With a combined phase-contrast and fluorescence video imaging system, changes in morphology and cytosolic [Ca2+]i were investigated of fura-2–loaded platelets during adhesion to fibrinogen or collagen matrices. The Ca2+ signals were, on the level of single platelets, compared to the secretion and procoagulant responses, using fluorescent-labeled AK-6 antibody against P-selectin and labeled annexin V for detection of surface-exposed phosphatidylserine (PS), respectively. Platelets in contact with fibrinogen developed filapods and spread over the matrix, in most of the cells without detectable Ca2+ signal. Thrombin induced repetitive spiking in [Ca2+]i , followed by the expression of P-selectin but not of PS on the platelet surface. Platelet interaction with collagen resulted in spreading and transformation of the cells into blebbing, “balloon”-like structures (diameter about 5 μm). The latter morphological changes were accompanied by high and prolonged increases in [Ca2+]i , by the exposure of both P-selectin and PS, and by the ability of the platelets to convert prothrombin into thrombin. Thrombin addition accelerated the onset of the Ca2+ signals and the appearance of surface-exposed PS. Collagen-induced PS exposure was slightly reduced by treatment of the platelets with aspirin, and strongly inhibited by suppression of the Ca2+ responses with prostaglandin E1 or the Ca2+ chelator, dimethyl-BAPTA. Inhibition of protein tyrosine phosphorylation with genistein, U73343, or wortmannin resulted in spiking Ca2+ responses in many of the platelets and in almost complete reduction of bleb formation and PS exposure. In contrast, genistein did not suppress bleb formation and PS exposure of platelets stimulated with the Ca2+ ionophore A23187. We conclude that a collagen but not fibrinogen matrix acts as a potent activator of the procoagulant response through activation of tyrosine kinases and subsequent generation of sustained intracellular Ca2+ signals.

2؉ responses, and also on integrin ␣ IIb ␤ 3 exposure and platelet aggregation, were abolished by pharmacological stimulation of cAMP-dependent protein kinase, and these effects were mimicked by inhibition of this activity. In permeabilized platelets, UK14304 and EPI potentiated InsP 3 -induced, CICR-mediated mobilization of Ca 2؉ from internal stores in a similar way as did inhibition of cAMP-dependent protein kinase. In summary, a G i␣ -mediated decrease in cAMP level appears to play a major role in the platelet-activating effects of ␣ 2A -adrenergic receptor stimulation. Thus, in platelets, unlike other cell types, occupation of the G i␣ -coupled ␣ 2A -adrenergic receptors does not result in phospholipase C activation but rather in modulation of the Ca 2؉ response by relieving cAMP-mediated suppression of InsP 3 -dependent CICR.In most cell types, the ␣ 2A -adrenergic receptor is linked to a G i protein, and thus receptor occupation inhibits adenylate cyclase activity in a pertussis toxin-sensitive manner. In human platelets, containing various isoforms of both ␣-and ␤-adrenergic receptors, it appears to be mainly the ␣ 2A -receptor type that is responsible for the platelet-activating effect of epinephrine (EPI) 1 and other catecholamines (1-4). Thus, in platelets, EPI causes activation of G i␣2 followed by adenylate cyclase inhibition (5, 6). Consequently, EPI efficiently antagonizes the cAMP-elevating effect of G s␣ -stimulating agents like prostacyclin and prostaglandin E 1 (PGE 1 ) (7-9). In addition, EPI evokes a range of functional platelet responses, such as activation of encrypted integrin ␣ IIb ␤ 3 (fibrinogen) receptors followed by platelet aggregation and, in the presence of other platelet agonists, increased exocytosis (10 -13). However, which signaling events, putatively downstream of G i , underlie these platelet reactions has long remained unclear. Earlier data from the literature suggest that a fall in cAMP level as such is insufficient to activate platelets (14 -16), implying that EPI may activate other, G i -independent pathways. For instance, EPI can induce a transient increase in cytosolic [Ca 2ϩ ] i in platelets charged with the Ca 2ϩ -sensitive photoprotein, aequorin, although this is not the case for platelets loaded with the Ca 2ϩ probes Quin-2 or Fura-2 (7, 17). In addition, EPI potentiates phosphoinositide hydrolysis evoked by other receptor agonists, due to a stimulation of ADP release and/or thromboxane A 2 formation (18 -20), or by enhancing the coupling of the other receptors with phospholipase C (16, 21). Another early proposal is that EPI may act through stimulation of the Na ϩ /H ϩ exchanger in the plasma membrane (22), although this effect could later be ascribed to an involvement of protein kinase C (23). A final suggestion is that EPI may act by inhibiting the GTPase-activating protein, Rap1B-GAP (24). Intriguingly, however, most or all of these EPI effects are also under control of the cAMP concentration (7,24), which may point to a possible G i -mediated effect.In a v...

Scott syndrome is an hereditary bleeding disorder characterized by a deficiency in platelet procoagulant activity. Unlike normal blood cells, Scott platelets, as well as erythrocytes and lymphocytes, are strongly impaired in their ability to scramble their membrane phospholipids when challenged with Ca2+. In normal cells this collapse of membrane asymmetry leads to surface exposure of phosphatidylserine. Here we report that Scott erythrocytes show an apparent defect in tyrosine phosphorylation on treatment with Ca2+-ionophore. Diminished tyrosine phosphorylation was also apparent in activated Scott platelets, but much less pronounced than observed in red blood cells. On the other hand, tyrosine phosphorylation profiles observed in Scott red blood cell ghosts after sealing in the presence of adenosine triphosphate (ATP) were indistinguishable from those obtained from normal ghosts. Several observations argue in favor of a mechanism in which tyrosine phosphorylation in red blood cells is facilitated by, rather than required for scrambling of membrane lipids. Staurosporin blocks tyrosine phosphorylation in normal red blood cells, but does not inhibit the lipid scrambling process. White ghosts from normal erythrocytes, resealed in the absence of ATP, exhibit Ca2+-induced lipid scrambling without tyrosine phosphorylation. A selective inhibitor of Ca2+-induced lipid scrambling also showed an apparent inhibition of tyrosine phosphorylation in ionophore-treated normal red blood cells, similar to that observed in Scott erythrocytes. While this inhibitor also suppressed Ca2+-induced lipid scrambling in ghosts that were sealed in the presence of ATP, it did not inhibit tyrosine kinase activity. We conclude that the apparent deficiency in tyrosine phosphorylation in Scott cells is an epiphenomenon, possibly associated with a defect in phospholipid scrambling, but not causal to this defect.