Background: The Regulation of Coagulation in Orthopedic Surgery to Prevent Deep Venous Thrombosis and Pulmonary Embolism (RECORD) 1 to 4 trials compared rivaroxaban 10 mg daily with commonly used doses of enoxaparin and demonstrated similar rates of VTE and bleeding. Objective: To evaluate bleeding events between patients who received enoxaparin or rivaroxaban for prevention of venous thromboembolism (VTE) following total hip arthroplasty (THA) or total knee arthroplasty (TKA). Methods: Retrospective cohort that compared patients undergoing THA and TKA who received enoxaparin (enoxaparin) with those who received rivaroxaban (rivaroxaban) and also with those who received enoxaparin in the RECORD 1 to 4 trials (enoxaparin RECORD). The primary outcome was any postoperative bleeding, defined as a composite of major and clinically relevant nonmajor bleeding based on the definitions in the RECORD 1 to 4 trials. Results: There was a lower rate of any postoperative bleeding (2.2% vs 6.8%, P = 0.004) in patients who received enoxaparin compared with rivaroxaban, and bleeding rates between the enoxaparin group and the enoxaparin RECORD groups were similar (2.2% vs 2.5%, P = 0.085). Major bleeding in the enoxaparin group (0.2%) was not significantly different from that in the rivaroxaban group (1.4%, P = 0.12) or the RECORD group (0.2%, P = 0.93). Clinically relevant nonmajor bleeding was also lower in the enoxaparin group compared with the rivaroxaban group (2.0% vs 5.5%, P = 0.012). Conclusions: The use of enoxaparin for VTE prophylaxis following THA and TKA was associated with a lower rate of the primary outcome (any postoperative bleeding) compared with the use of rivaroxaban in a similar cohort of patients.

Buerger disease is characterized by progressive distal extremity ischemia in persons with recent tobacco consumption. Typically, affected persons are young men. There have been case reports of Buerger disease in older men. Almost all of the reported cases were characterized with progressive, severe disease requiring amputation. We report a case of an older man with features of Buerger disease without significant proximal progression despite active smoking. Arteriographic and pathologic studies confirmed the diagnosis.

Le CD40 ligand (CD40L) est une molécule thrombo-inflammatoire qui prédit des événements cardiovasculaires. Les plaquettes constituent la principale source du CD40L soluble (sCD40L) dans la circulation, avec la capacité d’influencer la fonction des plaquettes par l'intermédiaire de sa liaison à ses récepteurs plaquettaires : CD40, αIIbβ3 et α5β1. Nous avons précédemment démontré que la stimulation des plaquettes humaines avec le sCD40L induit une activation du nuclear factor kappa B (NF-κB), qui pourra jouer un rôle non génomique en amorçant les plaquettes. En effet, l’amorçage des plaquettes par le sCD40L augmente fortement l’activation et l’agrégation plaquettaire en réponse à des doses sous-optimales d’agonistes. Cependant, l’implication des différents récepteurs dans l’amorçage des plaquettes suite à l’activation du NF-κB par le sCD40L demeure inconnue. De plus, le transforming growth factor-B (TGF-B)-activated Kinase (TAK1), un régulateur majeur de l’inflammation, est activé par le sCD40L dans les cellules nucléées, et pourra ainsi être impliqué dans la signalisation sCD40L/NF-κB plaquettaire. D’autre part, le sCD40L se trouve en quantité élevée dans la circulation sanguine des patients coronariens. De plus, le traitement antiplaquettaire à l’Aspirine (ASA) est inefficace chez certains patients et son efficacité est réduite chez les patients à hauts risques d’évènements coronariens, ce qui augmente leur risque de sur-activation plaquettaire et de développer une thrombose. Des taux sanguins élevés de médiateurs thrombo-inflammatoires, tels que le sCD40L, peuvent expliquer de telles variabilités. Ainsi, ce projet a été entrepris dans le but d’élucider l’impact de l’axe sCD40L/NF-κB sur la fonction plaquettaire et sa modulation par l’ASA. Dans le premier volet de mon projet, nous avons identifié les récepteurs de sCD40L impliqués dans l'activation du NF-κB plaquettaire, leur signalisation en aval et leur implication dans l'agrégation plaquettaire. Nous avons montré que les plaquettes exprimaient les récepteurs du CD40L : CD40, αIIbβ3 et α5β1 et libéraient du sCD40L. Le sCD40L seul a induit une activation de NF-κB plaquettaire. Cet effet était absent des plaquettes de souris déficientes en CD40 (CD40-/-) et inhibé par le blocage de CD40, mais n'était pas affecté par le blocage de αIIbβ3 ou α5β1. L’axe sCD40L/CD40 a aussi activé TAK1 en amont de NF-κB. Dans les études fonctionnelles, le sCD40L seul n’a pas eu d’effet sur l’agrégation plaquettaire, mais a potentialisé l’agrégation en présence de doses sous-optimales de thrombine; cet effet a été aboli par des inhibiteurs de CD40, TAK1 et NF-κB. Cette première étude nous a permis de conclure que le sCD40L amorce les plaquettes via des voies de signalisation impliquant CD40/TAK1/NF-κB, ce qui prédispose les plaquettes à une activation et agrégation accrues en réponse à des stimuli thrombotiques. Dans le deuxième volet de mon projet, nous avons émis l’hypothèse qu’en présence de taux élevés de sCD40L, l’efficacité de l’ASA peut varier et avons visé à déterminer les effets de l’ASA sur la signalisation et l’agrégation des plaquettes en présence de sCD40L. Les effets de l'ASA sur les plaquettes humaines traitées au sCD40L, en réponse à des concentrations sous-optimales de collagène ou de thrombine, ont été évalués sur l'agrégation, la sécrétion de thromboxane A2 (TxA2) et la phosphorylation de p38 MAPK, NF-κB, TAK1 et la chaîne légère de la myosine (MLC). Le sCD40L a significativement augmenté la sécrétion de TxA2 par les plaquettes, en réponse à des doses sous-optimales de collagène et de thrombine; cet effet a été inversé par l'ASA. L'ASA n'a pas inhibé la phosphorylation de p38 MAPK, NF-κB, TAK1, que ce soit avec une stimulation plaquettaire par le sCD40L seul ou en présence des agonistes. Le sCD40L a potentialisé l'agrégation plaquettaire, un effet complètement inversé et partiellement réduit par l'ASA en réponse à une dose sous-optimale de collagène et de thrombine, respectivement. Les effets de l'ASA sur les plaquettes traitées au sCD40L avec du collagène étaient liés à l'inhibition du changement de forme des plaquettes et à la phosphorylation de la MLC. En résumé, l'ASA n'affecte pas la signalisation plaquettaire du sCD40L, mais empêche son effet sur la sécrétion de TxA2 et l'agrégation plaquettaire en réponse au collagène, via un mécanisme impliquant l'inhibition de la MLC. En conclusion, ce projet nous a permis de déterminer que l’amorçage des plaquettes par le CD40L via l’activation de NF-κB dépend du récepteur CD40 et la signalisation via TAK1. Ainsi, l’axe sCD40L/CD40/TAK1/NF-κB potentialise l’activation et l’agrégation en réponse à des stimuli thrombotiques, ce qui peut favoriser l’occurrence d’événements athéro-thrombotiques chez les patients coronariens. De plus, étant donné que l’ASA n’a pas d’effet sur la signalisation via l’axe sCD40L, le ciblage de cet axe dans les plaquettes peut avoir un potentiel thérapeutique chez les patients coronariens présentant des taux élevés de sCD40L et ne réagissant pas ou moins à l'ASA. / CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets are the main source of soluble CD40L (sCD40L) in the circulation, with the ability to influence platelet function through its binding to platelet receptors: CD40, αIIbβ3, and α5β1. We have previously demonstrated that stimulation of human platelets with sCD40L induces activation of nuclear factor kappa B (NF-κB), which may play a non-genomic role in priming platelets. Indeed, platelet priming by sCD40L strongly enhances platelet activation and aggregation in response to suboptimal doses of agonists. However, the involvement of the different receptors in platelet priming following activation of NF-κB by sCD40L remains unknown. In addition, the transforming growth factor-B (TGF-B) -activated Kinase (TAK1), a major regulator of inflammation, is activated by sCD40L in nucleated cells, and may be involved in sCD40L/NF-κB signaling. On the other hand, sCD40L is high in the bloodstream of coronary patients, who receive Aspirin (ASA) for secondary prevention. However, antiplatelet therapy with ASA is ineffective in some patients and its efficacy is reduced in patients at high risk for coronary events, increasing their risk of platelet over-activation and developing thrombosis. High blood levels of thrombo-inflammatory mediators, such as sCD40L, may explain such variability. Thus, we undertake this project to elucidate the impact of the sCD40L/NF-κB axis on platelet function and its modulation by ASA. In the first part of our project, we aimed to identify the sCD40L receptors involved in the activation of platelet NF-κB, their downstream signaling and their involvement in platelet aggregation. We have shown that platelets express CD40L receptors: CD40, αIIbβ3 and α5β1 and release sCD40L. sCD40L alone induced activation of platelet NF-κB. This effect was absent in CD40-/- mouse platelets and inhibited by CD40 blockade, but was not affected by αIIbβ3 or α5β1 blockade. The sCD40L/CD40 axis has also activated TAK1 upstream of NF-kB. In functional studies, sCD40L alone had no effect on platelet aggregation but potentiated aggregation in the presence of suboptimal thrombin doses. The inhibitors of CD40, TAK1 and NF-κB abolished this effect. This first study concluded that sCD40L primes platelets via CD40/TAK1/NF-κB signaling pathways, which predisposes platelets to increased activation and aggregation in response to thrombotic stimuli. In the second part of our project, we hypothesized that in the presence of high levels of sCD40L; the effectiveness of ASA may vary. Accordingly, we aimed at determining the effects of ASA on signaling and aggregation of platelets in the presence of sCD40L. The effects of ASA on human platelets treated with sCD40L, in response to suboptimal concentrations of collagen or thrombin, were evaluated on aggregation, thromboxane A2 (TxA2) secretion and phosphorylation p38 MAPK, NF-κB, TAK1 and myosin light chain (MLC). sCD40L significantly increased TxA2 secretion by platelets in response to suboptimal doses of collagen and thrombin; this effect has been reversed by the ASA. ASA did not inhibit the phosphorylation of p38 MAPK, NF-κB, TAK-1, with platelet stimulation by sCD40L either alone or in the presence of agonists. sCD40L potentiated platelet aggregation, an effect completely reversed and partially reduced by ASA in response to a suboptimal dose of collagen and thrombin, respectively. In addition, the effects of ASA on sCD40L-treated platelets with collagen related to inhibition of platelet shape change and phosphorylation of MLC. In summary, ASA does not affect platelet signaling via sCD40L, but prevents its effect on TXA2 secretion and platelet aggregation in response to collagen, via a mechanism involving inhibition of MLC. In conclusion, this project allowed us to determine that platelet priming by sCD40L via activation of NF-κB depends on the CD40 receptor and signaling via TAK1. Thus, the sCD40L/CD40/TAK1/NF-κB axis potentiates activation and aggregation in response to thrombotic stimuli, which may promote occurrence of atherothrombotic events in coronary patients. In addition, since ASA has no effect on platelet signaling via the sCD40L axis, targeting of this axis in platelets may have therapeutic potential in coronary patients with high levels of sCD40L that are none or less responding to ASA.

This work describes the development and testing of a real-time three-dimensional computational fluid dynamics simulation of thrombosis and embolization to be used in the design of blood-contacting devices. Features of the model include the adhesion and aggregation of blood platelets on device material surfaces, shear and chemical activation of blood platelets, and embolization of platelet aggregates due to shear forces. As thrombus develops, blood is diverted from its regular flow field. If shear forces on a thrombus are sufficient to overcome the strength of adhesion, the thrombus is dislodged from the wall. Development of the model included preparing thrombosis and embolization routines to run in a parallel processing configuration, and estimating necessary parameters for the model including the adhesion strength of platelet conglomerations to the device surfaces and the criterion threshold for the coalescence of neighboring thrombi. Validation of the model shows that the effect of variations in geometry may be accurately predicted through computational simulation. This work is based on previous work by Paul Goodman, Daniel Lattin, Jeff Ashton, and Denzel Frost.

Habilitacijos procedūrai teikiamoje mokslo darbų apžvalgoje apibendrinama Vilniaus universiteto Širdies ir kraujagyslių ligų klinikos Širdies chirurgijos centro patirtis, pakartotinai operuojant ligonius po širdies vožtuvų protezavimo. Įvertintas mažai trombogeniškų protezų efektyvumas. Nustatyta, kad labai sunkių ligonių su infekuotais protezais mirštamumas nemažėja, nors pastaraisiais metais daugiau operuojame III funkcinės klasės ligonių ir jų operacijų rezultatai puikūs. Dėl pasiūlyto radikalesnio triburio vožtuvo nesandarumo gydymo pirminių operacijų metu, efektyviai mažėja pakartotinių šio vožtuvo operacijų. Įteisinta planinė seno rutulinio protezo pakeitimo operacija. Įdiegti nauji širdies pjūviai, įdiegtos krūtinės ląstos pjūvių atlikimo metodikos, farmakologiniai ir nefarmakologiniai perioperacinio kraujavimo stabdymo būdai efektyviai sumažina kraujavimo pavojų. Pasiūlytas originalus kairio skilvelio drenavimo būdas per atskira torakotominį pjūvį padeda sumažinti pooperacinį širdies nepakankamumą. Šiame darbe remiuosi kartu su bendradarbiais atliktų operacijų rezultatais. Šiuo metu kaip skyriaus, kuriame gydomi ligoniai su vožtuvų patologija, vadovas atlieku daugiau kaip pusę pakartotinių operacijų. Moksliniai pranešimai apie pakartotinių operacijų rezultatus buvo daryti Lietuvos, Pasaulio ir Europos kongresuose ir suvažiavimuose. / The review of scientific work presented for habilitation procedure summarizes the experience of redo operations of patients after replacement of heart valves accumulated at the Heart Surgery Centre of Vilnius University Clinic of Angiology and Cardiology. The efficacy of low thrombogenicity valve prostheses was evaluated. It was found out that the mortality rate of very severely ill patients with infected did not decrease; however the larger number of patients in functional class III were being operated during the period of recent years and the results of these operations were excellent. The rate of redo tricuspid valve operations had decreased effectively because of more radical surgery treatment of this valve during the primary operation. The operation of replacement of old ball prosthesis has been validated. The new incisions of the heart were introduced as well as new methods of performance of chest box incisions and pharmacologic and non-pharmacologic methods of perioperative hemostasis; the methods mentioned above effectively reduced the risk of bleeding. The original method of the left ventricle venting via separate thoracotomy incision enabled to reduce postoperative heart failure. This work is based on the results of operations performed in cooperation with co-workers. As a chief of the department where the patients with pathology of heart valves are treated, I am performing more than a half of redo operations. The scientific presentations concerning the results of... [to full text]

<p>UVOD: Komplikacije terminalne bubrežne isuficijencije (TBI) kada se jačina glomerularne filtracije (JGF) smanji ispod 10 mL/min moguće je lečiti jedino hroničnom dijalizom ili transplantacijom bubrega tj. nadoknadom potpuno ili delimično izgubljene bubrežne funkcije. Uz blagovremenu edukaciju bolesnika o progresivnom toku hronične bubrežne bolesti, mogućnostima dijaliznog tretmana i transplantacije bubrega, treba na vreme obezbediti stalni funkcionalni vaskularni pristup za hemodijalizu (HD) hirur&scaron;kom intervencijom kreiranja arteriovenske fistule (AVF), po mogućnosti najmanje 6 meseci pre anticipiranog započinjanja HD, jer je za njenu maturaciju potrebno 4 do 6 nedelja. Primarna AVF je op&scaron;tepreporučeni najbolji stalni vaskularni pristup za bolesnike kod kojih se planira hemodijaliza. Najče&scaron;ći razlog za disfunkciju vaskularnog pristupa za hemodijalizu su u 80% slučajeva trombozne komplikacije, koje se u 90% slučajeva javljaju na venskom segmentu AVF i posledica su progresivne venske neointimalne hiperplazije. Pored histolo&scaron;kih karakteristika zida venskog krvnog suda i hemodinamskih uslova, u etiopatogenezi ovog &raquo;adaptivnog odgovora&laquo; vrlo značajnu ulogu igraju endotel i ostale komponente hemostaznog sistema (trombocitna, koagulaciona i fibrinolizna), imunolo&scaron;ki i citolo&scaron;ki činioci i genetski faktori. Prevencija nastanka rane tromboze vaskularnog pristupa za hemodijalizu kod bolesnika sa TBI je moguća primenom antitromboznih lekova, tj. antitrombocitne ili antikoagulantne terapije. CILJ: Proceniti efikasnost primenjenih antitromboznih lekova (tiklopidina i nadroparin-kalcijuma) u prevenciji nastanka rane tromboze/afunkcionalnosti AVF za hemodijalizu za vreme njene maturacije unutar 6 nedelja od kreiranja u bolesnika sa TBI. Ispitati nivo biomarkera hemostaznog sistema i markere trombofilije u bolesnika sa TBI pre kreiranja AVF u cilju dopune uzroka nastanka rane tromboze/afunkcionalnosti arteriovenskih fistula za hemodijalizu. Ispitati učestalost trombofilije i njen uticaj na funkcionalnost AVF i uporediti efikasnost primenjenih preventivnih režima između bolesnika sa i bez trombofilije. MATERIJAL I METODE: U ispitivanje su uključene osobe oba pola sa prethodno postavljenom dijagnozom TBI kod kojih nisu postojale kontraindikacije za planirno hirur&scaron;ko kreiranje prvog stalnog vaskularnog pristupa za hemodijalizu u vidu autologne arteriovenske fistule (AAVF). Nakon hirur&scaron;kog kreiranja radiocefalične arteriovenske fisule u distalnoj trećini podlaktice nedominantne ruke (89/121), intermedijalne (4/121) ili proksimalne (28/121) AAVF u studiju je uključen 121 ispitanik, koji su u cilju procene uticaja različitih antitromboznih lekova na sprečavanje nastanka rane tromboze fistula za hemodijalizu kod bolesnika sa TBI ispitanici su podeljeni u 3 grupe: Grupa I, kontrolna; 40 ispitanika koji nakon kreiranja AVF nisu dobijali antitromboznu terapiju, Grupa II; 42 ispitanika kod kojih je dan nakon kreiranja AVF započeta primena antitrombocitnog leka iz grupe tienopiridina, Ticlodix&reg; (ticlopidin) tbl a 250 mg, 2 x &frac12; tbl dnevno tokom 6 nedelja i Grupa III; 39 ispitanika kod kojih je dan nakon kreiranja AVF započeta subkutana primena antikoagulantnog leka iz grupe niskomolekularnih heparina, Fraxiparine&reg; (nadroparin-kalcijum) 2850 anti Xa i.j. (0.3 ml) dnevno tokom 6 nedelja. Jednokratno određivanje laboratorijskih parametara pokazatelja bubrežne funkcije, metabolizma glukoze i hroničnog zapaljenja, funkcionalnosti hemostaznog sistema, trombofilnih markera i genskog polimorfizma vr&scaron;eno je unutar dve nedelje pre hirur&scaron;kog kreiranja AAVF. Kriterijum za utvrđivanje ishoda uticaja antitrombozne terpije predstavlja maturacija AVF koja je definisana kao uspe&scaron;na ako je započeto sprovođenje efikasne hemodijalize najranije 6 nedelja nakon njenog hirur&scaron;kog kreiranja po proceni nadležnog nefrologa. Dijagnoza prisustva tromboze AVF postavljena je od strane nadležnog vaskularnog hirurga/nefrologa fizikalnim pregledom tokom njene maturacije, koji je podrazumevao inspekciju, palpatorno utvrđivanje odsustva karakterističnog trila i auskultatornih karakteristika protočnosti AVF ili ultarsonografskim pregledom od strane radiologa. REZULTATI: Između ispitivanih grupa u odnosu na broj tromboziranih/ afunkcionalnih AVF tokom njene maturacije (12/40 vs. 4/42 vs. 5/39; P=0.033), ustanovljena je značajna statistička razlika kao i poređenjem broja tromboziranih/afunkcionalnih AVF tokom sazrevanja u kontrolnoj grupi u odnosu na grupu ispitanika (objedinjene Grupe II i Grupa III) koja je primala antitromboznu profilaksu (12/40 vs. 9/81; P=0.009). Daljom analizom ispitivanih grupa, utvrđena je statistički značajna razlika u broju tromboziranih/afunkcionih AV fistula između kontrolne Grupe I i Grupe II (P=0.019). Testiranjem razlike u broju tromboziranih/ afunkcionalnih AVF između ispitanika kontrolne Grupe I i Grupe III nije dobijena statistički značajna razlika, kao ni između Grupe II i Grupe III. Zastupljenost broja tromboziranih/afunkcionalnih distalnih AVF za vreme njihove maturacije (12/33 vs 2/31 vs. 3/24; P=0.008) se između ispitivanih grupa značajno statistički razlikovala kao i zastupljenost tromboziranih/afunkcionalnih distalnih AVF tokom sazrevanja u kontrolnoj grupi u odnosu na grupu ispitanika koja je primala antitromboznu profilaksu (12/34 vs. 5/55; P=0.002). Testiranjem statističke razlike u broju tromboziranih/afunkcionalnih distalnih AVF između ispitanika kontrolne Grupe I i Grupe II utvrđena je statistički značajna razlika (P=0.005), dok između Grupe I i Grupe III (P=0.051), kao ni između Grupe II i Grupe III (P=0.439) nije dobijena statistički značajna razlika. Između podgrupa ispitanika kod kojih je do&scaron;lo do tromboze/afunkcionalnosti AVF 21/121 (17.35%) i podgrupe ispitanika sa funkcionalno maturiranom AVF 90/121 (82.64%), značajna statistička razlika ispitanih hemostaznih parametara je bila prisutna u vrednostima agregabilnosti trombocita uz kolagen kao induktor (59.33&plusmn;33.1 vs. 75.04&plusmn;29.6; P=0.033). Značajna statistička razlika je zabeležena i u zastupljenosti sledećih trombofilnih markera: deficita PC (3/21 vs. 3/100; P=0.030), APC-R (4/21 vs. 5/100; P=0.026), prisustva antifosfolipidnih ACL IgM antitela (1/21 vs. 0/100; P=0.028), heterozigotnog polimorfizma FV G1691A (3/21 vs. 3/100; P=0.03) i homozigotne mutacije gena FII G20210A (1/21 vs. 0/100; P=0.028), između podgrupa bolesnika sa tromboziranom afunkcionalnom i funkcionalnom AVF. Takođe je značajna statistička razlika između podgrupa bolesnika kod kojih je do&scaron;lo tromboze/afunkcionalnosti AVF i podgrupe ispitanika sa funkcionalno maturiranom AVF bila prisutna u odnosu na postojanje ranijih tromboza (23/21 vs 19/100; P=0.000) kao i zastupljenosti izolovanih venskih tromboza (9/21 vs. 2/100; P=0.000). Prediktivni potencijal pojedinačnih parametara za maturaciju AVF ispitan je univarijantnom logističkom regresionom analizom. Prilikom ispitivanja uticaja pojedinačnih parametara na maturaciju fistule, zapazili smo da su ispitanici koji su primali antitromboznu terapiju imali 3 puta veću &scaron;ansu za funkcionalno maturiranu AVF [OR 3.45 (1.3-9.03)] u odnosu na bolesnike bez terapije. Ispitanici koji su imali prethodne tromboze su imali vi&scaron;estruko povi&scaron;en rizik [OR 6.92 (2.51-19.06)] za nastanak tromboze/afunkcionalnost AVF tokom maturacije. Prilikom ispitivanja uticaja pojedinačnih parametara na rizik od pojave tromboze/afunkcionalnosti distalne AVF, zapažamo da primena antitrombozne terapije [OR 5.4 (CI 1.7 - 17.35)] petostruko snižava rizik za nastanak tromboze/ afunkcionalnosti distalne AVF, odnosno da primena antitrombozne terapije petostruko povećava &scaron;ansu za adekvatnu maturaciju distalne AVF. Ispitanici koji su imali aterosklerotske KVB [OR 0.32 (0.1-0.98)] i ranije tromboze [OR 0.14 (0.04-0.44)] su imali za 68% i 86% manju verovatnoću za adekvatnu maturaciju distalne AVF (334). Trombofilija je bila prisutna u 59/121 (48.8%) ispitanika. U odnosu na markere aktivacije koagulacione komponente hemostaznog sistema i inflamatorne pokazatelje, između podgrupa ispitanika sa ili bez trombofilije statistički značajna razlika je bila prisutna u vrednostima koncentracije FVIII (170.35&plusmn;103.97 vs. 235.26&plusmn;124.80; P=0.02) i odnosa trombociti/limfociti (181&plusmn;64.58 vs. 148.11&plusmn;66.15; P=0.026). U odnosu na lokalizaciju AVF, u podgrupi ispitanika sa trombofilijom i tromboziranom/ afunkcionalnom AVF, njih 8/11 su pripadale distalnim AVF, 3/11 proksimalnim AVF, dok je u podgrupi ispitanika bez trombofilije i tromboziranom/afunkcionalnom AVF, njih 9/10 imalo distalnu, a 1/10 proksimalnu AVF. U grupi bolesnika sa trombofilijom nije zabeleženo prisustvo statistički značajne razlike u efikasnosti primenjenih antitromboznih režima merene učestalo&scaron;ću tromboza/afunkcionalnosti AVF u odnosu na bolesnike sa trombofilijom koji nisu primali antitromboznu terapiju (5/19 vs. 2/18 vs. 4/22; P=0.493). U grupi ispitanika bez trombofilije utvrđeno je postojanje statistički značajne razlike u učestalosti tromboza/afunkcionalnosti AVF između grupe sa i bez primene antitromboznih lekova kako u ukupnom broju tromboziranih/afunkcionalnih AVF (7/21 vs. 2/24 vs. 1/17; P=0.030). Iako je zastupljenost tromboza/afunkcionalnosti AVF u bolesnika sa kombinovanom trombofilijom če&scaron;ća u odnosu na ispitanike koji su imali drugu vrstu ili uop&scaron;te nisu imali trombofiliju (6/18 vs. 15/103; P=0.052), ona nije dostigla statistički značajnu vrednost. ZAKLJUČAK: Profilaktička primena antitromboznih lekova (tiklopidina i nadroparin-kalcijuma) smanjuje učestalost pojave rane tromboze i pojavu primarne nefunkcionalnosti AVF za hemodijalizu tokom njene maturacije. Primena antitrombozne terapije petostruko snižava rizik za nastanak tromboze/ afunkcionalnosti distalne AVF tokom njene maturacije. Bolesnici koji su imali prethodne tromboze imaju vi&scaron;estruko povi&scaron;en rizik za nastanak tromboze AVF tokom njene maturacije. Kod bolesnika koji su imali aterosklerotske KVB i ranije tromboze verovatnoća za adekvatnu maturaciju distalne AVF je niža za 68% , odnosno 86%. U na&scaron;em istraživanju nije utvrđeno postojanje superiornosti antikoagulantne u odnosu na antitrombocitnu profilaksu tj. oba primenjena režima su bila podjednako efikasna. U terminalnoj bubrežnoj insuficijenciji prisutan je značajan poremećaj funkcionalnosti hemostaznog sistema koji se očituje u disfunkciji endotela i poremećenoj (sniženoj) funkcionalnosti trombocita, prisustvu prokoagulantnog stanja koje se manifestuje povi&scaron;enom trombinskom aktivno&scaron;ću, povi&scaron;enom koncentracijom činilaca koagulacije i smanjenom fibrinoliznom aktivno&scaron;ću. Če&scaron;ća zastupljenost ukupnih ranijih tromboza (arterijskih i venskih), če&scaron;ća zastupljenost izolovanih venskih tromboza i učestalije prisustvo trombofilije prezentovano deficitom PC, prisustvom rezistencije na APC, prisusustvom antifosfolipidnih antikardiolipinskih antitela IgM, heterozigotnog polimorfizma FV G1691A, homozigotne mutacije FII G201210A i niža vrednost agregabilnosti trombocita uz kolagen kao induktor su markeri koji su u na&scaron;em ispitivanju signifikantno če&scaron;će zastupljeni kod ispitanika sa trombozom/ afunkcijom AVF za hemodijalizu tokom njenog sazrevanja. Trombofilija je prisutna kod 48.8% bolesnika saTBI, ali na&scaron;im ispitivanjem nije utvrđen njen uticaj na nastanak rane tromboze/afunkcionalnosti AVF izuzev u grupi bolesnika sa kombinovanom trombofilijom. Mali broj krvarećih komplikacija u na&scaron;oj studiji ukazuje na bezbednost primenjenog preventivnog režima. Na osnovu dobijenih rezultata može se preporučiti profilaktička primena tiklopidina ili nadroparin-kalcijuma u preventivnim dozama kod bolesnika sa TBI neposredno nakon kreiranja AVF. Primenu profilakse tromboznih komplikacija kod bolesnika sa novokreiranom AVF preporučujemo posebno kod bolesnika koji su imali prethodne tromboze i/ili kliničke manifestacije aterosklerotskih kardiovaskularnih bolesti.</p> / <p>INTRODUCTION: Complications in end stage renal disease (ESRD) when the glomerular filtration rate (GFR) decreases below 10mL/min can only be treated by chronic dialysis or kidney transplant ie. total or partial renal replacement therapy. With prompt education of the patient regarding the progressive course of the chronic kidney disease, possibilities of dialysis treatment and kidney transplantation, the patient should timely be granted permanent functional vascular hemodialysis (HD) access through surgical intervention by creating arteriovenous fistula (AVF), preferably at least 6 months prior to the anticipated start of HD, as period for its maturation is between 4 and 6 weeks. Primary AVF is the generally best recommended permanent vascular access for patients planned for dialysis. The most common reason for dysfunction of the vascular access for hemodialysis are thrombotic complications in 80% of the cases, 90% of which appear in the venous segment of AVF as the consequence of progressive venous neointimal hyperplasia. Beside the histological characteristics of the venous blood vessel wall and hemodynamic conditions, in the etiopathogenesis of this &ldquo;adaptive answer&rdquo;, endothel and other components of the hemostatic system (platelet, coagulation and fibrinolysis), immunological and cytological components as well as genetic factors play a very important role. Prevention of occurrence of early thrombosis of vascular access for hemodialysis in patients with ESRD is possible by treatment with antithrombotic drugs, ie. antiplatelet or anticoagulant therapy. OBJECTIVE: Estimate the efficiency of applied antithrombotic drugs (ticlopidine and nadroparincalcium) in prevention of occurrence of early thrombosis/dysfunction of AVF for hemodialysis during its time of maturation within the 6 week period. Examine the level of biomarkers of the hemostatic system and thrombophilic markers in patients with ESRD before the creation of AVF with the goal of finding additional causes of occurrence of early thrombosis/dysfunction of arteriovenous fistula for hemodialysis. Determine the incidence of thrombophilia and its impact on the functionality of AVF and compare the efficiency of applied preventive regimen between patients with and without thrombophilia. MATERIAL AND METHODS: The study included persons of both sexes with previously established diagnosis of ESRD in which there were no contraindications for the planned surgical creation of the first permanent vascular access for hemodialysis in the form of autologous arteriovenous fistula (AAVF). After the surgical creation of the radiocephalic arteriovenous fistula in the distal third of the forearm of the non-dominant hand (89/121), intermedial (4/121) or proximal (28/121) AAVF, the total number of 121 patients were included in the study and divided into three groups in order to estimate the influence of different antithrombotic drugs in prevention of early thrombosis for hemodialysis in patients with ESRD: Group I, control; 40 subjects which did not receive antithrombotic therapy after the creation of AVF, Group II; 42 subjects which started receiving an antithrombotic drug from the tienopiridine group, Ticlodix&reg; (ticlopidine) 2 x &frac12; of 250mg tbl, daily, during the period of 6 weeks, after the creation of AVF, and Group III; 39 subjects which started subcutaneously receiving a drug from the low-molecular weight herapin group, Fraxiparine&reg; (nadroparine-calcium) 2850 anti Xa i.j. (0.3 ml) daily, during the period of 6 weeks. One-time determination of laboratory parameters and renal function, glucose metabolism and chronic inflammation, hemostatic system functionality, thrombophilic markers and gene polymorphism was performed within two weeks prior to surgical creation of AAVF. The criteria for determining the outcome of the impact of antithrombotic therapy is the maturation of AVF, which is defined as successful if the implementation of effective hemodialysis started at least 6 weeks after its creation, where the effectiveness of hemodialysis is estimated by a competent nephrologist. The diagnosis of the presence of AVF thrombosis was set by a competent vascular surgeon/nephrologist through physical examination during its maturation, which included inspection, palpatory determination of absence of the characteristic thrill and auscultatory characteristics of the flow of AVF, or by ultrasonographic examination by the radiologist. RESULTS: Between the groups in terms of number of thrombosed/dysfunctional AVF during its maturation (12/40 vs. 4/42 vs. 5/39, P = 0.033), a significant statistical difference was established, as well as by comparing the number of thrombosed/dysfunctional AVF during maturation in the control group compared to the group of respondents (unified Group II and Group III) which received antithrombotic prophylaxis (12/40 vs. 9/81, P = 0.009). Through further analysis of the examined groups, a statistically significant difference was observed in the number of thrombosed/dysfunctional AV fistula between the control Group I and Group II (P = 0.019). There was no statistically significant difference noticed in the numbers of thrombosed/dysfunctional AVF between the subjects in the control Group I and Group III, as well as between Group II and Group III. Presence of the number of thrombosed/dysfunctional distal AVF during their maturation (12/33 vs 2/31 vs. 3/24, P = 0.008) between the groups statistically significantly varied, as well as the presence of the number of thrombosed/dysfunctional distal AVF during the maturation in the control group as compared to the group of subjects who received antithrombotic prophylaxis (12/34 vs. 5/55; P=0.002). By testing statistical differences in the number of thrombosed/dysfunctional distal AVF between the subjects in the control Group I and Group II a statistically significant difference (P = 0.005) was established, while there was no statistically significant difference between Group I and Group III (P = 0.051), nor between Group II and Group III (P = 0.439). Among the subgroup of patients with thrombosis/dysfunction of AVF 21/121 (17.35%) and the subgroup of subjects with functionally maturated AVF 90/121 (82.64%), a statistically significant difference of the examined hemostasis parameters was present in the values of platelet aggregation with collagen as the inducer (59.33 &plusmn; 75.04 vs. 33.1 &plusmn; 29.6; P = 0.033). A significant statistical difference was recorded in the presence of the following thrombophilic markers: deficit of PC (3/21 vs. 3/100; P = 0.030), APC-R (4/21 vs. 5/100; P = 0.026), the presence of antiphospholipid ACL IgM antibodies ( 1/21 vs. 0/100; P = 0.028), heterozygous FV G1691A polymorphism (3/21 vs. 3/100; P = 0.03) and homozygous gene mutation FII G20210A (1/21 vs. 0/100; P = 0.028), between the subgroups of patients with thrombosed/dysfunctional and functional AVF. There also was a significant statistical difference between the groups of patients which encountered thrombosis/dysfunction of AVF and subgroups of subjects with functional maturated AVF in relation to the existence of previous thrombosis (23/21 vs. 19/100; P = 0.000) and the presence of isolated venous thrombosis (9/21 vs. 2/100; P = 0.000). Predictive potential of individual parameters for AVF maturation was tested by univariate logistic regression analysis. During the examination of the influence of individual parameters on fistula maturation, we observed that subjects who received antithrombotic therapy were 3 times more likely to develop functionally maturated AVF [OR 3.45 (1.3-9.03)] as compared to subjects who did not receive any treatment. Subjects which previously had thrombosis had a multiple times increased risk [OR 6.92 (2:51 to 19:06)] of developing thrombosis/dysfunctional AVF during its maturation. When examining the influence of individual parameters on the risk of thrombosis/dysfunction of the distal AVF, we noted that the implementation of antithrombotic therapy [OR 5.4 (CI 1.7 - 17:35)] reduced risk of thrombosis/dysfunction of the distal AVF by five times, ie. that the implementation of antithrombotic therapy increases the chance for adequate distal AVF maturation by five times. The subjects that had atherosclerotic cardiovascular diseases (CVD) [OR 0.32 (0.1-0.98)] or previous thrombosis [OR 0.14 (0.04-00.44)] had a 68% or 86% less chance for adequate distal AVF maturation (334). Thrombophilia was present in 59/121 (48.8%) patients. In relation to the markers of activation of coagulation components of the hemostatic system and inflammatory markers, among subgroups of subjects with or without thrombophilia a statistically significant difference was present in the FVIII concentration (170.35 &plusmn; 103.97 vs. 235.26 &plusmn; 124.80; P = 0.02) and the platelets/lymphocytes ratio (181 &plusmn; 64.58 vs. 148.11 &plusmn; 66.15; P = 0.026). In relation to the localization of AVF, in the subgroup of subjects with thrombophilia and thrombosed/dysfunctional AVF, 8/11 of them belonged to distal AVF, 3/11 proximal AVF, while in the subgroup of subjects without thrombophilia and thrombosed/dysfunctional AVF, had 9/10 distal and 1/10 proximal AVF. In the group of subjects with thrombophilia there was no record of the presence of statistically significant differences in the efficiency of antithrombotic regimen which was measured by the frequency of thrombosis/dysfunction of AVF as compared to subjects with thrombophilia which did not receive antithrombotic therapy (5/19 vs. 2/18 vs. 4/22, P = 0.493). In the group of subjects without thrombophilia statistically significant differences were found in the frequency of thrombosis/dysfunctions of AVF among groups with and without the use of antithrombotic drugs in the total number of thrombosed/dysfunctional AVF (7/21 vs. 2/24 vs. 1/17, P = 0.030). Although the presence of thrombosis/dysfunction of AVF in patients with combined thrombophilia was more frequent compared to those who had other types of, or did not have thrombophilia (6/18 vs. 15/103; P = 0.052), it did not reach a statistically significant value. CONCLUSION: Prophylactic use of antithrombotic drugs (ticlopidine and nadroparin-calcium) reduces the incidence of early thrombosis and the occurrence of primary AVF dysfunction for hemodialysis during its maturation. Implementation of antithrombotic therapy reduced risk of thrombosis/ dysfunction of the distal AVF during its maturation by five times. Patients who have had previous thrombosis have multiple times greater risk of AVF thrombosis during its maturation. In patients who had atherosclerotic CVD or previous thrombosis, the probability for adequate maturation of distal AVF is lower by 68% or 86%. In our study there was no evidence of superiority of anticoagulant compared to antiplatelet prophylaxis ie. both regimens were equally effective. In ESRD there is significant disarrangement of hemostatic system functionality, which is reflected in endothelial dysfunction and disturbed (reduced) platelet functionality, the presence of procoagulant condition that is manifested by elevated thrombin activity, increased levels of clotting factors and reduced fibrinolytic activity. More frequent presence of total previous thrombosis (arterial and venous), higher frequency of isolated venous thrombosis and frequent presence of thrombophilia presented by the deficit of PC, the presence of resistance to APC, presence of anticardiolipin antiphospholipid antibodies IgM, heterozygous FV G1691A polymorphism, homozygous mutation FII G201210A and lower value of collagen induced platelet aggregation are the markers in our study which are significantly more frequent in patients with thrombosis/dysfunction of AVF for hemodialysis during its maturation. Thrombophilia is present in 48.8% of patients with ESRD, however our study does not determine its impact on early thrombosis/dysfunction of AVF except in the group of patients with combined thrombophilia. A small number of bleeding complications in our study points to the safety of the applied preventive regimen. Based on the obtained results, prophylactic use of ticlopidine or nadroparin-calcium in preventive doses can be recommended for patients with ESRD immediately after AVF creation. Prophylactic treatment of thrombotic complications in patients with newly created AVF is recommended especially in patients who have had previous thrombosis and/or clinical manifestations of atherosclerotic cardiovascular diseases.</p>

Les Cellules Endothéliales Progénitrices ("Endothelial Progenitor Cells", EPCs) sont des précurseurs endothéliaux qui jouent un rôle émergeant en biologie vasculaire. Les EPCs ont été localisées dans le cordon ombilical, la moelle osseuse, le sang périphérique et dans certains tissus régénérateurs. Les interactions des EPCs avec les cellules sanguines et vasculaires peuvent largement influencer leurs propriétés biologiques et dicter leur fonctionnement pendant la réparation endothéliale. Plus spécifiquement, les interactions des EPCs avec les plaquettes circulantes induisent leur migration, leur recrutement et leur différentiation en cellules endothéliales aux sites de lésions vasculaires. Cependant, l’impact d’une telle interaction sur la fonction plaquettaire n’a pas été recherché. Le but de mon projet était de :1) générer des EPCs à partir des cellules mononucléaires du sang humain périphérique ("Peripheral Blood Mononuclear Cells", PBMCs); 2) étudier les interactions adhésives entre les EPCs et les plaquettes; 3) déterminer leur impact sur la fonction plaquettaire et la formation du thrombus et 4) décrire le mécanisme d’action des EPCs sur les plaquettes et le thrombus. Mises en culture sur une surface de fibronectine dans un milieu conditionné, les PBMCs fraîchement isolées possédaient une morphologie ronde et une petite taille. Après cinq jours, les PBMCs adhérentes donnaient naissance à des colonies, puis formaient une monocouche de cellules aplaties caractéristiques des EPCs après dix jours de culture. Les EPCs différenciées étaient positives pour l’Ulex-lectine et l’Acétyle des lipoprotéines de faible densité ("Acetylated Low Density Lipoprotein", Ac-LDL), exprimaient les marqueurs progéniteurs (CD34, P-sélectine, VEGFR2, vWF et VE-Cadhérine) tandis que les marqueurs leucocytaires (CD14, PSGL-1 et L-sélectine) étaient absents. Ces EPCs interagissaient avec les plaquettes activées par un mécanisme dépendant de la P-sélectine plaquettaire, inhibaient l’activation et l’agrégation plaquettaire et réduisaient significativement l’adhésion plaquettaire, principalement par l’action de prostacycline (PGI2). En fait, ceci était associé avec une augmentation de l’expression de la cyclooxygénase-2 (COX-2) et du monoxyde d’azote (NO) synthéthase inductible (iNOS). Toutefois, les effets inhibiteurs des EPCs sur la fonction plaquettaire ont été renversés par une inhibition de la COX et non pas du NO. Bien que les EPCs fussent en mesure de lier les plaquettes via la P-sélectine, leurs effets prédominants étaient médiés essentiellement par une sécrétion paracrine, impliquant la PGI2. Néanmoins, un rapprochement étroit ou un bref contact entre les EPCs et les plaquettes était requis pour que cette fonction soit complètement réalisée. D’ailleurs, cet aspect a été investigué chez des souris déficientes en P-sélectine (P-sel-/-) et chez leurs congénères de phénotype sauvage (Wild Type, WT). Chez les souris WT, les EPCs inhibaient l’agrégation plaquettaire dans le sang complet de manière concentration-dépendante alors que dans les souris P-sel-/-, l’action des EPCs n’avait pas d’effet significatif. De plus, en utilisant un modèle murin de thrombose artérielle, nous avons démontré que l’infusion systémique des EPCs altéraient la formation du thrombus et réduisaient significativement sa masse chez les souris WT, mais non pas chez les souris P-sel-/-. En outre, le nombre des EPCs incorporées au niveau du thrombus et de la paroi vasculaire était visiblement réduit chez les P-sel-/- par rapport aux souris WT. Dans cette étude, nous sommes parvenus à différentier adéquatement des EPCs à partir des PBMCs, nous avons étudié les interactions adhésives entre les EPCs et les plaquettes, et nous avons décrit leur impact sur la fonction plaquettaire et la formation du thrombus. De plus, nous avons identifié la PGI2 comme étant le principal facteur soluble sécrété par les EPCs en culture et responsable de leurs effets inhibiteurs sur l’activation, l’adhésion et l’agrégation plaquettaire in vitro. De surcroît, nous avons élucidé le mécanisme d’action des EPCs sur l’agrégation plaquettaire et la formation du thrombus, in vivo, et nous avons souligné le rôle de la P-sélectine plaquettaire dans ce processus. Ces résultats ajoutent de nouvelles connaissances sur la biologie des EPCs et définissent leur rôle potentiel dans la régulation de la fonction plaquettaire et la thrombogenèse. / Endothelial Progenitor Cells (EPCs) are believed to contribute to vascular biology and endothelial repair. EPCs have been isolated from umbilical cord, bone marrow, peripheral blood and in some regenerative tissues. Interactions of EPCs with vascular and blood cells can largely influence their functional properties and predict their destiny in the target tissues. More specifically, interactions of EPCs with circulating platelets provide the critical signal to ensure their migration and homing at the sites of vascular injury and their differentiation into endothelial cells. However, the functional consequences of such interactions on platelets remain unknown. Accordingly, this project was designed to investigate the impact of EPCs on platelet function and the specific objectives of this study were to: 1) generate EPCs from human peripheral blood mononuclear cells (PBMCs); 2) characterize the adhesive interactions between EPCs and platelets; 3) determine their impact on platelet function and thrombus formation and 4) elucidate the mechanistic action of EPCs on platelets and thrombus. Cultured on fibronectin in conditioned media, PBMCs differentiated, within ten days of culture, into EPCs, which were positive for Ulex-lectin and Ac-LDL (Acetylated Low Density Lipoprotein), and expressed progenitor markers (CD34, VEGFR2, vWF, and VE-Cadherin). These EPCs bind activated platelets through P-selectin-dependent mechanism, inhibited platelet activation, aggregation and adhesion, mainly via prostacyclin (PGI2) secretion. Indeed, this was associated with up-regulation of cyclooxygenase-2 (COX-2) and inducible nitric oxide (NO) synthase (iNOS). However, the effects on platelets were reversed by COX, but not by NO inhibition. Although EPCs bound platelets via platelet P-selectin, their predominant effects occurred via a paracrine secretion, implying PGI2. Nevertheless, a transitory link or brief contact between EPCs and platelets was required for this function to be fully realized. This was further depicted using a murine arterial thrombosis model in P-selectin deficient mice (P sel-/-) and their wild-type counterparts (WT). EPCs significantly impaired, in a concentration dependent-manner, collagen-induced whole blood platelet aggregation in WT mice; whereas in P-sel-/- mice, EPCs had no significant effect. Moreover, in murin model of arterial thrombosis, infusion of EPCs altered thrombus formation and significantly reduced the mass of thrombi generated in WT, but not in P-sel-/- mice. Furthermore, the number of EPCs recruited within the thrombi and along the vascular wall was visually reduced in P-sel-/- mice as compared to WT mice. In this project, we succeeded in adequately differentiating EPCs from PBMCs, we characterized the adhesive interaction between EPCs and platelets, and we addressed the impact of EPCs on platelet function and thrombus formation. Moreover, we identified PGI2 as the principal soluble factor secreted by cultured EPCs and responsible of their inhibitory effects on platelet function in vitro. In addition, using a murine model of arterial carotid injury in WT and P-sel-/- mice, we elucidate the mechanistic action of EPCs on platelet aggregation and thrombus formation, in vivo, and we highlighted the role of platelet P-selectin in this process. These findings add new insights into the biology of EPCs and reveal a potential role for EPCs in regulating platelet function, which in turn may limit thrombogenesis and maintain hemostasis at the sites of vascular injury.

Les efforts investis pour diminuer les risques de développer un infarctus du myocarde sont nombreux. Aujourd’hui les médecins prennent connaissance des divers facteurs de risque connus prédisposant aux syndromes coronariens aigus (SCA) dans le but de prendre en charge les patients «à risque» [1]. Bien que le suivi rigoureux et le contrôle de certains facteurs de risque modifiables aient permis une meilleure gestion des cas de SCA, les cas d’infarctus persistent de manière encore trop fréquente dans le monde. Puisque d’importantes études ont démontré que les SCA pouvaient survenir sans même la présence des facteurs de risque conventionnels [2, 3], les chercheurs se sont penchés sur un autre mécanisme potentiellement responsable de l’avènement des SCA : l’inflammation. L’inflammation joue un rôle prépondérant dans l’initiation, la progression et les complications de l’athérosclérose [4, 5] mais aussi dans les situations post-infarctus [6, 7]. Au cours des dernières années, le contrôle du processus inflammatoire est devenu une cible de choix dans la prévention et le traitement des SCA. Cependant, malgré les efforts investis, aucun de ces traitements ne s’est avéré pleinement efficace dans l’atteinte du but ultime visé par une diminution de l’inflammation : la diminution de la mortalité. Le complément est un système complexe reconnu principalement pour son rôle primordial dans l’immunité [2]. Cependant, lorsqu’il est activé de manière inappropriée ou excessive, il peut être à l’origine de nombreux dommages cellulaires caractéristiques de plusieurs pathologies inflammatoires dont font partie les complications de l’athérosclérose et des événements post-infarctus. Le travail effectué dans le cadre de mon doctorat vise à établir les rôles physiopathologiques du complément dans les interactions de l’axe thrombose-inflammation caractéristiques des SCA dans le but ultime d’identifier des cibles thérapeutiques permettant le développement de nouvelles approches pour la prévention et le traitement de ces pathologies. Les principaux résultats obtenus durant mon cursus suggèrent d’abord que la voie alterne du complément peut représenter une cible thérapeutique de choix dans les maladies coronariennes aiguës puisque l’activation terminale du complément semble y être principalement causée par l’activation du cette voie. De faibles niveaux sériques de MBL (mannan-binding lectin) et une activation terminale négligeable du complément caractérisent plutôt la maladie coronarienne stable. En comparant l’activité relative de chacune des voies du complément chez des cohortes de patients traités ou non par un anticorps spécifique à la protéine C5 du complément (pexelizumab), un second volet démontre quant à lui qu’une inhibition de l’activation du C5 n’a pas d’effet bénéfique majeur sur l’inhibition de la formation du complexe sC5b-9 ou sur les événements cliniques subséquents. Par conséquent, nous avons exploré, à l’aide d’un modèle in vitro, les raisons de l’inefficacité du traitement. Les résultats révèlent que le blocage du C5 avec le pexelizumab inhibe la production de l’anaphylatoxine pro-inflammatoire C5a et du complexe terminal du complément sans toutefois avoir d’effet sur l’apoptose des cellules endothéliales produites induite par le sérum des patients atteints de STEMI. Finalement, une autre section stipule que l’atorvastatine diminue l’activation du complément induite par les plaquettes sanguines chez des patients hypercholestérolémiques, mettant en évidence l’importance du rôle de cette statine dans la réduction des effets délétères de l’activation du système du complément médié par les plaquettes. Ensemble, l’étude du rôle spécifique des différentes voies d’activation du complément dans des contextes pathologiques variés, l’analyse des effets d’une inhibition spécifique de la protéine C5 du complément dans la progression des SCA et la mise en évidence des interactions entre l’activation du complément et les plaquettes activées ont contribué au développement d’une meilleure connaissance des rôles physiopathologiques du complément dans la progression de la maladie coronarienne. / Many efforts have been made in lowering the risk of myocardial infarction in the general population. Most clinicians are knowledgeable of the several identified risk factors leading to the development of acute coronary syndromes (ACS), and in turn, insure a better follow-up for “at risk” patients [1]. Despite the fact that intensive efforts in controlling modifiable risk factors have led to a better management of new cases of ACS, myocardial infarction and its deleterious consequences are still a world plague. Because it as been shown that ACS can occur without the presence of traditional risk factors [3, 4], researchers have been interested in modifying new ACS biological pathways such as inflammation. Inflammation plays a key role in the initiation, progression, and complications of atherosclerosis [5, 6], but also in post-infarction situations [7, 8]. In the past years, inflammation markers have become important targets for the prevention and treatment of ACS. Despite intensive efforts, none of the yet tested drug was found to be effective in decreasing mortality. The complement system is mainly known for its fundamental role in innate and adaptive immunity [2]. However, excessive activation of the complement can lead to a significant number of deleterious effects such as inflammation, apoptosis, necrosis and cell lysis. Earlier findings have shown that complement is extensively activated in atherosclerotic lesions, particularly in vulnerable and ruptured plaques. The objective of my doctoral project was to establish the pathophysiological roles of complement in the axis inflammation-thrombosis of ACS with the ultimate goal of identifying new therapeutic targets leading to the development of new drugs for the prevention and treatment of these diseases. The main results obtained first suggest that the complement alternative pathway represents a potential therapeutic target in acute coronary disease since terminal complement activation occurs mainly by this specific pathway. Low MBL levels (mannan-binding lectin) in serum and negligible terminal complement activation rather characterize stable coronary artery disease. By comparing the relative activity of each pathway of the complement in patients treated or not by an antibody specific to the C5 protein of the complement (pexelizumab), other results show that an inhibition of C5 activation does not have a major beneficial effect on the inhibition of the sC5b-9 complex expression or on the subsequent clinical events. Consequently, we explored, using an in vitro model of endothelial cells, the reasons of this inefficiency. This work reveals that C5 inhibition by pexelizumab inhibits the production of the pro-inflammatory anaphylatoxin C5a and of the terminal complement complex without, however, effecting endothelial cell apoptosis induced by the serum of patients with STEMI. Finally, another section stipulates that atorvastatin decreases platelet-induced complement activation in hypercholesterolemic patients, highlighting the importance of statins in the reduction of the deleterious effects of platelets-induced complement activation. All together, the study of the specific role of the various pathways of complement activation in different pathological contexts, the analysis of the effects of a specific inhibition of the C5 complement protein in the progression of ACS and the highlighting of the interactions between complement and platelet activation contribute to the development of a better knowledge of the pathophysiological roles of the complement system in ACS.

Drs Dandachli and Arzamendi contributed equally to this work. / L’adhésion, l’activation et l’agrégation des plaquettes représentent les étapes initiales dans la formation du thrombus aux sites des lésions vasculaires. Malgré l’utilisation des médicaments antiplaquettaires comme l’Aspirine, le Plavix et les inhibiteurs de la glycoprotéine IIb/IIIa (GPIIb/IIIa), l’incidence de thrombus dans la maladie coronarienne aigue reste élevée. Le dommage aux artères coronaires induit l’exposition du collagène de la matrice sous-endothéliale et sa liaison au facteur Von-Willebrand (vWF). Ceci contribue au recrutement et à l’adhésion des plaquettes via la liaison du domaine A1 du vWF à la GPIb des plaquettes. Nous avons postulé que l’inhibition de la liaison vWF/GPIb pourra représenter une stratégie efficace pour inhiber l’adhésion initiale des plaquettes et ainsi réduire la propagation du thrombus. L’objectif de notre étude était de déterminer le potentiel anti-thrombotique d’un inhibiteur du vWF. L’aptamère dirigé contre le domaine A1 du vWF (ARC1779) a été développé et fourni par la compagnie Archemix. Son effet et celui du Reopro (abciximab, inhibiteur de la GPIIb/IIIa comme témoin positif) ont été testés en utilisant du sang provenant de 5 volontaires sains et de 27 patients coronariens traités avec l’Aspirine (inhibiteur du cyclo-oxygénase ou COX) et le Plavix (anti-récepteur de l’adénosine diphosphate ou ADP), en accord avec le comité d’éthique de l’ICM. Les plaquettes ont été marquées avec l’Indium-111 afin de pouvoir quantifier leur adhésion dans le sang complet sur des surfaces artérielles porcines dénudées. L’adhésion des plaquettes a été effectuée dans des chambres de perfusion sous des forces de cisaillement de 6974/sec pendant 15 minutes à 37 °C. L’activation plaquettaire, suite à l’étude de l’adhésion, a été évaluée par l’expression de la P-sélectine et du vWF par la cytométrie en flux. L’effet de l’ARC1779 a été également déterminé sur l’agrégation plaquettaire, dans le sang complet par impédance, induite par l’acide arachidonique (AA), l’ADP, la Ristocétine et le peptide agoniste du récepteur de la thrombine-1 (TRAP-1). L’adhésion des plaquettes a été également observée par microscopie électronique à balayage. Dans un premier temps, nous avons trouvé que l’adhésion des plaquettes des volontaires sains à l’artère endommagée était élevée (80 x 106/cm2). Cette adhésion a été réduite significativement de plus que 90% par l’abciximab (100 nM) et d’une façon dose dépendante avec l’ARC1779 (25-250 nM). La perfusion du sang avec ou sans ARC1779 n’entraine pas une activation plaquettaire, telle que déterminée par l’expression de la P-sélectine et du vWF à la surface des plaquettes. Suite à ces résultats, l’étude avec le sang des patients a été poursuivie avec des doses de 25, 83 et 250 nM d’ARC1779. L’agrégation plaquettaire du sang des patients a été complètement inhibée en réponse à l’AA et à l’ADP, ce qui confirme que ces patients étaient bien traités avec l’Aspirine et le Plavix. L’adhésion des plaquettes aux surfaces artérielles endommagées a été réduite, chez les volontaires sains et les patients, d’une manière dépendante de la dose d’ARC1779, lorsqu’il était incubé avant la perfusion. Cependant, l’ARC1779 et aussi l’abciximab étaient sans effets significatifs sur l’adhésion plaquettaire, lorsqu’ils étaient ajoutés 10 minutes après la perfusion. L’inhibition de l’adhésion avec 25 nM d’ARC1779 était comparable à celle obtenue avec l’abciximab. Cependant, l’agrégation plaquettaire en réponse au TRAP-1 n’était pas affectée par l’ARC1779, alors qu’elle était complètement inhibée par l’abciximab. L’ARC1779 est un inhibiteur spécifique de la liaison du vWF au GPIb des plaquettes. Il inhibe l’adhésion plaquettaire aux surfaces artérielles endommagées sans affecter l’agrégation plaquettaire et confère une protection anti-thrombotique similaire à l’abciximab. L’ARC1779 pourra être considéré comme un nouvel antiplaquettaire qui possède des propriétés anti-thrombotiques plus intéressantes que l’abciximab. / Anti-platelet therapy in coronary artery disease (CAD) patients reduces recurrent athero-thrombosis, but at the cost of increased risk of bleeding. Because von Willebrand factor (vWF) functions predominantly in a high-shear environment, the vWF-specific aptamer, ARC1779 that blocks the binding of vWF A1-domain to platelet glycoprotein Ib, may deliver a site-specific anti-thrombotic effect while minimizing bleeding risk. We investigated the efficiency of ARC1779 on platelet activation, adhesion, and aggregation in CAD patients on double anti-platelet therapy. Blood from 27 patients taking aspirin and clopidogrel and 5 normal volunteers was labeled with 111In-autologous platelets and perfused over denuded porcine arteries at high shear rate for 15 minutes. Blood was treated with either 25, 83 and 250 nM ARC1779; 100nM abciximab or placebo, 5 min before (upstream therapy) or 10 min after (downstream therapy) beginning the perfusion. Under upstream, but not downstream therapy, platelet adhesion was significantly reduced by ARC1779 at 83 and 250 nM and by abciximab vs. placebo (4.8, 3.8 and 2.9 vs. 7.3 platelets x 106/cm2, p <0.05). In contrast to abciximab, ARC1779 did not significantly affect platelet aggregation in response to thrombin receptor activating peptide-1, arachidonic acid and adenosine diphosphate. In addition, ARC1779 was without any effect on P-selectin expression and platelet-leukocyte binding. In conclusion, ARC1779 has comparable anti-thrombotic efficacy to abciximab among CAD patients receiving aspirin and clopidogrel, but with lesser systemic effects on platelet activation and aggregation. These important proof-of-concept data form the framework for randomized clinical investigations of this novel anti-platelet therapy among CAD patients.

Les cellules endothéliales progénitrices («Endothelial Progenitor Cells», EPCs) sont des précurseurs endothéliaux qui possèdent un potentiel considérable dans la réparation et la régénération vasculaire. Dans le contexte des maladies cardiovasculaires, la compréhension du rôle des EPCs dans la régulation de la thrombogenèse et la réparation endothéliale est pertinente et nécessaire pour comprendre leur potentiel thérapeutique. Nous avons rapporté que les EPCs interagissent avec les plaquettes via la P-sélectine et inhibent l’adhésion, l’activation et l’agrégation des plaquettes ainsi que la formation de thrombus. Plus récemment, nous avons démontré que les EPCs expriment le récepteur inflammatoire CD40 et il est bien connu que les plaquettes constituent la source principale de la forme soluble de son agoniste le CD40L («soluble CD40 Ligand», sCD40L). Ainsi, nous avons émis l’hypothèse principale que l’axe CD40L/CD40 dans les EPCs influence leurs fonctions anti-thrombotique et pro-angiogénique. Pour vérifier cette hypothèse, nous avons réussi à générer des «early» et «late» EPCs à partir de cellules mononucléaires du sang périphérique («Peripheral Blood Mononuclear Cells», PBMCs) en culture. Nous avons mis en évidence l’existence de l’axe CD40L/CD40 dans ces EPCs en démontrant l’expression des protéines adaptatrices, nommées les facteurs associés au récepteur du facteur de nécrose tumorale («TNF Receptor Associated Factors», TRAFs). Dans une première étude, nous avons investigué l’effet du sCD40L sur la fonction des «early» EPCs dans l’agrégation plaquettaire. En effet, nous avons démontré que le sCD40L renverse leur effet inhibiteur sur l’agrégation plaquettaire, et ce sans avoir un effet significatif sur la sécrétion de prostacycline (PGI2) et d’oxyde nitrique («Nitric Oxide», NO) par ces cellules. De plus, aucun effet du sCD40L n’a été noté sur l’apoptose et la viabilité de ces cellules. Par contre, nous avons noté une augmentation importante du stress oxydatif dans les «early» EPCs suite à leur stimulation avec le sCD40L. L’inhibition du stress oxydatif renverse l’effet du sCD40L sur les «early» EPCs dans l’agrégation plaquettaire. Ces résultats pourraient expliquer, en partie, la fonction réduite des EPCs chez les individus présentant des niveaux élevés de sCD40L en circulation. Dans une deuxième étude, nous avons étudié l’effet de sCD40L dans la fonction des «early» EPCs en relation avec l’angiogenèse. Nous avons identifié, dans un premier temps,les métalloprotéinases de la matrice («Matrix Metalloproteinases», MMPs) qui sont sécrétées par ces cellules. Nous avons trouvé que les «early» EPCs relâchent principalement la MMP-9 et que cette relâche est augmentée par le sCD40L. Le sCD40L induit aussi la phosphorylation de la p38 MAPK qui contribue à augmenter la sécrétion de MMP-9. Des études fonctionnelles ont démontré que le prétraitement des «early» EPCs au sCD40L potentialise la réparation endothéliale des HUVECs. En conclusion, l’ensemble de nos travaux, dans le cadre de ce projet de doctorat, nous a permis d’élucider les mécanismes responsables de l’action du sCD40L sur les effets inhibiteur et angiogénique des «early» EPCs dans l’agrégation plaquettaire et l’angiogenèse, respectivement. Ces résultats ajoutent de nouvelles connaissances sur le rôle des EPCs et pourront constituer la base pour des études futures permettant de corréler les niveaux élevés du sCD40L circulant et l’incidence des maladies cardiovasculaires, particulièrement l’athérothrombose. / Endothelial progenitor cells (EPCs) are endothelial precursors which possess a considerable therapeutic potential in vascular repair and regeneration. In the context of cardiovascular diseases, the understanding of the role of EPCs in the regulation of thrombogenesis and endothelial repair is relevant and necessary to the understanding of their therapeutic potential. We have shown that EPCs interact with platelets via P-selectin and inhibit the adhesion, activation and aggregation of platelets as well as thrombus formation. Recently, we have shown that EPCs express the inflammatory receptor CD40 and it is well known that platelets are the main source of the soluble form of its agonist CD40L («soluble CD40 ligand», sCD40L). Hence, we have hypothesized that the CD40L/CD40 axis in EPCs influences the anti-thrombotic and pro-angiogenic functions of EPCs. To verify this hypothesis, we have successfully generated early and late EPCs from peripheral blood mononuclear cells in culture. We have demonstrated the existence of the CD40L/CD40 axis in EPCs by showing the expression of adaptor proteins, named tumor necrosis factor associated factors (TRAFs). In our first study, we investigated the effect of sCD40L on the function of early EPCs in platelet aggregation. Indeed, we have shown that sCD40L reverses their inhibitory effect on platelet aggregation without having an effect on prostacyclin (PGI2) and nitric oxide (NO) secretion by these cells. Moreover, no effect of sCD40L has been noted on the apoptosis and viability of these cells. However, we have shown a significant increase in oxidative stress in early EPCs following sCD40L stimulation. The inhibition of oxidative stress reverses the effect of sCD40L on early EPCs in platelet aggregation. These results could partially explain the decreased function of EPCs in individuals displaying higher levels of sCD40L in circulation. In our second study, we have studied the effect of sCD40L on the function of early EPCs in relation to angiogenesis. First, we have identified the matrix metalloproteinases (MMPs) which are secreted by these cells. We have found that early EPCs mainly release MMP-9 and that this release is increased by sCD40L. The sCD40L also induces the phosphorylation of p38 MAPK which contributes to increase the secretion of MMP-9. In functional studies, we have shown that pretreatment of early EPCs with sCD40L can potentialize HUVEC endothelial repair. In conclusion, our work in the context of this doctoral research project has allowed us to study the mechanisms involved in the role of sCD40L in the inhibitory and angiogenic function of early EPCs in platelet aggregation and angiogenesis, respectively. These results add new insights to the role of EPCs and could constitute the basis for future studies allowing for the correlation between high levels of sCD40L and the incidence of cardiovascular disease, particularly atherothrombosis.