We earlier reported that the soluble form of the CD40 ligand (sCD40L), is involved in thrombosis by stabilizing platelet thrombi. In this article, we have determined the mechanism by which this protein affects platelet biology. Addition of sCD40L to washed platelets was found to activate the receptor function of ␣IIb␤3 as measured by the induction of fibrinogen binding and the formation of platelet microparticles. Mutation in the KGD sequence (D117E) of sCD40L, the ␣IIb␤3-binding domain in the N terminus of the protein resulted in a loss of the platelet-stimulatory activity of this protein. Integrilin, a ␣IIb␤3 antagonist, but not an antibody to CD40 that blocked the ligand-binding activity, inhibited these platelet-stimulatory events. CD40 ؊/؊ platelets bound fibrinogen and formed microparticles similar to WT platelets, again indicating that CD40 is not involved in sCD40L-induced platelet activation. Exposure of platelets to sCD40L, but not D117E-sCD40L-coated surfaces, induced platelet thrombi formation under arterial shear rate. sCD40L-induced platelet stimulation resulted in the phosphorylation of tyrosine-759 in the cytoplasmic domain of ␤3. Platelets from the diYF mouse strain, expressing ␤3 in which both cytoplasmic tyrosines are mutated to phenylalanine, were defective in sCD40L-induced platelet stimulation. These data indicate that sCD40L is a primary platelet agonist and that platelet stimulation is induced by the binding of the KGD domain of sCD40L to ␣IIb␤3, triggering outside-in signaling by tyrosine phosphorylation of ␤3.

The cholesterol-suppressive actions of Palmvitee and gamma-tocotrienol were assessed in hypercholesterolemic subjects after acclimation to the American Heart Association Step 1 dietary regimen for four and eight weeks, respectively. The four-week dietary regimen alone elicited a 5% decrease (P < 0.05) in the cholesterol level of the 36 subjects. Subjects continuing on the dietary regimen for a second four-week period experienced an additional 2% decrease in their cholesterol levels. Dietary assessments based on unanticipated recalls of 24-h food intake records suggest that significant reductions in energy and fat, predominantly in saturated fat, intakes are responsible. The subjects experienced significant Palmvitee- and gamma-tocotrienol-mediated decreases in cholesterol. The group of subjects acclimated to the dietary regimen for four weeks responded to Palmvitee (a blend of tocols providing 40 mg alpha-tocopherol, 48 mg alpha-tocotrienol, 112 mg gamma-tocotrienol, and 60 mg delta-to-cotrienol/day for four weeks) with a 10% decrease in cholesterol (P < 0.05). Dietary assessments showed no further change in energy and fat intakes. alpha-Tocopherol attenuated the cholesterol-suppressive action of the tocotrienols. The second group of subjects, acclimated to the dietary regimen for eight weeks, received 200 mg gamma-tocotrienol/d for four weeks. The cholesterol-suppressive potency of this alpha-tocopherol-free preparation was calculated to be equivalent to that of the mixture of tocotrienols (220 mg) used in the prior study. Cholesterol levels of the 16 subjects in the second group decreased 13% (P < 0.05) during the four-week trial. Plasma apolipoprotein B and ex vivo generation of thromboxane B2 were similarly responsive to the tocotrienol preparations, whereas neither preparation had an impact on high density lipoprotein cholesterol and apolipoprotein A-1 levels.

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Previous studies have shown that ligand or immunoaffinity chromatography can be used to purify the human platelet thromboxane A 2 (TXA 2 ) receptor-G␣ q complex. The same principle of co-elution was used to identify another G-protein associated with platelet TXA 2 receptors. It was found that in addition to G␣ q , purification of TXA 2 receptors by ligand (SQ31,491)-affinity chromatography resulted in the co-purification of a member of the G 12 family. Using an antipeptide antibody specific for the human G 13 ␣-subunit, this G-protein was identified as G␣ 13 . In separate experiments, it was found that the TXA 2 receptor agonist U46619 stimulated [35 S]guanosine 5-O-(3-thiotriphosphate) incorporation into G 13 ␣-subunit. Further evidence for functional coupling of G 13 to TXA 2 receptors was provided in studies where solubilized platelet membranes were subjected to immunoaffinity chromatography using an antibody raised against native TXA 2 receptor protein. It was found that U46619 induced a significant decrease in G␣ q and G␣ 13 association with the receptor protein.These results indicate that both G␣ q and G␣ 13 are functionally coupled to TXA 2 receptors and dissociate upon agonist activation. Furthermore, this agonist effect was specifically blocked by pretreatment with the TXA 2 receptor antagonist, BM13.505. Taken collectively, these data provide direct evidence that endogenous G␣ 13 is a TXA 2 receptor-coupled G-protein, as: 1) its ␣-subunit can be co-purified with the receptor protein using both ligand and immunoaffinity chromatography, 2) TXA 2 receptor activation stimulates GTP␥S binding to G␣ 13 , and 3) G␣ 13 affinity for the TXA 2 receptor can be modulated by agonist-receptor activation.

The present studies mapped the protein kinase A (PKA) phosphorylation site of G␣ 13 and studied the consequences of its phosphorylation. Initial experiments using purified human G␣ 13 and the PKA catalytic subunit established that PKA directly phosphorylates G␣ 13 . The location of this phosphorylation site was next investigated with a new synthetic peptide (G 13 SRI pep ) containing the PKA consensus sequence (Arg-Arg-ProThr 203 ) within the switch I region of G␣ 13 . G 13 SRI pep produced a dose-dependent inhibition of PKA-mediated G␣ 13 phosphorylation. On the other hand, the Thr-phosphorylated derivative of G 13 SRI pep possessed no inhibitory activity, suggesting that G␣ 13 Thr 203 may represent the phosphorylation site. Confirmation of this notion was obtained by showing that the G␣ 13 -T203A mutant (in COS-7 cells) could not be phosphorylated by PKA. Additional studies using co-elution affinity chromatography and co-immunoprecipitation demonstrated that G␣ 13 phosphorylation stabilized coupling of G␣ 13 with platelet thromboxane A 2 receptors but destabilized coupling of G␣ 13 to its ␤␥ subunits. In order to determine the functional consequences of this phosphorylation on G␣ 13 signaling, activation of the Rho pathway was investigated. Specifically, Chinese hamster ovary cells overexpressing human G␣ 13 wild type (G␣ 13 -WT) or G␣ 13 -T203A mutant were generated and assayed for Rho activation. It was found that 8-bromo-cyclic AMP caused a significant decrease (50%; p < 0.002) of Rho activation in G␣ 13 wild type cells but produced no change of basal Rho activation levels in the mutant (p > 0.4). These results therefore suggest that PKA blocks Rho activation by phosphorylation of G␣ 13 Thr 203 .Protein phosphorylation is a well established and ubiquitous mechanism for regulating protein function. Such regulation can impact cellular signaling at multiple levels including enzymatic activity, protein structure, protein translocation, and protein-protein interactions, among others. Regarding seventransmembrane receptors, evidence has been provided that phosphorylation of specific sites can serve to alter receptor-G protein coupling, initiate receptor internalization, and ultimately modulate cross-membrane receptor signaling (1). Whereas such effects of receptor phosphorylation have been well documented, considerably less is known regarding the prevalence or the possible signaling consequences of G protein phosphorylation (2-6). Nevertheless, several reports have provided evidence that G protein phosphorylation can alter heterotrimer complex formation and downstream signaling events. Specifically, Kozasa and Gilman (4) demonstrated that in vitro phosphorylation of G␣ 12 and G␣ z by PKC 1 results in conformational changes that inhibit interaction of the G␣ and G␤␥ subunits. A similar PKC-mediated phosphorylation effect on G␣-G␤␥ interaction was observed by Fields and Casey (3) for G␣ z and by Murthy et al. (6) for G␣ i1/2 . Offermanns et al. demonstrated that a PKC-dependent phosphorylation of G␣ 12 and G␣ 13 o...