Platelet function measured by VerifyNow identifies generalized high platelet reactivity in aspirin treated patients

Selected aspirin treated patients may exhibit high platelet reactivity to agonists other than arachidonic acid. This study aimed to determine whether the VerifyNow identifies generalized high platelet reactivity supported by correlations with other established methods that stimulate platelets with various agonists. Stable outpatients with coronary artery disease (n = 110) were treated with aspirin in a two 3 x 3 Latin square design (81, 162 and 325 mg/day for 4 weeks each). VerifyNow (arachidonic acid (AA) cartridge); light transmittance aggregometry; thrombelastography; PFA-100; flow cytometry; PlateletWorks; and urinary 11- dehydro thromboxane levels were measured. Multianalyte profiling measured fibrinogen and von Willebrand factor (vWF). Patients with >or=550 ARU by VerifyNow had increased 5 mM AA-, 5 microM ADP-, and 2 microg/mL collagen-induced platelet aggregation compared to patients with <550 ARU (p 相似文献   

Vitamin E inhibits collagen-induced platelet activation by blunting hydrogen peroxide.

In this study, we investigated whether vitamin E at concentrations achievable in blood after supplementation inhibits platelet function in humans. Gel-filtered platelets were incubated 30 minutes with scalar concentrations (50 to 250 mmol/L) of vitamin E and then stimulated with collagen. Compared with controls, vitamin E inhibited collagen-induced platelet aggregation and thromboxane A2 formation in a dose-dependent manner. Furthermore, vitamin E inhibited, in a dose-dependent manner, Ca(2+) mobilization and formation of inositol 1,4,5-triphosphate. Because it was previously shown that hydrogen peroxide formation mediates arachidonic acid metabolism and phospholipase C activation in collagen-induced platelet activation, we investigated whether vitamin E was able to blunt hydrogen peroxide. In experiments performed in unstimulated platelets supplemented with hydrogen peroxide and in collagen-stimulated platelets, vitamin E was able to blunt hydrogen peroxide. In 6 healthy subjects given vitamin E for 2 weeks (600 mg/d), we found a significant decrease of collagen-induced H(2)O(2) formation, platelet aggregation, and calcium mobilization. This study demonstrated in vitro and ex vivo that vitamin E inhibits collagen-induced platelet activation by blunting hydrogen peroxide formation.     

Platelet function measured by VerifyNow™ identifies generalized high platelet reactivity in aspirin treated patients

Selected aspirin treated patients may exhibit high platelet reactivity to agonists other than arachidonic acid. This study aimed to determine whether the VerifyNow? identifies generalized high platelet reactivity supported by correlations with other established methods that stimulate platelets with various agonists. Stable outpatients with coronary artery disease (n?=?110) were treated with aspirin in a two 3?×?3 Latin square design (81, 162 and 325?mg/day for 4 weeks each). VerifyNow? (arachidonic acid (AA) cartridge); light transmittance aggregometry; thrombelastography; PFA-100®; flow cytometry; PlateletWorks®; and urinary 11- dehydro thromboxane levels were measured. Multianalyte profiling measured fibrinogen and von Willebrand factor (vWF). Patients with ≥550 ARU by VerifyNow? had increased 5?mM AA-, 5?µM ADP-, and 2?µg/mL collagen-induced platelet aggregation compared to patients with <550 ARU (p?≤?0.001). The highest ADP- and collagen-induced aggregation was present in the upper quartile by VerifyNow? (p?<?0.05). Fibrinogen or vWF levels did not differ between VerifyNow? quartiles. Patients with high platelet reactivity to arachidonic acid identified by VerifyNow? also exhibit increased reactivity to other important platelet agonists that is independent of fibrinogen and vWF levels. These data suggest that VerifyNow? may identify a generalized high platelet reactivity phenotype.     

Release of arachidonic acid from human platelets. A key role for the potentiation of platelet aggregability in normal subjects as well as in those with nephrotic syndrome

Low (nonaggregating) concentrations of collagen that potentiate platelet aggregation did not induce the formation of measurable amount of malondialdehyde (MDA) but released small but significant amounts of radioactivity from 14C-arachidonic acid-labeled platelets. A major portion of the radioactive compounds released by nonaggregating concentrations of collagen existed as arachidonic acid and a minor part as thromboxane B2. The nephrotic syndrome enhances platelet aggregability, and this effect is abolished by correcting hypoalbuminemia in vitro and in vivo by the addition of albumin, which is the main carrier for free fatty acids, including arachidonic acid. Human albumin (fatty acid free) inhibited collagen-induced aggregation, MDA formation, and release of the radioactivity from 14C-arachidonic acid-labeled platelets in normals as well as in those with nephrotic syndrome. These data support our hypothesis that the main mechanism responsible for the potentiation of platelet aggregation is the release of arachidonic acid from platelet membrane phospholipids via the activation of phospholipase A2. Furthermore, enhanced platelet aggregation in the nephrotic syndrome was at least partly attributable to an increased availability of arachidonic acid released secondary to hypoalbuminemia. Albumin inhibits aggregation probably by binding to released arachidonic acid preventing arachidonic acid from being metabolized to potent aggregating substances, endoperoxides and thromboxane A2. The mechanism of release of arachidonic acid may play a key role in the potentiation of platelet aggregability in normals as well as in pathologic conditions such as the nephrotic syndrome.     

Arachidonic acid uptake by human platelets is mediated by CD36

The involvement of glycoprotein (GP) IV (CD36) in arachidonic acid uptake by human platelets was investigated using an anti-CD36 monoclonal antibody (MAB). The binding of [(14)C]arachidonic acid to MAB-treated platelets was significantly reduced compared with untreated platelets. The MAB also inhibited arachidonic acid-induced platelet aggregation and thromboxane A(2) synthesis in a dose-dependent manner. Pre-incubation of gel-filtered platelets with the MAB (10mg/I) inhibited arachidonic acid-induced platelet aggregation by 50% and collagen-induced platelet aggregation by 7-8% and the lag time was increased by 200%. Although the mechanism of platelet aggregation is not fully understood yet, the inhibition of arachidonic acid-induced platelet aggregation by the MAB could be the result of a reduced uptake of exogeneously added arachidonic acid by the MAB-treated platelets. Our data clearly indicate that arachidonic acid uptake by platelets is mediated, at least in part, by CD36.     

Arachidonic acid uptake by human platelets is mediated by CD36

The involvement of glycoprotein (GP) IV (CD36) in arachidonic acid uptake by human platelets was investigated using an anti-CD36 monoclonal antibody (MAB). The binding of [¹⁴C]arachidonic acid to MAB-treated platelets was significantly reduced compared with untreated platelets. The MAB also inhibited arachidonic acid-induced platelet aggregation and thromboxane A₂ synthesis in a dose-dependent manner. Pre-incubation of gel-filtered platelets with the MAB (10mg/I) inhibited arachidonic acid-induced platelet aggregation by 50% and collagen-induced platelet aggregation by 7-8% and the lag time was increased by 200%. Although the mechanism of platelet aggregation is not fully understood yet, the inhibition of arachidonic acid-induced platelet aggregation by the MAB could be the result of a reduced uptake of exogeneously added arachidonic acid by the MAB-treated platelets. Our data clearly indicate that arachidonic acid uptake by platelets is mediated, at least in part, by CD36.     

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.     

Identification of human platelet glycoprotein VI-specific IgG autoantibody and its fragments.

Glycoprotein VI (GPVI), as a major receptor for collagen on the platelet surface, mediates the initial platelet contact with collagen, and causes platelet aggregation and thrombosis. Agents of anti-GPVI can inhibit the adhesion, activation and aggregation function of platelets, which can be used for preventing thrombotic diseases. To make humanized monoclonal antibodies by phage surface display technology, we studied plasmas from 44 patients with chronic idiopathic thrombocytopenic purpura using modified monoclonal antibody immobilization of platelet antigen assays. GPVI-specific antibodies were found in six (13.6%) patients. Among these, only one showed significant inhibition of platelet aggregation induced by collagen. The IgG antibody and F(ab')2 fragments of this patient's plasma were further purified and their immunoreactivities and effects on platelet aggregation were reanalyzed. It was found that purified IgG and its F(ab')2 fragments from the patient not only retained the ability to bind to platelet GPVI, but also inhibited collagen-induced platelet aggregation. In this study, therefore, the specific anti-platelet GPVI autoantibody that inhibits collagen-induced platelet aggregation has been successfully screened out, which can be used to develop completely humanized anti-GPVI phage antibody.     

Altered platelet function in cirrhosis of the liver: impairment of inositol lipid and arachidonic acid metabolism in response to agonists

Hemorrhagic disorders are common in patients with liver cirrhosis and result from several factors including impaired platelet function. We evaluated platelet aggregation and arachidonic acid metabolism in response to standard agonists in platelet-rich plasma from 12 cirrhotic patients with mild impairment of liver function (Child A), 12 patients with severe liver dysfunction (Child B and C) and 12 healthy subjects. Platelet aggregation and thromboxane A2 production were consistently reduced in patients with severe liver impairment. To determine whether the platelet dysfunction is due to an intrinsic platelet defect or a circulating inhibitor, we measured platelet aggregation and thromboxane A2 synthesis on washed platelets in healthy subjects and in Child B and C patients. The aggregating response of washed platelets in response to thrombin, collagen and arachidonic acid was markedly reduced, suggesting an intrinsic platelet defect. The biochemical events underlying platelet aggregation were investigated by prelabeling platelets with [1-14C]arachidonic acid. Thrombin-induced activation of phospholipase C (measured as the release of [1-14C]phosphatidic acid) and phospholipase A2 (measured as the release of [1-14C]arachidonic acid and its metabolites) was greatly impaired in platelets from patients with severe liver impairment. We conclude that in advanced cirrhosis there is a severe reduction in platelet aggregatory response to physiologic agonists due to an intrinsic platelet defect which is related to an impairment of the platelet transmembrane signaling mechanism induced by receptor stimulation.     

Inhibitory effect of melatonin on platelet activation induced by collagen and arachidonic acid

Abstract: Melatonin, an indolamine synthesized in the pineal gland, is known to have antiprostanoid activity. The inhibition of platelet aggregation induced by melatonin has been proposed to take place through the cyclooxygenase pathway. In the present study, we found that melatonin has a marked inhibitory effect on collagen, arachidonic acid (AA), adenosine diphosphate (ADP), epinephrine, and A23187-induced aggregation in platelet-rich plasma. On the other hand, using metrizamide-filtered platelets resuspended in Tyrode's buffer, melatonin fails to suppress AA-induced platelet aggregation and ¹⁴C-5-HT release. Under the same conditions, melatonin inhibits collagen-induced platelet activation; however, the addition of threshold doses of AA (0.3 mM) abrogates this effect. These studies suggest that melatonin also inhibits platelet function at a stage preceding the cyclooxygenase-dependent pathway.     

High shear induces platelet dysfunction leading to enhanced thrombotic propensity and diminished hemostatic capacity

Thrombosis and bleeding are devastating adverse events in patients supported with blood-contacting medical devices (BCMDs). In this study, we delineated that high non-physiological shear stress (NPSS) caused platelet dysfunction that may contribute to both thrombosis and bleeding. Human blood was subjected to NPSS with short exposure time. Levels of platelet surface GPIbα and GPVI receptors as well as activation level of GPIIb/IIIa in NPSS-sheared blood were examined with flow cytometry. Adhesion of sheared platelets on fibrinogen, von Willibrand factor (VWF), and collagen was quantified with fluorescent microscopy. Ristocetin- and collagen-induced platelet aggregation was characterized by aggregometry. NPSS activated platelets in a shear and exposure time-dependent manner. The number of activated platelets increased with increasing levels of NPSS and exposure time, which corresponded well with increased adhesion of sheared platelets on fibrinogen. Concurrently, NPSS caused shedding of GPIbα and GPVI in a manner dependent on shear and exposure time. The loss of intact GPIbα and GPVI increased with increasing levels of NPSS and exposure time. The number of platelets adhered on VWF and collagen decreased with increasing levels of NPSS and exposure time, respectively. The decrease in the number of platelets adhered on VWF and collagen corresponded well with the loss in GPIbα and GPVI on platelet surface. Both ristocetin- and collagen-induced platelet aggregation in sheared blood decreased with increasing levels of NPSS and exposure time. The study clearly demonstrated that high NPSS causes simultaneous platelet activation and receptor shedding, resulting in a paradoxical effect on platelet function via two distinct mechanisms. The results from the study suggested that the NPSS could induce the concurrent propensity for both thrombosis and bleeding in patients.     

Adenosine diphosphate (ADP)-induced thromboxane A(2) generation in human platelets requires coordinated signaling through integrin alpha(IIb)beta(3) and ADP receptors.

Adenosine diphosphate (ADP) is a platelet agonist that causes platelet shape change and aggregation as well as generation of thromboxane A(2), another platelet agonist, through its effects on P2Y1, P2Y12, and P2X1 receptors. It is now reported that both 2-propylthio-D-beta gamma-dichloromethylene adenosine 5'-triphosphate (AR-C67085), a P2Y12 receptor-selective antagonist, and adenosine-2'-phosphate-5'-phosphate (A2P5P), a P2Y1 receptor-selective antagonist, inhibited ADP-induced thromboxane A(2) generation in a concentration-dependent manner, indicating that coactivation of the P2Y12 and P2Y1 receptors is essential for this event. SC49992, a fibrinogen receptor antagonist, blocked ADP-induced platelet aggregation and thromboxane A(2) production in a concentration-dependent manner. Similarly, P2 receptor antagonists or SC49992 blocked ADP-induced arachidonic acid liberation. Whereas SC49992 blocked arachidonic acid-induced platelet aggregation, it failed to inhibit thromboxane A(2) generation induced by arachidonic acid. Thus, ADP-induced arachidonic acid liberation, but not subsequent conversion to thromboxane A(2), requires outside-in signaling through the fibrinogen receptor. The Fab fragment of ligand-induced binding site-6 (LIBS6) antibody, which induces a fibrinogen-binding site on the integrin alpha(IIb)beta(3), caused both platelet aggregation and thromboxane A(2) generation. Inhibitors of phosphoinositide 3-kinase, Syk, Src kinases, or protein tyrosine phosphatases inhibited platelet aggregation but not thromboxane A(2) generation, indicating that these signaling molecules have no significant role in phospholipase A(2) activation. In the presence of P2 receptor antagonists A2P5P or AR-C67085, LIBS6 failed to generate thromboxane A(2), suggesting that inside-out signaling through ADP receptors is necessary for this event. It was concluded that both outside-in signaling from the fibrinogen receptor and inside-out signaling from the P2Y1 and P2Y12 receptors are necessary for phospholipase A(2) activation, resulting in arachidonic acid liberation and thromboxane A(2) generation.     

Further Studies on the Effects of the Intracellular Histamine Antagonist DPPE on Platelet Function

SUMMARY. The role of intracellular histamine in the activation of human platelets was explored using the novel histamine antagonist N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine (DPPE). DPPE inhibited aggregation, [(32)P]-phosphatidic acid production (an index of phospholipase C activity) and the increase in cytosolic calcium ([Ca(2+)]i) in response to collagen. In contrast, while at higher concentrations DPPE inhibited aggregation in response to ADP and the thromboxane mimetic EP171, it failed to inhibit EP171-induced [(32)P]-phosphatidic acid production or increases in [Ca(2+)]i elicted by either ADP or EP171. Collagen but neither ADP nor EP171 caused the significant formation of intracellular histamine. The biochemical changes induced by collagen have been shown previously to be, at least partly, secondary to thromboxane generation. Collagen-induced arachidonic acid release was therefore assessed. DPPE inhibited the release of arachidonic acid in response to collagen but had no effect on its subsequent conversion to thromboxane. The findings imply that intracellular histamine acts at an early stage in collagen-induced platelet activation, most likely prior to arachidonic acid release, and further that DPPE inhibits ADP and EP171-induced activation by a non-histamine related effect.     

Reduced platelet activation and platelet aggregation in patients with alcoholic liver cirrhosis

Results from previous studies regarding platelet function in liver cirrhosis are discordant. The aim was to investigate platelet activation and platelet aggregation in patients with alcoholic liver cirrhosis. We included 27 patients with alcoholic liver cirrhosis and 22 healthy individuals. A recently established flow cytometric approach was used to measure platelet activation and platelet aggregation independent of sample platelet count. Platelet aggregation was further investigated using light transmission aggregometry (LTA) (for platelet count >100 × 10⁹/L). Platelet agonists were adenosine diphosphate, thrombin receptor-activating peptide, arachidonic acid, collagen, and collagen-related peptide. Patients had lower median platelet count than healthy individuals, 125 × 10⁹/L (interquartile range [IQR] 90?185) versus 240 × 10⁹ (IQR 204?285), p < 0.001. Platelet activation levels in stimulated samples were lower in patients versus healthy individuals, e.g., after collagen-related peptide stimulation, the median percentage of platelets positive for activated glycoprotein IIb/IIIa was 85% (IQR 70–94) in patients versus 97% (IQR 94–99) in healthy individuals, p < 0.001; lower platelet activation capacity being associated with low platelet count and Child–Pugh class B/C cirrhosis. Flow cytometric platelet aggregation was reduced in patients for collagen-related peptide and for adenosine diphosphate, e.g., platelet aggregation (mean ± standard deviation) was 57% ± 4 in patients versus 70% ± 1 in healthy individuals for collagen-related peptide, p = 0.01. Light LTA showed reduced collagen-induced platelet aggregation in some patients compared with healthy individuals. In conclusion, platelet function was reduced in some patients with alcoholic liver cirrhosis and the severity was associated with platelet count and severity of liver cirrhosis.     

Platelet Aggregation in Primary Raynaud's Phenomenon and the Effect of Enalapril Administration

Platelet aggregation using a single platelet counting technique in whole blood, was determined on 18 patients with primary Raynaud's phenomenon and 17 age-matched controls. Platelet aggregation in the Raynaud's patients was also assessed during a double-blind, crossover trial to investigate the efficacy of the angiotensin converting enzyme (ACE) inhibitor, enalapril. There were no differences in platelet aggregation to collagen, arachidonic acid, ADP or PAF, or in plasma levels of beta-thromboglobulin (BTG), platelet factor 4 (PF4) or thromboxane B(2) (TxB(2)) between the Raynaud's group and the normal controls. Similarly, there were no differences in these parameters in the Raynaud's group during treatment with enalapril when compared to placebo. It is concluded that patients with primary Raynaud's phenomenon have no evidence of abnormal platelet aggregation or increased platelet activation, and that platelet aggregation is not affected by enalapril.     

3,3',5'-Triiodothyronine inhibits collagen-induced human platelet aggregation.

To clarify further the activity of rT3, we examined the effect of rT3 on collagen-induced platelet activation as reflected by aggregation, serotonin release, and protein phosphorylation. rT3, T4, T3, and triiodothyroacetic acid inhibited collagen-induced platelet aggregation and serotonin release from platelets in a dose-dependent manner. However, thyronine did not inhibit collagen-induced platelet aggregation. The concentration at which rT3 inhibited by 50% collagen-induced platelet aggregation was 30 +/- 4 (mean +/- SE) mumol/L. rT3, T4, and T3 did not differ significantly in their abilities to inhibit platelet aggregation. Moreover, rT3 inhibited collagen-induced phosphorylation of the 20-kilodalton protein (myosin light chain) in platelets. In contrast, rT3 did not inhibit 12-O-tetradecanoylphorbol 13-acetate (TPA)- or thrombin-induced platelet aggregation and inhibited only minimally TPA-induced 40-kilodalton protein phosphorylation. These results suggest that rT3 inhibits collagen-induced platelet activation by inhibiting the activity of myosin light chain kinase and that it may be interesting to investigate some kinds of activity of rT3.     

Reverse correlations of collagen-dependent platelet aggregation and adhesion with GPVI shedding during storage

Platelet receptor GPVI plays an important role in platelet firm adhesion to site of vascular injury. Receptor ligation with collagen, in company with other agonist/receptor interactions, augments inside out signaling pathways leading to platelet aggregation and thrombus formation. As GPVI expression is significantly modulated by ectodomain shedding, this study aimed to examine whether GPVI shedding functionally affects collagen-mediated platelet activation during storage. 6 PRP-platelet concentrates were subjected to adhesion analysis on collagen matrix under mild stirring condition as well as collagen-induced aggregation on day 1, 3 and 5 post-storage. Concurrently, platelet supernatants of same samples were fractionated by ultra-centrifugation and obtained micro-particle-free samples were subjected to western blot analysis for the evaluation of GPVI shedding. We showed a direct correlation between collagen-dependent platelet aggregation and adhesion (r?=?0.8, p?=?0.0001). The increasing levels of GPVI shedding during storage were in reverse correlation with collagen-induced platelet aggregation (r?=???0.82, p?=?0.0004) which was significantly reducing during storage. Platelet adhesion to collagen matrix significantly decreased post-storage while it was also reversely correlated with the levels of GPVI shedding during 5 days storage of platelets (r?=???0.69, p?=?0.002). Data presented here demonstrated that progressive shedding of surface adhesion receptor GPVI can affect its functional activities in stored platelets. Thereby considering the crucial role of GPVI in platelet adhesion to the site of injury, whether the therapeutic efficacy of banked platelet products could be influenced by storage-dependent shedding of this receptor, remains to be answered in future studies.     

Arachidonic Acid-induced Platelet Aggregation ex vivo in Patients with Acute Ischaemic Heart Disease

The threshold concentrations of arachidonic acid (AA) required to induce platelet aggregation were measured in platelet-rich plasma (PRP) from patients with acute ischaemic heart disease and healthy controls. The analytical precision of the test was very good (coefficient of variation 0-4%). Analytical accuracy was evaluated by comparing the results with threshold values for collagen-induced platelet aggregation, and a significant correlation was found (r = 0.56; p<0.01). When comparing the serum levels of thromboxane B(2) to threshold values for AA-induced platelet aggregation an inverse relationship was found (r = -0.37; p<0.01). In the clinical study significantly increased aggregability to AA was seen in patients with unstable angina pectoris (n = 13) compared to patients with stable angina pectoris (n = 14), (p<0.01), and both groups had hyperaggregating platelets compared to healthy controls (n = 27), (p<0.01). The patients with acute myocardial infarction (n = 10) had nearly normoaggregating platelets for the first 2-3 days after admission, but after a week and at day 14 their platelets showed significant hyperaggregability compared to healthy controls (p<0.01). Thus studies of AA-induced aggregation ex vivo suggest that patients with unstable angina pectoris and acute myocardial infarction, in whom coronary thrombus is frequently present, have increased platelet aggregability compared to patients with stable angina pectoris and healthy controls.     

Evaluation of platelet function in aspirin treated patients with CAD

Background: Studies of platelet function in the acute coronary syndrome (ACS) have revealed both increased and unchanged platelet activation. To obtain a better understanding of platelet function in coronary artery disease in the setting of aspirin therapy, we performed platelet functional testing in patients with ACS and compared results to patients without CAD. Methods: We measured platelet aggregation and activation in response to ADP and epinephrine in 80 age and gender matched hospitalized patients (40 with ACS, 40 with non-cardiac chest pain (NCCP)). All subjects received ASA (81–325 mg). Platelet aggregation was performed using standard light transmission in platelet-rich plasma and activation was measured via flow cytometric analyses. We also studied platelet function under high shear rates measured by the platelet function analyzer (PFA-100). Results: ASA effect was found to be present in all subjects by blunted platelet aggregation in response to arachidonic acid. Patients with ACS showed significantly higher levels of platelet aggregation to epinephrine compared to patients with NCCP (p = 0.001). Other measures of platelet function including ADP aggregation, Pselectin, activated glycoprotein IIb/IIIa expression, and PFA-100 were unchanged between the two groups. Conclusions: We have found conflicting results of platelet functional testing in ACS. Specifically aspirin therapy in patients with ACS is effective in suppressing the platelet release response and is effective in the partial suppression of platelet aggregation; however, it appears that ACS patients have increased platelet aggregation to adrenergic stimuli when compared to age and gender matched controls without CAD despite the use of aspirin. In some studies, because ACS patients have an accentuated response to adrenergic stimuli this might be interpreted as aspirin resistance. Our study suggests that depending on the assay used to determine aspirin resistance, not all patients with this label are resistant to the biological effects of aspirin but they may have higher than normal baseline platelet sensitivity to adrenergic stimuli. This research was supported by grant K23HL67066 from the National Institute of Health.     

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)