Summary The chemokine sink hypothesis pertaining to erythrocyte Duffy Antigen Receptor for Chemokines (DARC) during inflammation has received considerable attention, but lacks direct in vivo evidence. Here we demonstrate, using mice with a targeted deletion in CXCL5, that CXCL5 bound erythrocyte DARC and impaired its chemokine scavenging in blood. CXCL5 increased the plasma concentrations of CXCL1 and CXCL2 in part through inhibiting chemokine scavenging, impairing chemokine gradients and desensitizing CXCR2, which led to decreased neutrophil influx to the lung, increased lung bacterial burden and mortality in an Escherichia coli pneumonia model. In contrast, CXCL5 exerted a predominant role in mediating neutrophil influx to the lung during inflammation after LPS inhalation. Platelets and lung resident cells were the sources of homeostatic CXCL5 in blood and inflammatory CXCL5 in the lung respectively. This study presents a paradigm whereby platelets and red cells alter chemokine scavenging and neutrophil-chemokine interaction during inflammation.

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. IntroductionPlatelet factor 4 (PF4) is a cationic chemokine with high affinity for unfractionated heparin (UFH) and other large, negatively charged molecules. 1 PF4 is stored in platelet ␣-granules, released upon activation, when it then binds rapidly to glycosaminoglycan (GAG) side chains expressed on the surface of platelets 2 and other vascular cells, with little remaining free in the circulation. 3 Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies that recognize complexes of human (h) PF4 with heparin or other GAGs. 4,5 In solution, formation of antigenic complexes between PF4 and heparin is critically dependent on their molar ratio, with loss of antibody binding when the optimal ratio is disrupted by an excess of either component. 6,7 Antigen formation on the platelet surface also follows a bell-shaped curve as PF4 concentration is increased, with maximal binding of antibody seen at an exogenous PF4 concentration of 50 g/mL. 8 Chondroitin sulfates (CSs) are the predominant GAG side chains expressed on platelets. 9,10 We have shown that the binding of the HIT-like monoclonal antibody KKO 11 to platelets is abrogated by chondroitinase ABC, 8 indicating that HIT antibodies bind to PF4/CS complexes on this cell type. Therapeutic concentrations of UFH disrupt antibody binding in part by eluting PF4 from the platelet surface, which reduces formation of antigenic complexes and the potential for platelet activation 8 through platelet Fc␥RIIA. 12 Thus, variation in the expression of platelet-derived PF4 (or CS) might help to explain why only a small percentage of patients who generate antibodies to PF4/UFH develop HIT. 13 Although it has been generally accepte...

Two major pathways contribute to Rasproximate-1-mediated integrin activation in stimulated platelets. Calcium and diacyglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI, Ras-GRP2) mediates the rapid but reversible activation of integrin ␣IIb␤3, while the adenosine diphosphate receptor P2Y12, the target for antiplatelet drugs like clopidogrel, facilitates delayed but sustained integrin activation. To establish CalDAG-GEFI as a target for antiplatelet therapy, we compared how each pathway contributes to thrombosis and hemostasis in mice. Ex vivo, thrombus formation at arterial or venous shear rates was markedly reduced in CalDAG-GEFI ؊/؊ blood, even in the presence of exogenous adenosine diphosphate and thromboxane A 2 . In vivo, thrombosis was virtually abolished in arterioles and arteries of CalDAG-GEFI ؊/؊ mice, while small, hemostatically active thrombi formed in venules. Specific deletion of the C1-like domain of CalDAG-GEFI in circulating platelets also led to protection from thrombus formation at arterial flow conditions, while it only marginally increased blood loss in mice. In comparison, thrombi in the micro-and macrovasculature of clopidogrel-treated wild-type mice grew rapidly and frequently embolized but were hemostatically inactive. Together, these data suggest that inhibition of the catalytic or the C1 regulatory domain in CalDAG-GEFI will provide strong protection from atherothrombotic complications while maintaining a better safety profile than P2Y12 inhibitors like clopidogrel. (Blood. 2011; 117(3):1005-1013) IntroductionArterial thrombosis in the coronary or cerebrovascular circulation is the principal pathological process underlying acute coronary syndrome and ischemic stroke, which together represent the leading cause of morbidity and mortality in industrialized countries. 1 Platelet activation is a central event in the pathogenesis of arterial thrombosis. Currently, the most powerful antiplatelet agents used in the clinic are inhibitors of cyclooxygenase-1 (acetylsalicylic acid, aspirin), the platelet adenosine diphosphate (ADP) receptor P2Y12 (eg, clopiodgrel or Plavix), and integrin ␣IIb␤3 (eg, abciximab or Reopro). 2,3 These agents have all been shown to improve clinical outcomes in large-scale randomized controlled trials. However, all therapies have limitations that include uncertainty about optimal dosing, questions about resistance, and issues regarding the lack of reversibility in situations where bleeding risks are high.␣IIb␤3, the platelet fibrinogen receptor, is the best-studied member of the integrin family. 4,5 Like most integrins, especially those regulating adhesion and trafficking of blood cells, it is expressed in a low-affinity state on resting platelets. Engagement of agonist receptors on the platelet surface triggers intracellular signaling events, which lead to inside-out activation of ␣IIb␤3. Deficiency in ␣IIb␤3 completely inhibits the ability of platelets to aggregate and adhere to sites of injury. 6,7 Consequently, inhibitors to integrin ␣IIb␤3 show...