765 research outputs found
Study of the origin of platelets coagulation protein S by human megakaryocyte cultures and characterization of platelets protein S in patients with inherited protein S deficiency
No full textProtein S (PS) is a vitamin K dependent plasma glycoprotein with multiple functions in coagulation, inflammation and apoptosis. The molecular weight of PS is approximately 70 kDa and its concentration in plasma is about 25 mg/L. In human plasma 40% of PS circulates as free form and the remaining 60% is complexes with complement C4b-binding protein, a component of the complement system. PS circulating in plasma is mainly derived from liver synthesis but, in addition, endothelial cells, testicular Leydig cells and a megakaryocytic cell line (MEG 01) can synthesize PS. Platelet contain PS, but whether this is derived from megakaryocytic synthesis or from uptake of plasma PS by megakaryocyte (Mk) is not known. Free PS acts as a cofactor for activated protein C (APC) in the inactivation of procoagulant factors Va and VIIIa. However, PS also has APC-independent anticoagulant functions, probably through direct inhibition of both the prothrombinase and the tenase complexes. It is hypothesized that intra-platelets PS, release upon platelets stimulation, plays a crucial role in regulating thrombin generation and therefore controlling procoagulant activity. PS deficiency is inherited as an autosomal dominant disordered and is classified in three types: I) reduced plasma levels of total and free PS antigen (PSAg); II) normal concentration of total and free PSAg but with low PS activity; III) low free PSAg; and
normal total PSAg. Inherited PS deficiency is generally associated with increased risk of deep venous thrombosis, pulmonary embolism and some cases of arterial thrombosis. The risk of venous thrombosis in PS deficiency increased if 2 associated with other genetic or acquired conditions these includes factor V (FV) Leiden, HR2 aplotype of FV and prothrombin mutation. Several
factors influence the concentration of plasma PS, pregnancy, oral contraceptive and oral anticoagulant therapy decreased the levels of PS. To clarify the origin of intra-platelets PS, we development an in vitro model of human megakaryocyte cell culture. Hematopoietic stem cells were isolated by the histopaque system from whole blood of healthy and PS deficiency subjects. Mononuclear cells have been grown in a serum free medium in presence of thrombopoietin (TPO) and interleuchin-3 (IL-3) to stimulate the differentiation into megakaryocytes lineage. The morphology of differentiated mononuclear cells was similar to MKs, and their positive stain with anti-CD41 antibody allowed us to conclude that these cells were indeed Mk. Mk was labeled with ??-tubulin and ?-tubulin antibodies and we observed the cytoplasmatic extensions called proplatelets and the release of platelets. In addition, through immunofluorescence techniques,
we detected FV in their cytoplasm whereas protein C was not present as expected. As for PS, it was present in the cytoplasm of MKs obtained from healthy and PS deficiency individuals. Our study demonstrated the PS biosynthesis by megakaryocyte. To study the mechanisms that regulate the
concentration of plasma and platelets PS we analyzed plasma and platelets PS from normal and PS deficiency subjects. PS contained in platelets have the same immunoblotting pattern respect to plasma PS. Plasma and platelet PS immunoblotting pattern demonstrated different molecular weight of PS in some deficient PS individuals as compared to normal control, suggesting different mutations in PS gene. We analyzed the presence of mutation and the presence of PS Heerlen allele. We investigated platelets PS antigen levels in type I and type III PS deficient patients. In type I subjects total and plasma free PS antigen levels were (PSAg) 62±7% and 37±12% 3 respectively. In carries of type III defect total and free PSAg levels were 85±13% and 41±13% respectively. Platelets PSAg in type I and type III were 66 ±32% and 80±37%.In a subgroup of healthy individuals total, free and platelet PSAg levels were 119±17%, 110±17% and 101±30%, respectively. The results indicate that type I and III subject’s total and plasma free PSAg levels were lower than normal individuals. Intra-platelets PSAg levels in type I and type III were lower than of healthy individuals. Our analysis demonstrates a strict correlation between total and free plasma PS and Plts PS. The reduction of platelet PS mirrors the reduced levels of free and total PSAg present in carries of the defect even though PS levels in Plts appears unexpectedly higher than the free PS counterpart. Moreover, we study the interaction of anticoagulant drugs on PSAg levels on 35 patient treatments with warfarin. The levels of total and free plasma PS decreased during treatment with oral anticoagulant, since PS is a vitamin K-dependent protein. Our study demonstrated significant decreased levels of platelet PS respectively plasma free and total PS. We valuated the effect of
anticoagulant drugs (warfarin) and of vitamin K on Mk cells. The Mk were treatment with 1?g/ml of warfarin or 1?g/ml of vitamin K and analyze synthesis of PS. We observed decreased PS synthesis on MKs with warfarin than control MKs; on the contrary, MKs cultured under vitamin K treatment increase PS synthesis.La proteina S (PS) è una glicoproteina plasmatica, vitamina K-dipendente, con molteplici funzioni nell’ambito della coagulazione, infiammazione e apoptosi. Il suo peso molecolare è di 70 kDa e la sua concentrazione plasmatica di circa 25 mg/L. Nel plasma umano il 40% della PS circola in
forma libera, mentre il restante 60% è legato alla C4b-binding-protein, una proteina del sistema del complemento. La PS circolante nel plasma viene sintetizzata principalmente nel fegato ma anche le cellule endoteliali, le cellule di Leydig e una linea cellulare di megacariociti sono in grado di
sintetizzarla. Le piastrine contengono PS, anche se la sua origine non è ancora stata chiarita. Si ipotizza che derivi dalla sintesi dei megacariociti o che siano gli stessi megacariociti ad assumerla dal pool plasmatico mediante un meccanismo di endocitosi. La PS libera agisce da cofattore per la proteina C attivata (APC) nell’inattivazione dei fattori procoagulanti Va (FVa ) e VIIIa (FVIIIa). La
PS esercita anche un’azione anticoagulante APC-indipendente, probabilmente inibendo direttamente i complessi tenase e protrombinase. Si suppone che la PS rilasciata dalle piastrine in seguito alla loro attivazione regoli la generazione di trombina, controllando perciò l’attività
procoagulante. I difetti di PS sono a trasmissione autosomica dominante e vengono classificati in tre tipi: – difetto di tipo I, caratterizzato da ridotti livelli plasmatici di PS totale e libera; – difetto di tipo II, caratterizzato da livelli fisiologici di PS totale e libera associati ad una ridotta attività; 6 – difetto di tipo III, presenta una PS libera ridotta ed una PS totale nella norma. I difetti di PS sono generalmente associati ad un aumentato rischio di trombosi venosa profonda, embolismo polmonare ed, in qualche caso, a trombosi arteriosa. Nei deficit di PS il rischio di trombosi venosa aumenta se associato ad altre condizioni di carattere genetico o acquisito quali il FV Leiden, l’aplotipo HR2 del FV e mutazioni a carico del gene che codifica per la protrombina. Molteplici fattori, tra cui la gravidanza, la terapia anticoncezionale e anticoagulante orale, riducono la concentrazione plasmatica della PS. Al fine di chiarire l’origine della PS piastrinica, abbiamo messo a punto un
modello in vitro di colture di megacariociti umani. Le cellule staminali ematopoietiche sono state isolate con histopaque da sangue intero di soggetti sani e con difetto di PS. Le cellule mononucleate sono state coltivate in un terreno privo di siero ed in presenza di trombopoietina (TPO) e interleuchina 3 (IL3) per stimolarne la differenziazione in una linea magacariocitaria. Le cellule mononucleate differenziate presentavano una morfologia simile a quella dei megacariociti e risultavano positive all’anticorpo anti-CD41; questi elementi ci hanno permesso di confermare
che si trattasse effettivamente di megacariociti. Inoltre, la marcatura dei megacariociti con anticorpi anti ??-tubulina e ?-tubulina ha evidenziato sia la presenza di estensioni citoplasmatiche denominate “proplatelets” sia il rilascio di piastrine da parte dei megacariociti. In aggiunta, mediante
tecniche di immunofluorescenza, abbiamo rilevato la presenza del FV a livello citoplasmatico, mentre la PC era assente. La PS era presente nel citoplasma dei megacariociti isolati da individui sani e con difetto di PS. La nostra ricerca ha così dimostrato la sintesi di PS da parte dei megacariociti. 7 Per studiare il meccanismo che regola i livelli di PS presenti nel plasma e all’interno delle piastrine, abbiamo determinato la concentrazione di PS plasmatica e piastrinica in soggetti sani e portatori di difetto di PS. La PS piastrinica mostrava lo stesso pattern elettroforetico di quella isolata dal plasma. L’analisi immunologica ha inoltre evidenziato, per alcuni soggetti
portatori del difetto, una PS plasmatica con differente peso molecolare rispetto ai controlli sani; questo ci ha suggerito la presenza di mutazioni nel gene della PS. Abbiamo quindi testato la presenza di eventuali mutazioni e dell’allele Heerlen. In soggetti portatori di difetto di PS di tipo I i livelli di PS totale plasmatici, e libera erano: 62±7% e 37±12% . In soggetti portatori di difetto di PS di tipo III i livelli di PS totale e libera nel plasma erano di 85±13% e 41±13%. I livelli di PS nelle piastrine nei soggetti portatori di difetto di PS di tipo I e di tipo III erano di 66 ±32% e 80±37%. In un gruppo di persone sane i livelli di PS totale, libera e piastrinica erano di 119±17%, 110±17% e 101±30%, rispettivamente. Dall’analisi dei livelli plasmatici e piastrinici di PS in soggetti portatori del difetto di tipo I e III è emerso che a) nei pazienti con difetto i livelli di PS
totale e libera erano più bassi rispetto ai soggetti sani; b) i pazienti con difetto presentavano livelli di PS piastrinica ridotti rispetto agli individui sani utilizzati come controllo. La nostra analisi ha dimostrato una stretta correlazione tra la PS plasmatica (libera e totale) e quella piastrinica. La diminuzione della concentrazione di PS piastrinica, osservata negli individui portatori del difetto, riflette l’abbassamento del livello di PS plasmatica, sebbene la quota di PS all’interno delle piastrine risulti maggiore rispetto a quella della PS presente nel plasma in forma libera. In seguito abbiamo studiato l’effetto di sostanze anticoagulanti sui livelli plasmatici e piastrinici di PS in pazienti 8 sani in trattamento con warfarina. E’ noto che la warfarina abbassa i livelli plasmatici di PS in quanto quest’ultima è una proteina vitamina Kdipendente. Anche i livelli di PS plasmatica, (totale e libera), e piastrinica dei medesimi soggetti in terapia con warfarina risultano ridotti rispetto alla norma ma l’abbassamento della concentrazione di PS appare molto più marcata all’interno delle piastrine piuttosto che nel plasma. Infine abbiamo valutato l’effetto della warfarina e della vitamina K sulla sintesi di PS da parte dei megacariociti. Mediante tecniche di immunofluorescenza abbiamo osservato una ridotta sintesi della PS nei megacariociti trattati con warfarina rispetto alle cellule di controllo; al contrario, i megacariociti coltivati in un terreno supplementato con vitamina K mostravano un incremento della sintesi di PS
Low plasma levels of tissue factor pathway inhibitor in patients with congenital factor V deficiency.
No full textSevere factor V (FV) deficiency is associated with mild to severe bleeding diathesis, but many patients with FV levels lower than 1% bleed less than anticipated. We used calibrated automated thrombography to screen patients with severe FV deficiency for protective procoagulant defects. Thrombin generation in FV-deficient plasma was only measurable at high tissue factor concentrations. Upon reconstitution of FV-deficient plasma with purified FV, thrombin generation increased steeply with FV concentration, reaching a plateau at approximately 10% FV. FV-deficient plasma reconstituted with 100% FV generated severalfold more thrombin than normal plasma, especially at low tissue factor concentrations (1.36 pM) or in the presence of activated protein C, suggesting reduced tissue factor pathway inhibitor (TFPI) levels in FV-deficient plasma. Plasma TFPI antigen and activity levels were indeed lower (P < .001) in FV-deficient patients (n = 11; 4.0 +/- 1.0 ng/mL free TFPI) than in controls (n = 20; 11.5 +/- 4.8 ng/mL), while persons with partial FV deficiency had inter-mediate levels (n = 16; 7.9 +/- 2.5 ng/mL). FV immunodepletion experiments in normal plasma and surface plasmon resonance analysis provided evidence for the existence of a FV/TFPI complex, possibly affecting TFPI stability/clearance in vivo. Low TFPI levels decreased the FV requirement for minimal thrombin generation in FV-deficient plasma to less than 1% and might therefore protect FV-deficient patients from severe bleeding
Evaluation of a procoagulant phospholipid functional assay as a routine test for measuring circulating microparticle activity.
No full textThrombin activatable fibrinolysis inhibitor in cancer patients with and without venous thromboembolism.
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Enhancing myoblast proliferation by using myogenic factors: a promising approach for improving fiber regeneration in sport medicine and skeletal muscle diseases
No full textMacrophages drive muscle regeneration and repair by removing necrotic material and producing key signaling molecules. The array of cytokines/growth factors produced by
macrophages and myogenic cells stimulates the proliferation, migration and differentiation of satellite cells. Although the details of such processes are only partially understood, it is
known that the administration of purified growth factors can improve the final outcome after traumatic muscle injuries. Also, such approach has proved to be beneficial in myoblast
transplantation experiments in animal models. The translation of such procedures into therapeutic protocols is, however, hampered by high costs and the somewhat oversimplified
biochemical input compared to the physiological signal network. We have previously reported that peritoneal macrophages could secrete factors capable of
increasing the myoblast/myotube yield in cultures of primary rat myoblasts. Recently, we observed that a macrophage cell line could be stimulated to produce a conditioned medium
that specifically enhances the proliferation of cultured neonatal primary myoblasts from mouse, rat, chicken, and human fetal myoblasts. The factors did not inhibit differentiation and led to a striking increase in the rate of contractile myotube formation. The factors could
also enhance muscle regenerative processes in vivo, thereby suggesting a potential role as an economical and effective tool for the treatment of traumatic and disease-related muscle
injuries. Further experiments in this direction and the biochemical characterization of the macrophage-produced myogenic factors are presently underway. The possibility to use the macrophage factors to improve the myoblast yield from diseased-muscle biopsies is also under investigation
Thromboelastometry profiles after “in vitro” addition of a new plasma-derived factor V concentrate to whole blood from parahaemophilia patients
No full textAnti-platelet factor 4 antibody-mediated disorders: an updated narrative review
No full text: Anti-platelet factor 4 (PF4) antibody-mediated disorders are a heterogenous group of diseases characterized by the presence of highly pathogenic immunoglobulins G directed against PF4 and/or PF4/heparin complexes. These antibodies are able to activate platelets, neutrophils and monocytes, thus resulting in thrombocytopenia and a hypercoagulable state. Five different forms of anti-PF4 antibody-mediated disorders have been identified: i) classic heparin-induced thrombocytopenia (cHIT) mediated by heparin and certain polyanionic drugs; ii) autoimmune HIT (aHIT) characterized by the presence of anti-PFA/polyanion antibodies that can strongly activate platelets even in the absence of heparin; iii) spontaneous HIT (spHIT) characterized by thrombocytopenia and thrombosis without proximate exposure to heparin, with two subtypes: (a) post-total knee arthroplasty, and cardiac surgery using cardiopulmonary bypass or extracorporeal membrane oxygenation, and (b) post-infections; iv) vaccine-induced immune thrombotic thrombocytopenia (VITT) characterized by thrombocytopenia, arterial and venous thrombosis, or secondary hemorrhage after receiving adenoviral vector vaccines for COVID-19; v) VITT-like disorders triggered by adenoviral infections. Though extremely rare and largely unknown, there has been growing interest in the VITT syndrome in recent years due to its clinical relevance. Timely detection of these antibodies is crucial for the diagnosis and treatment of anti-PF4 antibody-mediated disorders, via anti-PF4 antibody immunoassays using several antibody-capture systems (e.g., ELISA based, particle gel, turbidimetry) and functional assays (e.g., serotonin release assay or heparin-induced platelet activation). We aimed to present the latest on laboratory findings, clinical characteristics and therapeutic approaches for anti-PF4 antibody-mediated disorders
New prothrombin mutation (Arg596Trp, Prothrombin Padua 2) associated with venous thromboembolism
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