Figure 1.
The formation of the second peak in PRP TGCs mediated by PGE1, MeS-AMP, or DMSO.
Thrombin generation in the PRP of a healthy volunteer (the final platelet concentration in the experimental wells was equal to 100·103 µl−1) was triggered by 2 pM of TF in the presence of different concentrations of PGE1 (A), MeS-AMP (B), or DMSO (C).
Figure 2.
Influence of PGE1 on TG in PRP.
A: TGCs in PRP supplemented with 1.6% DMSO in the presence of different PGE1 concentrations. PRP without PGE1 was used as a control. B, C: The mean values of areas (B) and amplitudes (C) of the first peak in PRP, PRP with 830 nM PGE1 addition, and PPP. Coagulation was activated with 2 pM of TF. The platelet concentration was 100·103 platelets per µl. Mean values and SD are presented; n = 18, 8, and 7 for PRP, PRP+PGE1, and PPP, correspondingly. Student's t-test with P values equal to 0.05 and 0.01 was used to obtain statistics. The difference is not significant in all the bars where P-value is not presented.
Figure 3.
Kinetics of platelet activation in the presence of PGE1, DMSO, and MeS-AMP.
A suspension of gel-filtered platelets (100·103 cells per µl) labeled with annexin V-RPE and anti-CD62P-FITC was activated with human α-thrombin solution (35 nM final concentration) containing PGE1 (A,B), DMSO (C,D), or MeS-AMP (E,F). The mean RPE (A,C,E) fluorescence and the percentage of CD62P-positive platelets (B,D,F) after different incubation times is presented.
Figure 4.
Increasing platelet concentration causes the integration of two peaks into one.
A: Curves of thrombin generation obtained in a typical experiment with the plasma of a healthy donor containing 1.6% of DMSO and different concentrations of platelets. Coagulation was triggered with 2 pM of TF. B: The dependence of the mean ETP values and SD on platelet concentration for healthy donors (n – number of donors; * - difference between PRP and PPP is significant (Student's t-test, P<0.01)).
Figure 5.
Analysis of thrombin generation peak parameters.
For approximation of TGCs with one peak, a type I Gumbel's distribution function was used (A). The TGCs with two peaks were described by superposition of two extreme distribution Gumbel's functions (B). In some cases, it was impossible to determine how many peaks the TGC contained (C). Fitting was performed until the thrombin concentration began to be comparable with the experimental error (see Fig. S1 in File S1).
Table 1.
Criteria for determination of the quantity of TGC peaks*.
Figure 6.
Contribution of phospholipid surfaces to the first and the second thrombin generation peaks.
Plasma phospholipids, α-granules and preactivated platelets form the first peak. PS expression on the platelets' surface can mediate the second one formation.