Comparison of Thrombin Generation of Sickle Cell Patients in Microparticle Rich and Microparticle Poor Plasma Using Thrombin Generation Assay (TGA).

The pathophysiology of sickle cell disease (SCD) is complex, with increasing evidence of abnormalities in nearly every component of hemostasis and a pronounced prothrombotic state. Many factors contribute to this prothrombotic state, including: 1) an increase in thrombin production and 2) an increase in circulating procoagulant microparticles. Currently little is known about procoagulant microparticles and their role in thrombin generation in SCD. We have therefore attempted to define the role of thrombin and procoagulant microparticles in SCD utilizing a thrombin generation assay (TGA) and to correlate the results with D-dimer levels, which are known to be abnormal in SCD. The use of TGA allows examination of lag phase, acceleration phase, peak thrombin, and endogenous thrombin potential (ETP).

The study included a total of 20 HbSS adult and pediatric patients (mean age=22 years, range 3–35) who were admitted for vaso-occlusive crisis. Patients were excluded if they had been admitted within the last 2 weeks, were on chronic transfusion, or were on antiplatelet or anticoagulant medications. Blood was drawn within 36 hours of admission (crisis) and again following discharge, at least 2 weeks later (steady state). Nine of the enrolled patients were followed from inpatient to an outpatient follow up visit. All plasma was promptly separated and frozen, stored at −80C, thawed once and subsequently tested within 4 hours. For each patient, D-dimer and TGA were performed on platelet poor plasma (PPP). In addition, following initial thaw of PPP, samples were spun for 30 minutes at 30,000g to remove microparticles and isolate particle free plasma (PFP). TGA was also performed on PFP from both inpatient and outpatient samples.

The mean D-dimer during inpatient crisis (5358 ± 2052 ng/ml) was significantly higher than during outpatient steady state (1256 ± 298, p=0.042). In comparing crisis and steady state by TGA, there was a significant increase in mean ETP (1267 ± 56 nM vs 923 ± 231, p=0.032) and mean acceleration phase (5.14 ± 0.34 min vs 14.21 ± 6.75, p=0.038). Both lag phase (p=0.066) and peak thrombin (p=0.057) were not statistically different between crisis and steady states. Analysis of PPP and PFP by TGA revealed that all phases including ETP (p=0.070) and peak thrombin (p=0.080) were not statistically different between microparticle rich and poor plasma. Linear regression models for inpatient D-dimer versus age were also performed and a significant decrease in D-dimer was seen with increasing age (r²=0.20, p=0.044). This decreasing trend with age was also seen with outpatient D-dimer, although it did not reach significance (r²=0.21, p=0.076). There was no significant trend seen in linear regression models performed for TGA phases versus age. Linear regression models were performed for D-dimer with each phase of TGA and no significant correlation or trend was seen.

Our initial results using TGA to evaluate the increase in hypercoagulability seen in patients with SCD during an acute crisis indicate that the expected increase is also seen with certain phases of the TGA, when compared to outpatient non-crisis state. ETP and acceleration phase were significantly elevated during crisis, indicating that the rate of thrombin production and total thrombin are abnormally increased during this time. Peak thrombin and lag phase also approached significance. In evaluating the significance of microparticles, we were unable to show a significant decrease in thrombin production in microparticle free plasma by use of the TGA. With inclusion of both pediatric and adult patients, it was also found that D-dimer significantly decreased with age, which is not consistent with other chronic disease states which exhibit a gradual increase with age. It is possible that a broader age range of patients may be needed to better determine this trend. Finally, comparison of D-dimer with each phase of TGA did not reveal a significant correlation. This may be due to other influencing factors on D-dimer which do not affect TGA. Thus, TGA appears to be a promising technique with which to assess thrombin generation and the hypercoagulable state in SCD; further studies of increased numbers of patients are needed to validate the use of TGA, and define the contribution of microparticles, in SCD.

Shah:Thrasher Foundation: Research Funding.