Engagement of αIIbß3 (GPIIb/IIIa) with ανß3 integrin mediates interaction of melanoma cells with platelets: a connection to hematogenous metastasis.

A mutual relationship exists between metastasizing tumor cells and components of the coagulation cascade. The exact mechanisms as to how platelets influence blood-borne metastasis, however, remain poorly understood. Here, we used murine B16 melanoma cells to observe functional aspects of how platelets contribute to the process of hematogenous metastasis. We found that platelets interfere with a distinct step of the metastasis cascade, as they promote adhesion of melanoma cells to the endothelium in vitro under shear conditions. Constitutively active platelet receptor GPIIb/IIIa (integrin αIIbß3) expressed on Chinese hamster ovary cells promoted melanoma cell adhesion in the presence of fibrinogen, whereas blocking antibodies to aνß3 integrin on melanoma cells or to GPIIb/IIIa significantly reduced melanoma cell adhesion to platelets. Furthermore, using intravital microscopy, we observed functional platelet-melanoma cell interactions, as platelet depletion resulted in significantly reduced melanoma cell adhesion to the injured vascular wall in vivo. Using a mouse model of hematogenous metastasis to the lung, we observed decreased metastasis of B16 melanoma cells to the lung by treatment with a mAb blocking the aν subunit of aνß3 integrin. This effect was significantly reduced when platelets were depleted in vivo. Thus, the engagement of GPIIb/IIIa with aνß3 integrin interaction mediates tumor cell-platelet interactions and highlights how this interaction is involved in hematogenous tumor metastasis.

Diagnostic and therapeutic potentials of platelet glycoprotein VI.

Platelets respond immediately to vascular injury by adhesion, aggregation, and thrombus formation. Disruption of the endothelial cell layer exposes extracellular matrix to the bloodstream. Collagen binding to platelet glycoprotein VI (GPVI) mediates the initial adhesion of the rolling platelet to the vascular wound. Signaling by GPVI leads to the onset of the platelet activation cascade that is finally crowned by a firm and shear-resistant integrin-based adhesive clot. Blockade of collagen binding to GPVI would prevent initial adhesion and further activation of the platelet and would have an enormous impact in antithrombotic therapy. Besides the therapeutical implication, radiolabeled GPVI gives us a valuable diagnostic means that may be used clinically for plaque imaging in the near future.

Oxidized LDL-activated platelets induce vascular inflammation.

Platelets are involved in the initiation of atherosclerosis by adherence to inflamed endothelium. Monocytes bind to these platelets and transmigrate into the vessel wall, transforming into macrophages and foam cells. We have previously shown that lipid-laden platelets are phagocytosed by macrophages. In this study we investigated the functional consequences of oxidized low-density lipoprotein (oxLDL) uptake on platelet function and interaction with the endothelium. Human platelets were isolated from healthy donors and activated by adenosine diphosphate. Immunofluorescence microscopy and flow cytometry revealed that oxLDL is located intracellularly in vesicles. With mepacrine costaining and confocal microtomography, we were able to identify dense granules as the vesicles that contain oxLDL. OxLDL-laden platelets induced intercellular adhesion molecule 1 expression in endothelial cells more than exogenous native LDL, oxLDL, and oxLDL-negative platelets. Furthermore, oxLDL-laden platelets induced foam cell development from CD34(+) progenitor cells. On endothelial regeneration, oxLDL-laden platelets had the opposite effect: The number of CD34(+) progenitor cells (colony-forming units) able to transform into endothelial cells was significantly reduced in the presence of oxLDL-platelets, whereas native LDL had no effect. Our results demonstrate that activated platelets internalize oxLDL and that oxLDL-laden platelets activate endothelium, inhibit endothelial regeneration, and promote foam cell development. Platelet oxLDL contributes significantly to vascular inflammation and is able to promote atherosclerosis.

Platelet derived bFGF mediates vascular integrative mechanisms of mesenchymal stem cells in vitro.

Patients with myocardial infarction reveal an altered number of circulating mesenchymal stem cells (MSCs). Recently, it was shown that MSCs are able to regenerate myocardial tissue and to differentiate into endothelial cells. The homing mechanisms of MSCs from the circulation into the target tissue, however, are not understood so far. In this study, we evaluated the impact of platelets on MSC recruitment, proliferation, migration and integration into the endothelium. MSCs expressing alpha(v)beta(3) integrin were recruited to human arterial endothelial cells exposed to isolated platelets or IL-1 beta under high shear conditions. Furthermore, induction of vascular injury in vivo resulted in increased recruitment of injected MSCs as assessed by intravital microscopy and depletion of platelets significantly reduced this adhesion. The interaction of platelets and MSCs was inhibited by pre-incubation with the mAb 7E3 or an RGD protein both blocking beta(3) integrin mediated adhesion. Platelets had a chemotactic effect on MSCs, promoted a migratory MSC phenotype and dose- and activation-dependently enhanced migration of MSCs, a process, which was mediated by basic fibroblast growth factor (bFGF). Similarly, platelet derived bFGF increased proliferation of MSCs. Coincubation of MSCs with platelets facilitated integration into an endothelial monolayer, which was significantly reduced by pre-incubation with a blocking mAb to bFGF. We conclude that platelets may play a critical part in the recruitment of MSCs to the endothelium, influence MSC function and promote integration of MSCs into the endothelium.

Influence of platelet count on the expression of platelet collagen receptor glycoprotein VI (GPVI) in patients with acute coronary syndrome.

Platelets play a key role in the development of an acute coronary syndrome (ACS) and contribute to cardiovascular events. Platelet collagen receptor glycoprotein VI (GPVI) contributes significantly to platelet adhesion and thrombus formation in ACS. We consecutively investigated both the platelet count and the platelet surface expression of GPVI in 843 patients with a symptomatic coronary artery disease verified by coronary angiography. Four hundred fourteen patients presented with stable angina pectoris and 429 patients with ACS. Platelet surface expression of GPVI and CD62P was determined by flow cytometry and platelet count with a coulter counter, plasmatic soluble GPVI was measured by ELISA. Platelet GPVI expression in patients with ACS was compared to platelet count. Patients with ACS showed significantly elevated GPVI expression levels in the first and second quartiles of platelet count compared to patients with higher platelet count [mean fluorescence intensity (MFI) +/- standard deviation): 1(st) vs. 4(th): 20.44 +/- 6.1 vs. 18.62 +/- 3.7; p=0.012; 2(nd)vs.3(rd):21.2+/-8.5vs.18.76+/-3.7;P=0.03; 2(nd)vs.4(th): 21.2+/-8.5vs.18.62+/-3.7;P=0.004], which was paralleled in trend for the CD62P expression [MFI: 1(st) vs. 4(th): 11.2 +/- 6.8 vs. 12.3 +/- 9; p=0.057; 2(nd) vs. 3(rd): 16.3 +/- 16 vs.12.7 +/- 5.3; p=0.138; 2(nd) vs. 4(th): 16.3 +/- 16 vs.11 +/- 4.4; p=0.043]. In a subgroup of 48 patients with ACS, determination of soluble GPVI showed similar results [plasma GPVI (ng/ml): 1(st)vs.4(th): 1.6 +/- 0.6 vs. 1.2 +/- 0.4; p=0.046; 1(st) vs. 3(rd): 1.6 +/- 0.6 vs. 1.1 +/- 0.5; p=0.038; 2(nd) vs. 3(rd): 1.9 +/- 0.8 vs. 1.1 +/- 0.5; p=0.04; 2(nd) vs. 4(th): 1.9 +/- 0.8 vs. 1.2 +/- 0.4; p=0.056]. Thus, a lower platelet count comes along with a higher GPVI surface expression and plasma concentration in patients with ACS, which potentially reflects increased activation and enhanced recruitment of platelets to the site of vascular injury.

EMMPRIN (CD147) is a novel receptor for platelet GPVI and mediates platelet rolling via GPVI-EMMPRIN interaction.

The Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147, basigin) is an immunoglobulin-like receptor expressed in various cell types. During cellular interactions homotypic EMMPRIN-EMMPRIN interactions are known to induce the synthesis of matrix metalloproteinases. Recently, we have identified EMMPRIN as a novel receptor on platelets. To our knowledge EMMPRIN has not been shown to serve as adhesion receptor, yet. Here we characterise platelet glycoprotein VI (GPVI) as a novel adhesion receptor for EMMPRIN. Human platelets were prestimulated with ADP and perfused over immobilised recombinant EMMPRIN-Fc or Fc-fragments under arterial shear conditions. ADP-stimulated platelets showed significantly enhanced rolling (but not enhanced firm adhesion) on immobilised EMMPRIN-Fc compared to Fc. Pretreatment of platelets with blocking mAbs anti-EMMPRIN or anti-GPVI leads to a significant reduction of rolling platelets on immobilised EMMPRIN-Fc, whereas pretreatment with blocking mAbs anti-p-selectin, anti-alpha4-integrin or anti-GPIIb/IIIa complex (20 microg/ml each) had no effect. Consistently, chinese hamster ovary (CHO) cells stably transfected with GPVI showed enhanced rolling (but not adhesion) on immobilised EMMPRIN-Fc in comparison to non-transfected CHO cells. Similarly, CHO cells stably transfected with EMMPRIN showed enhanced rolling on immobilised GPVI-Fc (or EMMPRIN-Fc) compared to non transfected CHO-cells. Finally, specific binding of EMMPRIN to GPVI was demonstrated by a modified ELISA and surface plasmon resonance technology with a dissociation constant of 88 nM. Platelet GPVI is a novel receptor for EMMPRIN and can mediate platelet rolling via GPVI-EMMPRIN interaction.

Unexpected abundance of HLA class II presented peptides in primary renal cell carcinomas.

PURPOSE: To elicit a long-lasting antitumor immune response, CD8+ and CD4+ T cells should be activated. We attempted to isolate HLA-DR-presented peptides directly from dissected solid tumors, in particular from renal cell carcinoma, to identify MHC class II ligands from tumor-associated antigens (TAA) for their use in peptide-based immunotherapy. EXPERIMENTAL DESIGN: Tumor specimens were analyzed by immunohistochemical staining for their HLA class II expression. HLA class II peptides were subsequently isolated and identified by mass spectrometry. Gene expression analysis was done to detect genes overexpressed in tumor tissue. Peptides from identified TAAs were used to induce peptide-specific CD4+ T-cell responses in healthy donors and in tumor patients. RESULTS: In the absence of inflammation, expression of MHC class II molecules is mainly restricted to cells of the immune system. To our surprise, we were able to isolate and characterize hundreds of class II peptides directly from primary dissected solid tumors, especially from renal cell carcinomas, and from colorectal carcinomas and transitional cell carcinomas. Infiltrating leukocytes expressed MHC class II molecules and tumor cells, very likely under the influence of IFNgamma. Our list of identified peptides contains ligands from several TAAs, including insulin-like growth factor binding protein 3 and matrix metalloproteinase 7. The latter bound promiscuously to HLA-DR molecules and were able to elicit CD4+ T-cell responses. CONCLUSIONS: Thus, our direct approach will rapidly expand the limited number of T-helper epitopes from TAAs for their use in clinical vaccination protocols.

MAGED4-expression in renal cell carcinoma and identification of an HLA-A*25-restricted MHC class I ligand from solid tumor tissue.

MAGE derived HLA ligands have repeatedly been shown to elicit T-cell responses against tumor cells. In renal cell carcinoma (RCC), however, only few T-cell epitopes from cancer testis antigens have been described. To identify potential candidates, we applied a combined approach of microarray/qPCR expression analysis and sequencing of HLA ligands from RCC by mass spectrometry. We analyzed the expression of 21 MAGE genes in ten RCC samples and two glioblastoma samples and could identify the first MHC class I ligand NIGDEALIGRW from MAGED4 presented by HLA-A*25 on RCC solid tumor tissue. MAGED4 was expressed in 30% of RCC and both glioblastoma samples. Among the other MAGE family members only MAGEB2 and -C1 and the broadly expressed MAGED1, -D2, -F1 and -H1 were expressed in RCC. Ligands from MAGED4 could thus be interesting tumor-associated antigens in a subset of RCC, even though the identified ligand is presented by a rather rare allele.

EMMPRIN and its ligand cyclophilin A regulate MT1-MMP, MMP-9 and M-CSF during foam cell formation.

UNLABELLED: Upon coincubation with platelets, CD34(+) progenitor cells have the potential to differentiate into foam cells, and thereby may promote the progression of atherosclerosis. The exact mechanism of MMP-regulation during the cellular differentiation process to foam cells is still unclear. Thus, we investigated the role of EMMPRIN (CD147) and its ligand cyclophilin A (CyPA) during foam cell formation originating from both monocytes/macrophages and CD34(+) progenitor cells. METHODS AND RESULTS: Differentiation of CD34(+) progenitor to foam cells was analyzed in a coculture model of progenitor cells and platelets. While CD34(+) cells did not express EMMPRIN or MT1-MMP, mature foam cells strongly expressed EMMPRIN, which was associated with MT1-MMP expression as well as MMP-9. Gene silencing of EMMPRIN by siRNA during the cell differentiation process hindered not only the upregulation of MMPs (MT1-MMP, MMP-9), but also the secretion of the cytokine M-CSF. During the differentiation process CyPA was substantially released into the supernatant. The presence of the CyPA inhibitor NIM811 significantly reduced MMP-9 secretion during the differentiation process. Similar results were obtained using the classical pathway of foam cell formation by coincubating human macrophages with AcLDL. Additionally, the presence of soluble EMMPRIN ligands (CyPA, recombinant EMMPRIN) further enhanced MMP-9 secretion by mature foam cells. Consistently, CyPA and EMMPRIN were found in atherosclerotic plaques of ApoE-deficient mice by immunohistochemistry. CONCLUSION: EMMPRIN is upregulated during the differentiation process from CD34(+) progenitor cells to foam cells, whereas its ligand, CyPA, is released. The CyPA/EMMPRIN activation pathway may play a relevant role in promoting the vulnerability of atherosclerotic plaques.

Challenges in the treatment of patients with essential thrombocythemia and acute coronary syndrome.

Essential thrombocythemia (ET) is an acquired clonal hematological stem-cell disorder that is characterized by a persistent increase in platelet count over 600,000/microl and elevated megakaryocyte levels in the bone marrow. Patients with ET are on the one hand at risk of thrombosis and on the other hand of hemorrhagic events especially in patients with very high platelet accounts. We report two illustrative cases with ET and acute coronary syndrome from our recent clinical experience illustrating the challenges in the antithrombotic treatment of these patients.

Molecular pathways used by platelets to initiate and accelerate atherogenesis.

PURPOSE OF REVIEW: The response to injury model in the development of atherosclerosis is broadly accepted by the scientific audience. Platelets are generally not believed to be involved in the initiation of atherosclerosis. New data imply, however, that the response to injury model is too simple for a complete understanding of the inflammatory disease atherosclerosis. The involvement of platelets in the initiation of atherosclerotic lesion formation is critical in directing the atherosclerotic process into regeneration or ongoing vascular injury. RECENT FINDINGS: Platelets internalize oxidized phospholipids and promote foam cell formation. Platelets also recruit circulating blood cells including progenitor cells to the vessel, that are able to differentiate into foam cells or endothelial cells depending on conditions. Platelets express various scavenger receptors that are able to regulate LDL-uptake. LDL-laden platelets are internalized by adherent progenitor cells that in turn differentiate into macrophages and foam cells. SUMMARY: An expanding body of evidence continues to build on the role of platelets as initial actors in the development of atherosclerotic lesions. Platelets bind to leukocytes, endothelial cells, and circulating progenitor cells and initiate monocyte transformation into macrophages. Therefore platelets regulate the initiation, development and total extent of atherosclerotic lesions.