Plasminogen on the surfaces of fibrin clots prevents adhesion of leukocytes and platelets

Abstract

Background and objectives: Although leukocytes and platelets adhere to fibrin with alacrity in vitro, these cells do not readily accumulate on the surfaces of fibrin clots in vivo. The difference in the capacity of blood cell integrins to adhere to fibrin in vivo and in vitro is striking and implies the existence of a physiologic antiadhesive mechanism. The surfaces of fibrin clots in the circulation are continually exposed to plasma proteins, several of which can bind fibrin and influence cell adhesion. Recently, we have demonstrated that adsorption of soluble fibrinogen on the surface of a fibrin clot results in its deposition as a soft multilayer matrix, which prevents attachment of blood cells. In the present study, we demonstrate that another plasma protein, plasminogen, which is known to accumulate in the superficial layer of fibrin, exerts an antiadhesive effect.

Results: After being coated with plasminogen, the surfaces of fibrin clots became essentially non-adhesive for U937 monocytic cells, blood monocytes, and platelets. The data revealed that activation of fibrin-bound plasminogen by the plasminogen-activating system assembled on adherent cells resulted in the generation of plasmin, which decomposed the superficial fibrin layer, resulting in cell detachment under flow. The surfaces generated after the initial cell adhesion remained non-adhesive for subsequent attachment of leukocytes and platelets.

Conclusion: We propose that the limited degradation of fibrin by plasmin generated by adherent cells loosens the fibers on the clot surface, producing a mechanically unstable substrate that is unable to support firm integrin-mediated cell adhesion.

Fig. 1

Effect of plasmin(ogen) on adhesion of U937 cells to fibrin clots. (A) U937 cells (5 × 10⁵ mL⁻¹) were first preincubated with 200 μg mL⁻¹ plasminogen (+, Pg) for 15 min at 22 °C without or with tranexamic acid (TA, 5.0 mM) or phenylalanyl-L-prolyl-L-arginine chlormethyl ketone (PPACK) (0.5 mM), and aliquots of cells were added to intact fibrin gels. Cell adhesion was determined as described in Materials and methods. A hatched bar shows adhesion of control untreated U937 cells to intact fibrin gels. (B) Plasminogen (+, 200 μg mL⁻¹) without or with tranexamic acid (5.0 mM) was preincubated with fibrin gels for 15 min at 22 °C. The liquid above the gels was aspirated, and the gels were washed with Hank’s balanced salt solution (HBSS); this was followed by the addition of a suspension of calcein-labeled U937 cells in the absence or presence of PPACK (0.5 mM). (C) Five micrograms per milliliter of active plasmin (Pm) or PPACK-inactivated plasmin (Pm inact.) was preincubated with fibrin gels for 15 min, the liquid above the gels was aspirated, the gels were washed, and adhesion of calcein-labeled U937 cells was examined. The lack of proteolytic activity in PPACK-inactivated plasmin was confirmed by using the plasmin-specific substrate S-2251. A dotted line, which shows control adhesion of U937 cells in (A), also applies to control cell adhesion in (B) and (C). The data shown are the average of six measurements at each experimental data point and are representative of four experiments.

Fig. 2

Effect of different concentrations of plasminogen (Pg) on adhesion of U937 cells to fibrin gel. (A) Fibrin gels were preincubated with different concentrations of plasminogen for 15 min, washed with Hank’s balanced salt solution (HBSS) to remove non-bound protein, and used as substrates for adhesion of calcein-labeled U937 cells. Inset: plasminogen (200 μg mL⁻¹) was added to fibrin gels in the presence of different concentrations of tranexamic acid, gels were washed, and cell adhesion was determined. (B) Correlation between the amount of plasminogen bound to the fibrin gel and cell adhesion. Solutions of ¹²⁵I-labeled plasminogen (100 μL, 50 μg mL⁻¹, 800 cpm μg⁻¹) were incubated with fibrin gels formed in the wells of 96-well microtiter plates for the indicated times. The wells were washed with HBSS, and calcein-labeled U937 cells were added. Bound plasminogen (closed circles) and cell adhesion (open circles) were determined as described in Materials and methods.

Fig. 3

Relationship between adhesion of platelets and U937 cells to plasminogen-coated fibrin gels and their ability to activate plasminogen. Suspensions of platelets (5 × 10⁵ per 0.1 mL) (A) or U937 cells (5 × 10⁴ per 0.1 mL) (B) were added to untreated (−) or plasminogen (Pg)-coated fibrin gels, and cell adhesion was determined as described in Materials and methods. Gray bars show the fluorescence of adherent cells (left ordinate). Hatched bars (right ordinate) show the plasmin activity of platelets and U937 cells incubated with or without plasminogen. Aliquots of cells (10⁶ per 0.2 mL) were incubated for 60 min without or with Pg (10 μg mL⁻¹), the samples were centrifuged, and the generated plasmin was determined by measuring the optical density (OD) at 405 nm with the plasmin substrate S-2251. In additional experiments, suspended platelets were preincubated with tissue-type plasminogen activator (t-PA) (0.1 μg mL⁻¹) and then added to plasminogen-coated fibrin gels (Pg + t-PA). Data are means of triplicate determinations, and error bars represent standard errors. The result shown is representative of three independent experiments.

Fig. 4

Inhibition of adhesion of peripheral blood monocytes to fibrin clots as a consequence of plasmin generation. (A) Suspensions of calcein-labeled monocytes were preincubated for 15 min with tissue-type plasminogen activator (t-PA) (0.1 μg mL⁻¹), diluted five-fold with Hank’s balanced salt solution, and centrifuged. Cells were resuspended in the same buffer containing plasminogen (Pg) (100 μg mL⁻¹), and aliquots of cells (5 × 10⁴ per 0.1 mL) were added to fibrin gels (Pg + t-PA). Control cells without (−) or in the presence of plasminogen are shown. Cell adhesion was determined as described in Materials and methods. Gray bars show the fluorescence of adherent cells (left ordinate). Hatched bars (right ordinate) show the plasmin activity of monocytes incubated with or without plasminogen. Plasmin activity was determined as in Fig. 3. (B) Plasmin activity associated with fibrin gels after cell adhesion is shown. Monocytes (treated with t-PA or not) were mixed with plasminogen as described in (A), allowed to adhere to fibrin clots for 30 min, and then removed by washing. The activity of fibrin-bound plasmin was determined by measuring the optical density (OD) at 405 nm with the plasmin substrate S-2251.

Fig. 5

Effect of plasminogen on adhesion of U937 cells to fibrin gel under flow conditions. The fibrin gels containing the inner capillary were prepared in glass tubes as described in Materials and methods. U937 cells were resuspended at 5 × 10⁵ mL⁻¹ (A) or 10⁷ mL⁻¹ (B) in Hank’s balanced salt solution (HBSS)–bovine serum albumin without (control) or with 50 μg mL⁻¹ plasminogen and perfused through fibrin tubes for 5 min at a flow rate of 0.1 mL min⁻¹ (A) and for 10 min at a flow rate of 1.5 mL min⁻¹ (B). After these times, the tubes were flushed for an additional 9 min at the respective flow rates to remove non-adherent cells (open arrow). Adherent cells were viewed with a Leica DMIL microscope with a × 10 objective and a Leica DFC 340 FX digital camera (A). To quantify cell adhesion (B), calcein-labeled U937 cells were perfused through fibrin tubes at 1.5 mL min⁻¹ in the absence (control) or presence of plasminogen (Pg). The fluorescence of adherent cells was determined using a CytoFluor plate reader. The results shown are from two separate experiments performed with quadruplicate measurements for each flow tube.

Fig. 6

Initial adhesion of U937 cells to plasminogen-coated gels renders them non-adhesive for subsequent cell adhesion. (A) To produce non-adhesive fibrin gels, non-labeled U937 cells were allowed to adhere to plasminogen-treated fibrin for 30 min at 37 °C (first adhesion). The cells were completely removed by washing gels with phosphate-buffered saline (PBS), and the gels were used for the second round of adhesion. Calcein-labeled U937 cells (left panel) and platelets (right panel) in the absence (−) or in the presence of phenylalanyl-L-prolyl-L-arginine chlormethyl ketone (PPACK, 0.2 mM) and tranexamic acid (TA, 5.0 mM) were added to the postadhesion fibrin gels. After 30 min at 37 °C, cell adhesion was measured (gray bars). Adhesion of control U937 cells and platelets to intact fibrin gels untreated with plasminogen was assigned a value of 100% (closed bars). (B) Restoration of the adhesive properties of fibrin gels. The surfaces of fibrin gels were first rendered non-adhesive as in (A). Different concentrations of sodium dodecylsulfate (SDS) (0–0.08%) were then added to the gels for 20 min at 22 °C. Gels were washed extensively with PBS, and the second round of adhesion of calcein-labeled U937 cells in the presence of PPACK was performed. When non-adhesive surfaces were cleaned with increasing concentrations of SDS, adhesion was gradually restored to the level observed with untreated fibrin gels (shown as a dashed line). As a control, rinsing the intact fibrin gels with 0.08% SDS did not alter cell adhesion.