Discordant fibrin formation in hemophilia

Abstract

Background: The conversion of fibrinogen to fibrin and its crosslinking to form a stable clot are key events in providing effective hemostasis.

Objectives: To evaluate the relationship of fibrinopeptide (FP) release and factor (F) XIII activation in whole blood from hemophiliacs.

Patients/methods: We investigated FPA and FPB release, FXIII activation and fibrin mass in tissue factor-initiated coagulation in whole blood from individuals with hemophilia and healthy subjects.

Results: In hemophiliacs, the rates of fibrin formation were delayed as compared to healthy individuals. FPA/FPB release and FXIII activation were decreased in hemophiliacs vs. healthy individuals: 5.4 +/- 0.7 microM min(-1) to 1.7 +/- 0.4 microM min(-1) (P = 0.003), 2.3 +/- 0.6 microM min(-1) to 0.5 +/- 0.1 microM min(-1) (P = 0.025), and 12.1 +/- 0.7 nM min(-1) to 3.1 +/- 0.7 nM min(-1) (P < 0.0005), respectively. More FPA was released in hemophiliacs (6.6 +/- 1.2 microM) prior to clot time (CT) than in healthy individuals (2.6 +/- 0.4 microM, P = 0.013), whereas FPB and activated FXIII levels remained comparable. FXIII activation, which normally coincides with FPA release, was delayed in hemophiliacs. At CT in normal blood, the FPA concentration was 2.6-fold higher than that of FPB (P = 0.003), whereas in hemophiliacs this ratio was increased to 6.6-fold (P = 0.001).

Conclusions: These data suggest that essential dynamic correlations exist between the presentations of fibrin I, fibrin II, and FXIIIa. The 'discordance' of fibrin formation in hemophiliacs results in clots that are more soluble than normal (43% lower mass; P = 0.02). The resulting poor physical clot strength probably plays a crucial role in the pathology of hemophilia.

Figure 1. Thrombin generation in hemophilia A and hemophilia B

Contact pathway inhibited whole blood coagulation is initiated to clot upon addition of 5 pM relipidated tissue factor in hemophilia A (n=6, ▲) and hemophilia B (n=4, ◇) individuals. Thrombin generation is determined as thrombin-antithrombin complex formation (TAT) and compared to a cumulative control of 35 healthy individuals (■)[12]. All data are shown as the mean±SEM. Clot times (CT) are indicated as an arrow below the x-axis.

Figure 2. Factor XIII activation

Quenched time points (0–20 minutes) were analyzed for FXIII activation in CTI whole blood from a healthy individual (panel A) and a hemophilia A individual (panel B). The polyclonal antibody used is specific for the unactivated FXIII, Mr=75,000 and activated FXIII species (FXIIIAa, Mr=71,000). Clot time (CT, arrow) in the healthy individual was 3.2 min and 9.8 minutes in the hemophilia individual. C) Quantitation of the appearance of the activated form of FXIII is compared between hemophilia (◇) and healthy (◆) whole blood. The mean clot time (CT) is shown below the graph with an arrow.

Figure 3. Comparison of fibrinopeptide release and fibrin mass in normal and hemophilia whole blood

Contact pathway inhibited whole blood was initiated to clot upon addition of 5 pM tissue factor and analyzed for: A) fibrinopeptide A (FPA) release as a function of time in healthy (■) and hemophilia whole blood (□); B) fibrinopeptide B (FPB) release as a function of time in healthy (▲) and hemophilia whole blood (△); C) fibrin mass (clot weights) at each time point in 1 mL of healthy (●) and hemophilia (○) whole blood. All data are shown as the mean±SEM. Clot time is shown below the x-axis as an arrow.

Figure 4. Correlative patterns of fibrinopeptide release and FXIII activation

Fibrinopeptide A (FPA, □) and fibrinopeptide B (FPB, △) release is compared with FXIII activation (◆) for the quenched time points (0–20 minutes) in contact pathway suppressed healthy (panel A) and hemophilia A and B blood (panel B). Data are shown as the mean±SEM with the clot time shown as an arrow below the x-axis. Panel C. Maximum values for the control data for FPA, FPB and FXIII was determined and each time point for controls (closed symbols) and hemophiliacs (open symbols) and was replotted as a percentage of the maximum values.