Increased soluble P-selectin in patients with haematological and breast cancer: a comparison with fibrinogen, plasminogen activator inhibitor and von Willebrand factor A. D. Blann, D. Gurney, M. Wadley, D. Bareford, P. Stonelake and G. Y. H. Lip (Received 2 August 2000 revised 12 October 2000 accepted 13 October 2000) Abnormal platelet activation and an increased risk of thrombosis are frequent findings in cancer. As soluble adhesion molecule P- selectin is being increasingly recognized as reflecting increased platelet activation, we hypothesized raised levels in patients with cancer, obtaining plasma from 24 patients with a cross-section of haematological cancers, 41 with breast cancer, and from an equal number of healthy controls for each patient group. Levels of soluble P-selectin were compared with those of von Willebrand factor (vWf), plasminogen activator inhibitor-1 (PAI-1) activity and fibrinogen (markers of endothelial integrity, fibrinolysis and coagulation, respectively). We found raised soluble P-selectin, fibrinogen and vWf in both patient groups compared with their controls (P , 0.01). vWf and soluble P-selectin were higher in the haematological cancers than in breast cancer patients (by 30 and 74%, respectively both P , 0.01). There was no significant difference in levels of PAI-1 between any group. There were no differences in soluble P-selectin or vWf when the data from the women with breast cancer were classified according to tumour size, lymph node involvement or presence of vascular invasion. We conclude that the platelet marker soluble P-selectin is raised in both haematological and breast cancer, and is higher in the former, but is unrelated to the type or stage of breast cancer. Blood Coagul Fibrinolysis 12:43���50 # 2001 Lippincott Williams & Wilkins. Keywords: soluble P-selectin, cancer, plasminogen activator inhibitor-1, von Willebrand factor, fibrinogen Introduction Cancer is a complex disease characterized by pro- found changes to physiological systems such as haemostasis and vascular function, and, as a conse- quence, often confers an increased risk of arterial and venous thrombosis [1���3]. Evidence of these changes include abnormalities in the levels of plasma molecules involved in coagulation and fibrinolysis [such as fibrinopeptide A, fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and D-dimer] [2���5], markers of endothelial cell integrity (soluble E- selectin, von Willebrand factor and soluble throm- bomodulin) [2���7] and of platelet function (beta- thromboglobulin) [8,9]. Some of these changes may be related to, or even partially responsible for, the increased risk of thrombogenesis in these patients. The adhesion molecule P-selectin is a component A. D. Blann, D. Gurney and G. Y. H. Lip are with the Haemostasis, Thrombosis and Vascular Biology Unit, University Department of Medicine, M. Wadley and P. Stonelake are with the Department of Surgery, and D. Bareford is with the Department of Haematology and Blood Transfusion, City Hospital, Birmingham, UK. Address correspondence to Dr A. D. Blann, Ph.D., M.R.C.Path., Haemostasis, Thrombosis and Vascular Biology Unit, University Department of Medicine City Hospital, Birmingham, B18 7QH, UK. Tel/fax: (��44) 121 507 5076 e-mail: a.blann@bham.ac.uk Blood Coagulation and Fibrinolysis 2001, Vol 12, No 1 43 Blood Coagulation and Fibrinolysis 2001, 12:43���50 0957���5235 # 2001 Lippincott Williams & Wilkins

of the membrane of the platelet alpha granule and of the membrane of the Weibel���Palade body, the endothelial cell organelle that stores the pro-coagu- lant molecule von Willebrand factor [10,11]. A soluble form of P-selectin has been described in the plasma of normal subjects, with increased levels in patients with atherosclerotic and thrombotic disor- ders [12,13] that seem likely to be related to excess platelet activation [14���16]. For example, soluble P- selectin is released from activated platelets in vivo during haemodialysis [17]. Increased levels of other soluble adhesion molecules, including soluble E- selectin, have also been described in various cancers [7], and upregulation of cell membrane adhesion molecule expression may be important in extravasa- tion and metastasis [18,19]. However, although raised soluble P-selectin has been described in pa- tients with melanoma [20], this has been disputed [21]. Of further interest is the report of the increased expression of P-selectin by endothelial cells within breast cancer tissues [22]. As increased levels of the platelet alpha granule matrix product beta-thromboglobulin are present in cancer [8,9], it was our primary hypothesis that soluble P-selectin would also be increased in this disease, when compared with healthy controls. However, as already mentioned, cancer is character- ized by changes in many plasma proteins. We therefore intended to determine whether such in- creases in soluble P-selectin would be similar to, or greater than, the expected increases of other plasma markers also relevant to thrombosis and haemosta- sis. We chose PAI-1, fibrinogen and von Willebrand factor because they are markers of fibrinolysis, coagulation and endothelial function, respectively, are increased in cancer, and may have pathophysio- logical relevance as contributors to an increased risk of thrombosis [1���5,9,11]. We tested our hypothesis in a cross-sectional study of patients with different haematological cancers, and others with breast cancer, each compared with healthy controls. Re- cruiting patients free of current chemotherapy/blood product support, we sought to avoid the issue of the effects of these interventions. Subjects and methods Subjects We recruited 24 patients (mean SD age, 52 11 years 14 women, 10 men) with haematological cancer (13 with lymphoid neoplasia and 11 with myeloid/granulocytic leukaemia) from outpatient clinics. Exact diagnoses were one acute lymphocytic leukaemia, two chronic lymphocytic leukaemia, eight chronic myeloid leukaemia, two chronic gran- ulocytic leukaemia, five non-Hodgkin���s lymphoma, two lymphoma, three malignant myeloma and one Waldenstrom���s macroglobulinaemia. These patients had been free of chemotherapy, radiotherapy, or blood transfusion for at least 4 weeks, and had haemoglobin, leukocyte and platelet counts within the normal range and so may be regarded as being in remission. Forty-one women (aged 59 14 years) with early, operable breast cancer were recruited as outpatients prior to surgery. They had also been free of chemotherapy or radiotherapy for 4 weeks. After surgery, the size of the excised tumour (grade T1, , 2 cm diameter grade T2, 2���5 cm grade T3, . 5 cm grade T4, fixed to chest wall), lymph node involvement (presence of tumour in at least one axilliary draining node) and the degree of vascular invasion were recorded according to estab- lished criteria. Individual diagnoses were made according to standard protocols, supported by laboratory or imaging services, as appropriate (e.g. immunophenotyping). As there was clear sex difference between the two patient groups, each had its own different healthy control group. Thus, the 41 women with breast cancer were compared with 41 healthy age-matched (55 8 years) women, and the 24 patients with haematological cancer were compared with 24 con- trol subjects (aged 56 6 years 10 men, 14 women). All controls were drawn from attenders for endo- scopy, hernia repair or for minor operations, their spouses, and from healthy hospital staff. Exclusion criteria for all subjects was diabetes, venous ulcera- tion, serological evidence of hepatitis B virus or HIV infection, atherosclerotic vascular disease, deep vein thrombosis, acute or chronic liver and kidney disease, connective tissue disease, acute infections or inflammatory conditions, or treatment with vaso- pressin or antibiotics. The approval of the Ethics Committee of West Birmingham Health Authority and informed consent from each subject was ob- tained. Methods Venous blood was obtained following non-traumatic venepuncture into 0.11 mol/l sodium citrate or ethylenediamine tetraacetic acid (EDTA). Citrated whole blood was kept cold (at 48C), and plasma was obtained within 2 h of venepuncture following centrifugation for 20 min at 1000 3 g and 48C, and was stored at ���708C until assayed. Soluble P-selectin was measured in citrated plasma by enzyme-linked immunosorbent assay using commercial reagents 44 Blood Coagulation and Fibrinolysis 2001, Vol 12, No 1 A. D. Blann et al.