Contribution of Platelets and Erythrocytes to Thrombin Generation in Patients with β-Thalassemia.

Abstract

Patients with β-thalassemia show an abnormally high proportion of red blood cells (RBCs) with exposed phosphatidyl serine (PS) molecules on the RBC membrane, which may contribute to thrombophilia in these patients (1). We have reported previously (2) that thromboxane A2 and free-arachidonate—substances released from activated platelets—induce PS exposure in a subpopulation of normal RBCs. The aims of the present study were first, to evaluate the presence of activated circulating platelets in patients with β-thalassemia, and second, to determine tissue factor (TF)-initiated thrombin generation in plasma and the influence of RBCs, platelets and their cooperation on this process in normal subjects and patients with β-thalassemia. Thrombin generation was assessed according to Hemker et al. (3) in 12 patients with β-thalassemia diagnosed by molecular biology, age 5–33 years and in 18 normal subjects. Two patients had thalassemia major (T-M) requiring blood transfusion every 3 weeks and the analysis were performed just prior transfusion. Two had thalassemia intermedia (T-I) with esplenectomy without transfusion requirements and eight had thalassemia minor (T-m). Thrombin generation was evaluated in platelet-poor plasma (PP), platelet-rich plasma (PRP), RBCs in the plasma (P-RBC) and in the presence of both cell types (PRP+RBC). The presence of activated platelets in circulation was evaluated in whole blood by flow cytometry. We monitored P-selectin (CD-62) and the activated αIIbβ3 receptor, defined as PAC-1 binding. The percentage of platelets exposing activation markers CD-62 (mean± SEM, 0.72± 0.17 vs. 4.03± 1.39) and PAC-1(0.75± 0.14 vs. 12.26± 6.47) were significantly higher in patients than in control subjects (P < 0.05). In normal subjects, the presence of erythrocytes in plasma (P-RBC) triggered a quicker thrombin generation and a significant elevation in the thrombin peak (32 ± 5 nM in PP vs. 86 ± 16 nM in P-RBC; P < 0.05), in the absence of cell lysis. These effects of RBCs were markedly higher in the patients with β-thalassemia, who had 2.5-fold higher thrombin generation at 3 min and higher peak thrombin levels (86 ± 16 nM in controls vs. 130 ± 15 nM in patients; P < 0.05). Thrombin generation in PRP was also higher in the patients with thalassemia at early times (3 min; 19 ± 3 nM in controls vs. 43 ± 8 nM in patients; P < 0.05). Importantly, the rate and peak of thrombin generation were significantly amplified by the presence of both cell types (PRP+RBCs), suggesting a biochemical cooperation between activated platelets and RBCs for thrombin generation. This was enhanced in patients with β-thalassemia, particularly those with T-I. In conclusion, chronic platelet activation in patients with β-thalassemia may influence TF-induced thrombin generation in platelets and erythrocytes. Thrombin generation appears to be a novel mode of platelet-erythrocyte cooperation in thrombogenesis.