Direct evidence for the interaction of the nucleotide affinity analog 5'-p-fluorosulfonylbenzoyl adenosine with a platelet ADP receptor

Previous reports have indicated that the nucleotide affinity analog 5'- p-fluorosulfonylbenzoyl adenosine (FSBA) at concentrations between 40 and 100 mumol/L and at times greater than 20 minutes covalently modifies a single protein component on the external platelet membrane surface and that adenosine diphosphate (ADP) protects against this reaction. That this protein is an ADP receptor linked to platelet activation is shown by FSBA inhibition of ADP-mediated platelet shape change, aggregation, and fibrinogen receptor exposure. In this report, further evidence for the interaction of FSBA with the ADP receptor on platelets is provided by the observation that FSBA at high concentrations (100 to 500 mumol/L) behaves as a weak agonist to produce platelet shape change within one minute as detected by spectroscopic assay and scanning electron microscopy with concomitant phosphorylation of the light chain of platelet myosin. The specificity of FSBA as an agonist is demonstrated by inhibition of FSBA-induced shape change by ATP and the covalent incorporation of SBA as well as the failure of 5'-fluorosulfonylbenozoyl guanosine (FSBG) to cause shape change. In contrast, incubation of platelets with low concentrations of [3H]-FSBA (40 mol/L) is not associated with stimulation of platelet shape change or myosin light chain phosphorylation.     

Thromboxane A2/prostaglandin endoperoxide (TXA2/PG-END) receptor binding properties in human platelets of ridogrel, a combined TXA2 synthase inhibitor--TXA2/PG-END receptor antagonist.

Ridogrel, a potent thromboxane A2 (TXA2) synthase inhibitor, also has thromboxane A2 prostaglandin endoperoxide (TXA2/PG-END) receptor antagonistic properties as documented in functional studies of human platelets. In the present study, the binding affinities of the TXA2 synthase inhibitors, ridogrel, dazoxiben, dazmegrel and pirmagrel, and the TXA2/PG-END receptor antagonists, GR32191, L670596, SQ29548, ICI159995, AH69212 and sulotroban, for the TXA2/PG-END receptor labelled with [3H]SQ29548 on intact human platelets were assessed. The potencies of the TXA2/PG-END receptor antagonists to inhibit specific [3H]SQ29548 binding to intact human platelets ranged between 1.2 nM and 6,200 nM and corresponded to the ability of the drugs to suppress human platelet aggregation induced by TXA2/PG-END receptor stimulation with U46619 and collagen. The TXA2 synthase inhibitors dazoxiben, dazmegrel and pirmagrel could not inhibit specific [3H]SQ29548 binding to intact human platelets, tested up to 10(-5) M, nor suppress human platelet aggregation, indicating lack of any receptor antagonistic properties. Ridogrel, however, directly bound to the TXA2/PG-END receptor with micromolar affinity (IC50 = 5.2 microM) and inhibited U46619-27, or collagen-induced platelet aggregation, with ED50-values of 27 microM and 4.7 microM respectively. The present study thus demonstrates that antagonism by ridogrel of TXA2/PG-END receptor activation on platelets as defined in functional tests, coincides with inhibition of specific ligand binding to the receptors.     

Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate.

This study characterizes a congenital hemorrhagic disorder caused by a platelet function defect with the following features: (1) severely impaired platelet aggregation and fibrinogen or von Willebrand factor (vWF) binding induced by adenosine diphosphate (ADP); (2) defective aggregation, release reaction, and fibrinogen or vWF binding induced by other agonists; (3) normal aggregation and release reaction induced by high concentrations of thrombin or collagen; (4) no further inhibition by ADP scavengers of aggregation, release reaction, and fibrinogen or vWF binding, comparable with those observed for normal platelets in the presence of ADP scavengers; (5) normal membrane glycoprotein (GP) composition and normal binding of the anti-GP IIb/IIIa monoclonal antibody 10E5; (6) no acceleration by ADP of binding of the anti-GP IIb/IIIa monoclonal antibody 7E3; (7) normal platelet-fibrin clot retraction if induced by thrombin or reptilase plus epinephrine, absent if induced by reptilase plus ADP; (8) no inhibition by ADP of the prostaglandin E1-induced increase in platelet cyclic adenosine monophosphate, but normal inhibition by epinephrine; (9) defective mobilization of cytoplasmic Ca2+ by ADP; (10) normal binding of 14C-ADP to fresh platelets, but defective binding of [2-3H]-ADP to formalin-fixed platelets. This congenital platelet function defect is characterized by selective impairment of platelet responses to ADP, caused by either decreased number of platelet ADP receptors or abnormalities of the signal-transduction pathway of platelet activation by ADP.     

Platelet aggregation by fibrinogen polymers crosslinked across the E domain

There is evidence that platelet interactions with artificial surfaces are mediated by plasma proteins, especially fibrinogen, adsorbed on the surfaces. Multiple site interactions between fibrinogen molecules adsorbed in high concentration and receptors in the unactivated platelet may be sufficient for platelet adhesion and subsequent activation. To examine this hypothesis, we prepared soluble polymers of fibrinogen. Polymers produced by interaction of fibrinogen with Fab'2 fragments of antibodies against fibrinogen's E (central) domain (Fg- Fab'2(E] induced, in gel-filtered platelets, aggregation and serotonin release, which were blocked by monoclonal antibodies against the GPIIb/IIIa complex, by Fab fragments against the D domain, and by metabolic inhibitors; aggregation was attenuated but not abolished by enzymatic removal of ADP (with CP/CPK) or by blockage of ADP binding sites (with FSBA), and when secretion was inhibited by aspirin. Fg- Fab'2(E) also induced a dose-dependent elevation in cytoplasmic Ca2+ (measured by Aequorin luminescence) which was attenuated by CP/CPK and by FSBA, and was eliminated by metabolic inhibitors and by anti- IIb/IIIa antibody. Fibrinogen complexes crosslinked with dimethylsuberimidate or Factor XIII neither aggregated gel-filtered platelets nor inhibited platelet aggregation by ADP and fibrinogen, probably because of inaccessibility of lysine residues in the D (terminal) domain of fibrinogen, which are thought to be required for platelet binding. Thus, soluble complexes of fibrinogen having multiple available platelet receptor recognition sites activate gel-filtered platelets and may provide a useful model for platelet-surface interactions mediated by adsorbed fibrinogen.     

ADP-dependent common receptor mechanism for binding of von Willebrand factor and fibrinogen to human platelets.

Human von Willebrand factor (vWF) and fibrinogen are adhesive plasma glycoproteins essential for formation of a platelet hemostatic plug. We investigated the role of ADP and fibrinogen in binding of vWF to platelets in vitro. Binding of 125I-labeled vWF to human platelets separated from plasma proteins and treated with ADP was specific, and time and concentration dependent, reaching equilibrium at 20 min and approaching saturation at 12 micrograms/ml. The binding was inhibited by EDTA and by prostaglandin I2, a known activator of platelet adenylate cyclase. A purine nucleotide affinity analog, 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), which covalently modifies the ADP binding sites on the human platelet membrane, prevented binding of vWF induced with ADP, as well as with human thrombin and with ionophore A23187, agents known to cause platelet ADP secretion. By comparison, FSBA did not inhibit binding of vWF induced by ristocetin, indicating that the ristocetin mechanism is not dependent on ADP. Human fibrinogen inhibited in a competitive manner the ADP-induced binding of 125I-labeled vWF (9 micrograms/ml) with an IC50 of 25 micrograms/ml. Conversely, unlabeled vWF inhibited ADP-induced binding of 125I-labeled fibrinogen (60 micrograms/ml) with an IC50 of 16 micrograms/ml. A synthetic dodecapeptide (Mr, 1188), analogous with the specific platelet receptor recognition site of human fibrinogen gamma chain (gamma 400-411), inhibited binding of both 125I-labeled vWF and 125I-labeled fibrinogen to ADP-treated platelets, whereas it was without effect on binding of 125I-labeled vWF to ristocetin-treated platelets. These data indicate that vWF and fibrinogen have a common receptor mechanism for their interaction with human platelets that is dependent on ADP occupancy of its binding sites and is recognized by the sequence of 12 amino acid residues at the carboxyl terminus of the human fibrinogen gamma chain.     

Constitutional Thrombocytopathy with Subnormal Response to Thromboxane A2

S ummary. A new type of congenital platelet dysfunction was found in a young woman presenting a life-long bleeding disorder. The known types of thrombopathia and von Willebrand's disease were excluded by appropriate investigations. The platelets were morphologically normal, underwent normal shape change and contraction and synthesized thromboxane A₂ (TXA₂) normally. The release reaction was abnormal and the aggregation response to ADP, adrenalin, collagen, thrombin, sodium arachidonate and vasopressin was depressed due to decreased sensitivity of the platelets to prostaglandin endoperoxides and TXA2. Platelet cAMP content was increased.     

Increased binding of fibrinogen to platelets in diabetes: the role of prostaglandins and thromboxane

Previous studies suggested a role for prostaglandins or thromboxane A2, or both in the exposure of fibrinogen receptors on normal platelets in response to several aggregating agents. Platelets from diabetics are known to be more sensitive to aggregating agents and to produce more prostaglandins and thromboxane than platelets from normal subjects. We compared fibrinogen binding to platelets from diabetic subjects with binding to platelets from normal subjects and determined whether aspirin (which inhibits the formation of prostaglandins and thromboxane) would inhibit the binding of fibrinogen to platelets from diabetic subjects and whether this correlated with its effects on platelet aggregation. We found the following: Aspirin suppressed thromboxane formation and rendered the platelets less sensitive to the induction of aggregation by adenosine diphosphate (ADP) or collagen. The amount of U-46619 [( 15s]-hydroxy-11-alpha, 9-alpha [epoxy-methano]- prosta[5Z,13E]-dienoic acid, a stable analog of prostaglandin endoperoxide/thromboxane A2) necessary to induce aggregation, was similar in normal and diabetic subjects and was unchanged after ingestion of aspirin. Binding of 125I-fibrinogen following stimulation of platelets by ADP or collagen was greater in diabetic (because more binding sites were exposed) than in normal subjects. However, following stimulation by U-46619, binding was similar in diabetic and normal subjects. Aspirin caused a reduction in the exposure of binding sites on both platelets from diabetic and normal subjects, so that (in this respect) platelets from diabetic subjects became more like those from normal subjects. Effects of the monoclonal antibody B59.2, which is specific for the platelet glycoprotein IIb-IIIa complex (the presumed receptor for fibrinogen on the platelet surface) were also studied. The amount of this antibody that bound to platelets was the same for normal and diabetic subjects both before and after aspirin and with or without stimulation by ADP or collagen. In addition, B59.2 inhibited aggregation and fibrinogen binding in both platelets from diabetic and normal subjects. The combined data suggest that the glycoprotein IIb- IIIa complex of platelets from diabetic subjects is similar to that of platelets from normal subjects and that the increased fibrinogen binding and aggregation of platelets from diabetic subjects in response to ADP or collagen is mediated by increased formation of prostaglandin endoperoxide or thromboxane A2, or both.     

Platelet shape change and Ca2+ mobilization induced by collagen, but not thrombin or ADP, are inhibited by phenylarsine oxide

Summary. In this report we have examined the effects of the protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO) on receptor-mediated platelet shape change, secretion and aggregation. PAO was found to inhibit platelet aggregation induced by collagen, thrombin, ADP and epinephrine at IC₅₀ values of 0.35 μmol/l, 2.5 μmol/l, 0.2 μmol/l and 0.3 μmol/l, respectively. Agonist-induced secretion of ATP was inhibited at similar or lower concentrations of PAO. The specificity of the interaction of PAO with platelet proteins was demonstrated by the ability of the disulfhydryl compound 2,3-dimercaptopropanol, which abstracts PAO from proteins to form a stable cyclic adduct, to reverse PAO inhibition of both agonist-induced platelet secretion and aggregation. Dimercaptopropanesulphonic acid, a membrane-impermeable analogue of dimercaptopropanol, did not reverse inhibition of collagen-induced shape change or aggregation by PAO, thereby demonstrating that PAO acted intracellularly. PAO inhibited collagen-induced shape change and internal Ca²⁺ mobilization but had no effect on these two phenomena when induced by thrombin or ADP. PAO was also unable to prevent arachidonic acid-induced shape change, indicating that PAO acts at a site prior to the phospholipase A₂-mediated release of arachidonic acid to inhibit collagen-induced shape change. PAO induced the accumulation of a number of phosphotyrosine-containing proteins and inhibited the collagen-induced phosphorylation of a 40 kD protein. The potency and agonist-specific effects of PAO on platelet activation suggest that this inhibitor will be of value in elucidation of signal transduction pathways involved in receptor-mediated platelet function.     

Disparate effects of the calcium-channel blockers, nifedipine and verapamil, on alpha 2-adrenergic receptors and thromboxane A2-induced aggregation of human platelets

Calcium-channel blockers inhibit human platelet aggregation in vitro and ex vivo. To further evaluate the mechanism(s) responsible for the inhibition induced by this structurally heterogeneous group of compounds, we studied the effect of nifedipine and verapamil on human platelet aggregation in vitro. Neither 10 microM nifedipine nor 10 microM verapamil consistently inhibited the aggregation response of platelet-rich plasma to threshold concentrations of ADP, sodium arachidonate, epinephrine, or collagen. However, both 10 microM nifedipine and 10 microM verapamil epinephrine-potentiated, thromboxane A2 (TXA2)-induced aggregation of aspirin-incubated, gel-filtered platelets. Aggregation of similarly prepared platelets induced by TXA2 alone was abolished by 10 microM nifedipine but not by 10 microM verapamil. Even 100 microM verapamil gave only partial and inconsistent inhibition of aggregation. Both drugs had essentially the same effects on platelet aggregation induced by the stable endoperoxide and TXA2 mimic, U46619, with or without epinephrine. Neither 10 microM nifedipine nor 10 microM verapamil elevated platelet cyclic AMP. Verapamil (10 microM) inhibited binding of [3H]-yohimbine (an alpha 2-adrenergic receptor antagonist) to intact human platelets (KD 10.5 nM vs 2.4 nM for control platelets) without altering the number of binding sites. In contrast, 10 microM nifedipine had no effect on KD or number of binding sites. These results indicate that nifedipine and verapamil inhibit epinephrine-potentiated, TXA2-induced human platelet aggregation by different mechanisms. Verapamil inhibits the epinephrine contribution to the aggregation response by blocking alpha 2-adrenergic receptor binding. Nifedipine blocks the platelet response to TXA2 without affecting alpha-adrenergic receptor binding. These observations have potential clinical implications with regard to the mechanisms by which calcium-channel blockers inhibit vascular spasm and myocardial ischemia.     

High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets.

In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)-induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin-induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma-thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma-thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.     

Glycoprotein VI-mediated platelet fibrinogen receptor activation occurs through calcium-sensitive and PKC-sensitive pathways without a requirement for secreted ADP

Collagen activates platelets by transducing signals through glycoprotein VI (GPVI). It is not clear whether collagen can directly activate fibrinogen receptors on the adherent platelets without a role for positive feedback agonists. We investigated the contribution of secondary G protein signaling to the mechanism of GPVI-stimulated platelet aggregation using the GPVI-selective agonists, convulxin and collagen-related peptide (CRP) as well as collagen. Adenosine diphosphate (ADP) scavengers or ADP receptor antagonists shifted the concentration-response curve slightly to the right at low concentrations of convulxin, whereas platelet aggregation at higher concentrations of convulxin was unaffected by these agents. ADP receptor antagonists shifted the concentration-response curve of collagen- or CRP-induced platelet aggregation to the right at all the concentrations. Protein kinase C inhibitor, Ro 31-8220, or a calcium chelator 5,5'-dimethyl-BAPTA shifted the concentration-response curve of convulxin-induced platelet aggregation to the right. In addition, pretreatment with both Ro 31-8220 and dimethyl-BAPTA resulted in total inhibition of convulxin-mediated aggregation. Blockade of either the calcium- or protein kinase C-regulated pathway leads to inhibition of fibrinogen receptor activation on platelets adherent to collagen, but inhibition of both pathways leads to abolished fibrinogen receptor activation. We conclude that collagen-induced activation of fibrinogen receptor on adherent platelets through GPVI signaling occurs without any significant role for secreted ADP or thromboxane A(2). Furthermore, protein kinase C- and calcium-regulated pathways independently contribute to GPVI-mediated platelet aggregation.     

Prostaglandin endoperoxide analogues stimulate phospholipase C and protein phosphorylation during platelet shape change

We have studied the effects of two stable prostaglandin endoperoxide analogues on platelet lipid metabolism in relation to specific platelet functional changes. During platelet shape change, the endoperoxide analogues induce the formation of 1,2-diacylglycerol and phosphatidic acid, indicating the activation of a phosphoinositide-specific phospholipase C. In parallel, they stimulate the phosphorylation of a 40-kd and a 20-kd protein. During platelet shape change, arachidonic acid is released, but not metabolized by platelet cyclo-oxygenase or lipoxygenase. Phospholipase C activation and platelet shape change are independent of extracellular Ca++ and Mg++, arachidonate metabolism, and release of adenosine diphosphate (ADP). Activation of phospholipase C during platelet aggregation seems, however, to be mediated partly by release of ADP. We conclude that endoperoxide analogues initially stimulate in platelets the formation of products derived from phospholipase C activation, which might serve as intracellular messengers for phosphorylation of specific proteins related to platelet shape change.     

Physiological role of an endoperoxide in human platelets: hemostatic defect due to platelet cyclo-oxygenase deficiency.

The endoperoxide prostaglandin G2 (PGG2) induced platelet aggregation as well as the platelet release reaction (release of ADP and serotonin) when added to human platelet-rich plasma. Formation of a metabolite of PGG2 [8-(l-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid] and a lipoxygenase product [12L-hydroxy-5,8,10,14-eicosatetraenoic acid] accompanied the release reaction caused by aggregating agents such as collagen, ADP, epinephrine, and thrombin. Indomethacin inhibited the release reaction and PGG2 formation induced by these agents but had no effect on PGG2-induced release reaction. The aggregating effect of PGG2 was abolished by furosemide, which is a competitive inhibitor of ADP-induced primary aggregation. These data indicate that the aggregating effect of PGG2 is due to release of ADP and that PGG2 synthesis is required for induction of the release reaction by various aggregating agents. A subject with a hemostatic defect due to abnormal release mechanism [decreased aggregation with epinephrine (second wave) and collagen and normal platelet ADP] had a deficiency of the cyclo-oxygenase that catalyzes formation of PGG2. Normal aggregation and release reaction were obtained with added PGG2. Ii is concluded that the endoperoxide (PGG2) is essential in normal hemostasis because of its role in initiating the release reaction required for aggregation by collagen and the second wave of aggregation caused by, e.g., ADP.     

The role of ADP secretion and thromboxane synthesis in factor VIII binding to platelets

Following stimulation with arachidonic acid, collagen, U-46619 (a stable analogue of prostaglandin endoperoxide/thromboxane-A2), thrombin, or adenosine diphosphate (ADP), unstirred human platelet suspensions bound labeled factor VIII in a reaction that reached equilibrium within 10 min. Apyrase inhibited binding induced by arachidonic acid, collagen, U-46619, and thrombin by less than 40%, but inhibited ADP-induced binding by 95%. Binding to aspirin-treated platelets was normal in response to U-46619, reduced by 60%-70% in response to ADP, collagen, and thrombin, and absent in response to arachidonic acid. Binding in response to U-46619 was not altered by the combination of apyrase and aspirin. Binding of factor VIII was decreased by 90% when 10 mM EDTA was added before each agonist, but it was inhibited less than 30% when EDTA was added following platelet stimulation. We conclude that arachidonic acid, collagen, and thrombin can expose binding sites for factor VIII independently of released ADP; that Ca++ is required for activation but probably not for binding of factor VIII to platelets; and that platelet thromboxane synthesis plays a major role in the binding of factor VIII to platelets induced by thrombin, ADP, or collagen.     

ADP-induced platelet shape change and mobilization of cytoplasmic ionized calcium are mediated by distinct binding sites on platelets: 5'- p-fluorosulfonylbenzoyladenosine is a weak platelet agonist

Platelet stimulation with ADP results in several responses, including shape change, increase in cytoplasmic ionized calcium concentration [Ca2+]i, an inhibition of adenylate cyclase. 5'-p-Fluorosulphonyl benzoyladenosine (FSBA), which covalently labels an ADP binding site on platelets, blocks platelet shape change but not the inhibition of cyclic AMP levels by ADP, whereas p-chloromercuribenzenesulfonate (pCMBS), a nonpenetrating thiol reagent, has the opposite effects. We examined the effect of FSBA and pCMBS on ADP-induced increase in [Ca2+]i using platelets loaded with fluorescent Ca2+ indicators quin2 and fura-2. FSBA (50 to 200 mumol/L) induced a dose-dependent rise in [Ca2+]i, indicating that it is a weak platelet agonist. Under conditions of covalent labeling of the ADP binding sites, FSBA (50 to 100 mumol/L) did not inhibit the ADP-induced increase in [Ca2+]i or its inhibition of adenylate cyclase, whereas pCMBS (up to 1 mmol/L) abolished both these responses but not shape change. These findings suggest that ADP-induced Ca2+ mobilization and inhibition of adenylate cyclase are mediated by platelet binding sites distinct from those mediating shape change.     

Evidence for cross-talk between glycoprotein VI and Gi-coupled receptors during collagen-induced platelet aggregation

Collagen-induced platelet aggregation is a complex process and involves synergistic action of integrins, immunoglobulin (Ig)-like receptors, G-protein-coupled receptors and their ligands, most importantly collagen itself, thromboxane A(2) (TXA(2)), and adenosine diphosphate (ADP). The precise role of each of these receptor systems in the overall processes of activation and aggregation, however, is still poorly defined. Among the collagen receptors expressed on platelets, glycoprotein (GP) VI has been identified to play a crucial role in collagen-induced activation. GPVI is associated with the FcRgamma chain, which serves as the signal transducing unit of the receptor complex. It is well known that clustering of GPVI by highly specific agonists results in platelet activation and irreversible aggregation, but it is unclear whether collagen has the same effect on the receptor. This study shows that platelets from Galphaq-deficient mice, despite their severely impaired response to collagen, normally aggregate on clustering of GPVI, suggesting this not to be the principal mechanism by which collagen activates platelets. On the other hand, dimerization of GPVI by a monoclonal antibody (JAQ1), which by itself did not induce aggregation, provided a sufficient stimulus to potentiate platelet responses to Gi-coupled, but not Gq-coupled, agonists. The combination of JAQ1 and adrenaline or ADP, but not serotonin, resulted in alpha(IIb)beta(3)-dependent aggregation that occurred without intracellular calcium mobilization and shape change in the absence of Galphaq or the P2Y(1) receptor. Together, these results provide evidence for a cross-talk between (dimerized) GPVI and Gi-coupled receptors during collagen-induced platelet aggregation. (Blood. 2001;97:3829-3835)     

The roles of alpha IIb beta 3-mediated outside-in signal transduction,thromboxane A2, and adenosine diphosphate in collagen-induced platelet aggregation

Collagen-induced activation of platelets in suspension leads to alpha(IIb)beta(3)-mediated outside-in signaling, granule release, thromboxane A2 (TxA2) production, and aggregation. Although much is known about collagen-induced platelet signaling, the roles of TxA2 production, adenosine diphosphate (ADP) and dense-granule secretion, and alpha(IIb)beta(3)-mediated outside-in signaling in this process are unclear. Here, we demonstrate that TxA2 and ADP are required for collagen-induced platelet activation in response to a low, but not a high, level of collagen and that alpha(IIb)beta(3)-mediated outside-in signaling is required, at least in part, for this TxA2 production and ADP secretion. A high level of collagen can activate platelets deficient in PLC gamma 2, G alpha q, or TxA2 receptors, as well as platelets treated with a protein kinase C inhibitor, Ro31-8220. Thus, activation of alpha(IIb)beta(3) in response to a high level of collagen does not require these signaling proteins. Furthermore, a high level of collagen can cause weak TxA2 and ADP-independent aggregation, but maximal aggregation induced by a high level of collagen requires TxA2 or secretion.     

野黄芩苷抗血小板活化效应的评价

目的评价野黄芩苷对血小板的聚集与活化的影响。方法在人洗涤血小板中进行血小板聚集实验,分别用胶原(1.00μg/ml)、U46619(0.30μM)、ADP(10.00μM)、凝血酶(0.04U/ml)作为诱导剂,对比在0.04mg/ml、0.10mg/ml、0.20mg/ml三种不同预孵浓度状态下野黄芩苷对血小板聚集的影响。并进一步研究三种不同浓度的野黄芩苷对血栓素类似物(U46619)引起的血小板纤维蛋白原结合的影响。结果胶原、U46619、ADP、凝血酶均可明显促进血小板聚集,而血小板预孵育野黄芩苷可以抑制诱导剂引起的聚集,并且抑制作用呈现浓度依赖关系。血小板预孵育野黄芩苷可以减少U46619引起的血小板纤维蛋白结合,并且抑制作用呈现浓度依赖关系。结论野黄芩苷可以抑制血小板的聚集和活化,很可能是通过阻断血栓素A2诱导血小板聚集的某一个中间环节产生作用。     

Epinephrine induces a late thromboxane-dependent platelet shape change and enhances synergistically the shape change induced by other platelet agonists

Epinephrine is the only physiological platelet activator which induces platelet aggregation without a preceding change in platelet shape. The reason why epinephrine cannot induce this shape change is not known. Electron microscopically, we could show that during the first phase of epinephrine-induced platelet aggregation, the platelet aggregate is composed of discoid platelets, lying in rather loose contact with neighbouring platelets. During the second wave of epinephrine-induced aggregation (this is when thromboxane (TX)A(2) production has taken place), platelets have completely lost their discoid shape and are very tightly bound. In EDTA-platelet rich plasma (PRP), we could demonstrate a clear synergistic action of epinephrine 10-20 μM on the first phase of shape change (disc-to-sphere transformation), induced by low concentrations of arachidonic acid (AA), collagen, adenosine diphosphate (ADP) and platelet activating factor (PAF). In combination with moderate concentrations of AA or collagen, epinephrine induced a clear aggregation-independent secretion of platelet granules, which in the absence of epinephrine, only takes place with higher inducer concentrations. All these synergistic actions could be demonstrated in the aggregometer and electron microscopically. To explain these findings, we hypothesize that the inability of epinephrine to induce a shape change that precedes aggregation is due to slow generation of TXA(2) which is only formed as a positive feedback mechanism of aggregation. This TXA(2) will bind to its own receptor and produce a shape change coinciding with the second wave of epinephrine-induced aggregation. Collagen, in contrast, induces very rapid TXA(2) generation, causing Ca(2+) mobilization and myosin light chain-phosphorylation, leading to shape change, clearly before aggregation starts.     

Investigation of the Role of Arachidonic Acid in Platelet Function and Mechanisms for Suppression of Prostaglandin Biosynthesis

S ummary . The effects of two hypolipidaemic agents (clofibrate and halofenate free acid, HFA) and aspirin on the release of the contents of different granules and on cyclic endoperoxide formation induced by sodium arachidonate and thrombin, were investigated. Aspirin and HFA completely blocked aggregation by arachidonate whereas clofibrate potentiated aggregation. Aspirin and HFA were able to inhibit the platelet shape change caused by arachidonate. Arachidonate at low concentrations released [³H]⁵HT; at a higher concentration there was release of β-glucuronidase accompanied by loss of cytoplasmic LDH into the supernatant, indicating lysis. Platelets converted exogenous sodium arachidonate to malonyldial-dehyde (MDA), the conversion being completely inhibited by aspirin and HFA but not by clofibrate. Thrombin which is believed to provide endogenous arachidonic acid for conversion to endoperoxides, caused MDA formation and this was inhibited by clofibrate as well as by HFA and aspirin. Despite inhibition of endoperoxide arachidonate were still able to release the contents of platelet granules. These findings suggest that all the effects on platelets of low concentrations of exogenous arachidonate may be due to its conversion to prostaglandin endoperoxides whereas the release reaction initiated by higher concentrations of thrombin or exogenous arachidonate involves endoperoxide-dependent and independent mechanisms. Moreover, the mechanisms of release from dense and α-granules of platelets may be similar in some respects. It is concluded that HFA, like aspirin, suppresses biosynthesis of endoperoxides by inhibiting formation of arachidonic acid from phospholipids.