Plasma Microparticles Have Different Effects on Thrombin Generation in Platelet-Rich and Platelet-Poor Plasma.
Authors: Ivanov Gleb E. , Macartney N. , Stephens E. , Bowen N. , Lees S. , Collins P. 
Published: October 2019

Abstract
Circulating peripheral blood microparticles (MPs) of various cell origin have been described and measured in physiological and a wide range of pathological conditions. MPs are likely to play a role in coagulation either by exposure of procoagulant phospholipids or expression of tissue factor (TF), but the degree of this contribution to global haemostasis is not yet clear. We studied thrombin generation (TG) parameters (lag, peak thrombin, initial velocity (Vini) and maximal velocity (Vmax) in platelet-free (PFP) and platelet-rich plasma (PRP) of normal volunteers (n=9) in presence of corn trypsin inhibitor, using calibrated automated thrombography (CAT). MP-rich plasma was prepared by ultracentrifugation of PFP and reconstitution of pelleted MPs in a reduced volume of autologous MP-free plasma. TG was also measured in MP-depleted supernatant and platelet-free plasma (PFP) filtered through 0.1 μm filter. In MP-rich plasma, triggered with 5pM TF, with no addition of exogenous phospholipids, we found significantly increased peak TG, compared with PFP and supernatant (70.8 +/− 6.3 vs 51.4 +/− 5.0 vs 28.4 +/− 2.2 nM/L thrombin, p=0.024 and p<0.0001 respectively). MP-rich fraction also produced raised Vini (10.3 +/− 0.9 vs 5.0 +/− 0.6 thrombin nM/L/min, p=0.019) and Vmax (18.3 +/− 2.4 vs 6.8 +/− 1.0 thrombin nM/L/min, p=0.004) compared with MP-depleted supernatant. Ultracentrifugation resulted in reduction of peak TG almost by half, compared with native PFP. The augmenting effect of MP-rich plasma on thrombin peak and velocity was shown to be abolished by filtration. In our experiments removal of MPs by filtration of PFP did not affect routine clinical coagulation tests, but resulted in a significant reduction of peak TG (from 51.4 +/− 5.0 to 23.9 +/− 1.4 thrombin nM, p=0.0002), Vini (from 10.2 +/− 0.4 to 5.6 +/− 0.6, p=0.02) and Vmax (from 15.2 +/− 1.8 to 5.9 +/− 0.2, p=0.02) as compared to PFP. In order to assess the contribution of MPs to TG in presence of platelets, MP-rich plasma was added to various dilutions of PRP, using low concentration of TF (0.5pM) as a trigger. Interestingly, addition of MP-rich fraction only marginally augmented PRP with a platelet concentration of 150x109/L, but the enhancement of peak and velocity of TG became more pronounced when platelet concentration was reduced to 1.5x109/L. In a separate set of experiments, we studied TG in PRP in which MP concentration was reduced by dilution with filtered MP-free plasma as compared to PRP diluted with MP-containing PFP. Reduction in PRP MP content did not lead to a significant decrease in TG even at a low platelet concentration (1.5x109/L), when MP concentration was reduced to about 100 times below the physiological level. Our results indicate that MPs contained in PFP of normal donors significantly affect thrombin generation peak and velocity when compared to PFP in which MPs were eliminated by either ultracentifugation or filtration. The in vitro effect of an increased number of MPs on TG is less noticeable in presence of near-physiological platelet count, but contribution of MPs to TG at low platelet concentrations may potentially protect from bleeding in thrombocytopenic states and explain differences in bleeding phenotype. CAT measurement of TG in MP-rich vs MP-poor plasma could serve as a useful tool in assessing these differences.