The cAMP-protein kinase A (PKA) pathway in platelets is important for both platelet activation and inactivation. We hypothesize that proteins/processes downstream of the cAMP-PKA pathway that are regulated after platelet activation ánd subsequent inactivation can serve as a "switch" in platelet activation and inhibition. We used a STRING-based protein-protein interaction network from proteins of interest distilled from publicly available quantitative platelet proteome datasets. The protein network was integrated with biological pathway information by functional enrichment analysis, phosphorylation by PKA, and drug-target information. Functional enrichment analysis revealed biological processes related to vesicle secretion and cytoskeletal reorganization to be overrepresented among these 30 proteins coinciding with topological clusters in the network. Our method identified proteins/processes with functions related to vesicle transport, cyclin-dependent protein kinases, tight junctions, and small GTPases as potential switches in platelet activation and inhibition. Next to established enzymes in cAMP-PKA signaling, such as PDE3A, proteins with an unknown/less well-known role in platelet biology, such as Stonin-2 and ABLIM-3, emerged from our analysis as interesting candidates for reversal of platelet activation. Our method can be used to repurpose existing datasets and provide a coherent overview of mechanisms involved to predict novel connections, by visually integrating multiple datasets. SIGNIFICANCE: This article presents a novel approach of visually incorporating multiple existing tools and proteomics datasets and in doing so provides novel insight into the complex molecular mechanisms involved in platelet activation. Using our approach, we also highlight several interesting candidates for future research into pathologies with high platelet reactivity.

Cerebral small vessel disease (cSVD) accounts for 25% of ischemic strokes and is a major cause of cognitive decline. Inflammatory processes, involving immune cells and platelets might drive development and progression of cSVD. The aim of the study was to identify potential novel biomarkers for cSVD, gaining new insights into its pathophysiology. We measured inflammation and platelet and neutrophil activation markers in patients with cSVD and age-matched controls. It was hypothesized that cSVD is accompanied by altered levels of these markers. The levels of interleukin 1 beta, CX(3)CL1, CXCL4, CXCL7, myeloperoxidase (MPO), MPO-DNA complex and S100A8/A9 were measured by ELISA in plasma samples of patients with cSVD presenting with mild vascular cognitive impairment (mVCI, n = 36) or lacunar stroke (Laci, n = 44), and controls (n = 38). To determine the relevance of these ELISA markers compared with patient-and MRI-based characteristics, all characteristics were entered into three machine learning models. Among the ELISA markers measured, MPO levels were significantly elevated in patients with cSVD (48.3 (27.8-80.1, interquartile range) ng/mL) compared with controls (32.2 (19.6-47.4) ng/mL, P = 0.023), particularly in the Laci group (56.8 (33.3-84.7) ng/mL, P = 0.004). Regularized logistic regression and random forest algorithms returned MPO levels as an important feature in the detection and prediction of cSVD. Of note, logistic regression and random forest analysis also highlighted levels of CXCL4, CXCL7, MPO-DNA and S100A8/A9 as features associated with cSVD. Taken together, the neutrophil activation marker MPO is elevated in patients with Laci and machine learning indicates platelet and neutrophil markers as interesting molecules for future investigation.& nbsp;Shortened abstract: Cerebral small vessel disease (cSVD) is a major cause of cognitive decline and stroke. We aimed to identify potential novel biomarkers for cSVD and to obtain new insights into its pathophysiology. Levels of markers reflecting neutrophil activation, neutrophil extracellular trap (NET) formation, platelet activation and vascular inflammation were measured in plasma samples of patients with cSVD, and controls. Only myeloperoxidase (MPO) levels were significantly altered. Regularized logistic regression and random forest algorithms returned MPO levels as an important feature in the detection and prediction of cSVD and highlighted platelet- and NET markers as cSVD associated.

Liver fibrosis, caused by fatty degeneration, is a major cause of liver failure, and is also associated with higher levels of hepatocellular carcinoma. Although the pathophysiologic process leading to liver fibrosis is not completely clarified, stellate cells appear to play an important role. In this study, we have investigated a possible interplay between hepatocytes under normal and steatotic conditions. Our hypothesis was that extracellular vesicles (EV) isolated from hepatocytes could influence the behavior and phenotype of stellate cells.

Ein wesentlicher Faktor des fibrotischen Mikromilieus bei Leberschädigung ist die Infiltration von Leukozyten. Interaktionen von Sinusoiden mit Leukozyten bleiben dabei weitgehend unverstanden. Niedriger portaler Fluss und der spezielle Phänotyp des Leberendothels (LSEC) konkretisieren sich jedoch in verändertem Adhäsionsverhalten. Selektinabhängiges Rolling ist selten. Analysen humaner Transkriptome ergaben eine vermehrte Expression der JAM-Adhäsionsmoleküle. Die Einflüsse von JAM-B und JAM-C sind bereits untersucht, für JAM-A liegen dagegen nur wenige Daten vor.

Atherothrombosis is a leading cause of cardiovascular mortality and long-term morbidity. Platelets and coagulation proteases, interacting with circulating cells and in different vascular beds, modify several complex pathologies including atherosclerosis. In the second Maastricht Consensus Conference on Thrombosis, this theme was addressed by diverse scientists from bench to bedside. All presentations were discussed with audience members and the results of these discussions were incorporated in the final document that presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following five topics: 1. Risk factors, biomarkers and plaque instability: In atherothrombosis research, more focus on the contribution of specific risk factors like ectopic fat needs to be considered; definitions of atherothrombosis are important distinguishing different phases of disease, including plaque (in) stability; proteomic and metabolomics data are to be added to genetic information. 2. Circulating cells including platelets and atherothrombosis: Mechanisms of leukocyte and macrophage plasticity, migration, and transformation in murine atherosclerosis need to be considered; diseasemechanism-based biomarkers need to be identified; experimental systems are needed that incorporatewhole-blood flow to understand how red blood cells influence thrombus formation and stability; knowledge on platelet heterogeneity and priming conditions needs to be translated toward the in vivo situation. 3. Coagulation proteases, fibrin(ogen) and thrombus formation: The role of factor (F) XI in thrombosis including the lower margins of this factor related to safe and effective antithrombotic therapy needs to be established; FXI is a key regulator in linking platelets, thrombin generation, and inflammatory mechanisms in a renin-angiotensin dependent manner; however, the impact on thrombin-dependent PAR signaling needs further study; the fundamental mechanisms in FXIII biology and biochemistry and its impact on thrombus biophysical characteristics need to be explored; the interactions of red cells and fibrin formation and its consequences for thrombus formation and lysis need to be addressed. Platelet-fibrin interactions are pivotal determinants of clot formation and stability with potential therapeutic consequences. 4. Preventive and acute treatment of atherothrombosis and arterial embolism; novel ways and tailoring? The role of protease-activated receptor (PAR)-4 vis a vis PAR-1 as target for antithrombotic therapy merits study; ongoing trials on platelet function test-based antiplatelet therapy adjustment support development of practically feasible tests; risk scores for patients with atrial fibrillation need refinement, taking new biomarkers including coagulation into account; risk scores that consider organ system differences in bleeding may have added value; all forms of oral anticoagulant treatment require better organization, including education and emergency access; laboratory testing still needs rapidly available sensitive tests with short turnaround time. 5. Pleiotropy of coagulation proteases, thrombus resolution and ischaemia-reperfusion: Biobanks specifically for thrombus storage and analysis are needed; further studies on novelmodified activated protein C-based agents are required including its cytoprotective properties; new avenues for optimizing treatment of patients with ischaemic stroke are needed, also including novel agents that modify fibrinolytic activity (aimed at plasminogen activator inhibitor-1 and thrombin activatable fibrinolysis inhibitor.

# 284 Platelet microvesicles in vascular inflammation {#article-title-2} Background: Microvesicles are gathering increasing attention as mediators of cell communication and as integral effectors of disease. Platelets present a major source of microvesicles and release these microvesicles either spontaneously or upon activation. Platelet microvesicles (PMVs) retain many features of their parent cells and have been shown to exert modulatory effects on vascular and immune cells. Purpose: We hypothesize that PMVs interact with vascular smooth muscle cells (SMCs) and modulate their function in the context of vascular remodeling. Methods: PMVs were isolated from aging human platelet concentrates by serial centrifugation steps. PMVs were quantified and characterized by flow cytometry using annexin A5/phosphatidylserine and antibodies against CD41a/GPIIb. Size calibrated micro beads were used to quantify the absolute amount of PMVs/mL. Cell migration experiments were performed using a boyden chemotaxis chamber. Platelet receptors implicated in PMV-SMC interaction were identified by blocking antibodies. Proliferation of SMCs was measured by the BrdU-cell proliferation kit. Adhesion of monocytic cells to SMCs was determined by a flow adhesion assay. Relative quantification of gene expression was determined by real time and quantitative PCR. Results: In the presence of PMVs, SMCs show increased migration. Under resting conditions, the PMV binding to SMCs was specifically abrogated by the integrin αIIbβ3 inhibitor (integrilin) indicating an integrin-dependent mechanism of interaction. A proliferative effect on SMCs was measured after 48 hours after incubation with PMVs and this proliferation relied on interactions via integrin αMβ2, CD40 and P-selectin. The firm adhesion of monocytic cells to PMVs stimulated SMCs under flow conditions was significantly increased compared to untreated, resting SMCs. The adhesion mainly depended on the integrin αIIbβ3 and P-selectin but also CD40 and fractalkine. PMVs decreased gene expression of contractile proteins, i.e. αSMA and calponin. Conclusion: Isolated PMVs have shown to exert an immunomodulatory activity on various cell types. The present data indicate a role of PMVs in inducing a phenotypic switch towards a synthetic inflammatory SMC phenotype, thus contributing to vascular atherogenesis, in particular vascular remodeling. # 285 Pharmacological depletion of serotonin promotes atherosclerotic plaque formation in apoE-/- mice {#article-title-3} Cardiovascular disease, like myocardial infarction and stroke, is the major cause of death in western countries, which mainly arise from atherosclerosis. This chronic disorder is characterized by inflammation of the vessel wall driving lesion formation. These so called plaques contain macrophages, T cells and other immune cells as well as an accumulation of lipids. In the early phase an activation of the endothelium induces adhesion molecule expression and secretion of pro-inflammatory cytokines and chemokines leading to the recruitment of leukocytes. Platelets also contribute to the leukocyte recruitment by interacting with the endothelium and secretion of granule content including serotonin. Platelet serotonin was recently shown to promote selectin-dependent leukocyte interaction and recruitment to post-capillary venules in a mouse model of acute inflammation. Clinically available serotonin reuptake inhibitors (SERT) used as anti-depressive drugs might be a useful strategy to impair peripheral serotonin storage in platelets and thus reduce serotonin-mediated endothelium activation in cardiovascular disease patients. We therefore hypothesized that treatment with serotonin transporter inhibitor fluoxetine (FLX) inhibits atherosclerotic plaque formation by limiting leukocyte adhesion. We fed apolipoprotein E-deficient (ApoE-/-) mice for 4 or 16 weeks with high-cholesterol diet (HCD) and treated them with FLX via the drinking water. We analyzed atherosclerotic lesion formation and composition via oil-red-O lipid staining and immunohistology of aortic root cross-sections. Leukocyte subsets in blood, bone marrow, spleen and the abdominal aorta were assessed by flow cytometry. Surprisingly, pharmacological serotonin depletion resulted in significantly increased plaque size after 4 weeks HCD (ctrl, 44592 ± 5920 µm2 vs fluoxetine, 68749 ± 6118 µm2; n=9-10; P=0.01), in part due to an increased macrophage infiltration (ctrl, 10965 ± 1931 µm2 vs FLX, 22424 ± 4906 n=14-15; P=0.058). After 16 weeks HCD feeding, we no longer observed a difference in lesion size. The enhanced arterial leukocyte recruitment was not due to enhanced myelopoiesis or leukocyte mobilization from bone marrow or spleen, but rather mediated by increased adhesion of myeloid cells to aortic lesions as evidenced by intravital microscopy of carotid arteries. The live imaging revealed increased numbers of adhering CD11b-stained myeloid cells in carotids of fluoxetine-treated mice compared to control mice. Further investigations of early and advanced plaque composition, adhesion molecule expression and role of endothelial versus leukocyte-dependent effects of serotonin-mediated activation are ongoing to explain the unexpected pro-atherogenic effect of pharmacological SERT inhibition in early plaque formation. Our findings might have important clinical implications in particular for cardiovascular risk patients treated with SERT inhibitors for depression. # 286 Deletion of junctional adhesion molecule a from platelets increases early stage neointima formation after wire injury in hyperlipidemic mice {#article-title-4} Objective: Platelets play an important role in the pathogenesis of vascular remodeling after injury. Junctional Adhesion Molecule A (JAM-A) was recently described to regulate platelet activation. Specific deletion of JAM-A from platelets resulted in increased reactivity and in accelerated progression of atherosclerosis. The aim of this study was to investigate the specific contribution of platelet-derived JAM-A to neointima formation after vascular injury. Approach and Results: Mice with or without platelet-specific (tr)JAM-A-deficiency in an apolipoprotein e (apoe–/–) background underwent wire-induced injury of the common carotid artery. Ex vivo imaging by 2-photon microscopy revealed increased platelet coverage at the site of injury in trJAM-A–deficient mice, 1 hour after wire injury (Figure 1A,B). Cell recruitment assays showed increased adhesion of monocytic cells to activated JAM-A–deficient platelets than to control platelets (Figure 1C). Up to 4 weeks after wire-injury, intimal neoplasia and neointimal cellular content were analyzed. Neointimal lesion area was increased in trJAM-A–/– apoe–/– mice (Figure 1D,E) and the lesions showed an increased macrophage accumulation and proliferating smooth muscle cells compared with trJAM-A+/+ apoe–/– littermates 2 weeks (Figure 1F,G), but not 4 weeks after injury. Re-endothelialization was decreased in trJAM-A–/– apoe–/– mice compared with controls 2 weeks after injury, yet it was complete in both groups after 4 weeks. Conclusions: A platelet gain-of-function by deletion of JAM-A accelerates neointima formation only during earlier phases after vascular injury, through an increased recruitment of mononuclear cells. Thus, the contribution of platelets might become less important when neointima formation progresses to later stages. ![Graphic][1] Figure 1 [1]: /embed/inline-graphic-1.gif

Einleitung: In den letzten Jahren konnte das von aktivierten Thrombozyten sezernierte Chemokin platelet factor 4 (PF4) als ein Mediator der experimentellen Leberfibrose identifiziert werden (Zaldivar et al, Hepatology, 2010). An der Aktivierung von Thrombozyten ist das junctional adhesion molecule A (JAM-A) beteiligt. Ziel dieser Studie war es, zu zeigen, dass die selektive Deletion von JAM-A auf Thrombozyten und hierdurch bedingte Inhibierung der Thrombozytenaktivierung zu einer verringerten Fibroserate führt.

We used a novel approach of cytostatically induced leucocyte depletion and subsequent reconstitution with leucocytes deprived of classical (inflammatory/Gr1(hi) ) or non-classical (resident/Gr1(lo) ) monocytes to dissect their differential role in atheroprogression under high-fat diet (HFD). Apolipoprotein E-deficient (Apoe(-/-) ) mice lacking classical but not non-classical monocytes displayed reduced lesion size and macrophage and apoptotic cell content. Conversely, HFD induced a selective expansion of classical monocytes in blood and bone marrow. Increased CXCL1 levels accompanied by higher expression of its receptor CXCR2 on classical monocytes and inhibition of monocytosis by CXCL1-neutralization indicated a preferential role for the CXCL1/CXCR2 axis in mobilizing classical monocytes during hypercholesterolemia. Studies correlating circulating and lesional classical monocytes in gene-deficient Apoe(-/-) mice, adoptive transfer of gene-deficient cells and pharmacological modulation during intravital microscopy of the carotid artery revealed a crucial function of CCR1 and CCR5 but not CCR2 or CX3 CR1 in classical monocyte recruitment to atherosclerotic vessels. Collectively, these data establish the impact of classical monocytes on atheroprogression, identify a sequential role of CXCL1 in their mobilization and CCR1/CCR5 in their recruitment.

Summary.  Background and Objectives: The non‐allelic variant of CXCL4/PF4, CXCL4L1/PF4alt, differs from CXCL4 in three amino acids of the C‐terminal α‐helix and has been characterized as a potent anti‐angiogenic regulator. Although CXCL4 structurally belongs to the chemokine family, it does not behave like a ‘classical’ chemokine, lacking significant chemotactic properties. Specific hallmarks are its angiostatic, anti‐proliferative activities, and proinflammatory functions, which can be conferred by heteromer‐formation with CCL5/RANTES enhancing monocyte recruitment. Methods and Results: Here we show that tube formation of endothelial cells was inhibited by CXCL4L1 and CXCL4, while only CXCL4L1 triggered chemokinesis of endothelial cells. The chemotactic response towards VEGF and bFGF was attenuated by both variants and CXCL4L1‐induced chemokinesis was blocked by bFGF or VEGF. Endothelial cell proliferation was inhibited by CXCL4 (IC50 6.9 μg mL−1) but not by CXCL4L1, while both chemokines bound directly to VEGF and bFGF. Moreover, CXCL4 enhanced CCL5‐induced monocyte arrest in flow adhesion experiments and monocyte recruitment into the mouse peritoneal cavity in vivo, whereas CXCL4L1 had no effect. CXCL4L1 revealed lower affinity to CCL5 than CXCL4, as quantified by isothermal fluorescence titration. As evidenced by the reduction of the activated partial thromboplastin time, CXCL4L1 showed a tendency towards less heparin‐neutralizing activity than CXCL4 (IC50 2.45 vs 0.98 μg mL−1). Conclusions: CXCL4L1 may act angiostatically by causing random endothelial cell locomotion, disturbing directed migration towards angiogenic chemokines, serving as a homeostatic chemokine with a moderate structural distinction yet different functional profile from CXCL4.

Objective: Hyperhomocysteinemia induces endothelial dysfunction and promotes atherosclerotic vascular disease. Infiltrates of activated macrophages and lymphocytes are observed in human and experimental atherosclerotic lesions, their emigration being guided by endothelial-leukocyte adhesion molecules and chemoattractants. The CXC-chemokine CXCL16 functions as an adhesion molecule by interacting with its receptor (CXCR6) and also as a scavenger for oxidized low density lipoprotein (oxLDL). We investigated the modulation of CXCL16 on cultured endothelial cells (EC) and the recruitment of CXCR6(+) lymphocytes in response to homocysteine (Hcy), in vitro and in vivo. Methods and Results: Hcy-stimulated EC show a significant increase in CXCL16 mRNA and protein expression. Incubation of EC with D,L-Hcy and L-Hcy significantly increased CXCR6(+) lymphocyte adhesion to EC while L-Cysteine (L-Cys) had no effect. Furthermore, EC stimulation with Hcy increased uptake of DiI-oxLDL. An anti-CXCL16 monoclonal antibody, antioxidants (Tiron) and PPAR-gamma agonists (Pioglitazone) considerably reduced CXCR6(+) lymphocyte adhesion and uptake of DiI-oxLDL. Upon injection in the peritoneal cavities of mice, L-Hcy and not L-Cys, increased the number of CXCR6(+) lymphocytes, which was reduced by coinjection with Pioglitazone or anti-human CXCL16 antibody. Conclusions: Hyperhomocysteinemia up-regulates CXCL16 leading to increased recruitment of CXCR6(+) lymphocytes and scavenging of modified lipids via a potential involvement of a PPAR-gamma-dependent mechanism. CXCL16 may therefore contribute to the formation and progression of atherosclerotic lesions under conditions of hyperhomocysteinemia.

Objective— Although junctional adhesion molecule-A (JAM-A) has recently been implicated in leukocyte recruitment on early atherosclerotic endothelium and after reperfusion injury, its role in neointima formation after arterial injury remains to be elucidated. Methods and Results— Here we show that the genetic deletion of JAM-A in apolipoprotein E–deficient (apoE −/− ) mice significantly reduced neointimal hyperplasia after wire injury of carotid arteries without altering medial area. This was associated with a significant decrease in neointimal macrophage content, whereas the relative content of smooth muscle cells and endothelial recovery was unaltered in JAM-A −/− apoE −/− compared with JAM-A +/+ apoE −/− lesions. In carotid arteries perfused ex vivo, deficiency in JAM-A significantly impaired the recruitment of monocytes 1 week, but not 1 day, after injury. These effects were paralleled by an attenuation of monocyte arrest and transmigration on activated JAM-A −/− apoE −/− versus JAM-A +/+ apoE −/− endothelial cells under flow conditions in vitro. A mechanism underlying reduced recruitment was implied by findings that the luminal expression of the arrest chemokine RANTES in injured arteries and its endothelial deposition by activated platelets in vitro were diminished by JAM-A deficiency. Conclusions— Our data provide the first evidence to our knowledge for a crucial role of JAM-A in accelerated lesion formation and monocyte infiltration in atherosclerosis-prone mice.

The extended family of chemokines regulates the activation of leukocytes and coordinates their trafficking to sites of inflammation and during immune surveillance. The heptahelical-receptor-binding and function of chemokines is thought to be governed by their interaction with cell surface proteoglycans, oligomer formation, naturally occurring antagonists and proteolytic processing. Recent studies reveal that heterophilic interactions between chemokines can significantly modify their biological activities and through these we can gain initial insights into the structural basis underlying this novel regulatory mechanism. Here, we propose the concept of a functional 'interactome', constituted by a variety of heterophilic chemokine-chemokine interactions in particular microenvironments. This model could establish how signals conferred by various chemokines are integrated for the combinatorial control of leukocyte responses.

The chemokines platelet factor 4 (PF4) and RANTES (regulated on activation normal T cell expressed and secreted) are secreted by activated platelets and influence multiple cell types and biologic processes. For instance, PF4 inhibits progenitor cell proliferation and angiogenesis, while platelet-derived RANTES is involved in vascular recruitment of monocytes. However, little is known about functional interactions of PF4 and RANTES. Here we show that the presence of PF4 enhanced the arrest of RANTES-stimulated monocytes and monocytic cells on activated endothelial cells under flow conditions, while binding of PF4 to the monocyte surface was increased by RANTES. Both RANTES-triggered arrest and PF4 binding involved monocytic chondroitin sulfate. Ligand blots and surface plasmon resonance revealed a robust heterophilic interaction of PF4 with RANTES but not with RANTES variants defective in higher order oligomerization. The tetrameric mutant E26A bound to the monocyte surface without increasing PF4 binding, and monocyte arrest induced by E26A-RANTES was not enhanced by PF4. Stimulation of monocytes with supernatants of activated platelets triggered arrest involving RANTES and PF4, as shown by inhibition studies. Our results suggest that heterophilic interactions with PF4 require structural motifs important in RANTES oligomerization and amplify RANTES-triggered effects on monocyte adhesion. This may have implications for the modulation of inflammatory recruitment by platelet-derived chemokines.

We determined anticoagulant parameters that depend on protein S function in plasma, i.e. the APC-independent anticoagulant activity of protein S (expressed as pSR) and APC resistance determined with thrombin generation-based tests (expressed as APCsr) as well as plasma levels of total and free protein S and prothrombin in men, women not using oral contraceptives (OC), and in women using second or third generation OC. Thrombin generation in the APC resistance assays was initiated either with factor Xa (Xa-APCsr) or tissue factor (TF-APCsr). The APC-independent anticoagulant activity of protein S was highest in men (pSR=1.69) and gradually decreased from women not using OC (pSR=1.49) via women using second generation (pSR=1.35) to women using third generation OC (pSR=1.27). The pSR correlated inversely with nAPCsr determined with the tissue factor-based APC resistance test (TF-APCsr) but not with nAPCsr determined with the factor Xa-based assay (Xa-APCsr). Multiple linear regression analysis in which sex, OC use, and protein S and prothrombin levels were included as independent variables and the pSR,TF-APCsr or Xa-APCsr as dependent variables indicated that plasma protein S levels poorly predict the pSR and the TF-APCsr, but are the main determinant of the Xa-APCsr. This indicates that OC use alters the expression of protein S activity. This phenomenon can be caused by differences in modulation of the activity of protein S by other plasma proteins that change during OC use or by OC-induced changes in the protein S molecule that impair its anticoagulant activity. Functional impairment of protein S as a result of hormonal influence may, at least in part, contribute to the thrombotic risk of OC users.

Objective— Platelet activation mediates multiple cellular responses, including secretion of chemokines such as RANTES (CCL5), and formation of platelet microparticles (PMPs). We studied the role of PMPs in delivering RANTES and promoting monocyte recruitment. Methods and Results— Here we show that PMPs contain substantial amounts of RANTES and deposit RANTES on activated endothelium or murine atherosclerotic carotid arteries. RANTES deposition is facilitated by flow conditions and more efficient than that conferred by PMP supernatants. Interactions of PMPs with activated endothelium in flow were mostly characterized by rolling. RANTES deposition showed a diffuse distribution pattern and was rarely colocalized with firmly adherent PMPs, substantiating that RANTES deposition occurs during transient interactions. Importantly, preperfusion with PMPs enhanced monocyte arrest on activated endothelium or atherosclerotic carotid arteries, which could be inhibited by a blocking antibody or a RANTES receptor antagonist. Blockade or deficiency of PMP-expressed adhesion receptors demonstrated differential requirement of P-selectin, glycoprotein Ib (GPIb), GPIIb/IIIa, and junctional adhesion molecule-A for PMP interactions with endothelium, PMP-dependent RANTES deposition, and subsequent monocyte arrest. Conclusion— Circulating PMPs may serve as a finely tuned transcellular delivery system for RANTES, triggering monocyte arrest to inflamed and atherosclerotic endothelium, introducing a novel mechanism for platelet-dependent monocyte recruitment in inflammation and atherosclerosis.