Here comes STREAM ……

The results of STREAM were presented at ACC meeting and study was published online in NEJM – “Fibrinolysis or Primary PCI in ST-Segment Elevation Myocardial Infarction”. In nutshell the results can be summarized as pre-hospital fibrinolysis with bolus tenecteplase in conjunction with timely coronary angiography was similar to primary PCI in patients with early STEMI who could not undergo primary PCI within 1 hour after the first medical contact. Patients who failed fibrinolysis underwent emergent PCI (36.3% of the fibrinolysis group). Cardiogenic shock and congestive heart failure occurred more often in the primary PCI group and intracranial hemorrhage and ischemic strokes were more frequent in the fibrinolysis group. The study findings are likely to be reassuring for some parts of the world while may change treatment strategies at other places.

Some Useful Shell Commands for Cluster

bkill
especially bkill 0 as it will kill all jobs submitted by the user

bjobs

bhosts

Farnesyl Pyrophosphate and ADP-mediated Platelet Aggregation

Read this article recently and it seems interesting. Farnesyl pyrophosphate (FPP) can itself activate platelets and can induce platelet aggregation. However, this study concludes that FPP can act as endogenous antithrombotic factor by acting as insurmountable antagonist of ADP-mediated platelet aggregation. FPP is an intermediate in the cholesterol biosynthetic pathway. FPP also serves as a donor in post-translational isoprenylation of proteins. The steady-state plasma level was reported to be 6.6 ng/ml,  but  even  the  mild  physiological alteration caused by eating a meal has been demonstrated to increase the plasma concentration 200 fold. FPP is a natural antagonist of the LPA₂  (lysophosphatidic acid type 2) and LPA₃  receptors, and an agonist at the LPA₄  and LPA₅ receptors. While these receptors are present in platelets, FPP doesn’t appear to act through these receptors. Hogberg et al realized that the structure of ADP and FPP is similar as are their receptors. Thus, through a series of experiments they should that FPP inhibits platelet aggregation by blocking ADP receptors.

Platelet Function and Subsequent MACE in ACS patients

An interesting substudy of TRILOGY ACS was published in JAMA recently in which a group of patients with ACS underwent evaluation of platelet function after prasugrel or clopidogrel. All patients were also on aspirin. Thus, platelet function studies were performed after treatment with aspirin + prasugrel and aspirin + clopidgorel. TRILOGY ACS trial was a randomized, double-blind, active-comparator trial comparing prasugrel with clopidogrel in patients with unstable angina or non–ST-segment elevation myocardial infarction (UA/NSTEMI) who were managed medically without planned revascularization.Platelet function was assessed in a subset of enrolled patients using VerifyNow kits. VerifyNow P2Y₁₂ is a whole-blood, turbidimetric-based assay that measures platelet agglutination to fibrinogen-coated polystyrene beads after platelet activation with adenosine diphosphate. The results are expressed in PRU (P2Y₁₂ reaction units). There was no significant association between platelet reactivity and occurrence of ischemic outcomes during a 30 month follow-up period.

The results of this study are important; it is a large study, enrolling a high-risk population and followed for a relatively longer period of time. While trials of antiplatelet agents have established, beyond doubt, that platelet play an important (if not essential) role in the pathogenesis of ACS and perhaps also in the development and progression of atherosclerosis, studies have generally failed to find an association of platelet function with future events. Probably the most likely explanation is that we have, so far, been unable to identify a platelet function test that ACTUALLY measures platelet function in a way which is important clinically. We can predict bleeding but not aggregability. We do need to explore existing platelet function tests for association with future events that have not yet been examined and we also need to develop newer tests that are more closer in measuring what happens inside the vessel.

Today’s Interesting Links - September 10th, 2012

 

Genetic polymorphisms of CYP2C19 influences the response to clopidogrel in ischemic heart disease patients in the South Indian Tamilian population.

Regulated granule trafficking in platelets and neurons: A common molecular machinery

Idiopathic Sudden Sensorineural Hearing Loss: Classic Cardiovascular and New Genetic Risk Factors

Panobinostat (LBH589)-induced acetylation of tubulin impairs megakaryocyte maturation and platelet formation.

Itga2b regulation at the onset of definitive hematopoiesis and commitment to differentiation

Effects of clopidogrel added to aspirin in patients with recent lacunar stroke

WOEST - Optimal Antiplatelet Treatment in Patient Already taking Anticoagulants

Dual antiplatelet therapy (aspirin and clopidogrel or another P2Y12 inhibitor) is almost always prescribed (and is beneficial in preventing future adverse cardiovascular events) to patients with coronary artery disease who undergo coronary intervention and have stents placed (although it is associated with increased risk of bleeding). For most patients with atrial fibrillation or mechanical heart valves, anticoagulation therapy is a the standard of care (although it is associated with increased bleeding risk).

Question remains as to what to do with patients who are on anticoagulation but then develop coronary artery disease, undergo percutaneous coronary intervention (PCI) and stent placement. Use of dual antiplatelet therapy along with anticoagulation increases the overall risk of bleeding considerably and whether such a high risk of bleeding overshadows the benefit associated with dual antiplatelet therapy remains unknown.

In an ideal situation, one would randomize patients who are taking anticoagulants and who have undergone PCI with stent placement in one of the three arms: dual antiplatelet therapy, aspirin alone, or clopidogrel (or another P2Y12 agent) alone. Of course, all the three groups should continue taking their anticoagulation treatment. While, we don’t have such an ideal study, we do have results of a study (reported today in the ESC Congress 2012) in which patients taking anticoagulants were randomized to dual antiplatelet agents or clopidogrel alone. The results are interesting; not only that dual antiplatelet therapy in addition to anticoagulation was associated with increased risk of bleeding, it was also associated with higher risk of all-cause mortality (6.4% vs. 2.5%; p = 0.027).

One thing is certain from this study and that is that dual antiplatelet therapy, in addition to anticoagulation, is not optimal treatment and a single antiplatelet agent is likely to do a better job in reducing not only bleeding but also in decreasing all-cause mortality. However, whether this single antiplatelet agent has to be aspirin or clopidogrel, that remains unclear. In fact, it is quite possible that aspirin may have larger benefit than clopidogrel when used in such population. Perhaps future studies may be better able to point us relative benefits of antiplatelet agents in patients with PCI and stents who are also taking anticoagulants.

Clopidogrel vs. Prasugrel in Medically Managed ACS

Clopidogrel (famous by its brand name Plavix) is now generic and while prices vary from one pharmacy to another, it is very likely that very soon its price will come down. On the other hand, Prasugrel (which works the same way as clopidogrel) has a long way to go before it becomes generic and thus is likely to remain costly for next several years.

Prasugrel has one advantage over clopidogrel; its metabolism is not dependent on CYP2C19 (a member of cytochrome P450 system). A mutation within the gene of this particular enzyme decreases its function and decreases the conversion of the pro-drug, clopidogrel, into its active metabolite. The metabolism is shunted towards inactive metabolites and hence individuals with certain mutations in this gene are associated with increased platelet reactivity while on clopidogrel. This was shown conclusively in a GWAS by Alan Shuldiner and his team.

Prasugrel does seem to be superior to clopidogrel in individuals who undergo percutaneous coronary intervention (PCI) however, it was not clear if prasugrel is also superior to clopidogrel in individuals who are managed conservatively.

The results of the TRILOGY-ACS trial (double blind, randomized, double-dummy, active control, event-driven), announced today at the ESC Congress 2012 suggest that there is no difference. In this study, investigators randomized >9,000 patients with acute coronary syndromes (ACS) to either clopidogrel+acetyl salicylic acid (ASA or aspirin) or prasugrel+ASA. The primary end point of the trial was cardiovascular death, myocardial infarction, or stroke. The study did not find a benefit of prasugrel over clopidogrel for either the primary outcome or individual components of the primary outcome (p = 0.21). Post-hoc analysis suggests some interesting hypothesis that may be worth looking into in future studies.

Ticagrelor, another anti-platelet agent that also works by inhibiting the same ADP receptors (P2Y12), on the other hand, was shown to be superior to clopidogrel in a similar type of medically managed population in the PLATO trial. It will be interesting to see how they do in their head-to-head comparison (if ever done).

GWAS of Correlated Traits

Korte et al has written an interesting paper which extends the analysis of correlated traits to genome-wide association studies (GWAS). (Korte A, Vilhjálmsson BJ, Segura V, Platt A, Long Q,Nordborg M. A mixed-model approach for genome-wide association studies of correlated traits in structured populations. Nat Genet. 2012 Aug 19. doi: 10.1038/ng.2376. [Epub ahead of print] PubMed PMID: 22902788.)

GWA studies have commonly employed a simple statistical model in which a single locus is tested for association with a single phenotype (usually in an additive model). There have been few attempts so far, if any to utilize correlated phenotypes and perhaps improve power of the studies. 

In the 2011 meeting of American Society of Human Genetics at Montreal, Canada, I have used one, relatively simple, method to combine results from two correlated traits with resulting increase in power of GWAS. (Qayyum R et al. Correlated meta-analysis of genome-wide association studies of agonist-mediated native platelet aggregation in African Americans). What we did was to conduct two separate GWAS studies of correlated platelet aggregation phenotypes and then combined the results using meta-analysis. However, the resulting p-values were adjusted for correlation between the two phenotypes using tetrachoric correlation. We further conformed our findings using the simulations from correlated distributions, confirming our findings. 

Korte et al, use linear mixed model approach to not only adjust for population stratification but also for correlation between phenotypes. They use ASReml and R for this analysis and provide R scripts on their website. Using their techniques, Korte et al unveil additional SNPs in a GWAS of LDL and triglycerides.

 

Some Interesting Articles to Read

This one claims that odds are against men because of natural selection

Extreme phenotypes are helpful in identifying genetic loci and this study provides such an example

Here is introduction to Personal Genome Project, an interesting resource.

Ornithine Decarboxylase, Aspirin, and Platelets

Ornithine Decarboxylase (gene name ODC1) is the rate limiting enzymes of the polyamine biosynthesis pathway and catalyzes ornithine to putrescine. The activity level for the enzyme varies in response to growth-promoting stimuli and exhibits a high turnover rate in comparison to other mammalian proteins. Interestingly, this enzyme (as well as its inhibitor, Ornithine decarboxylase antizyme, gene name OAZ1) is also differentially expressed at higher levels in platelets from individuals with sickle cell disease than in in those without sickle cell disease (PMCID: PMC2225987). Platelets are in a basally activated state in patients with sickle cell disease and may contribute to at least some of the long-term vascular complications seen in patients with sickle cell disease. In patients with CAD, variants in OAZ1 have shown to be associated with increased risk of 6-month in-stent restenosis, increase in carotid intima-media thickness over the a 4-year period, and an increased risk of CAD (PMID: 17761941).

Aspirin is widely used for its anti-platelet effects and is also shown to be beneficial in reducing the recurrence of colon adenomatous polyps. A recent study has found that variants located downstream of the 3’ end of ODC1 gene may influence the risk of colorectal adenoma and impact the efficacy of aspirin. (PMID: 21930798)Whether there is a similar relationship between the antiplatelet effect of aspirin and ODC1 gene variants is unknown. Although one may be tempted to postulate that such a relationship exists based on the above noted studies, there are other potential mechanisms that may play a role. For example, ornithine decarboxylase has been shown to affect proliferation of vascular smooth muscle cells (PMID: 21894530) and the function of endothelial cells (PMID: 20594968); both cell types important in the process of atherosclerosis.

Selective Genotyping

In selective genotyping, a large number of individuals are phenotyped; however, only individuals with extreme traits are genotyped. This design tends to be more efficient than pure random sampling because phenotypically extreme individuals are likely to be genetically more informative [Huang and Lin, 2007].

Several regression-based methods have been developed for mapping QTLs under selective genotyping. These include the prospective linear regression [Xiong et al.,
2002], the retrospective likelihood approach [Wallace et al., 2006] and the conditional likelihood method [Huang and Lin, 2007]. Tang Y [2010] showed that the prospective, retrospective and conditional likelihoods actually yield identical score tests for association between a quantitative trait and a candidate locus.

Some Lectures on Basic Statistics

 

Statistical Reasoning I

Statistical Reasoning II

Introduction to Biostatistics

Above are three courses from Johns Hopkins Bloomberg School of Public Health

Vancouver, Canada

All right, so I came here to Vancouver, Canada few days ago for a conference. Although I must say that I didn’t get enough time to go around and see the city, I still have only good feelings about the city. There were several things that impressed me. Probably the most striking feature was the diverse ethnic backgrounds of the people. It is very difficult, if not impossible to say where in the world you are by just looking at the people. Yes, I have been to New York many times but it was a much more diverse crowd than I have seen in Manhattan.

Weather was great; especially coming from Baltimore, from a very hot and humid weather, this was a nice break. Temperature was really moderate, pretty cold at night; cold enough that one needs an extra layer of clothing.

People were nice, polite, down to earth. City was clean, and scenery was beautiful. Stanley Park is a great place to hang out, Granville Island is a nice place for shopping. I was particularly struck by the view of snow covered peaks from the middle of the city; amazing view!

Coming back here? For sure, hopefully soon.

CARMIL or LRRC16A

CARMIL (protein CP ARp2/3 myosin I linker) was initially named Acan125 when it was discovered in amoeba where it was found to bind the SH3 domain of class I myosin molecules. Subsequently, this protein was found to bind capping protein (CP) and to Arp2/3 complex which to it being named as CARMIL. This is a large protein and has a long leucine-rich repeat (LRR) region. The function of this LRR region is unclear (PMID: 18544499).

CARMIL is important in actin-based cell motility. In addition to its role in many cellular process, actin is also involved in cell motility and shape change. Actin polymerization occurs mainly at the barbed ends of the actin filament. Addition of new actin molecules to the barbed ends of actin filaments are thought to be responsible for cell motility and change in shape. The assembly of actin filament by addition of actin monomers to the barbed ends is inhibited by Capping Proteins (CP). These capping proteins bind and thus hide the barbed ends of the actin filaments making them inaccessible to other actin molecules. CP is  a heterodimer of two subunits with a shape similar to a mushroom. CARMIL binds to the CP and this binding of CARMIL to CP results in decreased affinity of the CP to barbed ends of actin filament and dislodges itself from it. Thus barbed ends are exposed and actin polymerization starts resulting in cell-shape (PMID: 16434392).

Of note, the ability of CARMIL to expose barbed ends of actin filament is specific to CP and doesn’t include other proteins that inhibit actin polymerization. The CP binding region of CARMIL resides in the later part of the protein called CAH3 region (CARMIL homology domain 3; amino acid residues 940-1121). Within this region, a portion of 25 amino acids that is highly conserved from protozoa to flies, to worms to vertebrates. Point mutations in this region result in loss of CP-binding ability of CARMIL (PMID: 16434392).

A patch consisting of basic amino acids on CP is essential for its interaction with barbed ends of the actin. Using nuclear magnetic resonance (NMR), Zwolak et al  studied the interaction of the CAH3 domain and CP in a mouse model. They found that the highly basic mouse CAH3a subdomain binds with high affinity to a complementary “acidic groove” on CP.  This CAH3a-CP interaction orients the CAH3b subdomain directly adjacent to the basic patch of CP. The importance of specific residue interactions between CP and CAH3a/b was confirmed by site-directed mutagenesis of both proteins (PMID: 20630878).

In another study Hernandez-Valladares identified two similar regions  within the CAH3(965–1038) performing essentially the same two functions. The first region, Ile971–Cys1004, is CP interacting motif. The second region, Arg1021–Thr1035, binds to the underside of the CP mushroom cap on the opposite side of the CP mushroom stalk to which the CP interaction motif binds. Together, these two regions resemble a finger (CPI) and thumb (CSI) encircling the stalk on the underside of the mushroom cap (PMID: 20357771).

LRRC16A or CARMIL gene is located on chromosome 6p22.2, has 341,453 base-pairs, and 36 exons. As is also clear from the LD block of YRI (data from HapMap, software used Haploview) the later part of the gene is conserved and is present in a larger LD block.

First experience with cluster

I have been thinking about using our cluster but had been so far procrastinating until now. So this past week I thought I should submit a job myself instead of asking others to do this. Even if others submit jobs for me, I need to know some basics of working with clusters … and I mean real basics….

So, I borrowed a completely written script from a smart colleague in which I had to change few words only, basically location of files – input files and output files. And then I took that step … step of submitting a job to a computer cluster.

Result: complete failure; I have been so far unable to run that one… and I am talking about one only … job. Every time when I submit I get error “Requeued job is waiting for rescheduling”. I have still not figured out what and where the problem is. I have been asking friends and colleagues and they have been very forth coming but despite their best efforts and my repeated attempts (and prayers) I have gotten nothing but failure.

However, in the process I have learned few shell commands which I would probably have never learned … at least not this soon.

sh : submit your script

bjobs : submit your job

bjobs –lp : see why your job is still pending- this is the command that told me that my job was waiting for rescheduling or something… whatever!

bkill : get rid of the job that you have submitted

bkill –q <queue name> –u all 0 : this will kill all jobs in that queue

bhosts : shows hosts and load on them

busers : shows your submitted work/jobs

busers all : shows submitted jobs by everyone

bqueues : shows different queues

Alright, so this is enough learning so far. I have to get back to trying again and again and see if repetition of same silly things changes anything ……..

WOW! Interesting spin……

So I get these several newsletters in my email everyday …. Often I delete them without reading and sometimes I eyeball them. Today I got one from ‘MedPage Today’ (www.medpagetoday.com). The headline says “AHA: Drug-eluting stent safe in large artery disease” and then it goes on to say ...

“CHICAGO -- Among patients with coronary disease in large arteries, treatment with a drug-eluting stent was as safe as treatment with a bare-metal one, results of a large international study showed.”

The correct and realistic headline should be that “bare-metal stents are as good as drug-eluting stents (DES) in large artery disease”. In fact, this study is a win for bare-metal stent (BMS) proving that BMS can be used without any hesitation in large vessel disease. Headline on the other hand, tries to portray that it’s safe to continue using DES for large vessel disease.

Platelets and Apoptosis

Platelet Lifespan

Various in vivo and in vitro methods can be used to determine platelet lifespan. One often recommended is the use of 111indium-labelled platelets. In this method, labeled platelets are injected into the patient and samples are collected at various time intervals (at 45 min, 2, 3, 4 hours) after injection and then daily for up to 10 days. Recovered platelets are calculated from each sample, data is plotted on an arithmetic graph paper and survival time is calculated.

What Determines Platelet Lifespan?

The lifespan of a platelet in circulation is about 8-10 days and dying platelets are continuously replaced by new platelets formed in the bone marrow. Platelets are removed from circulation either by consumption during hemostasis or through uptake by reticuloendothelial (RE) system. For the later process, initially, it was thought that platelets die due to usual ‘wear and tear’ while they are in circulation. Various lines of evidence suggested this possibility. For example, older platelets are less responsive to physiological agonists as compared to younger platelets. More recently, there is increasing evidence that platelets undergo programmed cell death through apoptosis and during this process express certain receptors on their surface that lead to uptake by RE system.

Apoptosis

There are two pathways through which apoptosis can be triggered; extrinsic and intrinsic. Extrinsic pathway is activated by the stimulation of cell-surface receptors (death receptors) which leads to the formation of Death Inducing Signaling Complex (DISC) and ultimately of caspases. On the other hand, intrinsic pathway is activated when the intracellular balance of pro-apoptotic and anti-apoptotic proteins tilt in the favor of the pro-apoptotic proteins. The pro-apoptotic proteins then trigger mitochondrial damage, which initiates the apoptosis cascade.

BCL-2 Family of Proteins

BCL-2 family of proteins is mainly responsible for intrinsic pathway of apoptosis (also called programmed cell death). There are about 25 proteins in this family. Some of these are pro-apoptotic while others are anti-apoptotic. A third subgroup within this family (BH3-only) may be involved in sensing signals that trigger programmed cell death. Anti-apoptotic proteins include Bcl-2, Bcl-w, Bcl-XL, Mcl-1 and A1 while pro-apoptotic proteins are mainly Bak and Bax. . BH3-only proteins include Bim, Bad, Bmf, Hrk, Bik, Noxa, and Puma. Together these proteins regulate programmed cell death.

Platelet Apoptosis

In a series of elegant experiments Mason et al has shown that the main anti-apoptotic protein in platelets is Bcl-XL [17382885]. They showed that mutations in Bcl-XL gene resulted in dose dependent reductions in platelet lifespan. The found that the major pro-apoptotic protein in platelets is Bak and to a minor extent Bax. Experiments in which the genes for these proteins were deleted result in doubling of the platelet lifespan. BH3-only proteins also appear to play important pro-apoptotic role in platelet lifespan as BH3-only mimetic compounds trigger apoptosis and rapid development of thrombocytopenia. In contrast, platelets from double-null mice (mice knocked out for both Bak and bax) are refractory to BH3-only mimetic compounds suggesting that the effect of BH3-only proteins is mediated through Bak and Bax.

In a study recently published study Kelly et al [19936621] showed that Bad-deficient mice had elevated platelets in their blood. They further showed that Bad is present in platelets and that bone marrow of Bad deficient mice showed normal number of megakaryocytic. They further studied the half life of platelets from Bad-deficient mice and found it to be modestly increased. To be certain that this was the intrinsic property of the platelets and not due to other host factors, they injected platelets from Bad-deficient mice into wild-type mice and found increased platelet lifespan. These series of experiments suggest that Bad acts by increasing the lifespan of platelets. On the other hand, mice deficient of another BH3-only protein, Bim, has mild thrombocytopenia. Bim appears to be involved in platelet formation by megakaryocytic and therefore, its deficiency is associated with impaired platelet formation. Interestingly, mice deficient in both Bad and Bim have normal number of platelets in their blood.

Another recent study shed some more light on the role of Bad in platelet lifespan. Catani et al [19936621] showed that activation of type-1 cannabinoid receptor by either endocannabinoid anandamide or methanandamide result in prolongation of platelet lifespan. This effect appears to be mediated though activation of Akt which, in turn, phosphorylates Bad (a BH3-only protein). A phosphorylated Bad cannot enter mitochondria and thus unable to bind and inactivate the anti-apoptotic Bcl-XL protein. Thus cytosolic sequestration of Bad results in prolongation of platelet lifespan in this model.

Vitamin D and Arterial Pulse Wave Velocity

Ok, so here is a study that continues the discussion of vitamin D supplementation to enhance health (or a surrogate of health). In this randomized trial, authors took 49 Black teens with mean age of 16.3 years and gave one group 2000 IU of vitamin D while control group was given 400 IU daily. Investigators measured carotid-femoral pulse-wave velocity (PWV) before and after 16 weeks of therapy in both groups. Investigators noticed a significant decrease in PWV in subjects taking higher dose of vitamin D (experimental group) as compared to the control group (group that was given 400 IU/day). In fact, in the control group PWV increased during the 16 week period.

This was despite the fact that both groups received vitamin D. To be sure that oral vitamin D was enough to raise serum vitamin D levels, investigators checked serum levels of vitamin D. Interestingly, serum levels increased in both groups.

Let’s see experimental group first (in nmol/L):
Baseline: 33.1
4 weeks: 55.0
8 weeks: 70.9
16 weeks: 85.7

Now PWV at baseline in the experimental group was
Baseline: 5.41 m/s
16 weeks: 5.33 m/s
p-value: 0.03

Ok, so this is relatively straightforward story so far, vitamin D was given, serum levels increased, and there was decrease in PWV. But lets see what happens with the control group; serum vitamin D levels were as below (in nmol/L):
Baseline: 34
4 weeks: 44.9
8 weeks: 51.2
16 weeks: 59.8

There is a rise in vitamin levels although not as pronounced as with 2000 IU but there is a considerable increase which is statistically significant as well. Now, if vitamin D is really effective then there should be some decrease in PWV although it might not be as much as with the high dose group, right? Lets see what was the PWV in the control group:
Baseline: 5.38 m/s
16 weeks: 5.71 m/s
p-value: 0.02

Oops! There is an increase in PWV and this is despite the fact that the serum levels of vitamin D almost doubled. In other words, this group was better off without any vitamin D supplementation. Hum! How can we interpret these findings? Is it possible that there is a cut-off after which vitamin D is effective? We know that is not the case; there is no consensus about what is the optimum level of vitamin D but it is much closer to 35 than to 60 and certainly not 80 nmol/L. Is it possible that very high vitamin D acts differently? Or is it possible that this is simply a result that happened by chance alone, that because the effect was in the correct direction and that it was consistent with the currently accepted wisdom. We don’t know but this is likely as the sample size was small. It is possible that blinding was not adequate enough and an investigator with a belief that vitamin D is effective may have interpreted PWV studies differently. We don’t know and while we don't know it is difficult to understand these results.

Intra-individual Variability in Platelet Responsiveness to Clopidogrel

Clopidogrel, in combination with aspirin, is a commonly used anti-platelet agents after PCI (percutaneous coronary intervention) in CHD patients. In one study, about 70% of the inter-individual variability in platelet response to clopidogrel is due to hereditary factors. A polymorphism in the CYP2C19 has been shown to be significantly associated with poor platelet response to clopidogrel. Even more important, poor platelet responsiveness to clopidogrel in patients with PCI has been shown to be associated with increased MACE (major adverse cardiac events).

While focus has been generally on inter-individual variation, it is possible that there is a significant intra-individual variability. This difference can't be explained by stable factors such as genetics or gender but is likely to be due to factors that vary over a short period of time. These include inflammatory state.

In recently published study, Armero et al examined patients on two different occasions after they had clopidogrel 600 mg loading dose. Platelet reactivity was measured using VASP (vasodilator-stimulated phosphoprotein). Interestingly, they found that there was a poor intra-individual correlation between the two occasions (kappa 0.33). In 65% of patients, platelet inhibition increased on the second evaluation while in 35% of the patients, platelet inhibition decreased.

The implications of this finding are worthy of notice. This mean that there are some rapidly varying factors that can alter platelet responsiveness to clopidogrel. On potential candidate is inflammation, although this was not measured and tested as such in this study. Investigators did measure leukocyte count and fibrinogen (and there was no difference) but both are rather crude measures of low-grade inflammation. Other possibilities include poor glycemic control in diabetics; in fact, investigators did find a relationship between the presence of diabetes and poor clopidogrel responsiveness.

Mutation T@Ster: Evaluation For Disease-Causing DNA Sequence Alterations

This is a web-based bioinformatics tool for rapid evaluation for the disease-causing DNA sequence alterations. MutationT@ster integrates information from different biomedical databases and analyses evolutionary conservation, splice-site changes, loss of protein features and changes that might affect the amount of mRNA to predict consequences of a polymorphism. The test is quite fast and a typical query takes less than 0.3 seconds to complete.

Its limitations include 1) inability to analyze insertion-deletions greater than 12 base pairs, 2) inability to analyze alterations spanning an intron-exon border, and 3) analysis of non-exonic alterations is restricted to Kozak consensus sequence, splice sites and poly(A) signal.

Other software with similar capability are: 1) Panther, 2) Pmut, 3) PolyPhen and PolyPhen-2, and 4) SNAP (screening for non-acceptable polymorphisms).