Kamala Bhavaraju

Impaired activation of platelets lacking protein kinase C-θ isoform

Protein kinase C (PKC) isoforms have been implicated in several platelet functional responses, but the contribution of individual isoforms has not been thoroughly evaluated. Novel PKC isoform PKC-theta is activated by glycoprotein VI (GPVI) and protease-activated receptor (PAR) agonists, but not by adenosine diphosphate. In human platelets, PKC-theta-selective antagonistic (RACK; receptor for activated C kinase) peptide significantly inhibited GPVI and PAR-induced aggregation, dense and alpha-granule secretion at low agonist concentrations. Consistently, in murine platelets lacking PKC-theta, platelet aggregation and secretion were also impaired. PKC-mediated phosphorylation of tSNARE protein syntaxin-4 was strongly reduced in human platelets pretreated with PKC-theta RACK peptide, which may contribute to the lower levels of granule secretion when PKC-theta function is lost. Furthermore, the level of JON/A binding to activated alpha(IIb)beta(3) receptor was also significantly decreased in PKC-theta(-/-) mice compared with wild-type littermates. PKC-theta(-/-) murine platelets showed significantly lower agonist-induced thromboxane A(2) (TXA(2)) release through reduced extracellular signal-regulated kinase phosphorylation. Finally, PKC-theta(-/-) mice displayed unstable thrombus formation and prolonged arterial occlusion in the FeCl(3) in vivo thrombosis model compared with wild-type mice. In conclusion, PKC-theta isoform plays a significant role in platelet functional responses downstream of PAR and GPVI receptors.

Role of phosphoinositide 3-kinase β in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways

PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms alpha, beta, gamma and delta in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kbeta-selective inhibitor, but not by PIK75 (a PI3Kalpha inhibitor), AS252424 (a PI3Kgamma inhibitor) or IC87114 (a PI3Kdelta inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1-/- mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kbeta in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kbeta plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kbeta mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.

Protein Kinase Cδ Differentially Regulates Platelet Functional Responses

Objective—Protein Kinase C delta (PKC&dgr;) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors. The purpose of this study was to investigate the role of PKC&dgr; in platelets. Methods and Results—We evaluated the role of PKC&dgr; in platelets using two approaches—pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (&dgr; V1-1)TAT, and in the murine platelets lacking PKC&dgr;, PAR4-mediated dense granule secretion was inhibited, whereas GPVI-mediated dense granule secretion was potentiated. These effects were statistically significant in the absence and presence of thromboxane A2 (TXA2). Furthermore, TXA2 generation was differentially regulated by PKC&dgr;. However, PKC&dgr; had a small effect on platelet P-selectin expression. Calcium- and PKC-dependent pathways independently activate fibrinogen receptor in platelets. When calcium pathways are blocked by dimethyl-BAPTA, AYPGKF-induced aggregation in PKC&dgr; null mouse platelets and in human platelets pretreated with (&dgr; V1-1)TAT, was inhibited. In a FeCl3-induced injury in vivo thrombosis model, PKC&dgr;−/− mice occluded similar to their wild-type littermates. Conclusions—Hence, we conclude that PKC&dgr; differentially regulates platelet functional responses such as dense granule secretion and TXA2 generation downstream of PARs and GPVI receptors, but PKC&dgr; deficiency does not affect the thrombus formation in vivo.

Mechanism of Activation and Functional Role of Protein Kinase Cη in Human Platelets

The novel class of protein kinase C (nPKC) isoform η is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCη using pharmacological and gene knock-out approaches. nPKCη was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1 receptor antagonist, or YM-254890, a Gq blocker, abolished 2MeSADP-induced phosphorylation of nPKCη. Similarly, ADP failed to activate nPKCη in platelets isolated from P2Y1 and Gq knock-out mice. However, pretreatment of platelets with P2Y12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCη phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCη was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin αIIbβ3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a αIIbβ3 receptor antagonist, nPKCη dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cγ, a catalytic subunit of serine/threonine phosphatase, αIIbβ3 failed to dephosphorylate nPKCη. Thus, we conclude that ADP activates nPKCη via P2Y1 receptor and is subsequently dephosphorylated by PP1γ phosphatase activated by αIIbβ3 integrin. In addition, pretreatment of platelets with η-RACK antagonistic peptides, a specific inhibitor of nPKCη, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCη positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.

Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Calcium Mobilization and Thromboxane Generation in Platelets

Objective—Members of the protein kinase C (PKC) family are shown to positively and negatively regulate platelet activation. Although positive regulatory roles are extensively studied, negative regulatory roles of PKCs are poorly understood. We investigated the mechanism and specific isoforms involved in PKC-mediated negative regulation of ADP-induced functional responses. Methods and Results—A pan-PKC inhibitor, GF109203X, potentiated ADP-induced cPLA2 phosphorylation and thromboxane generation as well as ERK activation and intracellular calcium (Ca2+i) mobilization, 2 signaling molecules, upstream of cPLA2 activation. Thus, PKCs inhibit cPLA2 activation by inhibiting ERK and Ca2+i mobilization. Because the inhibitor of classic PKC isoforms, GO-6976, did not affect ADP-mediated thromboxane generation, we investigated the role of novel class of PKC isoforms. ADP-induced thromboxane generation, calcium mobilization, and ERK phosphorylation were potentiated in PKC&egr; null murine platelets compared with platelets from wild-type littermates. Interestingly, when thromboxane release is blocked, ADP-induced aggregation in PKC&egr; knockout and wild-type was similar, suggesting that PKC&egr; does not affect ADP-induced aggregation directly. PKC&egr; knockout mice exhibited shorter times to occlusion in an FeCl3-induced arterial injury model and shorter bleeding times in tail-bleeding experiments. Conclusion—We conclude that PKC&egr; negatively regulates ADP-induced thromboxane generation in platelets and offers protection against thrombosis.

Antagonism of P2Y12 reduces physiological thromboxane levels

Antiplatelet therapy for the management of patients with cardiovascular risks often includes a combination therapy of aspirin and clopidogrel, acting through inhibition of thromboxane generation and blockade of Gi-coupled P2Y12 receptor, respectively. We hypothesized that ADP acting through P2Y12 regulates physiological thromboxane levels. The serum thromboxane levels in mice (n = 3) dosed with clopidogrel and prasugrel were decreased by 83.1 ± 5.3% and 94.26 ± 1.75% respectively compared to untreated mice. Pre-treatment of human blood (n = 3) ex vivo with active metabolites of clopidogrel or prasugrel led to a reduction in thromboxane levels to 16.3 ± 3.2% and 4.9 ± 0.8% respectively, compared to untreated human serum. We also evaluated serum thromboxane levels in P2Y receptor null mice (n = 4). Whereas serum thromboxane levels in P2Y1 null mice were similar to those in wild type littermates, those in the P2Y12 null mice were inhibited by 83.15 ± 3.8%. Finally, in a pilot study, serum thromboxane levels were reduced by 76.05 ± 8.41% in healthy human volunteers (n = 6) upon dosing with clopidogrel, compared to the levels before dosing. In conclusion, P2Y12 antagonism alone can decrease physiological thromboxane levels. Thus, this study could pave way the for newer/modified treatment regimens for the management of patients with thrombotic complications who are allergic or non-responsive to aspirin.

G12/13 Signaling Pathways Substitute for Integrin αIIbβ3-Signaling for Thromboxane Generation in Platelets

Background We have previously shown that ADP-induced TXA2 generation requires signaling from αIIbβ3 integrin in platelets. Here we observed that, unlike ADP, protease-activated receptor (PAR)-mediated TXA2 generation occurs independently of αIIbβ3. PAR agonists, but not ADP, activate G12/13 signaling pathways. Hence, we evaluated the role of these pathways in TXA2 generation. Principal Findings Inhibition of ADP-induced thromboxane generation by fibrinogen receptor antagonist SC57101 was rescued by co-stimulation of G12/13 pathways with YFLLRNP. This observation suggested an existence of a common signaling effector downstream of integrins and G12/13 pathways. Hence, we evaluated role of three potential tyrosine kinases; c-Src, Syk and FAK (Focal Adhesion Kinase) that are known to be activated by integrins. c-Src and Syk kinase did not play a role in ADP-induced functional responses in platelets. Selective activation of G12/13 pathways resulted in the activation of FAK, in the absence of integrin signaling. Interestingly, αIIbβ3-mediated FAK activation occurred in a Src family kinase (SFK)-independent manner whereas G12/13 pathway caused FAK activation in a SFK and RhoA-dependent manner. A FAK selective inhibitor TAE-226, blocked TXA2 generation. However, in comparison to WT mice, Pf4-Cre/Fak-Floxed mice did not show any difference in platelet TXA2 generation. Conclusions Therefore, we conclude that differential activation of FAK occurs downstream of Integrins and G12/13 pathways. However, the common effector molecule, possibly a tyrosine kinase downstream of integrins and G12/13 pathways contributing to TXA2 generation in platelets remains elusive.