Anticoagulant and antithrombotic properties of intracellular protease-activated receptor antagonists.
BACKGROUND
Blockade of the thrombin receptors protease-activated receptor (PAR)1 and PAR4 with pepducins, cell-penetrating lipopeptides based on the third intracellular loop of PAR1 and PAR4, effectively inhibits platelet aggregation. We have previously shown that PAR1 pepducin also exerts an anticoagulant activity by partial inhibition of the thrombin plus collagen-induced externalization of phosphatidylserine (PS) at the platelet plasma membrane.


OBJECTIVE
In the present study we examined the effects of PAR1 and PAR4 pepducins on tissue factor (TF)-initiated thrombin generation in platelet-rich plasma (PRP) and the interaction between PAR4 pepducin-loaded mouse platelets and a growing thrombus to confirm the relevance of the in vitro data.


RESULTS
Localization of pepducins at the inner leaflet of the plasma membrane was confirmed with a fluorescence resonance energy transfer assay. Both the PAR1 pepducin, P1pal12, and the PAR4 pepducin, P4pal10, inhibited TF-initiated thrombin generation in PRP. Concentrations of P1pal12 and P4pal10, which blocked the thrombin-induced influx of extracellular calcium ions and inhibited platelet aggregation, reduced the rate of thrombin generation during the propagation phase by 38% and 36%, respectively. Whether this anticoagulant effect is relevant in inhibiting in vivo arterial thrombin growth is uncertain because P4pal10 prevented the incorporation of platelets in a growing thrombus.


CONCLUSIONS
Our findings suggest that in spite of their potential anticoagulant activities the in vivo antithrombotic effect of intracellular PAR pepducins is mainly based on inhibiting platelet-platelet interactions.
Wielders
S J H
SJ
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, the Netherlands.
Bennaghmouch
A
A
Reutelingsperger
C P M
CP
Bevers
E M
EM
Lindhout
T
T
D000818:Animals; D000925:Anticoagulants / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D017536:Carotid Artery, Common / Q000601:surgery; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D005343:Fibrinolytic Agents / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D066298:In Vitro Techniques; D008074:Lipoproteins / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D008297:Male; D051379:Mice; D018715:Microscopy, Video; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D044462:Receptors, Proteinase-Activated / Q000037:antagonists & inhibitors* / Q000378:metabolism; D018179:Receptors, Thrombin / Q000037:antagonists & inhibitors; D013917:Thrombin / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism; D013927:Thrombosis / Q000097:blood / Q000378:metabolism / Q000517:prevention & control; D013997:Time Factors
D000925:Anticoagulants; D005343:Fibrinolytic Agents; D008074:Lipoproteins; D010975:Platelet Aggregation Inhibitors; D044462:Receptors, Proteinase-Activated; D018179:Receptors, Thrombin; D013925:Thromboplastin; D013917:Thrombin; C112908:protease-activated receptor 4