The importance of the second messengers calcium (Ca 2؉ ) and diacylglycerol (DAG) in platelet signal transduction was established more than 30 years ago. Whereas protein kinase C (PKC) family members were discovered as the targets of DAG, little is known about the molecular identity of the main Ca 2؉ sensor(s). We here identify Ca 2؉ and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) as a critical molecule in Ca 2؉ -dependent platelet activation. CalDAG-GEFI, through activation of the small GTPase Rap1, directly triggers integrin activation and extracellular signalregulated kinase-dependent thromboxane A 2 (TxA 2 ) release. CalDAG-GEFIdependent TxA 2 generation provides crucial feedback for PKC activation and granule release, particularly at threshold agonist concentrations. PKC/P2Y12 signaling in turn mediates a second wave of Rap1 activation, necessary for sustained platelet activation and thrombus stabilization. Our results lead to a revised model for platelet activation that establishes one molecule, CalDAG-GEFI, at the nexus of Ca 2؉ -induced integrin activation, TxA 2 generation, and granule release. The preferential activation of CalDAG-GEFI over PKC downstream of phospholipase C activation, and the different kinetics of CalDAG-GEFI-and PKC/P2Y12-mediated Rap1 activation demonstrate an unexpected complexity to the platelet activation process, and they challenge the current model that DAG/PKC-dependent signaling events are crucial for the initiation of platelet adhesion. IntroductionPlatelet thrombus formation at sites of vascular injury is a complex and dynamic process that occurs in several phases. 1,2 Platelet preactivation and tethering (transient adhesion) are mediated by the interaction of glycoprotein (GP) lb␣ with matrix-bound von Willebrand factor, particularly in conditions of high shear. Platelet activation and firm adhesion are then triggered by collagen exposed in the vessel wall and/or locally generated thrombin. Sustained integrin activation and the formation of stable thrombi further depend on persistent costimulatory signaling provided by the second wave agonists, thromboxane A 2 (TxA 2 ) and adenosine 5Ј-diphosphate (ADP), which are released from activated platelets. [3][4][5] The central biochemical event in platelet aggregation is the agonist-induced inside-out activation of ␣ IIb ␤ 3 integrin. Most platelet agonists initiate intracellular signaling through the activation of phospholipase C (PLC), followed by the generation of the second messengers calcium (Ca 2ϩ ) and diacylgycerol (DAG). DAG is critical for protein kinase C (PKC) activation, a key event in platelet granule release and integrin activation. Platelets express at least 6 PKC isoforms, which have activating as well as inhibitory roles in platelet activation. [6][7][8][9][10] Early studies with calcium ionophores established a role of Ca 2ϩ in integrin activation, TxA 2 generation, and granule release. 11-13 Initially, Ca 2ϩ -binding classical isoforms of PKC were suggested as the cell's main signal integ...

The intracellular Ca 2؉ concentration of many nonexcitable cells is regulated by calcium store release and store-operated calcium entry (SOCE). In platelets, STIM1 was recently identified as the main calcium sensor expressed in the endoplasmic reticulum. To evaluate the role of the SOC channel moiety, Orai1, in platelet SOCE, we generated mice expressing a mutated, inactive form of Orai1 in blood cells only (Orai1 R93W ). Platelets expressing Orai1 R93W were characterized by markedly reduced SOCE and impaired agonistinduced increases in [Ca 2؉ ] i . Orai1 R93W platelets showed reduced integrin activation and impaired degranulation when stimulated with low agonist concentrations under static conditions. This defect, however, did not significantly affect the ability of Orai1 R93W platelets to aggregate or to adhere to collagen under arterial flow conditions ex vivo. In contrast, these adherent Orai1 R93W platelets were defective in surface phosphatidylserine exposure, suggesting that Orai1 is crucial for the platelets' procoagulant response rather than for other Ca 2؉ -dependent cellular responses. (Blood. 2009;113:675-678) IntroductionIn electrically nonexcitable cells such as lymphocytes and platelets, the major mechanism for Ca 2ϩ entry is store-operated calcium entry (SOCE), a process controlled by the Ca 2ϩ concentration in the endoplasmic reticulum (ER). Depletion of Ca 2ϩ stores triggers activation of SOC channels in the plasma membrane. 1 Two major players in lymphocyte SOCE have recently been identified 2-6 : the 4-transmembrane-spanning calcium release-activated (CRAC) channel moiety Orai1 (CRACM1), and STIM1, a Ca 2ϩ sensor expressed predominantly in the ER.Orai1 deficiency in mice results in impaired Ca 2ϩ influx and grossly defective degranulation in mast cells, 7 and impaired SOCE and cytokine production in T cells. 8 A naturally occurring mutation in Orai1 (R91W), found in patients with severe combined immunodeficiency (SCID), led to a marked reduction in SOCE in lymphocytes. 4 In addition to Orai1, Orai2 and Orai3 are widely expressed in mammalian cells, and both molecules, when overexpressed together with STIM1, can form ion channels with properties similar to those of CRAC channels. 9,10 In platelets, STIM1 was recently identified as the major Ca 2ϩ sensor expressed in the ER. 11 The nature of the SOC channel regulated by STIM1, however, remains elusive. Among the candidate proteins are several members of the canonical transient potential channel (TRPC) family, 12,13 as well as Orai1. 14 In this study, we provide evidence for Orai1 as the major SOC channel expressed in platelets. Methods Mice and generation of fetal liver chimerasGene targeting of the Orai1 gene is described in detail in Document S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). Fetal liver cells were obtained from E14.5 mouse embryos derived from matings of Orai1 ϩ/R93W mice on the C57BL/6 background. Orai1 R93W/R93W and littermate wild-type control cells (CD45.2 ϩ ) were ...

Two major pathways contribute to Rasproximate-1-mediated integrin activation in stimulated platelets. Calcium and diacyglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI, Ras-GRP2) mediates the rapid but reversible activation of integrin ␣IIb␤3, while the adenosine diphosphate receptor P2Y12, the target for antiplatelet drugs like clopidogrel, facilitates delayed but sustained integrin activation. To establish CalDAG-GEFI as a target for antiplatelet therapy, we compared how each pathway contributes to thrombosis and hemostasis in mice. Ex vivo, thrombus formation at arterial or venous shear rates was markedly reduced in CalDAG-GEFI ؊/؊ blood, even in the presence of exogenous adenosine diphosphate and thromboxane A 2 . In vivo, thrombosis was virtually abolished in arterioles and arteries of CalDAG-GEFI ؊/؊ mice, while small, hemostatically active thrombi formed in venules. Specific deletion of the C1-like domain of CalDAG-GEFI in circulating platelets also led to protection from thrombus formation at arterial flow conditions, while it only marginally increased blood loss in mice. In comparison, thrombi in the micro-and macrovasculature of clopidogrel-treated wild-type mice grew rapidly and frequently embolized but were hemostatically inactive. Together, these data suggest that inhibition of the catalytic or the C1 regulatory domain in CalDAG-GEFI will provide strong protection from atherothrombotic complications while maintaining a better safety profile than P2Y12 inhibitors like clopidogrel. (Blood. 2011; 117(3):1005-1013) IntroductionArterial thrombosis in the coronary or cerebrovascular circulation is the principal pathological process underlying acute coronary syndrome and ischemic stroke, which together represent the leading cause of morbidity and mortality in industrialized countries. 1 Platelet activation is a central event in the pathogenesis of arterial thrombosis. Currently, the most powerful antiplatelet agents used in the clinic are inhibitors of cyclooxygenase-1 (acetylsalicylic acid, aspirin), the platelet adenosine diphosphate (ADP) receptor P2Y12 (eg, clopiodgrel or Plavix), and integrin ␣IIb␤3 (eg, abciximab or Reopro). 2,3 These agents have all been shown to improve clinical outcomes in large-scale randomized controlled trials. However, all therapies have limitations that include uncertainty about optimal dosing, questions about resistance, and issues regarding the lack of reversibility in situations where bleeding risks are high.␣IIb␤3, the platelet fibrinogen receptor, is the best-studied member of the integrin family. 4,5 Like most integrins, especially those regulating adhesion and trafficking of blood cells, it is expressed in a low-affinity state on resting platelets. Engagement of agonist receptors on the platelet surface triggers intracellular signaling events, which lead to inside-out activation of ␣IIb␤3. Deficiency in ␣IIb␤3 completely inhibits the ability of platelets to aggregate and adhere to sites of injury. 6,7 Consequently, inhibitors to integrin ␣IIb␤3 show...

We are interested in the controlled assembly of photoelectronic materials using peptides as scaffolds and porphyrins as the conducting material. We describe the integration of a peptide-based polymer strategy with the ability of designed basic peptides to bind anionic porphyrins in order to create regulated photoelectronically active biomaterials. We have described our peptide system in earlier work, which demonstrates the ability of a peptide to form filamentous materials made up of self-assembling coiled-coil structures. We have modified this peptide system to include lysine residues appropriately positioned to specifically bind meso-tetrakis(4-sulfonatophenyl)porphine (TPPS(4)), a porphyrin that contains four negatively charged sulfonate groups at neutral pH. We measure the binding of TPPS(4) to our peptide using UV--visible and fluorescence spectroscopies to follow the porphyrin signature. We determine the concomitant acquisition of helical secondary structure in the peptide upon TPPS(4) binding using circular dichroism spectropolarimetry. This binding fosters polymerization of the peptide, as shown by absorbance extinction effects in the peptide CD spectra. The morphologies of the peptide/porphyrin complexes, as imaged by atomic force microscopy, are consistent with the coiled-coil polymers that we had characterized earlier, except that the heights are slightly higher, consistent with porphyrin binding. Evidence for exciton coupling in the copolymers is shown by red-shifting in the UV--visible data, however, the coupling is weak based on a lack of fluorescence quenching in fluorescence experiments.

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The intracellular Ca2+ concentration of non-excitable cells is regulated by calcium store release and store-operated calcium entry (SOCE). In platelets, STIM1 was recently identified as the main calcium sensor expressed in the endoplasmatic reticulum. To evaluate the role of the SOC channel moiety, Orai1, in platelet SOCE, we generated mice expressing a mutated, inactive form of Orai1 in blood cells only (Orai1R93W). Platelets expressing Orai1R93W were characterized by markedly reduced SOCE and impaired agonist-induced increases in [Ca2+]i. Orai1R93W platelets showed reduced integrin activation and impaired degranulation when stimulated with low agonist concentrations under static conditions. This defect, however, did not significantly affect the ability of Orai1R93W platelets to aggregate or to adhere to collagen under arterial flow conditions ex vivo. In contrast, these adherent Orai1R93W platelets were defective in surface phosphatidylserine exposure, suggesting that Orai1 is crucial for the platelets pro-coagulant response rather than for other Ca2+-dependent cellular responses.

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