M. Anna Kowalska

Disrupting functional interactions between platelet chemokines inhibits atherosclerosis in hyperlipidemic mice

Atherosclerosis is characterized by chronic inflammation of the arterial wall due to chemokine-driven mononuclear cell recruitment. Activated platelets can synergize with chemokines to exacerbate atherogenesis; for example, by deposition of the chemokines platelet factor-4 (PF4, also known as CXCL4) and RANTES (CCL5), triggering monocyte arrest on inflamed endothelium. Homo-oligomerization is required for the recruitment functions of CCL5, and chemokine heteromerization has more recently emerged as an additional regulatory mechanism, as evidenced by a mutual modulation of CXCL8 and CXCL4 activities and by enhanced monocyte arrest resulting from CCL5-CXCL4 interactions. The CCL5 antagonist Met-RANTES reduces diet-induced atherosclerosis; however, CCL5 antagonism may not be therapeutically feasible, as suggested by studies using Ccl5-deficient mice which imply that direct CCL5 blockade would severely compromise systemic immune responses, delay macrophage-mediated viral clearance and impair normal T cell functions. Here we determined structural features of CCL5-CXCL4 heteromers and designed stable peptide inhibitors that specifically disrupt proinflammatory CCL5-CXCL4 interactions, thereby attenuating monocyte recruitment and reducing atherosclerosis without the aforementioned side effects. These results establish the in vivo relevance of chemokine heteromers and show the potential of targeting heteromer formation to achieve therapeutic effects.

Megakaryocyte precursors, megakaryocytes and platelets express the HIV co‐receptor CXCR4 on their surface: determination of response to stromal‐derived factor‐1 by megakaryocytes and platelets

Thrombocytopenia is a late complication of human immunodeficiency virus (HIV) infection. The chemokine receptor CXCR4 has been shown to be a co‐receptor for lymphocyte‐tropic HIV‐1 strains. CXCR4 is also a natural receptor for the chemokine SDF‐1. We have previously shown that CXCR1 and CXCR2 are present on megakaryocytes and platelets. Although interleukin‐8 (IL‐8) and other chemokines that bind to these two receptors do not activate platelets, they are able to inhibit megakaryocytopoiesis, presumably through these receptors. We therefore examined whether CXCR4 is present on developing and mature megakaryocytes and on platelets. Reverse transcription‐polymerase chain reaction (RT‐PCR) demonstrated the presence of CXCR4 message. Immature and mature αIIbβ3+ megakaryocytes, and platelets were also positive for CXCR4 by flow cytometric studies using a CXCR4‐specific antibody. We then tested whether SDF‐1 can affect the biology of these cells. CD34+ cells and immature αIIbβ3+ cells responded to SDF‐1 as indicated by Ca2+ mobilization and chemotaxis. However, mature megakaryocytes failed to demonstrate either of these responses, in spite of their continued ability to bind 125I‐SDF‐1. Further, SDF‐1 failed to inhibit megakaryocyte colony growth. Platelets bound 125I‐SDF‐1 with a KD similar to the affinity seen for CXCR4 on other cells, yet SDF‐1 did not aggregate washed platelets nor augment aggregation by low‐dose ADP or thrombin. SDF‐1 also failed to stimulate Ca2+ mobilization, granular release or expression of P‐selectin in platelets. Accordingly, although our studies demonstrate that CD34+ precursors, megakaryocytes and platelets all express CXCR4 and bind SDF‐1, biological effects were only demonstrable of SDF‐1 on CD34+ precursors. The potential biological implications of CXCR4 expression on maturing megakaryocytes and platelets in normal individuals and following HIV infection are discussed.

Elimination of platelet factor 4 (PF4) from platelets reduces atherosclerosis in C57Bl/6 and apoE-/- mice

Activated platelets, which release platelet factor 4 (PF4) are present in patients with atherosclerosis. To date, no direct in-vivo evidence exists for the involvement of PF4 in atherogenesis. In the current study, we tested the hypothesis that PF4 is atherogenic, and that genetic elimination of PF4 would protect mice from atherosclerosis. We have bred PF4(-/-) mice onto two athero-susceptible backgrounds, WT-C57Bl/6(WT) and apoE(-/-) to examine the importance of PF4 in atherogenesis. In order to induce atherosclerosis, WT and PF4(-/-) mice were fed an atherogenic diet for 30 weeks, while apoE(-/-) and apoE(-/-) PF4(-/-) mice were fed a high-fat Western-style diet for 10 weeks. Examination of lesions in the aortic roots of atherogenic diet fed mice demonstrated reduced atherosclerosis in PF4(-/-) (20% compared to WT). Examination of apoE(-/-) mice demonstrated similar changes, with apoE(-/-) PF4(-/-) mice demonstrating 37% of the aortic atherosclerotic burden compared to apoE(-/-) mice. Although we found similar levels of total and non-HDL cholesterol in WT and PF4(-/-) mice, HDL-cholesterol levels were increased in PF4(-/-) on both backgrounds. These data demonstrate, for the first time, that the platelet specific chemokine PF4 promotes atherosclerotic lesion development in vivo.

Platelet factor 4 mediates inflammation in experimental cerebral malaria.

Cerebral malaria (CM) is a major complication of Plasmodium falciparum infection in children. The pathogenesis of CM involves vascular inflammation, immune stimulation, and obstruction of cerebral capillaries. Platelets have a prominent role in both immune responses and vascular obstruction. We now demonstrate that the platelet-derived chemokine, platelet factor 4 (PF4)/CXCL4, promotes the development of experimental cerebral malaria (ECM). Plasmodium-infected red blood cells (RBCs) activated platelets independently of vascular effects, resulting in increased plasma PF4. PF4 or chemokine receptor CXCR3 null mice had less severe ECM, including decreased T cell recruitment to the brain, and platelet depletion or aspirin treatment reduced the development of ECM. We conclude that Plasmodium-infected RBCs can directly activate platelets, and platelet-derived PF4 then contributes to immune activation and T cell trafficking as part of the pathogenesis of ECM.

Monocyte-bound PF4 in the pathogenesis of heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a life- and limb-threatening thrombotic disorder that develops after exposure to heparin, often in the setting of inflammation. We have shown previously that HIT is associated with antibodies to complexes that form between platelet factor 4 and glycosaminoglycan (GAG) side chains on the surface of platelets. However, thrombosis can occur in the absence of thrombocytopenia. We now show that platelet factor 4 binds to monocytes and forms antigenic complexes with their surface GAG side chains more efficiently than on platelets likely due to differences in GAG composition. Binding to monocytes is enhanced when the cells are activated by endotoxin. Monocyte accumulation within developing arteriolar thrombi was visualized by situ microscopy. Monocyte depletion or inactivation in vivo attenuates thrombus formation induced by photochemical injury of the carotid artery in a modified murine model of HIT while paradoxically exacerbating thrombocytopenia. These studies demonstrate a previously unappreciated role for monocytes in the pathogenesis of arterial thrombosis in HIT and suggest that therapies targeting these cells might provide an alternative approach to help limit thrombosis in this and possibly other thrombotic disorders that occur in the setting of inflammation.

CXC chemokine ligand 4 (Cxcl4) is a platelet‐derived mediator of experimental liver fibrosis

Liver fibrosis is a major cause of morbidity and mortality worldwide. Platelets are involved in liver damage, but the underlying molecular mechanisms remain elusive. Here, we investigate the platelet‐derived chemokine (C‐X‐C motif) ligand 4 (CXCL4) as a molecular mediator of fibrotic liver damage. Serum concentrations and intrahepatic messenger RNA of CXCL4 were measured in patients with chronic liver diseases and mice after toxic liver injury. Platelet aggregation in early fibrosis was determined by electron microscopy in patients and by immunohistochemistry in mice. Cxcl4−/− and wild‐type mice were subjected to two models of chronic liver injury (CCl4 and thioacetamide). The fibrotic phenotype was analyzed by histological, biochemical, and molecular analyses. Intrahepatic infiltration of immune cells was investigated by fluorescence‐activated cell sorting, and stellate cells were stimulated with recombinant Cxcl4 in vitro. The results showed that patients with advanced hepatitis C virus–induced fibrosis or nonalcoholic steatohepatitis had increased serum levels and intrahepatic CXCL4 messenger RNA concentrations. Platelets were found directly adjacent to collagen fibrils. The CCl4 and thioacetamide treatment led to an increase of hepatic Cxcl4 levels, platelet activation, and aggregation in early fibrosis in mice. Accordingly, genetic deletion of Cxcl4 in mice significantly reduced histological and biochemical liver damage in vivo, which was accompanied by changes in the expression of fibrosis‐related genes (Timp‐1 [tissue inhibitor of matrix metalloproteinase 1], Mmp9 [matrix metalloproteinase 9], Tgf‐β [transforming growth factor beta], IL10 [interleukin 10]). Functionally, Cxcl4−/− mice showed a strongly decreased infiltration of neutrophils (Ly6G) and CD8+ T cells into the liver. In vitro, recombinant murine Cxcl4 stimulated the proliferation, chemotaxis, and chemokine expression of hepatic stellate cells. Conclusion: The results underscore an important role of platelets in chronic liver damage and imply a new target for antifibrotic therapies. (HEPATOLOGY 2010.)

Infusion of mature megakaryocytes into mice yields functional platelets

Thrombopoiesis, the process by which circulating platelets arise from megakaryocytes, remains incompletely understood. Prior studies suggest that megakaryocytes shed platelets in the pulmonary vasculature. To better understand thrombopoiesis and to develop a potential platelet transfusion strategy that is not dependent upon donors, of which there remains a shortage, we examined whether megakaryocytes infused into mice shed platelets. Infused megakaryocytes led to clinically relevant increases in platelet numbers. The released platelets were normal in size, displayed appropriate surface markers, and had a near-normal circulating half-life. The functionality of the donor-derived platelets was also demonstrated in vivo. The infused megakaryocytes mostly localized to the pulmonary vasculature, where they appeared to shed platelets. These data suggest that it may be unnecessary to generate platelets from ex vivo grown megakaryocytes to achieve clinically relevant increases in platelet numbers.

Binding of stromal derived factor‐1α(SDF‐1α) to CXCR4 chemokine receptorin normal human megakaryoblasts butnot in platelets induces phosphorylationof mitogen‐activated protein kinase p42/44 (MAPK), ELK‐1 transcription factor and serine/threonine kinase AKT

Abstract: The aim of this study was to identify pathways which are involved in signal transduction from the CXCR4 receptor stimulated by stromal derived factor‐1α (SDF‐1α) in human malignant hematopoietic cells and normal megakaryoblasts. First, we found that activation of CXCR4 in human T cell lines (Jurkat and ATL‐2) rapidly induced phosphorylation of mitogen‐activated protein kinases (MAPK) (p44 ERK‐1 and p42 ERK‐2). Next, we became interested in CXCR4‐mediated signaling in normal hematopoietic cells, and employed human megakaryoblasts, which highly express CXCR4 as a model. We found that stimulation of these cells with SDF‐1α led to the phosphorylation of MAPK and serine/threonine kinase AKT as well. Activation of MAPK further led to the phosphorylation of the nuclear transcription factor ELK‐1. Phosphorylation of ELK‐1 in megakaryoblasts implies that phosphorylated MAPK translocate from cytoplasm into the nucleus where they may phosphorylate some nuclear proteins. Note that neither MAPK nor AKT was phosphorylated in normal human platelets after stimulation by SDF‐1. We conclude that both MAPK and AKT are involved in signal transduction pathways from the CXCR4 receptor in malignant and normal human hematopoietic cells. The biological consequences of MAPK, ELK‐1 and AKT phosphorylation in megakaryoblasts after stimulation with SDF‐1α require further studies.

Role of the platelet chemokine platelet factor 4 (PF4) in hemostasis and thrombosis.

Chemokines are a family of small proteins that have significant roles in inflammation, angiogenesis and cellular homing. Since inflammation and hemostasis/thrombosis have multiple overlapping roles and pathways, one could expect that some chemokines would also have biologically significant roles in hemostasis/thrombosis as well. This would especially be true for chemokines that are localized solely or predominantly within platelets and released in large amounts at sites of platelet activation such as platelet factor 4 (PF4, CXCL4) and its closely related chemokine, platelet basic protein (PBP, CXCL7). Our group and others have clearly demonstrated an in vivo role for PF4 in hemostasis/thrombosis, but not for PBP, which in contrast has clear proinflammatory properties. This review will focus on PF4 and its potential roles in hemostasis/thrombosis and the underlying pathways by which PF4 may be especially important in such pathologic thrombotic states as heparin-induced thrombocytopenia (HIT) and septic shock.

Sustained thromboprophylaxis mediated by an RBC-targeted pro-urokinase zymogen activated at the site of clot formation

Plasminogen activators (PAs) are used to treat life-threatening thrombosis, but not for thromboprophylaxis because of rapid clearance, risk of bleeding, and central nervous system (CNS) toxicity. We describe a novel strategy that may help to overcome these limitations by targeting a thrombin-activated PA pro-drug to circulating red blood cells (RBCs). We fused a single chain antibody (scFv Ter-119) that binds to mouse glycophorin A (GPA) with a variant human single-chain low molecular weight urokinase construct that can be activated selectively by thrombin (scFv/uPA-T). scFv/uPA-T bound specifically to mouse RBCs without altering their biocompatibility and retained its zymogenic properties until converted by thrombin into an active 2-chain molecule. As a result, RBC-bound scFv/uPA-T caused thrombin-induced fibrinolysis. One hour and 48 hours after intravenous (IV) injection in mice, approximately 70% and approximately 35% of scFv/uPA-T was retained in the blood, respectively, and approximately 95% of the circulating scFv/uPA-T remained bound to RBCs. A single IV injection of scFv/uPA-T provided effective prophylaxis against arterial and venous thrombosis for up to 24 hours. Thus, prophylactic delivery of RBC-targeted PA pro-drugs activated selectively at the site of clot formation represents a new approach to prevent thrombosis in clinical settings where the risk of clotting is high.