A. Phillip Owens

Microparticles in Hemostasis and Thrombosis

Blood contains microparticles (MPs) derived from a variety of cell types, including platelets, monocytes, and endothelial cells. In addition, tumors release MPs into the circulation. MPs are formed from membrane blebs that are released from the cell surface by proteolytic cleavage of the cytoskeleton. All MPs are procoagulant because they provide a membrane surface for the assembly of components of the coagulation protease cascade. Importantly, procoagulant activity is increased by the presence of anionic phospholipids, particularly phosphatidylserine (PS), and the procoagulant protein tissue factor (TF), which is the major cellular activator of the clotting cascade. High levels of platelet-derived PS+ MPs are present in healthy individuals, whereas the number of TF+, PS+ MPs is undetectable or very low. However, levels of PS+, TF+ MPs are readily detected in a variety of diseases, and monocytes appear to be the primary cellular source. In cancer, PS+, TF+ MPs are derived from tumors and may serve as a useful biomarker to identify patients at risk for venous thrombosis. This review will summarize our current knowledge of the role of procoagulant MPs in hemostasis and thrombosis.

Platelet ITAM signaling is critical for vascular integrity in inflammation

Platelets play a critical role in maintaining vascular integrity during inflammation, but little is known about the underlying molecular mechanisms. Here we report that platelet immunoreceptor tyrosine activation motif (ITAM) signaling, but not GPCR signaling, is critical for the prevention of inflammation-induced hemorrhage. To generate mice with partial or complete defects in these signaling pathways, we developed a protocol for adoptive transfer of genetically and/or chemically inhibited platelets into thrombocytopenic (TP) mice. Unexpectedly, platelets with impaired GPCR signaling, a crucial component of platelet plug formation and hemostasis, were indistinguishable from WT platelets in their ability to prevent hemorrhage at sites of inflammation. In contrast, inhibition of GPVI or genetic deletion of Clec2, the only ITAM receptors expressed on mouse platelets, significantly reduced the ability of platelets to prevent inflammation-induced hemorrhage. Moreover, transfusion of platelets without ITAM receptor function or platelets lacking the adapter protein SLP-76 into TP mice had no significant effect on vascular integrity during inflammation. These results indicate that the control of vascular integrity is a major function of immune-type receptors in platelets, highlighting a potential clinical complication of novel antithrombotic agents directed toward the ITAM signaling pathway.

Tissue factor and thrombosis: The clot starts here

Thrombosis, or complications from thrombosis, currently occupies the top three positions in the cardiovascular causes of morbidity and mortality in the developed world. There are a limited number of safe and effective drugs to prevent and treat thrombosis. Animal models of thrombosis are necessary to better understand the complex components and interactions involved in the formation of a clot. Tissue factor (TF) is required for the initiation of blood coagulation and likely plays a key role in both arterial and venous thrombosis. Understanding the role of TF in thrombosis may permit the development of new antithrombotic drugs. This review will focus on the role of TF in in vivo models of thrombosis.

Monocyte tissue factor-dependent activation of coagulation in hypercholesterolemic mice and monkeys is inhibited by simvastatin.

Hypercholesterolemia is a major risk factor for atherosclerosis. It also is associated with platelet hyperactivity, which increases morbidity and mortality from cardiovascular disease. However, the mechanisms by which hypercholesterolemia produces a procoagulant state remain undefined. Atherosclerosis is associated with accumulation of oxidized lipoproteins within atherosclerotic lesions. Small quantities of oxidized lipoproteins are also present in the circulation of patients with coronary artery disease. We therefore hypothesized that hypercholesterolemia leads to elevated levels of oxidized LDL (oxLDL) in plasma and that this induces expression of the procoagulant protein tissue factor (TF) in monocytes. In support of this hypothesis, we report here that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma microparticle (MP) TF activity. Furthermore, a high-fat diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor-deficient mice and African green monkeys. Genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte-derived TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity. Simvastatin treatment of hypercholesterolemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation, without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hypercholesterolemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6 complex.

Hematopoietic and nonhematopoietic cell tissue factor activates the coagulation cascade in endotoxemic mice

Tissue factor (TF) is the primary activator of the coagulation cascade. During endotoxemia, TF expression leads to disseminated intravascular coagulation. However, the relative contribution of TF expression by different cell types to the activation of coagulation has not been defined. In this study, we investigated the effect of either a selective inhibition of TF expression or cell type-specific deletion of the TF gene (F3) on activation of coagulation in a mouse model of endotoxemia. We found that inhibition of TF on either hematopoietic or nonhematopoietic cells reduced plasma thrombin-antithrombin (TAT) levels 8 hours after administration of bacterial lipopolysaccharide (LPS). In addition, plasma TAT levels were significantly reduced in endotoxemic mice lacking the TF gene in either myeloid cells (TF(flox/flox),LysM(Cre) mice) or in both endothelial cells (ECs) and hematopoietic cells (TF(flox/flox),Tie-2(Cre) mice). However, deletion of the TF gene in ECs alone had no effect on LPS-induced plasma TAT levels. Similar results were observed in mice lacking TF in vascular smooth muscle cells. Finally, we found that mouse platelets do not express TF pre-mRNA or mRNA. Our data demonstrate that in a mouse model of endotoxemia activation of the coagulation cascade is initiated by TF expressed by myeloid cells and an unidentified nonhematopoietic cell type(s).

Endothelial Cell–Specific Deficiency of Ang II Type 1a Receptors Attenuates Ang II–Induced Ascending Aortic Aneurysms in LDL Receptor−/− Mice

Rationale: Human studies and mouse models have provided evidence for angiotensin II (Ang II)–based mechanisms as an underlying cause of aneurysms localized to the ascending aorta. In agreement with this associative evidence, we have published recently that Ang II infusion induces aneurysmal pathology in the ascending aorta. Objective: The aim of this study was to define the role of angiotensin II type 1a (AT1a) receptors and their cellular location in Ang II–induced ascending aortic aneurysms (AAs). Methods and Results: Male LDL receptor−/− mice were fed a saturated fat–enriched diet for 1 week before osmotic mini-pump implantation and infused with either saline or Ang II (1000 ng/kg per minute) for 28 days. Intimal surface areas of ascending aortas were measured to quantify ascending AAs. Whole body AT1a receptor deficiency ablated Ang II–induced ascending AAs (P<0.001). To determine the role of AT1a receptors on leukocytes, LDL receptor−/−×AT1a receptor+/+ or AT1a receptor−/− mice were irradiated and repopulated with bone marrow–derived cells isolated from either AT1a receptor+/+ or AT1a receptor−/− mice. Deficiency of AT1a receptors in bone marrow–derived cells had no effect on Ang II–induced ascending AAs. To determine the role of AT1a receptors on vascular wall cells, we developed AT1a receptor floxed mice with depletion on either smooth muscle or endothelial cells using Cre driven by either SM22 or Tek, respectively. AT1a receptor deletion in smooth muscle cells had no effect on ascending AAs. In contrast, endothelial-specific depletion attenuated this pathology. Conclusions: Ang II infusion promotes aneurysms in the ascending aorta via stimulation of AT1a receptors that are expressed on endothelial cells.

Interferon-γ and the Interferon-Inducible Chemokine CXCL10 Protect Against Aneurysm Formation and Rupture

Background— Vascular disease can manifest as stenotic plaques or ectatic aneurysms, although the mechanisms culminating in these divergent disease manifestations remain poorly understood. T-helper type 1 cytokines, including interferon-&ggr; and CXCL10, have been strongly implicated in atherosclerotic plaque development. Methods and Results— Here, we specifically examined their role in the formation of abdominal aortic aneurysms in the angiotensin II–induced murine model. Unexpectedly, we found increased suprarenal aortic diameters, abdominal aortic aneurysm incidence, and aneurysmal death in apolipoprotein E– and interferon-&ggr;–deficient (Apoe−/−/Ifng−/−) mice compared with Apoe−/− controls, although atherosclerotic luminal plaque formation was attenuated. The interferon-&ggr;–inducible T-cell chemoattractant CXCL10 was highly induced by angiotensin II infusion in Apoe−/− mice, but this induction was markedly attenuated in Apoe−/−/Ifng−/− mice. Apoe−/−/Cxcl10−/− mice had decreased luminal plaque but also increased aortic size, worse morphological grades of aneurysms, and a higher incidence of death due to aortic rupture than Apoe−/− controls. Furthermore, abdominal aortic aneurysms in Apoe−/−/Cxcl10−/− mice were enriched for non–T-helper type 1–related signals, including transforming growth factor-&bgr;1. Treatment of Apoe−/−/Cxcl10−/− mice with anti-transforming growth factor-&bgr; neutralizing antibody diminished angiotensin II–induced aortic dilation. Conclusions— The present study defines a novel pathway in which interferon-&ggr; and its effector, CXCL10, contribute to divergent pathways in abdominal aortic aneurysm versus plaque formation, inhibiting the former pathology but promoting the latter. Thus, efforts to develop antiinflammatory strategies for atherosclerosis must carefully consider potential effects on all manifestations of vascular disease.

PAR-1 contributes to the innate immune response during viral infection

Coagulation is a host defense system that limits the spread of pathogens. Coagulation proteases, such as thrombin, also activate cells by cleaving PARs. In this study, we analyzed the role of PAR-1 in coxsackievirus B3-induced (CVB3-induced) myocarditis and influenza A infection. CVB3-infected Par1(-/-) mice expressed reduced levels of IFN-β and CXCL10 during the early phase of infection compared with Par1(+/+) mice that resulted in higher viral loads and cardiac injury at day 8 after infection. Inhibition of either tissue factor or thrombin in WT mice also significantly increased CVB3 levels in the heart and cardiac injury compared with controls. BM transplantation experiments demonstrated that PAR-1 in nonhematopoietic cells protected mice from CVB3 infection. Transgenic mice overexpressing PAR-1 in cardiomyocytes had reduced CVB3-induced myocarditis. We found that cooperative signaling between PAR-1 and TLR3 in mouse cardiac fibroblasts enhanced activation of p38 and induction of IFN-β and CXCL10 expression. Par1(-/-) mice also had decreased CXCL10 expression and increased viral levels in the lung after influenza A infection compared with Par1(+/+) mice. Our results indicate that the tissue factor/thrombin/PAR-1 pathway enhances IFN-β expression and contributes to the innate immune response during single-stranded RNA viral infection.

MyD88 Deficiency Attenuates Angiotensin II-Induced Abdominal Aortic Aneurysm Formation Independent of Signaling Through Toll-Like Receptors 2 and 4

Objective—The purpose of this study was to determine whether myeloid differentiation factor 88 (MyD88) and its related Toll-like receptors (TLRs) 2 and 4 contributed to the development of angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) and atherosclerosis. Methods and Results—AngII was infused into either apoE−/− or LDL receptor (LDLR)−/− male mice that were either MyD88+/+ or −/−. MyD88 deficiency profoundly reduced AngII-induced AAAs and atherosclerosis in both strains. To define whether deficiency of specific TLRs had similar effects, AngII was infused into LDLR−/− mice that were also deficient in either TLR2 or TLR4. TLR2 deficiency had no effect on AAA development but inhibited atherosclerosis. In contrast, TLR4 deficiency attenuated both AAAs and atherosclerosis. To resolve whether MyD88 and TLR4 exerted their effects through cells of hematopoietic lineage, LDLR−/− mice were lethally irradiated and repopulated with bone marrow-derived cells from either MyD88 or TLR4 strains. MyD88 deficiency in bone marrow-derived cells profoundly reduced both AngII-induced AAAs and atherosclerosis. However, TLR4 deficiency in bone marrow-derived cells had no effect on either pathology. Conclusion—These studies demonstrate that MyD88 deficiency in leukocytes profoundly reduces AngII-induced AAAs and atherosclerosis via mechanisms independent of either TLR2 or TLR4.

Angiotensin II Induces a Region-Specific Hyperplasia of the Ascending Aorta Through Regulation of Inhibitor of Differentiation 3

Rationale: Angiotensin II (Ang II) has diverse effects on smooth muscle cells (SMCs). The diversity of effects may relate to the regional location of this cell type. Objective: The aim of this study was to define whether Ang II exerted divergent effects on smooth muscle cells in the aorta and determine the role of blood pressure and specific oxidant mechanisms. Methods and Results: Ang II (1000 ng/kg per minute) infusion for 28 days into mice increased systolic blood pressure and promoted medial expansion of equivalent magnitude throughout the entire aorta. Both effects were ablated by angiotensin II type 1a (AT1a) receptor deficiency. Similar increases in systolic blood pressure by administration of norepinephrine promoted no changes in aortic medial thickness. Increased medial thickness was attributable to SMC expansion owing to hypertrophy in most aortic regions, with the exception of hyperplasia of the ascending aorta. Deficiency of the p47phox component of NADPH oxidase ablated Ang II–induced medial expansion in all aortic regions. Analysis of mRNA and protein throughout the aorta revealed a much higher abundance of the inhibitor of differentiation 3 (Id3) in the ascending aorta compared to all other regions. A functional role was demonstrated by Id3 deficiency inhibiting Ang II–induced SMC hyperplasia of the ascending aorta. Conclusions: In conclusion, Ang II promotes both aortic medial hypertrophy and hyperplasia in a region-specific manner via an oxidant mechanism. The ascending aortic hyperplasia is dependent on Id3.