Fibrin derived from patients with chronic thromboembolic pulmonary hypertension is resistant to lysis

Abstract

Rationale: Although acute pulmonary embolism is epidemiologically associated with chronic thromboembolic pulmonary hypertension, the factors responsible for resistance to thrombolysis and a shift toward vascular remodeling within the pulmonary arteries of patients with chronic thromboembolic pulmonary hypertension are unknown.

Objective: Determine whether fibrin from patients is more resistant to plasmin-mediated lysis than fibrin from healthy control subjects.

Methods: Fibrinogen purified from patients and control subjects was used to prepare fibrin clots, which were subsequently digested with plasmin for various periods of time. The degradation of the alpha-, beta-, and gamma-chains of fibrin and the appearance of peptide fragments over time were assessed by polyacrylamide gel electrophoresis and Western blotting.

Measurements and main results: Densitometry of Coomassie-stained gels revealed significantly slower cleavage of all three polypeptide chains of fibrin from patients compared with control subjects (p < 0.05). In particular, release of N-terminal fragments from the beta-chain of fibrin, which promote cell signaling, cell migration, and angiogenesis, was retarded in patients compared with control subjects (p < 0.01).

Conclusions: The relative resistance of patient fibrin to plasmin-mediated lysis may be due to alterations in fibrin(ogen) structure affecting accessibility to plasmin cleavage sites. The persistence of structural motifs of fibrin, such as the beta-chain N-terminus, within the pulmonary vasculature could promote the transition from acute thromboemboli into chronic obstructive vascular scars.

Figure 1.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of purified fibrinogen. Samples of purified fibrinogen (1 μg) from a representative patient with chronic thromboembolic pulmonary hypertension (CTEPH) and a healthy control subject were reduced with dithiothreitol and loaded onto a 4–12% SDS polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie blue and analyzed for purity by quantitative densitometry. Results indicated purities of 93.4 and 94.5%, respectively, for these samples. Molecular mass markers and the Aα-, Bβ-, and γ-chains of fibrinogen are indicated on the left and right side of the figure, respectively.

Figure 2.

Fibrin from patients with CTEPH is resistant to plasmin-mediated lysis. (A) Fibrin polymers from a representative control subject and patient with CTEPH were analyzed at the indicated times after digestion with plasmin. Samples (equivalent to 1 μg of fibrin) were applied to each lane and subjected to SDS-PAGE followed by Coomassie staining of the gel. Molecular mass markers and the intact α-, β-, and γ-chains of fibrin are indicated on the right and left side of the figure, respectively. (B) Summary data on control subjects and patients with CTEPH (n = 10 each) from densitometric analysis of Coomassie-stained gels showing delayed degradation of the intact α-, β-, and γ-chains of CTEPH fibrin. Results are expressed as the mean percentage of the band intensity at t = 0. Error bars indicate standard error of mean. Group differences were evaluated by two-way analysis of variance.

Figure 3.

Persistence of the β-chain N-terminus after plasmin-mediated lysis of CTEPH fibrin. (A) Proteins from the gel shown in Figure 2 were electroblotted and probed with an antibody specific for the fibrin β-chain N-terminus. The degradation of the intact β-chain and appearance of a pair of closely migrating pair of β-chain N-terminal fragments are shown. (B) Summary data on control subjects and patients with CTEPH (n = 10 each) from densitometric analysis of Western blots showing persistence of the intact β-chain N-terminus as well as N-terminal β-chain fragments of CTEPH fibrin. Results are expressed as the mean percentage of the band intensity at t = 0. Error bars indicate standard error of mean. Group differences were evaluated by two-way analysis of variance.

Figure 4.

The balance between plasmin-mediated lysis and vascular remodeling of the fibrin polymer in pulmonary emboli. Plasmin (triangles) lyses the fibrin polymer by cleaving the peptide coils connecting the E and D regions. Plasmin also cleaves the signaling peptide at the N-termini of the two β-chains (gray knobs), which protrudes from the central “E” regions. If fibrinolysis is delayed, endothelial cells and fibroblasts may migrate in response to the presence of the N-termini of the β-chains, remodeling the fibrin polymer into a vascular scar.