Modulation Of Calcium Fluxes In Human Platelets By Human Milk in vitro † 21

The effect of adding human milk (HM) to washed fura-2 loaded human platelets was investigated in vitro. The effect of thrombin (0.01 U/ml) to increase intracellular free calcium ([Ca²⁺]_i), when calcium was present in the medium, was inhibited approx. 60% by a brief (10 second) treatment with HM (6% produced the maximum inhibition). Similar inhibition of the ability of vasopressin to increase ([Ca²⁺]_i) was also observed. The effect of thrombin to mobilize intracellular calcium in platelets (5 mM EGTA in the medium) was inhibited approx. 70% by HM (4% produced the maximum inhibition). This result suggests the HM contains a factor or factors that prevent thrombin from increasing([Ca²⁺]_i) in platelets, perhaps by either stimulating calcium efflux from the platelet, inhibiting intracellular calcium release or stimulating calcium uptake into the intracellular stores. When calcium influx into platelets was measured using barium ions as a calcium surrogate, HM blocked the influx by approx. 70% (4% produced the maximum inhibition). This result also implies that HM also contains a factor that inhibits calcium influx across the plasma membrane. Significant effects could be observed with 1% HM on all three parameters. The inhibitory activity did not pass through a 10 KDa molecular weight cut off filter. The inhibitory effect of HM was substantially reduced by heating the milk for 15 min. at 100 C. Treating HM with 0.3 M HCl or 0.5M NaOH at room temperature for 10 min. had no effect on activity. It is concluded that HM contains a factors or factors that reduce the ability of thrombin to increase [Ca²⁺]_i in platelets by both blocking calcium influx and preventing mobilization from internal calcium stores, alternatively the plasma membrane calcium ATPase pump may be activated. The factor is stable to mild acid and base treatment is heat labile and has a molecular weight in excess of 10 KDa. Because platelets participate in propagation of the acute inflammatory response, human milk factors that modify intracellular signalling in platelets might contribute to the anti-inflammatory character of human milk.