Novel Strategy to Combat the Procoagulant Phenotype in Heparin-Induced Thrombocytopenia Using 12-LOX Inhibition.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a major concern for all individuals that undergo cardiac bypass surgeries or require prolonged heparin exposure. HIT is a life- and limb-threatening adverse drug reaction with an immune response following the formation of ultra-large immune complexes that drive platelet activation through the receptor FcγRIIA. Thrombotic events remain high following the standard of care treatment with anticoagulants, while increasing risk of bleeding complications. This study sought to investigate a novel approach to treatment of HIT. Recent reports demonstrate increased procoagulant activity in HIT; however, these reports required analysis ex vivo, and relevance in vivo remains unclear. METHODS: Using human and mouse model systems, we investigated the cooperativity of PARs (protease-activated receptors) and FcγRIIA in HIT. We challenged humanized FcγRIIA transgenic mice with or without endogenous mouse Par4 (denoted as IIA-Par4+/+ or IIA-Par4-/-, respectively) with a well-established model IgG immune complex (anti [α]-CD9). Furthermore, we assessed the procoagulant phenotype and efficacy to treat HIT utilizing inhibitor of 12-LOX (12[S]-lipoxygenase), VLX-1005, previously reported to decrease platelet activation downstream of FcγRIIA and PAR4, using the triple allele HIT mouse model. RESULTS: IIA-Par4+/+ mice given αCD9 were severely thrombocytopenic, with extensive platelet-fibrin deposition in the lung. In contrast, IIA-Par4-/- mice had negligible thrombocytopenia or pulmonary platelet-fibrin thrombi. We observed that pharmacological inhibition of 12-LOX resulted in a significant reduction in both platelet procoagulant phenotype ex vivo, and thrombocytopenia and thrombosis in our humanized mouse model of HIT in vivo. CONCLUSIONS: These data demonstrate for the first time the need for dual platelet receptor (PAR and FcγRIIA) stimulation for fibrin formation in HIT in vivo. These results extend our understanding of HIT pathophysiology and provide a scientific rationale for targeting the procoagulant phenotype as a possible therapeutic strategy in HIT.

Aspirin pharmacokinetics and pharmacodynamics through gestation.

BACKGROUND: Low dose aspirin is recommended for prevention of preeclampsia, however there is not consensus on the appropriate dose. Pregnancy specific changes have the potential to impact the pharmacology of aspirin in pregnancy, however there are very limited studies on aspirin pharmacokinetics in pregnancy and none linking pharmacokinetics (PK, drug dose and drug level) to pharmacodynamics (PD, drug dose and physiologic response) in pregnancy. As a result, we do not have a good understanding of the pharmacologic response to aspirin in pregnancy, which has important implications for clinical efficacy. We sought to describe the PK and PD of aspirin through pregnancy and to identify individual covariates that impacted aspirin PK/PD. OBJECTIVE: We sought to describe the PK and PD of aspirin through pregnancy (first and third trimester), to identify covariates that significantly impact aspirin PK and to identify the relationship between aspirin PK and PD. STUDY DESIGN: This is a prospective study of patients at high risk for preeclampsia recommended to take 81 mg aspirin daily. This study involved 3 visits as follows: (1) baseline, first trimester (10-16 weeks of gestation) 6-hour PK visit, done before initiation of aspirin; (2) follow-up 1: 2 to 4 weeks after aspirin initiation; and (3) follow-up 2: third trimester 6-hour PK visit (28-32 weeks of gestation). The following were assessed at each visit: weight or body mass index, platelet function analysis-100 (Siemens), urinary thromboxane B2, serum thromboxane B2, and plasma salicylic acid. The PK visits consisted of blood work done at baseline (predose), administration of 81 mg nonenteric coated aspirin, and then plasma blood level of salicylic acid assessed at 30 minutes and then hourly 1 to 6 hours after dose. Pearson correlation and multivariable regression were used to identify associations between parameters and identify relevant covariates. Log-adjusted values were used for regression analysis. P<.05 was considered statistically significant. RESULTS: Nineteen participants were included with first trimester data, and 16 with third trimester data. There was no statistically significant change in mean PK parameters between the first and third trimester, although there was a trend to lower peak concentration in the third than in the first trimester (P=.08). In multivariable regression, baseline obesity and current body mass index as a continuous measures were negatively associated with log-adjusted peak salicylic acid concentration (-0.28 [-0.46 to -0.11], P=.003 and -0.02 [-0.03 to -0.009], P=.001, respectively) and log-adjusted plasma salicylic acid area under the curve 0 to 6 hours postdose (-0.25 [-0.45 to 0.05], P=.02, -0.04 [-0.07 to -0.01], P=.008 respectively). There was a significant decrease in urinary thromboxane 2 to 4 weeks after aspirin initiation compared with baseline, which correlated with a concomitant increase in platelet function analysis-100 closure time. In multivariable regression model, there was a strong association between plasma salicylic acid concentration (area under the curve 0-6 hours postdose) and urinary thromboxane (B=-3.12 [-5.38 to -1.04], P=.006), and with urinary thromboxane suppression and platelet inhibition, platelet function analysis-100 (-0.23 [-0.31 to -0.14], P<.001). With progressive thromboxane suppression, platelet inhibition (platelet function analysis-100 closure time) increased. Individual comorbidities, including weight, hypertension, and pregestational diabetes (Type I or II), also impacted aspirin response. CONCLUSION: We have demonstrated the relationship between individual factors, plasma concentrations of salicylic acid, thromboxane suppression, and platelet inhibition at a single dose (81 mg) of aspirin taken through pregnancy. Our findings suggest that dose modification of aspirin in pregnancy may help to achieve the optimal response. Our results may be used to facilitate computational modeling to identify optimal dosing, taking into consideration individual factors.

Species Differences in Platelet Protease-Activated Receptors.

Protease-activated receptors (PARs) are a class of integral membrane proteins that are cleaved by a variety of proteases, most notably thrombin, to reveal a tethered ligand and promote activation. PARs are critical mediators of platelet function in hemostasis and thrombosis, and therefore are attractive targets for anti-platelet therapies. Animal models studying platelet PAR physiology have relied heavily on genetically modified mouse strains, which have provided ample insight but have some inherent limitations. The current review aims to summarize the notable PAR expression and functional differences between the mouse and human, in addition to highlighting some recently developed tools to further study human physiology in mouse models.

The Function of ASK1 in Sepsis and Stress-Induced Disorders.

Apoptosis signal-regulating kinase 1 (ASK1) is a serine-threonine kinase that is ubiquitously expressed in nucleated cells and is responsible for the activation of multiple mitogen-activated protein kinases (MAPK) to regulate cell stress. Activation of ASK1 via cellular stress leads to activation of downstream signaling components, activation of transcription factors, and proinflammatory cytokine production. ASK1 is also expressed in anucleate platelets and is a key player in platelet activation as it is important for signaling. Interestingly, the mechanism of ASK1 activation is cell type-dependent. In this review we will explore how ASK1 regulates a variety of cellular processes from innate immune function to thrombosis and hemostasis. We will discuss how ASK1 influences FcγRIIA-mediated platelet reactivity and how that reactivity drives platelet clearance. Furthermore, we will explore the role of ASK1 in thromboxane (TxA2) generation, which highlights differences in the way ASK1 functions in mouse and human platelets.

FcγRIIA expression accelerates nephritis and increases platelet activation in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease characterized by deposits of immune complexes (ICs) in organs and tissues. The expression of FcγRIIA by human platelets, which is their unique receptor for immunoglobulin G antibodies, positions them to ideally respond to circulating ICs. Whereas chronic platelet activation and thrombosis are well-recognized features of human SLE, the exact mechanisms underlying platelet activation in SLE remain unknown. Here, we evaluated the involvement of FcγRIIA in the course of SLE and platelet activation. In patients with SLE, levels of ICs are associated with platelet activation. Because FcγRIIA is absent in mice, and murine platelets do not respond to ICs in any existing mouse model of SLE, we introduced the FcγRIIA (FCGR2A) transgene into the NZB/NZWF1 mouse model of SLE. In mice, FcγRIIA expression by bone marrow cells severely aggravated lupus nephritis and accelerated death. Lupus onset initiated major changes to the platelet transcriptome, both in FcγRIIA-expressing and nonexpressing mice, but enrichment for type I interferon response gene changes was specifically observed in the FcγRIIA mice. Moreover, circulating platelets were degranulated and were found to interact with neutrophils in FcγRIIA-expressing lupus mice. FcγRIIA expression in lupus mice also led to thrombosis in lungs and kidneys. The model recapitulates hallmarks of human SLE and can be used to identify contributions of different cellular lineages in the manifestations of SLE. The study further reveals a role for FcγRIIA in nephritis and in platelet activation in SLE.

Cleavage of anti-PF4/heparin IgG by a bacterial protease and potential benefit in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is due to immunoglobulin G (IgG) antibodies, which bind platelet factor 4 (PF4) modified by polyanions, such as heparin (H). IgG/PF4/polyanion complexes directly activate platelets via Fc gamma type 2 receptor A (FcγRIIA) receptors. A bacterial protease, IgG-degrading enzyme of Streptococcus pyogenes (IdeS), cleaves the hinge region of heavy-chain IgG, abolishing its ability to bind FcγR, including FcγRIIA. We evaluated whether cleavage of anti-PF4/H IgG by IdeS could suppress the pathogenicity of HIT antibodies. IdeS quickly cleaved purified 5B9, a monoclonal chimeric anti-PF4/H IgG1, which led to the formation of single cleaved 5B9 (sc5B9), without any reduction in binding ability to the PF4/H complex. However, as compared with uncleaved 5B9, the affinity of sc5B9 for platelet FcγRIIA was greatly reduced, and sc5B9 was also unable to induce heparin-dependent platelet activation. In addition, incubating IdeS in whole blood containing 5B9 or HIT plasma samples led to cleavage of anti-PF4/H antibodies, which fully abolished the ability to induce heparin-dependent platelet aggregation and tissue factor messenger RNA synthesis by monocytes. Also, when whole blood was perfused in von Willebrand factor-coated microfluidic channels, platelet aggregation and fibrin formation induced by 5B9 with heparin was strongly reduced after IdeS treatment. Finally, IdeS prevented thrombocytopenia and hypercoagulability induced by 5B9 with heparin in transgenic mice expressing human PF4 and FcγRIIA receptors. In conclusion, cleavage of anti-PF4/H IgG by IdeS abolishes heparin-dependent cellular activation induced by HIT antibodies. IdeS injection could be a potential treatment of patients with severe HIT.

Platelets Disseminate Extracellular Vesicles in Lymph in Rheumatoid Arthritis.

OBJECTIVE: The lymphatic system is a circulatory system that unidirectionally drains the interstitial tissue fluid back to blood circulation. Although lymph is utilized by leukocytes for immune surveillance, it remains inaccessible to platelets and erythrocytes. Activated cells release submicron extracellular vesicles (EV) that transport molecules from the donor cell. In rheumatoid arthritis, EV accumulate in the joint where they can interact with numerous cellular lineages. However, whether EV can exit the inflamed tissue to recirculate is unknown. Here, we investigated whether vascular leakage that occurs during inflammation could favor EV access to the lymphatic system. Approach and Results: Using an in vivo model of autoimmune inflammatory arthritis, we show that there is an influx of platelet EV, but not EV from erythrocytes or leukocytes, in joint-draining lymph. In contrast to blood platelet EV, lymph platelet EV lacked mitochondrial organelles and failed to promote coagulation. Platelet EV influx in lymph was consistent with joint vascular leakage and implicated the fibrinogen receptor α2bß3 and platelet-derived serotonin. CONCLUSIONS: These findings show that platelets can disseminate their EV in fluid that is inaccessible to platelets and beyond the joint in this disease.

Platelets release pathogenic serotonin and return to circulation after immune complex-mediated sequestration.

There is a growing appreciation for the contribution of platelets to immunity; however, our knowledge mostly relies on platelet functions associated with vascular injury and the prevention of bleeding. Circulating immune complexes (ICs) contribute to both chronic and acute inflammation in a multitude of clinical conditions. Herein, we scrutinized platelet responses to systemic ICs in the absence of tissue and endothelial wall injury. Platelet activation by circulating ICs through a mechanism requiring expression of platelet Fcγ receptor IIA resulted in the induction of systemic shock. IC-driven shock was dependent on release of serotonin from platelet-dense granules secondary to platelet outside-in signaling by αIIbß3 and its ligand fibrinogen. While activated platelets sequestered in the lungs and leaky vasculature of the blood-brain barrier, platelets also sequestered in the absence of shock in mice lacking peripheral serotonin. Unexpectedly, platelets returned to the blood circulation with emptied granules and were thereby ineffective at promoting subsequent systemic shock, although they still underwent sequestration. We propose that in response to circulating ICs, platelets are a crucial mediator of the inflammatory response highly relevant to sepsis, viremia, and anaphylaxis. In addition, platelets recirculate after degranulation and sequestration, demonstrating that in adaptive immunity implicating antibody responses, activated platelets are longer lived than anticipated and may explain platelet count fluctuations in IC-driven diseases.

The antigenic complex in HIT binds to B cells via complement and complement receptor 2 (CD21).

Heparin-induced thrombocytopenia is a prothrombotic disorder caused by antibodies to platelet factor 4 (PF4)/heparin complexes. The mechanism that incites such prevalent anti-PF4/heparin antibody production in more than 50% of patients exposed to heparin in some clinical settings is poorly understood. To investigate early events associated with antigen exposure, we first examined the interaction of PF4/heparin complexes with cells circulating in whole blood. In healthy donors, PF4/heparin complexes bind preferentially to B cells (>90% of B cells bind to PF4/heparin in vitro) relative to neutrophils, monocytes, or T cells. Binding of PF4 to B cells is heparin dependent, and PF4/heparin complexes are found on circulating B cells from some, but not all, patients receiving heparin. Given the high proportion of B cells that bind PF4/heparin, we investigated complement as a mechanism for noncognate antigen recognition. Complement is activated by PF4/heparin complexes, co-localizes with antigen on B cells from healthy donors, and is present on antigen-positive B cells in patients receiving heparin. Binding of PF4/heparin complexes to B cells is mediated through the interaction between complement and complement receptor 2 (CR2 [CD21]). To the best of our knowledge, these are the first studies to demonstrate complement activation by PF4/heparin complexes, opsonization of PF4/heparin to B cells via CD21, and the presence of complement activation fragments on circulating B cells in some patients receiving heparin. Given the critical contribution of complement to humoral immunity, our observations provide new mechanistic insights into the immunogenicity of PF4/heparin complexes.

Platelet transactivation by monocytes promotes thrombosis in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is characterized by a high incidence of thrombosis, unlike other antibody-mediated causes of thrombocytopenia. We have shown that monocytes complexed with surface-bound platelet factor 4 (PF4) activated by HIT antibodies contribute to the prothrombotic state in vivo, but the mechanism by which this occurs and the relationship to the requirement for platelet activation via fragment crystallizable (Fc)γRIIA is uncertain. Using a microfluidic model and human or murine blood, we confirmed that activation of monocytes contributes to the prothrombotic state in HIT and showed that HIT antibodies bind to monocyte FcγRIIA, which activates spleen tyrosine kinase and leads to the generation of tissue factor (TF) and thrombin. The combination of direct platelet activation by HIT immune complexes through FcγRIIA and transactivation by monocyte-derived thrombin markedly increases Annexin V and factor Xa binding to platelets, consistent with the formation of procoagulant coated platelets. These data provide a model of HIT wherein a combination of direct FcγRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk.

Analysis of 13 cell types reveals evidence for the expression of numerous novel primate- and tissue-specific microRNAs.

Two decades after the discovery of the first animal microRNA (miRNA), the number of miRNAs in animal genomes remains a vexing question. Here, we report findings from analyzing 1,323 short RNA sequencing samples (RNA-seq) from 13 different human tissue types. Using stringent thresholding criteria, we identified 3,707 statistically significant novel mature miRNAs at a false discovery rate of ≤ 0.05 arising from 3,494 novel precursors; 91.5% of these novel miRNAs were identified independently in 10 or more of the processed samples. Analysis of these novel miRNAs revealed tissue-specific dependencies and a commensurate low Jaccard similarity index in intertissue comparisons. Of these novel miRNAs, 1,657 (45%) were identified in 43 datasets that were generated by cross-linking followed by Argonaute immunoprecipitation and sequencing (Ago CLIP-seq) and represented 3 of the 13 tissues, indicating that these miRNAs are active in the RNA interference pathway. Moreover, experimental investigation through stem-loop PCR of a random collection of newly discovered miRNAs in 12 cell lines representing 5 tissues confirmed their presence and tissue dependence. Among the newly identified miRNAs are many novel miRNA clusters, new members of known miRNA clusters, previously unreported products from uncharacterized arms of miRNA precursors, and previously unrecognized paralogues of functionally important miRNA families (e.g., miR-15/107). Examination of the sequence conservation across vertebrate and invertebrate organisms showed 56.7% of the newly discovered miRNAs to be human-specific whereas the majority (94.4%) are primate lineage-specific. Our findings suggest that the repertoire of human miRNAs is far more extensive than currently represented by public repositories and that there is a significant number of lineage- and/or tissue-specific miRNAs that are uncharacterized.

TULA-2 Protein Phosphatase Suppresses Activation of Syk through the GPVI Platelet Receptor for Collagen by Dephosphorylating Tyr(P)346, a Regulatory Site of Syk.

Protein-tyrosine phosphatase TULA-2 has been shown to regulate receptor signaling in several cell types, including platelets. Platelets are critical for maintaining vascular integrity; this function is mediated by platelet aggregation in response to recognition of the exposed basement membrane collagen by the GPVI receptor, which is non-covalently associated with the signal-transducing FcRγ polypeptide chain. Our previous studies suggested that TULA-2 plays an important role in negatively regulating signaling through GPVI-FcRγ and indicated that the tyrosine-protein kinase Syk is a key target of the regulatory action of TULA-2 in platelets. However, the molecular basis of the down-regulatory effect of TULA-2 on Syk activation via FcRγ remained unclear. In this study, we demonstrate that suppression of Syk activation by TULA-2 is mediated, to a substantial degree, by dephosphorylation of Tyr(P)346, a regulatory site of Syk, which becomes phosphorylated soon after receptor ligation and plays a critical role in initiating the process that yields fully activated Syk. TULA-2 is capable of dephosphorylating Tyr(P)346 with high efficiency, thus controlling the overall activation of Syk, but is less efficient in dephosphorylating other regulatory sites of this kinase. Therefore, dephosphorylation of Tyr(P)346 may be considered an important "checkpoint" in the regulation of Syk activation process. Putative biological functions of TULA-2-mediated dephosphorylation of Tyr(P)346 may include deactivation of receptor-activated Syk or suppression of Syk activation by suboptimal stimulation.

Anti-miR-148a regulates platelet FcγRIIA signaling and decreases thrombosis in vivo in mice.

Fc receptor for IgG IIA (FcγRIIA)-mediated platelet activation is essential in heparin-induced thrombocytopenia (HIT) and other immune-mediated thrombocytopenia and thrombosis disorders. There is considerable interindividual variation in platelet FcγRIIA activation, the reasons for which remain unclear. We hypothesized that genetic variations between FcγRIIA hyper- and hyporesponders regulate FcγRIIA-mediated platelet reactivity and influence HIT susceptibility. Using unbiased genome-wide expression profiling, we observed that human hyporesponders to FcγRIIA activation showed higher platelet T-cell ubiquitin ligand-2 (TULA-2) mRNA expression than hyperresponders. Silent interfering RNA-mediated knockdown of TULA-2 resulted in hyperphosphorylation of spleen tyrosine kinase following FcγRIIA activation in HEL cells. Significantly, we found miR-148a-3p targeted and inhibited both human and mouse TULA-2 mRNA. Inhibition of miR-148a in FcγRIIA transgenic mice upregulated the TULA-2 level and reduced FcγRIIA- and glycoprotein VI-mediated platelet αIIbß3 activation and calcium mobilization. Anti-miR-148a also reduced thrombus formation following intravascular platelet activation via FcγRIIA. These results show that TULA-2 is a target of miR-148a-3p, and TULA-2 serves as a negative regulator of FcγRIIA-mediated platelet activation. This is also the first study to show the effects of in vivo miRNA inhibition on platelet reactivity. Our work suggests that modulating miR-148a expression is a potential therapeutic approach for thrombosis.

Mice Expressing Low Levels of CalDAG-GEFI Exhibit Markedly Impaired Platelet Activation With Minor Impact on Hemostasis.

OBJECTIVE: The tight regulation of platelet adhesiveness, mediated by the αIIbß3 integrin, is critical for hemostasis and prevention of thrombosis. We recently demonstrated that integrin affinity in platelets is controlled by the guanine nucleotide exchange factor, CalDAG-GEFI (CD-GEFI), and its target, RAP1. In this study, we investigated whether low-level expression of CD-GEFI leads to protection from thrombosis without pathological bleeding in mice. APPROACH AND RESULTS: Cdg1(low) mice were generated by knockin of human CD-GEFI cDNA into the mouse Cdg1 locus. CD-GEFI expression in platelets from Cdg1(low) mice was reduced by ≈90% when compared with controls. Activation of RAP1 and αIIbß3 was abolished at low agonist concentrations and partially inhibited at high agonist concentrations in Cdg1(low) platelets. Consistently, the aggregation response of Cdg1(low) platelets was weaker than that of wild-type platelets, but more efficient than that observed in Cdg1(-/-) platelets. Importantly, Cdg1(low) mice were strongly protected from arterial and immune complex-mediated thrombosis, with only minimal impact on primary hemostasis. CONCLUSIONS: Together, our studies suggest the partial inhibition of CD-GEFI function as a powerful new approach to safely prevent thrombotic complications.

TULA-2 (T-Cell Ubiquitin Ligand-2) Inhibits the Platelet Fc Receptor for IgG IIA (FcγRIIA) Signaling Pathway and Heparin-Induced Thrombocytopenia in Mice.

OBJECTIVE: The objective of this study is to investigate the role of T-cell ubiquitin ligand-2 (TULA-2) in the platelet Fc receptor for IgG IIA (FcγRIIA) pathway and in the pathogenesis of heparin-induced thrombocytopenia (HIT). APPROACH AND RESULTS: HIT is a life-threatening thrombotic disease in which IgG antibodies against the heparin-platelet factor 4 complex activate platelets via FcγRIIA. We reported previously differential expression of TULA-2 in human population was linked to FcγRIIA responsiveness. In this study, we investigated the role of TULA-2, a protein phosphatase, in the FcγRIIA pathway and HIT pathogenesis by crossing TULA-2-/- mice with transgenic FcγRIIA +/+ mice. Ablation of TULA-2 resulted in hyperphosphorylation of spleen tyrosine kinase, linker for the activation of T cells, and phospholipase Cγ2 in platelets via FcγRIIA activation. Platelet integrin activation, granule secretion, phosphatidylserine exposure, and aggregation were also enhanced in TULA-2-/- murine platelets. Compared with wild-type mice, TULA-2-/- mice showed aggravated antibody-mediated thrombocytopenia, augmented thrombin generation, and shortened tail bleeding time. In contrast, there was no significant difference between TULA-2-/- and TULA-2+/+ platelets in platelet spreading and clot retraction. Of note, heterozygous TULA-2+/- mice, whose platelets contained 50% as much protein as the TULA-2+/+ platelets, showed significantly increased platelet reactivity and more severe thrombocytopenia in vivo compared with TULA-2+/+ mice. CONCLUSIONS: Together, the data demonstrate that not only the absence of TULA-2 but also the relative level of TULA-2 expression modulates FcγRIIA-mediated platelet reactivity and HIT in vivo. TULA-2 expression could be a valuable marker for HIT and inhibiting TULA-2 may serve as a potential therapy to reverse the bleeding adverse effect of anticoagulants.

Human platelet microRNA-mRNA networks associated with age and gender revealed by integrated plateletomics.

There is little data considering relationships among human RNA, demographic variables, and primary human cell physiology. The platelet RNA and expression-1 study measured platelet aggregation to arachidonic acid, ADP, protease-activated receptor (PAR) 1 activation peptide (PAR1-AP), and PAR4-AP, as well as mRNA and microRNA (miRNA) levels in platelets from 84 white and 70 black healthy subjects. A total of 5911 uniquely mapped mRNAs and 181 miRNAs were commonly expressed and validated in a separate cohort. One hundred twenty-nine mRNAs and 15 miRNAs were differentially expressed (DE) by age, and targets of these miRNAs were over-represented among these mRNAs. Fifty-four mRNAs and 9 miRNAs were DE by gender. Networks of miRNAs targeting mRNAs, both DE by age and gender, were identified. The inverse relationship in these RNA pairs suggests miRNAs regulate mRNA levels on aging and between genders. A simple, interactive public web tool (www.plateletomics.com) was developed that permits queries of RNA levels and associations among RNA, platelet aggregation and demographic variables. Access to these data will facilitate discovery of mechanisms of miRNA regulation of gene expression. These results provide new insights into aging and gender, and future platelet RNA association studies must account for age and gender.

Platelet 12-LOX is essential for FcγRIIa-mediated platelet activation.

Platelets are essential in maintaining hemostasis following inflammation or injury to the vasculature. Dysregulated platelet activity often results in thrombotic complications leading to myocardial infarction and stroke. Activation of the FcγRIIa receptor leads to immune-mediated thrombosis, which is often life threatening in patients undergoing heparin-induced thrombocytopenia or sepsis. Inhibiting FcγRIIa-mediated activation in platelets has been shown to limit thrombosis and is the principal target for prevention of immune-mediated platelet activation. In this study, we show for the first time that platelet 12(S)-lipoxygenase (12-LOX), a highly expressed oxylipin-producing enzyme in the human platelet, is an essential component of FcγRIIa-mediated thrombosis. Pharmacologic inhibition of 12-LOX in human platelets resulted in significant attenuation of FcγRIIa-mediated aggregation. Platelet 12-LOX was shown to be essential for FcγRIIa-induced phospholipase Cγ2 activity leading to activation of calcium mobilization, Rap1 and protein kinase C activation, and subsequent activation of the integrin αIIbß3. Additionally, platelets from transgenic mice expressing human FcγRIIa but deficient in platelet 12-LOX, failed to form normal platelet aggregates and exhibited deficiencies in Rap1 and αIIbß3 activation. These results support an essential role for 12-LOX in regulating FcγRIIa-mediated platelet function and identifies 12-LOX as a potential therapeutic target to limit immune-mediated thrombosis.

Cooperative integrin/ITAM signaling in platelets enhances thrombus formation in vitro and in vivo.

The integrin family is composed of a series of 24 αß heterodimer transmembrane adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. Adaptor molecules bearing immunoreceptor tyrosine-based activation motifs (ITAMs) have recently been shown to cooperate with specific integrins to increase the efficiency of transmitting ligand-binding-induced signals into cells. In human platelets, Fc receptor γ-chain IIa (FcγRIIa) has been identified as an ITAM-bearing transmembrane receptor responsible for mediating "outside-in" signaling through αIIbß3, the major adhesion receptor on the platelet surface. To explore the importance of FcγRIIa in thrombosis and hemostasis, we subjected FcγRIIa-negative and FcγRIIa-positive murine platelets to a number of well-accepted models of platelet function. Compared with their FcγRIIa-negative counterparts, FcγRIIa-positive platelets exhibited increased tyrosine phosphorylation of Syk and phospholipase Cγ2 and increased spreading upon interaction with immobilized fibrinogen, retracted a fibrin clot faster, and showed markedly enhanced thrombus formation when perfused over a collagen-coated flow chamber under conditions of arterial and venous shear. They also displayed increased thrombus formation and fibrin deposition in in vivo models of vascular injury. Taken together, these data establish FcγRIIa as a physiologically important functional conduit for αIIbß3-mediated outside-in signaling, and suggest that modulating the activity of this novel integrin/ITAM pair might be effective in controlling thrombosis.