Thrombin Generation Studies Research Articles

Analysis of Racial Disparity in Plasma of Healthy Volunteers Using Rotational Thromboelastometry Reveals Higher Prothrombotic Profile in African Americans

Racial differences in the incidence of arterial and venous thrombotic events are well established in literature, with a higher incidence noted in African Americans compared to Caucasians. Several studies have tried to explain this difference, by looking separately at chemical biomarkers, socioeconomic and clinical risk factors. Yet the exact reason behind this disparity remains unclear. At least one study of thrombin generation suggested that African ethnicity was associated with increased peak thrombin generation when compared to Caucasians. Rotational thromboelastometry is a visco-elastic methodology that offers a global assessment of hemostasis using either whole blood or plasma. We explored the racial differences in rotational thromboelastometry findings using plasma samples from healthy volunteers. We studied a cohort of 9 otherwise healthy adult volunteers with no history of cardiovascular nor thromboembolic events, 5 African Americans and 4 Caucasians. After informed consent, we collected citrated whole blood samples and processed them within 3 hours of phlebotomy. Platelet free plasma, obtained after centrifugation of whole blood for 20 minutes, was kept frozen at -70°C, and then thawed at 37°C for 5 minutes prior to testing. Samples were re-calcified with star-tem® reagent, and then the in-tem® reagent was added. The latter contains an optimized concentration of ellagic acid and partial thromboplastin phospholipid from rabbit brain. Thromboelastometry parameters including Clot Formation Time, Alpha Angle, and Maximum Clot Firmness were determined. We then compared the data between the two study populations using parametric unpaired Student’s t-test. Our results showed that the Clot Formation Time was higher in the plasma of Caucasians when compared to African Americans with a difference between means of 40.1 ± 4.4 seconds (p <.0001); the Alpha Angle was lower in Caucasians with a difference between means of 3.45 ± 0.3 degrees (p <.0001); and the Maximum Clot Firmness was lower in Caucasians with a difference between means of 9.75 ± 2 mm (p <.01). These findings demonstrated that the plasma of Caucasians took longer to reach the maximum firmness, and this maximum firmness was less than that reached in the plasma of African Americans. This reveals a significantly increased prothrombotic profile in the plasma of African Americans compared to Caucasians. Despite the limited number of participants, the striking observed differences in the thromboelastometry parameters suggest that global assays may offer benefit in assessing thrombotic risks in disparate patient populations, as they incorporate multiple components of the hemostatic system. Higher numbers of subjects and assessment of both whole blood and plasma are indicated. We are currently in the process of expanding our cohort to confirm these preliminary results. Disclosures No relevant conflicts of interest to declare.

Effects of a plasma-derived C1 esterase inhibitor on hemostatic activation, clot formation, and thrombin generation.

Hereditary angioedema (HAE) is a rare, autosomal dominant disease in which C1 esterase inhibitor (C1 INH) is deficient or dysfunctional. Package inserts for nanofiltered C1 esterase inhibitor (C1 INH-nf) products contain warnings about thrombotic events. The objective of this study was to evaluate the effect of C1 INH-nf on hemostatic activation, clot formation, and thrombin generation. Ten healthy volunteers provided blood samples for thromboelastometry using the ROTEM system. Platelet-poor samples were prepared for thrombin generation studies. C1 INH-nf was added to samples at final concentrations of 0.14, 0.7, 1.4, 2.8, and 7.0 U/ml. Recombinant factor VIIa and prothrombin complex concentrate were used as procoagulant controls, and antithrombin and desirudin were used as anticoagulant controls. C1 INH-nf had no procoagulant effect on hemostasis based on thromboelastometry, regardless of the final concentration or activating reagent used (P > 0.05 for all comparisons of C1 INH-nf versus negative control). C1 INH-nf 2.8 and 7.0 U/ml concentrations had a statistically significant anticoagulant effect on maximum clot firmness (P < 0.05 for all comparisons of C1 INH-nf versus negative control), with a magnitude similar to that observed with desirudin. C1 INH-nf had no effect on thrombin generation lag time, peak thrombin generation, or thrombin generation rate, regardless of the final concentration or activating reagent used (P < 0.05 for all comparisons of C1 INH-nf versus negative controls). We found no evidence of a procoagulant effect of C1 INH-nf when studied ex vivo at concentrations up to 10-fold higher than those achieved with clinical dosing in patients with HAE.

Defibrotide Interaction With Newer Oral Anticoagulant and Antiplatelet Drugs

Introduction Defibrotide represents a single stranded mammalian DNA derived agent originally developed for anti-thrombotic and anti-ischemic indications. Defibrotide is currently used to treat or prevent failure of normal blood flow (Veno-occlusive disease, VOD) in the liver of patients who had bone marrow transplants or received drugs such as oral estrogens and mercatopurine. Defibrotide is a polypharmocologic agent with multiple sites of actions, which include anti-inflammatory and vaso-facilitatory effects. The purpose of this investigation is to determine potential interactions of defibrotide and some of the newer oral anticoagulant drugs, such as dabigatran, apixaban and rivaroxaban and antiplatelet drugs such as ticagrelor. Materials &amp; Methods Defibrotide (lot no. DV0601) was obtained in powder form from Gentium s.p.A (Villa Guardia, Italy). The active form of dabigatran was purchased from Selleckcham (Houston, TX). Apixaban and rivaroxaban were obtained commercially and were of synthetic origin. The effect of defibrotide on dabigatran, apixaban, rivaroxaban and ticagrelor, on agonists (epinephrine, ADP, collagen, arachidonic acid and 2.5U thrombin) was measured using platelet aggregation techniques. The platelet rich plasma collected from normal healthy donors (n=15) were also supplemented with each of the individual anticoagulant drugs, alone in a range of 0-1000 ng/ml and in combination with defibrotide at 50-250ug/ml. Such clotting times as the PT, aPTT and Heptest and thrombin generation studies were carried out. In addition, whole blood activated clotting time (celite) studies were carried out by supplementing each of the individual oral anticoagulant agents at 1ug/ml, alone and with defibrotide at 100ug/ml. Results Neither defibrotide nor the newer anticoagulants and ticagrelor produced any effects on the epinephrine, ADP, collagen and arachidonic acid induced aggregation of PRP (p&gt;0.05). However, dabigatran at concentrations of &gt;62.5ng/ml produced inhibition of thrombin induced aggregation, all of the other agents did not have any effect on thrombin. Defibrotide at a concentration 100 ug/ml did not alter the aggregation profile in anticoagulant supplemented PRP. In both the plasma and whole blood anticoagulant assays, dabigatran produced stronger anticoagulant effects (306+30sec) than both apixaban (145+12sec) and rivaroxaban(160+15sec). Defibrotide exhibited minimal effects (135+10sec). Ticagrelor did not have any anticoagulant effect. Defibrotide in combination with dabigatran produced modest augmentation of the anticoagulant responses (360+42sec), however, it had much weaker effects on rivaroxaban (168+18sec) and apixaban(152+14sec). All of the anticoagulants in the TGA produced varying degrees of inhibition of thrombin generation in the following order; dabigatran &gt; rivaroxaban &gt; apixaban. Ticagrelor did not produce any inhibition of thrombin generation. Defibrotide did not produce any effects on TGA at concentrations up to 100ug/ml. When combined with oral anticoagulants, it did not show any augmentation of rivaroxaban and apixaban; however, it enhanced dabigatrans inhibitory effects. Conclusions These results suggest that defibrotide itself has weak or negligible anticoagulant effects in the plasma and whole blood assays. All of the new oral anticoagulants produced varying degrees of assay dependent anticoagulant effects in plasma and whole blood systems. However defibrotide did not show any interactions with apixaban and rivaroxaban, it showed some augmentation of the anticoagulant responses of dabigatran. Ticagrelor did not exhibit any interactions with defibrotide. These studies demonstrate that unlike heparins, defibrotide exhibits minimal interactions with newer oral anticoagulant and antiplatelet drugs. Disclosures: No relevant conflicts of interest to declare.

Oral Anti-Factor Xa and Factor IIa Agent Mediated Inhibition Of Tissue-Factor Mediated Generation Of Thrombin In Prothrombin Complex Concentrates

Introduction Several oral anti-factor IIa and factor Xa agents have recently been developed. These include the thrombin inhibitors Ximelagatran/Melagatran (M) and Dabigatran Etexilate/Dabigatran (D), which require endogenous conversion to the active agents D and M. The factor Xa inhibitors, Rivaroxaban (R) and Apixaban (A), are anti-Xa agents that do not require any endogenous activation. Ximelagatran was withdrawn from the market due to adverse reactions. Dabigatran, Rivaroxaban, and Apixaban are approved for various clinical indications. Antagonism of the anticoagulant effect may be required in bleeding complications. Contradictory results were reported for the efficacy of various prothrombin complex concentrates (PCCs) with these new oral anticoagulants (NOACs). The purpose of this study was to determine the differences in the thrombin generation inhibitory profiles of the newer oral anticoagulant agents. Methods Commercially available PCCs namely Octaplex and Beriplex, were used as a source of Factors II, VII, IX and X. To investigate the effect of each of these agents, a working solution of 1U/ml of both PCCs were supplemented in a graded concentration of 0-1250ng/ml with M, D, R and A. Thrombin generation studies were carried out using a thromboplastin activator (RC High, Technoclone Vienna, Austria). Total thrombin generated was measured in terms of nM’s. The IC-50 for each agent was calculated individually. The time course of thrombin generation was also measured following the kinetic profiles and AUC. Results Dabigatran and Melagatran produced relatively weaker inhibition of thrombin generation with the IC-50 values ranging from 410-110ng/ml in Beriplex and 350-1120ng/ml in Octaplex. Both Rivaroxaban and Apixaban produced strong inhibition of thrombin generation, with the IC-50 ranging from 58-62ng/ml in Octaplex; whereas, in Beriplex these values ranged from 48-50ng/ml. The onset time for thrombin generation and total thrombin formation was concentration dependent. The kinetics of thrombin generation with A and R were distinct from D and M. At concentrations below 310ng/ml the total amount of thrombin generated was comparable to the control; however, its formation was delayed. In both systems, D exhibited the weakest thrombin generation inhibitory potential. While the onset time of thrombin generation was delayed at concentrations below 310ng/ml the levels were comparable to or higher than the control. Discussion This data suggests that PCC’s such as Octaplex and Beriplex can be used to generate thrombin and it’s inhibition by new oral anticoagulant drugs. Octaplex generates much higher amount of thrombin than Beriplex at equivalent units. These results also show that in comparison to the oral anti-Xa agents, the oral anti-IIa agents are relatively weaker inhibitors of thrombin generation. These studies also suggest that the differential inhibition of the generation of thrombin through tissue factor by the anti-Xa and IIa agents may contribute to the potential neutralization profile of PCC’s for these drugs. Disclosures: No relevant conflicts of interest to declare.

Clinical utility of viscoelastic tests of coagulation in patients with liver disease

The prothrombin time (PT) and international normalised ratio (INR) are used in scoring systems (Child-Pugh, MELD, UKELD) in chronic liver disease and as a prognostic tool and for dynamic monitoring of hepatic function in acute liver disease. These tests are known to be poor predictors of bleeding risk in liver disease; however, they continue to influence clinical management decisions. Recent work on coagulation in liver disease, in particular thrombin generation studies, has led to a paradigm shift in our understanding and it is now recognised that haemostasis is relatively well preserved. Whole blood global viscoelastic tests (TEG(®) /ROTEM(®) ) produce a composite dynamic picture of the entire coagulation process and have the potential to provide more clinically relevant information in patients with liver disease. We performed a systematic review of all relevant studies that have used viscoelastic tests (VET) of coagulation in patients with liver disease. Although many studies are observational and small in size, it is clear that VET provide additional information that is in keeping with the new concepts of how coagulation is altered in these patients. This review provides the basis for large scale, prospective outcome studies to establish the clinical value of these tests.

Biochemical and pharmacological differentiation of dabigatran, apixaban and rivaroxaban

Dabigatran (D), apixaban (A) and rivaroxaban (R) have been approved for various indications at specific dosages. This study was designed to compare the biochemical and pharmacologic profile of these drugs in a battery of standardized tests. Powdered forms of commercially available active D, A and R were dissolved at 250 ug/ml and diluted for various testing. Global anticoagulant assays, interactions with fibrinolytic agents, effect on thrombin anti‐thrombin complex and prothrombin fragment F1.2 activation, thrombin generation studies using functional and immunologic methods, platelet aggregation, whole blood clotting studies and in vivo bleeding studies were carried out. In the global anticoagulant assays, D produced strong anticoagulant effects followed by R. A produced relatively weaker effects. In the thrombin generation assays D was a relatively weaker inhibitor in comparison to both A and R. None of these agents produced any inhibition of agonist induced aggregation except D which inhibited thrombin induced aggregation and activation responses in platelets. In the whole blood assays, each agent produced it's own profile. D was stronger than A and R. In the in vivo bleeding studies, D produced stronger effects in comparison to both A and R. The currently available new oral anticoagulant drugs exhibit distinct biochemical and pharmacologic profiles which may impact their safety and efficacy outcomes.

Differential inhibition of tissue factor mediated activation of thrombin generation by Dabigatran, Apixaban, Rivaroxaban and Otamaxaban. Pharmacologic implications

IntroductionNewer anticoagulant drugs Dabigatran (D), Rivaroxaban (R), Apixaban (A) and Otamaxaban (O) are monotherapeutic agents targeting thrombin and factor Xa. Beside inhibiting these enzymes these agents also inhibit the generation of thrombin. The purpose of this study was to investigate relative inhibitory effects of D, R, A and O on thrombin generation by using mass spec and functional methods.Materials and MethodsSeveral commercially available prothrombin complex concentrates (PCCs), and normal human plasma (NHP) were supplemented with each of the different inhibitors and proteomic profile was obtained in SELDI‐TOF analysis in the molecular weight (MW) range of 0–150,000. Functional thrombin generation studies were carried out using chromogenic and fleurogenic substrate methods.ResultsAll of the prothrombin complexes exhibited 2 major native peaks at 72 and 13.9 kDa and upon thromboplastin activation showed a 36 and 12.6 kDa peaks representing thrombin generation. D did not inhibit the generation of the 36 and 12.6 peaks whereas O,A and R inhibited the generation of these peaks in a conc dependent manner. Functional generation of thrombin was also inhibited by O, A and R but not by D.ConclusionsThe generation of 36 and 12.6 kDa peak signifies thrombin formation which is strongly inhibited by Xa inhibitors such as O, A and R. D is a relatively weaker inhibitor of this process.

Biomarker Profiling of Bladder Cancer Patients Undergoing Radical Cystectomy. Relevance of Thrombotic and Inflammatory Processes

Abstract 3405 Introduction:Postsurgical thrombosis represents a serious comorbidity in bladder cancer. Inflammation and hemostatic activation mediators contribute to thrombotic pathogenesis. This study profiled such biomarkers as antiphospholipid antibody, IgG subtype (APA), procoagulant microparticles (MP) and antiglycosaminoglycan antibody (AGA). Thrombin generation profile was also carried out. In addition protein and peptide biomarkers were profiled using SELDI-TOF mass spectrometry. Materials and Methods:This study was conducted retrospectively using an IRB approved protocol with 134 baseline serum samples collected from patients undergoing radical cystectomy. The samples were banked at −70C and thawed prior to analysis. The normal group was comprised of plasma samples collected from healthy male and female individuals (n=50). The APA were measured with an ELISA for IgG specific antibodies (American diagnostica, Stanford CT). The MP were measured using an anexin capture chromogenic substrate to quantify functional procoagulant activities (Hyphen Biomedical, Paris France). The AGA were measured with a commercially available method with a polysulfonate polymer capture probe for the antibodies (GTI Madison, WI). Thrombin generation studies were carried out by using a fluormetric method (Technoclone). The proteomic profile was analyzed on a GoldChip in the range of 5–150 kDa. Results:In comparison to the normal group, a wide scatter in the cancer group was noted, all of these exhibited a higher prevalence of elevated biomarkers compared to normal. The average MP value was 3.1+5.8nM (range 0.1–34.6nM) with 26 of 134 samples above the normal values which are (2.8+1.1). The average APA value was 6.7+11 (range 4–65nM) with 32 above normal. The average AGA value was 0.22+0.09 (range .01-.8OD) with 10 above normal. The SELDI analysis showed unique biomarkers in the area of 11–12kDa, 15.1–15.4kDa, and 15.8–16.2kDa which were absent in the normal group. The serum samples showed the presence of higher amidolytic activities for serine proteases. The thrombin generation studies in a modified method also showed a higher thrombin generation in the cancer group. Conclusion:The biomarker upregulation suggests that bladder cancer patients have subclinical activation of thrombotic and inflammatory processes. The MP may be generated in cellular damage whereas the APA and AGA suggest vascular endothelial damage and activation. The unique proteomic biomarkers in these patients suggest endogenous protease activation. The increased protease activity and higher thrombin generation suggest a role of microparticles in mediating thrombogenesis. A clinical correlation in the subset with higher markers may provide insight into the biomarker role in prognosis, risk stratification and treatment of bladder cancer. Disclosures:No relevant conflicts of interest to declare.

Population Based Differences in the Anticoagulant and Antiprotease Responses of Newer and Oral Anticoagulant Drugs

Abstract 3421 Introduction:Most of the screening of the newer anticoagulant drugs is usually carried out in normal human blood derived plasma and its products. Therefore endogenous compositional factors in different patient groups which may result in altering the anticoagulant and antiprotease effects of these drugs are not taken into account. Recently an oral anti-IIa agent Dabigatran and an anti-Xa agent namely Rivaroxaban are approved for clinical usage in the US. An additional anti-Xa drug namely Apixaban is approved in Europe and is under review by the US FDA. The pharmacologic properties of these drugs and their mechanism of action are distinct from one another. Therefore their endogenous interactions with blood and vascular sites also differ. The purpose of this study is to evaluate the differential anticoagulant responses of these agents in plasma samples from different patient populations groups such as the liver diseases (LD), acute coronary syndrome (ACS), end stage renal disease (ESRD), sepsis associated coagulopathy (SAC), cancer (C) and diabetes (D) in comparison to normal plasma. Materials and Methods:Plasma samples were collected from healthy normal male and female individuals (n=50) served as a control group. In addition plasma samples from various patient groups such as LD, ESRD, ACS, SAC, C and D and elevated aPTT, intrinsic defect (ID) and elevated PT extrinsic defect (ED) were collected ( n=30–50). These plasma samples were individually supplemented with Dabigatran, Rivaroxaban and Apixaban, at a concentration of 0.125 and 0.25ug/ml. Such tests as PT/INR, aPTT, TT and Heptest times and prothrombinase induced clotting time (PiCT) were measured using standard assays. The same samples were also analyzed for thrombin generation inhibition and anti-Xa and anti-IIa activities. Additionally pooled LD and SAC samples along with normal plasma were supplemented in a graded concentration of 0–1000 ng/ml with each of these anticoagulants. Concentration response curves were constructed for each of these agents in all the tests employed for the anticoagulant, antiprotease and thrombin generation studies. Results:In comparison to the normal plasma, different patient group plasma samples exhibited variable anticoagulant and thrombin generation inhibitory activities. In particular LD patients showed a stronger anticoagulant response with all agents, however Dabigatran produced the most profound effects. While these agents only produced a modest effect in the PT/INR assay, other coagulation tests were significantly elevated, in particular the PiCT tests. In comparison to both Apixaban and Rivaroxaban, Dabigatran produced the most pronounced prolongation of the aPTT, Heptest time, PiCT and TT. The results with Apixaban and Rivaroxaban were comparable. In the intrinsically defected plasma samples a similar trend was noted and Dabigatran produced the most pronounced prolongation in contrast to both Apixaban and Rivaroxaban. Similarly in the extrinsic defective plasma samples the Dabigatran produced the most pronounced prolongation of all of the tests in comparison to Apixaban and Rivaroxaban. The anti-protease (anti-Xa and anti-IIa) activities in different plasma matrices did not differ markedly. Population based differences were noted in other groups such as ACS, ESRD, SAC, C and D. The individual groups of the special patient populations showed wide scatter. The concentration response curves also differed from one population to another population. Conclusion:While all three anticoagulants produced stronger anticoagulant effects in LD, Dabigatran produced most markedly higher elevation of clotting responses. Rivaroxaban and Apixaban produced relatively weaker responses and differed from one another. Interestingly in the thrombin generation inhibition assays, Dabigatran produced relatively weaker effects in both normal and LD. Assay based variations among three agents were pronounced in patient populations. These studies clearly suggest that the anticoagulant and thrombin generation inhibitory effects of newer anticoagulants markedly differ in normal plasma and patient plasmas, however the antiprotease effects are similar. Therefore population based differences in the anticoagulant responses may alter their anticoagulant effects in different patient populations. These variations may account for the differential safety and efficacy of these drugs. Disclosures:No relevant conflicts of interest to declare.

Non-Pathogenic Anti-Heparin/PF4 Antibodies Generated by Low Molecular Weight Heparins Mediate Procoagulant Effects.

AbstractAbstract 1091 Introduction:While the incidence of symptomatic heparin-induced thrombocytopenia (HIT) is relatively low with the use of low molecular weights heparins (LMWHs), these agents do generate anti-heparin/PF4 antibodies in 10–20% of treated patients. Dosage, duration, and the pathologic predisposition of the patient influence the quantitative and qualitative nature of these antibodies. It has been suggested that these non-pathogenic antibodies (NPAs) which do not produce symptomatic HIT may, nevertheless, be biologically active and mediate thrombogenic responses. The overall pathophysiologic role of NPAs is unknown at this time. Hypothesis:NPAs generated by LMWHs cause coagulation activation and compromise the anticoagulant effects of the administered LMWH. Study Design:Blood plasma samples collected at baseline and day 10 from patients enrolled in orthopedic surgery clinical trials of LMWHs for the prophylactic management of deep vein thrombosis (Lovenox enoxaparin, sanofi-aventis, n=352; Clivarin reviparin, Abbott, n=380) were retrospectively screened for the presence of anti-heparin/PF4 antibodies using the GTI ELISA method (Waukesha, WI). Positive samples were tested by the 14C-SRA to determine if the antibodies were capable of functionally activating platelets. Both ELISA positive and negative samples were evaluated in an assay of thrombin generation (Technothrombin TGA kit, diaPharma, West Chester, OH). Result:In the enoxaparin study, the baseline pre-treatment samples only showed one patient in the heparin control group to be positive by ELISA. On day 10, 11 of 175 (6.3%) enoxaparin patients had a positive ELISA response, whereas 22 of 177 (12.4%) heparin patients were ELISA positive. None of the samples were 14C-SRA positive. In the thrombin generation assay, the ELISA positive samples showed a lesser inhibition of thrombin generation for both the enoxaparin and heparin groups (270 ± 27 nM TGA enoxaparin group; 220 ± 21 nM TGA heparin group) compared to the thrombin generation response of the ELISA negative samples (190 ± 18 nM TGA enoxaparin group; 160 ± 20 nM TGA heparin group). In the reviparin study, none of the patients were ELISA positive at baseline. On day 10, in the reviparin group 19 of 200 (9.5%) patients were ELISA positive, whereas 28 of 180 (15.6%) heparin control patients had a positive ELISA titer. None of the samples were 14C-SRA positive. In comparison to the baseline (pre-treatment), both the reviparin and heparin treated patients showed an inhibition of thrombin generation (410 ± 27 nM TGA baseline vs 180–290 nM with treatment). However, consistent with the above study, those samples that were ELISA antibody positive showed a lesser inhibition of thrombin generation (240 ± 21 nM TGA reviparin group; 210 ± 16 nM TGA heparin group) in comparison to the ELISA negative samples (190 ± 12 nM TGA reviparin group; 180 ± 14 nM TGA heparin group). Interestingly, the D-dimer levels were found to be higher in the ELISA positive samples in all groups for both studies (p<0.05). Conclusion:These studies suggest a potential pathologic role of NPAs. The results of the thrombin generation studies strongly suggest that the generation of NPAs may result in a reduction of the antithrombotic potential of both LMWH and heparin in treated patients. While the exact mechanism of this process is not clear, dosage adjustment may be useful in those patients who generate NPAs. Disclosures:No relevant conflicts of interest to declare.

Anticoagulation of a patient with hypertrophic cardiomyopathy and factor VII deficiency

A 50-year-old male patient with hypertrophic cardiomyopathy and atrial fibrillation was anticoagulated, with warfarin following insertion of a cardioverter defibrillator. He became markedly over anticoagulated after standard moderate induction doses of warfarin. His baseline prothrombin time was prolonged and further investigation showed the patient to have a mild factor VII deficiency. He was restarted on low-dose warfarin and successfully stabilized with a target international normalized ratio (INR) of 3.0 (range 2.5-3.5). We used the data from factor VII levels and thrombin generation studies before and after anticoagulation to control dosage and to decide on a suitable therapeutic range for the INR. Molecular studies showed him to have two separate mutations in the factor VII gene. This report highlights the importance of noting the baseline prothrombin time before initiating oral anticoagulation and describes how well tolerated anticoagulation can be achieved in a patient with congenital factor VII deficiency.

Potency Adjustment of Unfractionated Heparins to Harmonize the USP and WHO Standardization. Impact on Anticoagulant Practice in the US

To harmonize the potency of US heparin (UFH) supplies with the WHO standard, the USP has mandated use of a revised procedure resulting in a 10–13% reduction of anticoagulant potency. To assess the impact of 10% potency adjustment, stock solutions of UFH were prepared to represent a 10% decrease (90 U/ml), no change (100 U/ml) and a 10% increase (110 U/ml). Differences were evaluated in the surgical and interventional range (1–5 U/ml) using the ACT and in the therapeutic range (0–1.0 U/ml) using plasma clotting assays. Protamine titration studies were conducted in both studies. Thrombin generation studies were conducted at lower levels. Rabbit models of stasis thrombosis and ear bleeding were used to determine the impact on antithrombotic and hemorrhagic activities. Differences in the ACT were only noted at concentrations &gt;1.0 U/ml. No difference in protamine neutralization profile was noted. Supplementation to normal plasma did not result in differences in the PT, APTT, Heptest, thrombin time, anti‐IIa and anti‐Xa activities. Some differences were noted utilizing a thrombin generation assay. These studies suggest that a 10% change in heparin potency may not impact the anticoagulant practice in the therapeutic or prophylactic range. Closer monitoring may be needed in specialized populations and in those patients requiring surgical and prophylactic anticoagulation where higher concentrations of UFH are reached.

Comparative Studies on Branded Dalteparin with Two Generic Versions: Hepagumin and Fluzepamin

Dalteparin is a low molecular weight heparin (LMWH) prepared by nitrous acid digestion. Recently generic versions of the product have become available. The purpose of this study was to compare the molecular and functional profile of Dalteparin and two generic versions from Japan (Hepagumin and Fluzepamin). The molecular profile was determined using the HPLC before and after heparinase‐1 digestion. The anticoagulant activity was measured using the whole blood ACT‐Hemochron and plasma‐based APTT and Heptest, and amidolytic anti‐Xa and anti‐IIa assays. Thrombin generation studies were performed using a kinetic method. By HPLC, the molecular weight of the three products was comparable and ranged from 6.3 to 6.8 kDa. After heparinase digestion all three products give comparable result (molecular weight ranging from 2.1 to 2.3 kDa). The oligosaccharide components after digestion were also comparable. In the whole blood anticoagulant studies no differences were noted between products at the 1.25 U/ml. In the APPT and anti‐Xa assay, there were no differences between three products. However, minor differences were noted in the Heptest and factor IIa inhibition. These results show that Hepagumin and Fluzepamin do not significantly differ from the branded dalteparin. The minor differences noted in clot based assays were not statistically different.

Comparative study of coagulation and thrombin generation in the portal and jugular plasma of patients with cirrhosis

Portal vein thromboses are frequent in cirrhotic patients and may be favoured by hypercoagulability in the splanchnic venous system. The coagulation balance and thrombin generation (TG) were evaluated in platelet-free plasma obtained from portal and systemic blood samples in 28 cirrhotic patients while undergoing transjugular intrahepatic porto-systemic shunt. TG assay (TGA) was performed with all samples from cirrhotic patients and with plasma samples from 14 healthy controls, with varying concentrations of tissue factor and phospholipids, with or without thrombomodulin. Screening tests and specific assays were also performed and activated partial thromboplastin time was shorter in portal plasma samples with higher FVIII and lower protein C levels, well correlated with Child-Pugh scores, and higher D-dimers and F1+2 levels However, all TGA parameters were similar in portal and jugular samples, possibly due in part to similar concentrations of factor II and antithrombin in these two sites of plasma sampling. TGA showed lower thrombin peaks and endogenous thrombin potential values in cirrhotic plasma compared to those of healthy controls. Importantly, a resistance to thrombomodulin that well correlated with factor VIII and PC levels, was evidenced in all samples from patients with cirrhosis, and was more significant in those from severely affected cases. This study therefore supports the existence of a relative hypercoagulability in the portal vein of cirrhotic patients that is likely due to protein C/S deficiency and to high FVIII levels.

TRANSFUSION PRACTICE: Thrombin generation in trauma patients

BACKGROUND: Trauma patients are at risk of developing an acute coagulopathy of trauma (ACT) related to tissue injury, shock, and hemodilution. ACT is incompletely understood, but is similar to disseminated intravascular coagulation (DIC) and is associated with poor outcome.STUDY DESIGN AND METHODS: Thrombin generation assays were used to evaluate plasma hemostasis in 42 trauma patients, 25 normal subjects, and 45 patients on warfarin and in laboratory‐prepared factor reduced plasma.RESULTS: Prolonged prothrombin time (PT), more than 18 seconds, or an international normalized ratio of greater than 1.5 was present in 15 trauma patients indicating possible ACT. Native thrombin generation (no activator added, contact activation blocked) showed that Trauma with ACT patients had lag times 68% shorter and peak thrombin generation threefold higher than normal patients indicating the presence of circulating procoagulants capable of initiating coagulation systemically. Trauma patients had lower platelet counts and fibrinogen and Factor (F)II levels putting them at increased risk of bleeding. In laboratory‐prepared isolated factor‐reduced samples and in patients with vitamin K–dependent factor deficiency due to warfarin, thrombin generation decreased in direct proportion to FII levels. In contrast, in diluted plasma and in trauma patients with reduced factor levels, thrombin generation was increased and associated with slower inhibition of thrombin generation (prolonged termination time) and decreased antithrombin levels (43% of normal in Trauma with ACT).CONCLUSIONS: Thrombin generation studies indicate that Trauma with ACT patients show dysregulated hemostasis characterized by excessive non–wound‐related thrombin generation due to a combination of circulating procoagulants capable of activating coagulation systemically and reduced inhibitor levels allowing systemic thrombin generation to continue once started.

Defibrotide Augments the Anticoagulant Actions of Heparin and Low Molecular Weight Heparins

Defibrotide represents a polydeoxyribonucleotide derived antithrombotic and antiischemic drug, which has been used in the management of vascular disorders and is currently being developed in other clinical indications. Defibrotide is a polyelectrolyte-based agent with target effects on endothelium, platelets, and blood cells. In addition, the aptameric consensus sequences in the nucleotides exhibit inhibitory effects towards thrombin and related proteases. In the anticoagulant assays defibrotide exhibits relatively weak effects (<5 USP U/mg). These studies were undertaken to study whether there is an interaction between defibrotide and unfractionated heparin (UFH) in various systems of anticoagulation. The interaction of defibrotide with commercially available low molecular weight heparins (LMWHs), enoxaparin and dalteparin, was also studied. For the first investigation, to evaluate the effect of defibrotide on the anticoagulant effects of UFH, native whole blood freshly drawn from human volunteers (n = 20) was supplemented with UFH at a fixed concentration of 5 μg/mL (0.8 U/mL), and graded amounts of defibrotide were added in a concentration range of 12.5 – 100 μg/mL. The whole blood celite Activated Clotting Time test (ACT) and the thrombin generation markers fibrinopeptide A (FPA), thrombin-antithrombin complex (TAT), and prothrombin fragment 1.2 (F1.2) were measured. Parallel controls with saline were included. While defibrotide did not produce a significant prolongation of the ACT compared to saline (128 ± 9 s vs 132 ± 7 s), it produced a concentration-dependent increase in the heparinized whole blood leading to an almost doubling of the anticoagulant action of UFH (248 ± 19 s vs 418 ± 21 s). Additional studies carried out by varying the concentrations of the two agents also revealed supraadditive to synergistic effects. Defibrotide also augmented the inhibitory effects of UFH on thrombin generation markers in a concentration-dependent fashion. Similar studies carried out with the two LMWHs did not reveal a similar interaction in the anticoagulant assays such as the ACT; however, significant interactions between defibrotide and the LMWHs were observed in the thrombin generation studies. For the second investigation, studies were carried out using plasma samples collected from heparinized patients (aPTT of 50 – 100 s). These studies also revealed that supplementation of defibrotide augmented the anticoagulant effects of UFH in a concentration-dependent fashion. While defibrotide at 12.5 μg/mL did not significantly increase the aPTT of normal plasma, when supplemented to heparinized plasmas (n = 50 with aPTT of 64.6 ± 14.0 s) it produced a strong prolongation of the clotting time (96.1 ± 20.6 s). In the third investigation, animal models of thrombosis including the rat jugular vein clamping model, demonstrated an augmentation of the antithrombotic effects of intravenously administered UFH by defibrotide. However, no augmentation of the hemorrhagic effect was observed in the rat tail bleeding model. These studies demonstrate that defibrotide exhibits a strong anticoagulant interaction with UFH and to a lesser degree LMWH. While the combination of defibrotide and UFH exhibits enhanced anticoagulant/antithrombotic activities, it does not exhibit any alteration of the hemorrhagic profile. These studies clearly suggest that defibrotide can be combined with UFH to achieve a superior anticoagulant approach with better safety/efficacy profile.

Effects of ximelagatran, an oral direct thrombin inhibitor, r-hirudin and enoxaparin on thrombin generation and platelet activation in healthy male subjects

ObjectivesThe effects of ximelagatran, an oral direct thrombin inhibitor (DTI), recombinant hirudin (r-hirudin) and enoxaparin on thrombin generation and platelet activation were studied in humans. BackgroundRecombinant hirudin (parenteral DTI) and enoxaparin (low molecular weight heparin) have been demonstrated to be clinically effective in acute coronary syndromes. Ximelagatran is currently under investigation for the prevention and treatment of thromboembolism. The shed blood model allows for the study of thrombin generation and platelet activation in humans in vivo. MethodsThis was an open-label, parallel-group study involving 120 healthy male volunteers randomized to receive one of three oral doses of ximelagatran (15, 30 or 60 mg), r-hirudin (intravenous) or enoxaparin (subcutaneous) at doses demonstrated to be clinically effective in acute coronary syndromes, or to serve as a control. Thrombin generation (prothrombin fragment 1+2 [F1+2] and thrombin-antithrombin complex [TAT]) and platelet activation (β-thromboglobulin [β-TG]) biomarkers were studied using a shed blood model involving blood collection from skin incisions made using standardized bleeding time devices. ResultsOral ximelagatran, intravenous r-hirudin and subcutaneous enoxaparin rapidly and significantly (p < 0.05) decreased F1+2, TAT and β-TG levels in shed blood, indicating inhibition of thrombin generation and platelet activation. Statistically significant concentration (melagatran, the active form of ximelagatran)-response relationships for F1+2 (p = 0.005), TAT (p = 0.005) and β-TG (p < 0.001) levels, with IC50s of 0.376 (F1+2), 0.163 (TAT) and 0.115 (β-TG) μmol/l, were detected. Melagatran showed dose-proportional pharmacokinetics with low variability. All drugs were well tolerated. ConclusionsOral administration of the DTI ximelagatran resulted in a rapid inhibition of both thrombin generation and platelet activation in a concentration-dependent manner using a human shed blood model. The inhibition of thrombin generation by 60 mg ximelagatran was comparable to that observed with doses of r-hirudin and enoxaparin demonstrated to be effective for the treatment of acute coronary syndromes.

A high-fat meal does not activate blood coagulation factor VII in minipigs.

It is a matter of debate whether postprandial activation of blood coagulation factor VII (FVII) is associated with an increased risk of thrombosis. To clarify this question, an animal model in which consequences of dietary FVII activation can be studied in a more detailed way would be an important tool. We studied postprandial FVII activation in seven non-fasting Göttingen minipigs. Intralipid (4 g/kg) was administered through a gastric tube in two fractions at 9.00 a.m. (one-third of total dose) and 10.30 a.m. (two-thirds of total dose). Blood samples were drawn 0.5 h before (baseline) and 2, 3, 3.5, 4, 5, and 6 h after the first fat load. Triglycerides, activated FVII (FVIIa), FVII coagulant activity (FVIIc), FVII amidolytic activity (FVIIam) and prothrombin fragment I + 2 (F1 + 2) were analysed in plasma samples. Median plasma triglycerides were significantly raised from 0.67 mmol/l (baseline) to 2.56 mmol/l 5 h postprandially (P < 0.001). There were no significant changes in FVIIa (9.6 U/l at baseline), FVIIam (142% at baseline) and F1 + 2 (0.13 nmol/l at baseline). FVIIc decreased from 141% at baseline to 114% 6 h postprandially (P < 0.001). As a high-fat meal does not seem to activate blood coagulation FVII in minipigs, the pig is apparently not a relevant model for the study of dietary FVII activation and thrombin generation.

Thrombogenicity of vascular cells. Comparison between endothelial cells isolated from different sources and smooth muscle cells and fibroblasts.

When the endothelial cell layer is damaged, a thrombotic reaction starts on the cells' subendothelium and on the connective tissue deposited by smooth muscle cells in the deeper layers. When more severe vascular damage occurs, hemostasis will involve the vessel adventitia in which fibroblasts are found. In this article, the influence of in vitro cultured endothelial cells, smooth muscle cells, and fibroblasts on the hemostatic balance was studied. To do so, perfusions were performed with low molecular weight heparin anticoagulated blood over the extracellular matrix of the cells. This method allowed the study of tissue factor-dependent thrombin generation and its influence on formation of fibrin and platelet aggregates. The experiments described in this article show that endothelial cells isolated from different human organs interfere differently in the hemostatic response. Endothelial cells isolated from umbilical veins are nonthrombogenic; they do not synthesize tissue factor under unstimulated conditions. On their extracellular matrix, only adherent platelets are found, but no aggregates and no fibrin. Endothelial cells isolated from omentum and atrium contain tissue factor activity under unstimulated conditions. As a consequence, thrombin is generated on their surfaces, and platelet aggregates and fibrin deposition are found on the extracellular matrices after perfusions with whole blood. The matrix of smooth muscle cells and fibroblasts behaved similarly. Increase in shear rate and perfusion time resulted in an increase in platelet aggregate formation. Polymerized fibrin deposition decreased when perfusions were performed at higher shear. Both platelet aggregation and fibrin deposition were tissue factor dependent and could be blocked more than 70% by an antibody against tissue factor. Based on these results, we conclude that endothelial cells isolated from umbilical veins form the best nonthrombogenic surface in vitro. Moreover, coagulation-dependent hemostasis should be included when thrombogenicity of subendothelium is discussed, especially when it concerns matrix derived from cells present in the deeper layer of the vessel wall.