CalDAG-GEFI Deficiency Reduces Atherosclerotic Lesion Development in Mice.

OBJECTIVE: Platelets are important for the development and progression of atherosclerotic lesions. However, relatively little is known about the contribution of platelet signaling to this pathological process. Our recent work identified 2 independent, yet synergistic, signaling pathways that lead to the activation of the small GTPase Rap1; one mediated by the guanine nucleotide exchange factor, CalDAG-GEFI (CDGI), the other by P2Y12, a platelet receptor for adenosine diphosphate and the target of antiplatelet drugs. In this study, we evaluated lesion formation in atherosclerosis-prone low-density lipoprotein receptor deficient (Ldlr(-/-)) mice lacking CDGI or P2Y12 in hematopoietic cells. APPROACH AND RESULTS: Lethally irradiated Ldlr(-/-) mice were reconstituted with bone marrow from wild-type (WT), Caldaggef1(-/-) (cdgI(-/-)), p2y12(-/-), or cdgI(-/-)p2y12(-/-) (double knockout [DKO]) mice and fed a high-fat diet for 12 weeks. Ldlr(-/-) chimeras deficient for CDGI or P2Y12 developed significantly smaller atherosclerotic lesions in the aortic sinus and in aortas when compared with the Ldlr(-/-)/WT controls. We also observed a significant reduction in platelet-leukocyte aggregates in blood from hypercholesterolemic Ldlr(-/-)/cdgI(-/-) and Ldlr(-/-)/p2y12(-/-) chimeras. Consistently, fewer macrophages and neutrophils were detected in the aortic sinus of Ldlr(-/-)/cdgI(-/-) and Ldlr(-/-)/ p2y12(-/-) chimeras. Compared with controls, the plaque collagen content was significantly higher in Ldlr(-/-) chimeras lacking CDGI. Interestingly, no statistically significant additive effects were seen in Ldlr(-/-)/DKO chimeras when compared with chimeras lacking only CDGI. CONCLUSIONS: Our findings suggest that CDGI is critical for atherosclerotic plaque development in hypercholesterolemic Ldlr(-/-) mice because of its contribution to platelet-leukocyte aggregate formation and leukocyte recruitment to the lesion area.

GDF-15 prevents platelet integrin activation and thrombus formation.

BACKGROUND: Integrin-mediated platelet function plays an important role in primary hemostasis. Growth-differentiation factor 15 (GDF-15) has been shown to inhibit ß(2) -integrin activation in leukocytes. METHODS: We investigated the effect of GDF-15 on platelet integrin activation in vitro and in different in vivo models of thrombus formation. RESULTS: GDF-15(-/-) mice showed an accelerated thrombus formation and a reduced survival rate after collagen-induced pulmonary thromboembolism. In reconstitution experiments, recombinant GDF-15 decelerated thrombus formation and prolonged the bleeding time. In vitro experiments demonstrated that GDF-15 pretreated, agonist-stimulated platelets showed decreased binding to fibrinogen in flow chamber assays and reduced activation of ß(1) - and ß(3) -integrins in flow cytometry experiments. Pretreating human and mouse platelets with GDF-15 reduced platelet aggregation. Mechanistically, GDF-15 prevents agonist-induced Rap1- dependent α(II) (b) ß(3) activation by activating PKA. Platelet P-selectin expression and dense granule secretion after stimulation were unaffected by GDF-15, indicating a specific effect of GDF-15 on integrin activation. CONCLUSION: GDF-15 specifically inhibits platelet integrin activation. These findings may have profound clinical implications for the treatment of hemostatic conditions involving platelets.

CalDAG-GEFI and protein kinase C represent alternative pathways leading to activation of integrin alphaIIbbeta3 in platelets.

Second messenger-mediated inside-out activation of integrin alphaIIbbeta3 is a key step in platelet aggregation. We recently showed strongly impaired but not absent alphaIIbbeta3-mediated aggregation of CalDAG-GEFI-deficient platelets activated with various agonists. Here we further evaluated the roles of CalDAG-GEFI and protein kinase C (PKC) for alphaIIbbeta3 activation in platelets activated with a PAR4 receptor-specific agonist, GYPGKF (PAR4p). Compared with wild-type controls, platelets treated with the PKC inhibitor Ro31-8220 or CalDAG-GEFI-deficient platelets showed a marked defect in aggregation at low (< 1mM PAR4p) but not high PAR4p concentrations. Blocking of PKC function in CalDAG-GEFI-deficient platelets, how-ever, strongly decreased aggregation at all PAR4p concentrations, demonstrating that CalDAG-GEFI and PKC represent separate, but synergizing, pathways important for alphaIIbbeta3 activation. PAR4p-induced aggregation in the absence of CalDAG-GEFI required cosignaling through the Galphai-coupled receptor for ADP, P2Y12. Independent roles for CalDAG-GEFI and PKC/Galphai signaling were also observed for PAR4p-induced activation of the small GTPase Rap1, with CalDAG-GEFI mediating the rapid but reversible activation of this small GTPase. In summary, our study identifies CalDAG-GEFI and PKC as independent pathways leading to Rap1 and alphaIIbbeta3 activation in mouse platelets activated through the PAR4 receptor.

Identification of P2Y12-dependent and -independent mechanisms of glycoprotein VI-mediated Rap1 activation in platelets.

Glycoprotein (GP) VI is a critical platelet collagen receptor, yet the steps involved in GPVI-mediated platelet activation remain incompletely understood. Because activation of Rap1, an abundant small guanosine triphosphatase (GTPase) in platelets, contributes to integrin alpha(IIb)beta(3) activation, we asked whether and how GPVI signaling activates Rap1 in platelets. Here we show that platelet Rap1 is robustly activated upon addition of convulxin, a GPVI-specific agonist. Using a reconstituted system in RBL-2H3 cells, we found that GPVI-mediated Rap1 activation is dependent on FcRgamma but independent of another platelet collagen receptor, alpha(2)beta(1). Interestingly, GPVI-mediated Rap1 activation in human platelets is largely dependent on adenosine diphosphate (ADP) signaling through the P2Y(12) and not the P2Y(1) receptor. However, experiments with specific ADP receptor antagonists and platelets from knockout mice deficient in P2Y(1) or the P2Y(12)-associated G-protein, Galphai(2), indicate that human and murine platelets also have a significant P2Y(12)-independent component of GPVI-mediated Rap1 activation. The P2Y(12)-independent component is dependent on phosphatidylinositol 3-kinase and is augmented by epinephrine-mediated signaling. P2Y(12)-dependent and -independent components are also observed in GPVI-mediated platelet aggregation, further supporting a role for Rap1 in aggregation. These results define mechanisms of GPVI-mediated platelet activation and implicate Rap1 as a key signaling protein in GPVI-induced platelet signaling.

The protein kinase A in platelets from patients with panic disorder.

Although previous studies suggested that dysfunctions in the protein kinase A (PKA) and in some of its substrates are associated with several psychiatric disorders, there is no evidence regarding the possible involvement of such components in panic disorder (PD). Thus, the aim of the present study was to investigate the levels of PKA and Rap1 in platelets from patients with such disorder. Twenty-four drug free patients with PD and 24 healthy volunteers participated to the study. Employing the Western Blot analysis, immunostaining and computer-assisted imaging, the levels of the regulatory (R, type I and type II) and the catalytic (C) subunits of PKA, and those of Rap1 were assessed in platelets from the two groups. The data show that patients with PD have significantly higher levels of platelet RI and C subunits of PKA than controls, whereas the levels of RII were unchanged. No significant differences were found in the immunolabelling of Rap1 between groups. These findings may provide clues toward understanding the involvement of cAMP signalling in anxiety disorders.

The cAMP-dependent protein kinase substrate Rap1 in platelets from patients with obsessive compulsive disorder or schizophrenia.

Previous studies have reported that the cAMP-dependent protein kinase and one of its substrates, namely Rap1, are altered in patients with affective disorders. Abnormalities in the cAMP-dependent protein kinase have also been reported in platelets of patients with obsessive compulsive disorder and schizophrenia. However, it remains to be determined whether abnormalities in Rap1 are specifically related to affective disorders or may also be present in schizophrenia and obsessive compulsive disorder. Thus, we investigated Rap1 in platelets from 12 drug-free patients with obsessive compulsive disorder, ten drug-free patients with schizophrenia, and 20 healthy subjects. While no difference was observed in the levels of Rap1 between groups, the phosphorylation state of Rap1 was significantly lower in patients with obsessive compulsive disorder than in schizophrenic patients and controls. These data further support the idea that abnormalities of cAMP signalling pathway could be associated, albeit in a somewhat different way, with several psychiatric disorders.

Altered Rap1 endogenous phosphorylation and levels in platelets from patients with bipolar disorder.

Previous studies have reported abnormalities either in the cAMP-dependent endogenous phosphorylation or in the levels of Rap1 in platelets from bipolar patients. One limitation of these findings was that they come from different groups of patients in independent studies. To overcome this limitation, we designed the present study in which both these biochemicals parameters were assessed in the same cohort of euthymic bipolar patients and healthy subjects. The results showed that the cAMP-dependent phosphorylation of Rap1 was significantly higher in platelets of bipolar patients with respect to healthy subjects. Furthermore, immunoblotting experiments revealed that also the levels of Rap1 were significantly higher in bipolar patients than in control subjects, thus supporting that the abnormal phosphorylation can be ascribed to the increased levels of Rap1. Taken together the results of the present study further support that downstream components of the cAMP signal cascade could be involved in the pathophysiology of bipolar disorders.