Abstract
Coarse fibrin network clots display faster fibrinolysis than their fine network counterparts (
Collet et al, Arterioscler Thromb Vasc Biol 2000, 20: 1354–61
), and this is ostensibly of pathophysiologic relevance. Since relative to fine networks, coarse networks can be predicted to possess a relatively higher content of “B knob” (-”b” and/or -”a hole”) contacts, we investigated whether or not such contacts are more susceptible to plasmin than their A knob counterparts. Owing to much slower FpA than FpB release from Aα G16H fibrinogen, its clots acquire a high content of B knob contacts throughout polymerization (Galanakis et al, Bld Coag Fibrinol, 2007, 18: 731–737
) and were therefore used for this purpose. Plasminogen-free fibrinogen was obtained by Lys-Sepharose chromatography, and clots of isolates from one homophenotypic (HM) and one (unrelated) heterophenotypic (HT) Aα G16H proband were compared with normal controls. Plasmin digests of fibrinogen, monitored by PAGE-SDS, disclosed no differences from controls. Thrombin was added to fibrinogen (1–5 μM), pH 7.4, 8 mM CaCl2, with or without 20% afibrinogenemic plasma so as to achieve > 96% clottability. For lysis times, clots were suspended in 2 volumes of a lysis solution. For turbidity (340 nm) measurements the lysis solution was carefully layered on the cuvette clot top. To obtain clots of similar turbidities 0.8 to 1.2 thrombin U/ml were used. The lysis solution contained either 2 μM plasmin in buffer (37º) or 80 μM rtPA with or without 50 μM plasminogen. Selected clots were prepared in solutions containing either anti-β 15–42 IgG (Mab, 3 mols/mol fibrin) or the GlyHisProArgProOH peptide (50 mols/mol fibrin). Using fibrin (70 nM)-enhanced glu-plasminogen (250 nM) activation by rtPA (10 μM) and S2251 substrate (40 mM), no differences (405 nm) among control, HM, and HT fibrin could be shown in two separate sets of experiments. Clot lysis times (n=3) were 31% (HM) and 54% (HT) of controls, respectively. Turbidity measurements (n=3) also disclosed HM and HT clot lysis onset ~2 and 1.5 fold faster than controls, but similar lysis rates. Duplicate clots lacking >90% of intact αC (i.e. fraction I-9) yielded similar differences. Also assessed by turbidity, clots formed in Mab containing buffer lacked lysis differences from non-immune IgG controls. Using turbidity measurements of normal fibrin, lysis differences between coarse and fine clots were abolished when clots had been obtained from fibrinogen/GlyHisProArgProOH solutions (n=2). We conclude that B knob contacts are constitutively more susceptible to plasmin than their A knob counterparts. This property is attributable to faster release by plasmin of at least one of the two β 15–53 (B knob containing) fragments relative to release of either or both α 17–19 (A Knob) fragments from the DDE complex (Olexa et al, Biochemistry 1981, 20:6139–45
).Topics:
fibrin,
fibrinolysis,
cytokinesis,
fibrinogen,
plasmin,
plasminogen,
alteplase,
buffers,
immunoglobulin g,
mechlorethamine
Disclosures: No relevant conflicts of interest to declare.
Author notes
Corresponding author
2008, The American Society of Hematology
2008
