Fluoride-dependent calcium-induced platelet procoagulant activity shows that calpain is involved in increased phospholipid transbilayer movement.
Treatment of platelets with fluoride (10 mM) was found to result in a transient increase in Ca2+-permeability of the platelet plasma membrane. This phenomenon was used to provide supplementary evidence for the suggestions made earlier (Comfurius et al. (1985) Biochim. Biophys. Acta 815, 143; Verhallen et al. (1987) Biochim. Biophys. Acta 903, 206), that cytoskeletal disrupture by calpain is involved in the process leading to transbilayer movement of phosphatidylserine during expression of platelet procoagulant activity. This was achieved by relating both calpain activity and exposure of phosphatidylserine with platelet procoagulant activity. It was found that only upon addition of extracellular Ca2+ to fluoride-treated platelets, procoagulant activity, expressed as prothrombinase activity, and calpain activity, estimated from protein patterns after gel electrophoresis, were generated. Both Ca2+-inducible prothrombinase activity and calpain activity followed an identical time-course during incubation with fluoride: after a time-lag of about 10 min they sharply increased towards a peak level. Upon further incubation with fluoride, both activities decreased towards a final plateau, still above basal level. The presence of leupeptin during incubation with fluoride was found to inhibit Ca2+-inducible calpain activity and prothrombinase activity in an identical way. Ca2+-inducible exposure of phosphatidylserine, as determined with extracellular phospholipase A2, showed a similar pattern as Ca2+-inducible calpain activity and prothrombinase activity. From the strict parallelism between prothrombinase activity, calpain activity and exposure of phosphatidylserine, it is concluded that calpain plays an important role in the activation-dependent transbilayer movement of phosphatidylserine during expression of platelet procoagulant activity. It is suggested that degradation of the platelet membrane-skeleton by calpain disturbs the structural organization of the lipid bilayer of the platelet plasma membrane leading to enhanced transbilayer movement of phospholipids and appearance of phosphatidylserine at the platelet outer surface.
Verhallen
P F
PF
Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
Bevers
E M
EM
Comfurius
P
P
Zwaal
R F
RF
D001792:Blood Platelets / Q000187:drug effects*; D002118:Calcium / Q000097:blood; D002154:Calpain / Q000097:blood*; D005459:Fluorides / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007976:Leupeptins / Q000494:pharmacology; D008051:Lipid Bilayers; D008563:Membrane Lipids / Q000097:blood; D008565:Membrane Proteins / Q000097:blood; D009038:Motion; D010718:Phosphatidylserines / Q000097:blood; D013925:Thromboplastin / Q000097:blood*
D007976:Leupeptins; D008051:Lipid Bilayers; D008563:Membrane Lipids; D008565:Membrane Proteins; D010718:Phosphatidylserines; D013925:Thromboplastin; D002154:Calpain; C032854:leupeptin; D005459:Fluorides; D002118:Calcium