Eli Cohen

Effects of coagulation factor deficiency on plasma coagulation kinetics determined via thrombelastography®: critical roles of fibrinogen and factors II, VII, X and XII

Background:  Thrombelastography (TEG®) is used to assess coagulopathy. However, a comprehensive characterization of the effects of specific coagulation factor deficiencies and mode of activation on TEG® data does not exist.

Elastic modulus-based thrombelastographic quantification of plasma clot fibrinolysis with progressive plasminogen activation

Thrombelastographic detection of fibrinolysis has been critical in the identification and treatment of coagulopathy in many perioperative settings. However, the fibrinolytic assessments have been at best non-parametric, amplitude-based determinations (e.g. estimated % lysis, clot lysis time or clot lysis rate). Recognizing this limitation, a methodology was developed to measure the onset, speed and extent of clot disintegration by changes in elastic modulus derived from the amplitude. Using this approach, our goal was to characterize the clot disintegration kinetics of progressive plasminogen activation with tissue plasminogen activator (tPA) and to determine the extent of inhibition of fibrinolysis mediated by tPA with aprotinin and activated factor XIII. While the estimated % lysis and clot lysis time were significantly affected by tPA (0–300 U/ml), elastic modulus-based analyses in a more activity-specific fashion demonstrated significantly decreased onset, increased rate and increased extent of fibrinolysis. Furthermore, aprotinin was found to inhibit the onset, rate and extent of fibrinolysis in an activity-dependent fashion, whereas activated factor XIII was noted to enhance the speed of onset of clot growth and delay the onset of fibrinolysis. In summary, our results serve as the rational basis to utilize this elastic modulus-based approach to quantify the extent of fibrinolysis in clinical and laboratory settings, as well as potentially guiding antifibrinolytic therapy.

Measurement of functional fibrinogen levels using the Thrombelastograph

STUDY OBJECTIVE To validate a Thromboelastograph (Haemoscope Corporation, Niles, IL) assay for functional fibrinogen. DESIGN Correlation study of the Thromboelastograph assay with two conventional fibrinogen assays by the standard Clauss method. SETTING Research laboratory of a university medical center. PARTICIPANTS AND INTERVENTIONS Blood samples were obtained from 19 healthy volunteers. MEASUREMENT AND MAIN RESULTS Thromboelastograph assays, using heparinized whole blood from 19 healthy donors, indicated that reptilase-XIIIa mixture (Activatorf)-generated clot shear elasticity in dynes per square centimeter (Gf) correlated with fibrinogen (mg/dL). Blood from four donors was used to define the contribution of hematocrit (Hct) to Gf by titration with platelet-rich plasma. The Gf versus Hct gave linear correlations (r2 = 0.746) with Gf = 1258 - 17.8 x % Hct. A commercial collection of 19 normal, 10 borderline, and one deficient for functional fibrinogen-citrated plasmas was assayed for Gf after recalcification using Activatorf. Of the 30 plasma samples, four were from factor X- or factor VII-deficient donors and one was from a coumadin-treated donor. There was a linear correlation of Activatorf Gf with functional fibrinogen (r2 = 0.940) with Gf = -730 + 9.21 x fibrinogen (mg/dL). CONCLUSION Thrombelastography with Activatorf may be used to determine fibrinogen levels in whole blood.

A novel thrombelastograph® tissue factor/kaolin assay of activated clotting times for monitoring heparin anticoagulation during cardiopulmonary bypass

We used a thrombelastograph (TEG®) assay with tissue factor and kaolin (TEG® TF/K) to measure activated clotting time (ACT) in 31 patients during cardiopulmonary bypass. For comparison, ACTs were also determined by a Hemochron Jr. Signature® and a Hepcon® HMS. The TEG® TF/K correlated with both the Hepcon (r2 = 0.789) and Hemochron (r2 = 0.743) ACTs. The average ACT after heparin was 319 ± 119 s (mean ± SD) for the TEG® TF/K compared with 624 ± 118 s for the Hepcon instrument. To evaluate the effects of hemodilution on TEG® TF/K and Hemochron assays, ACT assays were performed on blood diluted to 50% and titrated with heparin from 0 to 6 U/mL. Both instruments showed significant (P < 0.01) changes in the ACT-versus-heparin slope, but the 0 heparin intercept for the TEG® TF/K ACTs was not significantly changed (P = 0.292), in contrast to that for the Hemochron device (P = 0.041). Both instruments also indicated the same 1.3:1 ratio of protamine to heparin for optimum heparin neutralization, with increasing ACTs at ratios >2.6:1. The TEG® TF/K ACT assay rapidly monitors heparin anticoagulation, in addition to the capabilities of this instrument to monitor platelet function, clotting factors, and fibrinolysis.

Measurement of patients' bivalirudin plasma levels by a Thrombelastograph® ecarin clotting time assay: A comparison to a standard activated clotting time

Standard activated clotting time (ACT) tests have a poor correlation to bivalirudin levels, leading to uncertainty regarding adequate anticoagulation in percutaneous coronary intervention patients. We tested a Thrombelastograph® (TEG®) ecarin clotting time (ECT) assay for sensitivity to bivalirudin using blood from 80 patients undergoing interventional cardiology procedures with bivalirudin anticoagulation. This was compared to a standard Hemochron ACT assay using diatomaceous earth. With the TEG® assay, the direct thrombin activator, ecarin, was used to initiate coagulation and measured as the reaction time. Plasma samples were evaluated for bivalirudin by a chromogenic assay at an independent hematological laboratory. Linear regression of the standard ACT versus bivalirudin level gave an r2 = 0.306 whereas the TEG® ECT gave a much higher r2 = 0.746 (both P < 0.0001). The TEG® ECT should prove more useful than the standard ACT for monitoring bivalirudin anticoagulation across the clinically therapeutic range.

Race and sex differences in thrombogenicity : risk of ischemic events following coronary stenting

Race and sex affect thrombogenicity. We have demonstrated that platelet–fibrin clot characteristics can be used to stratify patients for risk of ischemic events following percutaneous coronary intervention. We investigated race and sex differences in thrombogenicty and the relation to ischemic risk in 252 consecutive African–American and Caucasian men and women undergoing elective percutaneous coronary intervention. Platelet–fibrin clot characteristics were measured using the Thrombelastograph Hemostasis System. The incidence of adverse ischemic events was assessed over a 6-month follow-up period. Overall, 40 ischemic events (15.9%) occurred. Adverse events were higher in African–Americans than Caucasians (P = 0.14), and in women than men (P = 0.004). The incidence was highest in African–American women (37.5%) and lowest in African–American men (6.5%). Measured Thrombelastograph parameters were significantly different between ischemic and nonischemic patients (P < 0.05). African–American women in the ischemic group exhibited higher thrombogenicity than the other race and sex groups (P < 0.05). Multivariate logistic regression identified platelet–fibrin mediated clot strength (relative risk 2.52, P = 0.017) and sex (relative risk 2.56, P = 0.009) as significant independent predictors of ischemic events 6 months postpercutaneous coronary intervention. Thrombogenicity is a novel measurable cardiovascular risk factor that varies by race and sex, is highest in African–American women, and independently predicts the frequency of ischemic events following percutaneous coronary intervention. Point-of-service measurements of platelet–fibrin clot characteristics may lead to more intensified antithrombotic therapy and reduced mortality in selected patients.

First report of the point-of-care TEG: A technical validation study of the TEG-6S system

Abstract Thrombelastography (TEG) measured by the TEG5000 Hemostasis Analyzer is an established but the labor-intensive method for assessing global hemostasis. The first true point-of-care TEG, the TEG6s system, uses resonance-frequency viscoelasticity measurements and a disposable multi-channel microfluidic cartridge to assess hemostasis and response to antiplatelet therapy. TEG assays (n = 5,100) were performed on the blood of healthy volunteers (n = 157) and patients undergoing coronary revascularization at three hospitals (n = 300). The results from the TEG6s were compared with the conventional TEG5000 in accordance with Clinical and Laboratory Standards Institute (CLSI) and FDA recommendations. Precision testing was conducted using blood from healthy donors, all assays were run for 5 consecutive days in duplicate using multiple operators, lots, and instruments. Reference ranges were comparable between the TEG systems. Deming regression analysis demonstrated a strong correlation between the two systems for the standard hemostasis tests (R r = 0.932, MA r = 0.972, LY30 r = 0.938). Method comparison analysis showed an acceptable agreement between PlateletMapping (PM) assays for measuring arachidonic acid (indicator of aspirin response)- and adenosine diphosphate (indicator of P2Y12 inhibitor response)-induced platelet aggregation (total agreement = 90%, and 72%, respectively). TEG6s precision testing yielded low variability (CV 0–13%) in all measures. The new point-of-care TEG6s is associated with greater ease of use than the TEG5000 and provides precise results. The results correlated between methods for all variables. TEG6s is a promising device for near-patient hemostasis monitoring and future trials of personalized therapy designed to reduce bleeding and thrombosis.

Post-coronary intervention recurrent ischemia in the presence of adequate platelet inhibition by dual antiplatelet therapy: what are we overlooking?

Recurrent ischemia following percutaneous coronary intervention (PCI) despite therapy with aspirin and clopidogrel may be due to: (i) insufficient inhibition of P2Y12 or COX-1; or (ii) platelet activation and aggregation stimulated by agonists other than ADP and thromboxane A2, or hypercoagulability resulting from thrombin generation [1]. A key element in providing optimal antithrombotic therapy is the ability to diagnose all of the potential causes of pathological thrombus formation so that each can be therapeutically addressed to the appropriate degree. Results from the Platelet REactivity in Patients And Recurrent Events (PREPARE POST-STENTING) study demonstrated that rapid plateletfibrin clot generation (an indirect measure of the rate of thrombin generation), high thrombin-induced maximum platelet-fibrin clot strength, and high platelet reactivity to ADP were risk factors for post-PCI ischemic events. In this subanalysis of the PREPARE POST-STENTING study, we examined platelet-fibrin clot strength and the kinetics of platelet-fibrin clot formation in patients responsive to clopidogrel and aspirin who experienced ischemic events. We hypothesized that the recurrence of ischemic events in clopidogrel and aspirin responsive patients was secondary to a hypercoagulable state characterized by rapid plateletfibrin clot formation, and high thrombin-induced plateletfibrin clot strength function as measured by the TEG hemostatic analyzer (Haemoscope Corp., Niles, IL, USA). One hundred and ninety-two consecutive patients undergoing elective coronary stenting were prospectively studied. The study was conducted at the Sinai Center for Thrombosis Research. Patient recruitment, data acquisition and statistical analysis were performed at the Sinai Center for Thrombosis Research. The manufacturer had no role in these activities. Platelet fibrin-clot strength, time to platelet-fibrin clot generation and ADPand arachidonic acid-induced platelet aggregation were studied before treatment using the Platelet Mapping Assay as previously described [2]. Adequate platelet inhibition was defined as >75% inhibition of ADPand arachidonic acid-induced platelet aggregation as measured by the Platelet Mapping Assay. Patient demographics and recurrent ischemic events have been described previously. Thirty-six patients experienced recurrent ischemic events and were subdivided into two groups based on the presence or absence of adequate platelet inhibition (Table 1). Comparisons between the groups were performed using the ANOVA method using SAS software (Cary, NC, USA). Among the 36 patients, only seven (19%) demonstrated >75% platelet inhibition by both aspirin and clopidogrel treatment as compared with <75% inhibition in the remaining patients with ischemia (P < 0.05). Patients with ischemic events and adequate platelet inhibition (Group A) had a more rapid time platelet-fibrin clot generation and a greater thrombininduced clot strength compared with patients without ischemic events (P £ 0.001 for both). This is the first study to demonstrate the existence of a subset of patients whose platelets were markedly inhibited by aspirin and clopidogrel therapy and yet experienced the recurrence of an ischemic event within 6 months of elective PCI. Examination of these patients by thrombelastography revealed a hypercoagulable state that may explain the occurrence of ischemic events after stenting in the presence of adequate platelet inhibition. These results would indicate that the achievement of an adequate level of protection against a recurrent thrombotic event might require conventional antiplatelet therapywith aspirin and a P2Y 12 blocker together with long-term anticoagulant therapy in selected patients who can be identified by TEG. PAR-1 receptor blockers may also have benefits in these patients. Correspondence: Paul A. Gurbel, Sinai Center for Thrombosis Research, Hoffberger Building, Suite 56, 2401W. Belvedere Avenue, Baltimore, MD 21215, USA. Tel.: +410 601 9600; fax: +1 410 601 9601; e-mail: pgurbel@ lifebridgehealth.org

POINT-OF-CARE ASSESSMENT OF NOAC-INDUCED ANTICOAGULATION: THE NOVEL HAEMONETICS TEG 6S SYSTEM

New oral anticoagulants (NOACs) including direct thrombin inhibitors (DTIs) and Xa inhibitors offer major advantages over vitamin K antagonists. Although officially these agents do not require monitoring, bleeding and ischemic events occur raising concerns for over or under inhibition during NOAC

ASSESSMENT OF THROMBOGENICITY AND ANTITHROMBOTIC DRUG RESPONSE AT THE BEDSIDE IN PATIENTS PRESENTING WITH ISCHEMIC STROKE, TIA OR INTRACRANIAL HEMORRHAGE

Background: Early assessment of antithrombotic effects may be important for therapeutic decision making in patients presenting with stroke and TIA. Methods: In this prospective observational study, 80 patients presenting with symptoms suggestive of stroke and 45 patients with stroke-like symptoms