Although cryopreservation of platelets has been attempted for the last two decades, the results are not still satisfactory. To investigate the reasons for the functional loss of cryopreserved platelets, glycoproteins (GPs), such as GPIb, GPIb/IX, GPIa/IIa, GPIIb/IIIa, GPIIIa, GPIV and GMP-140 on the platelet membrane before and after cryopreservation were analyzed by monoclonal antibodies and flow cytometry (FCM). Platelets loaded with 5% dimethylsulfoxide (DMSO) were extracelluarly frozen at -5°C, cooled to -80°C at 0.5°C/min, and then thawed rapidly in a water bath (37°C). There was no change of expression of GPs except for GPIb. GPIb was lost from about half of the cell population after freezing and thawing. When platelets were extracellularly frozen without DMSO, GPIb was progressively lost from the population as temperature decreased. At -40°C or below, most of the platelets lost GPIb from the membrane. In contrast, the rapid cooling of platelets (-20°C/min) did not cause the loss of GPIb.
To investigate the mechanism of GPIb loss due to freezing and thawing, platelets were osmotically stressed in hyperosmotic plasma made by addition of NaCl, resuspended in isosmotic plasma, and then GPs were analyzed by FCM. GPIb was lost at osmotic stresses greater than 1000mOsm/kg. An enzyme immunoassay to quantify GPIb as glycocalicin, which was released to the medium by freezing-thawing or osmotic stress (1500mOsm/kg), showed a good correlation with the loss of GPIb on the platelet membrane detected by FCM. SDS-gel electrophoresis and immunoblotting analysis indicated that GPIb was released to the suspending medium as glycocalicin. It is known that membrane GPIb is cleaved to glycocalicin by intracellular Ca2+-dependent proteinases. Platelets frozen with proteinase inhibitors, such as N-ethylmaleimide, EDTA or EGTA, did not show the loss of GPIb. These results indicated that the loss of GPIb from the platelet membrane can be explained by the cleavage of GPIb to glycocalicin by the intracellular Ca2+-dependent proteinases which leaked from the osmotically damaged platelets as a result of freezing and thawing.
