Optimization of anticoagulation with warfarin for stroke prevention: pharmacogenetic considerations.

Warfarin is a cornerstone of oral anticoagulation for stroke prevention. Anticoagulation with warfarin in patients with atrial fibrillation is over twice as effective in secondary prevention of stroke as any other tested alternatives, including all other antithrombotic drugs or surgical interventions. General belief is that warfarin is capable of preventing 20 ischemic strokes for every hemorrhagic one it causes. However, warfarin is one of the most feared agents as a result of its woeful safety profile and difficulties in maintaining the proper daily dose. Recent research in pharmacogenetics predominantly focused on elucidating the influence of individual genetic predispositions to administered warfarin. Although the incorporation of genotype information improves the accuracy of adequate dose prediction, an improvement in anticoagulation control or a reduction in hemorrhagic complications has not been yet convincingly demonstrated. It is clear that identifying an individual patient's risk for hemorrhage on warfarin will require more broad clinical and genetic studies. Future research focused on patients with stroke should concentrate on defining the possible differences, especially focusing on predicting bleeding events in general and intracranial hemorrhages in particular. The purpose of this review is to summarize the existing evidence about pharmacogenetics of warfarin in general, especially focusing on stroke prevention.

Eltrombopag (Promacta), a thrombopoietin receptor agonist for the treatment of thrombocytopenia: current and future considerations.

Thrombocytopenia, a decreased platelet count, is a common clinical feature that may be caused by decreased platelet production or accelerated platelet removal. Accelerated platelet removal may result from various immunologic mechanisms, excessive consumption, or sequestration of platelets in the spleen. Thrombocytopenia can range from a transient, isolated finding to a severe, life-threatening condition. Eltrombopag (SB497115) is a novel, orally bioavailable, small-molecule thrombopoetin receptor agonist that induces differentiation and proliferation of megakaryocytes. Preclinical testing on healthy volunteers shows high drug bioavailability and efficacy in raising platelet counts. The 10-day treatment with eltrombopag increased platelet count up to 1.5 times. The drug is usually tolerated well and serious adverse events and discontinuation are rare. There is an unmet need for this agent, and probably eltrombopag is indeed the "drug of choice" for some limited categories of patients; however, its long-term safety profile is unknown. To evaluate the potential drug development, careful analysis of thrombocytopenia morbidity and prevalence of the applicable for treatment with eltrombopag clinical scenarios is highly warranted. Lack of vascular outcome data with regard to potential excessive activity of these next-generation novel platelets may result in increased thrombotic risks and cause worsened cardiovascular and stroke outcomes may be the major obstacle to the success of this promising agent.

Stability validation of paraformaldehyde-fixed samples for the assessment of the platelet PECAM-1, P-selectin, and PAR-1 thrombin receptor by flow cytometry.

Sample fixation for storage and/or transportation represents an unsolved challenge for multicenter clinical trials assessing serial changes in platelet activity, or monitoring various antiplatelet regimens. Whole blood flow cytometry represents a major advance in defining platelet function, although special training and expensive equipment is required. We sought to determine how fixation with 2% paraformaldehyde (PFA), and storage of blood samples over 1 week affects the flow cytometry readings for both intact and thrombin-activating four major surface platelet receptors. Whole blood platelet expression of PECAM-1, P-selectin, PAR-1 inactive receptor (SPAN-12), and cleaved (WEDE-15) epitope was assessed immediately after blood draw, after staining with 2% PFA, and at day 1, 3, 5, and 7. The study was performed in 6 volunteers with multiple risk factors for vascular disease, not receiving any antiplatelet agents. Staining with PFA resulted in a slight decrease of fluorescence intensity, especially for PECAM-1, while antigen expression at day 1, 3 and 5 remains consistent, and highly reproducible. At day 7 there was a small but inconsistent trend towards diminished fluorescence intensity. The platelet data were consistent while validated with the isotype-matched irrelevant antibody. These data suggest that there is a 5 day window to perform final flow cytometry readings of whole blood PFA-fixed inactivated platelet samples. In contrast, thrombin activation cause gradual loss of flow cytometry signal, and cannot be recommended for long-term storage. This is critical logistic information for conducting multicenter platelet substudies within the framework of major clinical trials.

Distribution of dipyridamole in blood components among post-stroke patients treated with extended release formulation.

Extended release dipyridamole (ERD) is widely used in patients after ischaemic stroke; however, the ability of this antithrombotic agent to be stored in different blood cells has never been explored in post-stroke patients. We hypothesised that since ERD is known to be highly lipophilic, the drug may be present not only in plasma, but also accumulated in platelets, leukocytes, and erythrocytes. Fifteen patients after documented ischaemic stroke were treated with Aggrenox (ERD and low-dose aspirin combination) BID for 30 days, and 12 of them completed the study. ERD concentrations in blood cells and platelet-poor plasma were measured by spectrofluorimetry at Baseline, Day 14, and Day 30 after the initiation of therapy. The background level of spectrofluorometry readings differs slightly among the blood components (132-211 ng/ml) due to the differences in the preparation of samples and cell isolation techniques. As expected, two weeks of ERD therapy produced steady-state plasma concentration of dipyridamole already at Day 14 (1,680 +/- 542 ng/ ml), followed by a slight not significant decrease at one month (1,619 +/- 408 ng/ml). Two weeks of therapy was sufficient to achieve a consistent dipyridamole accumulation in erythrocytes (361 +/- 43 ng/ml), but not in platelets (244 +/- 78 ng/ml), or leukocytes (275 +/- 49 ng/ml). In fact, white blood cells continued dipyridamole intake beyond 14 days period, and this increase (398 +/- 66 ng/ml) was significant (p = 0.02) at 30 days. Treatment with ERD in post-stroke patients resulted not only in achievement of therapeutic plasma dipyridamole concentrations, but also deposition of the drug in erythrocytes and leukocytes, but not in platelets. If confirmed, these data will affect our better understanding of dipyridamole pleiotropy, and may explain long-term benefit of ERD formulation.

Effects of Aggrenox and aspirin on plasma endothelial nitric oxide synthase and oxidised low-density lipoproteins in patients after ischaemic stroke. The AGgrenox versus aspirin therapy evaluation (AGATE) biomarker substudy.

Plasma endothelial nitric oxide synthase (eNOS), and oxidised low-density lipoproteins (oxLDL) are established biomarkers of atherosclerosis. We defined the time course and magnitude of changes of plasma eNOS and oxLDL after Aggrenox or aspirin in post-stroke patients. Baseline pretreatment eNOS levels were significantly diminished (110 ± 66pg /ml vs. 374 ± 88 pg/ml, p=0.0001), while oxLDL was twice higher (58 ± 9 mg/l vs. 23 ± 7 mg/l, p=0.004) in post-stroke survivors when compared to controls. Both Aggrenox and aspirin similarly increased plasma eNOS activity. However, oxLDL levels were static after aspirin, but inhibited late after Aggrenox. In the small randomised study, both aspirin and Aggrenox produced fast and sustained recovery of plasma eNOS levels, while only therapy with Aggrenox was associated with oxLDL inhibition late in the trial.

Antiplatelet 'resistance' and 'non-responders': what do these terms really mean?

The term 'resistance' should be restricted to very specialized physiologic circumstances, if not abandoned altogether. The term 'non-responder' needs to be placed in the context of the question: 'Non-responder to what?' Even if we would somehow magically know what an optimal response to antiplatelet therapy was, it will still be challenging to demonstrate an 'inadequate' response to antiplatelet therapy. At present there are two alternatives--give more drug or give additional drugs. Both strategies may work in further inhibiting platelet function, but both strategies can also be associated with an increased risk of bleeding. The trick, for the future, much as with our antihypertensive and lipid-lowering armamentaria, will be to know in whom to do what with which drug, and why. Single isolated measurements are not useful--if you don't know where you started, how we would know that antiplatelet drug is producing an 'adequate' clinical effect? There is no evidence of any sort of absolute 'threshold' that must be exceeded for treatment to be effective, and in the absence of this, if we are to evaluate the effect of a given drug, we have to have baseline values (off drug), therapeutic values (on drug), and some sort of assessment of both resting (unstimulated) and agonist-provoked (stimulated) platelet function. Moreover, given all of the different things that platelets do, the ideal assessment of platelet function and drug responsiveness will need to incorporate more than one agonist and some sort of assessment of both platelet activation and platelet aggregation. No one man (or test) tells us everything; it is the totality of the information that gives us the most complete picture. And, ultimately, we need to more firmly establish how the variability in platelet function and drug-associated changes in that function correlates with long-term, hard-endpoint clinical events.

AGI-1067, a novel vascular protectant, anti-inflammatory drug and mild antiplatelet agent for treatment of atherosclerosis.

Oxidation-sensitive signals play an important role in platelet activation. AGI-1067 is a novel, phenolic, intra- and extracellular antioxidant that inhibits the expression of a number of proinflammatory genes involved in atherosclerosis. AGI-1067 is the metabolically stable monosuccinic acid ester of probucol, and a potent phenolic antioxidant representing a novel class of orally bioavailable compounds termed vascular protectants. AGI-1067 exhibits antioxidant activity equipotent to probucol. In addition, animal studies have demonstrated dual pharmacological activities of AGI-1067: the ability to block the expression of oxidation-sensitive inflammatory genes including genes that code for vascular cell adhesion molecule-1 and monocyte chemotactic protein-1. Importantly, AGI-1067 also exhibits mild antiplatelet properties inhibiting surface expression of various key platelet receptors, the formation of platelet monocyte microparticles and PAR-1 thrombin receptors. AGI-1067 is currently being tested in the late trials, and if proven to improve clinical outcomes (ARISE trial), the drug will ultimately be used in patients with different manifestations of atherosclerosis and atherothrombosis.