Protein Kinase Cδ Differentially Regulates Platelet Functional Responses.

Abstract

Protein Kinase Cδ (PKCδ), a novel PKC isoform is expressed and activated in platelets downstream of PARs and GPVI receptors. In the current study, the role of PKCδ in regulating platelet functional responses was investigated using a pharmacological inhibitor, (δV1-1)TAT (a PKCδ inhibitor) in human platelets. These studies were further confirmed by a knockout approach using PKCδ+/+ and PKCδ−/− mice. In both human and murine platelets, PAR4-mediated dense granule secretions were inhibited, whereas GPVI-mediated dense granule secretions were potentiated. Furthermore, α-granule secretions and thromboxane A2 (TXA2) generation were differentially regulated in murine platelets.. These data suggest a differential role for this isoform in regulating dense granule secretion, α-granule secretion and TXA2 generation. Previous studies have shown that PAR-mediated fibrinogen receptor activation is regulated by a Calcium-dependent and a PKC-dependent pathway. The contribution of PKCδ to PAR-mediated fibrinogen receptor activation was studied by pretreating human and murine platelets with BAPTA. Our results showed a inhibition of AYPGKF-induced aggregation in human and murine platelets in the presence of BAPTA and fibrinogen. These results suggest a small contribution of PKCδ to PAR-4- mediated platelet aggregation and aIIbb3 activation. The in vivo significance of PKCδ was tested using a FeCl3 injury model. While the wildtype mice occluded in 7 minutes, PKCδ −/− mice occluded after 4 minutes of injury with 10 % FeCl3. Therefore, we conclude that PKCδ regulates platelet functional responses such as dense, α-granule secretions, TXA2 generation downstream of both PARs and GPVI receptors, contributes to PAR-4-mediated fibrinogen receptor activation ex vivo and plays a critical role in the thrombus formation in vivo. This study is supported by predoctoral fellowships to Ramya Chari and Swaminathan Murugappan from American Heart Association, Great Rivers affiliate.

Diverse functions of protein kinase C isoforms in platelet activation and thrombus formation

“…Mochly‐Rosen and colleagues [32,33] have proposed that there are unique RACKs for each PKC isoform, and that distinct cellular localizations of these RACKs would bring each PKC isoform into proximity with a distinct set of substrates. Peptides based on the RACK‐binding site, which thereby act as competitive inhibitors of RACK–PKC interactions, have been developed as isoform‐specific inhibitors for many cell types, including platelets [9,20,34].…”

“…In contrast, Chari et al. [34] found enhanced GPVI‐dependent dense granule secretion in PKCδ −/− platelets, an effect replicated in human platelets by δ(V1‐1)‐TAT, a cell‐permeant peptide designed to block the interaction between PKCδ and its RACK. The reasons for this apparent difference are not clear.…”

Platelet-Derived Inhibitors of Platelet Activation

“…PKCδ has also been shown to negatively regulate GPVI induced platelet responses, as platelets from PKCδ deficient mice have enhanced aggregation and dense granule secretion in comparison to WT controls. This was also confirmed in human platelets using a cell permeable peptide that is designed to block the interaction of PKCδ and its RACK, δ(V1-1)-TAT (Chari et al, 2009). Contrasting reports however, also exist as other groups found no abnormality in GPVI-dependent dense granule secretion in PKCδ −/− platelets (Pula et al, 2006).…”

“…Contrasting reports however, also exist as other groups found no abnormality in GPVI-dependent dense granule secretion in PKCδ −/− platelets (Pula et al, 2006). PKCδ deficient platelets generate larger thrombi when measured in vitro but not when measured in vivo (Gilio et al, 2010, Chari et al, 2009. The overall role of PKCδ has therefore been difficult to define, possibly as a consequence of diverse positive and negative regulatory roles played by this PKC isoform.…”

Platelet Physiology

“…In particular, protein C kinase isoforms α and β support granule secretion, 104 while others differentially affect granule release. [104][105][106]…”