Turmeric (Curcuma longa), a herbal remedy and culinary spice, has been used in traditional Indian culture for millennia. An active ingredient found in turmeric is curcumin (diferuloylmethane). In the current study, we investigated the antiplatelet properties of this naturally occurring compound. Curcumin inhibited human platelet aggregation and dense granule secretion induced by GPVI agonist convulxin in a concentration-dependent manner. At 50 microM, it effectively inhibited the maximal extent of aggregation and dense granule secretion to as much as 75%. It also dramatically inhibited the activation-dependent tyrosine phosphorylation of Y753 and Y759 on PLCgamma2, but did not affect the phosphorylation of Y145 residue on the cytosolic adaptor protein SLP-76. Interestingly, curcumin had no significant effect on the phosphorylation of Y525/Y526 present on the activation loop of Syk (spleen tyrosine kinase), but had a significant inhibitory effect on in vitro Syk kinase activity. Moreover, the inhibitory action of curcumin is not due to an inhibition of thromboxane generation because all our studies were performed using aspirin-treated platelets. We conclude that curcumin inhibits platelet activation induced by GPVI agonists through interfering with the kinase activity of Syk and the subsequent activation of PLCgamma2.

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Protein kinase C-zeta (PKCζ), an atypical isoform of the PKC family of protein serine/threonine kinases, is expressed in human platelets. However, the mechanisms of its activation and the regulation of its activity in platelets are not known. We have found that under basal resting conditions, PKCζ has a high phosphorylation status at the activation loop threonine 410 (T410) and the turn motif (autophosphorylation site) threonine 560 (T560), both of which have been shown to be important for its catalytic activity. After stimulation with agonist under stirring conditions, the T410 residue was dephosphorylated in a time- and concentration-dependent manner, while the T560 phosphorylation remained unaffected. The T410 dephosphorylation could be significantly prevented by blocking the binding of fibrinogen to integrin αIIbβ3 with an antagonist, SC-57101; or by okadaic acid used at concentrations that inhibits protein serine/threonine phosphatases PP1 and PP2A in vitro. The dephosphorylation of T410 residue on PKCζ was also observed in PP1cγ null murine platelets after agonist stimulation, suggesting that other isoforms of PP1c or another phosphatase could be responsible for this dephosphorylation event. We conclude that human platelets express PKCζ, and it may be constitutively phosphorylated at the activation loop threonine 410 and the turn motif threonine 560 under basal resting conditions, which are differentially dephosphorylated by outside-in signaling. This differential dephosphorylation of PKCζ might be an important regulatory mechanism for platelet functional responses.

Smooth muscle myosin gene products include two isoforms, SMA and SMB, differing by a 7-residue peptide in loop 1 (i7) at the myosin active site where ATP is hydrolyzed. Using chicken isoforms, previous work indicated that the i7 deletion in SMA prolongs strong actin binding by inhibiting active site ingress and egress of nucleotide when compared to i7 inserted SMB. Additionally, i7 deletion inhibits Pi release associated with the switch 2 closed → open transition in actin-activated ATPase. Switch 2 is far from loop 1 indicating i7 deletion has an allosteric effect on Pi release. Chicken SMA and SMB have unknown and robust nucleotide-sensitive tryptophan (NST) fluorescence increments, respectively. Human SMA and SMB both lack NST increments while Pi release in Ca2+ ATPase is not impacted by i7 deletion. The NST reports relay helix movement following conformation change in switch 2 but in the open → closed transition. The NST is common to all known myosin isoforms except human smooth muscle. Other independent works on human SMA and SMB motility indicate no functional effect of i7 deletion. Smooth muscle myosin is a stunning example of species-specific myosin structure/function divergence underscoring the danger in extrapolating disease-linked mutant effects on myosin across species.