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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

NP-EGTA uncaging triggers ICC events and platelet aggregation. Isolated platelets loaded with 10 μM NP-EGTA reconstituted in Tyrode's buffer with RBCs (50%) were perfused through vWf (100 μg/ml)-coated microcapillary tubes at a shear rate of 1,800 s−1. (A) Oregon green fluorescence images and real-time calcium flux recording demonstrating NP-EGTA–elicited ICC at a shear rate of 1,800 s−1. The gray box demarcates the point at which the secondary platelet tethers to the stationary adherent cell (→) activated by NP-EGTA uncaging. (B) Control experiments in which NP-EGTA–loaded platelets were allowed to tether and translocate across the vWf matrix (−1.2 and −0.6 s). The confocal field was subsequently exposed to a near-UV light source (350 nm ↑UV) for 0.6 s, leading to NP-EGTA uncaging and release of intracellular calcium (0 s). The zoomed boxes show the occurrence of platelet aggregation and ongoing ICC post-UV. The dashed circle demarks small aggregates forming on the vWf matrix. (C) NP-EGTA flow experiment conducted in the presence of 1.5 U/ml apyrase. The zoomed boxes demonstrate that in the presence of apyrase, ICC post-UV stimulation does not occur with the platelets in the field of view resuming translocation after a short-duration stationary adhesion.
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fig6: NP-EGTA uncaging triggers ICC events and platelet aggregation. Isolated platelets loaded with 10 μM NP-EGTA reconstituted in Tyrode's buffer with RBCs (50%) were perfused through vWf (100 μg/ml)-coated microcapillary tubes at a shear rate of 1,800 s−1. (A) Oregon green fluorescence images and real-time calcium flux recording demonstrating NP-EGTA–elicited ICC at a shear rate of 1,800 s−1. The gray box demarcates the point at which the secondary platelet tethers to the stationary adherent cell (→) activated by NP-EGTA uncaging. (B) Control experiments in which NP-EGTA–loaded platelets were allowed to tether and translocate across the vWf matrix (−1.2 and −0.6 s). The confocal field was subsequently exposed to a near-UV light source (350 nm ↑UV) for 0.6 s, leading to NP-EGTA uncaging and release of intracellular calcium (0 s). The zoomed boxes show the occurrence of platelet aggregation and ongoing ICC post-UV. The dashed circle demarks small aggregates forming on the vWf matrix. (C) NP-EGTA flow experiment conducted in the presence of 1.5 U/ml apyrase. The zoomed boxes demonstrate that in the presence of apyrase, ICC post-UV stimulation does not occur with the platelets in the field of view resuming translocation after a short-duration stationary adhesion.

Mentions: Finally, to provide more direct evidence that changes in cytosolic calcium flux in the primary adherent layer of platelets impacts on the efficiency of platelet aggregation, we examined the effect of inducing a single transient calcium spike in translocating platelets. This was achieved by loading platelets with the caged calcium chelator NP-EGTA, which upon UV exposure results in a rapid, transient increase in cytosolic calcium (Nesbitt et al., 2002). A significant advantage of this experimental approach is that it enables rapid release of calcium in a discrete population of primary adherent platelets only, immediately before the formation of platelet–platelet adhesion contacts. This maximizes the possibility of coordinating the initiation of calcium flux with subsequent ICC and, furthermore, minimizes potential experimental artifacts associated with platelet preadhesion, such as exhausted ADP release. To investigate the effects of calcium uncaging on platelet aggregate formation, NP-EGTA–loaded platelets were perfused through vWf-coated microcapillary tubes at physiological platelet concentrations (150 × 109/L) to increase the probability of platelet–platelet adhesion contact formation. Similar to our previous findings (Nesbitt et al., 2002), eliciting a transient calcium spike in platelets that were not interacting with other translocating platelets resulted in temporary arrest of these cells (unpublished data). In contrast, induction of a single calcium spike at the point of contact between two translocating platelets resulted in prolonged arrest of both cells, leading to the induction of a sustained oscillatory calcium flux. Moreover, these cells formed efficient nuclei for the recruitment of free-flowing platelets leading to ICC and the rapid formation of platelet aggregates (Fig. 6, A and B). Platelet aggregate formation and ICC under these experimental conditions were dependent on released ADP, as they were completely inhibited by pretreating the platelets with apyrase (Fig. 6 C) or a combination of both A3P5PS and AR-C6991MX (unpublished data). These findings provide further support for the concept that communication of calcium signals between platelets is important in regulating the efficiency of platelet aggregation under flow.


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)

NP-EGTA uncaging triggers ICC events and platelet aggregation. Isolated platelets loaded with 10 μM NP-EGTA reconstituted in Tyrode's buffer with RBCs (50%) were perfused through vWf (100 μg/ml)-coated microcapillary tubes at a shear rate of 1,800 s−1. (A) Oregon green fluorescence images and real-time calcium flux recording demonstrating NP-EGTA–elicited ICC at a shear rate of 1,800 s−1. The gray box demarcates the point at which the secondary platelet tethers to the stationary adherent cell (→) activated by NP-EGTA uncaging. (B) Control experiments in which NP-EGTA–loaded platelets were allowed to tether and translocate across the vWf matrix (−1.2 and −0.6 s). The confocal field was subsequently exposed to a near-UV light source (350 nm ↑UV) for 0.6 s, leading to NP-EGTA uncaging and release of intracellular calcium (0 s). The zoomed boxes show the occurrence of platelet aggregation and ongoing ICC post-UV. The dashed circle demarks small aggregates forming on the vWf matrix. (C) NP-EGTA flow experiment conducted in the presence of 1.5 U/ml apyrase. The zoomed boxes demonstrate that in the presence of apyrase, ICC post-UV stimulation does not occur with the platelets in the field of view resuming translocation after a short-duration stationary adhesion.
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Related In: Results  -  Collection

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fig6: NP-EGTA uncaging triggers ICC events and platelet aggregation. Isolated platelets loaded with 10 μM NP-EGTA reconstituted in Tyrode's buffer with RBCs (50%) were perfused through vWf (100 μg/ml)-coated microcapillary tubes at a shear rate of 1,800 s−1. (A) Oregon green fluorescence images and real-time calcium flux recording demonstrating NP-EGTA–elicited ICC at a shear rate of 1,800 s−1. The gray box demarcates the point at which the secondary platelet tethers to the stationary adherent cell (→) activated by NP-EGTA uncaging. (B) Control experiments in which NP-EGTA–loaded platelets were allowed to tether and translocate across the vWf matrix (−1.2 and −0.6 s). The confocal field was subsequently exposed to a near-UV light source (350 nm ↑UV) for 0.6 s, leading to NP-EGTA uncaging and release of intracellular calcium (0 s). The zoomed boxes show the occurrence of platelet aggregation and ongoing ICC post-UV. The dashed circle demarks small aggregates forming on the vWf matrix. (C) NP-EGTA flow experiment conducted in the presence of 1.5 U/ml apyrase. The zoomed boxes demonstrate that in the presence of apyrase, ICC post-UV stimulation does not occur with the platelets in the field of view resuming translocation after a short-duration stationary adhesion.
Mentions: Finally, to provide more direct evidence that changes in cytosolic calcium flux in the primary adherent layer of platelets impacts on the efficiency of platelet aggregation, we examined the effect of inducing a single transient calcium spike in translocating platelets. This was achieved by loading platelets with the caged calcium chelator NP-EGTA, which upon UV exposure results in a rapid, transient increase in cytosolic calcium (Nesbitt et al., 2002). A significant advantage of this experimental approach is that it enables rapid release of calcium in a discrete population of primary adherent platelets only, immediately before the formation of platelet–platelet adhesion contacts. This maximizes the possibility of coordinating the initiation of calcium flux with subsequent ICC and, furthermore, minimizes potential experimental artifacts associated with platelet preadhesion, such as exhausted ADP release. To investigate the effects of calcium uncaging on platelet aggregate formation, NP-EGTA–loaded platelets were perfused through vWf-coated microcapillary tubes at physiological platelet concentrations (150 × 109/L) to increase the probability of platelet–platelet adhesion contact formation. Similar to our previous findings (Nesbitt et al., 2002), eliciting a transient calcium spike in platelets that were not interacting with other translocating platelets resulted in temporary arrest of these cells (unpublished data). In contrast, induction of a single calcium spike at the point of contact between two translocating platelets resulted in prolonged arrest of both cells, leading to the induction of a sustained oscillatory calcium flux. Moreover, these cells formed efficient nuclei for the recruitment of free-flowing platelets leading to ICC and the rapid formation of platelet aggregates (Fig. 6, A and B). Platelet aggregate formation and ICC under these experimental conditions were dependent on released ADP, as they were completely inhibited by pretreating the platelets with apyrase (Fig. 6 C) or a combination of both A3P5PS and AR-C6991MX (unpublished data). These findings provide further support for the concept that communication of calcium signals between platelets is important in regulating the efficiency of platelet aggregation under flow.

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