Microtubule inhibitors alter the secretion of beta-glucuronidase by human blood platelets: involvement of microtubules in release reaction II

The effects of the antimicrotubular drugs colchicine and vinblastine on the blood platelet release reaction were studied by measuring release of ¹⁴C-5-hydroxytryptamine (¹⁴C-5-HT, release I) and β-glucuronidase (release II) from gel-filtered human platelets. β-glucuronidase release induced by thrombin was significantly inhibited by colchicine (0.01-1 mM) or vinblastine (0.05–0.1 mM). Release of ¹⁴C-5-HT, however, was unaffected at low concentrations of colchicine and only slightly inhibited at higher concentrations. Inhibition of β-glucuronidase release depended on colchicine or vinblastine concentrations and decreased with longer time intervals (1′, 5′, 20′) after thrombin stimulation. Levels of the cytoplasmic enzyme, lactic acid dehydrogenase, in supernatants of colchicine treated platelets were not significantly different from controls. Colchicine also inhibited β-glucuronidse release, but not ¹⁴C-5-HT release, induced by trypsin and sodium arachidonate. Binding of ¹⁴C-colchicine by platelets was measured and it was found that platelet aggregation and release of 5-HT induced by adenosine diphosphate, epinephrine and collagen proceeded without any alteration in colchicine binding. However, significant increases in the rate and degree of colchicine binding were observed when platelets were stimulated by thrombin, trypsin and arachidonic acid which induced aggregation, release of both 5-HT and β-glucuronidase. The results suggest that an alteration in platelet microtubules is correlated with the physiologic response resulting in release II and that the cellular mechanisms effecting release I and II by platelets differ qualitatively in that the microtubules may facilitate release II.     

Exaggerated polymerisation of beta-amyloid 40 stimulated by plasma lipoproteins results in fibrillar Abeta preparations that are ineffective in promoting ADP-induced platelet aggregation

The cytotoxic beta-amyloid peptide (Abeta) of Alzheimer's disease (AD) occurs in both plasma and platelets and may modulate platelet function. Its biological activity may relate to its fibril content and factors that promote Abeta fibrillogenesis, e.g., plasma lipoproteins could, therefore, have implications for Abeta action. We undertook a study in which structure-activity relationships were considered with respect to the actions of Abeta(1-40) on platelet function. Thus, the influence of soluble Abeta and various fibrillar Abeta preparations (0.1-10 microM) on platelet aggregation and endogenous 5-hydroxytryptamine (5-HT) efflux was investigated. Soluble Abeta(1-40) only enhanced platelet aggregation (+30%, P<0.05) and 5-HT release (+28%) stimulated by ADP (1 microM) at the highest concentration tested (10 microM). By contrast, fibrillar Abeta(1-40) at 1, 5 and 10 microM potentiated aggregation by 17.4%, 68.8% (P<0.05) and 99.5% (P<0.0001), respectively, and 5-HT efflux by 17.4%, 65% and 208% (P<0.001). Abeta(1-40) fibrils generated in the presence of native and oxidised very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) yielded platelet responses that did not differ from those seen with the lipoproteins alone. These responses were markedly lower than those obtained with homogeneous Abeta fibrils. Our data indicate that homogeneous Abeta(1-40) fibrils are more potent than soluble Abeta(1-40) in promoting platelet reactivity and that interactions with plasma lipoproteins result in the formation of Abeta fibrils that are ineffective. We suggest that lipoproteins may interfere with the recognition of Abeta by appropriate platelet receptors and/or cause Abeta to assume an "overaggregated" biologically inert state.     

Morphine-potentiated platelet aggregation in in vitro and platelet plug formation in in vivo experiments

The detailed mechanisms underlying morphine-signaling pathways in platelets remain obscure. Therefore, we systematically examined the influence of morphine on washed human platelets. In this study, washed human platelet suspensions were used for in vitro studies. Furthermore, platelet thrombus formation induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium was used for an in vivo thrombotic study. Morphine concentration dependently (0.6, 1, and 5 microM) potentiated platelet aggregation and the ATP release reaction stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Yohimbine (0.1 microM), a specific alpha(2)-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by agonists. Morphine also potentiated phosphoinositide breakdown and intracellular Ca(2+) mobilization in human platelets stimulated by collagen (1 microg/ml). Moreover, morphine (0.6-5 microM) markedly inhibited prostaglandin E(1) (10 microM)-induced cyclic AMP formation in human platelets, while yohimbine (0.1 microM) significantly reversed the inhibition of cyclic AMP by morphine (0.6 and 1 microM) in this study. The thrombin-evoked increase in pH(i) was markedly potentiated in the presence of morphine (1 and 5 microM). Morphine (2 and 5 mg/g) significantly shortened the time require to induce platelet plug formation in mesenteric venules. We concluded that morphine may exert its potentiation in platelet aggregation by binding to alpha(2)-adrenoceptors in human platelets, with a resulting inhibition of adenylate cyclase, thereby reducing intracellular cyclic AMP formation followed by increased activation of phospholipase C and the Na(+)/H(+) exchanger. This leads to increased intracellular Ca(2+) mobilization, and finally potentiation of platelet aggregation and of the ATP release reaction.     

Mechanism for antiplatelet effect of onion: AA release inhibition, thromboxane A(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade

Antiplatelet actions of aqueous extract of onion were investigated in rat and human platelet. IC(50)values of onion extract for collagen-, thrombin-, arachidonic acid (AA)-induced aggregations and collagen-induced thromboxane A(2)(TXA(2)) formation were 0.17 +/- 0. 01, 0.23 + 0.03, 0.34 +/- 0.02 and 0.12 +/- 0.01 g/ml, respectively. [(3)H]-AA release induced by collagen (10 microg/ml) in rat platelet was decreased by onion compared to control (22.1 +/- 2.13 and 5.2 +/- 0.82% of total [(3)H]-AA incorporated, respectively). In fura-2 loaded platelets, the elevation of intracellular Ca(2+)concentration stimulated by collagen was inhibited by onion. Onion had no cytotoxic effect in platelet. Onion significantly inhibited TXA(2)synthase activity without influence on COX activity. Platelet aggregation induced by U46619, a stable TXA(2)mimetic, was inhibited by onion, indicating its antagonism for TXA(2)/PGH(2)receptor. These results suggest that the mechanism for antiplatelet effect of onion may, at least partly, involve AA release diminution, TXA(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade.     

Characterization of an anti-thrombospondin monoclonal antibody (P8) that inhibits human blood platelet functions. Normal binding of P8 to thrombin-activated Glanzmann thrombasthenic platelets

Stimulated human blood platelets release thrombospondin, an alpha-granule glycoprotein of 450 kDa. The aim of this work was to characterize an anti-thrombospondin monoclonal antibody (P8) in order to study the role of thrombospondin in platelet functions. The presence of thrombospondin receptor sites on resting and thrombin-stimulated platelets of three Glanzmann's thrombasthenia patients and normal donors was investigated using the P8 monoclonal antibody. Monoclonal antibody P8 was extensively characterized using ELISA, immunoprecipitation, immunoadsorbent affinity chromatography combined with tryptic peptide map analysis and crossed immunoelectrophoretic techniques. Labelled P8 bound strongly to thrombin-stimulated normal platelets (n = 14917 +/- 420, mean +/- SD) (Kd = 9.2 +/- 3.0 nM) and poorly to resting platelets (n = 2697 +/- 1278) (Kd = 24.8 +/- 18.6 nM). Moreover, the number of binding sites for P8 on thrombin-stimulated platelets from three Glanzmann's thrombasthenia patients, lacking the IIb-IIIa glycoprotein complex, were found similar to normal samples. F(ab')2 fragments of P8 inhibited aggregation of, and reduced secretion from, washed platelets stimulated by low concentrations of thrombin (0.05-0.06 U/ml) and collagen (0.5-0.6 microgram/ml). F(ab')2 fragments of P8 inhibited thrombin-induced platelet aggregation, but did not reduce fibrinogen binding (n) nor affect its dissociation constant (Kd). Inhibition of platelet aggregation by P8 suggests that thrombospondin plays an active role in promoting platelet aggregation, at low concentrations of thrombin and collagen. Normal binding of P8 to thrombin-stimulated Glanzmann thrombasthenic platelets indicates the presence of a thrombospondin receptor on the platelet surface distinct from the GPIIb-IIIa complex.     

Nitric oxide and platelet energy metabolism

This study was undertaken to determine whether nitric oxide (NO) can affect platelet responses through the inhibition of energy production. It was found that NO donors: S-nitroso-N-acetylpenicyllamine, SNAP, (5-50 microM) and sodium nitroprusside, SNP, (5-100 microM) inhibited collagen- and ADP-induced aggregation of porcine platelets. The corresponding IC50 values for SNAP and SNP varied from 5 to 30 microM and from 9 to 75 microM, respectively. Collagen- and thrombin-induced platelet secretion was inhibited by SNAP (IC50 = 50 microM) and by SNP (IC50 = 100 microM). SNAP (20-100 microM), SNP (10-200 microM) and collagen (20 microg/ml) stimulated glycolysis in intact platelets. The degree of glycolysis stimulation exerted by NO donors was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or uncouplers (2,4-dinitrophenol). Neither the NO donors nor the respiratory chain blockers affected glycolysis in platelet homogenate. SNAP (20-100 microM) and SNP (50-200 microM) inhibited oxygen consumption by platelets. The effect of SNP and SNAP on glycolysis and respiration was not reduced by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, a selective inhibitor of NO-stimulated guanylate cyclase. SNAP (5-100 microM) and SNP (10-300 microM) inhibited the activity of platelet cytochrome oxidase and had no effect on NADH:ubiquinone oxidoreductase and succinate dehydrogenase. Blocking of the mitochondrial energy production by antimycin A slightly affected collagen-evoked aggregation and strongly inhibited platelet secretion. The results indicate that: 1) in porcine platelets NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase, 2) the inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.     

Modulating effects of adenosine on the process of human platelet activation]

The inhibitory effect of adenosine on aggregation of human platelets activated by platelet activating factor (PAF), ADP and serotonin (5-HT) were examined using native platelets from blood of volunteers. Platelet aggregation was determined by Born's method. Effective adenosine concentrations (IC50) which had inhibited platelet aggregation were found to be 0.63 +/- 0.11, 1.47 +/- 0.31 and 0.64 +/- 0.18 microM, respectively. It was shown that 10 microM adenosine inhibited PAF-induced platelet aggregation completely. The same adenosine concentration blocked ADP- and 5-HT-induced aggregation only partially. Adenosine is physiological inhibitor of human platelet aggregation in administration of PAF, ADP and 5-HT. Specific characteristics of adenosine modulating effect on these ligands was elicited.     

Inhaled NO inhibits platelet aggregation and elevates plasma but not intraplatelet cGMP in healthy human volunteers

Effects of inhaled nitric oxide (NO) on human platelet function are controversial. It is uncertain whether intraplatelet cGMP mediates the effect of inhaled NO on platelet function. We investigated the effect of 30 ppm inhaled NO on platelet aggregation and plasma and intraplatelet cGMP in 12 subjects. We performed platelet aggregation studies by using a photooptical aggregometer and five agonists (ADP, collagen, epinephrine, arachidonic acid, and ristocetin). During inhalation, the maximal extent of platelet aggregation decreased by 75% with epinephrine (P < 0.005), 56% with collagen (P < 0.005), and 20% with arachidonic acid (P < 0.05). Responses to ADP (8% P > 0.05) and ristocetin (5% P > 0.05) were unaffected. Platelet aggregation velocity decreased by 64% with collagen (P < 0.005), 60% with epinephrine (P < 0.05), 33% with arachidonic acid (P < 0.05), and 14% with ADP (P > 0.05). Plasma cGMP levels increased from 2.58 +/- 0.43 to 9.99 +/- 5.57 pmol/ml (P < 0.005), intraplatelet cGMP levels were unchanged (means +/- SD: 1.96 +/- 0.58 vs. 2.71 +/- 1.67 pmol/109 platelets; P > 0.05). Inhaled NO inhibits platelet aggregation via a cGMP independent mechanism.     

Effect of calcium concentration and inhibitors on the responses of platelets stimulated with collagen: contrast between human and rabbit platelets.

1. Variations in the concentration of Ca2+ [Ca2+] in the suspending medium have different effects on the responses of human and rabbit platelets to collagen. 2. When rabbit platelets are stimulated with a low concentration of collagen (0.5 micrograms/ml), aggregation, release of granule contents, and formation of thromboxane are maximal when the suspending medium contains [Ca2+] in the physiological range (0.5-2.0 mM), and very slight in a medium with no added Ca2+. 3. In contrast, human platelets respond most strongly when the suspending medium contains no added Ca2+ [( Ca2+] approx. 20 microM); this is attributable to the enhanced formation of thromboxane A2 (TXA2) upon close platelet-to-platelet contact in this medium. 4. When TXA2 formation is blocked by inhibition of cyclo-oxygenase with aspirin or indomethacin, rabbit platelet aggregation and release in response to 1.25-10 micrograms/ml collagen is also maximal at [Ca2+] of 0.5-2.0 mM and least at 20 microM; human platelets do not aggregate and the extent of release is relatively independent of [Ca2+]. 5. In 1 mM [Ca2+], use of apyrase and/or ketanserin with rabbit platelets in which TXA2 formation is blocked shows that released ADP and serotonin make large contributions to aggregation and release in response to high concentrations of collagen; human platelet aggregation is largely dependent on TXA2. 6. Use of fura-2-loaded platelets shows that the collagen-induced rise in cytosolic [Ca2+] is only slightly inhibited by aspirin or indomethacin in rabbit platelets, but almost completely inhibited in human platelets. 7. Responses of rabbit platelets to collagen are less dependent on TXA2 than those of human platelets. Released ADP and serotonin make major contributions to the responses of rabbit platelets to collagen.     

Inhibitory effect of ginkgolide B on platelet aggregation in a cAMP- and cGMP-dependent manner by activated MMP-9

Extracts from the leaves of the Ginkgo biloba are becoming increasingly popular as a treatment that is claimed to reduce atherosclerosis, coronary artery disease, and thrombosis. In this study, the effect of ginkgolide B (GB) from Ginkgo biloba leaves in collagen (10 microg/ml)- stimulated platelet aggregation was investigated. It has been known that human platelets release matrix metalloproteinase- 9 (MMP-9), and that it significantly inhibited platelet aggregation stimulated by collagen. Zymographic analysis confirmed that pro-MMP-9 (92-kDa) was activated by GB to form an MMP-9 (86-kDa) on gelatinolytic activities. And then, activated MMP-9 by GB dose-dependently inhibited platelet aggregation, intracellular Ca2+ mobilization, and thromboxane A2 (TXA2) formation in collagen-stimulated platelets. Activated MMP-9 by GB directly affects down-regulations of cyclooxygenase-1 (COX-1) or TXA2 synthase in a cell free system. In addition, activated MMP-9 significantly increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have the anti-platelet function in resting and collagen-stimulated platelets. Therefore, we suggest that activated MMP-9 by GB may increase the intracellular cAMP and cGMP production, inhibit the intracellular Ca2+ mobilization and TXA2 production, thereby leading to inhibition of platelet aggregation. These results strongly indicate that activated MMP-9 is a potent inhibitor of collagen-stimulated platelet aggregation. It may act a crucial role as a negative regulator during platelet activation.     

Nicotinic-type unitary currents in Helix neurons

1. The platelet aggregation response to several known platelet agonists was evaluated in four Asian elephants. The platelets were highly responsive to stimulation with platelet-activating factor (PAF) and collagen, less responsive to adenosine diphosphate (ADP) and non-responsive to arachidonic acid, serotonin and epinephrine. 2. Arachidonic acid (1 x 10(-4) M), while inducing no aggregation, caused the release of 1248 +/- 1147 pg/ul (mean +/- SD) of thromboxane B2 (TXB2), the stable metabolite of thromboxane A2 from stimulated platelet. The addition of 1 x 10(-4) M ADP to platelets caused suboptimal aggregation and the release of only 25 +/- 10 pg TXB2/microliters. 3. The calcium channel blocker, verapamil, produced a dose-dependent inhibition of PAF-induced but not collagen-induced aggregation. The cyclooxygenase inhibitor, acetylsalicylic acid, produced no inhibition of either collagen- or PAF-induced aggregation.     

Endogenous platelet serotonin release monitored during aggregation by means of a new electrochemical technique

Differential pulse voltammetry combined with electrochemically treated carbon fibre microelectrodes was used to monitor endogenous serotonin release occurring during platelet aggregation. After platelet stimulation by thrombin, an oxidation peak was recorded at +280 mV. HPLC analyses performed with fluorimetric detection have shown that this released electroactive compound was essentially serotonin. Moreover, serotonin measurements in the same samples by the technique reported here and by fluorimetry were found to be very similar (1.15 +/- 0.30 microM and 1.17 +/- 0.15 (mean +/- S.D., n = 6), respectively). Extracellular serotonin concentrations could be estimated either directly during aggregation or in supernatants obtained from stimulated or lysed platelets. Maximal serotonin concentrations have been found to be 6.93 +/- 0.37 and 3.28 +/- 0.39 nmol/10(9) platelets from rat and human, respectively. Using the reported procedure, we have observed that no serotonin was released from thrombin-stimulated platelets prepared from rats treated with reserpine. Our new technique represents a selective and performant tool for rapid determination of endogenous serotonin platelet secretion.     

Modulation of in vitro platelet 5-HT release by species of Erythrina and Cymbopogon

Extracts of Australian plants were screened to detect constituents affecting adenosine di-phosphate (ADP) induced platelet aggregation and [14C]5-hydroxytryptamine (5-HT) release. Extracts of four tested plants including, Eremophila gilesii, Erythrina vespertilio, Cymbopogon ambiguus, and Santalum acuminatum, were found to cause significant inhibition of platelet 5-HT release. Inhibition levels ranged from 56-98%, and was not due to the non-specific effects of protein binding tannins. These extracts, and those we have previously identified as being active, were examined further to determine if they affect epinephrine (EPN), arachidonic acid (A.A) or collagen stimulated platelet aggregation and 5-HT release. Among those extracts investigated, we found that both the methanolic extract of E. vespertilio and the dichloromethane (DCM) extract of C. ambiguus were most potent and caused significant inhibition of platelet activation induced by EPN, A.A and to a lesser extent by collagen. Inhibition of ADP induced platelet 5-HT release by both of these extracts, was dose-dependent, with IC50 values for E. vespertilio and C. ambiguus estimated to be 20.4 microl (1.855 mg/ml) and 8.34 microl (0.758 mg/ml), respectively. Overall, C. ambiguus exhibited most activity and also caused dose-dependent inhibition of A.A induced platelet activation. These results indicate that inhibition may occur specifically at a site within the A.A pathway, and suggest the presence of a cyclo-oxygenase inhibitor. Both E. vespertilio and C. ambiguus are reported to be traditional headache treatments, with the present study providing evidence that they affect 5-HT release.     

Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen

Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of beta1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of beta1-null platelets to native fibrillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, beta1-null platelets adhere to fibrillar, but not soluble collagen under static as well as low (150 s(-1)) and high (1000 s(-1)) shear flow conditions, probably through binding of alphaIIbbeta3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on fibrillar as well as soluble collagen. These data show that GPVI plays the central role in platelet-collagen interactions by activating different adhesive receptors, including alpha2beta1 integrin, which strengthens adhesion without being essential.     

Role of peroxynitrite in the process of vascular tone regulation by nitric oxide and prostanoids--a nanotechnological approach

The production of peroxynitrite (ONOO(-)) in the endothelium decreases NO bioavailability, decreases vasorelaxation and changes vascular tone. ONOO(-) can also influence the production of prostacyclin-another vasorelaxant. We used a nanotechnological approach (nanosensors) to elucidate the release of NO, O(2)(-), and ONOO(-) in endothelium and their effect on production of prostanoids. The basal ONOO(-) concentration near the endothelium (3-5 microm) varied from 1 to 50 nmol/L and maximal calcium ionophore stimulated ONOO(-), did not exceed 900 nmol/L. The highest ONOO(-) concentrations were produced in ischemia/reperfusion atherosclerosis, diabetes, aging and vary among different racial groups (higher in Blacks than in Whites). ONOO(-) decreased PGI(2) activity with IC(50) approximately 150 nmol/L for 8 min reaction time, but has no effect of short reaction time. Prostaglandin E(1) decreased NO, O(2)(-), and ONOO(-) by limiting Ca(2+) flux into endothelium, decreased edema and vasoconstriction during ischemia/reperfusion. In endothelium (HUVEC's) of Black's the ONOO(-) concentrations were high 750+/-50 nmol/L while the lowest concentrations of vasorelaxants were 275+/-25 nmol/L of NO, 150+/-15 pb/100 microg protein of 6-keto-PGF(1)(alpha) as compared to White's (420+/-30 and 470+/- nmol/L for ONOO(-) and NO respectively and 280+/-20 pg/100 mg protein for 6-keto-PGF(1)(alpha)).     

Physiological changes in platelet aggregation and nitric oxide levels during menstrual cycle in healthy women.

Hormonal levels, mainly those of estrogens, protect women from the appearance of cardiovascular diseases by an increasing nitric oxide (NO) activity. NO is an endogenous vasodilator and antiaggregating substance. We decided to investigate platelet function and plasma levels of nitric oxide during preovulatory and midluteal phases in young and healthy women with normal menstrual cycles (MCs). Nine young, healthy female subjects had recorded three consecutive MCs before entering this program. Platelet-rich plasma (PRP) was used for the determination of platelet aggregation and NO measurements. Moreover, platelet sensitivity to the inhibitory effect of exogenous NO was tested. The EC(50) of collagen showed no differences between the preovulatory (1.36+/-0.16 microg/mL) and the midluteal (1.31+/-0.08 microg/mL; P, NS) phases. However, the EC(90) during the preovulatory phase was higher (2.05+/-0.2 microg/mL) than during the midluteal phase (1.8+/-0.6 microg/mL). Plasma levels of NO were lower during the preovulatory phase (19.1+/-2 microM) in comparison to the midluteal phase (20.9+/-2.3 microM). Interestingly, the exogenous amount of NO to produce at least half of the inhibition of an EC(90) collagen-induced aggregation was higher at the preovulatory phase (323.3+/-60.9 nM) than during the midluteal phase (240.0+/-37.5 nM; P, NS). We propose that during the follicular phase platelets rather use NO produced by the endothelium; therefore, it is necessary to add more agonist to activate those, but it results in higher consumption of circulating NO, whereas during luteal-phase platelets are not able to use NO, requiring lower amounts of agonist and thus resulting in higher plasma levels of NO. This is an interesting fact in research on cardiovascular diseases of women.     

An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tick Haemaphysalis longicornis and its mechanism of action

Soluble materials of salivary glands from Haemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C_8 reverse phase HPLC. The purified activity, named longieornin, is a protein of moleeular weight 16 000 on SDS-PAGE under both reduced and nonredueed conditions. Collagen-mediated aggregation of platelets in plasma and of washed platelets (IC_(50) was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effeetors, including ADP, arachidonic acid, thrombin, ristocetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-O-phorbol-13-myristate acetate, was observed. Longieonin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intraeellar Ca~(2 ) level of platelets in response to collagen were com     

Stimulation of human platelets by collagen occurs by a Na+/H+ exchanger independent mechanism

In stimulated human platelets dense-granule secretion in response to the 'weak agonists' ADP, adrenaline, platelet activating factor and low concentrations of thrombin as well as Ca2+ mobilisation in response to thrombin are enhanced by a Na+/H+ exchanger. In the present study the role of this antiport in collagen stimulated human platelets was examined. While stimulation of platelets loaded with the fluorescent intracellular pH-sensitive dye, bis-carboxyethyl-5-(6)-carboxyfluorescein (BCECF) with thrombin resulted in the activation of the Na+/H+ exchanger, activation of this antiport did not occur in collagen-stimulated platelets. The lack of antiport activity in response to collagen using BCECF-loaded platelets correlated with the lack of any functional role of the antiport in collagen stimulated platelets. In the presence of a Na+/H+ exchange inhibitor, ethylisopropylamiloride, neither collagen-induced platelet aggregation or dense-granule secretion was affected. Furthermore, while the removal of extracellular Na+ (Na+ext), a condition that also prevents activation of the antiport, inhibited dense-granule secretion in response to a low concentration of thrombin, collagen-induced secretion was potentiated. This potentiatory effect could not be attributed to changes in either the membrane potential or in collagen-induced phospholipase C or protein kinase C activity. The present results indicate that in contrast to the 'weak agonists' (1) collagen-induced platelet activation does not require activation of the Na+/H+ exchanger and (2) Na+ext per se is an inhibitor of collagen-induced secretion.     

Thrombin-induced platelet microparticles improved the aggregability of cryopreserved platelets

Platelets were activated with freezing/thawing and thrombin stimulation, and platelet microparticles generated following platelet activation were isolated with ultracentrifugation. The effects of platelet microparticles on platelet activation were studied with annexin V assay, protein tyrosine phosphorylation, and platelet aggregation. Freezing-induced platelet microparticles decreased but thrombin-induced platelet microparticles increased platelet annexin V binding and aggregation. Freshly washed platelets were cryopreserved using epinephrine and dimethyl sulfoxide (Me(2)SO) as combined cryoprotectants, and stimulated with thrombin-induced platelet microparticles. Following incubation of thrombin-induced platelet microparticles, the reaction time of platelets to agonists decreased but the percentages of aggregation increased, such as washed platelets from 44% +/- 30 to 92% +/- 7, p < 0.001, and cryopreserved platelets from 66% +/- 10 to 77% +/- 7, p < 0.02. By increasing platelet aggregability, platelet microparticles recovered after thrombin stimulation improved platelet function for transfusion. A 53-kDa platelet microparticle protein showed little phosphorylation if it was released from resting platelets or platelets stimulated with ADP, epinephrine, propyl gallate or dephosphorylation if it was derived from ionophore A 23187-stimulated platelets. However, the same protein released from frozen platelets showed significant tyrosine phosphorylation. Since a microparticle protein with 53 kDa was compatible with protein tyrosine phosphatase-1B (PTP-1B), its phosphorylation suggests the inhibition of enzyme activity. The microparticle proteins derived from thrombin-stimulated platelets were significantly phosphorylated at 64 kDa and pp60c-src, suggesting that the activation of tyrosine kinases represents a possible mechanism of thrombin-induced platelet microparticles to improve platelet aggregation.     

Release of Phosphorylated HSP27 (HSPB1) from Platelets Is Accompanied with the Acceleration of Aggregation in Diabetic Patients

We investigated the relationship between HSP27 phosphorylation and collagen-stimulated activation of platelets in patients with diabetes mellitus (DM). Platelet-rich plasma was prepared from blood of type 2 DM patients. The platelet aggregation was analyzed in size of aggregates by an aggregometer using a laser scattering method. The protein phosphorylation was analyzed by Western blotting. Phosphorylated-HSP27 and PDGF-AB released from platelets were measured by ELISA. The phosphorylated-HSP27 levels at Ser-78 and Ser-82 induced by collagen were directly proportional to the platelet aggregation. Total HSP27 levels in platelets were decreased concomitantly with the phosphorylation. The released HSP27 levels were significantly correlated with the phosphorylated levels of HSP27 in the platelets stimulated by 0.3 μg/ml collagen. The low dose collagen-stimulated release of HSP27 was detected but relatively small in healthy donors. The released levels of PDGF-AB were in parallel with the levels of released HSP27. Area under the curve (AUC) of small aggregation (9-25 μm) induced by 0.3 μg/ml collagen was inversely proportional to the levels of released HSP27. AUC of large aggregation (50-70 μm) was directly proportional to the levels of released HSP27. Exogenous recombinant phosphorylated- HSP27 hardly affected the aggregation or the released levels of PDGF-AB induced by collagen. These results strongly suggest that HSP27 is released from human platelets accompanied with its phosphorylation induced by collagen, which is correlated with the acceleration of platelet aggregation in type 2 DM patients.