Defective PDI release from platelets and endothelial cells impairs thrombus formation in Hermansky-Pudlak syndrome.

Protein disulfide isomerase (PDI), secreted from platelets and endothelial cells after injury, is required for thrombus formation. The effect of platelet and endothelial cell granule contents on PDI-mediated thrombus formation was studied by intravital microscopy using a mouse model of Hermansky-Pudlak syndrome in which platelet dense granules are absent. Platelet deposition and fibrin generation were nearly absent, and extracellular PDI was significantly reduced in HPS6(-/-) mice after vascular injury. HPS6(-/-) platelets displayed impaired PDI secretion and impaired exocytosis of α granules, lysosomes, and T granules due to decreased sensitivity to thrombin, but these defects could be corrected by addition of subthreshold amounts of adenosine 5'-diphosphate (ADP). Human Hermansky-Pudlak syndrome platelets demonstrated similar characteristics. Infusion of wild-type platelets rescued thrombus formation in HPS6(-/-) mice. Human umbilical vein endothelial cells in which the HPS6 gene was silenced displayed impaired PDI secretion and exocytosis of Weibel-Palade bodies. Defective thrombus formation in Hermansky-Pudlak syndrome, associated with impaired exocytosis of residual granules in endothelial cells and platelets, the latter due to deficiency of ADP, is characterized by a defect in T granule secretion, a deficiency in extracellular PDI secretion, and impaired fibrin generation and platelet aggregation. Hermansky-Pudlak syndrome is an example of a hereditary disease whereby impaired PDI secretion contributes to a bleeding phenotype.

Both platelet- and endothelial cell-derived ERp5 support thrombus formation in a laser-induced mouse model of thrombosis.

Protein disulfide isomerase (PDI) and endoplasmic reticulum protein 57 (ERp57) are emerging as important regulators of thrombus formation. Another thiol isomerase, endoplasmic reticulum protein 5 (ERp5), is involved in platelet activation. We show here the involvement of ERp5 in thrombus formation using the mouse laser-injury model of thrombosis and a specific antibody raised against recombinant ERp5. Anti-ERp5 antibody inhibited ERp5-dependent platelet and endothelial cell disulfide reductase activity in vitro. ERp5 release at the thrombus site was detected after infusion of Alexa Fluor 488-labeled anti-ERp5 antibody at 0.05 µg/g body weight, a dose that does not inhibit thrombus formation. Anti-ERp5 at 3 µg/g body weight inhibited laser-induced thrombus formation in vivo by causing a 70% decrease in the deposition of platelets and a 62% decrease in fibrin accumulation compared to infusion of control antibody (P < .01). ERp5 binds to ß3 integrin with an equilibrium dissociation constant (KD) of 21 µM, measured by surface plasmon resonance. The cysteine residues in the ERp5 active sites are not required for binding to ß3 integrin. These results provide evidence for a novel role of ERp5 in thrombus formation, a function that may be mediated through its association with αIIbß3.

Compounds targeting disulfide bond forming enzyme DsbB of Gram-negative bacteria.

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond. These proteins include numerous bacterial virulence factors, and thus bacterial enzymes that promote disulfide bond formation represent targets for compounds inhibiting bacterial virulence. Here, we describe a new target- and cell-based screening methodology for identifying compounds that inhibit the disulfide bond-forming enzymes Escherichia coli DsbB (EcDsbB) or Mycobacterium tuberculosis VKOR (MtbVKOR), which can replace EcDsbB, although the two are not homologs. Initial screening of 51,487 compounds yielded six specifically inhibiting EcDsbB. These compounds share a structural motif and do not inhibit MtbVKOR. A medicinal chemistry approach led us to select related compounds, some of which are much more effective DsbB inhibitors than those found in the screen. These compounds inhibit purified DsbB and prevent anaerobic growth of E. coli. Furthermore, these compounds inhibit all but one of the DsbBs of nine other Gram-negative pathogenic bacteria tested.

Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b'x Domain.

Quercetin-3-rutinoside inhibits thrombus formation in a mouse model by inhibiting extracellular protein disulfide isomerase (PDI), an enzyme required for platelet thrombus formation and fibrin generation. Prior studies have identified PDI as a potential target for novel antithrombotic agents. Using a fluorescence enhancement-based assay and isothermal calorimetry, we show that quercetin-3-rutinoside directly binds to the b' domain of PDI with a 1:1 stoichiometry. The binding of quercetin-3-rutinoside to PDI induces a more compact conformation and restricts the conformational flexibility of PDI, as revealed by small angle x-ray scattering. The binding sites of quercetin-3-rutinoside to PDI were determined by studying its interaction with isolated fragments of PDI. Quercetin-3-rutinoside binds to the b'x domain of PDI. The infusion of the b'x fragment of PDI rescued thrombus formation that was inhibited by quercetin-3-rutinoside in a mouse thrombosis model. This b'x fragment does not possess reductase activity and, in the absence of quercetin-3-rutinoside, does not affect thrombus formation in vivo. The isolated b' domain of PDI has potential as an antidote to reverse the antithrombotic effect of quercetin-3-rutinoside by binding and neutralizing quercetin-3-rutinoside.

Platelets are required for enhanced activation of the endothelium and fibrinogen in a mouse thrombosis model of APS.

Antiphospholipid syndrome (APS) is defined by thrombosis, fetal loss, and the presence of antiphospholipid antibodies, including anti-ß2-glycoprotein-1 autoantibodies (anti-ß2GP1) that have a direct role in the pathogenesis of thrombosis in vivo. The cellular targets of the anti-ß2GP1 autoantibody/ß2GP1 complex in vivo were studied using a laser-induced thrombosis model of APS in a live mouse and human anti-ß2GP1 autoantibodies affinity-purified from APS patients. Cell binding of fluorescently labeled ß2GP1 and anti-ß2GP1 autoantibodies revealed their colocalization on the platelet thrombus but not the endothelium. Anti-ß2GP1 autoantibodies enhanced platelet activation, monitored by calcium mobilization, and endothelial activation, monitored by intercellular adhesion molecule-1 expression. When eptifibatide was infused to block platelet thrombus formation, enhanced fibrin generation and endothelial cell activation were eliminated. Thus, the anti-ß2GP1 autoantibody/ß2GP1 complex binds to the thrombus, enhancing platelet activation, and platelet secretion leads to enhanced endothelium activation and fibrin generation. These results lead to a paradigm shift away from the concept that binding of the anti-ß2GP1 autoantibody/ß2GP1 complex activates both endothelial cells and platelets toward one in which activation of platelets in response to anti-ß2GP1 autoantibody/ß2GP1 complex binding leads to subsequent enhanced endothelium activation and fibrin generation.

Protein disulfide isomerase capture during thrombus formation in vivo depends on the presence of ß3 integrins.

Extracellular protein disulfide isomerase (PDI) is required for platelet thrombus formation and fibrin generation after arteriolar wall injury in live mice. PDI is secreted from platelets and endothelial cells on cellular activation, but the mechanism of capture of secreted PDI within the injured vasculature is unknown. We establish that, like the endothelial ß3 integrin α(V)ß(3), the platelet integrin α(IIb)ß(3) binds PDI. PDI also binds to recombinant ß3. Using intravital microscopy, we demonstrate that PDI accumulation at the site of laser-induced arteriolar wall injury is markedly reduced in ß3-null (ß3(-/-)) mice, and neither a platelet thrombus nor fibrin is generated at the vessel injury site. The absence of fibrin after vascular injury in ß3(-/-) mice is because of the absence of extracellular PDI. To evaluate the relative importance of endothelial α(V)ß(3) versus platelet α(IIb)ß(3) or α(V)ß(3), we performed reciprocal bone marrow transplants on wild-type and ß3(-/-) mice. PDI accumulation and platelet thrombus formation were markedly decreased after vessel injury in wild-type mice transplanted with ß3(-/-) bone marrow or in ß3(-/-) mice transplanted with wild-type bone marrow. These results indicate that both endothelial and platelet ß3 integrins contribute to extracellular PDI binding at the vascular injury site.

ß2-Glycoprotein-1 autoantibodies from patients with antiphospholipid syndrome are sufficient to potentiate arterial thrombus formation in a mouse model.

Antiphospholipid syndrome is characterized by thrombosis, recurrent fetal loss, and the presence of the lupus anticoagulant, anticardiolipin antibodies, or anti-ß(2)-glycoprotein-1 (anti-ß(2)-GP1) antibodies. Although anti-ß(2)-GP1 antibodies have been documented as a biomarker for diagnosis of antiphospholipid syndrome, their direct role in the pathogenesis of thrombosis is unknown. We have demonstrated using intravital microscopy that anti-ß(2)-GP1 autoantibodies purified from the sera of patients with antiphospholipid syndrome complicated by thrombosis greatly amplify thrombus size after laser-induced vessel wall injury in live mice. Anti-ß(2)-GP1 autoantibodies from 3 patients with antiphospholipid syndrome were affinity-purified using human ß(2)-GP1 bound to agarose. The effects of purified anti-ß(2)-GP1 IgG autoantibodies, of anti-ß(2)-GP1-depleted IgG, and of IgG from normal human sera on thrombus formation were measured in mice after arterial injury in the cremaster muscle. Before injury, purified anti-ß(2)-GP1 IgG autoantibodies, anti-ß(2)-GP1 antibody-depleted IgG, or IgG from normal human sera were infused. Increasing amounts of purified anti-ß(2)-GP1 autoantibodies increased thrombus size in a dose-dependent manner, whereas neither anti-ß(2)-GP1 antibody-depleted IgG nor IgG from normal serum affected thrombus size. These results indicate that anti-ß(2)-GP1 IgG autoantibodies in antiphospholipid syndrome patient sera are not only a marker of antiphospholipid syndrome but are directly involved in the pathogenesis of thrombosis.

Structural basis for recognition of urokinase-type plasminogen activator by plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1), together with its physiological target urokinase-type plasminogen activator (uPA), plays a pivotal role in fibrinolysis, cell migration, and tissue remodeling and is currently recognized as being among the most extensively validated biological prognostic factors in several cancer types. PAI-1 specifically and rapidly inhibits uPA and tissue-type PA (tPA). Despite extensive structural/functional studies on these two reactions, the underlying structural mechanism has remained unknown due to the technical difficulties of obtaining the relevant structures. Here, we report a strategy to generate a PAI-1·uPA(S195A) Michaelis complex and present its crystal structure at 2.3-Å resolution. In this structure, the PAI-1 reactive center loop serves as a bait to attract uPA onto the top of the PAI-1 molecule. The P4-P3' residues of the reactive center loop interact extensively with the uPA catalytic site, accounting for about two-thirds of the total contact area. Besides the active site, almost all uPA exosite loops, including the 37-, 60-, 97-, 147-, and 217-loops, are involved in the interaction with PAI-1. The uPA 37-loop makes an extensive interaction with PAI-1 ß-sheet B, and the 147-loop directly contacts PAI-1 ß-sheet C. Both loops are important for initial Michaelis complex formation. This study lays down a foundation for understanding the specificity of PAI-1 for uPA and tPA and provides a structural basis for further functional studies.

Active site-labeled prothrombin inhibits prothrombinase in vitro and thrombosis in vivo.

Mouse and human prothrombin (ProT) active site specifically labeled with D-Phe-Pro-Arg-CH(2)Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProT(S195A) mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited ∼10-fold more potent anticoagulant activity compared with ProT(S195A) as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProT(S195A), the pathway of FPR-ProT cleavage by prothrombinase was redirected from meizothrombin toward formation of the FPR-prethrombin 2 (Pre 2)·F1.2 inhibitory intermediate. Localization of ProT labeled with Alexa Fluor® 660 tethered through FPR-CH(2)Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre 1, lacking the F1 membrane-binding domain did not bind or inhibit. Labeled F1.2 localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.

Endothelium-derived but not platelet-derived protein disulfide isomerase is required for thrombus formation in vivo.

Protein disulfide isomerase (PDI) catalyzes the oxidation reduction and isomerization of disulfide bonds. We have previously identified an important role for extracellular PDI during thrombus formation in vivo. Here, we show that endothelial cells are a critical cellular source of secreted PDI, important for fibrin generation and platelet accumulation in vivo. Functional PDI is rapidly secreted from human umbilical vein endothelial cells in culture upon activation with thrombin or after laser-induced stimulation. PDI is localized in different cellular compartments in activated and quiescent endothelial cells, and is redistributed to the plasma membrane after cell activation. In vivo studies using intravital microscopy show that PDI appears rapidly after laser-induced vessel wall injury, before the appearance of the platelet thrombus. If platelet thrombus formation is inhibited by the infusion of eptifibatide into the circulation, PDI is detected after vessel wall injury, and fibrin deposition is normal. Treatment of mice with a function blocking anti-PDI antibody completely inhibits fibrin generation in eptifibatide-treated mice. These results indicate that, although both platelets and endothelial cells secrete PDI after laser-induced injury, PDI from endothelial cells is required for fibrin generation in vivo.

Laser-induced endothelial cell activation supports fibrin formation.

Laser-induced vessel wall injury leads to rapid thrombus formation in an animal thrombosis model. The target of laser injury is the endothelium. We monitored calcium mobilization to assess activation of the laser-targeted cells. Infusion of Fluo-4 AM, a calcium-sensitive fluorochrome, into the mouse circulation resulted in dye uptake in the endothelium and circulating hematopoietic cells. Laser injury in mice treated with eptifibatide to inhibit platelet accumulation resulted in rapid calcium mobilization within the endothelium. Calcium mobilization correlated with the secretion of lysosomal-associated membrane protein 1, a marker of endothelium activation. In the absence of eptifibatide, endothelium activation preceded platelet accumulation. Laser activation of human umbilical vein endothelial cells loaded with Fluo-4 resulted in a rapid increase in calcium mobilization associated cell fluorescence similar to that induced by adenosine diphosphate (10 µM) or thrombin (1 U/mL). Laser activation of human umbilical vein endothelial cells in the presence of corn trypsin inhibitor treated human plasma devoid of platelets and cell microparticles led to fibrin formation that was inhibited by an inhibitory monoclonal anti-tissue factor antibody. Thus laser injury leads to rapid endothelial cell activation. The laser activated endothelial cells can support formation of tenase and prothrombinase and may be a source of activated tissue factor as well.

Tissue factor-bearing microparticles and thrombus formation.

Blood microparticles are vesicular structures with a diameter of 100 to 1000 nm that are present in the blood of normal subjects and in patients with various diseases. These microparticles are derived from cells that circulate in the blood and cells associated with the blood vessel wall. Microparticle membranes retain the protein receptors of their parent cells and may retain RNAs and other cytosolic content. On the basis of surface protein expression, microparticles are known to be derived from platelets, granulocytes, monocytes, endothelial cells, smooth muscle cells, and tumor cells. Only a subpopulation of these microparticles expresses tissue factor.

A critical role for extracellular protein disulfide isomerase during thrombus formation in mice.

Thiol isomerases, including protein disulfide isomerase (PDI), catalyze disulfide oxidation, reduction, and isomerization, thereby playing an important role in protein synthesis. To determine whether extracellular PDI mediates thrombus formation in an animal model, PDI expression, platelet accumulation, and fibrin generation were monitored in the blood vessels of mice by intravital fluorescence microscopy following laser-induced arteriolar injury. A time-dependent increase in PDI was observed in murine thrombi following injury. Infusion of the PDI inhibitor bacitracin or a blocking monoclonal antibody against PDI inhibited platelet thrombus formation and fibrin generation. Fibrin deposition is normal in mice lacking the G protein-coupled platelet receptor Par4, although there is no stable accumulation of platelets. Infusion of monoclonal antibodies against PDI into the circulation of Par4(-/-) mice prior to vessel wall injury inhibited fibrin generation. These results indicate that PDI is required in vivo in mice for both fibrin generation and platelet thrombus formation.

Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse.

We have used confocal and widefield microscopy to image thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and of a brightfield channel. Vascular injury is induced with a laser focused through the microscope optics. We observed platelet deposition, tissue factor accumulation and fibrin generation after laser-induced endothelial injury in a single developing thrombus. The initiation of blood coagulation in vivo entailed the initial accumulation of tissue factor on the upstream and thrombus-vessel wall interface of the developing thrombus. Subsequently tissue factor was associated with the interior of the thrombus. Tissue factor was biologically active, and was associated with fibrin generation within the thrombus.

Accumulation of tissue factor into developing thrombi in vivo is dependent upon microparticle P-selectin glycoprotein ligand 1 and platelet P-selectin.

Using a laser-induced endothelial injury model, we examined thrombus formation in the microcirculation of wild-type and genetically altered mice by real-time in vivo microscopy to analyze this complex physiologic process in a system that includes the vessel wall, the presence of flowing blood, and the absence of anticoagulants. We observe P-selectin expression, tissue factor accumulation, and fibrin generation after platelet localization in the developing thrombus in arterioles of wild-type mice. However, mice lacking P-selectin glycoprotein ligand 1 (PSGL-1) or P-selectin, or wild-type mice infused with blocking P-selectin antibodies, developed platelet thrombi containing minimal tissue factor and fibrin. To explore the delivery of tissue factor into a developing thrombus, we identified monocyte-derived microparticles in human platelet-poor plasma that express tissue factor, PSGL-1, and CD14. Fluorescently labeled mouse microparticles infused into a recipient mouse localized within the developing thrombus, indicating that one pathway for the initiation of blood coagulation in vivo involves the accumulation of tissue factor- and PSGL-1-containing microparticles in the platelet thrombus expressing P-selectin. These monocyte-derived microparticles bind to activated platelets in an interaction mediated by platelet P-selectin and microparticle PSGL-1. We propose that PSGL-1 plays a role in blood coagulation in addition to its known role in leukocyte trafficking.

Thrombin-initiated platelet activation in vivo is vWF independent during thrombus formation in a laser injury model.

Adhesion of platelets to an injured vessel wall and platelet activation are critical events in the formation of a thrombus. Of the agonists involved in platelet activation, thrombin, collagen, and vWF are known to induce in vitro calcium mobilization in platelets. Using a calcium-sensitive fluorochrome and digital multichannel intravital microscopy to image unstimulated and stimulated platelets, calcium mobilization was monitored as a reporter of platelet activation (as distinct from platelet accumulation) during thrombus formation in live mice. In the absence of vWF, platelet activation was normal, but platelet adherence and aggregation were attenuated during thrombus formation following laser-induced injury in the cremaster muscle microcirculation. In WT mice treated with lepirudin, platelet activation was blocked, and platelet adherence and aggregation were inhibited. The kinetics of platelet activation and platelet accumulation were similar in FcRgamma(-/-) mice lacking glycoprotein VI (GPVI), GPVI-depleted mice, and WT mice. Our results indicate that the tissue factor-mediated pathway of thrombin generation, but not the collagen-induced GPVI-mediated pathway, is the major pathway leading to platelet activation after laser-induced injury under the conditions employed. In the tissue factor-mediated pathway, vWF plays a role in platelet accumulation during thrombus formation but is not required for platelet activation in vivo.

Bile salt-dependent lipase interacts with platelet CXCR4 and modulates thrombus formation in mice and humans.

Bile salt-dependent lipase (BSDL) is an enzyme involved in the duodenal hydrolysis and absorption of cholesteryl esters. Although some BSDL is transported to blood, the role of circulating BSDL is unknown. Here, we demonstrate that BSDL is stored in platelets and released upon platelet activation. Because BSDL contains a region that is structurally homologous to the V3 loop of HIV-1, which binds to CXC chemokine receptor 4 (CXCR4), we hypothesized that BSDL might bind to CXCR4 present on platelets. In human platelets in vitro, both BSDL and a peptide corresponding to its V3-like loop induced calcium mobilization and enhanced thrombin-mediated platelet aggregation, spreading, and activated alpha(IIb)beta(3) levels. These effects were abolished by CXCR4 inhibition. BSDL also increased the production of prostacyclin by human endothelial cells. In a mouse thrombosis model, BSDL accumulated at sites of vessel wall injury. When CXCR4 was antagonized, the accumulation of BSDL was inhibited and thrombus size was reduced. In BSDL(-/-) mice, calcium mobilization in platelets and thrombus formation were attenuated and tail bleeding times were increased in comparison with those of wild-type mice. We conclude that BSDL plays a role in optimal platelet activation and thrombus formation by interacting with CXCR4 on platelets.

PTP-1B is an essential positive regulator of platelet integrin signaling.

Outside-in integrin alphaIIbbeta3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein-tyrosine phosphatase (PTP)-1B in this process. In resting platelets, c-Src forms a complex with alphaIIbbeta3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to alphaIIbbeta3 triggers PTP-1B recruitment to the alphaIIbbeta3-c-Src-Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B-deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from alphaIIbbeta3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B-deficient platelets are defective in outside-in alphaIIbbeta3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in alphaIIbbeta3 signaling in platelets.

Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex.

Factor VIII is a procofactor that plays a critical role in blood coagulation, and is missing or defective in hemophilia A. We determined the X-ray crystal structure of B domain-deleted human factor VIII. This protein is composed of five globular domains and contains one Ca(2+) and two Cu(2+) ions. The three homologous A domains form a triangular heterotrimer where the A1 and A3 domains serve as the base and interact with the C2 and C1 domains, respectively. The structurally homologous C1 and C2 domains reveal membrane binding features. Based on biochemical studies, a model of the factor IXa-factor VIIIa complex was constructed by in silico docking. Factor IXa wraps across the side of factor VIII, and an extended interface spans the factor VIII heavy and light chains. This model provides insight into the activation of factor VIII and the interaction of factor VIIIa with factor IXa on the membrane surface.

Peritoneal macrophages express both P-selectin and PSGL-1.

Macrophages, phagocytic cells involved in an early phase of host defense, are known to express the P-selectin ligand, PSGL-1. Heretofore, P-selectin has only been found on platelets and endothelial cells. Here, we demonstrate that peritoneal macrophages isolated by peritoneal lavage of unchallenged mice express P-selectin on the plasma membrane. The peritoneal macrophages synthesize P-selectin, as indicated by metabolic labeling experiments. P-Selectin is constitutively expressed on the extracellular surface of macrophages but is only partially colocalized with PSGL-1. P-Selectin is rapidly translocated from the macrophage plasma membrane to intracellular vesicles and to lysosomes. Peritoneal macrophages assemble into cell strings under flow conditions based upon macrophage-macrophage interactions mediated by P-selectin and PSGL-1. This is the first description of a leukocyte shown to express both P-selectin and PSGL-1.