent on integrin αIIbβ3 engagement. Platelets at a cell density of 150 × 109/L were perfused over the surface of immobilized vWF (100 μg/ml) for 1 min, and an initial population of stationary adherent cells undergoing sustained calcium oscillations was established (indicated by the arrow). (A) A secondary platelet population at a density of 150 × 109/L was subsequently perfused over this reactive population, and platelet–platelet interactions were monitored in real time. (B) Platelets pretreated with 200 nM aggrastat (dotted marques) for 10 min were perfused over the preadherent population, and platelet–platelet interactions were monitored. (C) Platelets pretreated with 10 μg/ml of the α2β1-blocking IgG BHA2.1 for 10 min were perfused over the preadherent population, and platelet–platelet interactions were monitored. In comparison with control cells (A) and BHA2.1-treated cells (C), aggrastat treatment effectively inhibited the propagation of the initial calcium signal and prevented aggregate formation at the surface of the vWF matrix (B).
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fig2: ICC requires integrin αIIbβ3 engagement. ICC is dependent on integrin αIIbβ3 engagement. Platelets at a cell density of 150 × 109/L were perfused over the surface of immobilized vWF (100 μg/ml) for 1 min, and an initial population of stationary adherent cells undergoing sustained calcium oscillations was established (indicated by the arrow). (A) A secondary platelet population at a density of 150 × 109/L was subsequently perfused over this reactive population, and platelet–platelet interactions were monitored in real time. (B) Platelets pretreated with 200 nM aggrastat (dotted marques) for 10 min were perfused over the preadherent population, and platelet–platelet interactions were monitored. (C) Platelets pretreated with 10 μg/ml of the α2β1-blocking IgG BHA2.1 for 10 min were perfused over the preadherent population, and platelet–platelet interactions were monitored. In comparison with control cells (A) and BHA2.1-treated cells (C), aggrastat treatment effectively inhibited the propagation of the initial calcium signal and prevented aggregate formation at the surface of the vWF matrix (B).

Mentions: To examine platelet calcium dynamics and translocation behavior, calcium dye–loaded platelets were perfused over the surface of preformed thrombi generated on a type I fibrillar collagen. In the course of examining platelet calcium dynamics, we consistently noted that platelets undergoing sustained calcium oscillations at the thrombus surface could act as effective nuclei for the further recruitment of freely translocating platelets. As such, the stationary platelets appeared to communicate their “calcium activation status” to subsequent tethering platelets, a process we term ICC. As demonstrated in Fig. 1 A, an initially adherent cell undergoing sustained (S) calcium oscillations at the surface of a developing thrombus induced a rapid increase in calcium flux in its tethering (T) counterparts. Examination of platelet aggregation at the surface of immobilized vWf demonstrated that a similar pattern of ICC was also observed during platelet aggregate formation on this adhesive substrate (Fig. 1 B; see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200207119/DC1). Propagation of platelet calcium signaling via ICC within the local population of platelets at the surface of immobilized vWf ultimately resulted in the formation of hot spots for platelet aggregation, with small, relatively stable platelet aggregates (6–10 cells) undergoing sustained oscillatory calcium flux throughout the observation period (Fig. 1 B). Fig. 1 C demonstrates that of the platelets tethering to the surface of a primary adherent platelet, only 53% subsequently exhibit concomitant calcium signaling. Detailed examination of these platelet–platelet tethering interactions demonstrated that the platelet contact must occur within a narrow temporal window (≤0.6 s), at the point when the primary adherent cell is expressing peak Δ[Ca2+]c (Fig. 2 D). Thus, the efficiency by which the platelet activation status is propagated by ICC is not only dependent on platelet–platelet contacts per se, but also on the timing of tether formation with Δ[Ca2+]c maxima. These studies suggest a potentially important role for ICC in dynamically regulating platelet accrual onto the surface of developing thrombi.


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)

ICC requires integrin αIIbβ3 engagement. ICC is dependent on integrin αIIbβ3 engagement. Platelets at a cell density of 150 × 109/L were perfused over the surface of immobilized vWF (100 μg/ml) for 1 min, and an initial population of stationary adherent cells undergoing sustained calcium oscillations was established (indicated by the arrow). (A) A secondary platelet population at a density of 150 × 109/L was subsequently perfused over this reactive population, and platelet–platelet interactions were monitored in real time. (B) Platelets pretreated with 200 nM aggrastat (dotted marques) for 10 min were perfused over the preadherent population, and platelet–platelet interactions were monitored. (C) Platelets pretreated with 10 μg/ml of the α2β1-blocking IgG BHA2.1 for 10 min were perfused over the preadherent population, and platelet–platelet interactions were monitored. In comparison with control cells (A) and BHA2.1-treated cells (C), aggrastat treatment effectively inhibited the propagation of the initial calcium signal and prevented aggregate formation at the surface of the vWF matrix (B).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172771&req=5

fig2: ICC requires integrin αIIbβ3 engagement. ICC is dependent on integrin αIIbβ3 engagement. Platelets at a cell density of 150 × 109/L were perfused over the surface of immobilized vWF (100 μg/ml) for 1 min, and an initial population of stationary adherent cells undergoing sustained calcium oscillations was established (indicated by the arrow). (A) A secondary platelet population at a density of 150 × 109/L was subsequently perfused over this reactive population, and platelet–platelet interactions were monitored in real time. (B) Platelets pretreated with 200 nM aggrastat (dotted marques) for 10 min were perfused over the preadherent population, and platelet–platelet interactions were monitored. (C) Platelets pretreated with 10 μg/ml of the α2β1-blocking IgG BHA2.1 for 10 min were perfused over the preadherent population, and platelet–platelet interactions were monitored. In comparison with control cells (A) and BHA2.1-treated cells (C), aggrastat treatment effectively inhibited the propagation of the initial calcium signal and prevented aggregate formation at the surface of the vWF matrix (B).
Mentions: To examine platelet calcium dynamics and translocation behavior, calcium dye–loaded platelets were perfused over the surface of preformed thrombi generated on a type I fibrillar collagen. In the course of examining platelet calcium dynamics, we consistently noted that platelets undergoing sustained calcium oscillations at the thrombus surface could act as effective nuclei for the further recruitment of freely translocating platelets. As such, the stationary platelets appeared to communicate their “calcium activation status” to subsequent tethering platelets, a process we term ICC. As demonstrated in Fig. 1 A, an initially adherent cell undergoing sustained (S) calcium oscillations at the surface of a developing thrombus induced a rapid increase in calcium flux in its tethering (T) counterparts. Examination of platelet aggregation at the surface of immobilized vWf demonstrated that a similar pattern of ICC was also observed during platelet aggregate formation on this adhesive substrate (Fig. 1 B; see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200207119/DC1). Propagation of platelet calcium signaling via ICC within the local population of platelets at the surface of immobilized vWf ultimately resulted in the formation of hot spots for platelet aggregation, with small, relatively stable platelet aggregates (6–10 cells) undergoing sustained oscillatory calcium flux throughout the observation period (Fig. 1 B). Fig. 1 C demonstrates that of the platelets tethering to the surface of a primary adherent platelet, only 53% subsequently exhibit concomitant calcium signaling. Detailed examination of these platelet–platelet tethering interactions demonstrated that the platelet contact must occur within a narrow temporal window (≤0.6 s), at the point when the primary adherent cell is expressing peak Δ[Ca2+]c (Fig. 2 D). Thus, the efficiency by which the platelet activation status is propagated by ICC is not only dependent on platelet–platelet contacts per se, but also on the timing of tether formation with Δ[Ca2+]c maxima. These studies suggest a potentially important role for ICC in dynamically regulating platelet accrual onto the surface of developing thrombi.

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