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Spleen tyrosine kinase (Syk) is expressed in a variety of hemopoietic cells. Upon phosphorylation of the platelet immunoreceptor-based activation motif of the glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor, both the tyrosine phosphorylation and activity of Syk are increased leading to downstream signaling events. Although it has been established that the activity of Syk is regulated by tyrosine phosphorylation, the specific roles of individual phosphorylation sites remain to be elucidated. We observed that Syk Y346 in mouse platelets was still phosphorylated when GPVIinduced Syk activity was inhibited. We then generated Syk Y346F mice and analyzed the effect this mutation exerts on platelet responses. Syk Y346F mice bred normally, and their blood cell count was unaltered. We did observe potentiation of GPVI-induced platelet aggregation and ATP secretion as well as increased phosphorylation of other tyrosines on Syk in the Syk Y346F mouse platelets when compared to WT littermates. This phenotype was specific for GPVI-dependent activation, since it was not seen when AYPGKF, a PAR4 agonist, or 2-MeSADP, a purinergic receptor agonist, was used to activate platelets. Despite a clear effect of Syk Y346F on GPVI-mediated signaling and cellular responses, there was no effect of this mutation on hemostasis as measured by tail-bleeding times, although the time to thrombus formation determined using the ferric chloride injury model was reduced. Thus, our results indicate a significant effect of Syk Y346F on platelet activation and responses in vitro and reveal its complex nature manifesting itself by the diversified translation of platelet activation into physiological responses. [ABSTRACT FROM AUTHOR]

Immune cells express receptors bearing an immune tyrosine activation motif (ITAM) containing two YXXL motifs or hemITAMs containing only one YXXL motif. Phosphorylation of the ITAM/hemITAM is mediated by Src family kinases allowing for the binding and activation of spleen tyrosine kinase (Syk). It is believed that Syk must be phosphorylated on tyrosine residues for activation, and Tyr342, а conserved tyrosine in the interdomain B region, has been shown to be critical for regulating Syk in FcεR1-activated mast cells. Syk is a key mediator of signaling pathways downstream of several platelet pathways including the ITAM bearing glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor and the hemITAM containing C-type lectin-like receptor-2 (CLEC-2). Since platelet activation is a crucial step in both hemostasis and thrombosis, we evaluated the importance of Syk Y342 in these processes by producing an Syk Y342F knock-in mouse. When using a CLEC-2 antibody as an agonist, reduced aggregation and secretion were observed in Syk Y342F mouse platelets when compared with control mouse platelets. Platelet reactivity was also reduced in response to the GPVI agonist collagen-related peptide. Signaling initiated by either GPVI or CLEC-2 was also greatly inhibited, including Syk Y519/520 phosphorylation. Hemostasis, as measured by tail bleeding time, was not altered in Syk Y342F mice, but thrombus formation in response to FeCl³ injury was prolonged in Syk Y342F mice. These data demonstrate that phosphorylation of Y342 on Syk following stimulation of either GPVI or CLEC-2 receptors is important for the ability of Syk to transduce a signal. [ABSTRACT FROM AUTHOR]

Platelets not only play an essential role in hemostasis after vascular injury but are also involved in the development of coronary artery disease (CAD) and cerebrovascular lesions. Patients with CAD and cerebral ischemia are recommended to undergo antiplatelet therapy, but they have an increased incidence of major bleeding complications. Both assessment of the platelet activation status and response to antiplatelet therapy in each patient are highly desired. β-Amyloid precursor protein (APP) 770 is expressed in vascular endothelial cells, and its extracellular region, a soluble form of APP770 (sAPP770, also called nexin-2), is proteolytically cleaved for shedding. Abundant sAPP770 is also released from activated platelets. In this study, we used peripheral blood samples from patients with CAD and control subjects and evaluated sAPP770 as a specific biomarker for platelet activation. First, the plasma levels of sAPP770 correlated well with those of the soluble form CD40 ligand (CD40L), an established biomarker for platelet activation. Additionally, flow cytometry analysis using peripheral blood cells showed that CD40L expression is up-regulated in activated T cells, whereas APP770 expression is negligible in all blood cell types except platelets. Following stimulation with collagen or ADP, aggregating platelets immediately released sAPP770. Finally, patients with dual antiplatelet therapy showed significantly lower levels of plasma sAPP770 than those with no therapy. Taken together, our data show that plasma sAPP770 could be a promising biomarker for platelet activation. [ABSTRACT FROM AUTHOR]

Proteinase-activated receptor (PAR)-4 is a member of the proteolytically-activated PAR family of G-protein-coupled receptors (GPCR) that represents an important target in the development of anti-platelet therapeutics. PARs are activated by proteolytic cleavage of their receptor N terminus by enzymes such as thrombin, trypsin, and cathepsin-G. This reveals the receptor-activating motif, termed the tethered ligand that binds intramolecularly to the receptor and triggers signaling. However, PARs are also activated by exogenous application of synthetic peptides derived from the tethered-ligand sequence. To better understand the molecular basis for PAR4-dependent signaling, we examined PAR4-signaling responses to a peptide library derived from the canonical PAR4-agonist peptide, AYPGKF-NH2, and we monitored activation of the Gαq/11-coupled calcium-signaling pathway, β-arrestin recruitment, and mitogen-activated protein kinase (MAPK) pathway activation. We identified peptides that are poor activators of PAR4-dependent calcium signaling but were fully competent in recruiting β-arrestin-1 and -2. Peptides that were unable to stimulate PAR4-dependent calcium signaling could not trigger MAPK activation. Using in silico docking and site-directed mutagenesis, we identified Asp230 in the extracellular loop-2 as being critical for PAR4 activation by both agonist peptide and the tethered ligand. Probing the consequence of biased signaling on platelet activation, we found that a peptide that cannot activate calcium signaling fails to cause platelet aggregation, whereas a peptide that is able to stimulate calcium signaling and is more potent for β-arrestin recruitment triggered greater levels of platelet aggregation compared with the canonical PAR4 agonist peptide. These findings uncover molecular determinants critical for agonist binding and biased signaling through PAR4. [ABSTRACT FROM AUTHOR]

Platelet factor 4 (PF4) is produced by platelets with roles in both inflammation and wound healing. PF4 is stored in platelet α-granules bound to the glycosaminoglycan (GAG) chains of serglycin. This study revealed that platelet serglycin is decorated with chondroitin/dermatan sulfate and that PF4 binds to these GAG chains. Additionally, PF4 had a higher affinity for endothelial-derived perlecan heparan sulfate chains than serglycin GAGchains. The binding of PF4 to perlecan was found to inhibit both FGF2 signaling and platelet activation. This study revealed additional insight into the ways in which PF4 interacts with components of the vasculature to modulate cellular events. [ABSTRACT FROM AUTHOR]

Extracellular fibrinogen-binding protein (Efb) from Staphylococcus aureus inhibits platelet activation, although its mechanism of action has not been established. In this study, we discovered that the N-terminal region of Efb (Efb-N) promotes platelet binding of fibrinogen and that Efb-N binding to platelets proceeds via two independent mechanisms: fibrinogen-mediated and fibrinogen-independent. By proteomic analysis of Efb-interacting proteins within platelets and confirmation by pulldown assays followed by immunoblotting, we identified P-selectin and multimerin-1 as novel Efb interaction partners. The interaction of both P-selectin and multimerin-1 with Efb is independent of fibrinogen. We focused on Efb interaction with P-selectin. Excess of P-selectin extracellular domain significantly impaired Efb binding by activated platelets, suggesting that P-selectin is the main receptor for Efb on the surface of activated platelets. Efb-N interaction with P-selectin inhibited P-selectin binding to its physiological ligand, P-selectin glycoprotein ligand-1 (PSGL-1), both in cell lysates and in cell-free assays. Because of the importance of P-selectin-PSGL-1 binding in the interaction between platelets and leukocytes, we tested human whole blood and found that Efb abolishes the formation of platelet-monocyte and platelet-granulocyte complexes. In summary, we present evidence that in addition to its documented antithrombotic activity, Efb can play an immunoregulatory role via inhibition of P-selectin-PSGL-1-dependent formation of platelet-leukocyte complexes. [ABSTRACT FROM AUTHOR]

Thrombin participates in coagulation, anticoagulation, and initiation of platelet activation. To fulfill its diverse roles and maintain hemostasis, this serine protease is regulated via the extended active site region and anion-binding exosites (ABEs) I and II. For the current project, amide proton hydrogen-deuterium exchange coupled with MALDI-TOF mass spectrometry was used to characterize ligand binding to individual exosites and to investigate the presence of exosite-active site and exositeexosite interactions. PAR3(44-56) and PAR1(49-62) were observed to bind to thrombin ABE I and then to exhibit long range effects over to ABE II. By contrast, Hirudin(54-65) focused more on ABE I and did not transmit influences over to ABE II. Although these three ligands were each directed to ABE I, they did not promote the same conformational consequences. D-Phe-Pro-Arg-chloromethyl ketone inhibition at the thrombin active site led to further local and long range consequences to thrombin-ABE I ligand complexes with the autolysis loop often most affected. When Hirudin(54-65) was bound to ABE I, it was still possible to bind GpIbα(269-286) or fibrinogen γ′(410-427) to ABE II. Each ligand exerted its predominant influences on thrombin and also allowed interexosite communication. The results obtained support the proposal that thrombin is a highly dynamic protein. The transmission of ligandspecific local and long range conformational events is proposed to help regulate this multifunctional enzyme. [ABSTRACT FROM AUTHOR]

The Src family kinases (SFKs) play essential roles in collagenand von Willebrand factor (VWF)-mediated platelet activation. However, the roles of SFKs in G protein-coupled receptor-mediated platelet activation and the molecular mechanisms whereby SFKs are activated by G protein-coupled receptor stimulation are not fully understood. Here we show that the thrombin receptor protease-activated receptor 4 agonist peptide AYPGKF elicited SFK phosphorylation in P2Y12 deficient platelets but stimulated minimal SFK phosphorylation in platelets lackingGq.We have previously shown that thrombin-induced SFK phosphorylation was inhibited by the calcium chelator 5,5'-dimethyl-bis- (o-aminophenoxy)ethane-N,N,Nα',N'-tetraacetic acid (dimethyl- BAPTA). The calcium ionophore A23187 induced SFK phosphorylation in both wild-type and Gq deficient platelets. Together, these results indicate that SFK phosphorylation in response to thrombin receptor stimulation is downstream from Gq/Ca2+ signaling. Moreover, A23187-induced thromboxane A2 synthesis, platelet aggregation, and secretion were inhibited by preincubation of platelets with a selective SFK inhibitor, PP2. AYPGKF-induced thromboxane A2 production in wild-type and P2Y12 deficient platelets was abolished by PP2, and AYPGKF-mediated P-selectin expression, integrin αIIbβ3 activation, and aggregation of P2Y12 deficient platelets were partially inhibited by the PKC inhibitor Ro-31-8220, PP2, dimethyl- BAPTA, or LY294002, but were abolished by Ro-31-8220 plus PP2, dimethyl-BAPTA, or LY294002. These data indicate that Ca2+/SFKs/PI3K andPKCrepresent two alternative signaling pathways mediating Gq-dependent platelet activation. [ABSTRACT FROM AUTHOR]

The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (Kd) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triplehelical (GPO)10-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ∼29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ∼39 and ∼44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation. [ABSTRACT FROM AUTHOR]

The platelet activation receptor CLEC-2 plays crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis, although its role in thrombosis/hemostasis remains controversial. An endogenous ligand for CLEC-2, podoplanin, is expressed in lymphatic endothelial cells (LECs). We and others have reported that CLEC-2-deficiency is lethal at mouse embryonic/ neonatal stages associated with blood-filled lymphatics, indicating that CLEC-2 is essential for blood/lymphatic vessel separation. However, its mechanism, and whether CLEC-2 in platelets is necessary for this separation, remains unknown. We found that specific deletion of CLEC-2 from platelets leads to the misconnection of blood/lymphatic vessels. CLEC-2+2+ platelets, but not by CLEC-2+2+ platelets, inhibited LEC migration, proliferation, and tube formation but had no effect on human umbilical vein endothelial cells. Additionally, supernatants from activated platelets significantly inhibited these three functions in LECs, suggesting that released granule contents regulate blood/lymphatic vessel separation. Bone morphologic protein-9 (BMP-9), which we found to be present in platelets and released upon activation, appears to play a key role in regulating LEC functions. Only BMP-9 inhibited tube formation, although other releasates including transforming growth factor-βand platelet factor 4 inhibited proliferation and/or migration. We propose that platelets regulate blood/lymphatic vessel separation by inhibiting the proliferation, migration, and tube formation of LECs, mainly because of the release of BMP-9 upon activation by CLEC-2/podoplanin interaction [ABSTRACT FROM AUTHOR]

The localization of the platelet glycoprotein GP Ib-IX complex (GP Ibα, GP Ibβ, and GP IX) to membrane lipid domain, also known as glycosphingolipid-enriched membranes (GEMs or raft) lipid domain, is essential for the GP Ib-IX complex mediated platelet adhesion to von Willebrand factor (vWf) and subsequent platelet activation. To date, the mechanism for the complex association with the GEMs remains unclear. Although the palmitate modifications of GP Ibβ and GP IX were thought to be critical for the complex presence in the GEMs, we found that the removal of the putative palmitoylation sites of GP Ibβ and GP IX had no effects on the localization of the GP Ib-IX complex to the GEMs. Instead, the disruption of GP Ibα disulfide linkage with GP Ibβ markedly decreased the amount of the GEM-associated GP Ibα without altering the GEM association of GP Ibβ and GP IX. Furthermore, partial dissociation with the GEMs greatly inhibited GP Ibα interaction with vWf at high shear instead of in static condition or under low shear stress. Thus, for the first time, we demonstrated that GP Ibβ/GP IX mediates the disulfide-linked GP Ibα localization to the GEMs, which is critical for vWf interaction at high shear. [ABSTRACT FROM AUTHOR]

In response to blood vessel injury, hemostasis is initiated by platelet activation, advanced by thrombin generation, and tempered by fibrinolysis. The primary fibrinolytic protease, plasmin, can be activated either on a fibrin-containing thrombus or on cells. Annexin A2 (A2) heterotetramer (A2p11)2 is a key profibrinolytic complex that assembles plasminogen and tissue plasminogen activator and promotes plasmin generation. We now report that, in endothelial cells, plasmin specifically induces activation of conventional PKC, which phosphorylates serine 11 and serine 25 of A2, triggering dissociation of the (A2 p11)2 tetramer. The resulting free p11 undergoes ubiquitin-mediated proteasomal degradation, thus preventing further translocation of A2 to the cell surface. In vivo, pretreatment of A2+/+ but not A2-/- mice with a conventional PKC inhibitor significantly reduced thrombosis in a carotid artery injury model. These results indicate that augmentation of fibrinolytic vascular surveillance by blockade of serine phosphorylation is A2-dependent. We also demonstrate that plasmin-induced phosphorylation of A2 requires both cleavage of A2 and activation of Toll-like receptor 4 on the cell surface. We propose that plasmin can limit its own generation by triggering a finely tuned "feedback" mechanism whereby A2 becomes serine-phosphorylated, dissociates from p11, and fails to translocate to the cell surface. [ABSTRACT FROM AUTHOR]

A pivotal step in the mitochondrial pathway of apoptosis is activation of Bak and Bax, although the molecular mechanism remains controversial. To examine whether mitochondrial apoptosis can be induced by just a lack of antiapoptotic Bcl-2-like proteins or requires direct activators of the BH3-only proteins including Bid and Bim, we studied the molecular requisites for platelet apoptosis induced by Bcl-xL deficiency. Severe thrombocytopenia induced by thrombocyte-specific Bcl-xL knock-out was fully rescued in a Bak and Bax double knock-out background but not with single knock-out of either one. In sharp contrast, deficiency of either Bid, Bim, or both did not alleviate thrombocytopenia in Bcl-xL knock-out mice. An in vitro study revealed that ABT-737, a Bad mimetic, induced platelet apoptosis in association with a conformational change of the amino terminus, translocation from the cytosol to mitochondria, and homo-oligomerization of Bax. ABT-737-induced Bax activation and apoptosis were also observed in Bid/Bim-deficient platelets. Human platelets, upon storage, underwent spontaneous apoptosis with a gradual decline of Bcl-xL expression despite a decrease in Bid and Bim expression. Apoptosis was attenuated in Bak/Bax-deficient or Bcl-xL-overexpressing platelets but not in Bid/Bim-deficient platelets upon storage. In conclusion, platelet lifespan is regulated by a fine balance between anti- and proapoptotic multidomain Bcl-2 family proteins. Despite residing in platelets, BH3-only activator proteins Bid and Bim are dispensable for Bax activation and mitochondrial apoptosis. [ABSTRACT FROM AUTHOR]

The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors. [ABSTRACT FROM AUTHOR]

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent a-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen. [ABSTRACT FROM AUTHOR]

Cbl-b, a member of the Cbl family of E3 ubiquitin ligases, plays an important role in the activation of lymphocytes. However, its function in platelets remains unknown. We show that Cbl-b is expressed in human platelets along with c-Cbl, but in contrast to c-Cbl, it is not tyrosine-phosphorylated upon glycoprotein VI (GPVI) stimulation. Cbl-b, unlike c-Cbl, is not required for Syk ubiquitylation downstream of GPVI activation. Phospholipase Cγ2 (PLCγ2) and Bruton's tyrosine kinase (BTK) are constituently associated with Cbl-b. Cbl-b-deficient (Cbl-b-/-) platelets display an inhibition in the concentration-response curve for GPVI-specific agonist-induced aggregation, secretion, and Ca2+ mobilization. A parallel inhibition is found for activation of PLCγ2 and BTK. However, Syk activation is not affected by the absence of Cbl-b, indicating that Cbl-b acts downstream of Syk but upstream of BTK and PLCγ2. When Cbl-b-/- mice were tested in the ferric chloride thrombosis model, occlusion time was increased and clot stability was reduced compared with wild type controls. These data indicate that Cbl-b plays a positive modulatory role in GPVI-dependent platelet signaling, which translates to an important regulatory role in hemostasis and thrombosis in vivo. [ABSTRACT FROM AUTHOR]

Integrin αIIbβ3 affinity regulation by talin binding to the cytoplasmic tail of β3 is a generally accepted model for explaining activation of this integrin in Chinese hamster ovary cells and human platelets. Most of the evidence for this model comes from the use of multivalent ligands. This raises the possibility that the activation being measured is that of increased clustering of the integrin rather than affinity. Using a newly developed assay that probes integrins on the surface of cells with only monovalent ligands prior to fixation, I do not find increases in affinity of αIIbβ3 integrins by talin head fragments in Chinese hamster ovary cells, nor do I observe affinity increases in human platelets stimulated with thrombin. Binding to a multivalent ligand does increase in both of these cases. This assay does report affinity increases induced by either Mn2+ a cytoplasmic domain mutant (D723R) in the cytoplasmic domain of β3, or preincubation with a peptide ligand. These results reconcile the previously observed differences between talin effects on integrin activation in Drosophila and vertebrate systems and suggest new models for talin regulation of integrin activity in human platelets. [ABSTRACT FROM AUTHOR]

Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase Cγ2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3Kα or -13 strongly and selectively suppressed GPVI-induced Ca2+ mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase Cγ2 by PI3Kαβ. That PI3Kα and -β have a non-redundant function in GPVI- induced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca2+ increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3Kα/β was blocked or p85α was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3Kδ or -γ. Furthermore, PI3Kα/β, but not PI3Kγ, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3Kα and -β isoforms are required for full GPVI-dependent platelet Ca2+ signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3Kα an interesting new target for anti-platelet therapy. [ABSTRACT FROM AUTHOR]

The cellular and molecular pathways that regulate platelet activation, blood coagulation, and inflammation are emerging as critical players in cancer progression and metastasis. Here, we demonstrate a novel signaling mechanism whereby protease-activated receptor 1 (PAR!) mediates expression of melanoma cell adhesion molecule MCAM/MUC18 (MUC18), a critical marker of melanoma metastasis, via activation of platelet-activating factor receptor (PAFR) and cAMP-responsive elementbinding protein (CREB). We found that PAR1 silencing with small hairpin RNA inhibits MUC18 expression in metastatic melanoma cells by inhibiting CREB phosphorylation, activity, and binding to the MUC18 promoter. We further demonstrate that the PAF/PAFR pathway mediates MUC18 expression downstream of PAR1. Indeed, PAR1 silencing down-regulates PAFR expression and PAF production, PAFR silencing blocks MUC18 expression, and re-expression of PAFR in PAR1-silenced cells rescues MUG 18 expression. We further demonstrate that the PAR1-PAFR-MUC18 pathway mediates melanoma cell adhesion to microvascular endothelial cells, transendothelial migration, and metastatic retention in the lungs. Rescuing PAFR expression in PAR1-silenced cells fully restores metastatic phenotype of melanoma, indicating that PAFR plays critical role in the molecular mechanism of PAR1 action. Our results link the two pro-inflammatory G-proteincoupled receptors, PAR1 and PAFR, with the metastatic dissemination of melanoma and suggest that PAR 1, PAFR, and MUG 18 are attractive therapeutic targets for preventing melanoma metastasis. [ABSTRACT FROM AUTHOR]

Lysophosphatidic acid (LPA) is a ligand for LPA[sub1-3] of the endothelial differentiation gene family G-protein-coupled receptors, and LPA[sub4-8] is related to the purinergic family G-protein-coupled receptor. Because the structure-activity relationship (SAR) of GPR92/LPA[sub5] is limited and whether LPA is its preferred endogenous ligand has been questioned in the literature, in this study we applied a combination of computational and experimental site-directed. mutagenesis of LPA[sub5] residues predicted to interact with the headgroup of LPA. Four residues involved in ligand recognition in LPA[sub5] were identified as follows R2.60N mutant abolished receptor activation, whereas H4.64E, R6.62A, and R7.32A greatly reduced receptor activation. We also investigated the SAR of LPA[sub5] using LPA analogs and other non-lysophospholipid ligands. SAR revealed that the rank order of agonists is alkyl glycerol phosphate > LPA > farnesyl phosphates ⪢ N-arachidonoylglycine. These results confirm LPA[sub5] to be a bonafide lysophospholipid receptor. We also evaluated several compounds with previously established selectivity for the endothelial differentiation gene receptors and found several that are LPA[sub5] agonists. A pharmacophore model of LPA[sub5] binding requirements was developed for in silico screening, which identified two non-lipid LPA[sub5] antagonists. Because LPA[sub5] transcripts are abundant in human platelets, we tested its antagonists on platelet activation and found that these non-lipid LPA[sub5] antagonists inhibit platelet activation, The present results suggest that selective inhibition of LPA[sub5] may provide a basis for future anti-thrombotic therapies. [ABSTRACT FROM AUTHOR]

ADP plays an integral role in the process of hemostasis by signaling through two platelet G-protein-coupled receptors, P2Y[sub1] and P2Y[sub12]. The recent use of antagonists against these two receptors has contributed a substantial body of data characterizing the ADP signaling pathways in human platelets. Specifically, the results have indicated that although P2Y[sub1] receptors are involved in the initiation of platelet aggregation, P2Y[sub12] receptor activation appears to account for the bulk of the ADP-mediated effects. Based on this consideration, emphasis has been placed on the development of a new class of P2Y[sub12] antagonists (separate from clopidogrel and ticlopidine) as an approach to the treatment of thromboembolic disorders. The present work examined the molecular mechanisms by which two of these widely used adenosine-based P2Y[sub1] antagonists (2-methylthioadenosine 5'-monophosphate triethylammonium salt (2MeSAMP) and ARC69931MX), inhibit human platelet activation. It was found that both of these compounds raise platelet cAMP to levels that substantially inhibit platelet aggregation. Furthermore, the results demonstrated that this elevation of cAMP did not require G[subi] signaling or functional P2Y[sub12] receptors but was mediated through activation of a separate G protein-coupled pathway, presumably involving G[subs]. However, additional experiments revealed that neither 2MeSAMP nor ARC69931MX (cangrelor) increased cAMP through activation of A2a, IP, DP, or EP[sub2] receptors, which are known to couple to G[subs]. Collectively, these findings indicate that 2MeSAMP and ARC69931MX interact with an unidentified platelet G protein-coupled receptor that stimulates cAMP-mediated inhibition of platelet function. This inhibition is in addition to that derived from antagonism of P2Y[sub12] receptors. [ABSTRACT FROM AUTHOR]

Platelet factor 4 (PF4) is an abundant platelet α-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the γ-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q/R679Q and FVa-R306Q/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg306 by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation. [ABSTRACT FROM AUTHOR]

Human plasma platelet-activating factor (PAF) acetylhydrolase functions by reducing PAF levels as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma, and allergic reactions. The enzyme has also been implicated in hydrolytic activities of other pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids. This plasma enzyme is tightly bound to low and high density lipoprotein particles and is also referred to as lipoprotein-associated phospholipase A2. The crystal structure of this enzyme has been solved from x-ray diffraction data collected to a resolution of 1.5Å. It has a classic lipase α/β-hydrolase fold, and it contains a catalytic triad of Ser273, His351, and Asp296. Two clusters of hydrophobic residues define the probable interface-binding region, and a prediction is given of how the enzyme is bound to lipoproteins. Additionally, an acidic patch of 10 carboxylate residues and a neighboring basic patch of three residues are suggested to play a role in high density lipoprotein/low density lipoprotein partitioning. A crystal structure is also presented of PAF acetylhydrolase reacted with the organophosphate compound paraoxon via its active site Ser273. The resulting diethyl phosphoryl complex was used to model the tetrahedral intermediate of the substrate PAF to the active site. The model of interface binding begins to explain the known specificity of lipoprotein-bound substrates and how the active site can be both close to the hydrophobic-hydrophilic interface and at the same time be accessible to the aqueous phase. [ABSTRACT FROM AUTHOR]

We previously demonstrated that human platelets activated with SFLLRN release PAR-1 activation peptide, PAR-1-(1-41), even in the presence of hirudin. This observation suggests that during their activation, platelets generate a protease that activates PAR-1. In this study, PAR-1 and -4 activation peptides were detected 10 s after ≤1.0 n α-thrombin, 10 μ SFLLRN, or 100 μ AYPGKF were added to platelets. When SFLLRN or AYGPKF were added to platelets, generation of PAR-1 and -4 activation peptides was complete at 10 s. Generation of both PAR-1 and -4 activation peptides in response to 1 n α-thrombin was significantly inhibited by affinity-purified anti-PAR-1-(35-62) IgY, anti-PAR-4-(34-54) IgY, and by the specific PAR-1 antagonistBMS200261, but not by the PAR-4 antagonist YD3. Effective inhibition of platelet aggregation in response to 1.0 n α-thrombin occurred only in the presence of both anti-PAR span antibodies. We conclude that platelet activation initiated with ≤1.0 n α-thrombin proceeds via simultaneous PAR-1 and -4 activation. Inhibiting the activation of either PAR inhibits activation of the other. Both PAR-1 and -4 activation must be inhibited to prevent platelet activation subsequent to thrombin binding to platelets. The more efficient generation of PAR activation peptides by platelets activated with SFLLRN or AYGPKF, compared with α-thrombin, indicates that a platelet-derived serine protease that is inactivated by soybean trypsin inhibitor propagates PAR-1 and -4 activation. [ABSTRACT FROM AUTHOR]

Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR! and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via α2A-adrenergic receptors to provoke aggregation, secretion, and Ca2+ mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pa1-il abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1- activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X1, P2Y1, and P2Y12 (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephnne combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR 1, interacts synergistically with α2A-adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated plate- lets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X1-receptor and the α2A-adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-fadiitated PAR4-mediated platelet activation. [ABSTRACT FROM AUTHOR]

Platelet-activating factor (PAP) is a potent lipid mediator playing various inflammatory and physiological roles. PAF is biosynthesized through two independent pathways called the de novo and remodeling pathways. Lyso-PAF acetyltransferase (lyso-PAF AT) was believed to biosynthesize PAF under inflammatory conditions, through the remodeling pathway. The first isolated lyso-PAF AT (LysoPAFAT/LPCAT2) had consistent properties. However, we show in this study the finding of a second lyso-PAF AT working under noninflammatory conditions. We partially purified a Ca2~-independent lyso-PAF AT from mouse lung. Immunoreactivity for lysophosphatidylcholine acyltransferase 1 (LPCAT1) was detected in the active fraction. Lpcatl-transfected Chinese hamster ovary cells exhibited both LPCAT and lyso-PAF AT activities. We confirmed that LPCAT1 transfers acetate from acetyl-CoA to lyso-PAF by the identification of an acetyl-CoA (and other acyl-CoAs) interacting site in LPCAT1. We further showed that LPCAT1 activity and expression are independent of inflammatory signals. Therefore, these results suggest the molecular diversity of lyso-PAF ATs is as follows: one (LysoPAFAT/LPCAT2) is inducible and activated by inflammatory stimulation, and the other (LPCAT1) is constitutively expressed. Each lyso-PAF AT biosynthesizes inflammatory and physiological amounts of PAF, depending on the cell type. These findings provide important knowledge for the understanding of the diverse pathologi- cal and physiological roles of PAF. [ABSTRACT FROM AUTHOR]

GTPase-activating proteins are required to terminate signaling by Rap1, a small guanine nucleotide-binding protein that controls integrin activity and cell adhesion. Recently, we identified Rap1GAP2, a GTPase-activating protein of Rap1 in platelets. Here we show that 14-3-3 proteins interact with phosphorylated serine 9 at the N terminus of Rap1GAP2. Platelet activation by ADP and thrombin enhances serine 9 phosphorylation and increases 14-3-3 binding to endogenous Rap1GAP2. Conversely, inhibition of platelets by endothelium-derived factors nitric oxide and prostacyclin disrupts 14-3-3 binding. These effects are mediated by cGMP- and cAMP-dependent protein kinases that phosphorylate Rap1GAP2 at serine 7, adjacent to the 14-3-3 binding site. 14-3-3 binding does not change the GTPase-activating function of Rap1GAP2 in vitro. However, 14-3-3 binding attenuates Rap1GAP2 mediated inhibition of cell adhesion. Our findings define a novel crossover point of activatory and inhibitory signaling pathways in platelets. [ABSTRACT FROM AUTHOR]

Podoplanin (aggrus), a transmembrane sialoglycoprotein, is involved in tumor cell-induced platelet aggregation, tumor metastasis, and lymphatic vessel formation. However, the mechanism by which podoplanin induces these cellular processes including its receptor has not been elucidated to date. Podoplanin induced platelet aggregation with a long lag phase, which is dependent upon Src and phospholipase Cγ2 activation. However, it does not bind to glycoprotein VI. This mode of platelet activation was reminiscent of the snake toxin rhodocytin, the receptor of which has been identified by us as a novel platelet activation receptor, C-type lectin-like receptor 2 (CLEC.-2) (Suzuki-Inoue, K., Fuller, G. L., Garcia, A., Eble, J. A., Pohlmann, S., Inoue, O., Gartner, T. K., Hughan, S. C., Pearce, A. C., Laing, G. D., Theakston, R. D., Schweighoffer, E., Zitzmann, N., Morita, T., Tybulewicz, V. L., Ozaki, Y., and Watson, S. P. (2006) Blood 107, 542-549). Therefore, we sought to evaluate whether CLEC-2 serves as a physiological counterpart for podoplanin. Association between CLEC-2 and podoplanin was confirmed by flow cytometry. Furthermore, their association was dependent on sialic acid on O-glycans of podoplanin. Recombinant CLEC-2 inhibited platelet aggregation induced by podoplanin-expressing tumor cells or lymphatic endothelial cells, suggesting that CLEC-2 is responsible for platelet aggregation induced by endogenously expressed podoplanin on the cell surfaces. These findings suggest that CLEC-2 is a physiological target protein of podoplanin and imply that it is involved in podoplanin-induced platelet aggregation, tumor metastasis, and other cellular responses related to podoplanin. [ABSTRACT FROM AUTHOR]

The protein kinase C (PKC) family is an essential signaling mediator in platelet activation and aggregation. However, the relative importance of the major platelet PKC isoforms and their downstream effectors in platelet signaling and function remain unclear. Using isolated human platelets, we report that PKCδ, but not PKCα or PKCβ, is required for collagen-induced phospholipase C-dependent signaling, activation of αIIbβ3, and platelet aggregation. Analysis of PKCδ phosphorylation and translocation to the membrane following activation by both collagen and thrombin indicates that it is positively regulated by αIIbδ3 outside-in signaling. Moreover, PKCδ triggers activation of the mitogen-activated protein kinase-kinase (MEK)/extracellular-signal regulated kinase (ERK) and the p38 MAPK signaling. This leads to the subsequent release of thromboxane A2, which is essential for collagen-induced but not thrombin-induced platelet activation and aggregation. This study adds new insight to the role of PKCs in platelet function, where PKCδ signaling, via the MEK/ERK and p38 MAPK pathways, is required for the secretion of thromboxane A2. [ABSTRACT FROM AUTHOR]

Regulation of platelet activation plays a central role in hemostasis and pathophysiological processes such as coronary artery disease. Thrombin is the most potent activator of platelets. Human platelets express two thrombin receptors, PAR1 and PAR4, both of which signal platelet activation. Evidence is lacking on the mechanism by which PAR1 and PAR4 may differentially signal platelet aggregation. Here we show that at the relatively high concentration of agonist most likely found at the site of a local thrombus, dual inhibition of the P2Y12 receptor and calcium mobilization result in a complete inhibition of PAR4-induced aggregation, while having no effect on either thrombin or PAR1-mediated platelet aggregation. Both PAR1- and PAR4-mediated aggregation are independent of calcium mobilization. Furthermore, we show that P2Y12 receptor activation is not required for protease-activated receptor-mediated aggregation at higher agonist concentrations and is only partially required for Rap1 as well as GPIIbIIIa activation. P2Y12 receptor inhibitors clinically in use such as clopidogrel are postulated to decrease platelet aggregation through partial inhibition of PAR1 signaling. Our data, however, indicate that at high local concentrations of thrombin, it is the signaling through PAR4 rather than PAR1 that may be regulated through purinergic feedback. Thus, our data identify an intra-platelet mechanism that may function as a future site for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Phosphoinositide 3-kinase (PI3K) and Akt play important roles in platelet activation. However, the downstream mechanisms mediating their functions are unclear. We have recently shown that nitric-oxide (NO) synthase 3 and cGMP-dependent protein kinase stimulate platelet secretion and aggregation. Here we show that PI3K-mediated Akt activation plays an important role in agonist- stimulated platelet NO synthesis and cGMP elevation. Agonist-induced elevation of NO and cGMP was inhibited by Akt inhibitors and reduced in Akt-1 knock-out platelets. Akt-1 knock-out or Akt inhibitor-treated platelets showed reduced platelet secretion and aggregation in response to low concentrations of agonists, which can be reversed by low concentrations of 8-bromo-cGMP or sodium nitroprusside (an NO donor). Similarly, PI3K inhibitors diminished elevation of cGMP and inhibited platelet secretion and the second wave platelet aggregation, which was also partially reversed by 8-bromo-cGMP. These results indicate that the NO-cGMP pathway is an important downstream mechanism mediating PI3K and Akt signals leading to platelet secretion and aggregation. Conversely, the PI3K-Akt pathway is the major upstream mechanism responsible for activating the NO-cGMP pathway in platelets. Thus, this study delineates a novel platelet activation pathway involving sequential activation of PI3K, Akt, nitric-oxide synthase 3, sGC, and cGMP-dependent protein kinase. [ABSTRACT FROM AUTHOR]

Platelets achieve bleeding arrest at sites of vascular injury via secretion of secretory proteins from their storage granules, termed α-granules. We have recently analyzed granule targeting of platelet factor 4 (PF4), a secretory α-granule chemokine, and demonstrated that PF4 α-granule storage relied upon determinants within PF4 mature sequence. To define these determinants, PF4 mutants fused to the fluorescent reporter protein green fluorescent protein were generated by progressive deletions and site-directed mutagenesis. They were then transfected in AtT20 cells and assessed for granule targeting by colocalization with ACTH-containing granules, using laser scanning confocal microscopy. This strategy identified the amino acid 41–50 (LIATLKNGRK) sequence as most critical for PF4 granule targeting and/or storage; its deletion from PF4 induced a marked decrease in granule storage (from 81 ± 2%te 17 ± 3%,p ≤ 0.0001).Ala-scanning mutagenesis of LIATLKNGRK narrowed down the targeting motif to LKNG. A direct role for LKNG in α-granule targeting was confirmed in the thrombopoietin-induced human megakaryocytic Dami cells, in which the LKNG. green fluorescent protein chimera exhibited an 82.5 ± 1.8% colocalization with the α-granule proteins yon Willebrand factor and P-selectin. LKNG is poorly conserved within the chemokine family. However three-dimensional alignments of the human α-granule chemokines Nap-2 (neutrophil-activating peptide) and RANTES (Regulated upon Activation Normal T Cell Expressed and Secreted) with PF4 revealed that LKNG, a surface-exposed hydrophilic turn/loop, matched Nap-2 (LKDG) and RANTES (TRKN) peptides with similar features. Moreover Nap-2 and RANTES peptides exhibited the same α-granule targeting efficiency than LKNG. We therefore postulate that the three-dimensional and physicochemical characteristics of PF4 LKNG are of general relevance to α-granule targeting of chemokines and possibly of other α-granule proteins. [ABSTRACT FROM AUTHOR]

Vav family proteins are guanine nucleotide exchange factors for the Rho/Rac family of small GTP-binding proteins. In addition, they have domains that mediate protein-protein interactions, including one Src homology 2 (SH2) and two Src homology 3 (SH3) domains. Vavl, Vav2, and Vav3 play a crucial role in the regulation of phospholipase Cγ (PLCγ) isoforms by immuno-tyrosine-based activation motif (ITAM)-coupled receptors, in- cluding the T- and B-cell antigen receptors. We have reported in platelets, however, that Vavl and Vav2 are not required for activation of PLCγ2 in response to stimulation of the ITAM-coupled collagen receptor glycoprotein VI (GPVI). Here we report that Vav3 is tyrosine-phosphorylated upon activation of GPVI but that Vav3-deficient platelets also exhibit a normal response upon activation of the ITAM receptor. In sharp contrast, platelets deficient in both Vavl and Vav3 show a marked inhibition of aggregation and spreading upon activation of GPVI, which is associated with a reduction in tyrosine phosphorylation of PLCγ2. The phenotype of Vavl/ 2/3 triple-deficient platelets is similar to that of Vavl/3 double-deficient cells. These results demonstrate that Vav3 and Vavl play crucial but redundant roles in the activation of PLCγ2 by GPVI. This is the first time that absolute redundancy between two protein isoforms has been observed with respect to the regulation of PLCγ2 in platelets. [ABSTRACT FROM AUTHOR]

Platelet secretion (exocytosis) is critical in amplifying platelet activation, in stabilizing thrombi, and in arteriosclerosis and vascular remodeling. The signaling mechanisms leading to secretion have not been well defined. We have shown previously that cGMP-dependent protein kinase (PKG) plays a stimulatory role in platelet activation via the glycoprotein Ib-IX pathway. Here we show that PKG also plays an important stimulatory role in mediating aggregation-dependent platelet secretion and secretion-dependent second wave platelet aggregation, particularly those induced via Gq-coupled agonist receptors, the thromboxane A2 (TXA2) receptor, and protease-activated receptors (PARs). PKG I knock-out mouse platelets and PKG inhibitor-treated human platelets showed diminished aggregation-dependent secretion and also showed a diminished secondary wave of platelet aggregation induced by a TXA2 analog and thrombin receptor-activating peptides that were rescued by the granule content ADP. Low dose collagen-induced platelet secretion and aggregation were also reduced by PKG inhibitors. Furthermore PKG I knockout and PKG inhibitors significantly attenuated activation of the Gi pathway that is mediated by secreted ADP. These data unveil a novel PKG-dependent platelet secretion pathway and a mechanism by which PKG promotes platelet activation. [ABSTRACT FROM AUTHOR]

ATP binding cassette transporter A1 (ABCA1) is involved in regulation of intracellular lipid trafficking and export of cholesterol from cells to high density lipoproteins. ABCA1 defects cause Tangier disease, a disorder characterized by absence of high density lipoprotein and thrombocytopenia. In the present study we have demonstrated that ABCA1 is expressed in human platelets and that fibrinogen binding and CD62 surface expression in response to collagen and low concentrations of thrombin, but not to ADP, are defective in platelets from Tangier patients and ABCA1-deficient animals. The expression of platelet membrane receptors such as GPVI, α2β1 integrin, and GPIIb/IIIa, the collagen-induced changes in phosphatidylserine and cholesterol distribution, and the collagen-induced signal transduction examined by phosphorylation of LAT and p72syk and by intracellular Ca2+ mobilization were unaltered in Tangier platelets. The electron microscopy of Tangier platelets revealed reduced numbers of dense bodies and the presence of giant granules typically encountered in platelets from Chediak­Higashi syndrome. Further studies demonstrated impaired release of dense body content in platelets from Tangier patients and ABCA1-deficient animals. In addition, Tangier platelets were characterized by defective surface exposure of dense body and lysosomal markers (CD63, LAMP-1, LAMP-2, CD68) during collagen- and thrombin-induced stimulation and by abnormally high lysosomal pH. We conclude that intact ABCA1 function is necessary for proper maturation of dense bodies in platelets. The impaired release of the content of dense bodies may explain the defective activation of Tangier platelets by collagen and low concentrations of thrombin, but not by ADP. [ABSTRACT FROM AUTHOR]

A critical role for the conserved α-integrin cytoplasmic motif, KVGFFKR, is recognized in the regulation of activation of the platelet integrin αIIbβ3. To understand the molecular mechanisms of this regulation, we sought to determine the nature of the protein interactions with this cytoplasmic motif. We used a tagged synthetic peptide, biotin-KVGFFKR, to probe a high density protein expression array (37,200 recombinant human proteins) for high affinity interactions. A number of potential integrin-binding proteins were identified. One such protein, a chloride channel regulatory protein, Icln, was characterized further because its affinity for the integrin peptide was highest as was its expression in platelets. We verified the presence of Icln in human platelets by PCR, Western blots, immunohistochemistry, and its co-association with αIIbβ3 by surface plasmon resonance. The affinity of this interaction was 82.82 ± 24.4 nM in a cell free assay. Icln co-immunoprecipitates with αIIbβ3 in platelet lysates demonstrating that this interaction is physiologically relevant. Furthermore, immobilized KVGFFKR peptides, but not control KAAAAAR peptides, specifically extract Icln from platelet lysates. Acyclovir (100 μM to 5 mM), a pharmacological inhibitor of the Icln chloride channel, specifically inhibits integrin activation (PAC-1 expression) and platelet aggregation without affecting CD62 P expression confirming a specific role for Icln in integrin activation. In parallel, a cell-permeable peptide corresponding to the potential integrin-recognition domain on Icln (AKFEEE, 10–100 μM) also inhibits platelet function. Thus, we have identified, verified, and characterized a novel functional interaction between the platelet integrin and Icln, in the platelet membrane. [ABSTRACT FROM AUTHOR]

Platelet activation triggers integrin αIIbβ3-dependent signals and the induction of tyrosine phosphorylation of the cytoskeletal protein α-actinin. We have previously reported that α-actinin is phosphorylated by the focal adhesion kinase (FAK) (Izaguirre, G., Aguirre, L., Hu, Y.-P., Lee, H. Y., Schlaepfer, D. D., Aneskievich, B. J., and Haimovich, B. (2001) J. Biol. Chem. 276, 28676-28685). In this study, a phosphatase of 68 kDa that dephosphorylated α-actinin in vitro was isolated from platelet lysates by three sequential chromatography steps. The phosphatase was identified as SHP-1 by electrospray tandem mass spectrometry. α-Actinin was dephosphorylated in vitro by recombinant SHP-1 and by SHP-1 immunoprecipitated from unstimulated or thrombin-stimulated platelet lysates. SHP-1 immunoprecipitated from lysates of platelets adherent to fibrinogen, however, failed to dephosphorylate α-actinin. In contrast, the activity of SHP-1 against a synthetic substrate was not affected by the mode of platelet activation. The robust and sustained phosphorylation of α-actinin detected in platelets adherent to fibrinogen thus correlates with a decrease in the activity of SHP-1 toward it. Tyrosine phosphorylation of α-actinin is seen in vanadate-treated COS-7 cells that are co-transfected with α-actinin and wild type FAK. Triple transfection of the cells with cDNAs encoding for α-actinin, FAK, and wild type SHP-1 abolished the phosphorylation of α-actinin. The phosphorylation of FAK, however, was barely affected by the expression of wild type SHP-I. Both α-actinin and FAK were phosphorylated in cells co-expressing α-actinin, FAK, and a catalytic domain mutant (C453S) of SHP-1. These findings establish that SHP-1 can dephosphorylate α-actinin in vitro and in vivo and suggest that SHP-1 may regulate the tethering of receptors to the cytoskeleton and/or the extent of cross-linking of actin filaments in cells such as platelets. [ABSTRACT FROM AUTHOR]

Activation of factor XI (FXI) by thrombin on stimulated platelets plays a physiological role in hemostasis, providing additional thrombin generation required in cases of severe hemostatic challenge. Using a collection of 53 thrombin mutants, we identified 16 mutants with <50% of the wild-type thrombin FXI-activating activity in the presence of dextran sulfate. These mutants mapped to anion-binding exosite (ABE) I, ABE-II, the Na[sup +]-binding site, and the 50-insertion loop. Only the ABE-II mutants showed reduced binding to dextran sulfate-linked agarose. Selected thrombin mutants in ABE-I (R68A, R70A, and R73A), ABE-II (R98A, R245A, and K248A), the 50-insertion loop (W50A), and the Na[sup +]binding site (E229A and R233A) with <10% of the wild-type activity also showed a markedly reduced ability to activate FXI in the presence of stimulated platelets. The ABE-I, 50-insertion loop, and Na[sup +]-binding site mutants had impaired binding to FXI, but normal binding to glycocalicin, the soluble form of glycoprotein Ibα (GPIbα). In contrast, the ABE-II mutants were defective in binding to glycocalicin, but displayed normal binding to FXI. Our data support a quaternary complex model of thrombin activation of FXI on stimulated platelets. Thrombin bound to one GPIbα molecule, via ABE-II on its posterior surface, is properly oriented for its activation of FXI bound to a neighboring GPIα molecule, via ABE-I on its anterior surface. GPIbα plays a critical role in the co-localization of thrombin and FXI and the resultant efficient activation of FXI. [ABSTRACT FROM AUTHOR]

Proposes a novel mechanism by which platelet activation may contribute to the development of atherosclerosis through the release of platelet factor 4 (PF4). Identification of accumulation of low-density lipoprotein (LDL) -derived cholesterol by macrophages in vessel walls as a pathogenic feature of atherosclerotic lesions; Conclusion that PF4 engances the binding of oxidized LDL to vascular wall cells.