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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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ICC drives thrombus growth. (A) Reconstructed platelet thrombi. Isolated platelets reconstituted with washed RBCs (50% hematocrit) and plasma perfused at a shear rate of 1,800 s−1 for 3 min over immobilized human vWf (100 μg/ml) or type I fibrillar collagen (2.5 mg/ml). Conditions: vWF, DiOC6-labeled thrombi on the surface of immobilized vWf; collagen, DiOC6-labeled thrombi on the surface of type I collagen fibrils; DM-BAPTA, calcium-chelated platelets translocating across the surface of type I collagen fibrils. Thrombus volumetric data demonstrating the marked difference in platelet thrombus size on the surface of immobilized vWf versus type I collagen. Note that in the case of DM-BAPTA–treated cells, thrombi did not form, and the platelets translocated freely across the collagen surface (n = 3). (B) The percentage of platelets coming into contact with an initially adherent platelet and undergoing concomitant calcium oscillations (ICC) was quantified at time points where the primary adherent cell was expressing maximal cytosolic calcium levels. The data indicate that ICC occurs in 53% of cells tethering to primary adherent cells at the surface of vWf, whereas 100% of tethering cells express sustained calcium oscillations at the surface of type I collagen (n = 25 platelets). (C) Δ[Ca2+]c population analysis showing the distribution of platelet calcium events occurring at the surface of immobilized vWf and type I collagen. The gray box indicates the 100-nM calcium threshold, below which platelets are considered to be in the resting state (n = 3). (D) Oregon green fluorescence images demonstrating intraplatelet calcium flux during real-time aggregate formation at the surface of immobilized vWf and a single collagen type I fibril at a platelet count of 150 × 109/L. The arrow indicates the site of initial platelet adhesion to both collagen and vWF matrices. Note that the images on type I collagen show aggregate formation on a single collagen fiber in comparison to the continuous surface of immobilized vWf.
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fig5: ICC drives thrombus growth. (A) Reconstructed platelet thrombi. Isolated platelets reconstituted with washed RBCs (50% hematocrit) and plasma perfused at a shear rate of 1,800 s−1 for 3 min over immobilized human vWf (100 μg/ml) or type I fibrillar collagen (2.5 mg/ml). Conditions: vWF, DiOC6-labeled thrombi on the surface of immobilized vWf; collagen, DiOC6-labeled thrombi on the surface of type I collagen fibrils; DM-BAPTA, calcium-chelated platelets translocating across the surface of type I collagen fibrils. Thrombus volumetric data demonstrating the marked difference in platelet thrombus size on the surface of immobilized vWf versus type I collagen. Note that in the case of DM-BAPTA–treated cells, thrombi did not form, and the platelets translocated freely across the collagen surface (n = 3). (B) The percentage of platelets coming into contact with an initially adherent platelet and undergoing concomitant calcium oscillations (ICC) was quantified at time points where the primary adherent cell was expressing maximal cytosolic calcium levels. The data indicate that ICC occurs in 53% of cells tethering to primary adherent cells at the surface of vWf, whereas 100% of tethering cells express sustained calcium oscillations at the surface of type I collagen (n = 25 platelets). (C) Δ[Ca2+]c population analysis showing the distribution of platelet calcium events occurring at the surface of immobilized vWf and type I collagen. The gray box indicates the 100-nM calcium threshold, below which platelets are considered to be in the resting state (n = 3). (D) Oregon green fluorescence images demonstrating intraplatelet calcium flux during real-time aggregate formation at the surface of immobilized vWf and a single collagen type I fibril at a platelet count of 150 × 109/L. The arrow indicates the site of initial platelet adhesion to both collagen and vWF matrices. Note that the images on type I collagen show aggregate formation on a single collagen fiber in comparison to the continuous surface of immobilized vWf.

Mentions: The demonstration that ICC plays an important role in regulating the formation of platelet aggregates under flow raised the possibility that the efficiency by which calcium signals are propagated throughout an aggregating population of platelets may be an important determinant regulating the rate and extent of thrombus growth. To investigate this possibility, real-time changes in cytosolic calcium levels were monitored during platelet thrombus formation on a type I fibrillar collagen or vWf matrix. These matrices were chosen for comparative analysis due to their marked difference in thrombogenic potential. As demonstrated in Fig. 5 A, thrombi forming on a vWf matrix tend to be relatively small (typically <20,000 μm3), forming discrete clusters over the vWf surface. In contrast, large thrombi (≥110,000 μm3) typically form on collagen fibrils, coalescing to form extremely large platelet masses (Fig. 5 A). Real-time analysis of platelet adhesion at the surface of vWf demonstrated that only 6.5 ± 1.7% of platelets maintained stationary adhesion contacts for a period >30 s. In contrast, all platelets interacting with collagen fibrils formed immediate stationary adhesion contacts that provided highly efficient nuclei for the recruitment of additional platelets, resulting in the rapid formation of very large platelet aggregates.


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 drives thrombus growth. (A) Reconstructed platelet thrombi. Isolated platelets reconstituted with washed RBCs (50% hematocrit) and plasma perfused at a shear rate of 1,800 s−1 for 3 min over immobilized human vWf (100 μg/ml) or type I fibrillar collagen (2.5 mg/ml). Conditions: vWF, DiOC6-labeled thrombi on the surface of immobilized vWf; collagen, DiOC6-labeled thrombi on the surface of type I collagen fibrils; DM-BAPTA, calcium-chelated platelets translocating across the surface of type I collagen fibrils. Thrombus volumetric data demonstrating the marked difference in platelet thrombus size on the surface of immobilized vWf versus type I collagen. Note that in the case of DM-BAPTA–treated cells, thrombi did not form, and the platelets translocated freely across the collagen surface (n = 3). (B) The percentage of platelets coming into contact with an initially adherent platelet and undergoing concomitant calcium oscillations (ICC) was quantified at time points where the primary adherent cell was expressing maximal cytosolic calcium levels. The data indicate that ICC occurs in 53% of cells tethering to primary adherent cells at the surface of vWf, whereas 100% of tethering cells express sustained calcium oscillations at the surface of type I collagen (n = 25 platelets). (C) Δ[Ca2+]c population analysis showing the distribution of platelet calcium events occurring at the surface of immobilized vWf and type I collagen. The gray box indicates the 100-nM calcium threshold, below which platelets are considered to be in the resting state (n = 3). (D) Oregon green fluorescence images demonstrating intraplatelet calcium flux during real-time aggregate formation at the surface of immobilized vWf and a single collagen type I fibril at a platelet count of 150 × 109/L. The arrow indicates the site of initial platelet adhesion to both collagen and vWF matrices. Note that the images on type I collagen show aggregate formation on a single collagen fiber in comparison to the continuous surface of immobilized vWf.
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Related In: Results  -  Collection

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fig5: ICC drives thrombus growth. (A) Reconstructed platelet thrombi. Isolated platelets reconstituted with washed RBCs (50% hematocrit) and plasma perfused at a shear rate of 1,800 s−1 for 3 min over immobilized human vWf (100 μg/ml) or type I fibrillar collagen (2.5 mg/ml). Conditions: vWF, DiOC6-labeled thrombi on the surface of immobilized vWf; collagen, DiOC6-labeled thrombi on the surface of type I collagen fibrils; DM-BAPTA, calcium-chelated platelets translocating across the surface of type I collagen fibrils. Thrombus volumetric data demonstrating the marked difference in platelet thrombus size on the surface of immobilized vWf versus type I collagen. Note that in the case of DM-BAPTA–treated cells, thrombi did not form, and the platelets translocated freely across the collagen surface (n = 3). (B) The percentage of platelets coming into contact with an initially adherent platelet and undergoing concomitant calcium oscillations (ICC) was quantified at time points where the primary adherent cell was expressing maximal cytosolic calcium levels. The data indicate that ICC occurs in 53% of cells tethering to primary adherent cells at the surface of vWf, whereas 100% of tethering cells express sustained calcium oscillations at the surface of type I collagen (n = 25 platelets). (C) Δ[Ca2+]c population analysis showing the distribution of platelet calcium events occurring at the surface of immobilized vWf and type I collagen. The gray box indicates the 100-nM calcium threshold, below which platelets are considered to be in the resting state (n = 3). (D) Oregon green fluorescence images demonstrating intraplatelet calcium flux during real-time aggregate formation at the surface of immobilized vWf and a single collagen type I fibril at a platelet count of 150 × 109/L. The arrow indicates the site of initial platelet adhesion to both collagen and vWF matrices. Note that the images on type I collagen show aggregate formation on a single collagen fiber in comparison to the continuous surface of immobilized vWf.
Mentions: The demonstration that ICC plays an important role in regulating the formation of platelet aggregates under flow raised the possibility that the efficiency by which calcium signals are propagated throughout an aggregating population of platelets may be an important determinant regulating the rate and extent of thrombus growth. To investigate this possibility, real-time changes in cytosolic calcium levels were monitored during platelet thrombus formation on a type I fibrillar collagen or vWf matrix. These matrices were chosen for comparative analysis due to their marked difference in thrombogenic potential. As demonstrated in Fig. 5 A, thrombi forming on a vWf matrix tend to be relatively small (typically <20,000 μm3), forming discrete clusters over the vWf surface. In contrast, large thrombi (≥110,000 μm3) typically form on collagen fibrils, coalescing to form extremely large platelet masses (Fig. 5 A). Real-time analysis of platelet adhesion at the surface of vWf demonstrated that only 6.5 ± 1.7% of platelets maintained stationary adhesion contacts for a period >30 s. In contrast, all platelets interacting with collagen fibrils formed immediate stationary adhesion contacts that provided highly efficient nuclei for the recruitment of additional platelets, resulting in the rapid formation of very large platelet aggregates.

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