Platelets in Translational Research – 2
Subtitle: Discovery of Potential Anti-platelet Targets
Larry H. Bernstein, MD, FCAP, Reviewer and Curator
http://phramaceuticalintelligence.com/2013-10-7/larryhbern/Platelets-in-Translational-Research-2/
This offering is the the second of a succession on Platelets in Translational Medicine:
Part I: В Platelet make, interactions between platelets and endothelium, and intracellular transcribing
Part II:В Discovery of Potential Anti-platelet Targets
Endothelium-contingent vasodilator effects of platelet activating broker on rat resistance vessels
1Katsuo Kamata, Tatsuya Mori, *Koki Shigenobu & Yutaka Kasuya Department of Pharmacology, School of Pharmacy, Hoshi University, Tokyo and *Department of Pharmacology, Toho University School of Pharmaceutical Sciences, Funabashi, Chiba, Jp Br. J. Pharmacol. (1989), 98, 1360-1364 1В To illustrate the mechanisms of the powerful and throughout-lasting hypotension produced by plateletВ activating middleman (PAF), its effects on perfusion press in the perfused mesenteric arterial hollow of the rat were examined. 2 Infusion of PAF (10-11 to 3 x 10-10M; EC50 = 4.0 x 10′ m; 95%CL = 1.6 x 10-11 — 9.4 x 10-11 M) and acetylcholine (ACh) (10′ to 10-6m; EC50 = 3.0 В± 0.1 x 10-9m) produced noted concentration-dependent vasodilatations which were significantly inhibited ~ means of treatment with detergents (0.1% Triton X-100 because of 30 s or 0.3% CHAPS by reason of 90 s). 3 Pretreatment with CV-6209, a PAF foe, inhibited PAF- but not ACh-induced vasodilaВtation. 4 Treatment through indomethacin (10-6m) had no tenor on PAF- or ACh-induced vasodilatation. 5 These results demonstrate that extremely low concentrations of PAF generate vasodilatation of resistance vessels through the quit of endothelium-derived relaxing factor (EDRF). This may narrative for the strong hypotension produced through PAF in vivo. Platelet activating constitutive element (PAF, acetyl glyceryl ether phosphorylcholine) has been shown to make strong and long-lasting hypotension in manifold animal species, e.g. normotensive and spontaneously hypertensive rats, rabbits, twenty-one shillings-pigs, and dogs (Tanaka et al., 1983). This affair of PAF is thought to subsist endothelium-dependent (Kamitani et al., 1984; Kasuya et al., 1984a,b; Shigenobu et al., 1985; 1987). In a antecedent study (Shigenobu et al., 1987), we erect that relatively low concentrations of PAF (10-9-10-7m) produced endothelium-hanging relaxation of the rat aorta in the mien of bovine serum albumin. This vasodilator representation of PAF at low concentrations might be the cause of its hypoВtensive performing in vivo. While the aorta faculty of volition offer a resistance to flow, it is visible that the contribution of vessels of smaller central chord to peripheral vascular resistance is a great quantity greater. In this regard, the mesenВteric dissemination of the rat receives approximately individual-fifth of the cardiac output (Nichols et al., 1985) and, so, regulation of this bed may compel a signifiВcant contribution towards systemic blade pressure and circulating blood volume. В Therefore, we examined the general intent of PAF on the resistance vessels of the rat mesenteric vascular stratum and found that extremely low concentrations (10 -11 to 3 x 10-16 m) can produce endothelium-dependent vasodilatation. Figure 1 Effects of PAF up~ the body the perfusion pressure of the methoxamine (10-3N)-constricted mesenteric vascuВlar depression. (a) Upper panel: relaxation induced ~ the agency of PAF (3 x 10-10 M). Lower body of jurors: effects of the PAF-antagonist, CV-6209 (3 x 10-914), forward the relaxation induced by PAF (3 x 10-“N). (b) Concentration-rejoinder curve for the relaxation produced ~ dint of. PAF (10-11 to 3 x 10-10N) in the methoxamine (10-51)-constricted mesenteric vascular channel. Each point is the mean and plumb bars represent the s.e.medium from 5 experiments. Figure 2 Effects of detergents without interrupti~ acetylcholine (ACh)-induced relaxation of the methoxamine (10-5M)-conВstricted mesentericВ vascular support. Concentration-response curves are shown by reason of ACh-induced vasodilatation before (0) and hinder treatment with 0.3% CHAPS (вќ‘) or 0.1% Triton X-100 (0). Each point is the grovelling and vertical bars represent the s.e.plebeian from 5 experiments. Infusions of extremely ~-minded concentrations of PAF (10-11 to 3.1 x 10-1В° m) produced a noted and long-lasting vasodilatation which was significantly suppressed ~ dint of. treatment with detergentsВ ar bed. Concentration-response curves are shown for ACh-induced vasodilatation in the van of (0) and after treatment with 0.3% CHAPS (вќ‘) or 0.1% Triton X-100 (0). Each single thing is the mean and vertical bars portray by action the s.e.mean from 5 experiments. Since Furchgott & Zawadzki (1980) demonstrated the obligatory role of endothelium in vascular relaxВation ~ means of ACh, many studies have suggested that endothelium-derived relaxing factor (EDRF) is reВleased from endothelial cells in replication to a large number of agonists (Furchgott, 1984). In the instant study with perfused resistance vessels, ACh produced vasodilatation in a concentration-dependent manner and the vasorelaxant responses were significantly suppressed through perfusion with detergents such as CHAPS or Triton X-100. В These premises strongly suggest the posВsible involvement of the endothelium in the relaxВation induced ~ the agency of PAF. CV-6209, a PAF adversary, inhibited PAF-induced but not ACh-induced vasodilatation in a compression into a small compass-dependent manner. Specific antagoВnism by CV-6209 has already been obtained through respect to PAF-induced hypotension or platelet aggregating (Terashita et al., 1987). An accumulating body of evidence suggests that hypotension resulting from endotoxin require is due to the endogenous exempt of PAF from endothelial cells (Camussi et al., 1983), leukocytes (Demopoules et al., 1979), macroВphages (Mencia-Huerta & Benveniste, 1979; Camussi et al., 1983) and platelets (Chingard et al., 1979). Indeed, PAF antagonists can reverse estabВlished endotoxin-induced hypotension (Terashita et al., 1985; Handley et al., 1985a,b). From the of rectitude too great for data and the results of the donation study, one posВsible explanation since endotoxin-induced hypotension may be that the extricate of PAF occurs, which then binds to its receptors located up~ the endothelial cells, stimulating production of EDRF. In termination, we demonstrated that extremely low concentrations of PAF bring long-lasting vasodilatation in a opposition vessel of the mesenteric vasculature. Moreover, we showed that this PAF-induced vasodilatation is mediated ~ means of a vasodilator substance released from endothelial cells (EDRF) which is not a prostaglandin. Since the PAF-induced endothelium-sustained by relaxation observed in the present study was elicited at feeble concentrations and was long-lasting, it may have ~ing the main mechanism by which PAF induces hypotension in vivo.
Static platelet sticking, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery sickness: a randomised cross-over study
Andreas C Eriksson*1, Lena Jonasson2, Tomas L Lindahl3, Bo Hedbäck2 and Per A Whiss11Divisions of Drug Research/Pharmacology and 2Cardiology, Department of Medical and Health Sciences, Linköping University, Linköpin, Sw, and 3Department of Clinical Chemistry, University Hospital, Linköping, Sw Journal of Translational Medicine 2009, 7:42   http:/dx.doi.org/10.1186/1479-5876-7-42  http://www.translational-drug.com/content/7/1/42
Abstract
Background: Despite the practice of anti-platelet agents such viewed like acetylsalicylic acid (ASA) and clopidogrel in coronary will disease, some patients continue to undergo from atherothrombosis. This has stimulated unfolding of platelet function assays to overseer treatment effects. However, it is yet not recommended to change treatment based in c~tinuance results from platelet function assays. This study aimed to evaluate the magnitude of a static platelet adhesion analysis to detect platelet inhibiting effects of ASA and clopidogrel. The adhering assay measures several aspects of platelet attachment. simultaneously, which increases the probability of verdict conditions sensitive for anti-platelet handling. Methods: With a randomised cross-from beginning to end design we evaluated the anti-platelet goods of ASA combined with clopidogrel to the degree that well as monotherapy with either medicine alone in 29 patients with a fresh acute coronary syndrome. Also, 29 matched well controls were included to evaluate intra-individual variability into the bargain time. Platelet function was measured through flow cytometry, serum thromboxane B2 (TXB2)-levels and ~ the agency of static platelet adhesion to different protein surfaces. The results were subjected to Principal Component Analysis followed ~ the agency of ANOVA, t-tests and linear regression analysis. Results: The majority of platelet tendency to adhere measures were reproducible in controls athwart time denoting that the assay be possible to monitor platelet activity. Adenosine 5′-diphosphate (ADP)-induced platelet adhesion decreased significantly upon treatment with clopidogrel compared to ASA. Flow cytometric measurements showed the same pattern (r2 = 0.49). In opposed, TXB2-levels decreased with ASA compared to clopidogrel. Serum TXB2 and ADP-induced platelet activation could the two be regarded as direct measures of the pharmacodynamic furniture of ASA and clopidogrel respectively. Indirect pharmacodynamic measures of that kind as adhesion to albumin induced ~ the agency of various soluble activators as well considered in the state of SFLLRN-induced activation measured by sweep along cytometry were lower for clopidogrel compared to ASA. Furthermore, adhering to collagen was lower for ASA and clopidogrel combined compared with either drug alone. Conclusion: The not directly to the point pharmacodynamic measures of the effects of ASA and clopidogrel potency be used together with ADP-induced activation and serum TXB2 as antidote to evaluation of anti-platelet treatment. This should be further evaluated in future clinical studies at what place screening opportunities with the adhesion test will be optimised towards increased sensitivity to anti-platelet handling. The benefits of ASA have been clearly demonstrated through the Anti-platelet Trialists’ Collaboration. They institute that ASA therapy reduces the exposure to harm by 25% of myocardial infarction, dash or vascular death in “elevated-risk” patients. When using the identical outcomes as the Anti-platelet Trialists’ Collaboration forward a comparable set of “vainglorious-risk” patients, the CAPRIE-study showed a disrespect benefit of clopidogrel over ASA. Furthermore, the confederacy of clopidogrel and ASA has been shown to have existence more effective than ASA alone on account of preventing vascuВlar events in patients with unstable angina and myoВcardial infarction being of the cl~s who well as in patients undergoing percutaneous coronary interposition (PCI). Despite the obvious benefits from anti-platelet therapy in coroВnary disorder, low response to clopidogrel has been described through several investigators. A lot of regard has also been drawn towards poor response to ASA, often called “ASA resistance”. The general of ASA resistance is complicated by reason of several reasons. First of all, deviating studВies have defined ASA hindrance in different ways. In its broadest soundness, ASA resistance can be defined any one as the inability of ASA to obstruct platelets in one or more platelet function tests (laboratory resistance) or as the incapacity of ASA to prevent recurrent thrombosis (i.e. method of treating failВure, here denoted clinical resistance). The lack of a general exact statement of the meaning of ASA resistance results in difficulties when trying to measure the prevalence of this phenomeВnon. Estimates of laboratory opposition range from approximately 5 to 60% depending forward the assay used, the patients deliberate and the way of defining ASA check. Likewise, lack of a standardized definition of low response to clopidogrel makes it beset with ~y to estimate the prevalence of this wonder as well. The principles of existing platelet assays, viewed like well as their advantages and disadvantages, require been described elsewhere. In short, assays potentially convenient for monitoring treatment effects include those commonly used in study such as platelet aggregometry and sweep along cytometry as well as immunoassays toward measuring metabolites of thromboxane A2 (TXA2). Also, the PFA-100TM, MultiplateTM and the VerifyNowTM are examples of instruments commercially developed during evaluation of anti-platelet therapy. HowВtill doomsday, no studies have investigated the serviceableness of alterВing treatment based attached laboratory findings of ASA resistance. Regarding clopidogrel, in that place are recent studies showing that reconciliation of clopidogrel loading doses according to vasodilator-stimulated phosphoprotein phosphorylation alphabetical table of references measured utilising flow cytometry decrease greater adverse cardiovascular events in patients by clopidogrel resistance. Static adhesion is ~y aspect of platelet function that has not been investigated in earlier studies of the furniture of platelet inhibiting drugs. Consequently, static platelet tendency to adhere is not measured by any of the current aspirant assays for clinical evaluation of platelet value derived. The static platelet adhesion assay offers ~y opportunity for simultaneous measurements of the combined movables of several different platelet activators up~ platelet function. In this study, platelet attachment. to albumin, collagen and fibrinogen was investigated in the neighborhood of soluble platelet activators including adenosine 5′-diphosphate (ADP), adrenaline, lysophosphatidic tart (LPA) and ris-tocetin. Collagen, fibrinogen, ADP and adrenaline are physiological agents that are well-known in quest of their interacВtions with platelets. Ristocetin is a farrago derived from bacteria that facilitates the interaction between von Willebrand factor (vWf) and glycoprotein (GP)-Ib-IX-V forward platelets, which otherwise occurs only at run condiВtions. The static nature of the trial therefore prompted us to include ristocetin in give an ~ to to get a rough estimate up~ GPIb-IX-V dependent events. LPA is a phospholipid that is produced and released ~ dint of. activated platelets and that also can be generated through mild oxiВdation of LDL. It was included in the donation study since it is present in atherosclerotic vessels and suggested to have ~ing important for platelet activation after enamelled plate rupВture. Finally, albumin was included for example a surface since the platelet activating efficiency of LPA can be detected at the time that measuring adhesion to such a outside. Thus, by the use of deviating platelet activators, several measures of platelet adhesion were obtained simultaneously This means that the possibilities to riddle for conditions potentially important for detecting furniture of platelet-inhibiting drugs far exceeds the screening abilities of other platelet value derived tests. Consequently, the static platelet attachment. assay is very well suited toward development into a clinically useful fanciful conception for monitoring platelet inhibiting treatment. Also, it has earlier been proposed that investiВgating the combined goods of two activators on platelet exercise might be necessary in order to detect effects of ASA and other antiplatelet agents [26]. This is a standard that can easily be met ~ the agency of the static platelet adhesion assay. Through the screening conduct we found different conВditions where the static adhesion was influenced ~ dint of. the drug given. The inclusion of patients and controls. Patients and controls were included consecutively. Blood samples from controls were drawn at two different occasions separated by 2–5.5 months. All patients entering the study current ASA combined with clopidogrel and hot spark sampling was performed 1.5–6.5 months succeeding initiating the treatment. This was followed by a randomised cross-over enabling every part of patients to receive monotherapy with the couple ASA and clopidogrel. The patients received monotherapy for at least 3 weeks and despite a maximum of 4.5 months before performing kindred sampling. A total of 33 patients and 30 controls entered the study. In the extreme point, 29 patients and 29 controls completed the study. Blood was drawn from patients at three contrary occaВsions (Figure 1). The ~ and foremost sample was drawn after all patients had accepted combined treatment with ASA (75 mg/sunshine) and clopidogrel (75 mg/day) with respect to 1.5–6.5 months on the model of the index event. The study in that case used a randomised cross-over design significance that half of the patients received ASA as monotherapy while half believed only clopidogrel (75 mg/day against both monotherapies). The monotherapy was sooner or later switched for every patient so that aggregate patients in total received all three therapies. Samples in spite of evaluation of the monotherapies were drawn for therapy for at least 3 weeks and at the in the greatest degree for 4.5 months. Most of the differences in handling length can be ascribed to the reality that the national recommendations for handling in this patient group were changed for the time of the course of the study. The allocation to monotherapy was blinded against the laboratory personnel. In general, the appliance of three different treatments for intra-individual comВparisons in a testy-over design is different from previous studies on ASA and clopidogrel, which have mainly been concerned with only two treatment alternatives.
Intra-individual mutation in healthy controls
Measurements of platelet clinging and serum TXB2-levels were performed forward healthy controls on two separate occaВsions (2–5.5 months term) in order to investigate the vicinity of intraindividual variation in platelet reactivity and clotting-induced TXB2-work. The standardised Z-scores from the simplified factors were used beneficial to analysis by Repeated Measures ANOVA of the premises from the healthy controls. We lay the ~ation of significantly decreased plateВlet adhesion at the side with compared to the first visit as being ADP-induced adhesion (Factor 1, p = 0.012) and in favor of adheВsion to fibrinogen (Factor 5, p = 0.012). This intra-indi-vidual variability to boot time makes it difficult to lengthen out any conclusions regarding effects of anti-platelet management. We therefore further analysed the individual variables constituting Factors 1 and 5 by Repeated Measures ANOVA in order to make celebrated the variables that varied significantly athwart time. Variables being significantly difВferent between visit 1 and visit 2 were soon afterward excluded and a new Repeated Measures ANOVA was performed ~ward the new factors. After this state, none of the factors corresponding to tendency to adhere showed variation over time and these factors were hereafter used for analysis on patients. Serum levels of TXB2, which constituted a separate factor, varied significantly in of a sound constitution controls at two separate occasions (Figure 2). 
Effect of platelet inhibiting treatment on serum TXB2-levels (Factor 13). Serum TXB2-levels (Factor 13) towards patients (n = 29) and healthy controls (n = 29) are presented since mean + SEM. ASA alone or in conspiracy with clopidogrel was signifВicantly manifold from clopidogrel alone and compared to the mode of the controls (p < 0.001). Also, the contention between controls at visit 1 and pay a ~ to 2 was significant. ***p < 0.001, ns = not important. When investigating possible effects of platelet-inhibiting manipulation with Repeated Measures ANOVA, significant goods were seen for four of the factors corresponding to platelet adhesion. The factors that were not able to find out significant treatment effects were adrenaline-induced adhesion (Factor 3), ristocetin-induced adhesion (Factor 4) and sticking to fibrinogen (Factor 5). Regarding adheВsion factors detecting handling effects, ADP-induced adhesion (Factor 1, Figure 3A inset) was significantly decreased by clopidogrel alone or by clopidogrel in addition ASA compared with ASA alone. Surprisingly, platelet tendency to adhere induced by ADP was lower during the term of the monotherapy with clopidogrel compared to dual therapy. ADP-induced adhering to albumin is shown as a agent example of the variables of Factor 1 (Figure 3A). Ristocetin-induced adhesion to albumin (Factor 6, Figure 3B inset) was signifВicantly decreased ~ dint of. clopidogrel alone compared with ASA alone. This quarrel was also seen for ristocetin combined by LPA, which is shown as each example of a variable belonging to Factor 6 (Figure 3B). In Factor 7 (Figure 3C inset), answering. to LPA-induced adheВsion to albumin, we build clopidogrel to decrease adheВsion compared with ASA and compared with ASA plus clopidogrel. These differences were reflected by the comВbined activation through LPA and adrenaline, which was a variable included in Factor 7 (Figure 3C). Finally, adheВsion to collagen (Factor 8, Figure 3D) was significantly decreased by dual therapy compared with ASA alone or clopidogrel alone. As be able to be seen from the above descripВtion, monotherapy by clopidogrel resulted in signifiВcantly decreased clinging compared to clopidogrel combined with ASA towards Factors 1 and 7. This was likewise observed for the variable shown in the same manner with a representative examВple of Factor 6 (Figure 3B). The pair factors corresponding to flow cytometric measurements (Factors 14 and 15, FigВure 4) both showed that ASA-treated platelets were other active than platelets treated with clopidogrel alone or clopidogrel plus ASA. Furthermore, serum TXB2-levels (Figure 2) was significantly decreased ~ dint of. ASA alone or by ASA plus clopidogrel compared with clopidogrel alone. Regarding the other measurements not soon measuring platelet function, significant differences were originate for Factor 10 including HDL and because platelet count (Factor 12) but nor one nor the other for the factor corresponding to inflammaВtion (Factor 9) nor instead of Factor 11 including LDL. Factor 10 including HDL was form in a mould to be elevated by both ASA and clopidogrel monotherapies compared with dual therapy (p = 0.003 on account of ASA, p = 0.019 for clopidogrel, premises not shown). Platelet count were construct to be increased after dual therapy compared with both monotherapies (p < 0.001, premises not shown). 
The influence of ASA and clopidogrel attached platelet adhesion. The main figures are acting for others examples of the variaВbles constituting the particular factors. The insets show the Z-scores by reason of each factor. Also shown in the insets are the comparВisons betwixt the control means of visit 1 and 2 and handling with ASA (A), clopidogrel (C) and the complot of ASA and clopidogrel (A+C). The several figures show the effect of platelet inhibiting manipulation on ADP-induced adhesion (Factor 1, Fig A), ristocetin-induced sticking to albumin (Factor 6, Fig B), LPA-induced adhering to albumin (Factor 7, Fig C) and adheВsion to collagen (Factor 8, Fig D) notwithstanding patients (n = 29) and healthy controls (n = 29). All values are presented being of the kind which mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significative. 
The influence of ASA and clopidogrel put ~ platelet activity measured by flow cytometry. The movables of platelet inhibiting treatment on platelet activation detected through flow cytometry induced by ADP (Factor 14, Fig A) and SFLLRN (Factor 15, Fig B) without ceasing patients (n = 29). The main figures are deputy examples of the variables constituting the respective facВtors. The insets show the Z-scores in spite of each factor. All values are presented because mean + SEM. ***p < 0.001, ns = not betokening. Platelets from patients (n = 29) were activated in vitro with adenosine 5′-diphosphate (ADP; 0.1 and 0.6 Ојmol/L) or SFLLRN (5.3 Ојmol/L) followed through flow cytometric measurements of fibrinogen-styptic or expression of P-selectin. Presented results are the pitiful-% of fibrinogen-binding and P-selectin play of features В± SEM. Reference values (obtained earlier for the time of routine analysis at the accredited Dept. of Clinical Chemistry at the University hospital in LinkГ¶ping) are shown because mean with reference interval within parenthesis. Stars show significant differences for patients compared to relation values. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not eminently expressive. В (Table not shown)
Discussion
With the bearing of finding variables sensitive to clopidogrel and ASA-handling, this study used a screening draw nigh and measured several different variables simultaneously. To subjugate the complexity of the material we performed PCA in degree to find correlating variables that steady the same property. In this street the 54 measurements of platelet sticking were reduced to 8 factors. Visual inspecВtion revealed that each factor represented a separate entity of platelet sticking and the factors could therefore have ~ing renamed according to the aspect they measured. We thus conclude that future studies fustiness not involve all 54 adheВsion variables, however instead, one variable from each determining element should be enough to cover 8 not the same aspects of platelet adhesion. In adding to the adhesion data, the remaining 15 variables in addition formed distinct factors that were feasible to rename according to measured property. It is noted that serum TXB2 formed a defined group not correlated to any of the other measurements.
It is serious that laboratory assays used for clinical purВposes are reproducible and that they adjust parameters that are not confounded through other variables. Some of the measurements performed in this study (clinical chemistry variables and platelet occupation measured by flow cytome-try) are used in favor of clinical analysis at accredited laboratories at the University hospital in LinkГ¶ping. However, the reproducibility of the platelet clinging assay was mostly unknown before this study. Our commencing results suggested that the factors answering. to ADP-induced adhesion and clinging to fibrinogen were not reproduciВble. We for that reason excluded the most varied variables conВstituting these factors, what one. resulted in no intra-individual furniture for healthy controls in the platelet adheВsion analyze . From this we conclude that many, but not all, measures of platelet sticking are reproducible. Moreover, the static estate might limit the possibilities for transВlating the results from the attachment. assay into in vivo platelet adhering occurring during flow conditions. HowВfor~, platelet adhesion to collagen and fibrinogen is at the disposal of on О±2131- and О±IIb133-receptors particularly in the current assay. This suggests that the static platelet sticking assay can measure important aspects of platelet exercise despite its simplicity. Furthermore, vWf dependВent adhering is not directly covered in the instant assay although ristocetin-induced adhesion appears to exist dependent on GPIb-IX-V and vWf . From this disputation it is evident that the adhesion assay as well as flow cytometry be possible to measure effects of clopidog-rel while using ADP as activating stimuli. It is also evident that serum-TXB2 levels rule the effects of ASA. HowВfor~, these measures focus on the primitive interaction between the drugs and the platelets, which could be probВlematic when dire to evaluate the complex in vivo treatВment event. It has previously been found that no other than 12 of 682 ASA-treated patients (≈ 2%) had residual TXB2 serum levels higher than 2 model deviations from the populaВtion indicate. Measurements of the effect of arachidonic pungent on platelet aggregometry have also led to the concluВsion that ASA check is a very rare phenomenon. Thus, our study supports these prior findings that assays measuring the pharmacodynamic spryness of ASA (to inhibit the COX-enzyme) rarely recognizes patients as ASA-resistant. This suggests that the inducement of ASA-resistance is not owing to an inability of ASA to act since a COX-inhibitor.
We suggest that put upon the right track measurements of ADP and TXA2-goods (in our case ADP-induced activation steady by adhesion or flow cytometry and serum TXB2-levels) be necessitated to be combined withВ measures that are solely partly dependent on ADP and TXA2 particularly. For instance, an adhesion variable partly dependent on TXA2 might be clever to detect ASA resistance caused through increased signalling through other activating pathways. Such a scenario would be characterВized by serum TXB2 values showing analogical COX-inhibiВtion while platelet attachment. is increased. This study employed a screening manner of proceeding in order to find such tortuous measures of the effects of ASA and clopidogrel. Our results simulation inhibiting effects of clopidogrel comВpared to ASA without interrupti~ adhesion to albumin in the presence-chamber of LPA or ristocetin. This was in addition observed for our flow cytometric measurements by SFLLRN as activator, which confirms that SFLLRN is adroit to induce release of granule subjects considered in platelets. SFLLRN- and ADP-induced platelet activation, because measured by flow cytometry, was moderately correlated to each other and adhesion induced by LPA in the same proportion that well as ristocetin showed weak correlaВtions with ADP-induced adhesion.В These results more distant confirm that these measures of platelet activity are partly dependent on ADP. We gain earlier shown that adhesion to albumin induced by simultaneous stimulation by LPA and adrenaline (a unsteady belonging to the LPA-factor in the at hand study) can be inhibited by interdict of ADP-signalling in vitro. This strengthens our conclusion that the effect on LPA-induced tendency to adhere observed for clopidogrel is caused ~ the agency of inhibition of ADP-signalling. Also, the presence-chamber of LPA in atherosclerotic plaques and its potential role in thrombus formation after brooch rupВture makes it especially attractive for the in vivo setВtinkle of myocardial infarction. Assays of static platelet sticking that have been used in preceding studies aimed at investigating treatment movables of platelet inhibiting drugs. Importantly, this study shows that the static platelet sticking assay is reproducible over time. We moreover showed that the static platelet adhering assay as well as flow cytometry detected the vigor of clopidogrel to inhibit platelet activation induced ~ means of ADP. Our results further suggest that other measures of platelet tendency to adhere and platelet activation measured by glide cytometry are indirectly dependent on secreted ADP or TXA2. One of that kind measure is adhesion to a collagen surface, which should be more thoroughly investigated by reason of its ability to detect effects of clopidogrel and ASA. Likewise, exactly to its connection to atherosclerosis and myocardial infarction, the LPA-induced tenor should be further evaluated for its sufficiency to detect effects of clopidogrel. In finale, the screening procedure undertaken in this study has revealed suggestions forward which measures of platelet activity to comВtwining-stem in order to evaluate platelet execution.
Effect of protein kinase C and phospholipase A2 inhibitors adhering the impaired ability of human platelets to incitement vasodilation
*,1Helgi J. Oskarsson, 1Timothy G. Hofmeyer, 1Lawrence Coppey & 1Mark A. Yorek 1Department of Internal Medicine, University of Iowa and VA Medical Center, Iowa City, IA British Journal of Pharmacology (1999) 127, 903-908 В В http://www.stockton-push.co.uk/bjp
1В В The bearing of this study was to put questions to the mechanism of impaired platelet-mediated endothelium-contingent vasodilation in diabetes. Exposure of human platelets to elevated glucose in vivo or in vitro impairs their energy to cause endothelium-dependent vasodilation. While antecedent data suggest that the mechanism in spite of this involves increased activity of the cyclo-oxygenase course of life, the signal transduction pathway mediating this meaning is unknown. 2 Platelets from diabetic patients in the same proportion that well as normal platelets and legitimate platelets exposed to high glucose concentrations were used to induce the role of the polyol footway, diacylglycerol (DAG) production, protein kinase C (PKC) spryness and phospholipase A2 (PLA2) activity ~ward vasodilation in rabbit carotid arteries. 3 We lay the ~ation of that two aldose-reductase inhibitors, tolrestat and sorbinil, caused but a modest improvement in the impairment of vasodilation by glucose exposed platelets. However, sorbitol and fructose could not have ~ing detected in the platelets, at any one normal or hyperglycaemic conditions. We fix that incubation in 17 mM grape-sugar caused a significant increase in DAG levels in platelets. Furthermore, the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused expressive impairment of platelet-mediated vasodilation. The PKC inhibitors calphostin C and H7 in the same manner with well as inhibitors of PLA2 agility normalized the ability of platelets from diabetic patients to object vasodilation and prevented glucose-induced impairment of platelet-mediated vasodilation in vitro. 4 These results advise that the impairment of platelet-mediated vasodilation caused ~ dint of. high glucose concentrations is mediated through increased DAG levels and stimulation of PKC and PLA2 nimbleness. Keywords: Glucose; signal-transduction; platelet; vasodilation; diabetes Abbreviations: ADP, adenosine diphosphate; DAG, diacyglycerol; DEDA, dimethyleicosadienoic pungent; EDNO, endothelium-derived nitric oxide; OAG, 1-oleoyl-2-acetyl-sn-glycerol; PKC, protein kinase C; PLA2, phospholipase A2; PMA, phorbol 12-myristate 13-acetate
Introduction
Activated perpendicular platelets produce vasodilation via release of platelet-derived adenosine diphosphate (ADP), which in turn stimulates the release of endothelium-derived nitric oxide (EDNO) . EDNO causes vascular varnish muscle relaxation and inhibits platelet act and excessive thrombus formation. Recent reports recommend that platelets from patients with diabetes mellitus be wanting the ability to produce EDNO-unable to exist without vasodilation. This platelet defect can be reproduced in vitro by exposure of normal human platelets to high glucose concentrations, in a time and condensation dependent manner. This glucose-induced platelet desert appears to involve activation of the cyclo-oxygenase track, including thromboxane synthase. However, it odds and ends unknown how exposure of platelets to admirable concentrations of glucose in vivo or in vitro, leads to increased agility of these enzymes. Previous studies designate that high glucose concentrations mediate more of their adverse biologic effects via the polyol pathwayВ high glucose increases intracellular diacylglycer-ol (DAG) levels, upregulates protein kinase C (PKC) activityВ and be able to lead to increased arachidonic acid freedom via PKC-mediated increase in phospholipase A2 sprightliness,В which in turn increases activity of cyclo-oxygenase. In this study we scrutinize the possible role of these metabolic pathways in mediating the incompetence of diabetic and hyperglycaemia-induced platelets to yield vasodilation. In this study we discover that in vitro incubation of vertical human platelets in high glucose causes a expressive increase in platelet DAG levels, what one. is evident after 30 min.
The role of protein kinase-C (PKC)
DAG and OAG are known activators of PKC. Data in Figure 2 simulation that normal human platelets incubated by the DAG analogue, (OAG), in kind to mimic the effect of increased intracellular DAG, lost their ability to cause vasodilation.  Next we pure whether enhanced PKC activity plays a role in the signalling track leading to impaired ability of diabetic platelets to efficient ~ vasodilation. We found that platelets from patients through diabetes mellitus that were treated by the PKC-inhibitor calphostin-C produced legitimate vasodilation, while untreated platelets from the identical patients lacked the ability to lead to vasorelaxation (Figure 3A). Similarly, while natural platelets incubated in high glucose puzzled their ability to cause vasorelaxation, co-incubation by calphostin-C prevented the glucose-mediated impairment of platelet-mediated vasodila-tion (Figure 3B). Calphostin-C did not arrogate the ability of normal platelets to arbitrate vasodilation: 35±3 vs 37±4% increase in utensil diameter, with or without the inhibitor (n=5), particularly. Similar results were obtained with the PKC-inhibitor H7 (50 ILM) (results not shown).  In addition, normal platelets  `primed’ ~ means of a 20 min incubation in Tyrode’s buffer containing PMA (80 nM) completely obdurate their ability to produce vasorelaxation (Figure 4). Figure 3 (A) Platelets were isolated from patients with diabetes mellitus (n=6). Platelets were incubated in Tyrode’s duffer for 2 h with or out of calphostin-C (50 nM). Subsequently the platelets were thrombin (0.1 U ml—1) activated and perfused through a phenylephrine (10 jIM) preconstricted erect rabbit carotid artery, and the modify in vessel diameter measured. *P<0.01. (B) Platelets solitary from healthy donors (n=6) were incubated in Tyrode’s concussion-guard containing either 6.6 mM (118 mg dl—1) [NL Plts] or 17 mM (300 mg dl—1) [Glucose Plts] grape-sugar for 4 h. For the ultimate 2 h the PKC-inhibitor calphostin-C (50 nM) was added to some of the high glucose treated platelets. Subsequently the three groups of platelets were thrombin (0.1 U ml—1) activated and perfused through a phenylephrine (10 jIM) preconstricted natural rabbit carotid artery, and the modify in vessel diameter measured. *P<0.01 vs NL-Plts and Gluc-Plts+Calp-C. (noy shown) Figure 4 Platelets from salutary donors (n=8) were isolated separated into couple groups and treated with or exclusively of phorbol 12-myristate 13-acetate (PMA) (80 nM) because of 20 min. After a washout revolution of time, treated and untreated platelets were thrombin (0.1 U ml—1) activated and perfused through a phenylephrine (10 jIM) precon-stricted cony carotid artery, and the change in canal diameter measured. *P<0.01 as far as concerns PMA-Plts vs NL-Plts. (not shown)
Conclusions
In abstract, the results of this study lengthwise with recently published data (Oskarsson & Hofmeyer 1997; Oskarsson et al., 1997) propose that high glucose levels cause ~y increase in platelet DAG that upregulates the briskness of PKC, which in turn increases the alertness of phospholipase A2 that causes exoneration of arachidonic acid which leads to increased spryness of cyclo-oxygenase and thromboxane synthase in platelets (Oskarsson et al., 1997). From a clinical perspective this pathway is of considerable part since it lends itself to therapeutic interventions with inhibitors both at the demolish of cyclo-oxygenase and the thromboxane-synthase.
References
OSKARSSON, H.J. & HOFMEYER, T.G. (1996). Platelet-mediated endothelium-at the disposal of vasodilation is impaired by platelets from patients with diabetes mellitus. J. Am. Coll. Cardiol., 27, 1464 – 1470. OSKARSSON, H.J. & HOFMEYER, T.G. (1997). Diabetic human platelets extricate a substance which inhibits platelet-mediated vasodilation. Am. J. Phys., 273, H371 – H379. OSKARSSON, H.J., HOFMEYER, T.G. & KNAPP, H.R. (1997). Malondialdehyde inhibits platelet-mediated vasodilation ~ the agency of interfering with platelet-derived ADP. JACC, 29 (Suppl A): 304A.
G-Proteinв€’Coupled Receptors as Signaling Targets for Antiplatele t Therapy
Susan S. Smyth, Donna S. Woulfe, Jeffrey I. Weitz, Christian Gachet, Pamela B. Conley, et al. Participants in the 2008 Platelet Colloquium Arterioscler Thromb Vasc Biol. 2009;29:449-457. В В http://dx.doi.org/10.1161/ATVBAHA.108.176388В В В Online ISSN: 1524-4636 В В http://atvb.ahajournals.org/make easy/29/4/449
Abstract—
Platelet G protein–coupled receptors (GPCRs) indoctrinate and reinforce platelet activation and thrombus constitution. The clinical utility of antagonists of the P2Y12 receptor during ADP suggests that other GPCRs and their intracellular signaling pathways may represent viable targets for novel antiplatelet agents. For example, thrombin stimulation of platelets is mediated ~ dint of. 2 protease-activated receptors (PARs), PAR-1 and PAR-4. Signaling downstream of PAR-1 or PAR-4 activates phospholipase C and protein kinase C and causes autoamplification ~ dint of. production of thromboxane A2, release of ADP, and stock of more thrombin. In addition to ADP receptors, thrombin and thromboxane A2 receptors and their downstream effectors—including phosphoinositol-3 kinase, Rap1b, talin, and kindlin—are promising targets for new antiplatelet agents. The mechanistic the why and the wherefore and available clinical data for drugs targeting disruption of these signaling pathways are discussed. The identification and progressive growth of new agents directed against especial platelet signaling pathways may offer every advantage in preventing thrombotic events in which case minimizing bleeding risk. (Arterioscler Thromb Vasc Biol. 2009;29:449-457.) Key Words: platelets . signaling . G proteins . receptors . thrombosis
Introduction
Since the foremost observations of agonist-induced platelet aggregating in 1962,В remarkable progress has been made in identifying elementary corpuscle surface receptors and intracellular signaling pathways that govern platelet function. These discoveries have translated into estabВlished, novel, and emerging therapeutics to treat and intercept acute ischemic events by targeting platelet notable transduction. В Indeed, antiplatelet therapy is a mainstay of commencing management of patients with ACS and those undergoing percutaneous coronary interposition (PCI). Evidence-based refinements in anticoagulant and antiplatelet therapies own played an important role in the proceeding decline in the death rate from coronary malady observed from 1994 to 2004. Despite these curative advances, however, ACS patients receiving “optimal” antithrombotic therapy calm sufВfer cardiovascular events. Platelet Signaling Pathways
Vascular injury—whether caused ~ dint of. spontaneous rupture of atherosclerotic plaque, brooch erosion, or PCI-related or other trauma—exposes tending to adhere proteins, tissue factor, and lipids promoting platelet tethering, clinging, and activation. Once bound and activated, platelets let go soluble mediators suchВ as ADP, thromboxane A2, and serotonin and render less difficult thromВbin generation. These mediators, in become acid, stimulate GPCRs on the platelet surface that are critical to initiation of numerous intracellular signaling pathways, including activaВtion of phospholipase C (PLC), protein kinase C (PKC), and phosphoinositide (PI)-3 kinase. Both calcium and PKC conВtoll to activation of the small G protein, В Recently, members of the kindlin household of focal adhesion proteins have been identified taken in the character of integrin activators,В perhaps functioning to make easy talin–integrin interactions. Figure. Role of G protein–coupled receptors in the thrombotic progress. In humans, protease-activated receptors (PAR)-1 and PAR-4 are coupled to intracellular signaling pathways through molecular switches from the Gq, G12, and Gi protein families. When thrombin (scissors) cleaves the amino-termination of PAR-l and PAR-4, particular signaling pathways are activated, one determination of which is ADP secretion. By covering to its receptor, P2Y12, ADP activates extra Gi-mediated pathways. In the defect of wounding, platelet activation is counteracted through signaling from PG I2 (PGI2). Adapted from references 26–28 by permission. Ca2 indicates calcium; CalDAG-GEF1, calcium and diacylglcerol-regulated guanine-nucleotide give and take reciprocally factor 1; GP, glycoprotein; IP, prostacyclin; PKC, proВtein kinase C; PLC, phospholipase C; RIAM, Rap1-GTP–interacting adapter molecule.
Future Directions: P2Y1 and P2X Inhibition
Given the clinical good luck of the P2Y12 antagonists, it is worthwhile to study other purinergic signaling pathways in platelets. Although platelets regard 2 P2Y receptors acting synergistically end different signaling pathways, the overall platelet rejoinder to ADP is relatively modest. For illustration, ADP alone elicits only reversible responses and does not encourage platelet secretion.В The low contain of ADP receptors on the platelet superficies also may limit signalВing.
Thrombin Signaling in Platelets
Thrombin, the ut~ potent platelet agonist, has diverse movables on various vascular cells. For model, thrombin promotes chemotaxis, adhesion, and violence through its effects on neutrophils and monocytes. Thrombin besides influВences vascular permeability through its furniture on endothelial cells and triggers assuage muscle vasoconstriction and mitogenesis.54 Thrombin interacts with 2 protease-activated receptors (PARs) on the surface of human platelets—PAR-1 and PAR-4. Signaling through the PARs is triggered by thrombin-mediated cleavage of the extracellular dominion of the receptor and exposure of a “tethered ligand” at the unaccustomed end of the receptor (Figure 1).В Signaling through either PAR can activate PLC and PKC and consideration autoamplification through the production of thromboxane A2, the discharge of ADP, and generation of other thing thrombin on the platelet surface.
PAR-1В Antagonists because Antithrombotic Therapy
The expression profiles of PARs without interrupti~ platelets differ between humans and nonprimates. Mouse platelets defectiveness PAR-1 and largely signal through PAR-4 in replication to thrombin, with PAR-3 serving a cofactor value derived. Platelets from cynomol-gus monkeys hold primarily PAR-1 and PAR-4, and a peptide-imitative PAR-1 antagonist extends the time to thromВbosis later than carotid artery injury.В The nonpeptide competitor SCH 530348 (described below) inhibits thrombin- and PAR-1 agonist peptide (TRAP)-induced platelet act (inhibitory concentrations of 47 nmol/L and 25 nmol/L, respectively), but it has no effect steady ADP, collagen, U46619, or PAR-4 agonist peptide stimulation of platelets.В SCH 530348 has of the best bioavailability in rodents and monkeys (82%; 1 mg/kg) and completely inhibits ex vivo platelet act in response to TRAP within 1 sixty minutes of oral administration in monkeys with no effect on prothrombin or activated interested thromboplastin times. Of the PAR-1 antagonists, SCH 530348 and E5555 are the compounds to along in development and clinical testing.В SCH 530348 is each oral reversible PAR-1 antagonist deВrived from himbacine, a combine found in the bark of the Australian magnolia tree.В In clinical trials, 68% of patients showed ~80% disallowance of platelet aggregation in response to thrombin receptor activating peptide (TRAP; 15 mol/L) 60 minutes ~wards receiving a 40-mg loading draught of SCH 530348. By 120 minutes, the distribution had risen to 96%.В In a Phase 2 unhappiness of SCH 530348, 1031 patients scheduled conducive to angiography and possible stenting were randomized to reВceive SCH 530348 or placebo more aspirin, clopidogrel, and antithrombin therapy (heparin or bivalirudin).В Major and petty bleeding did not differ substantially between the placebo and individual or combined SCH 530348 groups.
Future Directions: PAR-4 Inhibition
Activation and signaling of PAR-1 and PAR-4 impel a biphasic “spike and prolonged” answer, with PAR-1 actiВvated at thrombin concentrations 50% let down than those required to activate PAR-4.В A 4-amino pungent segment, YEPF, on the extracellular region of PAR-1 appears to reckoning for the receptor’s high-relationship interactions with thrombin. The YEPF order of succession has homology to the COOH-bound of hirudin and its synthetic GEPF analog, bivaliru-hurly-burly, which can interact with exosite-1 forward thrombin. Thus, thrombin may interact in tandem with PAR-1 and PAR-4, through the initial interactions involving exosite-1 and PAR-1, and after docking at PAR-4 via the thrombin in actual process site.56 PAR-1 and PAR-4 may mould a stable heterodimer that enables thrombin to act taken in the character of a bivalent functional agonist, rendering the PAR-1–PAR-4 heterodimer compage a unique target for novel antithrombotic therapies. Pepducins, or organic unit-permeable peptides derived from the third intracellular loop of either PAR-1 or PAR-4, disrupt signaling betwixt the receptors and G proteins and forbid thrombin-induced platelet aggregation. In mice, a PAR-4 pepducin has been shown to postpone bleeding times and attenuate platelet activation.В Combining bivalirudin with a PAR-4 pepducin (P4pal-i1) inhibited aggregation of human platelets from 15 sound volunteers, even in response to strong concentrations of thrombin.В In addition, although bivaliru-din and P4pal-i1 reaped ground delayed the time to carotid artery occlusion for ferric chloride-induced injury in guinea pigs, their combination prolonged the time to occlusion further than did bivalirudin alone. Additional blockade of the PAR-4 receptor may discourse a benefit beyond that achieved ~ the agency of inhibition of thrombin activity.
Targeting Thromboxane Signaling
Thromboxane A2 acts in successi~ the thromboxane A2/prostaglandin (PG) H2 (TP) receptor, causing PLC signaling and platelet activation. Several drugs be the subject of been tested and developed that prevent thromboxane synthesis—most notably, aspirin. BeВyond the documented issue of aspirin, however, results have been uniformly disappointing by a wide variety of thromboxane synthase inhibitors. В Likewise, a mass of TP receptor antagonists have been developed, end few have progressed beyond Phase 2 trials as of safety concerns.В More freshly, the thromboxane A2 receptor antagonist terutroban (S18886) showed fast, potent inhibition of platelet aggregation in a swinish model of in-stent thrombosis that was comparable to the combination of aspirin and clopidogrel ~-end with a more favorable bleeding side view.В Ramatroban, another TP inhibitor approved in Japan ~ the sake of treatment of allergic rhinitis, has shown antiaggre-gatory goods in vitro comparable to those of aspirin and cilostazol.
Novel Downstream Signaling Targets
Signaling pathways stimulated ~ means of GPCR activation are esВsential in quest of thrombus formation and may represent potential targets for drug development. One course of life involved in platelet activation is signaling through lipid kinases.В PI-3 kinases transduce signals ~ dint of. generating lipid secondВary messengers, that then recruit signaling proteins to the plasma membrane. A head target for PI-3K signaling is the protein kinase Akt (Figure 1).В Platelets contain both the Akt1 and Akt2 isoforms.28 In mice, the one and the other Akt1 and Akt2 are required for thrombus formation.В Mice lacking Akt2 possess aggregation defects in response to plain concentrations of thrombin or thromboxane A2 and answering. defects in dense and a-little grain secretion. The Akt isoforms have multiple substrates in platelets. Glycogen synthase kinase (GSK)-3(3 is phosphorylated ~ the agency of Akt in platelets and supВpresses platelet derivative and thrombosis in mice.В Akt-mediated phosphorylation of GSK-3(3 inhibits the kinase action of the enzyme, and with it, its concealment of platelet function. Akt activation besides stimulates nitric oxide producВtion in platelets, what one. results in protein kinase G–unable to exist without degranulation.В Finally, Akt has been implicated in activaВtion of cAMP-unable to exist without phosphodiesterase (PDE3A), which plays a role in reducing platelet cAMP levels subsequent to thrombin stimulation.67 Each of these Akt-mediated events is expected to grant to platelet activation. Rap1 members of the Ras kindred of small G proteins have been implicated in GPCR signaling and integrin activation. Rap1b, the greatest part abundant Ras GTPase in platelets, is activated quickly after GPCR stimulation and plays a key role in the activation of integrin aIIb(3)В Stimulation of Gq-linked receptors, in the same state as PAR-4 or PAR-1, activates PLC and, through consequent increases in intracellular calcium, PKC. These signals in suit activate calcium and diacylglcerol-regulated guanine-nucleotide reciprocity factor 1 (CalDAG-GEF1), which has been implicated in activation of Rap1 in silverware-lets.В Experiments in CalDAG-GEF1-insufficient platelets indicate that PKC- and CalDAG-GEF1–unable to exist without events represent independent synergistic pathways chief to Rap1-mediated integrin aIIb(33 activation.В Consistent by this concept, ADP can stimulate Rap1b activation in a P2Y12- and PI-3K-contingent, but calcium-independent, manner. A latest common step in integrin activation involves bindВing of the cytoskeletal protein talin to the integrin-(33-subunit cytoplasmic retinue.В Rap1 appears to be required to configuration an activation complex with talin and the Rap effector RIAM, that redistributes to the plasma membrane and unmasks theВ talin astringent site, resulting in integrin activation.В Mice that scantiness Rap1bВ or platelet talinВ have a venesection disorder with impaired platelet aggregation because of the lack of integrin aIIb( (33В activation. In exhibit the differences of, mice with a integrin-(33 subunit change that prevents talin binding have impaired agonist-induced platelet aggregating and are protected from thromВbosis, but do not display pathological bleeding,В suggestВing that this interaction may have existence an attractive therapeutic target. Recently, members of the kindlin clan of focal adhesion proteins, kindlin-2 and kindlin-3, bring forth been identiВfied as coactivators of integrins, required with regard to talin activation of integrins.В Kindlin-2 binds and synergistically enВhances talin activation of aIIb.В Of note, deficiency in kindlin-3, the prevailing kindlin family member found in hematopoietic cells, results in trenchant bleeding and protection from thrombosis in mice.
Conclusions
Antiplatelet therapy targeting thromboxane production, ADP effects, and fibrinogen binding to integrin aIIb(33 receive proven benefit in preventing or treating quick arterial thrombosis. New agents that bargain greater inhibition of ADP signaling and agents that clog thrombin’s actions on platelets are currently in clinical trials. Emerging strategies to inhibit platelet function include blocking alternative platelet GPCRs and their intracellular signaling pathways. The defy remains to determine how to most profitably combine the various current and during antiplatelet therapies to maximize benefit and minimize disadvantage. It is well documented that aspirin therapy increases bleeding compared with plaВcebo; that whenever clopidogrel is added to aspirin therapy, phlebotomy increases relative to the use of aspirin therapy alone; and that whenever even greater P2Y12 inhibition with prasugrel is added to aspirin therapy, venesection is further increased comВpared with the use of clopidogrel and aspirin league therapy. Does this mean that improved antiplatelet competency is mandated to come at the worth of increased bleeding? Not necessarily, still it will require a far more fully understanding of platelet signaling pathways and the sort of aspects of platelet function must exist blocked to minimize arterial thrombosis. One of the good in the highest degree clinical examples of the disconnect betwixt antiplatelet-related bleeding and antithrombotic efВficacy is the situation of the oral platelet glycoprotein (GP) IIb/IIIa antagonists. The employment of these agents uniformly led to significantly greater phlebotomy compared with aspirin but no greater virtue; in fact, mortality was increased mixed patients receiving the oral glycoprotein IIb/IIIa inhibitors.77 Through one improved understanding of platelet signaling pathways, antiplatelet therapies well-adapted can be developed not based in successi~ their ability to inhibit platelets from aggregating, taken in the character of current therapies are, but rather based in successi~ their ability to prevent the clinically meaningful consequences of platelet activation. What exactly these are scraps the greatest obstacle.
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