Insufficient Mobilization of Calcium by Early Breakdown of Phosphatidylinositol 4, 5-Bisphosphate for Aggregation of Human Platelets by Collagen1

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Abstract
When human platelets (5 × 108/ml) were stimulated by a threshold concentration of collagen (2 μg/ml), a lag period of about 60 s was seen before the initiation of release reaction and aggregation. Breakdown of [32P]phosphatidylinositol 4, 5-bisphosphate was seen within 10 s after the addition of collagen. The concentration of intra-cellular free Ca2+ (monitored by Quin II) rose from 80 μM to 145 nM within 10 s after stimulation by collagen. However, a lag period of about 50 s remained. The rise was not blocked by indomethacin. It was supposed that the initial Ca2+ mobilization by myo-inositol 1, 4, 5-trisphosphate was too small to cause aggregation. Thromboxane A2, was gradually accumulated during the lag period and then abruptly increased in parallel with aggregation. These events were completely inhibited by 10 μM indomethacin. Thus, aggregation appeared to be dependent on the generation of thromboxane A,. Addition of 25 nM A23187 at 10 s after stimulation by collagen shortened the lag period before initiation of the abrupt thromboxane As generation, secretion and aggregation, whereas 25 nM A23187 could not cause these reactions in the absence of collagen. Accordingly, the lag period is assumed to be required for accumulation of free Ca2+ to the threshold for aggregation of platelets. It is considered that thromboxane A4 plays a central role in Ca2+ mobilization during stimulation of human platelets by collagen.

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