Spiezia, Luca (2008) Studio della sintesi del fattore V della coagulazione attraverso il modello sperimentale delle colture megacariocitarie ed il modello clinico delle famiglie con deficit ereditario di fattore V. [Tesi di dottorato]
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Background. In humans, approximately 80% of the coagulation factor V (FV) circulates in plasma and the remainder is contained within the a-granules of platelets (Plts), in a partially activated form. The role of Plts FV is not clear. Its release at the site of injury, when Plts are activated, probably increases the local concentration of FV. This mechanism facilitates site-specific haemostasis. Plasma-derived FV is synthesized in the liver; however, the origin of platelet FV is still a matter of debate. Platelet-derived FV might originate from plasma through endocytosis or be synthesized by the precursor of platelets, the megakaryocytes (MKs). Although studies on isolated and cultured human MKs have provided evidence for both endogenous FV synthesis and secondary endocytosis of plasma FV, it is not clear in what proportion these two processes contribute to the platelet FV pool and what the mechanisms that regulate these processes are. To clarify the origin of Plts FV in humans and to evaluate the interactions between plasma and Plts FV we have developed an "experimental model" of human MKs cultures obtained from normal subjects and from severe (homozygous) FV deficient individuals and we studied a "clinical model" based on family bearing FV defect.
Materials and Methods. After informed consent we drew, from an antecubital vein, 30 ml of blood from 5 healthy subjects and from 5 severe (homozygous) FV deficient individuals. We isolated, from peripheral blood, the haematopoietic progenitor circulating cells that have been grown in a serum-free medium and have been induced to differentiate into the megakaryocytic lineage in the presence of thrombopoietin (TPO) plus interleukin 3 (IL3). With immunohistochemic and immunofluorescence techniques the presence of FV has been detected inside the cytoplasm of MKs at basal conditions and after supplementation of the colture medium with purified FV. As for the "clinical model", after informed consent, we drew, from an antecubital vein, 20 ml of peripheral blood from 20 subjects with heterozygous FV defect, from 55 relatives and 5 subjects with homozygous FV defect. In the group enrolled we determined FV plasma and intra-Plts levels, we have also identify FV Leiden mutation and HR2 aplotipe of FV gene and we have studied the possible influence of these genetic factors on plasma and intra-Plts FV levels.
Results. At day +5, +8, +10 from the seed, we obtained, in vitro, cellular elements with morphology analogous to MKs. The positive immunofluorescence test for CD41 confirmed our hypothesis that these cells were MKs. In the 5 healthy individuals we have demonstrated the presence of FV in the cytoplasm of MKs, while in the homozygous FV deficient individuals we have noted the absence of FV. Adding a note concentration of purified FV to the colture medium of Mks derived from homozygous FV defect subjects we observed the positive immunofluorescence test for FV. As for the "clinical model" we observed that subjects with heterozygous FV defect had intra-Plts FV levels similar to that of relatives without defect. Moreover in the relatives without defect, FV plasma levels correlated with intra-Plts.
Discussion. These findings are in agreement with previous observations and confirm that MKs, in healthy individuals, can synthesize FV. We have clarified conclusively that homozygous FV deficient individuals do not synthesize FV. For the first time we have demonstrated the capacity of Mks to endocyte FV when FV was added to the colture medium. In the "clinical model" we identified a trend of association between plasma and intra-Plts FV levels. The mild number od subjects, identified among our study population, bearing FV Leiden mutation and/or HR2 aplotipe of FV gene did not made any conclusion about the possible role of these genetic factor upon the regulation of plasma and intra-Plts FV levels.
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