Mice with Reduced PAR4 Reactivity show Decreased Venous Thrombosis and Platelet Procoagulant Activity

Abstract

Background: Hypercoagulation and thrombin generation are major risk factors for venous thrombosis. Sustained thrombin signaling through PAR4 promotes platelet activation, phosphatidylserine exposure, and subsequent thrombin generation. A single-nucleotide polymorphism in PAR4 (rs2227376) changes proline to leucine extracellular loop 3 (P310L), which decreases PAR4 reactivity and is associated with a lower risk for venous thromboembolism (VTE) in a GWAS meta-analysis.

Objective: The goal of this study is to determine the mechanism for the association of rs2227376 with reduced risk for VTE in using mice with a homologous mutation (PAR4-P322L).

Methods: Venous thrombosis was examined using our recently generated PAR4-P322L mice in the inferior vena cava stasis and stenosis models. Coagulation and clot stability was measured using rotational thromboelastometry (ROTEM). Thrombin generating potential was measured in platelet-rich plasma. Phosphatidylserine surface expression and platelet-neutrophil aggregates were analyzed using flow cytometry.

Results: PAR4^P/L and PAR4^L/L had reduced incidence and size of venous clots at 48 hours. PAR4^P/L and PAR4^L/L platelets had progressively decreased phosphatidylserine in response to thrombin and convulxin, which led to decreased thrombin generation and decreased PAR4-mediated platelet-neutrophil aggregation.

Conclusions: The leucine allele in extracellular loop 3, PAR4-322L leads to fewer procoagulant platelets and decreased endogenous thrombin potential. This decreased ability to generate thrombin offers a mechanism for PAR4's role in VTE highlighting a key role for PAR4 signaling.

Keywords: animal model; blood platelets; protease-activated receptor 4; single nucleotide polymorphisms; thrombin receptor.

Figure 1.. PAR4-P322L Reduces Venous Thrombosis after Complete IVC Stasis.

(A) Venous clots within the inferior vena cava (IVC) were harvested from wild-type, heterozygous PAR4–322^P/L, homozygous PAR4^L/L, and global PAR4^−/− mice 48 hours after complete IVC ligation. (B) Clot incidence and (C) thrombus weight were also recorded 24 hours after IVC ligation. Dots represent individual mice. **p<0.01

Figure 2.. PAR4-P322L Reduces Venous Thrombosis after Partial IVC Stenosis.

(A) The presence or absence of a thrombus was recorded 48 hours after IVC stenosis. (B) Clots were also excised from the surrounding tissue and weighed. Dots represent individual mice. *p<0.05

Figure 3.. PAR4-P322L Mice have Smaller Thrombi after IVC Stenosis.

Venous clots were excised from the surrounding tissue, embedded in paraffin, and sectioned for (A) haematoxylin and eosin (A and B) or carstairs (C and D) staining. Images were taken at 4x magnification and stitched together when necessary to display the entire clot (panels A and C). The stitching is indicated with the vertical dotted line. Higher resolution images (200x) are presented in panels B and D.

Figure 4.. Blood from PAR4^−/− Mice Shows Impaired Clot Formation.

ROTEM was performed to measure coagulation parameters by generating a thromboelastogram. (A) representative traces for each genotype are shown. (B) Clotting time, (C) clot formation time, (D) maximum clot firmness, (E) alpha angle, and the amplitudes at (F) 10 and (G) 20 minutes were extracted from the ROTEM traces. Dots represent individual mice. *p<0.05, ****p<0.0001.

Figure 5.. PAR4-P322L Decreases Platelet-Mediated Thrombin Generation.

Platelet rich plasma (PRP) was obtained from wild-type, PAR4–322^P/L, PAR4–322^L/L, and PAR4^−/− mice and assessed for thrombin generating potential (A-D). (A) Thrombin generation curves were created using a fluorogenic thrombin substrate over 1 hour. (B) The area under the curve for each genotype was compared to the provided control human plasma (control high and control low) and interpreted as the total thrombin generated over that hour. (C) The peak concentration of thrombin generated and (D) the time it took to reach that peak was also recorded.

Figure 6.. PAR4-P322L Reduces Platelet Phosphatidylserine Surface Expression.

PS exposure was measured with flow cytometry using FITC-tagged Lactadherin. Washed mouse platelets were stimulated with convulxin (1 nM) in combination with 200 μM of the PAR4-AP (AYPGKF), 1 nM of thrombin, and 5 nM of thrombin. Lactadherin-positive events were recorded as the percentage of CD41-positive cells that were also positive for FITC. Dots represent individual mice. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 7.. PAR4-P322L Decreases Platelet-Neutrophil Aggregation.

Platelet-Neutrophil complexes were measured using flow cytometry to record the percentage of LyG+ neutrophils that also expressed CD41. Lysed whole blood from wild-type, PAR4–322^P/L, PAR4–322^L/L, and PAR4^−/− mice were incubated with with (A) no agonist, or (B) 50 μM, (C) 200 μM, or (D) 400 μM of the PAR4-agonist peptide (AYPGKF). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.