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Hyperhomocysteinemia due to folate deficiency. Impact on haemostasis and vascular biology

Liselotte Sabroe Ebbesen  


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Accepted by: Faculty of Health Sciences University of Aarhus
Defended on: September 19, 2003
Official opponents: Overlæge, dr.med. Ottar Kjell Nygaard, Bergen, Norway , Overlæge, dr.med. Søren Risom Kristensen, Odense Universitetshospital , Professor, overlæge, dr.med. Ebba Nexø, Århus Universitetshospital
Tutors: Overlæge, dr.med. Jørgen Ingerslev , Professor, overlæge, dr.med. Torben Falck Ørntoft , Overlæge, ph.d. Ulrik Baandrup

Published in the PhD Database: August 24, 2004


English abstract
This thesis is based on experiments conducted at the Institute of Experimental Clincial Reseach, University of Aarhus, Centre for Hemophilia & Thrombosis, and the Molecular Diagnostic Laboratory at the Department of Clinical Biochemistry, University Hospital of Aarhus (Skejby Sygehus) and at the Institute of Pathology, University Hospital of Aarhus (Kommunehospitalet) in the period between 1999 and 2003.
Hyperhomocysteinemia (HH) has been identified as a risk factor for thrombosis both in arterial thrombosis and venous thromboembolism in epidemiologic studies, although the pathogenesis is still largely unresolved.
An in vivo model of HH rats induced by folate deficiency was used in three series of experiments. Experiment 1 (n=2x30) and experiment 2 (n=2x16) consisted of control and HH animals, while experiment 3 (n=3x12) consisted of control, HH and treated animals (treated animals were HH animals which were subsequently fed a folate containing diet). Whole blood coagulation analyses have demonstrated that the whole blood coagulation is changed in a thrombogenic direction, characterized by increased velocity, increased firmness of the formed clot, while the initiation phase was prolonged. Gene expression analysis of blood cells (buffy coat cells), measuring the expression of about 8,800 genes, revealed a plausible explanation for the changes in the whole blood coagulation. Up-regulation of integrin beta 3, PECAM 1, platelet glycoprotein V and rap 1 B contribute to increased platelet activation and thereby contribute to explaining the increased velocity of whole blood coagulation and the increased firmness of the formed clot, as detected. Down-regulation of renal kallikrein contribute to explain the prolonged initiation phase of coagulation through a diminished resting thrombin potential and reduced activities of coagulation factors contributing in the contact activation pathway of coagulation. FXII:C, FX:C and FII:C were reduced in HH animals. In contrast, the tissue factor dependent pathway of coagulation, FVII:C, was unchanged. The down-regulation of the contact activating pathway contributes to an impaired fibrinolysis, which may also contribute to increased risk of thrombosis. The gene expression analysis also demonstrated an up-regulation of PAI-1 and slight down-regulation of t-PA, both contributing in reduced capacity of fibrinolysis. Treatment of HH rats with the folate containing diet reversed the coagulation changes, as estimated by whole blood coagulation analysis and single coagulation factor functions. Addition of increasing dosages of Integrilin (inhibitor of the platelet GpIIb/IIIa receptor) did not change the HH induced increased velocity and the HH induced increased firmness. Indication of increased numbers of GpIIb/IIIa receptors was found in HH animals due to saturation kinetics. Up-regulation of uPAR, L-, E- and P-selectins was found by gene expression analysis and may contribute to increased tethering, rolling of leukocytes and migration of these to the vessel wall. The influence of HH on atherosclerosis was demonstrated by a model of balloon induced neointima formation in the left common carotid artery. A reduced amount of neointima was found in HH animlals, indicating HH does not contribute to the development of atherosclerosis. These findings may be explained by a reduced methylation capacity related to the severeness of folate depletion in experiment 1 animals



Danish abstract
Denne Ph.D. afhandling bygger på arbejder udført i regi af Institut for Eksperimentel Klinisk Forskning, Århus Universitet; Koagulationslaboratoriet samt Molekylær Diagnostisk Laboratorium ved Klinisk Biokemisk afdeling, Skejby Sygehus samt Patologisk Institut, Århus Kommunehospital i perioden 1999 ¿ 2003.
Hyperhomocysteinæmi (HH) er via epidemiologiske studier kendt som risiko faktor for både arteriel og venøs trombose. Den patogenetiske sammenhæng mellem øget indhold af homocystein i blodet og den dertil forøgede blodproprisiko er stadig uafklaret.
En in vivo dyre eksperimentel model med HH rotter, induceret ved fodring med folinsyre depleteret foder, blev anvendt. Der blev lavet undersøgelser på 3 dyre serier bestående af 2x30 dyr (kontrol og HH dyr), 2x16 dyr (kontrol og HH dyr) samt 3x12 dyr (kontrol, HH dyr samt HH dyr som efterfølgende blev behandlet med folinsyreholdigt foder).
Resultaterne af disse studier har vist, at fuldblodskoagulationen er ændret i trombogen retning karakteriseret ved en øget hastighed i blodets størkningsevne, øget elasticitet af det dannede blod koagel, men en forlænget initieringsfase af koagulationen.
Gen chip ekspressions studier, målende ca. 8800 geners ekspression, af buffy coat celler, har givet en plausibel forklaring på ændringerne i fuldblodskoagulationen. Opregulering af integrin beta 3, PECAM 1, platelet glycoprotein V og Rap 1b kan via en øget thrombocyt aktivering forklare den øgede hastighed af koagulationen samt til dels medvirke til den øgede elasticitet af det dannede koagel. Nedregulering af renal kallikrein genet kan forklare den forlængede initieringsfase via en nedsat trombin generering samt en nedsat aktivitet af kontakt aktiveringsvejen i koagulationen. Måling af aktiviteten af enkelt koagulationsfaktorerne viste, at kontakt aktiveringsvejen inklusiv den fælles vej¿s faktorer, (FXII:C, FX:C og FII:C) var nedregulerede i HH dyr, hvorimod den eksterne koagulationsvej var upåvirket (FVII:C). Den nedregulerede kontaktaktiveringsvej kan medvirke til en hæmmet fibrinolyse, som medvirkende faktor til den øgede trombose tendens i HH individer. Nedregulering af tPA samt opregulering af PAI-1 blev endvidere fundet i gen expressionsanalyserne, og fundet medvirkende til en hæmmet fibrinolyse. Behandling af HH rotter med folinsyre holdigt foder kunne revertere koagulationsændringerne målt ved enkelt faktoranalyse samt ved fuldblodskoagulationsmåling. Tilsætning af Integrilin i stigende doser ændrede ikke på, at HH havde øget hastighed og øget elasticitet af det dannede clot i forhold til control og behandlede dyr, indikerende at opregulation af thrombocyt GpIIb/IIIa receptoren ikke alene kan forklare den øgede hastighed og den øgede elasticitet af det dannede koagel. Tilsætning af Integrilin viste endvidere tegn til øget antal GpIIb/IIIa receptorer i HH dyrenes thrombocytter som følge af mætningskinetik.
Opregulation af uPAR, L-, E- og P-selectiner blev fundet og kan være medvirkende til øget tiltrækning og migration af leukocytter til karvæggen.
HH¿s indvirkning på åreforkalkningsprocessen blev belyst i model med ballon induceret endothelskade i arteria carotis communis, hvor den efterfølgende neointima dannelse blev anvendt som effekt mål. Der fandtes en mindre mængde neointima i HH dyr end i kontrol dyr. Dette skyldes formentlig hypomethylering pga. den svære folinsyre mangel i experiment 1 dyrene.