Abstract

Because of methodologic problems, platelet function in thrombocytopenic patients has never been adequately studied, and therefore individual differences in bleeding risk are poorly understood. Whole blood flow cytometry analyzes the function of individual platelets, thereby enabling assessment of platelet function in severe thrombocytopenia (Michelson, A. Platelets, 2nd ed, Elsevier, 2007). In this study, platelet function was compared in 31 ITP patients, 14 CIT patients, and 16 healthy controls. Clinical bleeding was related to platelet function testing. The immature platelet fraction (IPF, or reticulated platelets) was measured in a Sysmex XE-2100. Platelet surface P-selectin and activated integrin αIIbβ3 (reported by monoclonal antibody PAC1) were measured by whole blood flow cytometry in the presence and absence of 0.5 μM ADP, 20 μM ADP, 1.5 μΜ TRAP, or 20 μM TRAP. Bleeding was quantified by a comprehensive score that allocates grades of 0 (no), 1 (minor) or 2 (marked) bleeding at 10 anatomic sites (Page, L.K. Br J Haematol 2007). Mean platelet volume (MPV) and IPF were higher in ITP than CIT, reflecting, as expected, a higher rate of platelet production (Table 1). Platelet surface P-selectin without added agonist (i.e. circulating activated platelets) was significantly higher in both ITP and CIT patients than in controls (Table 2). However, upregulation of platelet surface P-selectin and activated αIIbβ3 in response to ADP and TRAP (platelet ‘activatability’) was reduced in CIT patients compared with ITP patients and controls (Table 2). Stratification of bleeding scores by platelet count showed that CIT patients had more GI, urinary, and pulmonary bleeding at platelet counts <20 × 109/L whereas ITP patients had more skin/oral bleeding (Table 1). In sum, the higher platelet surface P-selectin and activated αIIbβ3 in CIT and ITP patients than in controls is consistent with a role for circulating activated platelets in maintenance of vascular integrity in thrombocytopenia. However, platelet activation in response to ADP and TRAP is reduced in CIT compared with both ITP and controls, which may reflect the relative senescence (as evidenced by lower IPF) of CIT platelets and/or effects of chemotherapy or the underlying leukemia. These data demonstrate that bleeding in different thrombocytopenic conditions is not entirely explained by the thrombocytopenia per se. Reduced responses to ADP and TRAP in CIT patients compared with ITP patients may be clinically significant, given that, at equivalent degrees of thrombocytopenia, CIT patients had more significant bleeding (GI, urinary, pulmonary) than ITP patients.

Table 1.
All PatientsPatients with platelet counts <20 × 109/L
MPVIPF%Absolute IPF ×109/LSkin/oral bleedingBleeding other than skin/oral
*P <0.05 
CIT 7.2 11.0 1.7 6/10 5/10 
ITP 9.2* 18.1 5.8* 10/11 0/11* 
All PatientsPatients with platelet counts <20 × 109/L
MPVIPF%Absolute IPF ×109/LSkin/oral bleedingBleeding other than skin/oral
*P <0.05 
CIT 7.2 11.0 1.7 6/10 5/10 
ITP 9.2* 18.1 5.8* 10/11 0/11* 
Table 2.

(mean fluorescence intensity)

CITITPControls
a = P<0.05 for CIT vs ITP; b = P<0.05 for ITP or CIT vs controls 
No Agonist P-selectin 7.6b 6.2b 3.1 
 ActivatedαIIbβ3 7.0 10.2 7.2 
High ADP P-selectin 41.8a,b 114.0 87.5 
 ActivatedαIIbβ3 154.4a,b 390.2 381.7 
High TRAP P-selectin 69.9a,b 296.6 505.6 
 ActivatedαIIbβ3 46.9a,b 241.2b 457.7 
CITITPControls
a = P<0.05 for CIT vs ITP; b = P<0.05 for ITP or CIT vs controls 
No Agonist P-selectin 7.6b 6.2b 3.1 
 ActivatedαIIbβ3 7.0 10.2 7.2 
High ADP P-selectin 41.8a,b 114.0 87.5 
 ActivatedαIIbβ3 154.4a,b 390.2 381.7 
High TRAP P-selectin 69.9a,b 296.6 505.6 
 ActivatedαIIbβ3 46.9a,b 241.2b 457.7 

Author notes

Disclosure:Ownership Interests:; Dr. Bussel: Amgen and GlaxoSmithKline. Research Funding: Dr. Bussel, WMC, NY: Amgen, Biogen Idec, Cangene, Genentech, GlaxoSmithKline, and Sysmex. Dr. Michelson, Center for Platelet Function Studies, MA: GlaxoSmithKline. Membership Information: Dr. Bussel: Speakers Bureau: Baxter, Advisory Committees: Amgen, GlaxoSmithKline, Baxter.