In:
Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 4060-4060
Abstract:
Sickle cell disease (SCD) is considered to be a hypercoagulable state with chronic activation of coagulation and an increased incidence of thrombotic events. However, there is no consensus on whether global assays of thrombin generation in platelet poor (PPP) or platelet rich (PRP) plasma display an increased thrombin generation potential in SCD (reviewed in Lim et al. Curr. Opin. Hematol. 2013). Based on our recent observation that RBC contribute to thrombin generation in whole blood (Whelihan et al. Blood 2012), we hypothesized that the cellular components in blood (notably RBCs) contribute to enhanced thrombin generation in SCD. 25 SCD patients in a non-crisis, “steady state” and 25 healthy race–matched controls were recruited for study. Whole blood thrombin generation, thromboelastography (TEG) and plasma-based thrombin generation assays (TGA) were performed on contiguous blood samples from each individual. Complete blood counts, as well as quantification of phosphatidylserine (PS) exposure by RBCs (assayed by Annexin V binding in flow cytometry) were also performed. Whole blood thrombin generation was monitored by serial α-thrombin-antithrombin (TAT) complex formation following activation by both the extrinsic pathway (5 pM recombinant tissue factor (TF)) and intrinsic (native contact activation) pathways. Results. With extrinsic activation, controls clotted on average at 3.9±0.5 min (mean±SD), generated TAT at a rate of 38.8±22.2 nM/min and reached a maximum level of 261±58nM. In patients with SCD, clot time (4.4±0.8 min, ƿ=0.04) and the maximum rate of TAT generation (41.5±19.4 nM/min, ƿ=0.65) were similar to that observed in controls while the maximum level (369±123 nM) of TAT generated was significantly higher (ƿ=0.0026). With contact activation, there was no significant difference in clot time (7.2±1.2 min vs 6.4±1.0 min, ƿ=0.07) or the maximum rate of TAT generation (52.9.5±26.9 nM/min vs 42.3 ± 12.4 nM/min, ƿ=0.57) for the SCD and control cohorts, respectively. However, similar to what was observed with extrinsic activation, SCD patients generated a significantly higher (ƿ=0.024) maximum level of TAT (352±116 nM) than controls (276±21 nM). Interestingly, the SCD cohort showed a strong positive correlation (ƿ= 〈 0.001) between the maximum levels of TAT generated with either stimulus (extrinsic vs intrinsic). We also examined extrinsic- and intrinsic-initiated clot formation using TEG. Re-calcification of citrated blood from SCD in the presence of TF and Corn Trypsin Inhibitor (CTI) displayed similar R (8.1±0.3 min vs 8.4±0.6 min, ƿ=0.7) and MA (68±2 mm vs 65±2 mm, ƿ=0.16) values to those exhibited by controls, respectively. The -angle however, was significantly (ƿ=0.035) higher in the SCD cohort (61±3°) compared to controls (53±3°). Contact activated blood also displayed no significant difference in R time between the two groups (14.5±3 min for SCD vs 16.5±4 min for controls). On the other hand, the MA and -angle were significantly increased (ƿ=0.04 and ƿ=0.012) in SCD (62±2 mm and 41±3°) compared to the controls (51±3 mm and 26±5°), suggesting a significant increase in the overall rate and extent of clot formation as a result of contact activation. To examine thrombin generation in the absence of cellular components, PPP was activated using an identical 5pM TF stimulus. In contrast to whole blood, TGA displayed no significant differences in the peak thrombin or maximum rate of thrombin generation between the two groups. Lag time was marginally longer and time to peak thrombin generation shorter in SCD (ƿ=0.036 and ƿ=0.03, respectively). Surprisingly, a weak negative correlation (ƿ=0.15) between RBC PS expression and total TAT was present in SCD patient samples. Conclusions: While plasma-based assays exhibit no major differences in thrombin generation potential between SCD and controls, corresponding whole blood samples showed a significant increase in overall thrombin generation and clotting potential, regardless of the initiating stimulus (extrinsic or intrinsic). Interestingly, there was no significant correlation between absolute cell counts (i.e. RBCs, Retics, Neuts, PLTs) or parameters and TAT levels. Collectively, these data make a strong case for cellular involvement in the hypercoagulability observed in SCD, but a direct role for RBC PS expression in net thrombin generation is not apparent. Disclosures No relevant conflicts of interest to declare.
Type of Medium:
Online Resource
ISSN:
0006-4971
,
1528-0020
DOI:
10.1182/blood.V124.21.4060.4060
Language:
English
Publisher:
American Society of Hematology
Publication Date:
2014
detail.hit.zdb_id:
1468538-3
detail.hit.zdb_id:
80069-7
