Imaging and quantitation of calcium fluxes in platelets

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Imaging and quantitation of calcium fluxes in
platelets Application of FIMS in the area of
thrombosis and haemostasis
Johan Heemskerk
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Contents

• Haemostasis platelet vessel wall interaction.
• Ex vivo measurement of platelet calcium fluxes.

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Model of platelet thrombus formation
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Platelet activation
F II F V F VIII F IX F X F XI
vWf collagen fibrinogen fibrin cells
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Haemostasis platelet vessel wall interaction
adhesion activation aggregate form.
coagulation
vessel damage low high
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Thrombosis platelet vessel wall interaction
adhesion activation aggregate form.
coagulation
adhesion activation aggregate form.
Collagen Ca2 from stores ADP
elevation in Ca2i TxA2 Ca2 from
external Thrombin
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Haemostasis platelet vessel wall interaction
adhesion
activation aggregate coagulation
formation
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Thrombosis platelet vessel wall interaction
adhesion activation aggregate form.
coagulation
adhesion activation aggregate form.
Platelet receptors of (potential) clinical
relevance
Collagen GP VI a2b1 devel. vWf GP Ib-IX-V
-
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Thrombosis platelet vessel wall interaction
adhesion activation aggregate form.
coagulation
adhesion activation aggregate form.
Platelet receptors of (potential) clinical
relevance
Collagen GP VI a2b1 devel. vWf GP Ib-IX-V
- TxA2 TPa TPb aspirin
ADP P2Y12 P2Y1 clopidogrel,
etc. Fibrinogen aIIbb3 abciximab, etc.
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Thrombosis platelet vessel wall interaction
adhesion activation aggregate form.
coagulation
adhesion activation aggregate form.
Platelet receptors of (potential) clinical
relevance
Collagen GP VI a2b1 devel. vWf GP Ib-IX-V
- TxA2 TPa TPb aspirin ADP P2Y12
P2Y1 clopidogrel, etc. Fibrinogen aIIbb3
abciximab, etc. Thrombin PAR-1 PAR-4
heparin, hirudin, etc.
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Contents

• Haemostasis platelet vessel wall interaction.
• In vivo measurement of platelet calcium fluxes.
• Ex vivo measurement of platelet calcium fluxes.

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Glycoprotein VI
Integrin a2b1
Ca2
annexin V
Trp
Ca2
IP3
IP3-R
Ca2
Ca2
cytoskeleton
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Glycoprotein VI stimulating peptide is a more
potent stimulus of the calcium and procoagulant
responses than thrombin.
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Platelet adhesion stimulates the procoagulant
response elicited by glycoprotein VI ligands.
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• Glycoprotein VI receptor
• Platelet adhesion to collagen or other
  glycoprotein VI ligands causes a high and
  prolonged calcium response.
• This is followed by a procoagulant response,
  characterized by prolonged Ca2 signal, membrane
  blebbing and PS exposure.

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Mouse models for ex vivo perfusion studies
platelet properties and bleeding.
GPVI a2b1 GPIIb/IIIa
Bleeding Control GPVI
defic (JAQ1) - FcR g
chain -/- - b1 null
-
(EMBO J 202120)
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• Whole-blood flow experiments
• In high-shear flow experiments with mouse blood
  over a collagen surface, glycoprotein VI appear
  to be indispensable for
• - platelet adhesion
• - platelet activation
• - release of ADP and TxA2
• - surface generation of procoagulant PS.

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• Platelets for transfusion purposes
• Some platelets slowly increase in activation
  state.
• Many platelets progressively decrease in
  excitability.

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Model of platelet thrombus formation
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Contributors Dept. of Biochemistry
(CARIM, UM) Dept. of Haematology (AZM) Marion
Feijge Karly Hamulyak Johan Heemskerk
Elisabeth van Pampus Marijke
Kuijpers Imke Munnix Sanquin
Maastricht Theo Lindhout Joyce
Curvers Kristof Vanschoonbeek Eva
Rombouts-Sestrienkova Dept. of Physiology
(CARIM, UM) Synapse (CARIM, UM) Miriam van
Gestel Peter Giesen Mirjam oude
Egbrink Coenraad Hemker