Mohammad Urooj Zafar

Normalization of platelet reactivity in clopidogrel-treated subjects.

Summary.  Background: Aspirin (ASA) + clopidogrel are commonly used in acute coronary syndrome (ACS), but persistent antiplatelet effects may complicate surgery.Methods and Results: To study the possibility of normalizing platelet reactivity after ASA + clopidogrel treatment, 11 healthy subjects received a 325‐mg ASA + clopidogrel loading dose (300 or 600 mg dependent on study arm), followed by 81 mg of ASA + 75 mg of clopidogrel daily for 2 days. Platelet reactivity was assessed by light transmittance aggregometry (LTA) [challenged by adenosine diphosphate (ADP), arachidonic acid (AA), collagen, and thrombin receptor activating peptide (TRAP)] and flow cytometry for platelet activation by GPIIb/IIIa receptor exposure pretreatment, 4 and 72 h postload. To normalize platelet reactivity, increasing amounts of pooled platelets from five untreated volunteers [volunteers (V)‐platelet‐rich plasma (PRP)] were added ex vivo to the subjects PRP (S‐PRP). At both 4 and 72 h, 40% and 50% V‐PRP were needed to overcome platelet disaggregation in the 300 or 600 mg arms, respectively, after ADP challenge; an additional 10% V‐PRP fully normalized aggregation. Recovery of function was linear with each incremental increase of V‐PRP. ADP‐induced GPIIb/IIIa activation showed the same pattern as LTA (r = 0.74). Forty percent V‐PRP was required to normalize platelet function to AA, collagen, and TRAP.Conclusion: Our results suggest that the pre‐operative transfusion of 10 platelet concentrate units (the equivalent of 40% V‐PRP) after a 300‐mg clopidogrel loading or 12.5 units (50% V‐PRP) after a 600 mg loading may adequately reverse clopidogrel‐induced platelet disaggregation to facilitate postoperative hemostasis. An additional 2.5 units fully normalized platelet function. The potential clinical implications of our observations could include shorter hospitalizations and reduced bleeding complications. But these observations should be fully explored in an in vivo clinical setting with clopidogrel‐treated patients before and after surgery.

Antithrombotic effects of factor Xa inhibition with DU-176b : Phase-I study of an oral, direct factor Xa inhibitor using an ex-vivo flow chamber

Direct and specific inhibition of factor Xa is an emerging therapeutic strategy for atherothrombotic disease. Parenteral factor Xa inhibitors promise efficacy comparable to standard therapies, which could be extended to ambulatory patients with oral agents. We evaluated the antithrombotic effect of the oral, direct factor Xa inhibitor DU-176b in a phase-I study. Healthy subjects (n = 12) received a single, 60 mg dose of DU-176b. Antithrombotic effects were assessed by comparing ex-vivo thrombus formation at 1.5, 5, and 12 hours post-dose versus baseline, along with factor Xa activity, thrombin generation and clotting parameters. Under venous flow after 1.5 and 5 hours, the thrombus was 28% and 21% smaller versus baseline, respectively (p < 0.05). Under arterial condition, the reduction was 26% and 17% (p < 0.05). Thrombin generation decreased by 28% at 1.5 hours and 10% at 5 hours. Changes in PT and INR correlated well with plasma drug concentrations (R2 = 0.79 and 0.78). Direct and specific inhibition of factor Xa by DU-176b significantly reduced ex-vivo thrombus formation at both venous and arterial rheologies, up to 5 hours post-dose. The effects mirrored changes in clotting parameters, suggesting their potential usefulness for monitoring in a clinical setting.

Systems Pharmacology of Adverse Event Mitigation by Drug Combinations

Drug combinations that mitigate adverse events were identified using the FDA Adverse Event Reporting System, and one combination, exenatide and rosiglitazone, was tested in a rodent model. Two Drugs: Better Than One? Everyone has seen the commercial, where a drug is advertised as the much-awaited treatment for a disease. At the end of the commercial, there is a long list of adverse events (or side effects) that may affect anything from your heart to your eyesight. Surprisingly, the addition of a second drug can mitigate the side effects of the first drug, such that the combination is actually safer for the patient. To search for those mitigating combinations, Zhao and colleagues devised a computational method for scanning the Food and Drug Administration’s Adverse Event Reporting System (FAERS). This database is freely available and contains millions of records of drug-induced adverse events reported by patients, doctors, hospitals, lawyers, and drug companies. Thus, it represents a potentially useful source of beneficial drug combinations. The authors focused on rosiglitazone—a drug that effectively controls blood glucose levels in diabetic patients, but has been associated with myocardial infarction (MI). By searching through FAERS data, Zhao et al. found that when rosiglitazone was prescribed in combination with exenatide—another drug for treating type 2 diabetes—there were significantly fewer reports of MI as an adverse event. A cell biological interaction network was then developed to look for mechanisms by which rosiglitazone + exenatide affected the heart. The rosiglitazone target PPARγ was plugged into this network, and plasminogen activator inhibitor-1 (PAI-1) was obtained as a potential point of convergence between the two drugs. To test this hypothesis, the authors administered drugs to a mouse model of type 2 diabetes. Mice treated with rosiglitazone alone showed a marked increase in PAI-1 levels, whereas mice treated with the drug combination had significantly decreased levels of PAI-1, similar to those found in untreated mice. FAERS is self-reported and thus may contain some inaccurate data. Nevertheless, it could be a useful tool for generating meaningful hypotheses from human data and for then testing in vivo in clinically relevant disease models, as shown by Zhao et al. Animal models can provide information about drug mechanism of action, and prospective clinical trials will confirm that such combinations can indeed be translated to people to prevent adverse events. Drugs are designed for therapy, but medication-related adverse events are common, and risk/benefit analysis is critical for determining clinical use. Rosiglitazone, an efficacious antidiabetic drug, is associated with increased myocardial infarctions (MIs), thus limiting its usage. Because diabetic patients are often prescribed multiple drugs, we searched for usage of a second drug (“drug B”) in the Food and Drug Administration’s Adverse Event Reporting System (FAERS) that could mitigate the risk of rosiglitazone (“drug A”)–associated MI. In FAERS, rosiglitazone usage is associated with increased occurrence of MI, but its combination with exenatide significantly reduces rosiglitazone-associated MI. Clinical data from the Mount Sinai Data Warehouse support the observations from FAERS. Analysis for confounding factors using logistic regression showed that they were not responsible for the observed effect. Using cell biological networks, we predicted that the mitigating effect of exenatide on rosiglitazone-associated MI could occur through clotting regulation. Data we obtained from the db/db mouse model agreed with the network prediction. To determine whether polypharmacology could generally be a basis for adverse event mitigation, we analyzed the FAERS database for other drug combinations wherein drug B reduced serious adverse events reported with drug A usage such as anaphylactic shock and suicidality. This analysis revealed 19,133 combinations that could be further studied. We conclude that this type of crowdsourced approach of using databases like FAERS can help to identify drugs that could potentially be repurposed for mitigation of serious adverse events.

Prostanoid and TP-receptors in atherothrombosis: Is there a role for their antagonism?

Atherosclerosis and its clinical manifestations (i.e. myocardial infarction, stroke) are major causes of mortality and morbidity in Western countries. Endothelial dysfunction is considered the first step in the cascade leading up to coronary events. Increasing evidence suggests that direct inhibition of thromboxane A2/prostaglandin (TP)-receptors may not only have anti-platelet effects but also impact endothelial dysfunction as well as inflammatory component of atherosclerosis. While TP-receptor involvement in platelet function has received the greatest attention, more recent findings support the critical role of TP-receptor in other pathophysiological aspects of atherothrombosis. Prostanoids (i.e. TxA2, F2-isoprostanes, prostaglandins endoperoxides PGG2/PGH2) are known to promote the initiation and progression of atherosclerosis, not only via platelet activation, but through leukocyte-endothelial interactions and vasoconstriction. Dysfunctional endothelium, characterised by increased COX-activity, releases prostanoids that promote endothelial exposure to adhesion molecules and induce smooth muscle cell contraction. Plaque macrophages synthesise PGH2/PGG2 via COX-2; these potent prostanoids can trigger platelet activation and aggregation despite COX-1 inhibition by aspirin. TP-receptor inhibition has been reported to exert anti-atherosclerotic effects in pre-clinical model of disease. Reduction of plaque burden was associated with plaque stabilisation documented by the reduction in the content of macrophages, apoptotic cells, MMPs and endothelin-1, and the increase in smooth muscle cells content. TP-receptor blockade might have an anti-atherosclerotic and plaque stabilisation effect. The possibility of combining anti-platelet activity with an anti-atherosclerotic effect via selective TP-receptor inhibitors could have important implications especially in clinical conditions associated with increased production of prostanoids, such as diabetes.

Platelet function normalization after a prasugrel loading‐dose: time‐dependent effect of platelet supplementation

Summary.  Background: Hemostatic benefits of platelet transfusions in thienopyridine‐treated acute coronary syndrome (ACS) patients may be compromised by residual metabolite in circulation.Objectives: To estimate the earliest time after a prasugrel loading‐dose when added platelets are no longer inhibited by prasugrels active metabolite.Methods: Baseline platelet reactivity of healthy subjects (n = 25, 30 ± 5 years, 68% male) on ASA 325 mg was tested using maximum platelet aggregation (MPA, ADP 20 μm) and VerifyNow® P2Y12 and was followed by a 60 mg prasugrel loading‐dose. At 2, 6, 12 and 24 h post‐dose, fresh concentrated platelets from untreated donors were added ex‐vivo to subjects’ blood, raising platelet counts by 0% (control), 40%, 60% and 80%. To estimate the earliest time when prasugrels active metabolites inhibitory effect on the added platelets ceases, platelet function in supplemented samples was compared across time‐points to identify the time when effect of supplementation on platelet function stabilized (i.e. the increase in platelet reactivity was statistically similar to that at the next time‐point).Results: Supplemented samples showed concentration‐dependent increases in platelet reactivity vs. respective controls by both MPA and VerifyNow® at all assessment time‐points. For each supplementation level, platelet reactivity showed a sharp increase from 2 to 6 h but was stable (P = NS) between 6 and 12 h.Conclusions: The earliest measured time when supplemented platelets were not inhibited by circulating active metabolite of prasugrel was 6 h after a prasugrel loading‐dose. These findings may have important implications for prasugrel‐treated ACS patients requiring platelet transfusions during surgery.

A novel anti-ischemic nitric oxide donor (LA419) reduces thrombogenesis in healthy human subjects.

Summary.  Background: Platelet and endothelial production of nitric oxide (NO) is known to be impaired in coronary artery disease patients. Compounds that release NO (e.g. nitrates) have antiplatelet effects, but at supratherapeutic doses with hypotensive side effects. Objectives: To investigate the antithrombotic effect on human blood of a novel NO donor (LA419) with known anti‐ischemic properties but without hypotensive side effects and to compare with abciximab. Patients/methods: Healthy subjects (n = 8; 32 ± 3 years) received daily aspirin starting three days prior to the study day. Treatments (LA419 10 and 20 μm, and abciximab 4 μm) were added ex vivo to non‐anticoagulated blood, and the antithrombotic properties were assessed by measuring changes in thrombus size from pretreatment baseline in the Badimon perfusion chamber at low and high shear rates. Platelet surface adhesion using a Cone and Platelet Analyzer (CPA) and platelet fibrinogen‐receptor activation with flow cytometry were also evaluated. Results: At low shear rates, LA419 displayed a reduction in thrombus area of 43% ± 8% (10 μm) and 56% ± 6% (20 μm), whereas at high shear rates the reductions were 44% ± 3% (10 μm) and 62% ± 6% (20 μm). Platelet surface adhesion with the CPA was also reduced. Abciximab exhibited a strong inhibitory effect on thrombus formation, platelet surface adhesion and fibrinogen receptor activation. Conclusions: The novel NO donor, LA419, shows a strong antithrombotic effect in human blood, which is comparable to abciximab, especially under high shear rate conditions. Our observations suggest that the availability of an NO donor could prove beneficial in the prevention of thrombotic complications of cardiovascular disease. Further clinical studies are warranted.

A new oral antiplatelet agent with potent antithrombotic properties: Comparison of DZ-697b with clopidogrel in a randomised phase I study

DZ-697b is a new orally active antiplatelet agent that inhibits collagen and ristocetin-mediated platelet activation. It does not require metabolisation to generate its active compound and has a safer profile than clopidogrel in pre-clinical studies. We compared the antithrombotic effects and bleeding time prolongations of three DZ-697b doses with clopidogrel 300 mg. In a four-treatment, three-period, crossover-design, 20 healthy subjects (31 + or - 7 years, 85% males) were randomised to single oral doses of DZ-697b (60, 120 and 360 mg), and clopidogrel (300 mg) (n=15 in each treatment with crossing-over). Antithrombotic effects were assessed by measuring six-hour post-dose changes from baseline in thrombus size in the Badimon chamber and platelet adhesion using Diamed Impact-R platelet function assay. Bleeding times were also measured pre-dose and at six hours post-dose. DZ-697b caused dose-dependent reductions in thrombus size at both high- and low-shear rates (mean reductions at 60, 120 and 360 mg doses of: 13.0%, 18.7%, 26.4% and 11.4%, 12.7%, 22.1% respectively, p<0.05 for all). Effect of clopidogrel (reductions of 18.7% and 11.0% respectively, p<0.05 for both) was closest to DZ-697b 120 mg. Reductions in platelet adhesion were also dose-dependent. Bleeding time ratio from baseline were shorter with DZ-697b versus clopidogrel (1.3, 1.4 and 1.5 versus 1.9, p<0.05 for all). The oral agent DZ-697b shows potent, dose-dependent, antithrombotic effects that are comparable to 300 mg clopidogrel at the 120 mg dose. Despite having equal or greater antiplatelet potency than 300 mg clopidogrel, bleeding time prolongations are significantly shorter with DZ-697b.

Ovariectomy increases vascular calcification via the OPG/RANKL cytokine signalling pathway.

Background  Observational studies suggest a strong relationship between menopause and vascular calcification. Receptor activator of nuclear factor‐κΒ ligand (RANKL) and osteoprotegerin (OPG) are critical regulators of bone remodelling and modulate vascular calcification. We assessed the hypothesis that ovariectomy increases vascular calcification via the OPG/RANKL axis.

Antithrombotic potency of ticagrelor versus clopidogrel in type-2 diabetic patients with cardiovascular disease

Type-2 Diabetes Mellitus [T2DM] is associated with increased platelet reactivity and hypo-response to antiplatelet drugs. Ticagrelor, with its faster and more potent antiplatelet effects, was shown to reduce adverse events more than clopidogrel in the overall CAD patient population of PLATO trial, but the benefits did not reach statistical significance in the T2DM subgroup. To better understand these findings, we compared the antithrombotic effects of ticagrelor versus with clopidogrel in T2DM patients with cardiovascular disease. In a randomized, 2 treatment-sequence, crossover-design, T2DM patients (n=20, 57±8 years, 60 % male) received a loading-dose [LD] plus one week of daily-therapy [DT] of clopidogrel or ticagrelor. Treatment effects were assessed by measuring thrombus formation (Badimon Chamber) and platelet aggregation (Multiple Electrode Aggregometry (MEA) Analyzer and VerifyNow®) at 2- and 6-hour post-LD and on Day-7 of DT, in comparison with pre-treatment baseline. After 2 weeks of washout, patients switched to the second treatment under identical testing conditions. Ticagrelor significantly reduced thrombus formation versus baseline at 2- and 6-hour post-LD and Day-7 of DT (33 %, 40 % and 31 %, respectively, p<0.01 for all) whereas thrombus reductions with clopidogrel were much lower and significant only at 6-hour post-LD (16 %, 20 % and 17 %, respectively). Antithrombotic effect of ticagrelor at 6-hour was significantly stronger than clopidogrel (p<0.05). Platelet aggregation (MEA and VerifyNow®) was inhibited by both treatments but effects of ticagrelor were significantly stronger at each time-point. Ticagrelor exhibits a faster and more potent antithrombotic effect than clopidogrel in T2DM patients with cardiovascular disease, supporting its use in this population.

Ticagrelor reduces thrombus formation more than clopidogrel, even when co-administered with bivalirudin

Ticagrelor reduces thrombus formation more than clopidogrel, even when co-administered with bivalirudin -