Cytoskeletal changes in platelets induced by thrombin and phorbol myristate acetate (PMA).

Changes in shape, and aggregation that accompanies platelet activation, are dependent on the assembly and reorganization of the cytoskeleton. To assess the changes in cytoskeleton induced by thrombin and PMA, suspensions of aspirin-treated,32P-prelabeled, washed pig platelets in Hepes buffer containing ADP scavengers were activated with thrombin, and with PMA, an activator of protein kinase C. The cytoskeletal fraction was prepared by adding Triton extraction buffer. The Triton-insoluble (cytoskeletal) fraction isolated by centrifugation was analysed by SDS-PAGE and autoradiography. Incorporation of actin into the Triton-insoluble fraction was used to quantify the formation of F-actin. Thrombin-stimulated platelet cytoskeletal composition was different from PMA-stimulated cytoskeletal composition. Thrombin-stimulated platelets contained not only the three major proteins: actin (43 kDa), myosin (200 kDa) and an actin-binding protein (250 kDa), but three additional proteins of Mr56 kDa, 80 kDa and 85 kDa in the cytoskeleton, which were induced in by thrombin dose-response relationship. In contrast, PMA-stimulated platelets only induced actin assembly, and the 56 kDa, 80 kDa and 85 kDa proteins were not found in the cytoskeletal fraction. Exposure of platelets to thrombin or PMA induced phosphorylation of pleckstrin parallel to actin assembly. Staurosporine, an inhibitor of protein kinase C, inhibited actin assembly and platelet aggregation induced by thrombin or PMA, but did not inhibit the incorporation of 56 kDa, 80 kDa and 85 kDa into the cytoskeletal fraction induced by thrombin. These three extra proteins seem to be unrelated to the induction of protein kinase C. We conclude that actin polymerization and platelet aggregation were induced by a mechanism dependent on protein kinase C, and suggest that thrombin-activated platelets aggregation could involve additional cytoskeletal components (56 kDa, 80 kDa, 85 kDa) of the cytoskeleton, which made stronger actin polymerization and platelet aggregation more.     

Disaggregation of aggregated platelets by apyrase from the tick, Ornithodoros savignyi

Apyrase, secreted by ticks during feeding, is a platelet aggregation inhibitor that functions as a regulator of the host's hemostatic system. This present study concerns the disaggregation effect of salivary gland apyrase from the tick Ornithodoros savignyi. Secondarily aggregated platelets, disaggregated by apyrase, exhibited a reversal of shape from a spherical (aggregated) form to a discoid form, reminiscent of reversible aggregation at low ADP concentrations in citrated platelet-rich plasma. However, they showed a dilatory open canaliculary system and an absence of granules indicating disaggregation after degranulation had taken place. In contrast, disaggregation by the fibrin(ogen)olytic enzyme, plasmin, showed that platelets degranulated, but retained a spherical form with numerous extended pseudopods. While thrombin had no effect on aggregation or clotting of platelets disaggregated with plasmin, it did activate those platelets disaggregated with apyrase and clotted the plasma. This is the first study to describe the disaggregating effects of tick derived apyrase on aggregated platelets. It also shows that apyrase can disaggregate platelets even after secondary aggregation and degranulation of platelets has taken place. Platelet aggregation is one of the main barriers encountered by ticks during feeding and counteraction of this process by ticks is an important factor for successful feeding.     

Reversal of platelet aggregation by aortic microsomes

The microsomal fraction of dog aortas inhibited human platelet aggregation induced by arachidonic acid, ADP, or thrombin. When aortic microsomes were added to a preparation of irreversibly aggregated platelets, the aggregates dispersed after 4–6 minutes. The fact that aortic microsomes inhibit platelet aggregation induced by ADP suggests that its effect is probably on the cellular function of platelets and not in direct competition against thromboxane A₂.     

Movement of sodium into human platelets

Addition of ADP induces platelets in plasma to undergo shape change from a disc to a spiny sphere and to develop adhesiveness, i.e. to aggregate. The aggregation of human platelets by ADP is associated with a net uptake of Na⁺. The present experiments demonstrate that the induction of shape change by ADP in acidified or EGTA-treated plasma conditions which inhibit aggregation, is also associated with a movement of Na⁺ into platelets. When ADP-induced platelet shape change and aggregation is inhibited by prostaglandin E₁ Na⁺ uptake is also blocked. Platelets aggregated by epinephrine do not take up Na⁺. In a manner analogous to the effect of ADP, polylysine also induces Na⁺ uptake during aggregation. Vasopressin, in a manner analogous to epinephrine, induces aggregation without Na⁺ uptake. The increase in platelet Na⁺ resulting from ouabain inhibition of Na⁺ efflux induces an increase in the aggregation response to ADP and to epinephrine.     

Secretory mechanisms. Behaviour of adenine nucleotides during the platelet release reaction induced by adenosine diphosphate and adrenaline

1. Platelets containing adenine nucleotides labelled with ³H and ¹⁴C in vitro were aggregated biphasically with ADP and adrenaline. Amounts of ATP and ADP as well as the radioactivity of ATP, ADP, AMP, IMP, hypoxanthine and adenine were determined in platelets and plasma at different stages of aggregation. 2. ATP and ADP were released during the second aggregation phase and had a low specific radioactivity compared with the ATP and ADP retained by the cells. The specific radioactivity of intracellular nucleotides increased during release. The parameters observed with ADP and adrenaline as release inducers were the same as for collagen and thrombin. 3. Release induced by all four inducers was accompanied by conversion of cellular [³H]ATP into extracellular [³H]-hypoxanthine. By variation of temperature, inducer concentration, time after blood withdrawal and use of acetylsalicylic acid, the aggregation pattern caused by adrenaline and ADP could be made mono- or bi-phasic. Release or second-phase aggregation was intimately connected with the ATP–hypoxanthine conversion, whereas first phase aggregation was not. 4. The [³H]ATP–hypoxanthine conversion started immediately after ADP addition. With adrenaline it usually started with the appearance of the second aggregation phase. The conversion was present during first phase of ADP-induced aggregation only if a second phase were to follow. 5. When secondary aggregation took place while radioactive adenine was being taken up by the platelets, increased formation of labelled hypoxanthine still occurred, but there was either no change or an increase in the concentration of labelled ATP. 6. Biphasically aggregated platelets converted [³H]adenine more rapidly into [³H]-ATP and -hypoxanthine than non-aggregated platelets. Addition of [³H]adenine at different stages of biphasic aggregation showed that more [³H]hypoxanthine was formed during than after the release step. 7. We conclude that ADP and adrenaline, like thrombin and collagen, cause extrusion of non-metabolic granula-located platelet adenine nucleotides. During release metabolic ATP breaks down to hypoxanthine, and this process might reflect an ATP-requiring part of the release reaction.     

Differential effect of retinoic acid on ADP and collagen induced platelet aggregation

Retinoic acid (RA) was found to inhibit ADP induced but not collagen induced aggregation of human platelets and the differential action is related to intraplatelet Ca2+ reflux. RA was active at concentrations as low as 10(-7) M and required 20 min prior incubation with platelet suspension in order to inhibit aggregation by ADP. All the steps in ADP induced but not collagen induced platelet activation, viz. hydrolysis of phosphatidyl inositol, phosphorylation of 20, 47 and 250 kDa proteins as well as increased association of actin with Triton X-100 insoluble cytoskeletal matrix were inhibited by RA. RA when used as an agent for differentiation induction of cell progenitor is likely to affect the platelet aggregation and thereby the haemostatic process.     

Submaximal inhibition of protein kinase C restores ADP-induced dense granule secretion in platelets in the presence of Ca2+

Protein kinase C (PKC) is a family of serine/threonine kinases that play isoform-specific inhibitory and stimulatory roles in platelet activation. We show here that the pan-PKC inhibitor Ro31-8220 can be used to dissect these events following platelet activation by ADP. Submaximal concentrations of Ro31-8220 potentiated aggregation and dense granule secretion to ADP in plasma anticoagulated with citrate, in D-Phe-Pro-Arg-chloromethyl ketone-anticoagulated plasma, which has physiological levels of Ca(2+), and in washed platelets. Potentiation was retained on inhibition of cyclooxygenase and was associated with an increase in intracellular Ca(2+). Potentiation of aggregation and secretion was abolished by a maximally effective concentration of Ro31-8220, consistent with a critical role of PKC in secretion. ADP-induced secretion was potentiated in the presence of an inhibitor of PKCβ but not in the presence of available inhibitors of other PKC isoforms in human and mouse platelets. ADP-induced secretion was also potentiated in mouse platelets deficient in PKCε but not PKC. These results demonstrate that partial blockade of PKC potentiates aggregation and dense granule secretion by ADP in association with increased Ca(2+). This provides a molecular explanation for the inability of ADP to induce secretion in plasma in the presence of physiological Ca(2+) concentrations, and it reveals a novel role for PKC in inhibiting platelet activation by ADP in vivo. These results also demonstrate isoform-specific inhibitory effects of PKC in platelets.     

The roles of LAT in platelet signaling induced by collagen, TxA2, or ADP

The work presented here demonstrates that platelets from mice lacking LAT (linker for the activation of T cells) show reversible aggregation in response to concentrations of collagen that cause TxA2/ADP-dependent irreversible aggregation of control platelets. The aggregation defect of the LAT-deficient platelets was shown to be the result of almost no TxA2 production and significantly diminished ADP secretion. In contrast, the LAT deficiency does not affect aggregation induced by high concentrations of collagen because that aggregation is not dependent on TxA2 and/or ADP. Even though ADP and TxA2 provide amplification signals for platelet activation in response to low concentrations of collagen, LAT-deficient platelets hyperaggregate to low levels of U46619, a TxA2 analog, or ADP. Though the mechanism(s) of costimulatory signals by collagen, ADP, and TxA2 remains unidentified, it is clear that LAT plays a positive role in collagen-induced, TxA2/ADP-dependent aggregation, and a negative role in TxA2 or ADP-induced platelet aggregation.     

Platelet function testing in the pony

Platelet isolation techniques and platelet function were evaluated in 35 adult ponies. Platelet recovery from whole blood was consistent and the preparation of platelet rich plasma was facilitated by an enhanced erythrocyte sedimentation rate. All platelet samples aggregated in response to 10 microM ADP. However, concentrations of ADP as high as 100 microM did not elicit significant 14C-serotonin release. Collagen induced irreversible platelet aggregation and 14C-serotonin release in all samples. The threshold dose for collagen in most ponies was 1.5 micrograms. Arachidonic acid (500 microM) failed to induce irreversible platelet aggregation or 14C-serotonin release in any of the samples evaluated. Pony platelets were nonresponsive to epinephrine (5.5 microM).     

Activation of bovine platelets induced by long-chain unsaturated fatty acids at just below their lytic concentrations,and its mechanism

The effects of long chain unsaturated fatty acids such as linoleic acid on bovine platelets were examined. Not only linoleic acid, but also oleic and linolenic acid, at just below the concentrations causing marked cell lysis, induced an absorbance decrease of the platelet suspension in the presence of Ca²⁺. Since this absorbance decrease was reversed by the addition of EDTA and moreover aggregate formation was found by macroscopic and microscopic observation, it was concluded that unsaturated fatty acids at just below their lytic concentrations caused platelet aggregation. Unsaturated fatty acids also caused release of adenine nucleotides, but there was a lag time between the release and the aggregation, just as with ADP-induced release, suggesting that the aggregation was independent of the release of ADP. It was revealed that this activation of platelets by unsaturated fatty acids was caused by marked Ca²⁺ uptake into the cytoplasm, resulting from significant membrane perturbation.     

Inhibition of ADP-induced platelet activation by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole: Covalent modification of aggregin,a putative ADP receptor

ADP-induced platelet responses play an important role in the maintenance of hemostasis. There has been disagreement concerning the identity of an ADP receptor on the platelet surface. The chemical structure of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) shows considerable resemblance to that of the adenine moiety of adenine-based nucleotides. The reagent has been previously used by other investigators as an affinity label for adenine nucleotide-requiring enzymes, such as mitochondrial ATPase and the catalytic subunit of cAMP-dependent protein kinase. Since ADP-induced platelet responses depend on the binding of ADP to its receptor, we investigated the effect on ADP-induced platelet responses and the nature of ADP-binding protein modified by NBD-Cl. NBD-Cl inhibited ADP-induced shape change and aggregation of platelets in platelet-rich plasma in a concentration- and time-dependent manner. NBD-Cl also inhibited ADP-induced shape change, aggregation, exposure of fibrinogen binding sites, secretion, and calcium mobilization in washed platelets. NBD-Cl did not act as an agonist for platelet shape change and aggregation. Covalent modification of platelets by NBD-Cl blocked the ability of ADP to antagonize the increase in intracellular levels of cAMP mediated by iloprost (a stable analogue of prostaglandin I₂). NBD-Cl was quite specific in inhibiting platelet aggregation by those agonists, e.g., ADP, collagen, and U44619 (a thromboxane mimetic), that completely or partially depend on the binding of ADP to its receptor. Autoradiogram of the gel obtained by SDS-PAGE of solubilized platelets modified by [¹⁴C]-NBD-Cl showed the presence of a predominant radiolabeled protein band at 100 kDa corresponding to aggregin, a putative ADP receptor. The intensity of this band was considerably decreased when platelets were either preincubated with ADP and ATP or covalently modified by a sulfhydryl group modifying reagent before modification by [¹⁴C]-NBD-Cl. These results (1) indicate that covalent modification of aggregin by NBD-Cl contributed to loss of the ADP-induced platelet responses, and (2) suggest that there is a sulfhydryl group in the ADP-binding domain of aggregin. © 1996 Wiley-Liss, Inc.     

Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice.

Platelet activation is characterized by shape change, induction of fibrinogen receptor expression and release of granular contents, leading to aggregation and plug formation. While this response is essential for hemostasis, it is also important in the pathogenesis of a broad spectrum of diseases, including myocardial infarction, stroke and unstable angina. Adenosine 5'-diphosphate (ADP) induces platelet aggregation, but the mechanism for this has not been established, and the relative contribution of ADP in hemostasis and the development of arterial thrombosis is poorly understood. We show here that the purinoceptor P2Y1 is required for platelet shape change in response to ADP and is also a principal receptor mediating ADP-induced platelet aggregation. Activation of P2Y1 resulted in increased intracellular calcium but no alteration in cyclic adenosine monophosphate (cAMP) levels. P2Y1-deficient platelets partially aggregated at higher ADP concentrations, and the lack of P2Y1 did not alter the ability of ADP to inhibit cAMP, indicating that platelets express at least one additional ADP receptor. In vivo, the lack of P2Y1 expression increased bleeding time and protected from collagen- and ADP-induced thromboembolism. These findings support the hypothesis that the ATP receptor P2Y1 is a principal receptor mediating both physiologic and pathological ADP-induced processes in platelets.     

Nucleotide exchange between cytosolic ATP and F-actin-bound ADP may be a major energy-utilizing process in unstimulated platelets

About 40% of the cytosolic ADP of human platelets is tightly bound to protein and the complex is precipitated from the cells by 43% ethanol. We show here that this ADP is bound to F-actin by three criteria (a) copurification with F-actin, (b) specific extraction with water and (c) by specific interaction with DNase I. Platelets contain 0.3 mumol/10(11) cells of this F-actin--ADP complex compared to the total actin content of 0.8 mumol/10(11) cells, which is consistent with the view that actin is maintained in different pools (F-actin--ADP, profilactin, G-actin). In intact platelets the F-actin-bound ADP turns over rapidly and we have determined a turnover rate at 37 degrees C of 0.1 +/- 0.025 s-1 by using a double-labelling procedure. This rapid turnover indicates that F-actin in intact platelets is in a very dynamic state, which may be necessary for rapid responses to stimuli. If it is assumed that the source of the ADP bound to F-actin is cytosolic ATP, the turnover of F-actin ADP measured represents an ATP-consuming process that would account for up to 50% of total ATP consumption in resting platelets.     

Clq inhibition of the interaction of collagen with human platelets.

Human Clq, isolated in pure state after affinity chromatography on IgG-Sepharose, inhibited collagen-induced aggregation and release of 14C-Serotonin from prelabeled human platelets. Platelet aggregation induced by ADP or thrombin was not inhibited by Clq. Also, the adherence of platelets to glass surfaces was significantly diminished by Clq. In contrast, aggregated Clq mimicked the effect of collagen in causing platelet aggregation and release of serotonin. It appears that monomeric Clq, which has structural similarities to collagen competes with collagen for specific sites on the platelet surface.     

Effect of heparin on platelet aggregation in vitro

Heparin added to citrated platelet rich plasma influences shape change and aggregation of platelets in different ways. In the presence of heparin neither ADP nor collagen induces shape change, while shape change after thrombin or arachidonic acid remains unaltered. Heparin potentiates the first aggregation step induced by ADP and epinephrine but inhibits aggregation induced by thrombin and ristocetin. The second phase of aggregation and the release reaction are not directly influenced by heparin no matter which aggregation agent is used.     

Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F- actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.     

Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen-binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto-ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP-induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P less than 0.01) was found between the extent of platelet aggregation and the amount of 125I-fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP-induced platelet aggregation and fibrinogen binding. Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP-induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP-induced platelet aggregation.     

The role of thrombocyte prostanoids and products secreted by dense granules in the platelet attachment, spreading and aggregation on collagen substrates

The role of platelet prostanoids and substances released from dense bodies (ADP and serotonin) in the initial attachment, spreading and aggregation of platelets on surfaces coated with I, III, IV and V genetic types of collagen was investigated. A positive linear correlation was found to exist between thrombi-like aggregate formation on collagen substrates and platelet prostanoid synthesis. No correlation was established between platelet aggregate formation and 14C-serotonin release. The cyclooxygenase inhibitor indomethacin and the antagonists of PG endoperoxides and TXA2 (13-APA and BM 13.177) completely block thrombi-like aggregate formation. Neither 13-APA nor BM 13.177 affect platelet spreading, while indomethacin inhibits this process by 25%. The ADP-scavenger CP/CPK inhibits platelet aggregation and spreading by 25-30%. The inhibitors of cyclooxygenase and CP/CPK do not influence the initial attachment of platelets. The data obtained suggest that thrombi-like aggregate formation on collagen substrates is mediated by the synthesis of PG endoperoxides and TXA2; however, in platelet spreading this synthesis plays a limited role. Spreading and aggregation of platelets on collagen substrates is only partly mediated by ADP and serotonin. Initial attachment of platelets does not depend on ADP and serotonin release and PG endoperoxide/TXA2 synthesis.     

Electronic particle size measurements of platelet aggregates formed in vitro.

The aggregation of human platelets induced by adenosine diphosphate (ADP) was used to evaluate electronic particle size analyzer measurements of platelet aggregates in plasma. As platelets began to clump in plasma, the total volume and the diameter of individual aggregates increased; after a time dependent on experimental conditions, the diameter increased but the total volume remained unchanged. Similar but opposite changes in size distribution occurred during platelet deaggregation. The total volume of aggregates formed in plasma varied (linear correlation coefficient = 0.99) with the total volume of platelets which were available to clump and with simultaneous changes in optical density. The diameter of the aggregates varied with the concentration of, and time of exposure to, ADP and with the total volume of platelets and aggregates in plasma was not different from that of control platelets in untreated plasma, the individual platelets aggregated without an accompanying increase in size. This study demonstrates that platelet aggregation can be characterized by electronic measurements of the size distribution of platelet aggregates.     

Binding of adenosine diphosphate to human blood platelets and to isolated blood platelet membranes

The equilibrium binding of 14C-labeled ADP to intact washed human blood platelets and to platelet membranes was investigated. With both intact platelets and platelet membranes a similar concentration dependence curve was found. It consisted of a curvilinear part below 20 microM and a rectilinear part above this concentration. At high ADP concentrations, the rectilinear part appeared to be saturable. Because of this, two classes of saturable ADP binding sites were proposed. ADP was partly converted to ATP and AMP with intact platelets while this conversion was virtually absent in isolated platelet membranes. ADP was bound to platelet membranes with the same type of curves found for intact platelets. The ADP binding to the high affinity system, which was stimulated by calcium ions, was nearly independent of temperature and had a pH optimum at 7.8. A number of agents were investigated for inhibiting properties. Of the sulfhydryl reagents only p-chloromercuribenzene sulfonate inhibited both high and low affinity binding systems while iodoacetamide and N-ethylmaleimide were without effect. Compounds acting via cyclic AMP on platelet aggregation, such as adenosine and cyclic AMP itself, had no influence on binding. Some nucleosidediphosphates and nucleotide analogs at a concentration of 100 microM had no, or only a slight, effect on high affinity ADP binding. For some other nucleotides inhibitor constants were determined for both platelet ADP aggregation and ADP binding. The inhibitor constants of ATP, adenyl-5'-yl-(beta,gamma-methylene)diphosphate, IDP, adenosine-5'(2-O-thio)diphosphate, for aggregation and high affinity binding were in good correlation with each other. Exceptions formed fluorosulfonylbenzoyl adenosine and AMP. The ATP formation found with intact platelets could be attributed to a nucleosidediphosphate kinase. It was investigated in some detail. The enzyme was magnesium dependent, had a Q10 value of 1.41, a pH optimum at 8.0, was competitively inhibited by AMP and reacted via a ping pong mechanism. All findings described in this paper indicate that platelets as well as platelet membranes bind ADP with the same characteristics and they suggest that the high affinity binding of ADP is involved in platelet aggregation induced by ADP. The results on nucleosidediphosphate kinase did not permit a firm conclusion about the role of the enzyme in induction of platelet aggregation by ADP.