Patho- physiological role of BDNF in fibrin clotting

Abstract

Circulating levels of Brain Derived Neurotrophic Factor (BDNF) are lower in coronary heart disease (CHD) than in healthy subjects and are associated with coronary events and mortality. However, the mechanism(s) underling this association is not fully understood. We hypothesize that BDNF may influence fibrin fiber structure and clot stability, favoring clot lysis and thrombus resolution. We showed that recombinant BDNF (rh-BDNF) influenced with clot formation in a concentration-dependent manner in both purified fibrinogen and plasma from healthy subjects. In particular, rh-BDNF reduced the density of fibrin fibers, the maximum clot firmness (MCF) and the maximum clot turbidity, and affected the lysis of clot. In addition, both thrombin and reptilase clotting time were prolonged by rh-BDNF, despite the amount of thrombin formed was greater. Intriguingly, CHD patients had lower levels of BDNF, greater fibrin fibers density, higher MCF than control subjects, and a negative correlation between BDNF and MCF was found. Of note, rh-BDNF markedly modified fibrin clot profile restoring physiological clot morphology in CHD plasma. In conclusion, we provide evidence that low levels of BDNF correlate with the formation of bigger thrombi (in vitro) and that this effect is mediated, at least partially, by the alteration of fibrin fibers formation.

Figure 1

rh-BDNF reduces fibrin fibers density in vitro. Purified fibrin/thrombin clots were formed with recombinant BDNF (rh-BDNF: 100–250 pg/ml) or with BSA (1 mg/ml; control: point 0). (a) Representative image of clots using Alexa Fluor 488–labeled fibrinogen (20X magnification), and (b) percentage of fibrin fibers versus control. Fibrin fibers were analyzed using Image J software. All samples were performed in triplicate. Data are expressed as mean ± SEM; n = 9 *p < 0.05 ***p < 0.005.

Figure 2

rh-BDNF influences fibrin density and polymerization, and in vitro clot dimension in healthy subjects’ plasma. Recombinant BDNF (rh-BDNF; 60, 120 pg/ml) or BSA (1 mg/ml: control) was added to plasma pools from healthy subjects before induction of coagulation with thrombin, then fibrin density and polymerization, and viscoelastic property of clot were analyzed. (ai) Visualization images (20X magnification) with Alexa Fluor 488–labeled method and (aii) quantization of fibrin fibers using Image J software. (b) Maximum Clot firmness (MCF) assessed by thromboelastographic analyses. All samples were performed in triplicate. (c) Representative kinetic and (d) maximum turbidity detected at A350 nm at 37 °C and monitored every 23 sec by spectrophotometric method. Data are expressed as mean ± SEM; horizontal bars indicate variation of BDNF levels measured in plasma pools analyzed; n = 5 different pools. **p < 0.01.

Figure 3

Effect of rh-BDNF on lysis of fibrin clot in healthy subjects’ plasma. Recombinant BDNF (rh-BDNF; 60, 120 pg/ml) or BSA (1 mg/ml: control) were added to five plasma pools from healthy subjects before induction of coagulation with thrombin and tPA, consequently polymerization of clot were analyzed. (a) Representative turbidity curves monitored by spectrophotometric method every 23 sec (A350 nm at 37 °C), (b) % of Lysis at 60 minutes and (c) Lysis time. All samples were performed in triplicate. Data are expressed as mean ± SEM; n = 5 different pools.

Figure 4

rh-BDNF influences thrombin (TCT) and e) reptilase (RCT) clotting time in healthy subjects’ plasma. Recombinant BDNF (rh-BDNF; 60, 120 pg/ml) or BSA (1 mg/ml: control) were added to plasma pools from healthy subjects, then (a) thrombin (TCT) and (b) reptilase (RCT) clotting time were measured. All samples were performed in triplicate. Data are expressed as mean ± SEM; horizontal bars indicate variation of BDNF levels measured in plasma pools analyzed; n = 5 different pools. *p < 0.05 and **p < 0.01.

Figure 5

Effect of BDNF on thrombin generation. Recombinant BDNF (rh-BDNF; 120 pg/ml) or BSA (1 mg/ml: control) was added to platelet-free plasma and thrombin formation was measured by CAT assay. Thrombin generated (a) by the concomitant activation of both intrinsic and extrinsic coagulation pathways and (b) only by the extrinsic pathway. (i) Representative curves of the kinetic of thrombin formation. (ii) Endogenous thrombin potential (ETP, area under the curve), (iii) Peak Height (maximum concentration of generated thrombin) and (iv) Velocity Index (velocity of thrombin formation) were used as main parameters describing thrombin generation. Data are expressed as mean ± SEM; horizontal bars indicate variation of BDNF levels measured in plasma pools analyzed n = 5 different pools. *p < 0.05 and **p < 0.01.

Figure 6

Low circulating BDNF levels are associated with higher density of fibrin fibers and greater MCF in CHD patients. Plasma pools of two or three CHD patients or healthy subjects (Control) were obtained according to similar BDNF levels (±20 pg/ml): (a) BDNF levels, (b) fibrin fibers density, and (c) Maximum Clot firmness (MCF) have been analysed by ELISA kit, Alexa Fluor 488–labeled method (20X magnification) and thromboelastographic analyses, respectively. Correlation between BDNF concentrations and (d) MCF. All samples were performed in triplicate, and representative images are shown. Data are expressed as mean ± SEM. n = 12 and 9 pools of CHD and healthy subjects, respectively.

Figure 7

rh-BDNF reduces fibrin clot profile of CHD patients in vitro. Recombinant BDNF (rh-BDNF; 5, 25, 135 and 270 pg/ml) or BSA (1 mg/ml: control) was added to plasma from CHD patients before induction of coagulation with thrombin, and fibrin fibers were (a) visualized with Alexa Fluor 488–labeled (20X magnification) and (b) quantified using Image J software. All samples were performed in triplicate and representative images are shown. Data are expressed as mean ± SEM; horizontal bars indicate variation of BDNF levels measured in plasma pools analyzed. n = 12 plasma sample from CHD patients with BDNF < 100 pg/ml. *p < 0.05, **p < 0.01 and ***p < 0.005.