The effects of in vitro phosphorylation and dephosphorylation on the thrombin-induced gelation and plasmin degradation of fibrinogen.
The alpha-chain of human fibrinogen was found to be phosphorylated in EDTA-anticoagulated whole blood when trace amounts of (gamma-32P)ATP and 7.5 mM Mg2+ ions were added. Fibrinogen was not phosphorylated if only the ATP was added. The thrombin-induced gelation of fibrinogen phosphorylated by protein kinase A, casein kinase I or II was studied spectrophotomerically. It was found that phosphorylation by protein kinase A caused the formation of thinner fibrin fibres, whereas phosphorylation by casein kinase II resulted in fibres slightly thicker than those of the control fibrinogen (equivalent to a 20% increase in the control fibrinogen concentration). Phosphorylation with casein kinase I did not significantly affect the fibrin fibre thickness. Dephosphorylation by alkaline phosphatase removed 50% of the 32P-labelled phosphate from protein kinase A-phosphorylated fibrinogen and over 90% from the casein kinase I or II-phosphorylated fibrinogens. This dephosphorylation resulted in a general increase in fibre thickness in the gelation assay in all samples, although the fibres of the phosphorylated fibrinogens remained substantially thinner than the dephosphorylated control fibrinogen. Plasmin digestion of the phosphorylated fibrinogens showed that they were more resistant to cleavage, being cleaved at only 30% to 70% of the rate of control fibrinogen and that this resistance was unaltered by dephosphorylation, in contrast to the thrombin gelation experiments.
Martin
S C
SC
Department of Medical and Physiological Chemistry, Uppsala University, Sweden.
Forsberg
P O
PO
Eriksson
S D
SD
D000469:Alkaline Phosphatase / Q000494:pharmacology; D001777:Blood Coagulation / Q000502:physiology*; D047388:Casein Kinases; D005338:Fibrin Fibrinogen Degradation Products / Q000032:analysis; D005340:Fibrinogen / Q000187:drug effects*; D005341:Fibrinolysin / Q000494:pharmacology; D005342:Fibrinolysis / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D010766:Phosphorylation; D011494:Protein Kinases / Q000494:pharmacology*; D013918:Thrombin Time
D005338:Fibrin Fibrinogen Degradation Products; D005340:Fibrinogen; D011494:Protein Kinases; D047388:Casein Kinases; D000469:Alkaline Phosphatase; D005341:Fibrinolysin