Dependence of Platelet Thrombus Stability on Sustained Glycoprotein IIb/IIIa Activation Through Adenosine 5'-Diphosphate Receptor Stimulation and Cyclic Calcium Signaling
Shinya Goto, MD*,*,
Noriko Tamura, MS*,
Hideyuki Ishida, MD and
Zaverio M. Ruggeri, MD
* Department of Medicine, Tokai University School of Medicine, Kanagawa, Japan
Department of Basic Medicine, Tokai University School of Medicine, Kanagawa, Japan
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California
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Figure 1 Three-dimensional projection imaging of platelet thrombi. A piezo-electric motor (a) moved the objective lens at a constant speed of 0.4 µmol/l/s to provide scanning images of the platelet thrombi (a’). The sum of the confocal images in a bottom to top stack (z-axis) was projected on planes at 10° intervals relative to the x axis to obtain the three-dimensional projection images shown on the right, including projections at 0 degrees (top view; A), 60° (B), and 90° (front view; C). The maximum height (h) of the platelet thrombi was calculated from the front view projection, as shown in C.
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Figure 2 Three-dimensional projection images of thrombi formed in the presence or absence of adenosine diphosphate receptor antagonists. Blood with fluoresceinated platelets was perfused over immobilized collagen type I fibrils for 6 min at the wall shear rate of 1,500 s–1 in the absence (Control) or presence of MRS2179 (100 µmol/l) or AR-C69931MX (100 nmol/l), as indicated. The platelet thrombi were scanned in the z-axis by confocal microscopy, and the resulting images were projected on planes rotated around the x-axis at 10° intervals (please see the online version of this article for supplemental videos). The figure shows projection images from the top (A), 60° (B), and the front (C). These images are representative of the results obtained in eight separate experiments.
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Figure 3 Reduction in the size of platelet thrombi exposed to blood containing different antagonists of platelet function. These experiments were performed as described in the caption of Figure 2, with the difference that the surface was exposed to two subsequent aliquots of blood each perfused for 6 min. (A) Representative images (0-degree and 90° projections) of platelet thrombi after perfusion of the first (before) or second (after) control blood aliquot (please see the online version of this article for supplemental videos). (B) Representative images of platelet thrombi after perfusion of the first control blood aliquot (before) or a second blood aliquot containing the P2Y12 inhibitor AR-C69931MX (100 nmol/l) (please see the online version of this article for supplemental videos). (C) Cross-sectional area occupied by fluorescent platelets in horizontal planes passing through the thrombi at the indicated distance from the collagen surface, calculated as percentage of the area in the plane closest to collagen surface. The bars labeled "Before" show the area of thrombi formed after perfusion of untreated blood for 6 min. The bars labeled "No Addition," AR-C, MRS, and LAN show the area of thrombi remaining on the surface after an additional 6 min perfusion of untreated blood, or blood treated with the P2Y12 inhibitor AR-C69931MX (100 nmol/l), or the P2Y1 inhibitor MRS2179 (100 µmol/l), or the putative Ca2+ channel inhibitor lanthanum chloride (LAN) (1 mmol/l), respectively. Mean and SEM of eight experiments are shown.
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Figure 4 Changes in the two-dimensional and three-dimensional structure of platelet thrombi exposed to blood containing antiplatelet agents. These experiments were performed essentially as described in the caption of Figure 3. (A) Representative two-dimensional fluorescence microscopy images of platelet thrombi immediately after perfusion of the first control blood aliquot (0 min) or at different times after beginning the second perfusion with either untreated blood (No Addition) or blood containing tirofiban (0.5 µmol/l), as indicated (please see the online version of this article for supplemental videos). (B) Cross-sectional area of thrombi at the indicated distances from the collagen surface after perfusion of untreated blood for 6 min (No Addition), or after perfusion for an additional 6 min of blood containing aspirin (100 µmol/l) or tirofiban (0.5 µmol/l), as indicated. See Figure 3C for additional details.
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Figure 5 Changes in the intracytoplasmic Ca2+ concentration ([Ca2+]i) of platelet thrombi caused by adenosine diphosphate receptor antagonists. These experiments were conducted as described in the caption of Figure 3, except that blood was replaced with a cell suspension containing fluo-3AM–loaded platelets, washed erythrocytes, and homologous platelet-poor plasma with the specific thrombin inhibitor argatroban (100 µmol/l) as the anticoagulant. The cell suspension was perfused over type I collagen fibrils at the shear rate of 1,500 s–1 for 4 min to form platelet thrombi. Then, the same cell suspension without or with the addition of the P2Y12 antagonist AR-C69931MX (100 nmol/l), or the P2Y1 antagonist MRS2179 (100 µmol/l), was perfused for an additional 4 min. The [Ca2+]i of platelets incorporated into thrombi was measured during the second perfusion. (Left panels) Intracytoplasmic Ca2+ concentration of five randomly selected platelets recorded for 10 s beginning 2 min after the start of the second perfusion. (Right panels) Images reflecting the concentration of Ca2+ ions in platelets within thrombi formed during perfusion of untreated blood (Control) or blood containing 100 nmol/l AR-C69931MX (ARC).
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Figure 6 Changes in PAC-1 binding to activated platelets caused by adenosine diphosphate (ADP) receptor antagonists. To activate platelets, 25 µl of HEPES buffer containing ADP and epinephrine (400 µmol/l each) was mixed with 375 µl of platelet-rich plasma and incubated for 20 min. Fifty µl of fluorescein isothiocyanate-conjugated PAC-1 (25 µg/ml) was then added, and the fluorescence of individual platelets was measured 5, 15, 30, 45, and 60 min after the addition of 50 µl of HEPES buffer containing or not the P2Y12 antagonist AR-C69931MX (100 nmol/l). (Upper panel) Mean and SEM of the median fluorescence of 10,000 platelets in eight experiments. (Lower panels) Representative flow cytometric results at selected time points in one experiment without (solid lines) or with (dotted lines) the addition of AR-C69931MX.
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Figure 7 Effect of different platelet inhibitors on PAC-1 binding to activated platelets. These experiments were performed as described in the caption for Figure 6, with the only difference that the P2Y1 inhibitor, MRS2179, or the putative Ca2+ channel blocker, lanthanum chloride, was also added to platelets after activation by adenosine diphosphate and epinephrine. The upper panel shows the mean and SEM of the median fluorescence of 10,000 platelets measured 30 min after addition of AR-C69931MX (final concentration: 100 nmol/l), MRS2179 (final concentration: 100 µmol/l), or lanthanum chloride (final concentration: 1 mmol/l) as compared to control in which only buffer was added (n = 8). The lower panels show the actual flow cytometric results of one representative experiment. Solid lines represent the results in the presence of the inhibitor shown in each panel, while dotted lines represent the results in the absence of inhibition.
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binding to von Willebrand factor (VWF) under high shear rate conditions. Full activation depends on released adenosine diphosphate (ADP) and leads to the binding of soluble adhesive ligands such as VWF and fibrinogen. These form the new substrate for the recruitment of circulating platelets, again initiated under high shear rate conditions by GP Ib