The role of calcium and magnesium ions in platelet aggregation and the release reaction

The aim of the thesis is to determine the requirement of human platelet aggregation for extracellular divalent cations, and the role of these ions in modifying platelet reactivity. Collagen-induced aggregation was studied in H-PRP and in GFP suspensions. ADP-induced aggregation in H-PRP, and collageninduced release of 14c-5HT from GFP, were also studied. The effect of chelating agents on platelet aggregation was investigated. EDTA inhibited aggregation more effectively than EGTA, while Mg 2+ overcame the inhibition. The differential effects of the chelating agents indicated that the platelet surictce 111e111uram:: (.01-,ta~ii; uii ::DT,11, ::;c;;::;~t1·:c c.Ct~'.'~ty, :'::::'ri::>c:i:>nti:>rl as l,n.M2+, whose function is necessary for the promotion of primary and secondary aggregation. The results of deaggregation studies indicated that the EDTA-sensitive activity was also reauired to maintain adhesiveness in the primary aggregate. The evidence favours Mg 2+ as the divalent cation associated with this activity, although other possibilities are r.ot excluded, Collagen induced aggregation in H-PRP containing excess EGTA and in GFP suspensions deficient in extracellular divalent cations, indicating a lack of requirement for these ions in the suspending medium. The presence of either ext. ca 2+ or ext. Mg 2+ shortened the lag period and enhanced the rate and extent of aggregation, while a physiological concentration of both ions enhanced the latter, but prolonged the lag period. Threshold aggregation was most sensitive to the composition of extracellular divalent cations. The results indicate that a platelet surface activity functions during the lag period of collagen-induced aggregation and plays a role in the mechanism leading to onset of aggregation. After onset, this activity ceases to function, and a second platelet surface activity which is enhanced by ext. ca2+, becomes functional. The second activity may be associated with the ca 2+ uptake mechanism. The transition metal ions Ni 2+ and Co2+, did not inhibit the lag period activity, but did inhibit the aggregation phase activity. Extracellular ca2+ and Mg 2+ modified collagen-induced release of 14c-5HT from GFP in a manner complementary to aggregation studies, indicating that secondary aggregation and release are interrelated phenomena, subject to the same regulatory mechanisms. A study of the effect of ATP on platelet aggregation and deaggregation, indicated that the mechanism of collageninduced aggregation includes an ADP-associated function. The species of ATP responsible for its anti-aggregating activity appears to be [ATPJ 4- and not [M2+.ATPJ 2-. The thesis describes several platelet surface activities responsive to extracellular divalent cations, and the physiological implications of these activities are discussed. ABBREVIATIONS PRP Platc1et~~~ch plasma H-PRP Heparinized-PRP C-PRP Citrated-PRP GFP Gel-filtered platelets EDTA Ethylenediaminetetra-acetic acid EGTA Ethyleneglycol-bis-(S-amino-ethyl ether)N,N 1 -tetra-acetic acid ADP Adenosine-5 1 -diphosphate ATP Adenosine-5 1-triphosphate 5HT 5-hydroxytryptamine Lm membrane ligand 0..L rr· divalent metal cation ext. extracellula