Limits...
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.

Show MeSH

Related in: MedlinePlus

Intercellular calcium communication. Isolated platelets reconstituted in Tyrode's buffer and RBCs (50% hematocrit) were perfused over the surface of preformed thrombi at the surface of type I fibrillar collagen (2.5 mg/ml)– or vWf (100 μg/ml)-coated microcapillary slides at a shear rate of 1,800 s−1. (A) Single-channel Oregon green fluorescence images demonstrating ICC during platelet–platelet collisions on the surface of preformed thrombi in vitro at a platelet count of 150 × 109/L. Associated single-cell calcium flux recordings demonstrating the duration and amplitude of the calcium response in an initially adherent cell (S) and a tethering cell (T) at the surface of immobilized vWF at a shear rate of 1,800 s−1. The arrow indicates the point at which platelet–platelet interactions and propagation of the calcium signal first occur (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200207119/DC1). Δ[Ca2+]c recordings are representative of three independent experiments. (B) Oregon green fluorescence images demonstrating intraplatelet calcium flux during aggregate formation on the surface of immobilized vWf at a platelet count of 150 × 109/L. S, stationary adherent cell; T, tethering cell. Associated single-cell calcium flux recordings demonstrating the duration and amplitude of the calcium response in an initially adherent cell (S) and a tethering cell (T) at the surface of immobilized vWF at a shear rate of 1,800 s−1. The arrow indicates the point at which platelet–platelet interactions and propagation of the calcium signal first occur. Δ[Ca2+]c recordings are representative of four independent experiments. (C) The percentage of platelets tethering to an initially adherent platelet and undergoing concomitant calcium oscillations (ICC) was quantified at time points where the primary adherent cell was either expressing maximal cytosolic calcium levels (High Δ[Ca2+]c) or minimal cytosolic calcium levels (Low Δ[Ca2+]c). (D) Single-channel Oregon green fluorescence images showing the association between platelet–platelet collisions and intracellular calcium flux (Δ[Ca2+]c). Platelet–platelet contact occurs when the primary adherent cell (arrow) is expressing low intracellular calcium (21.0 s), resulting in no ICC and aggregation. The arrow indicates an initial transiently adherent cell; the dotted mark denotes a tethering cell.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172771&req=5

fig1: Intercellular calcium communication. Isolated platelets reconstituted in Tyrode's buffer and RBCs (50% hematocrit) were perfused over the surface of preformed thrombi at the surface of type I fibrillar collagen (2.5 mg/ml)– or vWf (100 μg/ml)-coated microcapillary slides at a shear rate of 1,800 s−1. (A) Single-channel Oregon green fluorescence images demonstrating ICC during platelet–platelet collisions on the surface of preformed thrombi in vitro at a platelet count of 150 × 109/L. Associated single-cell calcium flux recordings demonstrating the duration and amplitude of the calcium response in an initially adherent cell (S) and a tethering cell (T) at the surface of immobilized vWF at a shear rate of 1,800 s−1. The arrow indicates the point at which platelet–platelet interactions and propagation of the calcium signal first occur (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200207119/DC1). Δ[Ca2+]c recordings are representative of three independent experiments. (B) Oregon green fluorescence images demonstrating intraplatelet calcium flux during aggregate formation on the surface of immobilized vWf at a platelet count of 150 × 109/L. S, stationary adherent cell; T, tethering cell. Associated single-cell calcium flux recordings demonstrating the duration and amplitude of the calcium response in an initially adherent cell (S) and a tethering cell (T) at the surface of immobilized vWF at a shear rate of 1,800 s−1. The arrow indicates the point at which platelet–platelet interactions and propagation of the calcium signal first occur. Δ[Ca2+]c recordings are representative of four independent experiments. (C) The percentage of platelets tethering to an initially adherent platelet and undergoing concomitant calcium oscillations (ICC) was quantified at time points where the primary adherent cell was either expressing maximal cytosolic calcium levels (High Δ[Ca2+]c) or minimal cytosolic calcium levels (Low Δ[Ca2+]c). (D) Single-channel Oregon green fluorescence images showing the association between platelet–platelet collisions and intracellular calcium flux (Δ[Ca2+]c). Platelet–platelet contact occurs when the primary adherent cell (arrow) is expressing low intracellular calcium (21.0 s), resulting in no ICC and aggregation. The arrow indicates an initial transiently adherent cell; the dotted mark denotes a tethering cell.

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)

Intercellular calcium communication. Isolated platelets reconstituted in Tyrode's buffer and RBCs (50% hematocrit) were perfused over the surface of preformed thrombi at the surface of type I fibrillar collagen (2.5 mg/ml)– or vWf (100 μg/ml)-coated microcapillary slides at a shear rate of 1,800 s−1. (A) Single-channel Oregon green fluorescence images demonstrating ICC during platelet–platelet collisions on the surface of preformed thrombi in vitro at a platelet count of 150 × 109/L. Associated single-cell calcium flux recordings demonstrating the duration and amplitude of the calcium response in an initially adherent cell (S) and a tethering cell (T) at the surface of immobilized vWF at a shear rate of 1,800 s−1. The arrow indicates the point at which platelet–platelet interactions and propagation of the calcium signal first occur (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200207119/DC1). Δ[Ca2+]c recordings are representative of three independent experiments. (B) Oregon green fluorescence images demonstrating intraplatelet calcium flux during aggregate formation on the surface of immobilized vWf at a platelet count of 150 × 109/L. S, stationary adherent cell; T, tethering cell. Associated single-cell calcium flux recordings demonstrating the duration and amplitude of the calcium response in an initially adherent cell (S) and a tethering cell (T) at the surface of immobilized vWF at a shear rate of 1,800 s−1. The arrow indicates the point at which platelet–platelet interactions and propagation of the calcium signal first occur. Δ[Ca2+]c recordings are representative of four independent experiments. (C) The percentage of platelets tethering to an initially adherent platelet and undergoing concomitant calcium oscillations (ICC) was quantified at time points where the primary adherent cell was either expressing maximal cytosolic calcium levels (High Δ[Ca2+]c) or minimal cytosolic calcium levels (Low Δ[Ca2+]c). (D) Single-channel Oregon green fluorescence images showing the association between platelet–platelet collisions and intracellular calcium flux (Δ[Ca2+]c). Platelet–platelet contact occurs when the primary adherent cell (arrow) is expressing low intracellular calcium (21.0 s), resulting in no ICC and aggregation. The arrow indicates an initial transiently adherent cell; the dotted mark denotes a tethering cell.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Intercellular calcium communication. Isolated platelets reconstituted in Tyrode's buffer and RBCs (50% hematocrit) were perfused over the surface of preformed thrombi at the surface of type I fibrillar collagen (2.5 mg/ml)– or vWf (100 μg/ml)-coated microcapillary slides at a shear rate of 1,800 s−1. (A) Single-channel Oregon green fluorescence images demonstrating ICC during platelet–platelet collisions on the surface of preformed thrombi in vitro at a platelet count of 150 × 109/L. Associated single-cell calcium flux recordings demonstrating the duration and amplitude of the calcium response in an initially adherent cell (S) and a tethering cell (T) at the surface of immobilized vWF at a shear rate of 1,800 s−1. The arrow indicates the point at which platelet–platelet interactions and propagation of the calcium signal first occur (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200207119/DC1). Δ[Ca2+]c recordings are representative of three independent experiments. (B) Oregon green fluorescence images demonstrating intraplatelet calcium flux during aggregate formation on the surface of immobilized vWf at a platelet count of 150 × 109/L. S, stationary adherent cell; T, tethering cell. Associated single-cell calcium flux recordings demonstrating the duration and amplitude of the calcium response in an initially adherent cell (S) and a tethering cell (T) at the surface of immobilized vWF at a shear rate of 1,800 s−1. The arrow indicates the point at which platelet–platelet interactions and propagation of the calcium signal first occur. Δ[Ca2+]c recordings are representative of four independent experiments. (C) The percentage of platelets tethering to an initially adherent platelet and undergoing concomitant calcium oscillations (ICC) was quantified at time points where the primary adherent cell was either expressing maximal cytosolic calcium levels (High Δ[Ca2+]c) or minimal cytosolic calcium levels (Low Δ[Ca2+]c). (D) Single-channel Oregon green fluorescence images showing the association between platelet–platelet collisions and intracellular calcium flux (Δ[Ca2+]c). Platelet–platelet contact occurs when the primary adherent cell (arrow) is expressing low intracellular calcium (21.0 s), resulting in no ICC and aggregation. The arrow indicates an initial transiently adherent cell; the dotted mark denotes a tethering cell.
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