Granzyme B, a novel mediator of allergic inflammation: its induction and release in blood basophils and human asthma

Histamine, leukotriene C4, IL-4, and IL-13 are major mediators of allergy and asthma. They are all formed by basophils and are released in particularly large quantities after stimulation with IL-3. Here we show that supernatants of activated mast cells or IL-3 qualitatively change the makeup of granules of human basophils by inducing de novo synthesis of granzyme B (GzmB), without induction of other granule proteins expressed by cytotoxic lymphocytes (granzyme A, perforin). This bioactivity of IL-3 is not shared by other cytokines known to regulate the function of basophils or lymphocytes. The IL-3 effect is restricted to basophil granulocytes as no constitutive or inducible expression of GzmB is detected in eosinophils or neutrophils. GzmB is induced within 6 to 24 hours, sorted into the granule compartment, and released by exocytosis upon IgE-dependent and -independent activation. In vitro, there is a close parallelism between GzmB, IL-13, and leukotriene C4 production. In vivo, granzyme B, but not the lymphoid granule marker granzyme A, is released 18 hours after allergen challenge of asthmatic patients in strong correlation with interleukin-13. Our study demonstrates an unexpected plasticity of the granule composition of mature basophils and suggests a role of granzyme B as a novel mediator of allergic diseases.

Continent urinary diversion

During the last decade continent urinary diversion, especially orthotopic bladder substitution has become increasingly popular following radical cystectomy for bladder cancer. In general, if sphincter sparing surgery is possible, orthotopic bladder substitution is performed, if not then continent catheterisable reservoirs are a viable option. Strict patient selection criteria and improved surgical technique have had a positive influence on outcome, not only on survival but also on quality of life issues. It is becoming increasingly obvious, that a nerve sparing surgical technique not only improves sexual function but also continence. In addition, the length of the intestinal segment has an influence on continence and the degree of metabolic consequences, which are discussed in detail. Postoperative surveillance and instruction of patients is of utmost value for good functional results. Overall patient satisfaction and quality of life seem comparable in the various types of continent urinary diversions, and improved when compared to a urinary stoma. Continent urinary diversion offers a good quality of life with few long-term complications and should be considered the treatment of choice in the majority of patients, independent of sex.

Hematopoietic and endothelial progenitor cell trafficking in patients with myeloproliferative diseases

BACKGROUND AND OBJECTIVES. The presence of circulating hematopoietic progenitor cells in patients with myeloproliferative diseases (MPD) has been described. However, the exact nature of such progenitor cells has not been specified until now. The aim of this work was to investigate the presence of endothelial precursor cells in the blood of patients with MPD and to assess the role of the endothelial cell lineage in the pathophysiology of this disease. DESIGN AND METHODS. Endothelial progenitor cell marker expression (CD34, prominin (CD133), kinase insert domain receptor (KDR) or vascular endothelial growth factor receptor 2 (VEGFR2), and von Willebrand factor) was assessed in the blood of 53 patients with MPD by quantitative polymerase chain reaction. Clonogenic stem cell assays were performed with progenitor cells and monocytes to assess differentiation towards the endothelial cell lineage. The patients' were divided according to whether they had essential thrombocythemia (ET, n=17), polycythemia vera (PV, n=21) or chronic idiopathic myelofibrosis (CIMF, n=15) and their data compared with data from normal controls (n=16) and patients with secondary thrombo- or erythrocytosis (n=17). RESULTS. Trafficking of CD34-positive cells was increased above the physiological level in 4/17 patients with ET, 5/21 patients with PV and 13/15 patients with CIMF. A subset of patients with CIMF co-expressed the markers CD34, prominin (CD133) and KDR, suggesting the presence of endothelial precursors among the circulating progenitor cells. Clonogenic stem cell assays confirmed differentiation towards both the hematopoietic and the endothelial cell lineage in 5/10 patients with CIMF. Furthermore, the molecular markers trisomy 8 and JAK2 V617F were found in the grown endothelial cells of patients positive for trisomy 8 or JAK2 V617F in the peripheral blood, confirming the common clonal origin of both hematopoietic and endothelial cell lineages. INTERPRETATION AND CONCLUSIONS. Endothelial precursor cells are increased in the blood of a subset of patients with CIMF, and peripheral endothelial cells bear the same molecular markers as hematopoietic cells, suggesting a primary role of pathological endothelial cells in this disease.

Antisense molecules for targeted cancer therapy

The efficacy of traditional anti-cancer agents is hampered by toxicity to normal tissues, due to the lack of specificity for malignant cells. Recent advances in our understanding of molecular genetics and tumor biology have led to the identification of signaling pathways and their regulators implicated in tumorigenesis and malignant progression. Consequently, novel biological agents were designed which specifically target key regulators of cell survival and proliferation activated in malignant cells and thus are superior to unspecific cytotoxic agents. Antisense molecules comprising conventional single-stranded antisense oligonucleotides (ASO) and small interfering RNA (siRNA) inhibit gene expression on the transcript level. Thus, they specifically target the genetic basis of cancer and are particularly useful for inhibiting the expression of oncogenes the protein products of which are inaccessible to small molecules or inhibitory antibodies. Despite the somewhat disappointing results of recent antisense oncology trials, the identification of new cancer targets and ongoing progress in ASO and siRNA technology together with improvements in tumor targeted delivery have raised new hopes that this fascinating intervention concept will eventually translate into enhanced clinical efficacy.

ADAMTS13 activity in sickle cell disease

Sickle red blood cell (SRBC)-endothelial adhesion plays a central role in sickle cell disease (SCD)-related vaso-occlusion. As unusually large von Willebrand factor (ULVWF) multimers mediate SRBC-endothelial adhesion, we investigated the activity of ADAMTS13, the metalloprotease responsible for cleaving ULVWF multimers, in SCD. ADAMTS13 activity was determined using a quantitative immunoblotting assay. VWF:Ag and VWF:RCo were determined using commercial assays. The high-molecular-weight VWF multimer percentage was determined by employing gel electrophoresis. ADAMTS13 activity was similar among asymptomatic patients (n = 8), patients at presentation with a painful crisis (n = 23), and healthy controls. ADAMTS13/VWF:Ag ratios were lower in patients compared to healthy HbAA controls, with the lowest values at presentation with a painful crisis (P = 0.02). Division of samples in those with VWF:RCo/VWF:Ag ratios < 0.70 and those with ratios >or= 0.70 revealed significantly more samples with ratios >or= 0.70 (P = 0.01) collected during painful crises. ULVWF multimers were detected in 6 patient samples and in 1 control sample. ADAMTS13/VWF:Ag ratios were inversely related to the duration of symptoms at presentation with an acute vaso-occlusive event (r(s)-0.67, P = 0.002). Although SCD is characterized by elevated VWF:Ag levels, no severe ADAMTS13 deficiency was detected in our patients.

Platelet activation induces metalloproteinase-dependent GP VI cleavage to down-regulate platelet reactivity to collagen

Glycoprotein (GP) VI, the primary collagen receptor on platelets, has been shown to have variable expression, possibly as a consequence of immune modulation. The present study was designed to determine the mechanism by which GP VI clearance occurs. We found that direct activation of GP VI both by a GP VI-specific antibody and by GP VI ligands (collagen and convulxin) reduced binding of biotinylated convulxin to the stimulated platelets. Analysis of immunoblots of platelets and supernatants showed that the stimulated platelets contained less GP VI, while the soluble fraction contained a 57-kDa cleavage product. Stimulation of platelets with PAR-1 agonists (TRAP peptide and thrombin) also caused GP VI cleavage, although the amount of GP VI loss was less than that observed with direct GP VI ligands. The metalloproteinase (MMP) inhibitors GM6001 and TAPI prevented both the clearance of GP VI from the platelet surface and the appearance of the soluble cleavage product. Induction of GP VI cleavage caused specific down-regulation of collagen-induced platelet aggregation, providing a mechanism for the modulation of platelet responsiveness to this important platelet agonist.

The GPIb thrombin-binding site is essential for thrombin-induced platelet procoagulant activity

The role of the platelet glycoprotein (GP) Ib-V-IX receptor in thrombin activation of platelets has remained controversial although good evidence suggests that blocking this receptor affects platelet responses to this agonist. The mechanism of expression of procoagulant activity in response to platelet agonists is also still obscure. Here, the binding site for thrombin on GPIb is shown to have a key role in the exposure of negatively charged phospholipids on the platelet surface and thrombin generation, in response to thrombin, which also requires protease-activated receptor-1, GPIIb-IIIa, and platelet-platelet contact. Von Willebrand factor binding to GPIb is not essential to initiate development of platelet procoagulant activity. Inhibition of fibrinogen binding to GPIIb-IIIa also failed to block platelet procoagulant activity. Both heparin and low molecular weight heparin block thrombin-induced platelet procoagulant activity, which may account for part of their clinical efficacy. This study demonstrates a new, critical role for platelet GPIb in hemostasis, showing that platelet activation and coagulation are tightly interwoven, which may have implications for alternative therapies for thrombotic diseases.

Surface expression and functional characterization of alpha-granule factor V in human platelets: effects of ionophore A23187, thrombin, collagen, and convulxin

Factor V (FV) present in platelet alpha-granules has a significant but incompletely understood role in hemostasis. This report demonstrates that a fraction of platelets express very high levels of surface-bound, alpha-granule FV on simultaneous activation with 2 agonists, thrombin and convulxin, an activator of the collagen receptor glycoprotein VI. This subpopulation of activated platelets represents 30.7% +/- 4.7% of the total population and is referred to as convulxin and thrombin-induced-FV (COAT-FV) platelets. COAT-FV platelets are also observed on activation with thrombin plus collagen types I, V, or VI, but not with type III. No single agonist examined was able to produce COAT-FV platelets, although ionophore A23187 in conjunction with either thrombin or convulxin did generate this population. COAT-FV platelets bound annexin-V, indicating exposure of aminophospholipids and were enriched in young platelets as identified by the binding of thiazole orange. The functional significance of COAT-FV platelets was investigated by demonstrating that factor Xa preferentially bound to COAT-FV platelets, that COAT-FV platelets had more FV activity than either thrombin or A23187-activated platelets, and that COAT-FV platelets were capable of generating more prothrombinase activity than any other physiologic agonist examined. Microparticle production by dual stimulation with thrombin and convulxin was less than that observed with A23187, indicating that microparticles were not responsible for all the activities observed. These data demonstrate a new procoagulant component produced from dual stimulation of platelets with thrombin and collagen. COAT-FV platelets may explain the unique role of alpha-granule FV and the hemostatic effectiveness of young platelets. (Blood. 2000;95:1694-1702)

Adenosine diphosphate (ADP) and ADP receptor play a major role in platelet activation/aggregation induced by sera from heparin-induced thrombocytopenia patients

The molecular basis for heparin-induced thrombocytopenia (HIT), a relatively common complication of heparin therapy, is not yet fully understood. We found that pretreatment of platelets with AR-C66096 (formerly FPL 66096), a specific platelet adenosine diphosphate (ADP) receptor antagonist, at a concentration of 100 to 200 nmol/L that blocked ADP-dependent platelet aggregation, resulted in complete loss of platelet aggregation responses to HIT sera. AR-C66096 also totally inhibited HIT serum-induced dense granule release, as judged by measurement of adenosine triphosphate (ATP) release. Apyrase, added to platelets at a concentration that had only minor effects on thrombin- or arachidonic acid-induced aggregation, also blocked completely HIT serum-induced platelet aggregation. Furthermore, AR-C66096 inhibited platelet aggregation and ATP release induced by cross-linking Fc gamma RIIA with specific antibodies. These data show that released ADP and the platelet ADP receptor play a pivotal role in HIT serum-induced platelet activation/aggregation. The thromboxane receptor inhibitor, Daltroban, had no effect on HIT serum-induced platelet activation whereas GPIIb-IIIa antagonists blocked platelet aggregation but had only a moderate effect on HIT serum-induced dense granule release. Pretreatment of platelets with chondroitinases but not with heparinases resulted in concentration dependent inhibition of HIT serum-induced platelet aggregation. These novel data relating to the mechanism of platelet activation induced by HIT sera suggest that the possibility should be examined that ADP receptor antagonists or compounds that inhibit ADP release may be effective as therapeutic agents for the prevention or treatment of complications associated with heparin therapy.

Glycoprotein VI is a major collagen receptor for platelet activation: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein IIb/IIIa, and von Willebrand factor do not

Simple collagen-related peptides (CRPs) containing a repeat Gly-Pro-Hyp sequence are highly potent platelet agonists. Like collagen, they must exhibit tertiary (triple-helical) and quaternary (polymeric) structure to activate platelets. Platelet signaling events induced by the peptides are the same as most of those induced by collagen. The peptides do not recognize the alpha 2 beta 1 integrin. To identify the signaling receptor involved, we have evaluated the response to the CRP, Gly-Lys-Hyp(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly of platelets with defined functional deficiencies. These studies exclude a primary recognition role for CD36, von Willebrand factor (vWF), or glycoprotein (GP) IIb/IIIa. Thus, both CD36 and vWF-deficient platelets exhibited normal aggregation, normal fibrinogen binding, and normal expression of CD62 and CD63, measured by flow cytometry, in response to the peptide, and there was normal expression of CD62 and CD63 on thrombasthenic platelets. In contrast, GPVI-deficient platelets were totally unresponsive to the peptide, indicating that this receptor recognizes the Gly-Pro-Hyp sequence in collagen. GPVI-deficient platelets showed some fibrinogen binding in response to collagen but failed to aggregate and to express CD62 and CD63. Collagen, but not CRP-XL, contains binding sites for alpha 2 beta 1. Therefore, it is possible that collagen still induces some signaling via alpha 2 beta 1, leading to activation of GPIIb/IIIa. Our findings are consistent with a two-site, two-step model of collagen interaction with platelets involving recognition of specific sequences in collagen by an adhesive receptor such as alpha 2 beta 1 to arrest platelets under flow and subsequent recognition of another specific collagen sequence by an activatory receptor, namely GPVI.

All platelets are not equal: COAT platelets

Collagen- and thrombin-activated (COAT) platelets were first described in 2000 and have attracted considerable interest, changing the interpretation of the way in which platelets contribute to thrombin generation and how their procoagulant activity is organized. Platelets activated by two agonists coming from glycoprotein VI or Fc gamma-receptor IIA agonists on the one hand and thrombin on the other produce a population of approximately 50% highly procoagulant active platelets. This subgroup is formed by tissue transglutaminase and factor XIIIa linking of serotonin to the procoagulant proteins from granules or plasma, and these serotonylated proteins bind to fibrinogen or thrombospondin on the platelet surface. Serotonylation in the platelet cytoplasm has recently been shown to be an important regulating mechanism governing the activation of small GTPases and their function in granule release. Recent studies with Tph-/- mice in which the peripheral serotonin, including that in platelets, is very strongly reduced, have shown a prolonged bleeding time, suggesting it has an important hemostatic role in the release of platelet von Willebrand factor. More knowledge about how COAT platelets are formed will be important for a better understanding of the physiology and pathology of hemostasis.

Echicetin, a GPIb-binding snake C-type lectin from Echis carinatus, also contains a binding site for IgMkappa responsible for platelet agglutination in plasma and inducing signal transduction

Echicetin, a heterodimeric snake C-type lectin from Echis carinatus, is known to bind specifically to platelet glycoprotein (GP)Ib. We now show that, in addition, it agglutinates platelets in plasma and induces platelet signal transduction. The agglutination is caused by binding to a specific protein in plasma. The protein was isolated from plasma and shown to cause platelet agglutination when added to washed platelets in the presence of echicetin. It was identified as immunoglobulin Mkappa (IgMkappa) by peptide sequencing and dot blotting with specific heavy and light chain anti-immunoglobulin reagents. Platelet agglutination by clustering echicetin with IgMkappa induced P-selectin expression and activation of GPIIb/IIIa as well as tyrosine phosphorylation of several signal transduction molecules, including p53/56(LYN), p64, p72(SYK), p70 to p90, and p120. However, neither ethylenediaminetetraacetic acid nor specific inhibition of GPIIb/IIIa affected platelet agglutination or activation by echicetin. Platelet agglutination and induction of signal transduction could also be produced by cross-linking biotinylated echicetin with avidin. These data indicate that clustering of GPIb alone is sufficient to activate platelets. In vivo, echicetin probably activates platelets rather than inhibits platelet activation, as previously proposed, accounting for the observed induction of thrombocytopenia.

Alboaggregin A activates platelets by a mechanism involving glycoprotein VI as well as glycoprotein Ib

The snake venom C-type lectin alboaggregin A (or 50-kd alboaggregin) from Trimeresurus albolabris was previously shown to be a platelet glycoprotein (GP) Ib agonist. However, investigations of the signal transduction induced in platelets showed patterns of tyrosine phosphorylation that were different from those of other GPIb agonists and suggested the presence of an additional receptor. In this study, the binding of biotinylated alboaggregin A to platelet lysates, as well as affinity chromatography evaluations of platelet lysates on an alboaggregin A-coated column, indicated that this other receptor is GPVI. Additional experiments with reagents that inhibit either GPIb or GPVI specifically supported this finding. These experiments also showed that both GPIb and GPVI have a role in the combined signaling and that the overall direction this takes can be influenced by inhibitors of one or the other receptor pathway.