Vox Sang. 40: suppl. 1, pp. 69-71 (1981)

Complex Platelet Reactions

Scott Murphy Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, Pa., USA

In discussing complex platelet reactions, I shall concentrate on changes that occur in platelets during storage of platelet concen- trates at 22 "C. First, the effect of the pH of the medium is critical in determining whether or not the platelets will be viable after storage. 10 years ago we learned that there was a rapid and progressive drop-off in viability, if pH fell from starting levels of 7.0 to levels of 6.0 or below [l]. Initially, this relationship was not understood, but it was subsequently learned that the pH after stor- age correlated with the platelet count prior to storage. The lowest pH values were seen in the concentrates with the highest platelet counts [2]. Furthermore, it was found that there was a correlation between the pH and the lactate concentration at any period dur- ing storage, implying that pH fell due to pro- duction of lactate acid by platelet glycolysis. Finally, it was discovered that in units with platelet counts higher than 2,000,000 per mm3, the oxygen tension was unmeasurably low [2]. Because of the Pasteur effect there is a three-fold production of lactic acid per platelet in such hypoxic units. This is the major cause for the fall in pH in units with high platelet counts.

We have looked more closely into the metabolic events which occur during such storage. There is a direct relationship be- tween pH at any point during storage and glucose concentration. In those units in which pH falls to 6 or below, the glucose concentration falls to 5-10 mM from initial levels of 20-25 mM. There is nearly a 2 to 1 relationship between lactate production and glucose consumption with very little glucose consumption that is not accounted for by lactate production. Indirect calculations suggests that oxygen consumption is 0.5 mmol per 10" platelets per day. Although the measurements are not precise enough to be certain, these results suggest that some of the oxygen consumption is utilized for oxi- dizing fuels other than glucose, presumably fatty acids.

In any event, in platelet concentrates with high platelet counts, the oxygen tension falls to essentially 0 within 2 h and the oxygen tension remains at that level until the pH falls to 6.0. As the pH falls below 6.0 there is a dramatic morphological change with loss of integrity of the cells and a gradual rise in oxygen tension as pH falls further. It seems certain that this rise in oxygen tension re-

70 Murphy

flects the inability of the nonviable cells to consume oxygen that is transported through the walls of the plastic container. We have looked further into any possible effect that might occur prior to the arrival of the pH at 6.0. Using the Coulter counter, we have found that the platelets begin to swell at approximately pH 6.6-6.8. The swelling is progressive and reaches levels of twice the control as the pH falls below 6.0. Much of this swelling is reversible if the platelets are returned to physiologic pH and oxygen ten- sion, but with advanced changes some of the swelling is not reversible. This phenome- non, ‘cloudy swelling’, appears to be typical of many cell types when they are exposed to prolonged hypoxia.

In addition, we have developed a quanti- tative method for estimating platelet shape. This method was described in detail in a recent publication [3]. In using this method, we have found that platelets change from their normal discoid shape to a more spher- ical shape as pH falls to 6.1-6.6. Again, with incubation at physiologic pH and oxygen tension, this change is reversible. However, if pH falls to below 6.0, there is complete disc-to-sphere transformation, and this change is not reversible with return to phys- iologic circumstances. Thus, this change in shape correlates with in vivo viability re- sults.

We have also continued studies designed to define the functional change which occurs in platelets as they are stored at room tem- perature. It has been noted by many inves- tigators that there is diminution in response to many aggregating agents. We have con- centrated our recent attention on adenosine diphosphate (ADP) as a stimulating agent. Even with concentrations of 100 pM, there is often no change in light transmission when

aggregation is studied in an aggregometer. However, we have been interested to find that the initial shape change response to ADP is relatively intact after storage. There is a modest increase in the dose of ADP which is required to induce shape change, but shape change is complete. This suggests that the cell is capable of detecting stimuli and of making an initial response to these stimuli. Rather, the problem seems to be either an inability to develop ‘stickiness’ or a block at some point between the receipt of the initial stimulus and the development of stickiness. It is now known that ADP causes the uncovering of a surface receptor for fibrinogen which is necessary for platelet aggregation to occur [4]. The results are sug- gestive although hardly conclusive that some abnormality in this receptor takes place during storage. Holmsen has already reported in an earlier presentation [in this volume] that energy metabolism is required for platelet aggregation to take place. Mea- surements in our laboratory suggest that the adenylate energy charge of stored platelets is normal, suggesting that the cells are capable of maintaining high energy phosphate bonds intact. Thus, whatever the aggregation de- fect is, it appears not to be related to the cells’ inability to maintain a normal energy sta- tus.

The relationship between this in vitro defect and ultimate in vivo function remains a matter for conjecture. In our experience, this in vitro defect develops within 24 h of storage and then remains stable. There is lit- tle doubt that platelets stored for only 24 h at room temperature have acceptable in vivo function, at least after several hours of cir- culation. The matter for concern is whether there is an in vitro defect which is then cor- reted in vivo during circulation, as some

Complex Platelet Reactions 71

studies have suggested [5 ] . Patients are often transfused when they are bleeding acutely and in need of an immediate hemostatic response. It may not be optimal to wait for several hours for optimal in vivo function to return. The answers to these questions can only be determined with further in vivo stud- ies in thrombocytopenic recipients of trans- fusions of stored platelets.

References

I Murphy, S.; Sayar, S.; Gardner, F.H.: Storage of platelet concentrates at 22'C. Blood 35: 549-557 (1970).

2 Murphy, S . ; Gardner, F.H.: Platelet storage at 22 "C: Role ofgas transport across plastic containers

in maintenance of viability. Blood 46: 209-218 (1975).

3 Holme, S.; Murphy, S.: Quantitative measurements of platelet shape by light transmission studies; ap- plication to storage of platelets for transfusion. J. Lab. clin. Med. 92: 53-64 (1978).

4 Marguerie, G. A. ; Edgington, T. S. ; Plow, E. F.: In- teraction of fibrinogen with its platelet receptor as part of a multistep reaction in ADP-induced plate- let aggregation. J. biol. Chem. 255: 154-161 (1980).

5 Murphy, S.; Gardner, F.H.: Platelet storage at 22 "C; metabolic, morphologic, and functional stud- ies. J. din. Invest. 50: 370-377 (1971).

Scott Murphy, Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, Pa. (USA)