Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We have developed a direct test for activated platelets in whole blood that utilizes dual-color flow cytometry and requires no washing steps. Platelets were distinguished from erythrocytes and white blood cells in the flow cytometer by labeling the platelets with biotin-AP1, an antibody specific for membrane glycoprotein lb, and analyzing the cells for phycoerythrin-streptavidin fluorescence. Membrane surface changes resulting from platelet activation were detected with three different FITC-labeled monoclonal antibodies: 1) PAC1, an antibody specific for the fibrinogen receptor on activated platelets; 2) 9F9, which binds to the D-domain of fibrinogen and detects platelet-bound fibrinogen; and 3) S12, which binds to an alpha-granule membrane protein that associates with the platelet surface during secretion. Unstimulated platelets demonstrated no PAC1, 9F9, or S12-specific fluorescence, indicating that they did not bind these antibodies. Upon stimulation with agonists, however, the platelets demonstrated a dose-dependent increase in FITC-fluorescence. The binding of 9F9 to activated platelets required fibrinogen. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 or 9F9 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate that evokes full platelet aggregation and secretion induced maximal binding of all three antibodies. When blood samples containing activated and non-activated platelets were mixed, as few as 0.8% activated platelets could be detected by this technique. There was a direct correlation between ADP-induced FITC-PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = 0.99; p < 0.001). These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This method may be useful to assess the degree of platelet activation and the efficacy platelet inhibitor therapy in thrombotic disorders.

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SUMMARY Data from two community studies on men from South Wales and the west of England suggest that the effects of smoking on the haemostatic system remain for many years after giving up. Long term correlations between several variables, including plasma fibrinogen and white cell count, and the length of time after giving up were seen in ex-smokers. Dose response relations were apparent in current smokers in terms of the white cell count and two haematological variables, the packed and mean cell volumes. These long term correlations probably reflect the toxicity of other agents in tobacco smoke besides nicotine and carbon monoxide, which act only in the short term. Identification of these agents may further our understanding of the mechanism by which cigarette smoking is associated with atherosclerotic disease.Evidence is accumulating that haemostatic factors have a pathogenetic role in ischaemic heart disease' 2 and stroke.3 Smoking habit is known to affect substantially several haemostatic factors4 I and is also a major risk factor for ischaemic heart disease.6 7 This paper examines the activity of several haemostatic factors in non-smokers, smokers, and ex-smokers in two community studies on men from South Wales and the west of England. Material and methods STUDY POPULATIONSAll men aged between 45 and 59 years resident in the town of Caerphilly and five outlying villages (total population 41000) were included in the study. Subjects were selected principally from electoral registers and a private census carried out by letter and houseto-house survey. Age and sex registers and practice records were also used as an additional check on the eligible population. Twenty one general practitioners served the area covered by the survey working in seven independent surgeries. Accepted for publication 13 March 1987 In Bristol computerised age and sex registers of patients were available for all 16 general practitioners who worked from two health centres serving Speedwell, a largely residential district of east Bristol (total population 32 000). All male subjects aged 45 to 59 years of age on September 1 1979 were selected and invited by letter from their general practitioner to participate in the survey. SURVEY METHODSIn Caerphilly a total of 2818 men were included and invited to attend one of seven local clinics for a medical examination. A total of 2512 (89%) subjects were examined. A questionnaire was filled in by all subjects for details of medical history, occupation, and smoking habit, and other data.In the Bristol population 2550 men were included in the study, and 2348 (92%) subjects were seen at the Speedwell clinic. Questionnaires used in this survey were identical in essential respects with those used in Caerphilly. LABORATORY METHODSAll subjects were seen at a morning haematology clinic, and a venous blood sample was obtained with 909 on 11 May 2018 by guest. Protected by copyright.