Hindered dissolution of fibrin formed under mechanical stress

Abstract

Background: Recent data indicate that stretching forces cause a dramatic decrease in clot volume accompanied by gross conformational changes of fibrin structure.

Objective: The present study attempts to characterize the lytic susceptibility of fibrin exposed to mechanical stress as a model for fibrin structures observed in vivo.

Methods and results: The relevance of stretched fibrin models was substantiated by scanning electron microscopic (SEM) evaluation of human thrombi removed during surgery, where surface fibrin fibers were observed to be oriented in the direction of shear forces, whereas interior fibers formed a random spatial meshwork. These structural variations were modeled in vitro with fibrin exposed to adjustable mechanical stress. After two- and three-fold longitudinal stretching (2 × S, 3 × S) the median fiber diameter and pore area in SEM images of fibrin decreased two- to three-fold. Application of tissue plasminogen activator (tPA) to the surface of model clots, which contained plasminogen, resulted in plasmin generation which was measured in the fluid phase. After 30-min activation 12.6 ± 0.46 pmol mm(-2) plasmin was released from the non-stretched clot (NS), 5.5 ± 1.11 pmol mm(-2) from 2 × S and 2.3 ± 0.36 pmol mm(-2) from 3 × S clot and this hampered plasmin generation was accompanied by decreased release of fibrin degradation products from stretched fibrins. Confocal microscopic images showed that a green fluorescent protein-fusion variant of tPA accumulated in the superficial layer of NS, but not in stretched fibrin.

Conclusion: Mechanical stress confers proteolytic resistance to fibrin, which is a result of impaired plasminogen activation coupled to lower plasmin sensitivity of the denser fibrin network.

Fig. 1

Fibrin structure on the surface and in the core of thrombi. (A) After thrombectomy thrombi were washed, fixed and dehydrated as detailed in Methods. Scanning electron microscopic (SEM) images were taken from the surface and transverse section of the same thrombus sample, scale bar = 2 μm. DG: a thrombus from popliteal artery, SJ: a thrombus from aorto-bifemoral by-pass Dacron graft. (B) Fiber diameter (upper graphs) and fibrin pore area (lower graphs) were measured from the SEM images of the DG thrombus shown in (A) using the algorithms described in Methods. The graphs present the probability density function (PDF) of the empiric distribution (black histogram) and the fitted theoretical distribution (gray curves). The numbers under the location of the observed fibrin structure show the median, as well as the bottom and the top quartile values (in brackets) of the fitted theoretical distributions. The parameters of the fitted distributions differ between the interior and exterior data sets at P< 0.01 level according to Kuiper’s test-based evaluation as described in Methods.

Fig. 2

Changes in fibrin network structure caused by mechanical stretching. (A) Scanning electron microscopic (SEM) images of fibrin clots prepared from 30 μmol L⁻¹ fibrinogen clotted with 30 nmol L⁻¹ thrombin. Fibrin samples were fixed with glutaraldehyde before stretching or after two- and three-fold stretching as indicated, scale bar = 2 μm. (B) Fiber diameter (upper graphs) and fibrin pore area (lower graphs) were measured from the SEM images illustrated in (A) using the algorithms described in Methods. The graphs present the probability density function (PDF) of the empiric distribution (black histogram) and the fitted theoretical distribution (gray curves). The numbers under the fibrin type show the median, as well as the bottom and the top quartile values (in brackets) of the fitted theoretical distributions. The parameters of the fitted distributions differ between any two data sets at P< 0.001 level according to Kuiper’s test-based evaluation as described in Methods.

Fig. 3

Plasminogen activation on the surface of fibrin. (A) Plasminogen (200 nmol L⁻¹) was added to fibrinogen before clotting performed as in Fig. 2. After stretching, the buffer around the retracted fibrin in the rubber tube was replaced with 1 nmol L⁻¹ tissue-type plasminogen activator (tPA) and after 30-min incubation at 37 °C the plasmin activity in the fluid phase was measured on 0.1 mmol L⁻¹ Spectrozyme-PL. Using a series of accurately known plasmin concentrations as a reference, the amount of generated plasmin is shown (normalized for unit surface area of the fibrin clots as described in Methods). (B) Plasminogen activation was initiated under the same conditions as in (A), but the tPA solution contained 0.2 mmol L⁻¹ Spectrozyme-PL. After 150-min incubation the fluid surrounding the fibrin was withdrawn and its volume and absorbance at 405 nm were measured. The amount of p-nitroaniline released from the plasmin substrate is shown (normalized for unit surface area of the fibrin clots as described in Methods). Data are presented as mean and SD (n= 6–9), the P-values refer to Kolmogorov–Smirnov test for the linked pairs of data sets (NS indicates P> 0.05).

Fig. 4

Release of soluble fibrin degradation products (FDP) from the surface of clots. (A) Fibrin containing 200 nmol L⁻¹ plasminogen was prepared as in Fig. 3 and fibrinolysis was initiated with 15 nmol L⁻¹ tissue-type plasminogen activator (tPA). (B) Plasminogen-free fibrin was prepared as in Fig. 2 and fibrinolysis was initiated with 1 μmol L⁻¹ plasmin. At 15-min intervals the fluid surrounding the fibrin was withdrawn and its ethanol-soluble FDP content was measured as described in Methods. The amount of released FDP is shown (normalized for unit surface area of the fibrin clots) for the 1^st (light gray bars) and 3^rd (dark gray bars) 15-min period of the lysis. Data are presented as mean and SD (n= 4) and the differences between the non-stretched and stretched fibrins are significant at the P <0.01 level according to the Kolmogorov–Smirnov test. (Inset A) After adjustment for protein concentration the samples in (A) were subjected to SDS electrophoresis on 12.5% polyacrylamide gel under non-reducing conditions and silver-stained. (Inset B) After withdrawal of the fluid phase after 45-min digestion the samples in B were fixed in glutaraldehyde and SEM images were taken as described in Methods; truncated fibers are indicated by white arrows, scale bar = 2 μm.

Fig. 5

Lysis of fibrin monitored with confocal laser microscopy. Fibrin clots were prepared from 30 μmol L⁻¹ fibrinogen containing 50 nmol L⁻¹ Alexa⁵⁴⁶-labeled fibrinogen and 200 nmol L⁻¹ plasminogen, clotted with 30 nmol L⁻¹ thrombin and stretched as indicated. Thereafter 60 nmol L⁻¹ tissue-type plasminogen activator (tPA)- green fluorescent protein (GFP) was added to fibrin and the fluid/fibrin interface was monitored with a confocal laser scanning microscope using dual fluorescent tracing: green channel for tPA and red channel for fibrin (the third panel in each image presents the overlay of the green and red channels), scale bar = 50 μm. The time after addition of tPA-GFP is indicated.