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Platelet integrin αIIbβ3must be activated via intracellular mechanisms before it binds soluble ligands, and it is thought to be activated at its extracellular site by surface-bound ligands. Integrin activation is associated with rearrangement of the cytoskeleton and phosphorylation of proteins that become localized in focal contacts. In these processes, the cytoplasmic tail of the β-subunit plays a central role. We introduced peptides homologous to the E749ATSTFTN756domain (E–N peptide) and the T755NITYRGT762domain (T–T peptide) of β3in streptolysin O-permeabilized platelets and analyzed the initial interaction with soluble fibronectin, fibrinogen and PAC-1 after stimulation with thrombin. E–N peptide left the initial binding of fibronectin intact but interfered with stable receptor occupancy. E–N peptide also inhibited fibrinogen binding, thereby reducing the formation of large aggregates. Strikingly, E–N peptide did not disturb the binding of PAC-1, which is known to reflect activation of the integrin. E–N peptide also inhibited tyrosine phosphorylation of focal adhesion kinase, a response known to be dependent on αIIbβ3. T–T peptide did not affect these processes. In a model for outside-in integrin activation, E–N peptide disrupted the binding of CHO cells expressing αIIbβ3to surface-bound ligand. Again, T–T peptide had no effect. We conclude that the E749ATSTFTN756region of the β3-tail stabilizes the binding of soluble and surface-bound ligand to integrin αIIbβ3via a mechanism that involves the phosphorylation of FAK.

Stimulation of human platelets increases cytoplasmic pH (pH i) via activation of Na +/H +exchange. We have determined the effect of inhibiting Na +/H +exchange on (i) thrombin-induced Ca 2+mobilization and (ii) turnover of 32P-labelled phospholipids. Blocking Na +/H +exchange by removal of extracellular Na +or by ethylisopropylamiloride (EIPA) inhibited Ca 2+mobilization induced by 0.2 Umlthrombin, whereas increasing pH iby NH 4C1 enhanced the thrombin-induced increase in cytosolic free Ca 2+. The effect of EIPA was bypassed after increasing pH iby moneasin. The thrombin-induced cleavage of phosphatidylinositol 4,5-bisphosphate (PIP 2) was unaffected by treatments that blocked Na +/H +exchange or increased pH i. It is concluded that activation of Na +/H +exchange is a prerequisite for Ca 2+mobilization in human platelets but not for the stimulus-induced hydrolysis of PIP 2.

We have studied the regeneration of adenosine triphosphate (ATP) in the glycolytic pathway in platelets with a 75% reduction in hexokinase (HK) activity and have investigated aggregation and Ca2+ secretion. HK- deficient platelets had a normal glycolytic flux in the resting state, but responded insufficiently to stimulation with thrombin (5 U/ml). In contrast, glycogen contents and glycogenolysis were normal. When the metabolic adenine nucleotides were labeled with 14C-adenine, the patient's platelets showed a normal adenylate energy charge and a normal level of 14C-ATP. However, the inhibitor of mitochondrial energy generation, CN-, induced a weaker fall in 14C-ATP in the patient's platelets than in the controls. Analysis of secretion markers revealed decreased amounts of granule-bound ATP and secretable Ca2+, whereas granule-bound adenosine diphosphate (ADP), beta-thromboglobulin, N- acetyl-beta-D-glucosaminidase, and beta-glucuronidase were within the normal range. Aggregation and Ca2+ secretion induced by 5 U/ml thrombin were normal and were not changed in the presence of inhibitors of mitochondrial and glycogenolytic energy generation. Aggregation was also normal at 0.1 U/ml thrombin and was independent of these inhibitors, but Ca2+ secretion was greatly impaired when mitochondrial and glycogenolytic ATP resynthesis was abolished. These findings indicate that a severe reduction in HK activity causes insufficient acceleration of the glycolytic flux during stimulation with thrombin. This leads to impaired dense granule secretion in conditions where secretion depends on concurrent ATP resynthesis and glycolysis is rate limiting.

Stimulation of human platelets increases cytoplasmic pH (pHi) via activation of Na+/H+exchange. We have determined the effect of inhibiting Na+/H+exchange on (i) thrombin‐induced Ca2+mobilization and (ii) turnover of 32P‐labelled phospholipids. Blocking Na+/H+exchange by removal of extracellular Na+or by ethylisopropylamiloride (EIPA) inhibited Ca2+mobilization induced by 0.2 thrombin, whereas increasing pHiby NH4C1 enhanced the thrombin‐induced increase in cytosolic free Ca2+. The effect of EIPA was bypassed after increasing pHiby moneasin. The thrombin‐induced cleavage of phosphatidylinositol 4,5‐bisphosphate (PIP2) was unaffected by treatments that blocked Na+/H+exchange or increased pHi. It is concluded that activation of Na+/H+exchange is a prerequisite for Ca2+mobilization in human platelets but not for the stimulus‐induced hydrolysis of PIP2.

Among the different platelet responses, secretion requires the greatest amount of metabolic energy. The velocities of dense, alpha- and acid hydrolase granule secretion vary in parallel with the increase in energy consumption seen in thrombin-stimulated cells. This covariance is preceded by a phase in which energy consumption is increased without the extracellular appearance of secretion markers. By treating the platelets with thrombin and hirudin we have stimulated the platelets for short intervals and succeeded in separating shape change, single platelet disappearance and secretion to a great extent. In this report we show that the early increase in energy consumption reflects the energy requirement of aggregation but not of shape change. The cost of 100% of single platelet disappearance is 2.8 mumol of ATPeq. X (10(11) platelets)-1. Concurrent analysis of phosphorylation of Mr 20 000 and 47 000 proteins and of 32P-labelled phosphatidylinositol metabolites led to the following observations. Firstly, shape change is neither accompanied by an increase in protein phosphorylation nor by changes in the steady state levels of 32P-labelled phosphatidylinositol metabolites. Secondly, when aggregation occurs both proteins are phosphorylated, but the phosphatidylinositol metabolites do not change. Thirdly, when secretion follows, more phosphorylation of the Mr 47 000 protein occurs and initially only phosphatidic acid accumulates. At a later stage of the secretion responses, more protein phosphorylation and phosphatidic acid accumulation become evident, and are now accompanied by alterations in the steady state levels of 32P-labelled (poly)phosphoinositides. Hence, the early increase in energy consumption coincides with protein phosphorylation and, at a later stage, with alterations in (poly)phosphoinositides metabolites. This demonstrates that metabolic energy is directly involved in stimulus-response coupling in aggregating platelets.

A novel method has been developed for rapid and quantitative determination of the rate of energy consumption in platelets. In platelets suspended in a cyanide-containing medium. ATP resynthesis is abruptly blocked by addition of 2-deoxyglucose and D-glucono-1,5-lactone. We demonstrate that the subsequent changes in the levels of cytoplasmic ATP and ADP reflect the velocity of energy consumption in the platelets immediately before addition of the inhibitors. Despite the arrest in ATP resynthesis the platelets remain responsive to stimulation by thrombin (5 units x ml-1) which triggers the secretion of the contents of dense, alpha- and acid hydrolase granules. Unstimulated platelets were found to consume about 3.5 and 0.5 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at 37 degrees C and 15 degrees C, respectively; the thrombin-treated platelets consumed respectively 16 and 2 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at these temperatures. When the velocity of energy consumption was varied by (a) changing the temperature and (b) preincubation with glyco(geno)lytic inhibitors, it was found to be linearly related to the initial rate of secretion from the three types of granules. The precise nature of this relationship differed between the three types of secretion responses and indicated an increasing requirement for metabolic energy for secretion from the three types of granules in the order: dense granule less than alpha-granule less than acid hydrolase granule. The results obtained with changes in temperature were superimposable on those obtained with the glyco(geno)lytic inhibitors for dense granule secretion and alpha-granule secretion, suggesting an apparent coupling between energy consumption and the rate of these secretion responses. The rate of secretion of acid hydrolase was always higher when energy consumption was varied by temperature changes than when glyco(geno)lytic inhibitors were used, probably as a result of metabolic changes prior to induction of secretion. On the basis of these experiments, we calculated an incremental energy consumption during complete secretion of dense, alpha- and acid hydrolase granule contents of 2.5, 4.2 and 6.7 mumol of ATP equivalents x (10(11) platelets)-1, respectively.