Albrecht Leo

Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cgamma2 regulation in platelets.

The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin alphaIIbbeta3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cgamma2 (PLCgamma2). Similar pathways are activated downstream of immune receptors in lymphocytes, where they have been localized in part to glycolipid-enriched membrane domains (GEMs). Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCgamma2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells). In sharp contrast, although fibrinogen binding to platelets stimulates alphaIIbbeta3-dependent activation of Syk and tyrosine phosphorylation of SLP-76 and PLCgamma2, it does not utilize GEMs to promote these responses or to support platelet aggregation. These results establish that GPVI and alphaIIbbeta3 trigger distinct patterns of receptor signalling in platelets, leading to tyrosine phosphorylation of PLCgamma2, and they highlight the role of GEMs in compartmentalizing signalling reactions involved in haemostasis.

Adapters in lymphocyte signalling

Adapter proteins are well recognised as important molecular switches connecting immunoreceptors with intracellular signalling pathways. However, recent data suggest that homeostasis within the lymphatic system also depends on the coordinated activities of negative regulatory adapter proteins. These prevent activation of lymphocytes in the absence of externally applied signals and regulate termination/limitation of ongoing immune responses via different mechanisms.

Influence of Platelet-Rich Plasma on Osteogenic Differentiation of Mesenchymal Stem Cells and Ectopic Bone Formation in Calcium Phosphate Ceramics

Platelet-rich plasma (PRP) contains a mixture of growth factors that play an important role in wound and fracture healing. While PRP enhanced bone formation by autogenous cancellous bone grafts, its influence in combination with different bone substitutes remained unknown. This study evaluated the effect of PRP on osteogenic differentiation and ectopic bone formation of human mesenchymal stem cells (MSC) in distinct resorbable calcium phosphate ceramics. Calcium-deficient hydroxyapatite (CDHA) blocks with a large specific surface area (48 m2/g) and β-tricalcium phosphate (β-TCP) with a low specific surface area (<0.5 m2/g) were loaded with 2 × 105 bone marrow-derived MSC. Half of the specimens were treated with 5-fold concentrated PRP. Biocomposites were implanted subcutaneously into SCID mice or kept under osteogenic culture conditions for 2 weeks before implantation. The addition of PRP increased the specific alkaline phosphatase (ALP) activity (p = 0.012) in undifferentiated MSC/CDHA composites but not in MSC/β-TCP composites. Osteogenic preinduction was ineffective for CDHA and reduced ALP activity of β-TCP composites significantly at explantation. Ectopic bone formation was stronger in MSC/CDHA (7/32) compared to MSC/β-TCP (2/30) composites, but no influence of PRP was evident. In conclusion, the effect of PRP depended on the type of ceramic and the differentiation status of the MSC, and enhanced ALP activity of MSC on the high surface scaffold CDHA only, but PRP did not improve osteogenesis in our setting.

Adapters in lymphocyte signaling

In order to exert their effector functions, lymphocytes need to be activated. This process requires at least two stimuli, a primary stimulus, which is mediated via an immunoreceptor (e.g., the T cell receptor [TCR], the B cell receptor [BCR], or the Fc receptors [FcRs]), and a costimulatory signal (see Frauwirth and Thompson, this Perspective series, ref. 1), which is mediated via so-called accessory receptors (e.g., CD28 on T cells and CD19 on B cells). These two types of signals, as well many others that are generated by triggering of regulatory receptors expressed on the lymphocyte surface, are transmitted to the intracellular compartment, where they initiate cascades of biochemical events that finally produce a cellular response. How, then, are these externally applied signals integrated to yield an appropriate immune response? Here we review the current knowledge about the membrane-adjacent signaling events that are generated immediately after triggering of immunoreceptors, focusing in particular on the functions of one group of signaling proteins, collectively termed “adapter” or “linker” proteins. In addition, we offer a (very subjective) discussion of key unanswered questions related to the connection of signal-transducing receptors with the cellular environment.

Effect of platelet-rich plasma on the in vitro proliferation and osteogenic differentiation of human mesenchymal stem cells on distinct calcium phosphate scaffolds: the specific surface area makes a difference.

The in vitro effect of platelet-rich plasma (PRP) on cell loading, proliferation, and osteogenic differentiation of human mesenchymal stem cells (MSC) is assessed on distinct resorbable and synthetic calcium phosphate scaffolds. A high specific surface area scaffold composed of calcium-deficient hydroxyapatite (CDHA; 48m2/g) is compared with one made out of β-tricalcium phosphate (β-TCP; surface area <0.5 m2/g). Fivefold concentrated fresh PRP is applied to scaffolds loaded with 2 × 105 MSC (n = 5). These constructs are kept in a medium with osteogenic supplements for 3 weeks. The addition of PRP leads to a higher cell loading efficiency of MSC on CDHA (p = 0.0001), that reaches the values of β-TCP. Proliferation over 21 days is improved by PRP both on CDHA (p = 0.0001) and β-TCP (p = 0.014) compared to MSC/calcium phosphate composites. Without the addition of PRP, CDHA has a lower cell loading efficiency (p= 0.0001) and proliferation (p= 0.001) than β-TCP. The ALP activity is higher in the MSC/ceramics groups than in the monolayer controls (p<0.05). The addition of PRP does not significantly affect ALP activity. However, ALP activity varies considerably within the cell donors and different PRP-pools (p = 0.001), while the cell numbers do not vary within these two parameters. PRP generates a positive effect on the loading efficiency of MSC on the high specific surface scaffold CDHA that thereby reaches the loading efficiency of β-TCP. PRP improved proliferation, but its osteogenic properties on both calcium phosphate scaffolds are weak.

AB0 blood group and prognosis in patients with pancreatic cancer

BackgroundAlthough blood group 0 is associated with a reduced risk of pancreatic cancer, little is known about the role of AB0 blood group antigens in disease progression. We assessed the prognostic relevance of AB0 blood status in a large cohort of patients with resected pancreatic cancer.MethodsA total of 627 patients, who underwent resection for pancreatic ductal adenocarcinoma between October 2001 and December 2008 were enrolled. The relationship between AB0 blood group status and outcome was analyzed using univariate and multivariate Cox regression analyses.ResultsIn patients with pancreatic cancer the incidence of blood group 0 (31%) was lower compared to 13.044 patients without pancreatic cancer (38%) (p = 0.0005). There were no significant differences in clinicopathologic characteristics among patients with different AB0 blood groups. The 3-year and 5-year overall survival rates were 29% and 14%. On univariate analysis AB0 blood group status did not correlate with survival (p = 0.39). Multivariate analysis, however, revealed a favorable and independent impact of blood group 0 on survival (Hazard ratio 0.78; 95% confidence interval 0.62 – 0.99; p = 0.037).ConclusionAB0 blood group status is associated independently with the prognosis of patients with resected pancreatic cancer.

Structural and functional dissection of the cytoplasmic domain of the transmembrane adaptor protein SIT (SHP2-interacting transmembrane adaptor protein)

SIT (SHP2‐interacting transmembrane adaptor protein) is a recently identified transmembrane adaptor protein, which is expressed in lymphocytes. Its structural properties, in particular the presence of five potential tyrosine phosphorylation sites, suggest involvement of SIT in TCR‐mediated recruitment of SH2 domain‐containing intracellular signaling molecules to the plasma membrane. Indeed, it has recently been demonstrated that SIT inducibly interacts with the SH2‐containing protein tyrosine phosphatase 2 (SHP2) via an immunoreceptor tyrosine‐based inhibition motif (ITIM). Moreover, SIT is capable to inhibit TCR‐mediated signals proximal of activation of protein kinase C. However, inhibition of T cell activation by SIT occurs independently of SHP2 binding. The present study was performed to further characterize the molecular interaction between SIT and intracellular effector molecules and to identify the protein(s) mediating its inhibitory function. We demonstrate that SIT not only interacts with SHP2 but also with the adaptor protein Grb2 via two consensus YxN motifs. However, mutation of both Grb2‐binding sites also does not influence the inhibitory function of SIT. In contrast, mutation of the tyrosine‐based signaling motif Y168 ASV completely abrogates the ability of SIT to inhibit T cell activation. Co‐precipitation experiments revealed that the tyrosine kinase p50csk could represent the negative regulatory effector molecule that binds to this motif.

Laboratory Diagnosis of Heparin-Induced Thrombocytopenia and Monitoring of Alternative Anticoagulants

The major complication in the therapeutic or prophylactic use of heparin in medical treatment is type II heparin-induced thrombocytopenia (HIT II), a unique form of drug-induced immune-mediated thrombocytopenia. Due to the extensive use of heparin, this side effect is widespread; up to 3% of patients treated with unfractionated heparin (UFH) develop HIT II (69). Clinically, HIT II is characterized by thrombocytopenia which paradoxically is associated with thrombosis (HIT IIinduced thrombosis [HITTS]) (i.e., deep vein thrombosis, pulmonary embolism, and venous gangrene) in about 50% of the cases. In the pathogenesis of HIT II, the formation of platelet factor 4 (PF4)/heparin complexes seems to be the major determinant (2, 3, 20). The binding of PF4 to heparin induces antibody production directed against a neoepitope created by the three-dimensional assembly of PF4 and heparin. Subsequently, PF4/heparin-antibody complexes induce platelet activation, followed by the shedding of platelet microparticles, thrombin generation, and the involvement of endothelial cells (Fig. 1). Remarkably, the incidence of HIT II, HIT II-associated thrombotic complications, and PF4/heparin-antibodies (HIT II antibodies) in patients receiving therapy with UFH is clearly dependent on the clinical setting of the patient. Cardiosurgical patients are at a high risk to develop HIT II antibodies, while orthopedic patients undergoing hip replacement have a high risk for developing thromboembolic complications (Iceberg model [66]). In medical patients the risk for developing HIT II is substantially lower. Comparing low-molecular-weight heparins (LMWH) with UFH, the use of LMWH coincides with a substantially lower risk of HIT II, but this effect holds true only within the same group of patients (77, 78). It is generally accepted that the clinical diagnosis of HIT II should be followed by immediate cessation of heparin therapy and initiation of therapy with alternative anticoagulants. Negative results in the concomitant laboratory testing should not serve as the sole criterion for restarting heparin therapy. In almost every case of HIT II, administration of alternative anticoagulants is essential either to continue prophylactic anticoagulation or to treat and prevent thrombotic complications due to HIT II. Three substances (danaparoid, hirudin, and argatroban) are available. Since these substances have not been compared directly with each other in the treatment of HIT II, criteria such as half-life and/or route of excretion should be considered for selection. This review focuses on (i) the present laboratory methods for diagnosing HIT II, including their principles, time consumption, and significance, and (ii) the alternative anticoagulants, including their characteristics and indications in certain clinical settings.

Significant thrombocytopenia associated with the addition of rituximab to a combination of fludarabine and cyclophosphamide in the treatment of relapsed follicular lymphoma.

Abstract:  Fludarabine in combination with cyclophosphamide is an effective treatment for newly diagnosed as well as relapsed follicular lymphoma. The anti‐CD20 antibody rituximab has been employed successfully for the same indications. No such data were available on a combined use of these agents. Therefore, we conducted a phase II study to evaluate the safety and efficacy of a combination of rituximab (375 mg/m2), fludarabine (4 × 25 mg/m2) and cyclophosphamide (1 × 750 mg/m2), for the treatment of relapsed follicular lymphoma. An unexpected, severe hematologic toxicity with significant, prolonged thrombocytopenias WHO grade III/IV in 6 (35%) of 17 patients treated in total occurred, leading to early termination of the trial. Cytologic and serologic analyses point toward a direct toxic effect. Older patients (mean age 64.7 vs. 56.5 yr) were significantly (P = 0.02) more likely to suffer from this toxicity, whereas no other clinical or hematologic parameter differed statistically between the patients suffering from thrombocytopenia and those who did not. The addition of rituximab to fludarabine/cyclophosphamide employed at doses given above in relapsed follicular lymphoma may have led to this increase in thrombocytopenias. Therefore, caution should be exercised when combining these drugs for the treatment of patients with relapsed follicular lymphoma, especially when treating older patients.

Networks in signal transduction: the role of adaptor proteins in platelet activation

In multicellular organisms, the translation of externally applied signals into appropriate cellular responses is mediated by a multitude of complex intracellular signalling cascades. The accurate function of these signalling pathways is based on the sound interaction of proteins of different categories such as transmembrane receptors, protein kinases, protein phosphatases and g-proteins in three-dimensional signalling complexes. During the past 10 years it has became evident that a new class of proteins termed adaptor proteins is indispensable for the assembly of these intracellular signalling scaffolds. The primary function of adaptor proteins is to mediate protein–protein and protein–lipid interactions and thus to integrate receptor-mediated signals at the intracellular level and to couple signalling receptors to cytosolic signalling pathways. In order to perform this task, adapter proteins are equipped with particular protein–protein and/or protein–lipid interaction modules allowing them to communicate with other signalling proteins. While the essential function of adaptor proteins is clearly established in a variety of cell types (e.g. immune cells), the current knowledge about their role in platelet activation is still in the beginning. Numerous adaptor proteins have been shown to be expressed in platelets and many of them seem to be involved in the assembly of signalling complexes after engagement of platelet receptors such as the collagen receptor glycoprotein VI (GPVI), thrombin receptors, integrin receptors and the GP Ib receptor. This review will focus on the functional role of the most extensively studied adaptor proteins during platelet activation.