Modulation of platelet activation and initial cytokine release by alloplastic bone substitute materials.

OBJECTIVES: Platelet-derived cytokines play a crucial role in tissue regeneration. In regenerative dental medicine, bone substitute materials (BSM) are widely used. However, initial interactions of BSM and platelets are still unknown. The aim of this study was to evaluate the potential of platelet activation and subsequent initial cytokine release by different commercial alloplastic BSM. MATERIAL AND METHODS: Eight commercial BSM of different origins and chemical compositions (tricalcium phosphate, hydroxyapatite, bioactive glass: SiO(2) and mixtures) were incubated with a platelet concentrate (platelet-rich plasma, PRP) of three healthy volunteers at room temperature for 15 min. Platelet count, aggregation, degranulation (activated surface receptor CD62p) and cytokine release (Platelet-derived growth factor, Vascular endothelial growth factor) into the supernatant were quantified. Highly thrombogenic collagen served as a reference. RESULTS: The investigated PRP samples revealed different activation patterns when incubated with different BSM. In general, SiO(2)-containing BSM resulted in high platelet activation and cytokine release. In detail, pure bioactive glass promoted platelet activation most significantly, followed by hybrid BSM containing lower ratios of SiO(2). Additionally, we found indications of cytokine retention by BSM of large specific surfaces. CONCLUSIONS: Platelet activation as well as consecutive storage and slow release of platelet-derived cytokines are desirable attributes of modern BSM. Within the limits of the study, SiO(2)-containing BSM were identified as promising biomaterials. Further investigations on cytokine adsorption and cytokine release kinetics by the respective BSM have to be conducted.

Diagnosis and Management of Inherited Platelet Disorders.

In clinical daily practice the definition of a bleeding tendency is rather subjective. Clinical manifestations usually include hematoma, epistaxis, menorrhagia, and severe bleeding episodes after surgery or injuries. The most common causes are disorders of primary hemostasis that occur sometimes due to platelet function disorders. Inherited thrombocytopathies are much less frequent in comparison to acquired platelet function disorders. However, congenital disorders can lead to severe bleeding tendency and are often not diagnosed. They are induced by different platelet defects based on disorders of platelet adhesion, receptors, secretion, and signal transduction. In some cases, they are associated with thrombocytopenias, giant platelets, and various comorbidities. This article gives an overview of the different defects, their diagnosis, and treatment options.

Novel Mutations in the GPIIb and GPIIIa Genes in Glanzmann Thrombasthenia.

BACKGROUND: Glanzmann thrombasthenia (GT) is an inherited autosomal recessive platelet disorder characterized by a complete or partial lack, or mutation, of the GPIIb/IIIa complex (integrin α(IIb)ß(3)) on the thrombocytes' surface, leading to a severe bleeding syndrome. MATERIAL AND METHODS: Molecular genetic analysis was performed in patients with suspected GT. The aim of the present study was the identification of new natural variants, their impact on platelet function, and their relation to the risk of bleeding. RESULTS: Expression of the platelet integrin α(IIb)ß(3) was determined by flow cytometry. Mutations were identified through sequencing of cDNA and genomic DNA. In addition, platelet function studies (PAC-binding, aggregations) were implemented. The study included 25 patients revealing 13 mutations (GPIIb: n = 9; GPIIIa: n = 4). Two of the 13 mutations were previously described (T207I; L214P). The remaining mutations have not been published yet, whereas 1 mutation in 2 unrelated families was identical (3062 T→C). CONCLUSION: All patients with less than 25% of present α(IIb)ß(3) have a medical history of bleeding.

Successful treatment of catastrophic antiphospholipid antibody syndrome (CAPS) associated with splenic marginal-zone lymphoma with low-molecular weight heparin, rituximab and bendamustine.

CASE REPORT: A 69-year-old woman with splenic marginal-zone lymphoma was admitted with progressive abdominal pain and splenomegaly as the suspected cause of pain. Rituximab treatment (375 mg/m) had been initiated on the day of admission. Abdominal computerized tomography revealed splenic infarction. Laboratory tests showed elevation of liver enzymes and creatinine, low platelet count, prolonged partial thromboplastin time, and lupus anticoagulant positivity. The diagnosis of catastrophic antiphospholipid antibody syndrome was made. Weight-adjusted low-molecular weight heparin therapy was initiated. Freedom from symptoms and normalization of liver enzymes and creatinine occurred within 4 weeks. Treatment was continued with 6 cycles of bendamustine monotherapy (90 mg/m) and heparin, leading to partial remission of lymphoma and lupus anticoagulant negativity. CONCLUSIONS: In case of multiorgan failure in patients suffering from lymphoma and showing features of disseminated intravascular coagulation, catastrophic antiphospholipid antibody syndrome should be considered. In our patient, rituximab followed by weight-adjusted low-molecular weight heparin and bendamustine therapy led to recovery.

[Clinical assessment of potential fields of application of recombinant factor VIIa in internal and pediatric diseases. Recommendations of an expert group]. / Klinische Bewertung potentieller Einsatzbereiche von rekombinantem Faktor VIIa bei internistischen und pädiatrischen Erkrankungen: Empfehlungen einer Expertengruppe.

Recombinant factor VIIa (rFVIIa) is increasingly used outside the labeled indications for treatment of life-threatening bleeding episodes after failure of the respective standard therapy. An interdisciplinary group of experts summarizes the state of knowledge of the use of rFVIIa in gastroenterology and hepatology, thrombocytopenia and -pathia, coagulation factor deficiencies, von Willebrand's disease, periinterventional bleeding without specific bleeding diathesis, drug-induced bleeding, disseminated intravascular coagulation, and neonatology. The most commonly used dose is 90 microg/kg body weight rFVIIa as bolus, if necessary followed by additional injections at intervals of 2-3 h. In factor VII deficiency lower dosages of 15-30 microg/kg body weight of rFVIIa are given every 4-6 h, whereas higher doses of 150-200 microg/kg body weight are used in neonates.

Multicenter study on the diagnostic value of a new RIA for the detection of free plasma metanephrines in the work-up for pheochromocytoma.

Available laboratory test methods for the detection of elevated concentrations of catecholamines and their metabolites in urine and/or plasma are not always sensitive enough for the detection of pheochromocytoma. High-quality immunoassays for these compounds appear to be as accurate as high-pressure liquid chromatography (HPLC) or gas chromatography/mass spectrometry (GC-MS). Therefore, the current project aims to establish a new sensitive radioimmunoassay (RIA) for the measurement of free metanephrines in the plasma of patients in the work-up for pheochromocytoma. We report first results of an ongoing multicenter clinico-chemical evaluation study in hypertensive patients and normotensive volunteers. After an overnight fast plasma samples were collected on ice in EDTA- and heparin-coated tubes after insertion of an indwelling venous line and resting in the supine (patients) or sitting position (normal volunteers) for 30 min. Plasma metanephrines were measured by a newly developed RIA from IBL, Hamburg, Germany. Good agreement of the assay with the tandem mass spectrometry (LC-MS/MS) method for normetanephrine (r2=0.975) and for metanephrine (r2=0.985) could be demonstrated. Both specimens, EDTA and heparin plasma, can be used with the same results. The RIA has a good precision of <15% in the normal range and of <10% in the elevated concentration range. Our preliminary data suggest a high validity of the newly developed RIA for measuring free metanephrine and normetanephrine in hypertensive subjects in both EDTA and heparin plasma. Further work is required to determine the accuracy of the test in larger patient populations and in patients with pheochromocytoma.

Glanzmann thrombasthenia Frankfurt I is associated with a point mutation Thr176Ile in the N-terminal region of alpha IIb subunit integrin.

In this study, we report on the characterization of a patient with Glanzmann thrombasthenia (GT). Immunochemical analysis on platelets from the patient showed that the expression of alpha IIb beta 3 was only 25% of that in normal healthy controls, suggesting a case of GT. Functional analysis revealed a total lack of fibrinogen binding capacity. Molecular genetic analysis of the full-length cDNA sequences of alpha IIb and beta 3 subunits showed a novel point mutation C621T in alpha IIb cDNA, leading to a missense substitution of threonine for isoleucine at position 176. Coexpression of normal beta 3 and mutant alpha IIb(1176) isoform in mammalian cells showed a marked reduction in the expression of alpha IIb beta 3 heterodimer when compared to the wild-type and a decreased intracellular level of alpha IIb. The T176 I mutation is located in the N-terminal region in the W3:1-2 connecting strand of the beta-propeller. These data suggest that the N-terminal alpha IIb domain plays an important structural role in the formation of heterodimer and that it is also involved in fibrinogen binding.

Thrombin-induced interleukin 1beta synthesis in platelet suspensions: impact of contaminating leukocytes.

A controversial discussion as to whether human platelets are capable of regulated protein synthesis has been ongoing for over half a century. A previous study has suggested that human platelets synthesize large amounts of interleukin 1beta (IL-1beta) in response to external cues and in a physiologically significant manner. However, cytokines such as IL-1beta are generally considered to be products of leukocytes and it could not be completely excluded that contaminating leukocytes may have contributed to the IL-1beta results in platelet preparations. It was therefore our intention to investigate whether residual leukocytes had an impact on thrombin-induced IL-1beta synthesis. Using various methods to reduce the level of contaminating leukocytes, we found that IL-1beta production in platelet-rich suspensions is dependent on the presence of leukocytes, as it was decreased by reducing the number of leukocytes. In addition, we found that thrombin-induced IL-1beta synthesis was completely eliminated in leukocyte-free platelet preparations and could be restored by adding leukocytes. IL-1beta synthesis could be detected in platelet suspensions contaminated with at least 1 leukocyte per 10(5) platelets. This study demonstrated that platelets are incapable of synthesizing detectable amounts of IL-1beta on their own. We suggest that any IL-1beta synthesis detected is a by-product of leukocytes contaminating the platelet preparations. Thus, the hypothesis that platelets producing IL-1beta, provide a new link between thrombosis and inflammation needs to be reconsidered.

Pharmacodynamic characterization of the interaction between the glycoprotein IIb/IIIa inhibitor YM337 and unfractionated heparin and aspirin in humans.

AIMS: To investigate the pharmacodynamic interaction of unfractionated heparin (UFH) and acetylic salicylic acid (ASA) on YM337, a monoclonal humanized antibody of the platelet GPIIb/IIIa receptor. METHODS: In a randomized, placebo-controlled study three treatment groups each with six healthy volunteers received the following medication: group 1, ASA (3 days) + UFH + YM337 (placebo); group 2, ASA (placebo) + UFH (placebo) + YM337; group 3, ASA + UFH + YM337. Assessments were made over 24 h and included bleeding time (BT), ADP (20 microm)- and collagen (5 microg ml-1)-induced platelet aggregation and PAC1 and CD62 expression measured by flow cytometry. RESULTS: In group 3 BT was prolonged to 35 [median, 16-45 min (1,3 quartile)] after UFH administration, increasing to 45 [median, 42-45 min (1,3 quartile)] after YM infusion (6 h). BT remained elevated to 26 [median, 14-45 min (1,3 quartile)] at 24 h, while groups 1 and 2 returned to normal values. Collagen-induced aggregation was 73% [median, 70-80% (1,3 quartile)] under YM337 alone, 79% [median, 72-80% (1,3 quartile)] under ASA + UFH and reduced only in group 3 to 24% [median, 18-29% (1,3 quartile)]. In both groups receiving active YM337, PAC1 expression showed a reduction to < 20% after 6 h of infusion. CD62 expression was not significantly affected by any treatment. CONCLUSION: UFH and YM337 have strong synergistic effects on BT, while coadministration of ASA strongly augments inhibitory effects of YM337 on collagen-induced platelet aggregation.

Close relationship between the platelet activation marker CD62 and the granular release of platelet-derived growth factor.

The expression of CD62 on the surface of platelets is considered to be an indicator of platelet degranulation and secretion. We characterized the relationship between CD62 expression and platelet-derived growth factor (PDGF)(AB) and PDGF(BB) secretion in response to thrombin-receptor activating peptide (TRAP). The principal findings were 1) expression of CD62 as a constituent of platelet alpha-granule membrane and secretion of PDGF, an important ingredient of alpha-granules, can be stimulated by TRAP-induced activation in a dose-dependent fashion; 2) the activation marker and secretion product are closely correlated with each other; and 3) changes in the CD62 expression induced by a drug, namely clopidogrel, or by a disease, namely diabetes, are paralleled by changes in PDGF secretion. Although CD62 is perceived as an activation marker of platelets indicating enhanced aggregability and secretion of alpha-granular content, the proof that the CD62 status and its modifications reflect directly the actual secretion of the most important platelet mitogen, PDGF, has so far not been given. This ex vivo-in vitro study shows that at least for the activation pathway provided by the PAR-1 receptor for which TRAP is the selective agonist, CD62 expression on platelets could be a surrogate for their secretory activity.