Plasmin generation analysis in patients with bleeding disorder of unknown cause

Abstract

Bleeding disorder of unknown cause (BDUC) is a diagnosis of exclusion after evaluation of plasma coagulation and platelet function. Patients with BDUC (n = 375) recorded in the Vienna Bleeding Biobank were analyzed in comparison with healthy controls (HCs; n = 100) in this case-control study. Plasmin generation (PG) parameters were analyzed using calibrated fluorescence detection in citrated plasma. Turbidimetric plasma clot formation/lysis of 293 (78%) patients with BDUC and confocal microscopy of clots from representative patients with BDUC (n = 6) and HCs (n = 9) were assessed. In the PG analysis, patients with BDUC exhibited lower velocity and peak plasmin levels but a higher endogenous plasmin potential than HCs. Peak plasmin levels correlated with maximum clot absorbance but not with clot lysis time. Clot absorbance is an indicator of clot fiber density. Confocal microscopy analysis revealed a tendency towards thicker fibers in clots of patients with BDUC, which negatively correlated with peak plasmin (r = -0.561; P = .030). Peak plasmin correlated weakly with factor XIII, but not with other fibrinolytic factors (alpha2-antiplasmin, thrombin activatable fibrinolysis inhibitor, or plasminogen activator inhibitor 1) or bleeding severity. A model comprising fibrinogen and parameters of PG yielded high predictive power in discriminating between patients with BDUC and HCs across a fivefold stratified cross validation (80% of data; mean area under the curve [AUC], 0.847). The model generalized well to unseen data (20% of data; AUC, 0.856). Overall, patients with BDUC counterintuitively exhibited reduced peak plasmin levels, potentially related to altered clot structure.

Graphical abstract

Figure 1.

PG assay experiment setup and representative curves of PG in patients with BDUC and HCs. Summary of the setup of the PG assay. PG curves represent single measurements of 1 representative patient with BDUC and 1 representative HC. rtPA, recombinant tPA. Figure created with BioRender.com

Figure 2.

PG parameters in patients with BDUC and HCs. Procoagulant activity was initiated by the addition of exogenous TF (PPP-reagent LOW, ∼1pM TF, Diagnostica Stago) to recalcified plasma, and PG was triggered by the addition of exogenous tPA (0.62 μg/mL). PG was detected via fluorogenic substrate cleavage over time. Black lines represent the median and IQR. Significance (adjusted for age, sex, blood group O, and fibrinogen levels) is indicated as follows: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 3.

Association between peak plasmin level and maximum plasma clot absorbance and clot lysis time. After addition of calcium chloride (20 mM, final), phospholipids (4 μmol/L, final), TF (Innovin, 2 pmol/L final), and tPA (333 ng/mL final) to citrated plasma, turbidity was monitored by absorbance (OD) on a Thermo Scientific microplate reader. Clot lysis time was defined as the time from 50% of the peak OD during clot formation to 50% decrease in turbidity from peak OD.

Figure 4.

Fibrin network structure and its correlations with peak plasmin in patients with BDUC (n = 6) and HCs (n = 9). (A) Fibrin network structure of a representative patient with BDUC and an HC. The clot structure was analyzed using confocal microscopy (Zeiss LSM700) with a 63×/1.4 oil immersion objective. The settings included a pinhole aperture of 1 Airy unit (0.7 μm section), an image/frame size of 101.5 μm × 101.5 μm at a resolution of 1024 × 1024 pixels, and a pixel size of 0.1 μm. (B) Comparison of the fibrinogen levels, fiber density, and fiber diameters and the association of fiber density and diameter with the peak plasmin level in 6 representative patients with BDUC and 9 HCs. The 2 representative participants showed in panel A are marked as light red (BDUC) and light blue (HC). Significance is indicated as follows: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; ns, not significant.

Figure 5.

Association between peak plasmin level and bleeding severity. Correlation between PG potential and bleeding severity reflected by the Vicenza and ISTH BAT bleeding scores, as well as the number of bleeding manifestations in patients with BDUC. The pathologic Vicenza bleeding score cutoff is ≥5 for women and ≥3 for men; the pathologic ISTH BAT bleeding score cutoff is ≥5 for women aged 18 to 30 years, >6 for women aged 31 to 51 years, ≥7 for women aged ≥52 years, and ≥4 for men. BS, bleeding score; ns, not significant.

Figure 6.

ROC curve analysis (BDUC vs HC). ROC curve analysis for the tested model, including fibrinogen, PG velocity, peak plasmin, EPP, and ST time, in the validation data set (20% of data). FPR, false positive rate; TPR, true positive rate.