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ABSTRACT: To determine the role of platelets in stimulating mouse and human neutrophil activation and pulmonary injury in sickle cell disease (SCD).
Both platelet and neutrophil activation occur in SCD, but the interdependence of these events is unknown. Platelet activation and binding to leukocytes were measured in mice and patients with SCD and in controls. Relative to controls, blood obtained from mice or patients with SCD contained significantly elevated platelet-neutrophil aggregates (PNAs). Both platelets and neutrophils found in sickle PNAs were activated. Multispectral imaging (ImageStream) and conventional flow cytometry revealed a subpopulation of activated neutrophils with multiple adhered platelets that expressed significantly more CD11b and exhibited greater oxidative activity than single neutrophils. On average, wild-type and sickle PNAs contained 1.1 and 2.6 platelets per neutrophil, respectively. Hypoxia/reoxygenation induced a further increase in PNAs in mice with SCD and additional activation of both platelets and neutrophils. The pretreatment of mice with SCD with clopidogrel or P-selectin antibody reduced the formation of PNAs and neutrophil activation and decreased lung vascular permeability.
Our findings suggest that platelet binding activates neutrophils and contributes to a chronic inflammatory state and pulmonary dysfunction in SCD. The inhibition of platelet activation may be useful to decrease tissue injury in SCD, particularly during the early stages of vaso-occlusive crises.
Arteriosclerosis Thrombosis and Vascular Biology 11/2010; 30(12):2392-9. · 6.37 Impact Factor
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ABSTRACT: Shiga toxins (Stxs) produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli are the most common cause of hemolytic-uremic syndrome (HUS). It is well established that vascular endothelial cells, mainly those located in the renal microvasculature, are targets for Stxs. The aim of the present research was to evaluate whether E. coli-derived Shiga toxin 2 (Stx2) incubated with human microvascular endothelial cells (HMEC-1) induces release of chemokines and other factors that might stimulate platelet function. HMEC-1 were exposed for 24 h in vitro to Stx2, lipopolysaccharide (LPS), or the Stx2-LPS combination, and chemokine production was assessed by immunoassay. More interleukin-8 was released than stromal cell-derived factor 1alpha (SDF-1alpha) or SDF-1beta and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1alpha at 24 h than LPS or Stx2-LPS did. In the presence of low ADP levels, HMEC-1 supernatants activated platelet function assessed by classical aggregometry, single-particle counting, granule secretion, P-selectin exposure, and the formation of platelet-monocyte aggregates. Supernatants from HMEC-1 exposed only to Stx2 exhibited enhanced exposure of platelet P-selectin and platelet-THP-1 cell interactions. Blockade of platelet cyclooxygenase by indomethacin prevented functional activation. The chemokine RANTES enhanced platelet aggregation induced by SDF-1alpha, macrophage-derived chemokine, or thymus and activation-regulated chemokine in the presence of very low ADP levels. These data support the hypothesis that microvascular endothelial cells exposed to E. coli O157:H7-derived Stx2 and LPS release chemokines and other factors, which when combined with low levels of primary agonists, such as ADP, cause platelet activation and promote the renal thrombosis associated with HUS.
Infection and Immunity 01/2006; 73(12):8306-16. · 4.16 Impact Factor
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ABSTRACT: Platelet and monocyte activation may contribute to hemolytic anemia, thrombocytopenia and renal failure associated with the hemolytic uremic syndrome (HUS) caused by Escherichia coli O157:H7. Since Shiga toxins (Stxs) and lipopolysaccharide (LPS) from this bacterium are implicated in the pathogenesis of HUS, we examined whether stimulation of THP-1 human monocytic cells by Shiga toxin 2 (Stx2) and LPS can lead to the activation of platelet function. We now show that Stx2 causedTHP-1 cells to release the chemokines IL-8, MDC, and RANTES and that the presence of LPS further stimulated this release. IL-8 was produced in greatest amount and was an effective co-agonist for inducing platelet aggregation. Primary human monocytes also released large amounts of IL-8 in response to LPS and Stx2. Factors released byTHP-1 cells exposed to Stx2 and LPS activated platelet function as evidenced by increased aggregation, serotonin secretion, P-selectin exposure and by the formation of stable platelet-monocyte aggregates. Our data therefore show that monocytes exposed to E.coli-derived Stx2 and LPS release factors which activate platelet function.
Thrombosis and Haemostasis 12/2005; 94(5):1019-27. · 5.04 Impact Factor
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ABSTRACT: The in vitro oxidation of low-density lipoprotein (LDL) by hypochlorous acid produces a modified form (HOCl-LDL) capable of stimulating platelet function. We now report that HOCl-LDL is highly effective at inducing platelet function, causing stable aggregation and alpha-granule secretion. Such stimulation depended on the presence of low levels of primary agonists such as adenosine diphosphate (ADP) and thrombin, or others like epinephrine (EPI) and macrophage-derived chemokine (MDC, CCL22). Agonist levels, which by themselves induced little or reversible aggregation, caused strong stable aggregation when combined with low levels of HOCl-LDL. Platelet activation by HOCl-LDL and ADP (1 microM) caused P-selectin (CD62P) exposure, without serotonin or adenosine triphosphate (ATP) secretion. Intracellular calcium levels rose slowly (from 100 to 200 nM) in response to HOCl-LDL alone and rapidly when combined with ADP to about 300 nM. p38 mitogen-activated protein kinase (MAPK) became phosphorylated in response to HOCl-LDL alone. This phosphorylation was not blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, which reduced the extent of aggregation and calcium increase. However, the p38 MAPK inhibitor SB203580 blocked platelet aggregation and phosphorylation of p38 MAPK. These findings suggest that HOCl-LDL exposed during atherosclerotic plaque rupture, coupled with low levels of primary agonists, can rapidly induce extensive and stable thrombus formation.
Blood 08/2004; 104(2):380-9. · 9.90 Impact Factor
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Methods in molecular biology (Clifton, N.J.) 02/2004; 272:153-63.
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ABSTRACT: Platelet stimulation by collagen and collagen-related peptides (CRPs) is associated with activation of protein tyrosine kinases. In the present study, we investigated the role of Src family tyrosine kinases in the initial adhesion events of human platelets to collagen and cross-linked CRP.
Under arterial flow conditions, a glycoprotein VI-specific substrate, cross-linked CRP, caused rapid (<2 second) platelet retention and protein tyrosine phosphorylation that were markedly decreased by the Src family kinase inhibitor pyrozolopyrimidine (PP2) or by aggregation inhibitor GRGDSP. CRP-induced platelet retention was transient, and 90% of single platelets or aggregates detached within seconds. PP2, although having no effect on RGD peptide-binding to CRP, completely blocked aggregation and tyrosine phosphorylation of Syk and phospholipase Cgamma2 (PLCgamma2). In contrast, PP2 weakly (<30%) suppressed firm adhesion to collagen mediated primarily by the alpha2beta1 integrin. Although PP2 prevented activation of Syk and PLCgamma2 in collagen-adherent platelets, tyrosine phosphorylation of several unidentified protein bands persisted, as did autophosphorylation of pp125FAK.
These findings indicate that activation of Src-tyrosine kinases Syk and PLCgamma2 is not required for the initial stable attachment of human platelets to collagen and for FAK autophosphorylation. However, Src-tyrosine kinases are critical for glycoprotein VI-mediated signaling leading to platelet aggregation.
Arteriosclerosis Thrombosis and Vascular Biology 10/2003; 23(10):1934-40. · 6.37 Impact Factor
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ABSTRACT: Blood platelets play critical roles in hemostasis, providing rapid essential protection against bleeding and catalyzing the important slower formation of stable blood clots via the coagulation cascade. They are also involved in protection from infection by phagocytosis of pathogens and by secreting chemokines that attract leukocytes. Platelet function usually is activated by primary agonists such as adenosine diphosphate (ADP), thrombin, and collagen, whereas secondary agonists like adrenalin do not induce aggregation on their own but become highly effective in the presence of low levels of primary agonists. Current research has revealed that chemokines represent an important additional class of agonists capable of causing significant activation of platelet function. Early work on platelet alpha-granule proteins suggested that platelet factor 4, now known as CXCL4, modulated aggregation and secretion induced by low agonist levels. Subsequent reports revealed the presence in platelets of messenger RNA for several additional chemokines and chemokine receptors. Three chemokines in particular, CXCL12 (SDF-1), CCL17 (TARC), and CCL22 (MDC), recently have been shown to be strong and rapid activators of platelet aggregation and adhesion after their binding to platelet CXCR4 or CCR4, when acting in combination with low levels of primary agonists. CXCL12 can be secreted by endothelial cells and is present in atherosclerotic plaques, whereas CCL17 and CCL22 are secreted by monocytes and macrophages. Platelet activation leads to the release of alpha-granule chemokines, including CCL3 (MIP-1alpha), CCL5 (RANTES), CCL7 (MCP-3), CCL17, CXCL1 (growth-regulated oncogene-alpha), CXCL5 (ENA-78), and CXCL8 (IL-8), which attract leukocytes and further activate other platelets. These findings help to provide a direct linkage between hemostasis, infection, and inflammation and the development of atherosclerosis.
Microcirculation 07/2003; 10(3-4):335-50. · 2.57 Impact Factor
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ABSTRACT: Heat-shock protein 90 (hsp90) is a chaperone important for the function of many signaling proteins. In this study, we show that hsp90 exists in resting platelets as a complex with the heat-shock cognate protein 70 (hsc70), the alpha- and beta-subunits of protein kinase CK2, and other unidentified phosphoproteins. Platelet activation by thrombin caused the rapid dissociation of hsc70 and CK2alpha from the hsp90 complex, the ex vivo phosphorylation of many protein components, and the stimulation of protein kinase(s) associated with the hsp90 complex. These results suggest that the hsp90 complex, with its associated protein kinase(s), which may include CK2, and their substrates, is involved in thrombin-induced platelet activation.
Biochemical and Biophysical Research Communications 10/2002; 297(1):129-33. · 2.48 Impact Factor
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ABSTRACT: We have previously shown that adhesion of human platelets to immobilized collagen is extremely rapid, with initial rates approaching 3% of single particles adhering per 10 ms. Here, we have investigated adhesion efficiency to collagen as a function of platelet density. Platelet subpopulations: low-density (1.040d< 1.065 g/ml), intermediatedensity (1.065 d< 1.070 g/ml) and high-density (1.070 d< 1.080 g/ml) were separated by Percoll density gradient centrifugation. They constituted 24%, 47% and 29% of the total platelet population and had mean volumes of 6.01, 7.37 and 8.21 fl, respectively. Using a continuous-flow, micro-affinity column, we found that the most dense (large) platelets exhibited initial rate of adhesion 4 times greater than the least dense (small) platelets. They were also less sensitive to inhibition by prostacyclin (PGIJ. In contrast, there was no significant difference in aggregation induced by high doses of ADP and collagen, indicating that the most dense platelets were not preferentially involved in aggregation induced by high doses of agonists. These results suggest that normal circulating platelets can be distinctly heterogeneous in their ability to adhere to collagen under arterial-flow conditions. The greater efficiency of high-density platelets may be related to increased content of the glycoprotein Ia/IIa (GPIa/IIa) complex.
British Journal of Haematology 11/1992; 82(4):715 - 720. · 4.94 Impact Factor
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ABSTRACT: Since vascular complications in diabetes mellitus are attributed in part to blood platelets, our study tested the hypothesis that adhesion of platelets to collagen is enhanced in diabetic subjects. Platelet adhesion kinetics to type I collagen in the presence of plasma were evaluated by a new continuous-flow, micro-adhesion assay combined with resistive-particle counting to detect the loss of single platelets between 0.3 and 2.3 sec. Adhesion was also studied in a magnesium-containing Krebs-Ringer buffer to help assess whether the platelets themselves might be abnormal. We did not observe any differences in adhesion kinetics to collagen between the insulin-dependent (type I), the non-insulin dependent (type II) diabetics and the control subjects for platelets suspended in plasma or in washed platelets (p > 0.05). These findings suggest that platelet adhesiveness to type I collagen is not enhanced in diabetic subjects and is unlikely to contribute to the development of vascular complications.
Thrombosis Research.
