Effects of a monoclonal antibody to glycoprotein IIb/IIIa (P256) and of enzymically derived fragments of P256 on human platelets.

The anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10(-9)-3 x 10(-8) M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10(-7) M. P256-induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In-111-labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications.

Measurement of fibrinogen binding to platelets in whole blood by flow cytometry: a micromethod for the detection of platelet activation.

Platelet fibrinogen binding in whole blood has been measured in vitro by flow cytometry using a commercially available, fluorescein isothiocyanate (FITC)-conjugated polyclonal antifibrinogen antibody. Fibrinogen-antifibrinogen immune complexes were formed in experimental conditions approaching antigen-antibody equivalence, but optimal reaction conditions in which their formation was prevented or minimized could be achieved. Immune complex formation was associated with fibrinogen binding to unstimulated platelets but did not significantly affect ADP-induced fibrinogen binding. Half-maximal fibrinogen binding occurred at about 0.4 microM ADP, and ADP-induced fibrinogen binding continued progressively during 20 min incubation with 10 microM ADP. Fibrinogen binding correlated closely with platelet glycoprotein IIb-IIIa expression in members of a family with Glanzmann's thrombasthenia, and, in double labelling experiments, with the binding of PAC1, a monoclonal antibody that binds to GP IIb-IIIa only after the exposure of fibrinogen receptors. These studies show that platelet fibrinogen binding can be reliably measured in whole blood by means of a polyclonal antifibrinogen antibody which does not discriminate between plasma and platelet-bound fibrinogen, despite the presence of an approximately 100-fold excess of the former.

The action of ticlopidine on human platelets. Studies on aggregation, secretion, calcium mobilization and membrane glycoproteins.

The effects on platelet function of a 5-day course of Ticlopidine (Tcl) have been studied in two groups of volunteers receiving different dosage schedules. Tcl had a relatively greater inhibitory effect on aggregation induced by ADP than by other agonists, and a greater effect, in contrast to that of an ADP receptor antagonist, on the second phase than on the initial rate of aggregation. Tcl inhibited ATP secretion in response to ADP and 0.05 u/ml thrombin, but not to higher concentrations of thrombin or to calcium ionophores. No inhibitory effect was observed on Ca2+ influx or intracellular mobilization, on the binding of monoclonal antibodies to the glycoprotein IIb-IIIa complex or on the state of association of the complex. We suggest that Tcl neither inhibits the binding of ADP to its receptor nor acts directly on the fibrinogen binding site, but that it may inhibit a step in signal transduction between these two events.

Potentiation by adrenaline of Ca2+ influx and mobilization in stimulated human platelets: dissociation from thromboxane generation and aggregation.

In a medium containing 1 mM extracellular Ca2+ (Ca2+o), the prior addition of 0.5 microM adrenaline to quin 2-loaded human platelets increased both the rate and amplitude of the rise in cytosolic free Ca2+ (Ca2+i) in response to sub-threshold concentrations of thrombin and PAF and these effects were not prevented by blocking either fibrinogen binding and aggregation or cyclo-oxygenase. In the presence of 2 mM EGTA [( Ca2+o] less than 100 nM), the rate, but not the extent of rise of [Ca2+i] was enhanced by adrenaline, and this was also unaffected by blockade of cyclo-oxygenase. Addition of adrenaline 1 min after the other agonist in the presence of 1 mM Ca2+o resulted in aggregation without further elevation of [Ca2+i]. Adrenaline thus enhances both influx and intracellular mobilization of Ca2+ by a mechanism independent of both fibrinogen binding and thromboxane production, but these effects do not fully explain its potentiation of aggregation by other agonists.

Grey platelet syndrome: studies on platelet alpha-granules, lysosomes and defective response to thrombin.

The platelets of a young man with the grey platelet syndrome were severely depleted of all seven alpha-granule proteins assayed as well as partially deficient in alpha-mannosidase and alpha-fucosidase; four other lysosomal enzymes were present in normal concentrations. Total platelet 5-hydroxytryptamine (5HT) and adenine nucleotides were normal, and 14C-5HT uptake reached normal levels only slightly more slowly than a control. Aggregation and dense body secretion occurred normally in response to ADP, adrenaline, collagen, PAF-acether, sodium arachidonate, A23187, Ionomycin, TPA and U44069, but were very delayed in response to thrombin. The increase in cytosolic free calcium in response to thrombin was very slow and much reduced in amplitude, whether in the presence or absence of extracellular Ca2+. These defects in response to thrombin were not corrected by the separate addition of purified alpha-granule proteins or by a whole releasate from normal platelets. It is suggested that these platelets, in addition to their alpha-granule deficiency, may have a specific defect of thrombin receptor-mediated activation of phospholipase C.

Differential effects of 12-O-tetradecanoylphorbol 13-acetate on platelet responses to various agonists in the presence and absence of extracellular Ca2+.

In the presence of 1 mM extracellular Ca2+ (Ca2+o), incubation of washed, quin 2 loaded platelets with a low concentration (1 nM) of 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited platelet shape change, aggregation, ATP secretion and cytosolic calcium (Ca2+i) fluxes in response to thrombin, platelet activating factor (PAF), adenosine diphosphate (ADP), vasopressin (VP) and the thromboxane mimetic U46619, but potentiated platelet activation induced by the calcium ionophores, A23187 and ionomycin, the Ca2+ flux remaining unaltered. In the presence of less than 100 nM Ca2+o, TPA again inhibited shape change but potentiated ATP secretion induced by all agonists, even those in which the cytosolic calcium flux was inhibited. Addition of TPA 30 seconds after a low dose of agonist, sufficient to elevate [Ca2+i] to about 250 nM without causing aggregation, induced substantial aggregation and dense granule secretion without affecting Ca2+ flux. These results confirm that very slight elevation of [Ca2+i] above resting levels greatly enhances the effect of low concentrations of TPA, and suggest that protein kinase C activation may exert a feedback inhibition of receptor-mediated shape change, aggregation, ATP secretion and Ca2+ mobilization.

Glycoprotein IIb-IIIa complex and Ca2+ influx into stimulated platelets.

Changes in intracellular Ca2+ concentrations [( Ca2+]i) in platelets stimulated with aggregating agents were measured with the fluorescent indicator dye quin 2. Ca2+ influx, but not intracellular mobilization, in response to adenosine diphosphate (ADP), platelet aggregating factor (PAF-acether), and sodium arachidonate was significantly inhibited by monoclonal antibodies against the glycoprotein (GP) IIb-IIIa complex; inhibition of thrombin-stimulated influx was inhibited to a lesser extent and reached statistical significance only at thrombin concentrations of 0.1 U/mL and below. Anti-GP Ib and HLA-ABC monoclonal antibodies had no effect on Ca2+ influx in response to any agonist. Thrombasthenic platelets gave normal [Ca2+]i responses to ADP and thrombin, which were not inhibited by an anti-GP IIb-IIIa antibody. It is suggested that Ca2+ influx in response to weak agonists occurs predominantly via a channel closely adjacent to the GP IIb-IIIa complex, but that higher concentrations of thrombin and A23187 also stimulate influx via another pathway.