Fibrin clot analysis
Plasma fibrin clot permeability was determined as described previously12. Briefly, 20 mM
calcium chloride and 1 U/mL human thrombin (Merck, Darmstadt, Germany)
were added to citrated plasma. Tubes containing the clots were connected
to a reservoir of a Tris-buffered saline. Its volume flowing through the
gels was measured within 60 min. A permeation coefficient reflected by
Ks, which indicates the average size of pores formed in
the fibrin network with low values indicating tightly packed fibrin
structure, was calculated from the equation:
Ks=Q×L×η/t×A×Δp, where Q is the flow rate in time t, L
is the length of a fibrin gel, η is the viscosity of liquid (in poise),
t is percolating time, A is the cross-sectional area (in
cm2), and Δp is a differential pressure (in
dyne/cm2).
CLT was measured using the method of Pieters M, et al13 . Briefly, citrated
plasma was mixed with 20 mM calcium chloride, 0.5 U/mL thrombin (Merck),
15 µM phospholipid vesicles (Rossix) and 18 ng/mL recombinant tissue
plasminogen activator (Actilyse 20 mg, Boerhinger Ingelheim, Germany).
The mixture was transferred to a microtiter plate and its turbidity was
measured at 405 nm at 37°C. CLT was defined as the time from the
midpoint of the clear-to-maximum-turbid transition, which represents
clot formation, to the midpoint of the maximum-turbid-to-clear
transition. The interassay coefficients of variation for lysis variables
were <8%.