Procoagulant and profibrinolytic activities of cryopreserved human monocytes.
The purpose of this study was to compare the ability of fresh and cryopreserved mononuclear cells to generate thrombin, induce fibrin formation and finally resolve the fibrin formed, when exposed to plasma. Peripheral blood mononuclear cells (PBM) from 4 donors were collected by gradient centrifugation on Lymfoprep, and cryopreserved in fetal calf serum and 10% dimethyl sulfoxide. Viability was tested by exclusion of trypan blue, as well as green/red fluorescence of fluorescein-diacetate and ethidium bromide (FDA/EB). Fresh and frozen-thawed cells were seeded, stimulated with lipopolysaccharide(LPS), and exposed to a standard heparinized overlay plasma. Plasma was harvested at intervals (0-7 days). Thrombin generation and fibrin formation were measured by quantification of prothrombin fragment (F1 + 2) and fibrinopeptide A (FPA) and the fibrinolytic capacity of the cells as the amount of fibrin (ogen) degradation products (FbDP and FgDP). Recovery of cells after thawing was about 80%, and the viability of fresh and cryopreserved PBM was > 95%. Compared to fresh, frozen cells fully retained their capability of Tissue Factor synthesis, leading to prothombinase activity (F1 + 2) and fibrin formation (FPA). In contrast, the fibrinolytic capacity of frozen-thawed cells were significantly reduced. As expected there were significant variations between the donors in all the parameters measured. We conclude that cryopreservation of human blood mononuclear cells is possible with maintainance of the potential of the cells to mediate coagulation in plasma upon LPS stimulation, whereas the fibrin resolving capacity apparently is reduced by the preservation procedure.
Osnes
L T
LT
Dept. of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norway.
Westvik
A B
AB
Kierulf
P
P
D001777:Blood Coagulation; D001793:Blood Preservation; D015925:Cryopreservation; D005337:Fibrin / Q000096:biosynthesis; D005338:Fibrin Fibrinogen Degradation Products / Q000032:analysis; D005342:Fibrinolysis; D005344:Fibrinopeptide A / Q000032:analysis; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000187:drug effects / Q000502:physiology*; D010446:Peptide Fragments / Q000032:analysis; D011516:Prothrombin / Q000032:analysis; D013917:Thrombin / Q000096:biosynthesis
D005338:Fibrin Fibrinogen Degradation Products; D008070:Lipopolysaccharides; D010446:Peptide Fragments; C045879:prothrombin fragment 1.2; D005344:Fibrinopeptide A; D011516:Prothrombin; D005337:Fibrin; D013917:Thrombin