Urinary 8-epi-PGF2 alpha may represent a noninvasive, quantitative index of oxidant stress in vivo. Elevated levels of 8-epi-PGF2 alpha in smokers may be modulated by quitting cigarettes and switching to nicotine patches or by antioxidant vitamin therapy.

Heparin-induced thrombocytopenia and thrombosis (HITT) is a severe complication of heparin therapy caused by antibodies to complexes between unfractionated heparin (UFH) and platelet factor 4 (PF4) that form over a narrow molar range of reactants and initiate antibody-induced platelet activation. We observed that UFH and tetrameric PF4 formed ultralarge (> 670 kDa) complexes (ULCs) only over a narrow molar range with an optimal ratio of PF4 to heparin of approximately 1:1. These ULCs were stable and visible by electron microscopy, but they could be dissociated into smaller complexes upon addition of heparin. ULCs formed inefficiently when PF4 was incubated with low-molecular-weight heparin, and none formed with the pentasaccharide fondaparinux sodium. In addition, mutation studies showed that formation of ULCs depended on the presence of PF4 tetramers. The ULCs were more reactive as determined by their capacity to bind to a HITTlike monoclonal antibody and showed greater capacity to promote platelet activation in an antibody-and Fc␥RIIA-dependent manner than were the smaller complexes. The capacity of PF4 to form ULCs composed of multiple PF4 tetramers arrayed in a lattice with several molecules of UFH may play a fundamental role in autoantibody formation, antibody-dependent platelet activation, and the propensity for thrombosis in patients with HITT.

Heparin-induced thrombocytopenia/thrombosis (HIT/HITT) is a severe, life-threatening complication that occurs in 1% to 3% of patients exposed to heparin. Interactions between heparin, human platelet factor 4 (hPF4), antibodies to the hPF4/heparin complex, and the platelet Fc receptor (FcR) for immunoglobulin G, Fc␥RIIA, are the proposed primary determinants of the disease on the basis of in vitro studies. The goal of this study was to create a mouse model that recapitulates the disease process in humans in order to understand the factors that predispose some patients to develop thrombocytopenia and thrombosis and to investigate new therapeutic approaches. Mice that express both human platelet Fc␥RIIA and hPF4 were generated. The Fc␥RIIA/hPF4 mice and controls, transgenic for either Fc␥RIIA or hPF4, were injected with KKO, a mouse monoclonal antibody specific for hPF4/heparin complexes, and then received heparin (20 U/d). Nadir platelet counts for KKO/heparin-treated Fc␥RIIA/hPF4 mice were 80% below baseline values, significantly different (P < .001) from similarly treated controls. Fc␥RIIA/hPF4 mice injected with KKO IntroductionHeparin is one of the most widely used anticoagulants during invasive vascular procedures and to treat thromboembolic diseases. Among patients who receive therapeutic courses of heparin, 1% to 3% will develop an antibody-mediated thrombocytopenia, 1-3 and 30% to 70% of these patients will develop potentially lifethreatening thrombosis. There is abundant evidence that more than 95% of patients with heparin-induced thrombocytopenia (HIT) alone and those with thrombocytopenia and thrombosis (HITT) develop antibodies that recognize complexes between platelet factor 4 (PF4) and heparin. 4-7 PF4, a major component of platelet ␣-granules, is released when platelets are activated. 8,9 It is currently believed that complexes between PF4 and heparin form on the surface of activated platelets, where they are positioned to be recognized by anti-PF4/heparin antibodies. [10][11][12] Antibodies to the PF4/heparin complex have been shown to activate human platelets in vitro via the platelet Fc receptor (FcR) for immunoglobuin (Ig)-G, Fc␥RIIA. [13][14][15][16] The binding of HIT antibodies to activated platelets probably promotes microparticle release as well as platelet-platelet and platelet-vessel wall interactions, predisposing to thrombosis. 15,17 However, the mechanism by which HIT antibodies activate platelets and promote thrombosis is uncertain. It has been proposed that HIT antibodies bind to cell-surface-associated PF4/heparin complexes via the Fab end of the molecule, providing an opportunity to transduce platelet-activating signals through the interaction between the Fc portion of the bound IgG and Fc␥RIIA. 12,13 Fc␥RIIA, the sole Fc␥ receptor expressed on platelets, 18 has been shown to transduce signals without the requirement for another transmembrane partner. 19 A monoclonal antibody to Fc␥RIIA blocks platelet aggregation and secretion induced by HIT antibodies. 12,13 Although increased...

Heparin-induced thrombocytopenia (HIT)is a major cause of morbidity and mortality resulting from the associated thrombosis. Extensive studies using our transgenic mouse model of HIT have shown that antibodies reactive with heparinplatelet factor 4 complexes lead to Fc␥RIIA-mediated platelet activation in vitro as well as thrombocytopenia and thrombosis in vivo. We tested PRT-060318 (PRT318), a novel selective inhibitor of the tyrosine kinase Syk, as an approach to HIT treatment. PRT318 completely inhibited HIT immune complex-induced aggregation of both human and transgenic HIT mouse platelets. Transgenic HIT model mice were treated with KKO, a mouse monoclonal HIT-like antibody, and heparin. The experimental group received orally dosed PRT318, whereas the control group received vehicle. Nadir platelet counts of PRT318-treated mice were significantly higher than those of control mice. When examined with a novel thrombosis visualization technique, mice treated with PRT318 had significantly reduced thrombosis. The Syk inhibitor PRT318 thus prevented both HIT immune complex-induced thrombocytopenia and thrombosis in vivo, demonstrating its activity in HIT. (Blood. 2011;117(7): 2241-2246) IntroductionHeparin-induced thrombocytopenia (HIT), characterized by antibodies to macromolecular complexes formed by heparin and platelet factor 4 (PF4), is the most frequent drug-induced immune thrombocytopenia. Patients with HIT are at an increased risk for thrombosis, a major cause of morbidity and mortality in treated patients. Despite this potential side effect, heparins (unfractionated or low molecular weight) remain the drug of choice in clinical situations where high-intensity therapy is needed along with the ability to rapidly modulate the anticoagulant level. 1 The incidence of HIT has therefore not decreased, notwithstanding the introduction of new anticoagulants, primarily because no drug has replaced heparin for the immediate therapy of acute deep vein thrombosis, arterial thrombosis, or extracorporeal circuits during surgery. In addition, indications for its use in the aging population continue to increase.Multiple factors influence the incidence and severity of HIT. The pathogenesis of the disease is well understood, 2-5 although additional progress is being made. Extensive studies in vitro 4,6,7 and in vivo using our transgenic mouse model of HIT 8 show that antibodies reactive with heparin-PF4 complexes lead to Fc receptormediated platelet activation. This activation leads to platelet aggregation, a procoagulant surface, and release of prothrombotic microparticles. In addition, monocytes and other leukocytes bearing Fc␥ receptors can become activated by the HIT immune complex (IC), generating tissue factor and resulting in other prothrombotic and proadhesive changes. [9][10][11] Blocking Fc␥RIIA signaling is an attractive target for therapeutic intervention because Fc␥RIIA-mediated platelet activation (and possibly concurrent monocyte activation) is central to the disease.Fc␥RIIA, like other activating receptors, i...

Objective-Biologically significant amounts of two procoagulant molecules, phosphatidylserine (PS) and tissue factor (TF), are transported by monocyte/macrophage-derived microvesicles (MVs). Because cellular cholesterol accumulation is an important feature of atherosclerotic vascular disease, we now examined effects of cholesterol enrichment on MV release from human monocytes and macrophages. Methods and Results-Cholesterol enrichment of human THP-1 monocytes, alone or in combination with lipopolysaccharide (LPS), tripled their total MV generation, as quantified by flow cytometry based on particle size and PS exposure. The subset of these MVs that were also TF-positive was likewise increased by cellular cholesterol enrichment, and these TF-positive MVs exhibited a striking 10-fold increase in procoagulant activity. Moreover, cholesterol enrichment of primary human monocyte-derived macrophages also increased their total as well as TF-positive MV release, and these TF-positive MVs exhibited a similar 10-fold increase in procoagulant activity. To explore the mechanisms of enhanced MV release, we found that cholesterol enrichment of monocytes caused PS exposure on the cell surface by as early as 2 hours and genomic DNA fragmentation in a minority of cells by 20 hours. Addition of a caspase inhibitor at the beginning of these incubations blunted both cholesterol-induced apoptosis and MV release.

The genes for the related human (h) chemokines, PBP (platelet basic protein) and PF4 (platelet factor 4), are within 5.3 kilobases (kb) of each other and form a megakaryocyte-specific gene locus. The hypothesis was considered that the PBP and PF4 genes share a common distal regulatory region(s) that leads to their high-level megakaryocyte-specific expression in vivo. This study examined PBP and PF4 expression in transgenic mice using 4 distinct human PBP/PF4 gene locus constructs. These studies showed that within the region studied there was sufficient information to regulate tissue-specific expression of both hPBP and hPF4. Indeed this region contained sufficient DNA information to lead to expression levels of PBP and PF4 comparable to the homologous mouse genes in a position-independent, copy number-dependent fashion. These studies also indicated that the DNA domains that led to this expression were distinct for the 2 genes; hPBP expression is regulated by a region that is 1.5 to 4.4 kb upstream of that gene. Expression of hPF4 is regulated by a region that is either intergenic between the 2 genes or immediately downstream of the hPF4 IntroductionPlatelet basic protein (PBP) and platelet factor 4 (PF4) are related, platelet-specific chemokines that are expressed at high levels during megakaryopoiesis and stored in platelet ␣-granules. 1 They represent 3% to 5% of total protein releasate from platelets on a molar basis. PBP is N-terminally cleaved to ␤-thromboglobulin (␤-TG) and then to neutrophil-activating peptide-2 (NAP-2). [2][3][4] This final product binds and activates the chemokine receptor CXCR2 on neutrophils. 5 What role this chemokine has in thrombosis and inflammation is unclear. Unlike PBP and other chemokines, PF4 appears to bind mostly to large, negatively charged molecules such as heparin, and though many biologic functions have been attributed to PF4, its true role in vivo is unknown. [6][7][8][9] Although the biologic roles of these chemokines need further investigation, their genes offer an opportunity to understand megakaryocyte-specific expression. Like many other genes encoding chemokines, both the PBP and PF4 genes are encoded by 3 exons, and are preceded by a TATA box in the immediate 5Ј-flanking region. 10,11 Transient transcriptional studies with the immediate 5Ј-flanking region defined a PU.1-binding promoter region upstream of the PBP gene, 12 similar to a thrombopoietininduced enhancer domain upstream of the platelet-specific [alpha]IIb gene. 13 Several silencers and promoter domains have been similarly defined in the immediate 5Ј-flanking region of the PF4 gene, including one GATA-1 binding site. 14,15 Reporter gene constructs with 245 bp of the immediate 5Ј-flanking region of the hPF4 gene showed that this region contained sufficient information to drive tissue-specific expression of a LacZ reporter construct 16 and that 1.1 kb of rat PF4 promoter can drive vigorous tissue-specific expression. 17 However, neither of these constructs had sufficient information to drive positionind...

Platelet activation via Fc␥ receptor IIA (Fc␥RIIA) is a critical event in immunemediated thrombocytopenia and thrombosis syndromes (ITT). We recently identified signaling by the guanine nucleotide exchange factor CalDAG-GEFI and the adenosine diphosphate receptor P2Y12 as independent pathways leading to Rap1 small GTPase activation and platelet aggregation. Here, we evaluated the contribution of CalDAG-GEFI and P2Y12 signaling to platelet activation in ITT. Mice transgenic for the human Fc␥RIIA (hFcR) and deficient in CalDAG-GEFI ؊/؊ (hFcR/CDGI ؊/؊ ) were generated. Compared with controls, aggregation of hFcR/ CDGI ؊/؊ platelets or P2Y12 inhibitortreated hFcR platelets required more than 5-fold and approximately 2-fold higher concentrations of a Fc␥RIIA stimulating antibody against CD9, respectively. Aggregation and Rap1 activation were abolished in P2Y12 inhibitor-treated hFcR/ CDGI ؊/؊ platelets. For in vivo studies, a novel model for antibody-induced thrombocytopenia and thrombosis was established. Fc␥RIIA-dependent platelet thrombosis was induced by infusion of Alexa750-labeled antibodies to glycoprotein IX (CD42a), and pulmonary thrombi were detected by near-infrared imaging technology. Anti-GPIX antibodies dosedependently caused thrombocytopenia and pulmonary thrombosis in hFcRtransgenic but not wild-type mice. CalDAG-GEFI-deficient but not clopidogreltreated hFcR-transgenic mice were completely protected from ITT. In summary, we established a novel mouse model for ITT, which was used to identify CalDAG-GEFI as a potential new target in the treatment of ITT. (Blood. 2011;118(4): 1113-1120) IntroductionPlatelets are essential components of the hemostatic response to vascular injury. However, platelets also play a role in pathologic conditions, such as atherothrombosis, and in immune-mediated thrombocytopenia and thrombosis (ITT). Several ITT syndromes, including heparin-induced thrombocytopenia and thrombosis (HIT), 1-3 bacterial sepsis-associated thrombocytopenia and disseminated intravascular coagulation, 4,5 and the thrombotic manifestations of antiphospholipid syndromes 6 are characterized by immunemediated platelet activation through the platelet Fc␥ receptor, Fc␥RIIA. In addition, thrombotic complications have been observed with the expanded use of therapeutic IgG antibodies, such as bevacizumab. [7][8][9] One of the barriers to successful treatment of these thrombotic syndromes is that therapeutic targeting of platelet activation pathways to prevent thrombosis is either not effective or comes with an inherent risk of bleeding complications.In humans, Fc␥RIIA is expressed on platelets, neutrophils, monocytes, and macrophages and activates these cells following the binding of the Fc region of IgG-coated cells or IgG-containing immune complexes. 10 Mice lack the genetic equivalent of human Fc␥RIIA and, indeed, do not express a platelet Fc receptor. Consequently, most of the studies on the role of Fc␥RIIA in platelet activation were entirely dependent on the use of inhibitors, and they did not provide in...

Heparin-induced thrombocytopenia (HIT) is the most common drug-induced, antibody-mediated cause of thrombocytopenia and thrombosis. HIT is caused by IgG antibodies that bind to epitopes on platelet factor 4 (PF4) released from activated platelets that develop when it forms complexes with heparin. Anti-PF4/antibodies develop in over 50% of patients undergoing surgery involving cardiopulmonary bypass (CPB), an incidence 20-fold higher than HIT. Why might this occur? Binding of HIT IgG occurs only over a narrow molar ratio of reactants, being optimal at 1 mol PF4 tetramer to 1 mol unfractionated heparin (UFH). At these ratios, PF4 and UFH form ultralarge (>670 kD) complexes that bind multiple IgG molecules/complex, are highly antigenic, and promote platelet activation. Low molecular weight heparin (LMWH), which is less antigenic, forms ultralarge complexes less efficiently and largely at supratherapeutic concentrations. In transgenic mice that vary in expression of human PF4 on their platelets, antigenic complexes form between PF4 and endogenous chondroitin sulfate. Binding of HIT IgG to platelets and induction of thrombocytopenia in vivo is proportional to PF4 expression. Heparin prolongs the duration and exacerbates the severity of the thrombocytopenia. High doses of heparin, as used in CPB, or protamine, which competes with PF4 for heparin, disrupts antigen formation and prevents thrombocytopenia induced by HIT antibody. These studies may help explain the disparity between the incidence of antibody formation and clinical disease and may help identify patients at risk for HIT (high platelet PF4). They also demonstrate that this autoimmune disease can be modulated at the level of autoantigen formation and point to rational means to intervene proximal to thrombin generation.