Geometry of normal mammalian platelets by quantitative microscopic studies

Abstract

The shape distributions of normal and hardened human and rabbit erythrocytes and platelets were obtained for edge-on orientations of a few hundred freely rotating cells from analyses of microphotographs obtained similarly as by Ponder(1930, Q. J. Exp. Physiol. 20:29) by phase-contrast microscopy at 800 X magnification. Major average diameters (d) and thicknesses (t) were estimated for both normal and hardened cells, and were used to calculate an average geometric axis ratio, rp = t/d, which increases to unity as cells become more spherical. Our fixation procedure did not alter these shape parameters: rp was unchanged for erythrocytes, with d and t values similar to those reported by Ponder (1930); platelets had d X t = 3.6 +/- 0.7 mum X 0.9 +/- 0.3 mum and 3.1 +/- 0.4 mum X 0.6 +/- 0.3 mum, respectively, for human and rabbit cells, with rp = 0.26 and 0.20, respectively. Agreement in rp was found with data obtained by a novel rheo-optical method which allows for a direct statistical averaging for large populations (greater than 100 X 10(3) cells). Histograms and linear correlation studies were made of the above three parameters (d,t,rp), as well as volume (V), total surface area are (S), and sphericity index (S.I.) calculated for both "prolate ellipsoid" and "disc with rounded edges" models. Results indicate very high linear correlations between rp - t, rp - S. I., and d -S, with high correlations for t - V,d -V and S. Data are in agreement with the few reports in the literature determined by other methods, with the best model for platelets appearing to be an oblate spheroid.

Transient kinetics of the rapid shape change of unstirred human blood platelets stimulated with ADP.

“…Specifically, our analysis indicates that these are +25% for the disc to sphere and -10% for the sphere to spiny sphere (pseudopod formation) transformations; scattering theory predicts +24% and -9% for these same changes, assuming a platelet volume of [5][6][7][8][9][10] ,um3 and an axial ratio of0.26 for the initial disc (7,8). The assumed volume and axial ratio are consistent with values estimated for the discoid form by direct observation ofresting platelets (12), and the agreement of the predicted and fitted values argues that the intermediate state is indeed roughly spherical and has an effective scattering volume more closely resembling the initial disc than the final spiny sphere.…”

“…All further operations were done at 370C. After incubating the cell suspension with gentle swirling to allow time for any stressed cells to spontaneously revert to the flat discoid form characteristic ofthe resting state [axial ratio 0.26 (12)], it was diluted to 40,000 platelets per ul with 26 mM 2-{[tris(hydroxymethyl)methyl]-amino}ethane sulfonate (Tes) buffer containing 128 mM NaCl, 3 mM KCI, 0.06 mM CaCl2, 0.02 mM MgCl2, and glucose at 1 mg/ml, pH 7.4, and reincubated with gentle swirling.…”

Prothrombin is activated on vascular endothelial cells by factor Xa and calcium.

“…6) indicates that vascular endothelium may also be important for physiological hemostasis. It must be recognized, however, that 6 X 105 cells have =75-fold greater surface area than do an equivalent number of platelets (24). The observation that sparse ABAE cells generate more thrombin than do confluent cells (Fig.…”

Kinetics of ADP-induced human platelet shape change: apparent positive cooperativity

“…1978) have utilized this measurement to estimate V. (b) Phase-contrast microscopy At 5 s following the addition of ADP to stirred PRP, 0.05-mL samples were withdrawn and fixed with four volumes of 1.3% glutaraldehyde at 37°C as described previously (Frojmovic and Panjwani 1976;Milton and Frojmovic 1979). This sampling time was chosen as it was found that for all [ADP]' s used in this study, the increase in the fraction of shape-changed platelets,!.…”