Platelets play a key role in atherogenesis and its complications. Both hypercholesterolemia and increased platelet production promote athero-thrombosis; however, a potential link between altered cholesterol homeostasis and platelet production has not been explored. Transplantation of bone marrow (BM) deficient in ABCG4, a transporter of unknown function, into Ldlr−/− mice resulted in thrombocytosis, accelerated thrombosis and atherosclerosis. While not detected in lesions, Abcg4 was highly expressed in BM megakaryocyte progenitors (MkP). Abcg4−/− MkPs displayed defective cholesterol efflux to HDL, increased cell surface levels of thrombopoietin (TPO) receptor (c-MPL) and enhanced proliferation. This appeared to reflect disruption of the negative feedback regulation of c-MPL levels and signaling by E3 ligase c-CBL and cholesterol-sensing LYN kinase. HDL infusions reduced platelet counts in Ldlr−/− mice and in a mouse model of myeloproliferative neoplasm, in a completely ABCG4-dependent fashion. HDL infusions may offer a novel approach to reducing athero-thrombotic events associated with increased platelet production.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by systemic microvascular thrombosis caused by adhesion of platelets to ultra-large vWF (ULVWF) multimers. These multimers accumulate because of a deficiency of the processing enzyme ADAMTS13. vWF protein forms long multimers from homodimers that first form through C-terminal disulfide bonds and then join through their N termini by further disulfide bonding. N-acetylcysteine (NAC) is an FDA-approved drug that has long been used to treat chronic obstructive lung disease and acetaminophen toxicity and is known to function in the former disorder by reducing mucin multimers. Here, we examined whether NAC could reduce vWF multimers, which polymerize in a manner similar to mucins. In vitro, NAC reduced soluble plasma-type vWF multimers in a concentration-dependent manner and rapidly degraded ULVWF multimer strings extruded from activated ECs. The effect was preceded by reduction of the intrachain disulfide bond encompassing the platelet-binding A1 domain. NAC also inhibited vWF-dependent platelet aggregation and collagen binding. Injection of NAC into ADAMTS13-deficient mice led to the rapid resolution of thrombi produced by ionophore treatment of the mesenteric venules and reduced plasma vWF multimers. These results suggest that NAC may be a rapid and effective treatment for patients with TTP.

Clinical studies suggest that diets rich in ω-3 polyunsaturated fatty acids (PUFAs) provide beneficial anti-inflammatory effects, in part through their conversion to bioactive metabolites. Here we report on the endogenous production of a previously unknown class of ω-3 PUFA–derived lipid metabolites that originate from the crosstalk between endocannabinoid and cytochrome P450 (CYP) epoxygenase metabolic pathways. The ω-3 endocannabinoid epoxides are derived from docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to form epoxyeicosatetraenoic acid-ethanolamide (EEQ-EA) and epoxydocosapentaenoic acid-ethanolamide (EDP-EA), respectively. Both EEQ-EAs and EDP-EAs are endogenously present in rat brain and peripheral organs as determined via targeted lipidomics methods. These metabolites were directly produced by direct epoxygenation of the ω-3 endocannabinoids, docosahexanoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA) by activated BV-2 microglial cells, and by human CYP2J2. Neuroinflammation studies revealed that the terminal epoxides 17,18-EEQ-EA and 19,20-EDP-EA dose-dependently abated proinflammatory IL-6 cytokines while increasing anti-inflammatory IL-10 cytokines, in part through cannabinoid receptor-2 activation. Furthermore the ω-3 endocannabinoid epoxides 17,18-EEQ-EA and 19,20-EDP-EA exerted antiangiogenic effects in human microvascular endothelial cells (HMVEC) and vasodilatory actions on bovine coronary arteries and reciprocally regulated platelet aggregation in washed human platelets. Taken together, the ω-3 endocannabinoid epoxides’ physiological effects are mediated through both endocannabinoid and epoxyeicosanoid signaling pathways. In summary, the ω-3 endocannabinoid epoxides are found at concentrations comparable to those of other endocannabinoids and are expected to play critical roles during inflammation in vivo; thus their identification may aid in the development of therapeutics for neuroinflammatory and cerebrovascular diseases.

No abstract

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder caused by maternal antibody-mediated destruction of fetal/neonatal platelets. It is the most common cause of severe thrombocytopenia in neonates, but the frequency of FNIT-related miscarriage is unknown, and the mechanism(s) underlying fetal mortality have not been explored. Furthermore, although platelet αIIbβ3 integrin and GPIbα are the major antibody targets in immune thrombocytopenia, the reported incidence of anti-GPIbα-mediated FNIT is rare. Here, we developed mouse models of FNIT mediated by antibodies specific for GPIbα and β3 integrin and compared their pathogenesis. We found, unexpectedly, that miscarriage occurred in the majority of pregnancies in our model of anti-GPIbα-mediated FNIT, which was far more frequent than in anti-β3-mediated FNIT. Dams with anti-GPIbα antibodies exhibited extensive fibrin deposition and apoptosis/necrosis in their placentas, which severely impaired placental function. Furthermore, anti-GPIbα (but not anti-β3) antiserum activated platelets and enhanced fibrin formation in vitro and thrombus formation in vivo. Importantly, treatment with either intravenous IgG or a monoclonal antibody specific for the neonatal Fc receptor efficiently prevented anti-GPIbα-mediated FNIT. Thus, the maternal immune response to fetal GPIbα causes what we believe to be a previously unidentified, nonclassical FNIT (i.e., spontaneous miscarriage but not neonatal bleeding) in mice. These results suggest that a similar pathology may have masked the severity and frequency of human anti-GPIbα-mediated FNIT, but also point to possible therapeutic interventions.

Summary. The role of vitronectin (Vn) in thrombosis is currently controversial; both inhibitory and supportive roles have been reported. To monitor directly the function of Vn in thrombotic events at the site of vascular injury, we studied Vn-deficient (Vn-/-) and wild-type (WT) control mice with two real-time intravital microscopy thrombosis models. In the mesenteric arteriole model, vessel injury was induced by ferric chloride. We observed unstable thrombi and a significantly greater number of emboli in Vn-/-mice. Vessel occlusion was also delayed and frequent vessel re-opening occurred. In the cremaster muscle arteriole model, vessel injury was induced by a nitrogen dye laser. We observed significantly fewer platelets, lower fibrin content, and unstable fibrin within the thrombi of Vn-/-mice. To define further the role of Vn in thrombus growth, we studied platelet aggregation in vitro. Consistent with our in vivo data, the second wave of thrombin-induced aggregation of gel-filtered platelets was abolished at a low concentration of thrombin in Vn-/-platelets. Interestingly, adenosine diphosphate (ADP)-induced platelet aggregation was significantly increased in Vn-/-platelet-rich plasma (PRP) and this effect was attenuated by adding purified plasma Vn. We also observed increased platelet aggregation induced by shear stress in Vn-/-whole blood. These data demonstrate that Vn is a thrombus stabilizer. However, in contrast to released platelet granule Vn which enhances platelet aggregation, plasma Vn inhibits platelet aggregation.

Summary.  Background: Fibrinogen (Fg) has been considered essential for platelet aggregation. However, we recently demonstrated formation of occlusive thrombi in Fg‐deficient mice and in mice doubly deficient for Fg and von Willebrand factor (Fg/VWF−/−). Methods and results: Here we studied Fg/VWF‐independent platelet aggregation in vitro and found no aggregation in citrated platelet‐rich plasma of Fg/VWF−/− mice. Surprisingly, in Fg/VWF−/− plasma without anticoagulant, adenosine diphosphate induced robust aggregation of Fg/VWF−/− platelets but not of β3‐integrin‐deficient (β3−/−) platelets. In addition, β3−/− platelets did not significantly incorporate into thrombi in Fg/VWF−/− mice. This Fg/VWF‐independent aggregation was blocked by thrombin inhibitors (heparin, hirudin, PPACK), and thrombin or thrombin receptor activation peptide (AYPGKF‐NH2) induced aggregation of gel‐filtered Fg/VWF−/− platelets in 1 mm Ca2+ PIPES buffer. Notably, aggregation in PIPES buffer was only 50–60% of that observed in Fg/VWF−/− plasma. Consistent with the requirement for thrombin in vitro, hirudin completely inhibited thrombus formation in Fg/VWF−/− mice. These data define a novel pathway of platelet aggregation independent of both Fg and VWF. Although this pathway was not detected in the presence of anticoagulants, it was observed under physiological conditions in vivo and in the presence of Ca2+in vitro. Conclusions: β3 integrin, thrombin, and Ca2+ play critical roles in this Fg/VWF‐independent aggregation, and both plasma and platelet granule proteins contribute to this process.

We previously showed that platelet aggregation and thrombus formation occurred in mice lacking both fibrinogen (Fg) and von Willebrand factor (VWF) and that plasma fibronectin (pFn) promoted thrombus growth and stability in injured arterioles in wild-type mice. To examine whether pFn is required for Fg/VWFindependent thrombosis, we generated Fg/VWF/conditional pFn triple-deficient (TKO; Cre ؉ , Fn flox/flox , Fg/VWF ؊/؊ ) mice and littermate control (Cre ؊ , Fn flox/flox , Fg/ VWF ؊/؊ ) mice. Surprisingly, TKO platelet aggregation was not abolished, but instead was enhanced in both heparinized platelet-rich plasma and gel-filtered platelets. This enhancement was diminished when TKO platelets were aggregated in pFn-positive control platelet-poor plasma (PPP), whereas aggregation was enhanced when control platelets were aggregated in pFn-depleted TKO PPP. The TKO platelet aggregation can be completely inhibited by our newly developed mouse anti-mouse ␤ 3 integrin antibodies but was not affected by anti-mouse GPIb␣ antibodies. Enhanced platelet aggregation was also observed when heparinized TKO blood was perfused in collagen-coated perfusion chambers. Using intravital microscopy, we further showed that thrombogenesis in TKO mice was enhanced in both FeCl 3 -injured mesenteric arterioles and laser-injured cremaster arterioles. Our data indicate that pFn is not essential for Fg/VWF-independent thrombosis and that soluble pFn is probably an important inhibitory factor for platelet aggregation. IntroductionPlatelet adhesion and subsequent aggregation at the site of vascular injury are key events required for hemostasis. However, excessive platelet accumulation and thrombus formation may result in myocardial infarction or stroke, the 2 leading causes of morbidity and mortality worldwide. 1,2 It has been shown that fibrinogen (Fg) and von Willebrand factor (VWF) are the 2 key molecules required for platelet adhesion and aggregation. 3 Interestingly, we found that platelet aggregation and thrombus formation still occur in mice lacking Fg or VWF or both, 4,5 suggesting that additional molecule(s) can promote platelet aggregation and thrombus formation.We subsequently found that platelet fibronectin (Fn) content was markedly increased in Fg-deficient mice 4 and a severe hypofibrinogenemic human patient, 6 although no alteration of plasma Fn (pFn) was observed in their Fg-deficient blood. The increase in platelet Fn content was found to be due to enhanced pFn internalization via ␤ 3 integrin. 7 Further experiments with pFn conditional deficient mice and Fn heterozygous mice showed that pFn promoted thrombus growth and stability in injured arterioles. 8,9 These data are consistent with recent in vitro studies that showed that Fn assembly on the platelet surface, 10,11 supported thrombus growth. [12][13][14][15] However, the role of pFn in Fg/VWF-independent platelet aggregation and thrombus formation has not been studied. It was expected that pFn may be required for these processes.To address this question, we generated Fg...