Targeted siRNA Delivery and mRNA Knockdown Mediated by Bispecific Digoxigenin-binding Antibodies.

Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G (IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3'end was bound in a 2:1 ratio to the bsAbs. These bsAb-siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC(50) siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.Molecular Therapy - Nucleic Acids (2012) 1, e45; doi:10.1038/mtna.2012.39; published online 18 September 2012.

Bispecific digoxigenin-binding antibodies for targeted payload delivery.

Bispecific antibodies that bind cell-surface targets as well as digoxigenin (Dig) were generated for targeted payload delivery. Targeting moieties are IgGs that bind the tumor antigens Her2, IGF1R, CD22, or LeY. A Dig-binding single-chain Fv was attached in disulfide-stabilized form to C termini of CH3 domains of targeting antibodies. Bispecific molecules were expressed in mammalian cells and purified in the same manner as unmodified IgGs. They are stable without aggregation propensity and retain binding specificity/affinity to cell-surface antigens and Dig. Digoxigeninylated payloads were generated that retain full functionality and can be complexed to bispecific antibodies in a defined 21 ratio. Payloads include small compounds (Dig-Cy5, Dig-Doxorubicin) and proteins (Dig-GFP). Complexed payloads are targeted by the bispecifics to cancer cells and because these complexes are stable in serum, they can be applied for targeted delivery. Because Dig bispecifics also effectively capture digoxigeninylated compounds under physiological conditions, separate administration of uncharged Dig bispecifics followed by application of Dig payload is sufficient to achieve antibody-mediated targeting in vitro and in vivo.

PI3 kinase-dependent stimulation of platelet migration by stromal cell-derived factor 1 (SDF-1).

Platelets have been regarded as static cells that do not move once they adhere to a matrix. The present study explored, whether platelets are able to migrate. In contrast to the current opinion, we found that platelets were mobile, able to migrate over a surface, and transmigrate through a transwell membrane and endothelium toward a source of stromal cell-derived factor 1 (SDF-1). Platelet migration was stimulated by SDF-1, which led to the downstream activation and phosphorylation of Wiskott-Aldrich syndrome protein. SDF-1 signaling and subsequent platelet migration could be inhibited by CXCR4-receptor blocker AMD3100, pertussis toxin, inhibition of phosphoinositol 3-kinase (PI3 kinase) with LY294002 or wortmannin, and disruption of actin polymerization with cytochalasin B. The potential of platelets to migrate in an SDF-1-mediated fashion may redefine the role of platelets in the pathophysiology of vascular inflammation, subsequent atherosclerotic degeneration, and vascular regeneration.

Inhibition of foam cell formation using a soluble CD68-Fc fusion protein.

The appearance of lipid-rich foam cells is a major feature of vulnerable atherosclerotic plaque formation. The transformation of macrophages into foam cells results from excessive uptake of cholesterol-rich particles by scavenger receptors such as CD68. We cloned a CD68-Fc immunoadhesin, a fusion protein consisting of the extracellular domain of the human CD68 and a human Fc domain, and investigated the function in vitro. Specific binding of CD68-Fc to OxLDL with an affinity of 10 nmol/L was determined by surface plasmon resonance and increased binding to lipid-rich human and ApoE(-/-) mice plaque tissue. This was confirmed both by immunohistochemical staining of CD68-Fc-treated paraffin sections from human plaques and by ELISA-based quantification of CD68-Fc binding to human atherosclerotic plaque extracts. In an in vitro model of macrophage/foam cell formation, CD68-Fc reduced foam cell formation significantly. This was caused both by interference of CD68-Fc with OxLDL uptake into macrophages and platelets and by the inhibition of platelet/OxLDL phagocytosis. Finally, expression of metalloproteinases by macrophages/foam cells was inhibited by CD68-Fc. In conclusion, CD68-Fc seems to be a promising new tool for preventing macrophage/foam cell formation. Thus, CD68-Fc might offer a novel therapeutic strategy for patients with acute coronary syndrome by modulating the generation of vulnerable plaques.

Capture of endothelial progenitor cells by a bispecific protein/monoclonal antibody molecule induces reendothelialization of vascular lesions.

Tissue injury is inevitably accompanied by disruption of the endothelium and exposure of the subendothelial matrix. To generate a guidance molecule directing progenitor cells to sites of vascular lesions, we designed a bifunctional protein. The protein consists of the soluble platelet collagen receptor glycoprotein VI and an antibody to CD133 (hereafter called GPVI-CD133). In vitro and in vivo, this construct substantially mediates endothelial progenitor cell (EPC) homing to vascular lesions. Exposure of EPCs to GPVI-CD133 did not impair their capability to differentiate toward mature endothelial cells as verified by the formation of colony-forming units, the upregulation of endothelial markers CD31 and CD146 analyzed by flow cytometry or von Willebrand factor and endoglin assessed by immunofluorescence microscopy, as well as the presence of Weibel-Palade bodies using transmission electron microscopy. In vivo, GPVI-CD133 augments reendothelialization of vascular lesions. Thus, this bifunctional protein could be a potential new therapeutic option for cardiovascular diseases.

Platelet aggregates-induced human CD34+ progenitor cell proliferation and differentiation to macrophages and foam cells is mediated by stromal cell derived factor 1 in vitro.

The chemokine stromal cell derived factor 1 (SDF-1) regulates chemotactic recruitment (homing) and differentiation of CD34 (+) stem cells. Platelets express substantial amounts of SDF-1 upon activation. The aim of the present study was to evaluate the role of SDF-1 in platelet-induced proliferation and differentiation of human CD34 (+) cells to macrophages and foam cells, as well as in regulation of matrix metalloproteinase (MMP)-9 secretion. Co-culture experiments of platelet thrombi (2 x 10(8)/mL) with human CD34 (+) progenitor cells resulted in platelet phagocytosis by the latter, causing their differentiation into CD68-positive macrophages and subsequent Sudan red III-positive foam cells. Platelet aggregates-induced foam cell generation and MMP-9 secretion were attenuated by a neutralizing monoclonal antibody against platelet-derived SDF-1, as evaluated by immunofluorescence microscopy and gelatin zymography. Co-culture experiments of human arterial endothelial cells and human CD34 (+) progenitor cells resulted in a fourfold increased proliferation of CD34 (+) cells in the presence of platelets being mainly regulated by platelet-derived SDF-1 in vitro. These findings imply that in the presence of platelet thrombi, CD34 (+) progenitor cells phagocytize platelets in an SDF-1 dependent manner, causing their differentiation into macrophages and then foam cells, a mechanism most likely contributing to atherogenesis and atheroprogression.

CD36 and macrophage scavenger receptor a modulate foam cell formation via inhibition of lipid-laden platelet phagocytosis.

CD34 (+) progenitor cells are a promising source of regeneration in atherosclerosis or ischemic heart disease. However, as recently published, CD34(+) progenitor cells have the potential to differentiate not only into endothelial cells but also into foam cells upon interaction with platelets. The mechanism of platelet-induced differentiation of progenitor cells into foam cells is as yet unclear. In the present study we investigated the role of scavenger receptor (SR)-A and CD36 in platelet-induced foam cell formation. Human CD34(+) progenitor cells were freshly derived from human umbilical veins and were co-incubated with platelets (2 x 10(8)/mL) up to 14 days resulting in large lipid-laden foam cells. Developing macrophages expressed SR-A, CD36, and Lox-1 as measured by fluorescent-activated cell sorting analysis. The presence of a blocking anti-CD36 or anti-SR-A antibody nearly abrogated foam cell formation, whereas anti-Lox-1 did not affect foam cell formation. Consistently blocking either anti-CD36 or anti-SR-A antibody significantly reduced the phagocytosis of lipid-laden platelets by macrophages. We conclude that CD36 and SR-A play an important role in platelet-induced foam cell formation from CD34(+) progenitor cells and thus represent a promising target to inhibit platelet-induced foam cell formation.

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.

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.

Platelet collagen receptor glycoprotein VI as a possible novel indicator for the acute coronary syndrome.

BACKGROUND: Platelet collagen receptor glycoprotein VI (GPVI) plays a critical role in acute coronary thrombosis. This prospective study examined the predictive value of GPVI for acute coronary syndromes (ACS) in a large consecutive group of patients with symptomatic coronary artery disease to identify the high-risk cohort with imminent coronary events. METHODS: We evaluated 1,003 patients with symptomatic coronary artery disease, verified by coronary angiography, and determined the surface expression of GPVI using flow cytometry. In a subgroup of 471 patients, who were treated with aspirin plus clopidogrel for coronary stenting, adenosine disphosphate (20 micromol/L)-induced platelet aggregation was evaluated. RESULTS: Patients with ACS (n = 485) showed a significantly enhanced GPVI expression compared to patients with stable angina pectoris (SAP; n = 518) (mean fluorescence intensity for ACS 19.8 +/- 5.9; SAP 18.7 +/- 8.5, P = .01). Patients with elevated GPVI levels on admission (GPVI cutoff value > or =18.6 mean fluorescence intensity) had a 1.4-fold relative risk for ACS. Logistic regression analysis showed that an elevated platelet GPVI level may indicate ACS independent of biomarkers of myocardial necrosis including troponin, creatine kinase, and creatine kinase-MB. Patients with increased platelet activation (GPVI expression level > or =18.6) showed significant enhanced residual platelet aggregation despite dual antiplatelet therapy compared to patients with low GPVI levels (P = .028). CONCLUSIONS: Surface expression of GPVI is enhanced in patients with ACS and indicates an imminent acute coronary event before irreversible myocardial necrosis is evident. High GPVI levels are associated with increased residual platelet aggregation despite antiplatelet therapy. Therefore, GPVI is useful to identify the subgroup of patients with a high risk for coronary stent thrombosis and thromboischemic events.

Platelet lipoprotein interplay: trigger of foam cell formation and driver of atherosclerosis.

In the last decade, it was recognized that platelets and lipoproteins play a pivotal role in both early and late atherogenesis. Beside cellular interactions of platelets with other blood cells and vascular cells, interactions with lipoproteins seem to be quite important. Lipoproteins are fundamental 'players' in atherogenesis since they change the properties of different cells involved in atherosclerosis and thrombosis. Several studies have already shown that low density lipoproteins (LDL) are involved in the initiation of platelet signalling pathways. Platelets of hypercholesterolemic patients show hyperaggregability in vitro and enhanced activity in vivo. This review elucidates the major aspects concerning how native and modified lipoproteins influence the activation and metabolic behaviour of platelets, and shows a new way by which platelet-mediated lipoprotein transfer might contribute to foam cell formation. In hyperlipidaemia, circulating platelets are activated. This is accompanied by increased platelet aggregation, platelet-leukocyte aggregate formation, and platelet-induced superoxide anion production. Furthermore, oxidized LDL induces monocyte adhesion to the endothelium, migration and proliferation of smooth muscle cells, injures cells, interferes with nitric oxide release, and promotes procoagulant properties of vascular cells. New data about platelet-mediated lipoprotein transport and consequent foam cell formation, however, provide proof of how platelets might contribute to atheromatous lesion formation.

Platelet-derived stromal cell-derived factor-1 regulates adhesion and promotes differentiation of human CD34+ cells to endothelial progenitor cells.

BACKGROUND: Peripheral homing of progenitor cells in areas of diseased organs is critical for tissue regeneration. The chemokine stromal cell-derived factor-1 (SDF-1) regulates homing of CD34+ stem cells. We evaluated the role of platelet-derived SDF-1 in adhesion and differentiation of human CD34+ cells into endothelial progenitor cells. METHODS AND RESULTS: Adherent platelets express substantial amounts of SDF-1 and recruit CD34+ cells in vitro and in vivo. A monoclonal antibody to SDF-1 or to its counterreceptor, CXCR4, inhibits stem cell adhesion on adherent platelets under high arterial shear in vitro and after carotid ligation in mice, as determined by intravital fluorescence microscopy. Platelets that adhere to human arterial endothelial cells enhance the adhesion of CD34+ cells on endothelium under flow conditions, a process that is inhibited by anti-SDF-1. During intestinal ischemia/reperfusion in mice, anti-SDF-1 and anti-CXCR4, but not isotype control antibodies, abolish the recruitment of CD34+ cells in microcirculation. Moreover, platelet-derived SDF-1 binding to CXCR4 receptor promotes platelet-induced differentiation of CD34+ cells into endothelial progenitor cells, as verified by colony-forming assays in vitro. CONCLUSIONS: These findings imply that platelet-derived SDF-1 regulates adhesion of stem cells in vitro and in vivo and promotes differentiation of CD34+ cells to endothelial progenitor cells. Because tissue regeneration depends on recruitment of progenitor cells to peripheral vasculature and their subsequent differentiation, platelet-derived SDF-1 may contribute to vascular and myocardial regeneration.

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.

The evil in atherosclerosis: adherent platelets induce foam cell formation.

Platelet interaction with circulating progenitor cells plays an important role for repair mechanisms at sites of vascular lesions. Foam cell formation represents a key process in atherosclerotic plaque formation. We revealed that platelets regulate recruitment and differentiation of CD34 (+) progenitor cells into foam cells and endothelial cells. Adhesion studies showed that platelets recruit CD34 (+) progenitor cells via specific adhesion receptors, including P-selection/P-selectin glycoprotein ligand 1, and beta (1) and beta (2) integrins. CD34 (+) progenitor cells were coincubated with human platelets for 1 week. We demonstrated that a substantial number of CD34 (+) cells differentiated into foam cells. Hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) and agonists of peroxisome proliferator-activated receptor-alpha and -gamma (PPAR-alpha and -gamma agonists) reduced this foam cell generation via inhibition of matrix metalloproteinase 9 secretions. Foam cell formation is also induced by low-density lipoproteins (LDLs). It was revealed that platelets take up modified LDL (fluorochrome-conjugated acetylated LDL) that is stored in the dense granules and internalized rapidly into the foam cells. These findings emphasize that the balance between endothelial cell regeneration and platelet-mediated foam cell generation derived from CD34 (+) progenitor cells may play a critical role in atherogenesis and atheroprogression.

Platelets recruit human dendritic cells via Mac-1/JAM-C interaction and modulate dendritic cell function in vitro.

OBJECTIVE: Thrombotic events and immunoinflammatory processes take place next to each other during vascular remodeling in atherosclerotic lesions. In this study we investigated the interaction of platelets with dendritic cells (DCs). METHODS AND RESULTS: The rolling of DCs on platelets was mediated by PSGL-1. Firm adhesion of DCs was mediated through integrin alphaMbeta2 (Mac-1). In vivo, adhesion of DCs to injured carotid arteries in mice was mediated by platelets. Pretreatment with soluble GPVI, which inhibits platelet adhesion to collagen, substantially reduced recruitment of DCs to the injured vessel wall. In addition, preincubation of DCs with sJAM-C significantly reduced their adhesion to platelets. Coincubation of DCs with platelets induced maturation of DCs, as shown by enhanced expression of CD83. In the presence of platelets, DC-induced lymphocyte proliferation was significantly enhanced. Moreover, coincubation of DCs with platelets resulted in platelet phagocytosis by DCs, as verified by different cell phagocytosis assays. Finally, platelet/DC interaction resulted in apoptosis of DCs mediated by a JAM-C-dependent mechanism. CONCLUSIONS: Recruitment of DCs by platelets, which is mediated via CD11b/CD18 (Mac-1) and platelet JAM-C, leads to DC activation and platelet phagocytosis. This process may be of importance for progression of atherosclerotic lesions.

Platelets induce differentiation of human CD34+ progenitor cells into foam cells and endothelial cells.

Recruitment of human CD34+ progenitor cells toward vascular lesions and differentiation into vascular cells has been regarded as a critical initial step in atherosclerosis. Previously we found that adherent platelets represent potential mediators of progenitor cell homing besides their role in thrombus formation. On the other hand, foam cell formation represents a key process in atherosclerotic plaque formation. To investigate whether platelets are involved in progenitor cell recruitment and differentiation into endothelial cells and foam cells, we examined the interactions of platelets and CD34+ progenitor cells. Cocultivation experiments showed that human platelets recruit CD34+ progenitor cells via the specific adhesion receptors P-selectin/PSGL-1 and beta1- and beta2-integrins. Furthermore, platelets were found to induce differentiation of CD34+ progenitor cells into mature foam cells and endothelial cells. Platelet-induced foam cell generation could be prevented partially by HMG coenzyme A reductase inhibitors via reduction of matrix metalloproteinase-9 (MMP-9) secretion. Finally, agonists of peroxisome proliferator-activated receptor-alpha and -gamma attenuated platelet-induced foam cell generation and production of MMP-9. The present study describes a potentially important mechanism of platelet-induced foam cell formation and generation of endothelium in atherogenesis and atheroprogression. The understanding and modulation of these mechanisms may offer new treatment strategies for patients at high risk for atherosclerotic diseases.

Expression of platelet collagen receptor glycoprotein VI is associated with acute coronary syndrome.

AIMS: Platelet collagen receptor glycoprotein VI (GPVI) is critical for the formation of arterial thrombosis. In this observational study, we examined the platelet surface expression of GPVI in patients with symptomatic coronary artery disease (CAD). METHODS AND RESULTS: We evaluated a consecutive cohort of 367 patients with symptomatic CAD, who underwent coronary angiography. The surface expression of platelet activation markers (GPVI, CD62P, and CD42b) was determined by flow cytometry. Patients with acute coronary syndrome (ACS) showed a significantly enhanced GPVI expression on admission when compared with patients with stable angina pectoris (SAP) (ACS: 21.4+/-9.7; SAP: 18.6+/-7.1 mean fluorescence intensity+/-SD; P=0.004). The expression of GPVI correlated with CD62P (r=0.702; P=0.001). Logistic regression analysis demonstrated that on admission, elevated platelet GPVI expression was associated with ACS, independent of markers of myocardial necrosis such as troponin and creatine kinase. CONCLUSION: Platelet GPVI surface expression is elevated in patients with ACS and is associated with imminent acute coronary events. The determination of the platelet-specific thrombotic marker GPVI may help to identify patients at risk before myocardial ischaemia is evident.

Adherent platelets recruit and induce differentiation of murine embryonic endothelial progenitor cells to mature endothelial cells in vitro.

The homing and differentiation mechanisms of endothelial progenitor cells (EPCs) at sites of vascular lesions are unclear. To investigate whether platelets play a role in the recruitment and differentiation of EPCs, we made use of a robust mouse embryonic EPC (eEPC) line that reliably differentiates to a mature endothelial phenotype. We found that platelets stimulate chemotaxis and migration of these murine eEPCs. Further, the substantial adhesion of murine eEPCs on immobilized platelets that occurs under dynamic flow conditions is inhibited by neutralizing anti-P-selectin glycoprotein ligand-1 and anti-VLA-4 (beta1-integrin) monoclonal antibodies but not by anti-CD11b (aM-integrin; macrophage antigen-1). Coincubation of murine eEPCs with platelets for 5 days induced differentiation of EPCs to mature endothelial cells as verified by positive von Willebrand factor immunofluorescence and detection of Weibel Palade bodies through electron microscopy. We conclude that platelets may play a critical part in the capture and subsequent differentiation of murine eEPCs at sites of vascular lesions, revealing a possible new role of platelets in neoendothelization after vascular injury.