Tuesday, November 2, 2010

The Missing Link for Lupus Anticoagulants

For years now we have dealt with an unknown concerning the fact that the Lupus Anticoagulants are physiologically linked to thrombotic conditions and not to bleeding as could be expected from its name. The mechanism by which this autoimmune condition caused a thrombosis was always very speculative. In a recent article (1), the link has finally been described and it involves the interaction of vonWillebrand Factor (vWF) with its activation inhibitor Beta-2-Glycoprotein I.(B2GPI).  B2GPI is a common circulating glycoprotein which regulates the activity of vWF to interact with platelets.  Thus, if B2GPI is decreased by the presence of a Lupus Anticoagulant antibody, vWF will bind to platelets and cause clumping.  The article describes how vWF is produced in high molecular weight forms by the endothelium and how it’s broken down by ADAMTS13 and circulates as a globular complex.  This globular complex does not interact with platelets unless it is exposed to high shear in the circulation which opens the complex and allows the vWF to interact with the platelet glycoprotein (Gp)-Ib/IX/V and form platelet clumps.  Therefore, our knowledge of deficiencies of ADAMTS13 which leads to Thrombotic Thrombocytopenia(TTP) due to high molecular weight forms of  vWF binding to platelets, is now enhanced by how normal molecular weight forms can be activated or induced to bind to platelets and cause thrombosis.  This also fits with the finding that certain forms of Lupus Anticoagulants that are associated with a high titer anti-B2-glcoprotein I antibody, are 20 fold more likely to cause thrombosis or be associated with spontaneous abortions than Lupus Anticoagulants which are linked to Prothrombin antibodies. This article provides exciting reading combing the knowledge of Lupus Anticoagulants, vWF disease (specifically Type IIB) and platelet interactions.

(1)     Regulation of von Willebrand factor-platelet interactions. Lenting, P.J., Pegon, J.N., Groot, E., de Groot, P.G.. Thrombosis and Haemostasis 104. 3, 2010 p449-454.  Site reference: http://www.schattauer.de/en/magazine/subject-areas/journals-a-z/thrombosis-and-haemostasis/issue/special/manuscript/13198/show.html

Thursday, September 16, 2010

Optical Platelet Aggregation drug monitoring for P2Y12 inhibitors (e.g. Plavix or Clopidogrel)

Due to a number of inquiries concerning the monitoring of platelet inhibitors used in cardiovascular procedures, such as percutaneous coronary interventions (PCI), we would like to call your attention to some studies in this area and what we do to answer the concerns of physicians to monitor the effectiveness of the drugs currently used to reduce the incidence of non-responders to the therapy.  Current recommendations for PCI are to use Clopidogrel (Plavix) and Aspirin prior to and post procedurally to inhibit platelets from coagulating in the newly inserted stents. There is a high incidence of  patients who do not respond well to these inhibitors and we can differentiate them from the normal responders using a classical thoroughly validated technique, known as Optical Platelet Aggregation or Platelet Function analysis. Other techniques have been used and their problems have been discussed in the paper cited below along with a comparison with the classical method of Optical Platelet Aggregation (OPA). This paper compares a population study of 530 patients undergoing PCI and studied using the techniques of OPA, Verify Now and PFA-100. It evaluated the parameters that influenced the determination of who responded and who didn’t respond to the therapy (e.g. age, gender, inflammation, BMI, Diabetes and malignancy which are typical aspects seen as variables with OPA). It noted unexpected variables seen with the Verify Now P2Y12 analyzer of hematocrit and hemoglobin levels, while the PFA was influenced by B-blockers.

I would also like to mention that  “P2Y12” is the  name of the membrane glycoprotein that is specifically inhibited by Plavix and affects the platelet’s response to ADP, an agonist used in OPA to monitor platelet aggregation. Aspirin is also monitored in platelet aggregation by using the agonist Arachidonic acid. To confuse the story a little more, the researchers have coined the term “High Responders” referring to the fact that patients are poor responders to the drugs or their platelet are still reactive to the ADP or Arachidonic Acid agonists.

The reference I refer you to is in the Journal of Haemostasis and Thrombosis, 2009,102:719-727.  The Influence of Clinical Characteristics, Laboratory and Inflamatory Markers on “High on Treatment Platelet Reactivity” as Measured with Different Platelet Function Tests.
Ellen H.A.M. Elsenberg, Jochen W van Werkum, Ruud M.A. van de Wal, A.Carla, Zomer, Heleen J. Bouman, Freek W.A. Verheugt, Jurrien M. ten Berg, Christian M. Hackeng.

In conclusion, if anyone asks for P2Y12, Plavix or Clopidogrel monitoring, we do it daily with an order of a Platelet Function Analysis, same day turn around time and the whole blood samples (3-4 Blue top tubes) should be sent with 2 hours of the phlebotomy at room temperature. Please make a note of the type of medications the patient is on so that our comments can be specific as to their response.

Recent Genetic testing at Coagulation Consultants Lab.

Please be advised that our laboratory is able to supply you with the currently available genetic testing for Coumadin( Warfarin) and shortly Clopidogrel (Plavix) which are currently listed and recommended in the drug’s labeling (by FDA mandate) along with dosage recommendations.

 In terms of available population studies for Coumadin, there is a significant reduction in hospitalizations (approximately 30%) for over and under dosages as a result of genetic pharmacologic guidance for this drug (900 patients).  Many other studies were used to derive the specific genetic mutations and how they are involved with metabolism of this drug and the regeneration of Vitamin K(1,2) by the liver enzymes.  The importance of  this type of genetic anaylsis can be circumvented by routine (daily) monitoring of the Prothrombin Times to provide the needed adjustments to achieve a stable dosage that fits with the patients metabolism, drugs and diet. Published articles have established that self monitoring is a satisfactory way to achieve a successful patient routine without having the patient to visit the clinic every day.

 Similarly, diminished antiplatelet  responses to Clopidogrel (Plavix) have been described in 21 studies (4,500 subjects) demonstrating the genetic variables involved with the metabolic conversion of Clopidogrel to its active form (3). Many of the problems associated with Clopidogrel responses can be attributed to its metabolism from the prodrug to its active form, and these can arise from genetic mutations or from drug-drug interactions in the liver enzymes that metabolize the drugs. The genetic testing will anticipate the defects in its metabolism and therefore establish a faster response to the desired dosage. However, the final decision of the desired antiplatelet effect will be assessed by an Optical Platelet Function Analysis and how the platelets respond to ADP agonists. This is especially important when Plavix is combined with Aspirin, which can be associated with a bleeding diathesis in some patients. For more details on the Optical Platelet function analysis see the article entitled Plavix monitoring.

Since we can foresee a new demand for these genetic tests as a result of published reports, we would like to inform you that we can perform these tests in a timely fashion and at a significant cost savings over most alternatives. Please call us for information since our new requisitions are not available yet.


1) Epstein, R., et al. (2010, March 30). Warfarin Genotyping Reduces Hospitalization Rates: Results from the Medco-Mayo Warfarin Effectiveness Study (MM-WES). Retrieved from Journal Of American College Of Cardiology website: http://content.onlinejacc.org/cgi/content/full/jacc;j.jacc.2010.03.009v1

2) Bristol-Myers Squibb Company. (2010, January 22). COUMADIN Tablets (Warfarin Sodium Tablet, USP) Crystalline COUMADIN FOR INJECTION (Warfarin Sodium for Injection, USP). Retrieved from U.S. Food & Drug Administration website: http://www.accessdata.fda.gov/drugsatfda_docs/label/2010/009218s108lbl.pdf

3) Bristol-Myer Squibb/Sanofi Pharmaceuticals Partnership. (2010, March 12). PLAVIX clopidogrel bisulfate tablets. Retrieved from U.S. Food & Drug Administration website:  http://www.accessdata.fda.gov/drugsatfda_docs/label/2010/020839s042lbl.pdf

Friday, August 20, 2010

Delayed HIT and their antibodies

Publications (1,2) demonstrated that HIT antibodies can sometimes cause platelet activation in a Serotonin Release Assay and possibly in the related Heparin Induced Platelet Aggregation assay in the absence of added heparin. This phenomenon is not related to the presence of heparin in the serum sample. Therefore the HIT antibodies can interact with platelets when there is no heparin “in vivo”. This may lend significant importance to the finding of Delayed HIT in patients who have received Heparin weeks to months earlier. There also seems to be a significant increase in the titer of antibody in the PF4/Heparin Elisa assays for patients that have Delayed HIT thrombosis, which may represent a clinical edge to establish the diagnosis since Delayed HIT is not always associated with thrombocytopenia.

Although the paper shows that no heparin could be detected in these samples by either Anti-Xa assays or by using heparinase-1 to digest any heparin, some previous reports have shown that some positive platelet aggregation assays (in the absence of added heparin) can be reversed by heparinase treatment. It was also noted that the antibody (IgG) responsible for the positive platelet aggregation was heterogeneous in that some gave strong positive SRA reactions but not PF4/Heparin Elisa’s and vice versa.