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Dynamic analysis of platelet deposition to resolve platelet adhesion receptor activity in whole blood at arterial shear rate.

Pugh N, Bihan D, Perry DJ, Farndale RW - Platelets (2014)

Bottom Line: On collagen, PM falls exponentially to a low level, corresponding to firm platelet adhesion, while on other substrates, PM remains high.Different receptor-specific thrombogenic surfaces reveal that the PM time constant reflects real-time changes in integrins αIIbβ3 and α2β1 activity.This ensemble kinetic analysis has the potential to provide valuable diagnostic information about platelet thrombus formation with both academic and clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge , Cambridge , UK .

ABSTRACT
Platelet activation is traditionally quantified using turbidimetric aggregometry, which reflects integrin αIIbβ3 activity, an important determinant of platelet function during pathophysiological thrombus formation. However, aggregometry does not recreate the shear conditions prevailing during thrombosis in vivo. Here we describe novel whole-frame analysis of real-time video microscopy to quantify platelet adhesion receptor activity under shear in parallel-plate flow chambers. We demonstrate that the rate of change of surface coverage (designated Platelet Population Mobility, PM) quantifies platelet mobility. On collagen, PM falls exponentially to a low level, corresponding to firm platelet adhesion, while on other substrates, PM remains high. Different receptor-specific thrombogenic surfaces reveal that the PM time constant reflects real-time changes in integrins αIIbβ3 and α2β1 activity. This ensemble kinetic analysis has the potential to provide valuable diagnostic information about platelet thrombus formation with both academic and clinical applications.

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PM quantifies integrin activation under flow conditions. Whole blood was pre-treated with carrier (A), 300 µM DM-BAPTA-AM (B) or 5 µM GR144053 (C), before perfusion at 1000 s−1 over coverslips coated with combinations of CRP, VWF-III and one of a panel of integrin α2β1-adhesive peptides of varying affinities. PM was calculated as before. For clarity, only GFOGER, (○), GMOGER (•) and GPP10 (•) are shown. Other data sets (GLOGER and GAOGER) are shown in Supplementary Figure S2. End-point measurements were calculated as described in the text, Plateau (D), Decay constant (E) and ZV50 (F). Blood was pre-treated with: Carrier (□), 5 µM GR144053 (▪), 300 µM DM-BAPTA-AM (▪).
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Figure 2: PM quantifies integrin activation under flow conditions. Whole blood was pre-treated with carrier (A), 300 µM DM-BAPTA-AM (B) or 5 µM GR144053 (C), before perfusion at 1000 s−1 over coverslips coated with combinations of CRP, VWF-III and one of a panel of integrin α2β1-adhesive peptides of varying affinities. PM was calculated as before. For clarity, only GFOGER, (○), GMOGER (•) and GPP10 (•) are shown. Other data sets (GLOGER and GAOGER) are shown in Supplementary Figure S2. End-point measurements were calculated as described in the text, Plateau (D), Decay constant (E) and ZV50 (F). Blood was pre-treated with: Carrier (□), 5 µM GR144053 (▪), 300 µM DM-BAPTA-AM (▪).

Mentions: Within this overall framework, PM profiles can be used to resolve the role of the collagen-binding integrin α2β1 in thrombus deposition. Experiments were conducted using surfaces coated with a series of GxOGER-containing peptides with differing affinity for α2β1, in combination with VWF-III and the GpVI-specific collagen-related peptide (CRP) [6]. The different PM profiles reflected the known affinities of the peptides for α2β1 (Figure 2A and D; Supplementary Figure S1). Perfusion of whole blood over surfaces including GPP10, the inert control peptide, or GAOGER, the low-affinity integrin ligand, resulted in gradual reductions of PM. PM fell more rapidly using surfaces including the higher affinity peptides, GFOGER and GLOGER, indicating stable adhesion, while the mid-affinity GMOGER yielded an intermediate PM profile. PM curves from the different surfaces converged, showing Plateau (an end-point parameter) to be unsuitable to resolve the role of α2β1 (Figure 2D). The differences in PM are reflected in significantly higher decay constants with GFOGER and GLOGER than on the lower affinity peptides, GMOGER, GAOGER or GPP10 (Figure 2E).


Dynamic analysis of platelet deposition to resolve platelet adhesion receptor activity in whole blood at arterial shear rate.

Pugh N, Bihan D, Perry DJ, Farndale RW - Platelets (2014)

PM quantifies integrin activation under flow conditions. Whole blood was pre-treated with carrier (A), 300 µM DM-BAPTA-AM (B) or 5 µM GR144053 (C), before perfusion at 1000 s−1 over coverslips coated with combinations of CRP, VWF-III and one of a panel of integrin α2β1-adhesive peptides of varying affinities. PM was calculated as before. For clarity, only GFOGER, (○), GMOGER (•) and GPP10 (•) are shown. Other data sets (GLOGER and GAOGER) are shown in Supplementary Figure S2. End-point measurements were calculated as described in the text, Plateau (D), Decay constant (E) and ZV50 (F). Blood was pre-treated with: Carrier (□), 5 µM GR144053 (▪), 300 µM DM-BAPTA-AM (▪).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389707&req=5

Figure 2: PM quantifies integrin activation under flow conditions. Whole blood was pre-treated with carrier (A), 300 µM DM-BAPTA-AM (B) or 5 µM GR144053 (C), before perfusion at 1000 s−1 over coverslips coated with combinations of CRP, VWF-III and one of a panel of integrin α2β1-adhesive peptides of varying affinities. PM was calculated as before. For clarity, only GFOGER, (○), GMOGER (•) and GPP10 (•) are shown. Other data sets (GLOGER and GAOGER) are shown in Supplementary Figure S2. End-point measurements were calculated as described in the text, Plateau (D), Decay constant (E) and ZV50 (F). Blood was pre-treated with: Carrier (□), 5 µM GR144053 (▪), 300 µM DM-BAPTA-AM (▪).
Mentions: Within this overall framework, PM profiles can be used to resolve the role of the collagen-binding integrin α2β1 in thrombus deposition. Experiments were conducted using surfaces coated with a series of GxOGER-containing peptides with differing affinity for α2β1, in combination with VWF-III and the GpVI-specific collagen-related peptide (CRP) [6]. The different PM profiles reflected the known affinities of the peptides for α2β1 (Figure 2A and D; Supplementary Figure S1). Perfusion of whole blood over surfaces including GPP10, the inert control peptide, or GAOGER, the low-affinity integrin ligand, resulted in gradual reductions of PM. PM fell more rapidly using surfaces including the higher affinity peptides, GFOGER and GLOGER, indicating stable adhesion, while the mid-affinity GMOGER yielded an intermediate PM profile. PM curves from the different surfaces converged, showing Plateau (an end-point parameter) to be unsuitable to resolve the role of α2β1 (Figure 2D). The differences in PM are reflected in significantly higher decay constants with GFOGER and GLOGER than on the lower affinity peptides, GMOGER, GAOGER or GPP10 (Figure 2E).

Bottom Line: On collagen, PM falls exponentially to a low level, corresponding to firm platelet adhesion, while on other substrates, PM remains high.Different receptor-specific thrombogenic surfaces reveal that the PM time constant reflects real-time changes in integrins αIIbβ3 and α2β1 activity.This ensemble kinetic analysis has the potential to provide valuable diagnostic information about platelet thrombus formation with both academic and clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge , Cambridge , UK .

ABSTRACT
Platelet activation is traditionally quantified using turbidimetric aggregometry, which reflects integrin αIIbβ3 activity, an important determinant of platelet function during pathophysiological thrombus formation. However, aggregometry does not recreate the shear conditions prevailing during thrombosis in vivo. Here we describe novel whole-frame analysis of real-time video microscopy to quantify platelet adhesion receptor activity under shear in parallel-plate flow chambers. We demonstrate that the rate of change of surface coverage (designated Platelet Population Mobility, PM) quantifies platelet mobility. On collagen, PM falls exponentially to a low level, corresponding to firm platelet adhesion, while on other substrates, PM remains high. Different receptor-specific thrombogenic surfaces reveal that the PM time constant reflects real-time changes in integrins αIIbβ3 and α2β1 activity. This ensemble kinetic analysis has the potential to provide valuable diagnostic information about platelet thrombus formation with both academic and clinical applications.

Show MeSH
Related in: MedlinePlus