Neutrophil Cathepsin G Enhances Thrombogenicity of Mildly Injured Arteries via ADP-Mediated Platelet Sensitization.

Inhalation of particulate matter in polluted air causes direct, size-restricted passage in the circulation and pronounced lung inflammation, provoking platelet activation and (non)-fatal cardiovascular complications. To determine potency and mechanism of platelet sensitization via neutrophil enzymes, we performed in vitro aggregation studies in washed human platelets and in murine and human blood, in the presence of elastase, cathepsin G and regular platelet agonists, present in damaged arteries. The impact of both enzymes on in vivo thrombogenicity was studied in the same thrombosis mouse model, previously having demonstrated that neutrophil activation enhances peripheral thrombogenicity. At 0.05 U/mL, cathepsin G activated washed human platelets via PAR1, whereas at 0.35 U/mL, aggregation occurred via PAR4. In Swiss mouse platelet-rich plasma no aggregation occurred by cathepsin G at 0.4 U/mL. In human and murine blood, aggregations by 0.05-0.1 U/mL cathepsin G were similar and not PAR-mediated, but platelet aggregation was inhibited by ADP antagonists, advocating cathepsin G-released ADP in blood as the true agonist of sustained platelet activation. In the mouse thrombosis model, cathepsin G and elastase amplified mild thrombogenicity at blood concentrations that activated platelets in vitro. This study shows that cathepsin G and elastase secreted in the circulation during mild air pollution-induced lung inflammation lyse red blood cell membrane proteins, leading to ADP-leakage into plasma, sensitizing platelets and amplifying their contribution to cardiovascular complications of ambient particle inhalation.

Successful Infliximab Treatment is Associated With Reversal of Clotting Abnormalities in Inflammatory Bowel Disease Patients.

BACKGROUND AND GOALS: Active inflammatory bowel diseases (IBD) represent an independent risk factor for venous thromboembolism. The authors investigated the hemostatic profile of IBD patients before and after induction treatment with infliximab, vedolizumab, and methylprednisolone. STUDY: This prospective study included 62 patients with active IBD starting infliximab, vedolizumab, and/or methylprednisolone, and 22 healthy controls (HC). Plasma was collected before (w0) and after induction therapy (w14). Using a clot lysis assay, amplitude (marker for clot intensity), time to peak (Tmax; marker for clot formation rate), area under the curve (AUC; global marker for coagulation/fibrinolysis), and 50% clot lysis time (50%CLT; marker for fibrinolytic capacity) were determined. Plasminogen activator inhibitor-1 (PAI-1) and fibronectin were measured by ELISA. Clinical remission was evaluated at w14. RESULTS: At baseline, AUC, amplitude, and 50%CLT were significantly higher in IBD patients as compared with HC. In 34 remitters, AUC [165 (103-229)% vs. 97 (78-147)%, P=0.001], amplitude [119 (99-163)% vs. 95 (82-117)%, P=0.002], and 50%CLT [122 (94-146)% vs. 100 (87-129)%, P=0.001] decreased significantly and even normalized to the HC level. Vedolizumab trough concentration correlated inversely to fibronectin concentration (r, -0.732; P=0.002). The increase in Tmax for infliximab-treated remitters was significantly different from the decrease in Tmax for vedolizumab-treated remitters (P=0.028). The 50%CLT increased (P=0.038) when remitters were concomitantly treated with methylprednisolone. CONCLUSIONS: Control of inflammation using infliximab most strongly reduced those parameters that are associated with a higher risk of venous thromboembolism.

Staphylococcus aureus endocarditis: distinct mechanisms of bacterial adhesion to damaged and inflamed heart valves.

AIMS: The pathogenesis of endocarditis is not well understood resulting in unsuccessful attempts at prevention. Clinical observations suggest that Staphylococcus aureus infects either damaged or inflamed heart valves. Using a newly developed endocarditis mouse model, we therefore studied the initial adhesion of S. aureus in both risk states. METHODS AND RESULTS: Using 3D confocal microscopy, we examined the adhesion of fluorescent S. aureus to murine aortic valves. To mimic different risk states we either damaged the valves with a surgically placed catheter or simulated valve inflammation by local endothelium activation. We used von Willebrand factor (VWF) gene-deficient mice, induced platelet and fibrinogen depletion and used several S. aureus mutant strains to investigate the contribution of both host and bacterial factors in early bacterial adhesion. Both cardiac valve damage and inflammation predisposed to endocarditis, but by distinct mechanisms. Following valve damage, S. aureus adhered directly to VWF and fibrin, deposited on the damaged valve. This was mediated by Sortase A-dependent adhesins such as VWF-binding protein and Clumping factor A. Platelets did not contribute. In contrast, upon cardiac valve inflammation, widespread endothelial activation led to endothelial cell-bound VWF release. This recruited large amounts of platelets, capturing S. aureus to the valve surface. Here, neither fibrinogen, nor Sortase A were essential. CONCLUSION: Cardiac valve damage and inflammation predispose to S. aureus endocarditis via distinct mechanisms. These findings may have important implications for the development of new preventive strategies, as some interventions might be effective in one risk state, but not in the other.

Beyond Haemostasis and Thrombosis: Platelets in Depression and Its Co-Morbidities.

Alongside their function in primary haemostasis and thrombo-inflammation, platelets are increasingly considered a bridge between mental, immunological and coagulation-related disorders. This review focuses on the link between platelets and the pathophysiology of major depressive disorder (MDD) and its most frequent comorbidities. Platelet- and neuron-shared proteins involved in MDD are functionally described. Platelet-related studies performed in the context of MDD, cardiovascular disease, and major neurodegenerative, neuropsychiatric and neurodevelopmental disorders are transversally presented from an epidemiological, genetic and functional point of view. To provide a complete scenario, we report the analysis of original data on the epidemiological link between platelets and depression symptoms suggesting moderating and interactive effects of sex on this association. Epidemiological and genetic studies discussed suggest that blood platelets might also be relevant biomarkers of MDD prediction and occurrence in the context of MDD comorbidities. Finally, this review has the ambition to formulate some directives and perspectives for future research on this topic.

A novel mechanism regulating human platelet activation by MMP-2-mediated PAR1 biased signaling.

Platelets contain and release several matrix metalloproteinases (MMPs). Among these, active MMP-2 enhances platelet aggregation by favoring the activation of phosphatidylinositol 3- kinase (PI3K) and contributes to arterial thrombosis. The platelet surface target of MMP-2 and the mechanism through which it primes platelets to respond to subsequent stimuli are still unknown. We show that active MMP-2 enhances platelet activation induced by weak stimuli by cleaving PAR1 at a noncanonical extracellular site different from the thrombin-cleavage site and thus initiates biased receptor signaling, triggering only some of the signaling pathways normally activated by full PAR1 agonism. The novel PAR1-tethered ligand exposed by MMP-2 stimulates PAR1-dependent Gq and G12/13 pathway activation, triggering p38-MAPK phosphorylation, Ca+2 fluxes, and PI3K activation, but not Gi signaling; this is insufficient to cause platelet aggregation, but it is enough to predispose platelets to fully respond to Gi-activating stimuli. Integrin αIIbß3 is a necessary cofactor for PAR1 cleavage by MMP-2 by binding the MMP-2 hemopexin domain, thus favoring the interaction of the enzyme with PAR1. Our studies unravel a novel mechanism regulating platelet activation that involves the binding of MMP-2 to integrin αIIbß3 and the subsequent cleavage of PAR1 by active MMP-2 at a noncanonical site, exposing a previously undescribed tethered ligand that triggers biased G-protein agonism and thus predisposes platelets to full activation by other stimuli. These results identify the MMP-2-αIIbß3-PAR1 interaction as a potential target for the prevention of arterial thrombosis.

Allele-specific DNA methylation reinforces PEAR1 enhancer activity.

Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (CpG-SNP), is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells, vs control HEK-293 cells show a 3-fold higher affinity for the methylated G allele compared with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation, the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first 5'-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that reinforces PEAR1 enhancer activity through allele-specific DNA methylation.

Cell-Specific PEAR1 Methylation Studies Reveal a Locus that Coordinates Expression of Multiple Genes.

Chromosomal interactions connect distant enhancers and promoters on the same chromosome, activating or repressing gene expression. PEAR1 encodes the Platelet-Endothelial Aggregation Receptor 1, a contact receptor involved in platelet function and megakaryocyte and endothelial cell proliferation. PEAR1 expression during megakaryocyte differentiation is controlled by DNA methylation at its first CpG island. We identified a PEAR1 cell-specific methylation sensitive region in endothelial cells and megakaryocytes that showed strong chromosomal interactions with ISGL20L2, RRNAD1, MRLP24, HDGF and PRCC, using available promoter capture Hi-C datasets. These genes are involved in ribosome processing, protein synthesis, cell cycle and cell proliferation. We next studied the methylation and expression profile of these five genes in Human Umbilical Vein Endothelial Cells (HUVECs) and megakaryocyte precursors. While cell-specific PEAR1 methylation corresponded to variability in expression for four out of five genes, no methylation change was observed in their promoter regions across cell types. Our data suggest that PEAR1 cell-type specific methylation changes may control long distance interactions with other genes. Further studies are needed to show whether such interaction data might be relevant for the genome-wide association data that showed a role for non-coding PEAR1 variants in the same region and platelet function, platelet count and cardiovascular risk.

Topographic analysis by atomic force microscopy of proteoliposomes matrix vesicle mimetics harboring TNAP and AnxA5.

Atomic force microscopy (AFM) is one of the most commonly used scanning probe microscopy techniques for nanoscale imaging and characterization of lipid-based particles. However, obtaining images of such particles using AFM is still a challenge. The present study extends the capabilities of AFM to the characterization of proteoliposomes, a special class of liposomes composed of lipids and proteins, mimicking matrix vesicles (MVs) involved in the biomineralization process. To this end, proteoliposomes were synthesized, composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS), with inserted tissue-nonspecific alkaline phosphatase (TNAP) and/or annexin V (AnxA5), both characteristic proteins of osteoblast-derived MVs. We then aimed to study how TNAP and AnxA5 insertion affects the proteoliposomes' membrane properties and, in turn, interactions with type II collagen, thus mimicking early MV activity during biomineralization. AFM images of these proteoliposomes, acquired in dynamic mode, revealed the presence of surface protrusions with distinct viscoelasticity, thus suggesting that the presence of the proteins induced local changes in membrane fluidity. Surface protrusions were measurable in TNAP-proteoliposomes but barely detectable in AnxA5-proteoliposomes. More complex surface structures were observed for proteoliposomes harboring both TNAP and AnxA5 concomitantly, resulting in a lower affinity for type II collagen fibers compared to proteoliposomes harboring AnxA5 alone. The present study achieved the topographic analysis of lipid vesicles by direct visualization of structural changes, resulting from protein incorporation, without the need for fluorescent probes.

PEAR1 is not a major susceptibility gene for cardiovascular disease in a Flemish population.

BACKGROUND: Platelet Endothelial Aggregation Receptor 1 (PEAR1), a membrane protein highly expressed in platelets and endothelial cells, plays a role in platelet contact-induced activation, sustained platelet aggregation and endothelial function. Previous reports implicate PEAR1 rs12041331 as a variant influencing risk in patients with coronary heart disease. We investigated whether genetic variation in PEAR1 predicts cardiovascular outcome in a white population. METHODS: In 1938 participants enrolled in the Flemish Study on Environment, Genes and Health Outcomes (51.3% women; mean age 43.6 years), we genotyped 9 tagging SNPs in PEAR1, measured baseline cardiovascular risk factors, and recorded Cardiovascular disease incidence. For SNPs, we contrasted cardiovascular disease incidence of minor-allele heterozygotes and homozygotes (variant) vs. major-allele homozygotes (reference) and for haplotypes carriers vs. non-carriers. In adjusted analyses, we accounted for family clusters and baseline covariables, including sex, age, body mass index, mean arterial pressure, the total-to-HDL cholesterol ratio, smoking and drinking, antihypertensive drug treatment, and history of cardiovascular disease and diabetes mellitus. RESULTS: Over a median follow-up of 15.3 years, 238 died and 181 experienced a major cardiovascular endpoint. The multivariable-adjusted hazard ratios of eight PEAR1 SNPs, including rs12566888, ranged from 0.87 to 1.07 (P ≥0.35) and from 0.78 to 1.30 (P ≥0.15), respectively. The hazard ratios of three haplotypes with frequency ≥10% ranged from 0.93 to 1.11 (P ≥0.49) for mortality and from 0.84 to 1.03 (P ≥0.29) for a cardiovascular complications. These results were not influenced by intake of antiplatelet drugs, nonsteroidal anti-inflammatory drugs, or both (P-values for interaction ≥ 0.056). CONCLUSIONS: In a White population, we could not replicate previous reports from experimental studies or obtained in patients suggesting that PEAR1 might be a susceptibility gene for cardiovascular complications.

A Human Platelet Receptor Protein Microarray Identifies the High Affinity Immunoglobulin E Receptor Subunit α (FcεR1α) as an Activating Platelet Endothelium Aggregation Receptor 1 (PEAR1) Ligand.

Genome-wide association studies to identify loci responsible for platelet function and cardiovascular disease susceptibility have repeatedly identified polymorphisms linked to a gene encoding platelet endothelium aggregation receptor 1 (PEAR1), an "orphan" cell surface receptor that is activated to stabilize platelet aggregates. To investigate how PEAR1 signaling is initiated, we sought to identify its extracellular ligand by creating a protein microarray representing the secretome and receptor repertoire of the human platelet. Using an avid soluble recombinant PEAR1 protein and a systematic screening assay designed to detect extracellular interactions, we identified the high affinity immunoglobulin E (IgE) receptor subunit α (FcεR1α) as a PEAR1 ligand. FcεR1α and PEAR1 directly interacted through their membrane-proximal Ig-like and 13th epidermal growth factor domains with a relatively strong affinity (KD ∼ 30 nm). Precomplexing FcεR1α with IgE potently inhibited the FcεR1α-PEAR1 interaction, and this was relieved by the anti-IgE therapeutic omalizumab. Oligomerized FcεR1α potentiated platelet aggregation and led to PEAR1 phosphorylation, an effect that was also inhibited by IgE. These findings demonstrate how a protein microarray resource can be used to gain important insight into the function of platelet receptors and provide a mechanistic basis for the initiation of PEAR1 signaling in platelet aggregation.

Shear-Resistant Binding to von Willebrand Factor Allows Staphylococcus lugdunensis to Adhere to the Cardiac Valves and Initiate Endocarditis.

BACKGROUND: Staphylococcus lugdunensis is an emerging cause of endocarditis. To cause endovascular infections, S. lugdunensis requires mechanisms to overcome shear stress. We investigated whether platelets and von Willebrand factor (VWF) mediate bacterial adhesion to the vessel wall and the cardiac valves under flow. METHODS: S. lugdunensis binding to VWF, collagen, and endothelial cells was studied in a parallel flow chamber in the absence and presence of platelets. In vivo adhesion of S. lugdunensis was evaluated in a mouse microvasculature perfusion model and a new mouse model of endocarditis. RESULTS: Contrary to other coagulase-negative staphylococci, S. lugdunensis bound to VWF under flow, thus enabling its adhesion to endothelial cells and to the subendothelial matrix. In inflamed vessels of the mesenteric circulation, VWF recruited S. lugdunensis to the vessel wall. In a novel endocarditis mouse model, local inflammation and the resulting release of VWF enabled S. lugdunensis to bind and colonize the heart valves. CONCLUSIONS: S. lugdunensis binds directly to VWF, which proved to be vital for withstanding shear forces and for its adhesion to the vessel wall and cardiac valves. This mechanism explains why S. lugdunensis causes more-aggressive infections, including endocarditis, compared with other coagulase-negative staphylococci.

Adhesion of Staphylococcus aureus to the vessel wall under flow is mediated by von Willebrand factor-binding protein.

Adhesion of Staphylococcus aureus to blood vessels under shear stress requires von Willebrand factor (VWF). Several bacterial factors have been proposed to interact with VWF, including VWF-binding protein (vWbp), a secreted coagulase that activates the host's prothrombin to generate fibrin. We measured the adhesion of S aureus Newman and a vWbp-deficient mutant (vwb) to VWF, collagen, and activated endothelial cells in a microparallel flow chamber. In vivo adhesion of S aureus was evaluated in the mesenteric circulation of wild-type (WT) and VWF-deficient mice. We found a shear-dependent increase in adhesion of S aureus to the (sub)endothelium that was dependent on interactions between vWbp and the A1-domain of VWF. Adhesion was further enhanced by coagulase-mediated fibrin formation that clustered bacteria and recruited platelets into bacterial microthrombi. In vivo, deficiency of vWbp or VWF as well as inhibition of coagulase activity reduced S aureus adhesion. We conclude that vWbp contributes to vascular adhesion of S aureus through 2 independent mechanisms: shear-mediated binding to VWF and activation of prothrombin to form S aureus-fibrin-platelet aggregates.

Dextran sulfate triggers platelet aggregation via direct activation of PEAR1.

UNLABELLED: Dextran sulfate (DxS; Mr 500 kD) induces fibrinogen receptor (αIIbß3) activation via CLEC-2/Syk signaling and via a Syk-independent SFK/PI3K/Akt-dependent tyrosine kinase pathway in human and murine platelets. The platelet surface receptor, responsible for the DxS-induced Syk-independent Akt-activation, has hitherto not been identified. We found that DxS elicited a concentration-dependent aggregation of human platelets resulting from direct PEAR1 activation by DxS. Blocking the PEAR1 receptor, in combination with a selective Syk-inhibitor, completely abrogated the DxS-driven platelet aggregation. The DxS-induced Syk-phosphorylation was not affected in Pear1(-/-) platelets, but Akt-phosphorylation was largely abolished. As a result, the aggregation of Pear1(-/-) platelets was reduced and reversible, i.e. aggregates were less stable compared to wild-type platelet aggregates. Moreover, DxS-induced Pear1(-/-) platelet aggregation was fully abrogated by Syk inhibition, indicating that the remaining platelet aggregation of Pear1(-/-) platelets was Syk dependent. Hence, the Pear1/c-Src/PI3K/Akt- and CLEC-2/Syk-signaling pathways are independently and additively activated during platelet aggregation by DxS. CONCLUSION: The DxS-induced aggregation of human and murine platelets is the result of activation of PI3K/Akt through direct PEAR1 phosphorylation and parallel Syk-signaling through CLEC-2.

Thrombin Generation by Fetoscopic Trauma to the Fetal Membranes: An in vivo and in vitro Study.

OBJECTIVE: We first aimed to investigate in vivo thrombin generation induced by fetoscopy, and second we used term membrane explants for measurement of thrombin generation, thrombin receptor location and induction of selected matrix metalloproteinases (MMPs) in tissue culture. MATERIALS AND METHODS: In vivo study (37 cases): samples of amniotic fluid were taken at the beginning and end of fetoscopy (mean gestational age 26.7 weeks) and analyzed by ELISA for thrombin-antithrombin complexes. In vitro study: fetal membranes were put in culture and punctured for measurement of thrombin generation by calibrated automated thrombography and ELISA. Induction of MMP-9 and MMP-2 was analyzed by zymography. PAR-1 was localized by immunohistochemistry. RESULTS: No significant increase in thrombin-antithrombin was measured in amniotic fluid obtained during fetoscopy. In vitro, thrombin generation induced by needle trauma of membrane cultures is correlated to the amount of plasma. Activity of MMP-9 but not MMP-2 was elevated in cultured membranes but could not be inhibited by a thrombin inhibitor. On histology, the thrombin receptor PAR-1 was located in the chorion and decidua, but not in the amnion. DISCUSSION: Despite the influence of thrombin on punctured fetal membranes in vitro, the role of thrombin in iatrogenic preterm premature rupture of membranes is questionable.

PEAR1 attenuates megakaryopoiesis via control of the PI3K/PTEN pathway.

Platelet endothelial aggregation receptor-1 (PEAR1) participates in platelet aggregation via sustaining αIIbß3 activation. To investigate the role of PEAR1 in platelet formation, we monitored and manipulated PEAR1 expression in vitro in differentiating human CD34(+) hematopoietic stem cells and in vivo in zebrafish embryos. PEAR1 expression rose during CD34(+) cell differentiation up to megakaryocyte (MK) maturation. Two different lentiviral short hairpin knockdowns of PEAR1 did not affect erythropoiesis in CD34(+) cells, but increased colony-forming unit MK cell numbers twofold vs control in clonogenic assays, without substantially modifying MK maturation. The PEAR1 knockdown resulted in a twofold reduction of the phosphatase and TENsin homolog (PTEN) phosphatase expression and modulated gene expression of several phosphatidylinositol 3-kinase (PI3K)-Akt and Notch pathway genes. In zebrafish, Pear1 expression increased progressively during the first 3 days of embryo development. Both ATG and splice-blocking PEAR1 morpholinos enhanced thrombopoiesis, without affecting erythropoiesis. Western blots of 3-day-old Pear1 knockdown zebrafish revealed elevated Akt phosphorylation, coupled to transcriptional downregulation of the PTEN isoform Ptena. Neutralization by morpholinos of Ptena, but not of Ptenb, phenocopied the Pear1 zebrafish knockdown and triggered enhanced Akt phosphorylation and thrombocyte formation. In summary, this is the first demonstration that PEAR1 influences the PI3K/PTEN pathway, a critical determinant of Akt phosphorylation, itself controlling megakaryopoiesis and thrombopoiesis.

Proteoliposomes with the ability to transport Ca(2+) into the vesicles and hydrolyze phosphosubstrates on their surface.

We describe the production of stable DPPC and DPPC:DPPS-proteoliposomes harboring annexin V (AnxA5) and tissue-nonspecific alkaline phosphatase (TNAP) and their use to investigate whether the presence of AnxA5 impacts the kinetic parameters for hydrolysis of TNAP substrates at physiological pH. The best catalytic efficiency was achieved in DPPS 10%-proteoliposomes (molar ratio), conditions that also increased the specificity of TNAP hydrolysis of PPi. Melting behavior of liposomes and proteoliposomes was analyzed via differential scanning calorimetry. The presence of 10% DPPS in DPPC-liposomes causes a broadening of the transition peaks, with AnxA5 and TNAP promoting a decrease in ΔH values. AnxA5 was able to mediate Ca(2+)-influx into the DPPC and DPPC:DPPS 10%-vesicles at physiological Ca(2+) concentrations (∼2 mM). This process was not affected by the presence of TNAP in the proteoliposomes. However, AnxA5 significantly affects the hydrolysis of TNAP substrates. Studies with GUVs confirmed the functional reconstitution of AnxA5 in the mimetic systems. These proteoliposomes are useful as mimetics of mineralizing cell-derived matrix vesicles, known to be responsible for the initiation of endochondral ossification, as they successfully transport Ca(2+) and possess the ability to hydrolyze phosphosubstrates in the lipid-water interface.

Supporting roles of platelet thrombospondin-1 and CD36 in thrombus formation on collagen.

OBJECTIVE: Platelets abundantly express the membrane receptor CD36 and store its ligand thrombospondin-1 (TSP1) in the α-granules. We investigated whether released TSP1 can support platelet adhesion and thrombus formation via interaction with CD36. APPROACH AND RESULTS: Mouse platelets deficient in CD36 showed reduced adhesion to TSP1 and subsequent phosphatidylserine expression. Deficiency in either CD36 or TSP1 resulted in markedly increased dissolution of thrombi formed on collagen, although thrombus buildup was unchanged. In mesenteric vessels in vivo, deficiency in CD36 prolonged the time to occlusion and enhanced embolization, which was in agreement with earlier observations in TSP1-deficient mice. Thrombi formed using wild-type blood stained positively for secreted TSP1. Releasate from wild-type but not from TSP1-deficient platelets enhanced platelet activation, phosphatidylserine expression, and thrombus formation on collagen. The enhancement was dependent on CD36 because it was without effect on thrombus formation by CD36-deficient platelets. CONCLUSIONS: These results demonstrate an anchoring role of platelet-released TSP1 via CD36 in platelet adhesion and collagen-dependent thrombus stabilization. Thus, the TSP1-CD36 tandem is another platelet ligand-receptor axis contributing to the maintenance of a stable thrombus.

Plasminogen activation by staphylokinase enhances local spreading of S. aureus in skin infections.

BACKGROUND: Staphylococcus aureus (S. aureus) is a frequent cause of skin and soft tissue infections. A unique feature of S. aureus is the combined presence of coagulases that trigger fibrin formation and of the plasminogen activator staphylokinase (SAK). Whereas the importance of fibrin generation for S. aureus virulence has been established, the role of SAK remains unclear. We studied the role of plasminogen activation by SAK in a skin infection model in mice and evaluated the impact of alpha-2-antiplasmin (α2AP) deficiency on the spreading and proteolytic activity of S. aureus skin infections. The species-selectivity of SAK was overcome by adenoviral expression of human plasminogen. Bacterial spread and density was assessed non-invasively by imaging the bioluminescence of S. aureus Xen36. RESULTS: SAK-mediated plasmin activity increased the local invasiveness of S. aureus, leading to larger lesions with skin disruption as well as decreased bacterial clearance by the host. Even though fibrin and bacterial surfaces protected SAK-mediated plasmin activity from inhibition by α2AP, the deficiency of α2AP resulted in increased bacterial spreading. SAK-mediated plasmin also induced secondary activation of gelatinases, shown both in vitro and in lesions from the in vivo model. CONCLUSION: SAK contributes to the phenotype of S. aureus skin infections by enhancing bacterial spreading as a result of fibrinolytic and proteolytic activation.

A novel mechanism of sustained platelet αIIbß3 activation via PEAR1.

Because single nucleotide polymorphisms (SNPs) in platelet endothelial aggregation receptor 1 (PEAR1) are associated with differential functional platelet responses in healthy subjects, we studied the function of PEAR1 in human platelets. During platelet aggregation by various agonists, the membrane expression of PEAR1 and its tyrosine phosphorylation increased. The recombinant PEAR1 EMI domain (GST-EMI) competitively reduced platelet adhesion to surface-coated PEAR1, diminished platelet aggregation, and eliminated PEAR1 phosphorylation. Polyclonal antibodies against the extracellular PEAR1 domain triggered PEAR1 phosphorylation in a src family kinase (SFK)-dependent manner. Such resulted in downstream signaling, culminating in extensive platelet degranulation and irreversible aggregation reactions interrupted by excess monovalent anti-GST-EMI F(ab) fragments. In resting platelets, the cytoplasmic tail of PEAR1 was found complexed to c-Src and Fyn, but on its phosphorylation, phospho-PEAR1 recruited p85 PI3K, resulting in persistent activation of PI3K and Akt. Thus, αIIbß3 activation was amplified, hence stabilizing platelet aggregates, a signaling cascade fully interrupted by the SFK inhibitor PP1 and the PI3K inhibitor LY294002. This study is the first demonstration of a functional role for PEAR1 in platelet activation, underpinning the observed association between PEAR1 and platelet function in genome-wide association studies.

Identification of a selective inhibitor of murine intestinal alkaline phosphatase (ML260) by concurrent ultra-high throughput screening against human and mouse isozymes.

Alkaline phosphatase (AP) isozymes are present in a wide range of species from bacteria to man and are capable of dephosphorylation and transphosphorylation of a wide spectrum of substrates in vitro. In humans, four AP isozymes have been identified-one tissue-nonspecific (TNAP) and three tissue-specific-named according to the tissue of their predominant expression: intestinal (IAP), placental (PLAP) and germ cell (GCAP) APs. Modulation of activity of the different AP isozymes may have therapeutic implications in distinct diseases and cellular processes. For instance, changes in the level of IAP activity can affect gut mucosa tolerance to microbial invasion due to the ability of IAP to detoxify bacterial endotoxins, alter the absorption of fatty acids and affect ectopurinergic regulation of duodenal bicarbonate secretion. To identify isozyme selective modulators of the human and mouse IAPs, we developed a series of murine duodenal IAP (Akp3-encoded dIAP isozyme), human IAP (hIAP), PLAP, and TNAP assays. High throughput screening and subsequent SAR efforts generated a potent inhibitor of dIAP, ML260, with specificity for the Akp3-, compared to the Akp5- and Akp6-encoded mouse isozymes.