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Homozygous sickle-cell disease (HbSS; SCD) is associated with vaso-occlusive manifestations of varying severity [1]. Pain crises are generally believed to result from obstruction of the microvasculature secondary to adhesion of red blood cells (RBCs) and other cellular elements, and decreased deformability of hypoxia-induced sickled RBCs, with ensuing activation of coagulation and inflammatory pathways [2, 3]. One of the proposed contributors to thrombosis in SCD is the loss of normal phospholipid asymmetry on sickled RBCs due to the repeated process of hypoxia-induced sickling and unsickling [4]. The exposure of anionic phosphatidylserine (PS) on the outer membrane supports the assembly of enzymatic clotting reactions, leading to a sub-population of RBCs with a prothrombotic phenotype [5, 6]. We recently demonstrated that in healthy individuals, a subpopulation (∼0.5%) of PS-expressing RBCs contribute a significant fraction (∼40%) of the total thrombin generating potential of blood [7]. Prothrombin activation requires factor Xa to perform two proteolytic cleavages at Arg 271 and Arg 320 to release the active α-thrombin (αIIa) product [8]. Depending on the order of proteolysis, prothrombin activation can occur via two possible intermediates; meizothrombin (mIIa), an active enzyme; or prethrombin-2 (pre2), a non-enzymatic intermediate [9]. Unlike platelets, which support thrombin generation exclusively through the pre2 intermediate [10], this subpopulation of procoagulant RBCs supports prothrombin activation via the mIIa intermediate in a manner similar to that on synthetic phospholipids [7]. mIIa is of interest because it exhibits the anticoagulant functions of αIIa towards protein C activation, while lacking any significant activity towards procoagulant substrates like fibrinogen, FV and platelets [11]. Due to the markedly enhanced PS expression by sickled RBCs, we therefore hypothesized that the total αIIa generation potential, as well as mIIa production, would be significantly increased in the whole blood of SCD patients compared to healthy controls. To test this hypothesis, we recruited 7 outpatients (4 female and 3 male, age 28-51) with HbSS, in their non-crisis, “steady states,” and 6 healthy African-American controls (3 female and 3 male age 24-38). None of the patients or controls was being treated currently with anticoagulant or anti-platelet therapy, and all SCD patients were at least 3 months remote from red cell transfusion or hospital admission for pain crisis. We utilized our immunoassays that are capable of selective quantitation of αIIa-antithrombin (αTAT) and mIIa-antithrombin (mTAT) to measure the relative production of the two species produced in SCD vs. control individuals after tissue factor (TF)-initiated coagulation. To examine the thrombin generation potential of the HbSS cohort vs. that of the control group, whole blood was subjected to a 5 pM TF stimulus in the presence of 0.1 mg/mL of corn trypsin inhibitor. Quenched time course samples were subsequently analyzed using αTAT and mTAT ELISAs. Figure 1A displays the time course data for αTAT generation in the HbSS and control cohorts. The control cohort clotted on average at 3.8±0.2 min (mean±SEM), generated αTAT at a rate of 63±3.9 nM/min and reached a maximum αTAT level of 502±14 nM. Unexpectedly, clot time (3.98±0.2 min) and the maximum level (515±49 nM) of αTAT generated in the HbSS cohort were similar to that observed in the controls while the rate of αTAT generation at 74.1±7.9 nM/min was only 15% higher (P>0.05). Figure 1 αTAT and mTAT ELISA analyses of HbSS and Control groups. Figure 1B displays the time course data for mTAT generation. As reported previously [7], the observed mTAT levels were significantly less than αTAT due to the lability of the mIIa intermediate. The control cohort generated mTAT at an average maximum rate of 1.02±0.13 nM/min and reached an average maximum level of 6.5±0.52 nM. The HbSS cohort generated mTAT 33% faster (1.51±0.12 nM/min) and reached a maximum level of mTAT (10.1±0.44 nM) that was 36% higher than that observed in the control group. Thus, unlike the case with αTAT, a significant difference (mTAT max level P

Prothrombin activation can proceed through the intermediates meizothrombin or prethrombin-2. To assess the contributions that these 2 intermediates make to prothrombin activation in tissue factor (Tf)–activated blood, immunoassays were developed that measure the meizothrombin antithrombin (mTAT) and α-thrombin antithrombin (αTAT) complexes. We determined that Tf-activated blood produced both αTAT and mTAT. The presence of mTAT suggested that nonplatelet surfaces were contributing to approximately 35% of prothrombin activation. Corn trypsin inhibitor–treated blood was fractionated to yield red blood cells (RBCs), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and buffy coat. Compared with blood, PRP reconstituted with PPP to a physiologic platelet concentration showed a 2-fold prolongation in the initiation phase and a marked decrease in the rate and extent of αTAT formation. Only the addition of RBCs to PRP was capable of normalizing αTAT generation. FACS on glycophorin A–positive cells showed that approximately 0.6% of the RBC population expresses phosphatidylserine and binds prothrombinase (FITC Xa·factor Va). These data indicate that RBCs participate in thrombin generation in Tf-activated blood, producing a membrane that supports prothrombin activation through the meizothrombin pathway.