Background: Erythrocyte disorders, such as polycythemia vera, sickle cell disease and malaria are often associated with an increased risk of venous thrombosis

Aims: To investigate the hypothesis that high erythrocyte numbers diminished protein C function and thereby increase the risk of thrombosis through impaired regulation of coagulation

Methods: The effects of activated protein C (APC) and thrombomodulin (TM) on calibrated thrombin generation (TG) were tested in whole blood (WB), platelet rich plasma (PRP), and platelet poor plasma (PPP), supplemented with various concentrations of erythrocytes. The effects of synthetic phospholipids supplementation on the activity of APC and TM in WB-TG were determined. Furthermore, the correlation between erythrocyte count and TM modulated WB-TG was tested in 119 healthy donors.

Results: APC and TM dose-dependently prolonged the lag time and reduced the endogenous thrombin potential until the Peak (ETPp) and thrombin Peak in both PRP-TG and WB-TG (Fig. 1A & 1B). Interestingly, the anticoagulant effects of APC and TM on ETPp and thrombin Peak were 3 times less effective in WB than in PRP. Washed erythrocytes added to PPP dose-dependently suppressed the inhibitory effect of APC on ETPp and thrombin Peak (Fig. 2A). The addition of synthetic phospholipids into WB dose-dependently enhanced the inhibitory effect of APC (Fig. 2B). Erythrocyte counts were inversely correlated with the inhibitory effect of TM on the ETPp (r = -0.41, p < 0.001) measured in WB of 119 healthy donors, while platelet or leukocyte counts were not associated with APC activity.

Conclusion(s): We have shown that high erythrocyte numbers are inversely associated with the anticoagulant function of both TM and APC. This dose-dependent inhibition of the protein C pathway may be an explanation for the high thrombosis risk in diseases, such as polycythemia vera, sickle cell disease and malaria.

Figure 1

The anticoagulant effect of activated protein C -APC- in thrombin generation -TG-. The effects of erythrocytes and synthetic phospholipids on the activity of activated protein C -APC-. TG in platelet rich plasma -panel A- and whole blood -panel B- was triggered with 1 pM TF, 16.7 mM CaCl2, 417 µM ZGGR-AMC, in the presence or absence of APC -0, 4, 8, 16, 32 or 64 nM-. The relative changes of TG parameters are shown. PRP and WB was from the same donor.

Figure 2

The effects of erythrocytes and synthetic phospholipids on the activity of activated protein C -APC-. Plasma was reconstituted with washed erythrocytes -0, 1750, 3500 or 5000*109/L-, and thrombin generation -TG- was triggered with 1 pM tissue factor -TF-, 16.7 mM CaCl2, 1 µM phospholipids -PL, 60%PC/20%PS/20%PE-, 417 µM ZGGR-AMC, in the presence or absence of 8 nM APC -panel A-. Of note, the reference erythrocyte count is 4000 to 5500*109/L. In panel B, whole blood TG was triggered with 1 pM TF, 16.7 mM CaCl2, different concentrations of PL -0, 1, 2 or 4 µM-, 417 µM ZGGR-AMC, in the presence or absence of 8 nM APC. The inhibition rate of ETPp and thrombin peak are shown.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/erythrocytes-impair-the-anticoagulant-function-of-the-protein-c-system-in-whole-blood-thrombin-generation/