Calpain-dependent proteolysis of VASP and the PKA/PKG pathway modulate COAT platelet generation and phenotype

Introduction: Decreased or enhanced procoagulant platelet generation may lead to bleeding or thrombotic events, respectively. The role of the actin system in regulating platelet activation is only partially described. In this study, we investigated the role of Vasodilator-Stimulated Phosphoprotein (VASP), a key player in actin filament formation, in the dichotomous aggregating (AGG) and procoagulant (COAT) platelet generation.

Method: Platelets from healthy controls were activated in vitro with convulxin (CVX, collagen analogue) plus thrombin (THR) in 2.5 mM calcium while monitoring AGG and COAT platelet generation using PAC1 and Annexin V (AnV). Platelets were fixed and permeabilized at baseline and at different timepoints up to 8 min after activation. VASP protein and phosphorylation levels were assessed by flow cytometry using antibodies recognizing total VASP and VASP phosphorylation at serine 239 (pVASP-S239). COAT platelet generation was monitored after addition of proteasome/calpain inhibitors and protein kinase A (PKA) and G (PKG) activators/inhibitors.

Results: Upon CVX+THR stimulation, VASP protein level in COAT platelets significantly decreased, reaching 10.9%±2.9% of the baseline level 8 min after activation. However, pVASP-S239 decreased only down to 43.1%±8.9% of baseline. Calpain inhibitors (MG-132, calpeptin) but not proteasome inhibitors (bortezomib, carfilzomib) prevented the decrease in VASP protein level. Of note, COAT platelets generated in presence of calpain inhibitors were quantitatively unchanged. However, they retained 2.5 times higher AnV surface level and produced 3 times less AnV-positive microparticles. PKA/PKG activators (sodium nitroprusside, cilostazol, prostaglandin E1) dose-dependently increased pVASP-S239 and reduced COAT generation. High concentrations of PKA/PKG inhibitor (H89) decreased pVASP-S239 and induced spontaneous generation of COAT-like platelets ([Fig. 1]).

Fig. 1 Monitoring of VASP protein and phosphorylation levels in COAT platelets (PLTs) Temporal analysis by phosphoflow of (A) protein level and (B) phosphorylation at S239 of VASP in aggregatory (black) versus procoagulant COAT PLTs (grey). PLT incubation with calpain inhibitors MG-132 and calpeptin but not proteasome inhibitors bortezomib and carfilzomib prevents (C) VASP proteolysis, increases (D) Annexin V levels and (E) decreases microparticle generation in COAT PLTs. Incubation with PKA/PKG activators (F) inhibits COAT PLT generation while PKA/PKG inhibitor H89 increases COAT PLT generation at high dose. Data are shown as mean±SD (n=4-6) .

Conclusion: Calpain inhibition not only prevents the decrease in VASP protein level in COAT platelets, but it also maintains higher level of AnV on COAT platelet surface concomitantly to lowering microparticle generation. Thus, calpain inhibition could be used to maintain quantitative COAT platelet generation, localizing procoagulant surfaces at the site of clot formation while preventing unwanted coagulation/inflammation spreading through procoagulant microparticles. Moreover, increased PKA/PKG activity (as shown by high pVASP-S239 levels in treated resting platelets) inhibits COAT platelet generation, while decreased PKA/PKG activity in resting platelets spontaneously generates COAT-like platelets. This suggests that COAT platelet generation might be the consequence of the suppression of an intrinsic platelet inhibition mechanism. Here, we propose that modulation of calpain activity and PKA/PKG activity could be a promising strategy for fine-tuning platelet procoagulant phenotype.

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Artikel online veröffentlicht:
13. Februar 2025