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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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Secreted ADP drives ICC and platelet aggregation at the surface of vWf. Oregon green fluorescence images demonstrating platelet calcium flux during reconstituted blood flow at the surface of immobilized vWf at a platelet count of 150 × 109/L. (A) Isolated platelets at a density of 150 × 109/L were incubated with 1.5 U/ml apyrase before perfusion over immobilized vWf. The arrow indicates a single platelet undergoing transient calcium flux that correlates with transient adhesion formation. Images taken at 10.2 and 11.9 s demonstrate that although platelet–platelet contacts occur in the shear field, these do not undergo sustained ICC. (B) Isolated platelets at a density of 150 × 109/L were incubated with 100 μM of the P2Y1-specific antagonist A3P5PS before perfusion over immobilized vWf. The arrow indicates a single platelet undergoing vWf-dependent calcium flux. The images taken at 10.2 and 46.6 s demonstrate that in the presence of P2Y1 blockade, ICC can still take place at sites of primary platelet adhesion. (C) Isolated platelets at a density of 150 × 109/L were incubated with 200 nM of the P2Y12-specific antagonist AR-C69931MX before perfusion over immobilized vWf. The arrow indicates a single platelet undergoing vWf-dependent calcium flux. The image taken at 18.2 s demonstrates that in the presence of P2Y12 blockade, platelet–platelet contact results in a transient intraplatelet calcium spike that is not sustained, resulting in complete inhibition of aggregation. (D) Single-platelet calcium flux profiles showing the calcium spike response (red profile) of a transiently tethering cell at the surface of a stationary adherent platelet undergoing sustained calcium oscillations (black profile).
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fig3: Secreted ADP drives ICC and platelet aggregation at the surface of vWf. Oregon green fluorescence images demonstrating platelet calcium flux during reconstituted blood flow at the surface of immobilized vWf at a platelet count of 150 × 109/L. (A) Isolated platelets at a density of 150 × 109/L were incubated with 1.5 U/ml apyrase before perfusion over immobilized vWf. The arrow indicates a single platelet undergoing transient calcium flux that correlates with transient adhesion formation. Images taken at 10.2 and 11.9 s demonstrate that although platelet–platelet contacts occur in the shear field, these do not undergo sustained ICC. (B) Isolated platelets at a density of 150 × 109/L were incubated with 100 μM of the P2Y1-specific antagonist A3P5PS before perfusion over immobilized vWf. The arrow indicates a single platelet undergoing vWf-dependent calcium flux. The images taken at 10.2 and 46.6 s demonstrate that in the presence of P2Y1 blockade, ICC can still take place at sites of primary platelet adhesion. (C) Isolated platelets at a density of 150 × 109/L were incubated with 200 nM of the P2Y12-specific antagonist AR-C69931MX before perfusion over immobilized vWf. The arrow indicates a single platelet undergoing vWf-dependent calcium flux. The image taken at 18.2 s demonstrates that in the presence of P2Y12 blockade, platelet–platelet contact results in a transient intraplatelet calcium spike that is not sustained, resulting in complete inhibition of aggregation. (D) Single-platelet calcium flux profiles showing the calcium spike response (red profile) of a transiently tethering cell at the surface of a stationary adherent platelet undergoing sustained calcium oscillations (black profile).

Mentions: The release of ADP from platelet-dense granules has a well-established role in promoting platelet aggregation in response to multiple physiological agonists, including vWf (Moritz et al., 1983; Peterson et al., 1987; Moake et al., 1988; Ikeda et al., 1991; Chow et al., 1992; Oda et al., 1995; Gachet, 2001). To investigate the contribution of secreted ADP to ICC, platelet calcium dynamics were examined during platelet adhesion and aggregation on a vWf matrix in the presence of the ADP-scavenging enzyme apyrase (1.5 U/ml ADPase activity). Consistent with previous findings, primary adherent platelets could elicit a sustained oscillatory calcium response in the presence of 1.5 U/ml apyrase (Nesbitt et al., 2002) (Fig. 3 A). However, the ability of these primary adherent cells to act as nuclei for subsequent ICC was completely blocked (Fig. 3 A), resulting in an inability of platelets to form stable aggregates. Similarly, pretreating platelets with the specific purinergic receptor antagonists, A3P5PS and AR-C69931MX in combination, to selectively inhibit both the P2Y1 and P2Y12 receptors, respectively, (Boyer et al., 1996; Ingall et al., 1999; Humphries, 2000) also completely eliminated ICC and platelet aggregate formation on vWf (unpublished data). Further examination of the individual contribution of the P2Y1 and P2Y12 signaling pathways demonstrated that blockade of ADP binding to the P2Y1 receptor alone had no effect on ICC and did not inhibit platelet aggregate formation (Fig. 3 B). Specific blockade of the P2Y12 receptor with 200 nM AR-C69931MX did not inhibit the initial calcium spiking associated with platelet–platelet contact (Fig. 3, C and D, dotted mark), but did completely inhibit ongoing/oscillatory ICC and stable aggregation (Fig. 3 C). The demonstration that P2Y12 blockade still allowed for a transient ICC spike suggested that the P2Y1 signaling pathway may be involved in initiating early calcium spiking events associated with platelet–platelet contact. Alternatively, the transient calcium spike observed in the presence of P2Y12 blockade alone may have been triggered by the release of a secondary secreted agonist, such as thromboxane A2 (TXA2). To examine this possibility, platelets were pretreated with 1.5 mM aspirin before perfusion over the surface of immobilized vWf. Although 1.5 mM aspirin was found to completely inhibit platelet aggregation responses to arachidonic acid (unpublished data), this treatment did not have any effect on initial platelet adhesive events nor on ICC at the surface of vWf under flow conditions (unpublished data). These studies indicate that activation of the P2Y12 purinergic receptor pathway is a key event in the establishment of effective ICC and platelet aggregation.


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)

Secreted ADP drives ICC and platelet aggregation at the surface of vWf. Oregon green fluorescence images demonstrating platelet calcium flux during reconstituted blood flow at the surface of immobilized vWf at a platelet count of 150 × 109/L. (A) Isolated platelets at a density of 150 × 109/L were incubated with 1.5 U/ml apyrase before perfusion over immobilized vWf. The arrow indicates a single platelet undergoing transient calcium flux that correlates with transient adhesion formation. Images taken at 10.2 and 11.9 s demonstrate that although platelet–platelet contacts occur in the shear field, these do not undergo sustained ICC. (B) Isolated platelets at a density of 150 × 109/L were incubated with 100 μM of the P2Y1-specific antagonist A3P5PS before perfusion over immobilized vWf. The arrow indicates a single platelet undergoing vWf-dependent calcium flux. The images taken at 10.2 and 46.6 s demonstrate that in the presence of P2Y1 blockade, ICC can still take place at sites of primary platelet adhesion. (C) Isolated platelets at a density of 150 × 109/L were incubated with 200 nM of the P2Y12-specific antagonist AR-C69931MX before perfusion over immobilized vWf. The arrow indicates a single platelet undergoing vWf-dependent calcium flux. The image taken at 18.2 s demonstrates that in the presence of P2Y12 blockade, platelet–platelet contact results in a transient intraplatelet calcium spike that is not sustained, resulting in complete inhibition of aggregation. (D) Single-platelet calcium flux profiles showing the calcium spike response (red profile) of a transiently tethering cell at the surface of a stationary adherent platelet undergoing sustained calcium oscillations (black profile).
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Related In: Results  -  Collection

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fig3: Secreted ADP drives ICC and platelet aggregation at the surface of vWf. Oregon green fluorescence images demonstrating platelet calcium flux during reconstituted blood flow at the surface of immobilized vWf at a platelet count of 150 × 109/L. (A) Isolated platelets at a density of 150 × 109/L were incubated with 1.5 U/ml apyrase before perfusion over immobilized vWf. The arrow indicates a single platelet undergoing transient calcium flux that correlates with transient adhesion formation. Images taken at 10.2 and 11.9 s demonstrate that although platelet–platelet contacts occur in the shear field, these do not undergo sustained ICC. (B) Isolated platelets at a density of 150 × 109/L were incubated with 100 μM of the P2Y1-specific antagonist A3P5PS before perfusion over immobilized vWf. The arrow indicates a single platelet undergoing vWf-dependent calcium flux. The images taken at 10.2 and 46.6 s demonstrate that in the presence of P2Y1 blockade, ICC can still take place at sites of primary platelet adhesion. (C) Isolated platelets at a density of 150 × 109/L were incubated with 200 nM of the P2Y12-specific antagonist AR-C69931MX before perfusion over immobilized vWf. The arrow indicates a single platelet undergoing vWf-dependent calcium flux. The image taken at 18.2 s demonstrates that in the presence of P2Y12 blockade, platelet–platelet contact results in a transient intraplatelet calcium spike that is not sustained, resulting in complete inhibition of aggregation. (D) Single-platelet calcium flux profiles showing the calcium spike response (red profile) of a transiently tethering cell at the surface of a stationary adherent platelet undergoing sustained calcium oscillations (black profile).
Mentions: The release of ADP from platelet-dense granules has a well-established role in promoting platelet aggregation in response to multiple physiological agonists, including vWf (Moritz et al., 1983; Peterson et al., 1987; Moake et al., 1988; Ikeda et al., 1991; Chow et al., 1992; Oda et al., 1995; Gachet, 2001). To investigate the contribution of secreted ADP to ICC, platelet calcium dynamics were examined during platelet adhesion and aggregation on a vWf matrix in the presence of the ADP-scavenging enzyme apyrase (1.5 U/ml ADPase activity). Consistent with previous findings, primary adherent platelets could elicit a sustained oscillatory calcium response in the presence of 1.5 U/ml apyrase (Nesbitt et al., 2002) (Fig. 3 A). However, the ability of these primary adherent cells to act as nuclei for subsequent ICC was completely blocked (Fig. 3 A), resulting in an inability of platelets to form stable aggregates. Similarly, pretreating platelets with the specific purinergic receptor antagonists, A3P5PS and AR-C69931MX in combination, to selectively inhibit both the P2Y1 and P2Y12 receptors, respectively, (Boyer et al., 1996; Ingall et al., 1999; Humphries, 2000) also completely eliminated ICC and platelet aggregate formation on vWf (unpublished data). Further examination of the individual contribution of the P2Y1 and P2Y12 signaling pathways demonstrated that blockade of ADP binding to the P2Y1 receptor alone had no effect on ICC and did not inhibit platelet aggregate formation (Fig. 3 B). Specific blockade of the P2Y12 receptor with 200 nM AR-C69931MX did not inhibit the initial calcium spiking associated with platelet–platelet contact (Fig. 3, C and D, dotted mark), but did completely inhibit ongoing/oscillatory ICC and stable aggregation (Fig. 3 C). The demonstration that P2Y12 blockade still allowed for a transient ICC spike suggested that the P2Y1 signaling pathway may be involved in initiating early calcium spiking events associated with platelet–platelet contact. Alternatively, the transient calcium spike observed in the presence of P2Y12 blockade alone may have been triggered by the release of a secondary secreted agonist, such as thromboxane A2 (TXA2). To examine this possibility, platelets were pretreated with 1.5 mM aspirin before perfusion over the surface of immobilized vWf. Although 1.5 mM aspirin was found to completely inhibit platelet aggregation responses to arachidonic acid (unpublished data), this treatment did not have any effect on initial platelet adhesive events nor on ICC at the surface of vWf under flow conditions (unpublished data). These studies indicate that activation of the P2Y12 purinergic receptor pathway is a key event in the establishment of effective ICC and platelet aggregation.

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