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Interaction between major pathways for platelet activation in mouse models of arterial thrombosis

Periodic Report Summary 1 - in vivo thrombosis (Interaction between major pathways for platelet activation in mouse models of arterial thrombosis)

Platelets play a central role in arterial thrombosis, causing life-threatening and disabling diseases like myocardial infarction and ischemic stroke. A better understanding of the multiple mechanisms by which platelets can be activated is the basis for the rational development of new antithrombotic drugs.In the outgoing hosting institution (Dr. Coughlin's laboratory) it was previously demonstrated that protease-activated receptor (PAR) signaling in platelets is necessary for platelet activation by thrombin, a major platelet agonist, and it is important for hemostasis and thrombosis in mouse models. Dr. Coughlin and coworkers also demonstrated that thrombin signaling through PAR4 is critical for propagation of the platelet thrombus away from the vessel wall, but not for the formation of juxtamural platelet thrombi at the site of a vascular injury. The objectives of the study are: i) to identify the pathway triggering the iuxtamural platelet thrombi formation in the absence of PAR4 function and ii) to explore how the thrombin-PAR4 pathway relates to other effectors of hemostasis and thrombosis. The work performed since the beginning of the project has been focused on the interplay between the PAR4-thrombin pathway and the Glycoprotein (GP) VI-collagen pathway. Collagen exposed by vascular damage can interact with platelets via the receptors GPVI and a2ß1 integrin, but available data suggest that GPVI engagement may be the main activating signal. The originality of the project is mainly in the cutting-edge use of genetic engeneering techniques to address the question stated in the objectives of the study. Mice knock out for the genes encoding for PAR4 and GPVI were used. The following genotypes were compared to each other in the models of hemostasis and thrombosis detailed below:
Par4 -/-:Gp6I wt; Par4 -/-:Gp6+/-; Par4 -/-:Gp6 -/-
Par4 wt:Gp6 -/-; Par4 +/-:Gp6 -/-; Par4 -/-: Gp6 -/-
Hemostasis and thrombosis models and end points: Hemostasis
Bleeding time - blood loss
Neonatal hemorrage - survival, appearance
Maternal hemorrage (placentation) – survival
Maternal hemorrage (placental or vaginal at parturition) - survival, appearance
Occult blood in stools - guaiac positivity
Anemia – hematocrit
Thrombosis
Ferric-chloride-induced carotid thrombosis – Flow
Main results of the project. Some results are reported as graphs attached to this section. In summary:
- Par4/Gp6 double deficient animals are born at expected Mendelian distribution (Fig.1)
- Double deficient females are able to support pregnancies
- 35% of pups are born with transient abdominal bleeding (Fig.2)
- A group of Par4/Gp6 double deficient mice did show a mild anemia (Fig.3)
- Par4/Gp6 double deficient adult mice do not have spontaneous mucocutaneous bleeding (Fig.4)
- Deficiency of PAR4 alone causes an increased bleeding risk but also a better protection against thrombosis than deficiency of GPVI alone (fig 5 and 6). The combined deficiency of GPVI and PAR4 provides better protection against thrombosis in this model but at expenses of a higher bleeding risk after challenge (Fig.5 and 6).
Impact and social implications of the project. Despite antiplatelet therapies have been proven efficacious for primary and secondary prevention of cardio-and cerebrovascular diseases, arterial thrombosis remain a major health problem being the first cause of mortality and morbidity in western countries.The combination of two antiplatelet drugs, targeting different agonist/receptor pathways, is becoming a standard for antithrombotic therapies. This project will provide informations on in vivo safety (bleeding risk) and efficacy (protection against thrombosis) of a combination of drugs inhibiting the platelet activation mediated by the PAR/thrombin and GPVI/collagen pathways. Important informations are expected with an impact on the management of diseases of interest for cardiologists, internal medicine physicians, neurologists …