Original Articles
Thrombogenesis in sickle cell disease,

https://doi.org/10.1067/mlc.2001.115450Get rights and content

Abstract

Thirty-three subjects with sickle cell disease (SCD), 11 during episodes of pain and 22 during periods without pain, were evaluated for in vivo thrombogenic activities as compared with 10 normal black control subjects. Measurements were performed for (1) platelet surface activation, assessing flow cytometric expression of activated integrin αIIbβ3 receptor (GPIIb/IIIa, CD41a) and P-selectin (CD62p); (2) platelet and erythrocyte surface procoagulant activities, measuring flow cytometric binding of activated factor (FVa) and annexin V; (3) plasma levels of platelet-specific secreted proteins platelet factor 4 (PF4) and β-thromboglobulin (βTG); (4) plasma markers of thrombin generation, prothrombin activation fragment (F1.2), and thrombin: antithrombin complex (TAT); and (5) plasma markers of fibrinolysis, D -dimer, and plasmin:antiplasmin complex (PAP). As compared with control subjects, asymptomatic subjects with SCD demonstrated significantly increased platelet activation (P < .01 for P-selectin and annexin V binding), elevated plasma levels of PF4 and βTG (P < .01 and P < .03, respectively), and increased plasma concentrations of F1.2, TAT, PAP, and D -dimer (P < .05 in all cases). During episodes of SCD pain, platelet activation was increased as compared with periods without pain (P < .01 for expression of activated integrin αIIbβ3 receptor and P-selectin and binding of FVa and annexin V), erythrocytes expressed procoagulant activities (P < .01 for FVa and annexin V binding), and platelet microparticles appeared in the circulation (3% to 30%; P < .001). SCD pain episodes were associated with elevated plasma levels of F1.2, TAT, PAP, and D -dimer (P < .05 as compared with asymptomatic intervals). The frequency of pain episodes correlated with enhanced platelet procoagulant activity (r = 0.61, P < .05) and elevated plasma fibrinolytic activity (r = 0.74, P < .01) measured during periods without pain. Plasma fibrinolytic activity was inversely correlated with time to the next pain episode (r = –0.50, P < .05). Thus, asymptomatic subjects with SCD exhibit ongoing platelet activation, thrombin generation, and fibrinolysis that increases during episodes of pain. These changes are predictive of frequency of pain and interval to next pain episode, thereby implicating thrombogenic activity in the development of SCD pain episodes. (J Lab Clin Med 2001;137:398-407)

Section snippets

Subjects

Thirty-three subjects with SCD (hemoglobin SS), 11 during episodes of pain and 22 while pain-free, were studied for in vivo platelet activation and platelet and red blood cell thrombogenic activities by using flow cytometry. Concurrent studies for circulating plasma markers of platelet secretion, thrombin generation, and fibrinolysis were done with enzyme-linked immunosorbant assays.

Pain episodes were defined as acute events characterized by diffuse pain occurring in the extremities, back,

Flow cytometric methods for detecting activated platelets

Three-color flow cytometric analysis of periph-eral blood labeled with lineage-specific probes readily discriminated erythrocytes, leukocytes, and platelets by immunofluorescence and light-scatter properties. The flow cytometric detection of platelets activated in vitro is shown in Fig 1.

. Flow cytometric analysis of activated platelets. Platelet-rich plasma was treated with 0.0, 0.5, and 10 μmol/L ADP (A , B , and C , respectively) and incubated with anti-LIBS1 mAb directed against activated αIIb

DISCUSSION

The present study establishes that patients with SCD demonstrate increased platelet activation, thrombin generation, and fibrinolysis during clinically quiescent periods and that during SCD pain episodes these thrombogenic activities become amplified. Both platelets and erythrocytes contribute procoagulant activities promoting the generation of thrombin, leading to the development of thrombo-occlusive complications of SCD. This interpretation is supported by significant correlations among the

Acknowledgements

We thank Scott Clark, PhD, for assistance with the statistical analysis.

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    Supported by grants 5 P60 HL48482 and 1R01 HL61703 from the National Institutes of Health.

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