Essential athrombia: study of a new case.

A constitutional platelet function disorder in a twelve year-old girl characterized by a lifelong bleeding tendency, prolonged bleeding time, normal platelet count, normal clot retraction, normal platelet factor 3 activity and impaired platelet aggregation was reported. Platelet aggregation, studied turbidimetrically, was absent in the presence of usual doses of ADP (1-4 MUM) although a small wave of primary aggregation was obtained by very large ADP concentrations (25-50 muM). The platelets were also unresponsive to epinephrine, thrombin and diluted collagen suspensions. But an almost normal aggregation response occurred with strong collagen suspensions. The platelets responded to Ristocetin. Release of platelet ADP was found to be normal by collagen and thrombin, but impaired by kaolin. Platelet fibrinogen content was normal. The present case, investigated with recent methods, confirms the existence of a type of primary functional platelet disorder characterized solely by an aggregation defect, described in 1955 and 1962 under the name of "essential athrombia."     

Decreased nucleotide and serotonin storage associated with defective function in Chediak-Higashi syndrome cattle and human platelets

Prolonged mean template bleeding time of 14 min observed in seven cattle with the Chediak-Higashi syndrome (CHS) prompted the examination of platelet function in these animals. There was no distinguishable difference in concentration of circulating platelets between CHS and control cattle. CHS bovine platelets failed to aggregate in vitro in response to concentrations of acid-soluble collagen which induced aggregation in all normal samples. The primary platelet response to 5 muM ADP was normal in CHS cattle. A markedly decreased amount of serotonin (1.2% of normal) was detected in CHS bovine platelets. Bovine CHS platelet ATP and ADP contents were significantly less than normal, and the ATP/ADP ratios were 5.04 in normal and 29.38 in CHS platelets. Results of these animal investigations prompted a similar study of two patients with CHS. In humans, an increased bleeding time greater than 15 min and an in vitro impaired aggregation response to acid-soluble collagen and 5 muM adrenaline were discovered. Both ATP and ADP were reduced in CHS human platelets, and the ATP/ADP ratio was 3.96, compared to a ratio of 1.52 for platelets of two normal subjects. These findings suggested the presence of a "storage pool disease" of CHS platelets.     

The in-vitro effect of tirofiban, glycoprotein IIb/IIIa antagonist, on various responses of porcine blood platelets

The present study systematically evaluates the in-vitro effect of tirofiban, glycoprotein IIb/IIIa (integrins alphaIIbbetaIII) antagonist, on porcine blood platelets. It was found that tirofiban at concentrations up to 5,000 ng/ml did not affect the calcium signal produced by thrombin. Tirofiban, in a concentration-dependent manner reduced platelet aggregation evoked by ADP (IC50 approximately 70 ng/ml), collagen (IC50 approximately 200 ng/ml), and thrombin (IC50 approximately 5,000 ng/ml). Substantial thrombin-evoked platelet aggregation still occurred at high (5,000 ng/ml) tirofiban concentrations. The concentrations of tirofiban completely blocking the optical aggregation evoked by ADP or collagen failed to eliminate microaggregate formation totally. Tirofiban strongly inhibited the dense-granule and lysosome secretion induced by ADP (IC50 approximately 70-170 ng/ml), moderately inhibited that induced by collagen (IC50 approximately 420-500 ng/ml) and very poorly inhibited that elicited by thrombin (IC50 approximately 1,500-5,000 ng/ml). The extent of the inhibition of aggregation and secretion rose as concentrations of the stimulus lowered. Tirofiban was a moderate inhibitor (IC50 approximately 200 ng/ml) of adhesion and a poor inhibitor of platelet procoagulant response induced by collagen. Thromboelastography measurements indicate that, in whole blood, tirofiban, up to concentrations of 2,000 ng/ml, did not affect the kinetics of tissue factor induced clot formation. The obtained results reveal that in porcine platelets, the maximal concentrations of tirofiban used in human medicine (250 ng/ml), effectively block platelet responses triggered by ADP, partly block those induced by collagen and very poorly block those evoked by thrombin. The reason for this phenomenon seems to be the inability of tirofiban to reduce platelet secretion completely.     

Levosimendan has an inhibitory effect on platelet function

Levosimendan enhances cardiac contractility by increasing myocyte sensitivity to calcium, and induces vasodilatation. Although studies have evaluated the efficacy of levosimendan in heart failure, it is not clear whether it might produce functional influence on platelet response. In this study, the effect of levosimendan on platelet aggregation was investigated. Platelet function tests were performed in 12 healthy male volunteers. Three concentrations of levosimendan solution were prepared that would result in 10, 25, and 45 ng/ml levosimendan concentrations in the blood similar to that observed after clinical therapeutic intravenous application of 0.05-0.1 microg/kg/min. Each concentration of levosimendan solution and a control diluent without levosimendan were incubated with whole blood at 37 degrees C. After incubation for 15 min, aggregation responses were evaluated with adenosine diphosphate (ADP) (5 and 10 microM) and collagen (2 and 5 microg/ml) in platelet-rich plasma. Preincubation with all dilutions of levosimendan inhibited aggregation of platelets induced by ADP and collagen significantly. Levosimendan also inhibited significantly the secondary wave of platelet aggregation induced by ADP. The results showed that there was a relationship between levosimendan concentration and inhibition of platelet aggregation. In conclusion, this study with an in vitro model showed that levosimendan had a significant inhibitory effect on platelets in clinically relevant doses.     

Adenosine diphosphate strongly potentiates the ability of the chemokines MDC, TARC, and SDF-1 to stimulate platelet function

Platelet activation is normally induced by primary agonists such as adenosine diphosphate (ADP), thrombin, and collagen, whereas other agonists, such as epinephrine, can play important accessory roles. It is now reported that the macrophage-derived chemokine (MDC), thymus activation-regulated chemokine (TARC), and stromal cell-derived factor one (SDF-1) are highly effective activators of platelet function under a variety of conditions, stimulating platelet shape change, aggregation, and adhesion to collagen or fibrinogen. Chemokine-mediated platelet activation was rapid and maximal (less than 5 seconds) under arterial flow conditions and depended strongly on the presence of low levels of primary agonists such as ADP or thrombin. Concentrations of ADP (0.05-0.25 microM) or thrombin (0.005-0.02 U/mL) that induced minimal aggregation caused major aggregation acting in combination with the chemokines. The ability of apyrase to block chemokine-dependent aggregation or adhesion was consistent with an important role for ADP. Chemokine-stimulated aggregation was also insensitive to indomethacin, suggesting that the activation of cyclo-oxygenase is not involved. TARC, MDC, and SDF-1 increased intracellular calcium concentrations [Ca(2+)](i) when combined with low levels of ADP. The MDC and TARC receptor CCR4 was expressed on platelets, and an anti-CCR4 antibody blocked aggregation induced by TARC or MDC. Treatment of platelets with SDF-1 and MDC rapidly exposed P-selectin (CD62P) on the cell surface but did not induce the secretion of serotonin. These findings suggest that the chemokines MDC, TARC, and SDF-1, which may be produced during inflammatory responses, coupled with low levels of ADP or thrombin, can serve as strong stimuli for activating platelet function.     

Antiaggregatory Properties of Beta-adrenoceptor Blocking Drugs are Related to their Physico-chemical Properties

The effects of the beta-adrenoceptor blockers (BAB) alprenolol, atenolol, metipranolol, oxprenolol, practolol and propranolol on platelet aggregation stimulated by ADP, thrombin and collagen were investigated. The BAB were divided into three groups according to their liposolubility and antiaggregatory activity. The first group consists of the highly active drugs alprenolol and propranolol; metipranolol and oxprenolol are in the second group; the least active atenolol and practolol are in the third group. Collagen induced platelet aggregation was inhibited by BAB to the highest extent, followed by inhibition of thrombin stimulated aggregation. ADP-induced aggregation was inhibited to the lowest extent. Consistent with the rank order of their partition coefficients, BAB inhibited aggregation with the following order of potency: propranolol > alprenolol > metipranolol > oxprenolol > practolol > atenolol. A positive correlation was found between inhibition of thrombin and collagen stimulated platelet aggregation and the partition coefficients of the individual drugs. A significant time-dependent inhibition of platelet aggregation developed within 30 s of exposure of blood platelets to BAB.     

Platelet inhibiton by sodium nitroprusside, a smooth muscle inhibitor.

The effects of sodium nitroprusside (N.P.), a pure smooth muscle inhibitor, on platelet function were studied. Platelet-rich plasmas (PRP) from normal controls and from patients receiving N.P. were studied in vitro for aggregation in response to adenosine diphosphate (ADP), epinephrine, and collagen. Platelet ADP release (release reaction) was also investigated. Normal platelets demonstrated marked inhibition of aggregation when incubated with N.P. for 3 min. Prolonging the incubation was without additional effect. ADP and ATP release from platelets in response to collagen was also inhibited. PRP from patients receiving nitroprusside at concentrations between 25 mug/min an 165 mug/min showed inhibition of aggregation when compared to findings prior to the administration of N.P. N.P. acts by inhibiting contractile proteins and thus platelet ADP release and aggregation may depend on contraction of platelet smooth muscle-like protein, thrombosthenin.     

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.     

Inhibitory effect of Ginkgo biloba extract on human platelet aggregation

The effect of pure flavonoids and Gingko biloba extract (GBE) on human platelet aggregation was investigated. Most of the flavonoids and vitamin E did not affect platelet aggregation in platelet-rich plasma (PRP); however some of these flavonoids inhibited platelet aggregation in gel-filtered platelets (GFP). GBE inhibited both ADP- and collagen-induced platelet aggregation in PRP, GFP and in whole blood in a dose-dependent manner. GBE at very low concentrations inhibited whole blood aggregation induced by ADP compared with those used for PRP or GFP. Flavonoids and GBE decreased the production of TxA(2) induced by collagen and ADP in PRP. However, no correlation was observed between the inhibition of platelet aggregation and the decrease of TxA(2) synthesis. GBE and flavonoids did not affect platelet membrane fluidity. However, the incubation of PRP with GBE increased cAMP levels in platelets, which is known to inhibit platelet activation by lowering intracellular Ca2+ levels. GBE is a mixture of many compounds, including flavonoids and gingkoglides, which affect metabolism of cAMP, TxA(2) and Ca2+ in platelets. It is effective in the inhibition of platelet aggregation, both in PRP and whole blood, and thus may be potentially used as an effective oral anti-platelet therapeutic agent.     

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.     

Disaggregatory effects of prostaglandin E 1 , amrinone and milrinone on platelet aggregation in human whole blood

Prostaglandin E 1 (PGE 1 ), amrinone and milrinone not only inhibit platelet aggregation, but also potentially induce disaggregation. We examined the disaggregatory effects of PGE 1 , amrinone and milrinone on platelet aggregation, and measured the platelet cyclic adenosine monophosphate (cAMP) levels using a standard radioimmunoassay. Platelet aggregation was induced by 10 w M adenosine diphosphate (ADP) or 0.625 w g/ml collagen that induces a secondary aggregation, and measured by whole blood aggregometry. PGE 1 (0.7-10.0 w M), amrinone (106.8-801.3 w M), or milrinone (9.5-190.0 w M) was added when aggregation reached 5 m z of impedance, and the percentage of disaggregation was measured 5 min after initiating disaggregation. Disaggregatory effects were also examined using a light transmission aggregometer and gel-filtered platelets. In platelet aggregation induced by ADP, the drug concentrations required to cause 50% disaggregation (EC 50 ) were PGE 1 ; 3.1 - 1.2 w M, amrinone; 313.1 - 128.8 w M, milrinone; 57.1 - 20.8 w M. In platelet aggregation induced by collagen, the maximum disaggregation percentages were 15.6 - 5.0% for 10 w M PGE 1 , 7.8 - 2.4% for 801.3 w M amrinone, and 5.9 - 2.3% for 190.0 w M milrinone. The EC 50 concentrations of platelet cAMP that caused 50% disaggregation in ADP-induced platelet aggregation were 67 - 13 pmoles/10 8 platelets for PGE 1 , 54 - 12 pmoles/10 8 platelets for amrinone, and 52 - 12 pmoles/10 8 platelets for milrinone. In gel-filtered platelets, the percentages of disaggregation in ADP- or collagen-induced platelet aggregation were 33.1 - 5.2% or 11.2 - 3.1% for PGE 1 (10 w M), 22.3 - 4.1% or 15.2 - 3.2% for amrinone (801.3 w M), and 23.5 - 4.3% or 14.6 - 3.5% for milrinone (190.0 w M).     

Zinc-induced platelet aggregation is mediated by the fibrinogen receptor and is not accompanied by release or by thromboxane synthesis

We demonstrate that zinc (0.1 to 0.3 mmol/L) induces aggregation of washed platelet suspensions. Higher concentrations (1 to 3 mmol/L) of zinc were needed to aggregate platelets in platelet-rich plasma obtained from blood anticoagulated with low-molecular-weight heparin, probably due to the binding of zinc to the plasma proteins. Zinc- induced aggregation of normal washed platelets required added fibrinogen and no aggregation occurred with thrombasthenic platelets or with normal platelets pretreated with a monoclonal antibody (10E5) that blocks the platelet fibrinogen receptor. These data indicate that the platelet membrane fibrinogen receptor-glycoproteins IIb and IIIa mediate the effect of zinc. Zinc-induced aggregation was blocked by the agent TMB-8, which interferes with the internal calcium flux, and by prostacyclin, which elevates platelet cyclic adenosine monophosphate levels. Zinc-induced aggregation was not accompanied by thromboxane synthesis or by the secretion of dense-body serotonin and was not affected by preexposure of platelets to acetylsalicylic acid. Experiments with creatine phosphate/creatine phosphokinase showed that the zinc effect on platelets was independent of extracellular adenosine diphosphate (ADP). Zinc had an additive effect when platelet aggregation was stimulated with subthreshhold concentrations of collagen or ADP. Together with the known effects of nutritional zinc on in vivo bleeding, on platelet aggregation, and on lipid metabolism, the results suggest that zinc may have an important bearing on normal hemostasis, thrombosis, and atherosclerosis.     

Disaggregatory effects of prostaglandin E1, amrinone and milrinone on platelet aggregation in human whole blood

Prostaglandin E1 (PGE1), amrinone and milrinone not only inhibit platelet aggregation, but also potentially induce disaggregation. We examined the disaggregatory effects of PGE1, amrinone and milrinone on platelet aggregation, and measured the platelet cyclic adenosine monophosphate (cAMP) levels using a standard radioimmunoassay. Platelet aggregation was induced by 10 microM adenosine diphosphate (ADP) or 0.625 microg/ml collagen that induces a secondary aggregation, and measured by whole blood aggregometry. PGE1 (0.7-10.0 microM), amrinone (106.8-801.3 microM), or milrinone (9.5-190.0 microM) was added when aggregation reached 5 mohm of impedance, and the percentage of disaggregation was measured 5 min after initiating disaggregation. Disaggregatory effects were also examined using a light transmission aggregometer and gel-filtered platelets. In platelet aggregation induced by ADP, the drug concentrations required to cause 50% disaggregation (EC50) were PGE1; 3.1+/-1.2 microM, amrinone; 313.1+/-128.8 microM, milrinone; 57.1+/-20.8 microM. In platelet aggregation induced by collagen, the maximum disaggregation percentages were 15.6+/-5.0% for 10 microM PGE1, 7.8+/-2.4% for 801.3 microM amrinone, and 5.9+/-2.3% for 190.0 microM milrinone. The EC50 concentrations of platelet cAMP that caused 50% disaggregation in ADP-induced platelet aggregation were 67+/-13 pmoles/10(8) platelets for PGE1, 54+/-12 pmoles/10(8) platelets for amrinone, and 52+/-12 pmoles/10(8) platelets for milrinone. In gel-filtered platelets, the percentages of disaggregation in ADP- or collagen-induced platelet aggregation were 33.1+/-5.2% or 11.2+/-3.1% for PGE1 (10 microM), 22.3+/-4.1% or 15.2+/-3.2% for amrinone (801.3 microM), and 23.5+/-4.3% or 14.6+/-3.5% for milrinone (190.0 microM).     

Platelet Aggregation and Release by ADP and Thrombin: Evidence for Two Separate Effects of ADP on Platelets, Involvement of Fibrinogen in Release, and Mechanism of Inhibitory Action of Acetylsalicylic Acid

S ummary . The previously demonstrated roles of thrombin and fibrinogen in platelet interaction (Ardlie & Han, 1974) suggested a possible relationship between platelet aggregation and the release reaction. The present results show that a product of the action of thrombin on fibrinogen induced the release of platelet constituents. This activity was not inhibited by heparin. The thrombin-fibrinogen reaction which occurs when platelets come into contact could be the cause of the subsequent release reaction. ADP was shown to be essential for the thrombin-induced aggregation, but not the alteration in platelet morphology. Evidence of involvement of ADP in cell contact was obtained and it is possible that its role may be to facilitate aggregation through an effect on platelet surface charge. Thus, ADP induces aggregation through two distinct actions: (1) alteration of platelet morphology; and (2) reduction in net negative surface charge of platelets. Incubation of thrombin with aspirin inhibited its action on platelets and it is suggested that acetylation of thrombin (or prothrombin) by aspirin may account for the action of this compound on platelets. The significance of these observations and a possible mechanism for the release reaction is discussed.     

Two Platelet Aggregation Inhibitors in Tsetse (Glossina) Saliva with Studies of Roles of Thrombin and Citrate in in Vitro Platelet Aggregation

Two inhibitors of platelet aggregation have been identified in saline extracts of Glossina morsitans (tsetse) salivary glands. A protein fraction (MW > 30000) inhibited primary and secondary aggregation to ADP, secondary aggregation to adrenalin, and aggregation to collagen. It also caused disaggregation of platelets stimulated by ADP and adrenalin. These properties could be explained by ADP hydrolytic activity. A previously identified antithrombin fraction (MW 11 000-13 000) abolished thrombin-induced aggregation. It did not affect platelet aggregation to ADP, adrenalin or collagen, as compared to aggregation in citrate, when used as sole anticoagulant for platelet rich plasma or when added to citrated platelet rich plasma. These results fail to support hypotheses (i) that thrombin plays an important role in platelet aggregation by other agonists and (ii) that secondary platelet aggregation is an artefact induced by citrate. It is proposed that these inhibitors may be important in maintaining mouthpart and crop patency during feeding. Their discovery suggests that other arthropods may also have antiplatelet agents in their saliva which might be entomologically important, provide useful tools for platelet studies, and be of potential therapeutic interest.     

NAD(P)H oxidase-dependent platelet superoxide anion release increases platelet recruitment

Platelets, although not phagocytotic, have been suggested to release O. Since O-producing reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases can be specifically activated by certain agonists and are found in several nonphagocytotic tissues, we investigated whether such an enzyme is the source of platelet-derived O. We further studied which agonists cause platelet O release and whether platelet-derived O influences thrombus formation in vitro. Collagen, but not adenosine 5'-diphosphate (ADP) or thrombin, increased O formation in washed human platelets. This was a reduced nicotinamide adenine dinucleotide (NADH)-dependent process, as shown in platelet lysates. Consistent with a role of a platelet, NAD(P)H oxidase expression of its subunits p47(phox) and p67(phox) and inhibition of platelet O formation by diphenylene-iodoniumchloride (DPI) and by the specific peptide-antagonist gp91ds-tat were observed. Whereas platelet-derived O did not influence initial aggregation, platelet recruitment to a preformed thrombus following collagen stimulation was significantly attenuated by superoxide dismutase (SOD) or DPI. It was also inhibited when ADP released during aggregation was cleaved by the ectonucleotidase apyrase. ADP in supernatants of collagen-activated platelets was decreased in the presence of SOD, resulting in lower ADP concentrations available for recruitment of further platelets. Exogenous O increased ADP- concentrations in supernatants of collagen-stimulated platelets and induced irreversible aggregation when platelets were stimulated with otherwise subthreshold concentrations of ADP. These results strongly suggest that collagen activation induces NAD(P)H oxidase-dependent O release in platelets, which in turn enhances availability of released ADP, resulting in increased platelet recruitment.     

ADP induces partial platelet aggregation without shape change and potentiates collagen-induced aggregation in the absence of Galphaq

Platelets from Galphaq knockout mice are unable to aggregate in response to physiological agonists like adenosine 5'-diphosphate (ADP), thromboxane A(2), thrombin, or collagen, although shape change still occurs in response to all of these agonists except ADP. ADP-induced platelet aggregation results from simultaneous activation of the purinergic P2Y(1) receptor coupled to calcium mobilization and shape change and of a distinct P2 receptor, P2cyc, coupled through Gi to adenylyl cyclase inhibition, which is responsible for completion and amplification of the response. P2cyc could be the molecular target of the antithrombotic drug clopidogrel and the adenosine triphosphate (ATP) analogs AR-C69931MX, AR-C67085, and AR-C66096. The aim of the present study was to determine whether externally added ADP could still act through the Gi pathway in Galphaq-deficient mouse platelets and thereby amplify the residual responses to agonists such as thrombin or collagen. It was found that (1) ADP and adrenaline still inhibited cyclic AMP accumulation in Galphaq-deficient platelets; (2) both agonists restored collagen- but not thrombin-induced aggregation in these platelets; (3) the effects of ADP were selectively inhibited in vitro by the ATP analog AR-C69931MX and ex vivo by clopidogrel and hence were apparently mediated by the P2cyc receptor; and (4) high concentrations of ADP (100 micromol/L) induced aggregation without shape change in Galphaq-deficient platelets through activation of P2cyc. Since adrenaline was not able to induce platelet aggregation even at high concentrations, we conclude that the effects of ADP mediated by P2cyc are not restricted to the inhibition of adenylyl cyclase through Gi(2).     

野木瓜多糖对家兔血小板聚集的影响

目的研究野木瓜多糖(SCP)对血小板聚集的影响及可能机制。方法选择成年家兔30只,随机分为空白对照组、SCP 17 g/L组、SCP 34 g/L组、SCP 68 g/L组、阿司匹林组,每组6只。制备家兔富血小板及贫血小板血浆,用Born's比浊法检测17、34、68 g/LSCP对胶原、花生四烯酸(AA)、二磷酸腺苷(ADP)及凝血酶诱导的血小板聚集的影响。检测SCP对胶原诱导后血小板中5-羟色胺含量、肝素凝血酶凝固时间(HTCT)及对凝血酶作用后血小板释放丙二醛含量的影响。结果与空白对照组比较,不同浓度SCP组、阿司匹林组的胶原、ADP、凝血酶诱导的血小板聚集明显降低(P<0.05,P<0.01),SCP 1 7 g/L组AA诱导的血小板聚集无明显变化(P>0.05),SCP34 g/L组、SCP 68 g/L组、阿司匹林组AA诱导的血小板聚集明显降低(P<0.05,P<0.01)。与空白对照组比较,不同浓度SCP组丙二醛含量明显降低、HTCT延长,呈浓度依赖性(P<0.01),除SCP 1 7 g/L组外,其余各组5羟色胺明显降低,呈浓度依赖性(P<0.05,P<0.01)。结论 SCP可以抑制由胶原、ADP、AA和凝血酶诱导的血小板聚集,其机制与抑制血小板释放5-羟色胺、血小板因子Ⅳ及减少丙二醛生成有关。     

Resveratrol decreases early signaling events in washed platelets but has little effect on platelet in whole blood

Resveratrol, a polyphenolic compound found in red wines, is believed to be a contributor in decreasing the incidence of coronary heart disease. Although its primary target is unknown, it blocks aggregation of washed platelets by an ill-defined mechanism. We show that resveratrol, at 10-50 microM, blocked aggregation induced by collagen (5 microg/ml), thrombin (0.2 units/ml), and ADP (10 microM). This affect was not overcome by adding exogenous human fibrinogen to the assay, suggesting that an early (wave I) signaling step in the alpha(IIb)beta(3) activation cascade was impaired. To explore this possibility we examined the effect of resveratrol on activation of MAP kinases. In the platelet, MAP kinases become activated as a consequence of agonist binding and not of aggregation, which itself induces signaling events. In fact, we find that collagen-induced activation of MAP kinases is superinduced in the presence of RGDS, an aggregation-blocking peptide. Resveratrol, at concentrations of 10 microM and greater, inhibited MAP kinase activation induced by collagen (in the absence and presence of RGDS peptide), thrombin, and ADP. These data indicate that resveratrol blocks receptor-mediated signaling events in washed platelets. In comparison, resveratrol has poor antiplatelet activity in whole blood. Under these conditions aggregation was not affected by 50-100 microM resveratrol. Concentrations of 200 microM resveratrol were needed to cause a 30-60% decrease in platelet aggregation in whole blood. Together these studies suggest that resveratrol is a potent inhibitor of platelet signaling responses, but its antiplatelet activity is weakened or masked in circulation. Thus, although resveratrol may function as a protective agent of coronary heart disease, its affects are not solely attributed to its effects on platelets in circulation.     

Inhibition of collagen-induced platelet activation by 5'-p- fluorosulfonylbenzoyl adenosine: evidence for an adenosine diphosphate requirement and synergistic influence of prostaglandin endoperoxides

The relative roles of platelet autacoids such as adenosine diphosphate (ADP), prostaglandin endoperoxides, and thromboxane A2 (TXA2) in collagen-induced platelet activation are not fully understood. We reexamined this relationship using the ADP affinity analogue, 5'-p- fluorosulfonylbenzoyl adenosine (FSBA), which covalently modifies a receptor for ADP on the platelet surface, thereby inhibiting ADP- induced platelet activation. Collagen-induced shape change, aggregation, and fibrinogen binding were each fully inhibited under conditions in which FSBA is covalently incorporated and could not be overcome by raising the collagen used to supramaximal concentrations. In contrast, TXA2 synthesis stimulated by collagen under conditions that produced maximum aggregation was only minimally inhibited by FSBA. Since covalent incorporation of FSBA has been previously shown to specifically inhibit ADP-induced activation of platelets, the present study supports the contention that ADP is required for collagen-induced platelet activation. Under similar conditions, indomethacin, an inhibitor of cyclooxygenase, inhibited collagen-induced shape change, indicating that endoperoxides and/or TXA2 also play a role in this response. Shape change induced by low concentrations (10 nmol/L) of the stable prostaglandin endoperoxide, azo-PGH2, was also inhibited by FSBA. These observations indicate a role for ADP in responses elicited by low concentrations of endoperoxides. However, at higher concentrations of azo-PGH2 (100 nmol/L), inhibition by FSBA could be overcome. Thus, the effect of collagen apparently has an absolute requirement for ADP for aggregation and fibrinogen binding and for both ADP and prostaglandins for shape change. Aggregation and fibrinogen binding induced by prostaglandin endoperoxides also required ADP as a mediator, but ADP is not absolutely required at high endoperoxide concentration to induce shape change.