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Increased reactivity of platelets induced by fibrinogen independent of its binding to the IIb-IIIa surface glycoprotein:

a potential contributor to cardiovascular risk

^a Department of Medicine, The University of Vermont College of Medicine, Burlington, Vermont, USA
^b Department of Pathology, The University of Vermont College of Medicine, Burlington, Vermont, USA

View larger version (20K): [in a new window]   Figure 1 The effect of addition of fibrinogen to blood on ADP-induced expression of P-selectin. Blood was anticoagulated with CTI (32 µg/ml). Degranulation of -granules was determined with flow cytometry and based on detection of surface expression of P-selectin. Values are means ± SEM determined in blood samples from 10 subjects. A significant (p < 0.001) interaction between fibrinogen and ADP was observed. The addition of fibrinogen (100 mg/dl) significantly increased P-selectin expression in response to 1.5 µmol/liter ADP.

View larger version (17K): [in a new window]   Figure 2 The effect of addition of fibrinogen to blood on ADP-induced (1.5 µmol/liter) expression of P-selectin. Values are expression of P-selectin expressed as a percentage of control values and are means ± SEM determined in blood samples from 19 subjects for samples with addition of 500 mg/dl, 12 subjects for samples with addition of 50 mg/dl and 300 mg/dl, and 10 subjects for samples with addition of 100 mg/dl and 200 mg/dl. Results with the addition of each concentration of fibrinogen greater than 50 mg/dl showed significant increases (p < 0.001) compared with control.

View larger version (24K): [in a new window]   Figure 3 The effect of addition of fibrinogen, albumin and ferritin to blood on ADP-induced (average of results with all concentrations of ADP used) expression of P-selectin. Values are P-selectin expressed as a percentage of control values and are means ± SEM determined in blood samples from four subjects. Equimolar concentrations of albumin and ferritin were selected to permit determination of the specificity of results with the addition of 100 mg/dl, 200 mg/dl and 500 mg/dl of fibrinogen.

View larger version (19K): [in a new window]   Figure 4 The binding of fibrinogen (FITC labeled) to platelets in response to ADP. Blood was anticoagulated with CTI (32 µg/ml) and results were compared with those in blood anticoagulated with CTI and spiked with ReoPro (4, 10 and 100 µg/ml). The binding of 10 mg/dl of FITC-fibrinogen to platelets was determined with the use of flow cytometry. Values are means ± SEM of results of triplicate determination in blood from two representative subjects.

View larger version (22K): [in a new window]   Figure 5 The effect of ReoPro on -granule degranulation induced by ADP after addition of fibrinogen to the sample. Fibrinogen (500 mg/dl) was added to blood that had been anticoagulated with CTI or anticoagulated with CTI and spiked with ReoPro (4, 10 and 100 µg/ml). Values are means ± SEM of results with blood samples from 10 to 19 subjects. No significant differences were observed when results under the two sets of conditions were compared.

View larger version (34K): [in a new window]   Figure 6 The association of FITC-fibrinogen with quiescent platelets in blood anticoagulated with CTI (32 µg/ml) and exposed to FITC-fibrinogen (300 mg/dl) for 15 min (micrographs on left: A, C, and E). The platelets were fixed in Optilyse-C and exposed subsequently to 0.1% Triton X-100 and anti-P-selectin IgG. After washing, platelets were exposed to a secondary Alexa-conjugated anti-mouse IgG. The concentration of fibrinogen in blood from this subject was 360 mg/dl. The total concentration of fibrinogen (endogenous plus FITC-fibrinogen) to which the platelets were exposed was 330 mg/dl. The micrographs on the right (B, D, and F) show results with blood drawn into CTI spiked with ReoPro (10 µg/ml). For each set of observations, the micrograph on the top (A and B) is a representative platelet imaged with phase contrast with the transmitted light detector of the confocal microscope merged with fluorescence (excitation 488 nm) demonstrating FITC-fibrinogen (green signal). The micrograph in the center (C and D) is the same platelet visualized with phase-contrast transmitted light and fluorescence (excitation 568 nm) demonstrating P-selectin expression (red signal). The red signal localizes -granules. The micrograph on the bottom shows the same platelet viewed by multiplying fluorescent images (excitation 488 and 568) and thus the degree of co-localization of fibrinogen-FITC and P-selectin (yellow color). Fluorescence in images A and C are multiplied to create E, and fluorescence in images B and D are multiplied to create F. Yellow demonstrates localization of fibrinogen in -granules. Bar = 5 µm; all images are same magnification.