Renata Polanowska-Grabowska

P-Selectin–Mediated Platelet-Neutrophil Aggregate Formation Activates Neutrophils in Mouse and Human Sickle Cell Disease*

Objective—To determine the role of platelets in stimulating mouse and human neutrophil activation and pulmonary injury in sickle cell disease (SCD). Methods and Results—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. Conclusion—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.

Interaction of the cysteine‐rich domain of snake venom metalloproteinases with the A1 domain of von Willebrand factor promotes site‐specific proteolysis of von Willebrand factor and inhibition of von Willebrand factor‐mediated platelet aggregation

Snake venom metalloproteinases (SVMPs) have recently been shown to interact with proteins containing von Willebrand factor A (VWA) domains, including the extracellular matrix proteins collagen XII, collagen XIV, matrilins 1, 3 and 4, and von Willebrand factor (VWF) via their cysteine‐rich domain. We extended those studies using surface plasmon resonance to investigate the interaction of SVMPs with VWF, and demonstrated that jararhagin, a PIII SVMP containing a metalloproteinase domain followed by disintegrin‐like and cysteine‐rich domains, catrocollastatin C, a disintegrin‐like/cysteine‐rich protein, and the recombinant cysteine‐rich domain of atrolysin A (A/C) all interacted with immobilized VWF in a dose‐dependent fashion. Binding of VWF in solution to immobilized A/C was inhibited by ristocetin and preincubation of platelets with A/C abolished ristocetin/VWF‐induced platelet aggregation, indicating that the interaction of A/C with VWF is mediated by the VWA1 domain. Jararhagin cleaved VWF at sites adjacent to the VWA1 domain, whereas atrolysin C, a SVMP lacking the cysteine‐rich domain, cleaved VWF at dispersed sites. A/C and catrocollastatin C completely inhibited the digestion of VWF by jararhagin, demonstrating that the specific interaction of jararhagin with VWF via the VWA1 domain is necessary for VWF proteolysis. In summary, we localized the binding site of PIII SVMPs in VWF to the A1 domain. This suggests additional mechanisms by which SVMPs may interfere with the adhesion of platelets at the site of envenoming. Thus, specific interaction of cysteine‐rich domain‐containing SVMPs with VWF may function to promote the hemorrhage caused by SVMP proteolysis of capillary basements and surrounding stromal extracellular matrix.

Shiga Toxin 2 and Lipopolysaccharide Induce Human Microvascular Endothelial Cells To Release Chemokines and Factors That Stimulate Platelet Function

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 1α (SDF-1α) or SDF-1β and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1α 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-1α, 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.

Shiga toxin binds to activated platelets.

Summary.  Hemolytic uremic syndrome (HUS) is associated with acute renal failure in children and can be caused by Shiga toxin (Stx)‐producing Escherichia coli. Thrombocytopenia and formation of renal thrombi are characteristic of HUS, suggesting that platelet activation is involved in its pathogenesis. However, whether Shiga toxin directly activates platelets is controversial. The present study evaluates if potential platelet sensitization during isolation by different procedures influences platelet interaction with Shiga toxin. Platelets isolated from sodium citrate anticoagulated blood were exposed during washing to EDTA and higher g forces than platelets prepared from acid‐citrate‐dextrose (ACD) plasma. Platelet binding of Stx was significantly higher in EDTA‐washed preparations relative to ACD‐derived platelets. Binding of Stx was also increased with ACD‐derived platelets when activated with thrombin (1 U mL−1) and exposure of the Gb3 Stx receptor was detected only on platelets subjected to EDTA, higher g forces or thrombin. EDTA‐exposed platelets lost their normal discoid shape and were larger. P‐selectin (CD62P) exposure was significantly increased in EDTA‐washed preparations relative to ACD‐derived platelets, suggesting platelet activation. Taken together, these results suggest that direct binding of Stx occurs only on ‘activated’ platelets rather than on resting platelets. The ability of Stx to interact with previously activated platelets may be an important element in understanding the pathogenesis of HUS.

Mechanisms of cyclic AMP compartmentation revealed by computational models.

### Introduction Cyclic adenosine 3′, 5′-monophosphate (cAMP) is a widely used biochemical messenger, transducing extracellular stimuli into a myriad of cellular responses. In the late 1970s, data emerged showing that hormone receptors acting via cAMP have distinct effects on cardiac function.

Heat-shock proteins and platelet function

Heat-shock proteins are found in organisms as diverse as slime moulds, bacteria, plants and higher eukarycotes. They play fundamental roles in cell function, ranging from protein folding to transmembrane protein movement, to serving as scaffolds or frameworks for the assembly of enzyme signalling complexes such as the steroid receptors. Intracellular concentrations may be high, in the range of structural proteins such as actin, with which they often interact. Therefore, it is not surprising that heat-shock proteins are present in blood platelets, and recent studies point to important roles in platelet function. The small heat-shock protein, hsp27, becomes phosphorylated following cell stimulation with thrombin and associates with the actin-rich cytoskeleton. Phosphorylation results from activation of a protein kinase cascade involving the p38 mitogen-activated protein kinase (MAPK), the MAPKAP-K2 kinase, as well as PRAK, or p38-regulated protein kinase. Intriguingly, platelet hsp27 can associate with platelet factor XIII, suggesting a role for regulation of transglutaminase activity in stabilizing fibrin-platelet clots. The higher molecular-weight heat-shock proteins hsc70 and hsp90 are also present in platelets, being found in a large phosphorylated complex that contains the catalytic and myosin-targeting subunits of protein phosphatase 1 (PP1). Platelet adhesion to collagen via the alpha 2 beta 1 integrin causes the rapid dissociation of this complex and dephosphorylation of components. These results suggest that hsc70 and hsp90 can serve as signalling scaffolds, helping regulate function, including platelet adhesion and spreading via modulation of protein phosphatase activity. Hsp27, on the other hand, may be more involved in controlling actin polymerization during the platelet shape change and subsequent aggregation.Heat-shock proteins are found in organisms as diverse as slime moulds, bacteria, plants and higher eukarycotes. They play fundamental roles in cell function, ranging from protein folding to transmembrane protein movement, to serving as scaffolds or frameworks for the assembly of enzyme signalling complexes such as the steroid receptors. Intracellular concentrations may be high, in the range of structural proteins such as actin, with which they often interact. Therefore, it is not surprising that heat-shock proteins are present in blood platelets, and recent studies point to important roles in platelet function. The small heat-shock protein, hsp27, becomes phosphorylated following cell stimulation with thrombin and associates with the actin-rich cytoskeleton. Phosphorylation results from activation of a protein kinase cascade involving the p38 mitogen-activated protein kinase (MAPK), the MAPKAP-K2 kinase, as well as PRAK, or p38-regulated protein kinase. Intriguingly, platelet hsp27 can associate with platelet factor XIII, suggesting a role for regulation of transglutaminase activity in stabilizing fibrin-platelet clots. The higher molecular-weight heat-shock proteins hsc70 and hsp90 are also present in platelets, being found in a large phosphorylated complex that contains the catalytic and myosin-targeting subunits of protein phosphatase 1 (PP1). Platelet adhesion to collagen via the alpha 2 beta 1 integrin causes the rapid dissociation of this complex and dephosphorylation of components. These results suggest that hsc70 and hsp90 can serve as signalling scaffolds, helping regulate function, including platelet adhesion and spreading via modulation of protein phosphatase activity. Hsp27, on the other hand, may be more involved in controlling actin polymerization during the platelet shape change and subsequent aggregation.

PKA catalytic subunit compartmentation regulates contractile and hypertrophic responses to β-adrenergic signaling.

β-Adrenergic signaling is spatiotemporally heterogeneous in the cardiac myocyte, conferring exquisite control to sympathetic stimulation. Such heterogeneity drives the formation of protein kinase A (PKA) signaling microdomains, which regulate Ca(2+) handling and contractility. Here, we test the hypothesis that the nucleus independently comprises a PKA signaling microdomain regulating myocyte hypertrophy. Spatially-targeted FRET reporters for PKA activity identified slower PKA activation and lower isoproterenol sensitivity in the nucleus (t50=10.6±0.7 min; EC50=89.0 nmol/L) than in the cytosol (t50=3.71±0.25 min; EC50=1.22 nmol/L). These differences were not explained by cAMP or AKAP-based compartmentation. A computational model of cytosolic and nuclear PKA activity was developed and predicted that differences in nuclear PKA dynamics and magnitude are regulated by slow PKA catalytic subunit diffusion, while differences in isoproterenol sensitivity are regulated by nuclear expression of protein kinase inhibitor (PKI). These were validated by FRET and immunofluorescence. The model also predicted differential phosphorylation of PKA substrates regulating cell contractility and hypertrophy. Ca(2+) and cell hypertrophy measurements validated these predictions and identified higher isoproterenol sensitivity for contractile enhancements (EC50=1.84 nmol/L) over cell hypertrophy (EC50=85.9 nmol/L). Over-expression of spatially targeted PKA catalytic subunit to the cytosol or nucleus enhanced contractile and hypertrophic responses, respectively. We conclude that restricted PKA catalytic subunit diffusion is an important PKA compartmentation mechanism and the nucleus comprises a novel PKA signaling microdomain, insulating hypertrophic from contractile β-adrenergic signaling responses.

Identification and Characterization of Poly(I:C)-induced Molecular Responses Attenuated by Nicotine in Mouse Macrophages

To further our understanding of the effects of nicotine on the molecular responses of macrophages during virus or virus-like infections, poly(I:C)-stimulated macrophage-like RAW264.2 cells or mouse primary peritoneal macrophages were challenged with nicotine; and their molecular responses were evaluated using a qRT-PCR array, antibody array, ELISA, Western blotting, and Ca2+ imaging. Of 51 genes expressed in the Toll-like receptor (TLR) and RIG-I-like receptor (RLR) pathways, mRNA expression of 15 genes in RAW264.7 cells was attenuated by nicotine, of which mRNA expression of IL-6, TNF-α, and IL-1β was confirmed to be attenuated in peritoneal macrophages. Concurrently, nicotine treatment attenuated the release of IL-6 and TNF-α from poly(I:C)-stimulated macrophages. However, when poly(I:C)-stimulated macrophages were challenged with nicotine plus α-bungarotoxin (α-BTX), secretion of IL-6 and TNF-α was found to be in a level seen with poly(I:C) stimulation only, indicating that α7-nAChR, a highly Ca2+ permeable ion channel sensitive to blockade by α-BTX, is involved in this process. Furthermore, results from an antibody array indicated that nicotine treatment attenuated the phosphorylation of 82 sites, including Thr286 on CaMKIIα, from poly(I:C)-stimulated RAW264.7 cells, of which 28 are expressed in the downstream cascade of Ca2+ signaling. Coincidentally, poly(I:C)-stimulated macrophages showed attenuated expression of phosphorylated CaMKIIα when pretreated with nicotine. In addition, nicotine attenuated intracellular Ca2+ signal from poly(I:C)-stimulated RAW264.7 cells. Collectively, these results indicate that poly(I:C)-induced molecular responses of macrophages could be significantly attenuated by nicotine.

Shiga toxin 2 and lipopolysaccharide cause monocytic THP-1 cells to release factors which activate platelet function.

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.

Platelet Adhesion to Collagen and Collagen-Related Peptide Under Flow

Objective—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. Methods and Results—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 C&ggr;2 (PLC&ggr;2). In contrast, PP2 weakly (<30%) suppressed firm adhesion to collagen mediated primarily by the &agr;2&bgr;1 integrin. Although PP2 prevented activation of Syk and PLC&ggr;2 in collagen-adherent platelets, tyrosine phosphorylation of several unidentified protein bands persisted, as did autophosphorylation of pp125FAK. Conclusions—These findings indicate that activation of Src-tyrosine kinases Syk and PLC&ggr;2 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.