Thrombin generation mediators and markers in sepsis-associated coagulopathy and their modulation by recombinant thrombomodulin.

Severe sepsis remains the most common cause of death in critically ill patients, and thrombin plays a crucial role in the pathogenesis of sepsis-associated disseminated intravascular coagulation (DIC). The purpose of this study was to profile prothrombin fragment (F1.2), thrombin-antithrombin complex (TAT), and d-dimer (DD) throughout the course of hospital stay in patients identified with sepsis. Plasma samples from patients enrolled in the ART-123 study, a phase 2b, international, multicenter, randomized placebo-controlled trial were analyzed for various parameters using enzyme-linked immunosorbent assay methods. Plasma levels of F1.2, DD, and TAT were measured at several time points following administration of recombinant thrombomodulin or placebo, and the results were tabulated. In the group treated with thrombomodulin, the median F1.2 levels demonstrated a 16% decrease from the baseline to day 7, while the placebo group showed an 8% increase. Both the treatment groups showed a gradual decrease in the TAT and DD, with the group treated with thrombomodulin demonstrating twice the decrease over the 7-day period. Although the data were widely scattered, these results show that DIC represents a hypercoagulable state along with other hemostatic abnormalities and the activation of the inflammatory process. Modulation of these activation processes through targets such as DD, F1.2, and TAT may play an important regulatory role in the pathogenesis of sepsis-associated coagulopathy. Moreover, this study validates the hypothesis that thrombomodulin downregulates the thrombin generation mediators/markers in sepsis-associated DIC.

Inflammatory and procoagulant cytokine levels during pregnancy as predictors of adverse obstetrical complications.

INTRODUCTION: Disturbances in cytokine networks are believed to be associated with increased risk of adverse pregnancy complications. METHODS: Plasma samples collected from pregnant women with preterm deliveries, high-risk pregnancy complications including postpartum hemorrhage, hypertensive disorders, and multiple gestations, and normal pregnancies were analyzed at different periods throughout gestation and postpartum. Interleukin (IL) 1ß , IL-6, IL-8, IL-10, tumor necrosis factor α, and antiprotein Z antibody levels were measured by enzyme-linked immunosorbent assay. RESULTS: The IL-6 levels in preterm delivery patients were elevated during pregnancy with statistically significant differences observed at 21 to 32 weeks (P < .01) and 33+ weeks (P < .001). The IL-10 levels were increased in normal pregnancy at all time points compared to the other patient groups (P < .05). The TNF-α levels were elevated in the high-risk pregnancy group versus normal controls (P < .001 at <21 weeks and P < .05 at 21-32 weeks). CONCLUSION: Analysis of the maternal plasma for elevated IL-6 and reduced IL-10 levels may be of value in the early prediction of pregnancy complications.

Variations in the circulating heparin levels during maintenance hemodialysis in patients with end-stage renal disease.

Unfractionated heparin has remained the anticoagulant of choice in patients undergoing hemodialysis. However, wide variations in the heparinization responses have been observed in patients anticoagulated with this drug. The purpose of this investigation was to measure circulating heparin levels in patients with end-stage renal disease (ESRD) prior to and after maintenance hemodialysis. This study included 119 patients with ESRD undergoing maintenance hemodialysis who received heparin during dialysis. Citrated blood samples were collected prior to and immediately after the dialysis session and analyzed utilizing clot-based methods such as activated partial thromboplastin time (APTT), Heptest, and prothrombinase-induced clotting time (PiCT). Circulating anti-Xa levels, antithrombin III levels, and thrombin generation (TG) were also measured. The circulating heparin levels ranged from 0 to 1.08 IU/mL with a mean of 0.07 ± 0.11 for the APTT and a range of 0 to 1.98 for the Heptest with a mean of 0.09 ± 0.26 U/mL. There was no significant difference in circulating levels of heparin between pre- and post-hemodialysis samples using APTT, Heptest, and PiCT, whereas the TG and anti-Xa tests showed a statistically significant P value <0.05 when comparing the 2 groups. The presence of detectable levels of heparin in the predialysis plasma samples for almost two-thirds (87 of 119) of the patients suggests that residual heparin circulates in these patients for a longer period of time. In all, 5% of postdialysis samples, 6 of 119, contained >0.25 U/mL of heparin, which may be related to a central catheter vascular access flushed with heparin. These findings suggest that patients on maintenance hemodialysis may accumulate a detectable amount of heparin due to the decreased renal clearance.

Comparative studies on branded enoxaparin and a US generic version of enoxaparin.

Enoxaparin, a complex, biologically derived low-molecular-weight heparin, is approved for a range of clinical indications. This study was carried out to compare the potency profile and pharmacodynamic responses of branded enoxaparin (Lovenox; Sanofi, US) with a generic enoxaparin (enoxaparin sodium injection, USP). Five batches of each product were tested. Although the average molecular weight, anti-factor Xa, and anti-factor IIa potencies were similar for the two products, differences were observed in the in vitro thrombin generation and kinetics of clot formation (P = .01) and in the ex vivo pharmacodynamics regarding thrombin generation inhibition (P = .029), tissue factor pathway inhibitor release (P = .006), and inhibition of the active form of thrombin-activated fibrinolysis inhibitor (P = .023). These findings suggest that simple analytical characterization can establish good quality control in manufacturing, but they may not assure similarity in biological performance between the branded and the generic enoxaparin.

Cross-reactivity of various thrombin products with anti-rabbit antibodies to bovine, human, and recombinant thrombin.

JMI-thrombin is used as topical hemostatic agent. While earlier clinically available JMI were reported to produce immunologic responses upon repeated exposure, the improved JMI, Recothrom?, and Evithrom? are claimed to be less immunogenic. Recothrom, despite its reduced immunogenic nature, upon repeated administration may result in the generation of antibodies (Abs) and that may cross react with bovine and human thrombin. Therefore, groups of rabbits were challenged repeatedly with Recothrom, Evithrom, and JMI over a 9-month period. Pre-immune blood and antiserum were collected from each rabbit on different time point. To determine their relative cross reactivity, JMI, Recothrom, and Evithrom were evaluated by western blotting using the rabbit IgG fractions. The results suggest that anti-Recothrom Abs cross-react with Evithrom and JMI in a time dependent fashion. Anti-JMI Abs did not cross-react with Recothrom, and Evithrom. Also, anti-Evithrom did not show any cross-reactivity with Recothrom and JMI at any time.

Gender-based differences in hemostatic responses.

AIM: Recent reports indicate increased mortality in women owing to cardiovascular diseases necessitating more gender-based studies. It is hypothesized that women have variable hemostatic responses to anticoagulant drugs. MATERIALS & METHODS: The hemostatic responses in healthy males (n = 10) and females (n = 10) were evaluated by performing various assays in the presence of anticoagulant drugs. Citrated whole blood from healthy volunteers (n = 20) was supplemented with rivaroxaban (final concentration [FC] = 0.3 µg/ml) and enoxaparin (FC =5 µg/ml). RESULTS: Differences between males and females were noted in the whole blood activated partial thromboplastin time (p = 0.0442) and Heptest® (p = 0.0345) assays in the saline control values. In the plasma system, rivaroxaban at a FC of 0.3 µg/ml and enoxaparin at 5 µg/ml showed a gender-based difference in the Heptest (p = 0.0423). Females showed faster fibrin formation than males. In the plasma system, plasminogen activator inhibitor-1 and domain-dimer assays (American Diagnostica, CT, USA) were performed with domain-dimer showing differences (p = 0.035). In the von Willebrand factor multimers, only band 5 showed differences (p = 0.032). Gender-based differences were observed. CONCLUSION: Careful adjustment of the dosages of anticoagulant drugs may be necessary to avoid bleeding or thrombosis.

A common standard is inappropriate for determining the potency of ultra low molecular weight heparins such as semuloparin and bemiparin.

INTRODUCTION: Lower low-molecular-weight heparins are being developed to improve on the safety and efficacy of antithrombotic therapy. Semuloparin and bemiparin are two depolymerized heparins produced by distinct manufacturing processes. The objective of this investigation was to determine whether a common standard could be used to define their potency. MATERIALS AND METHODS: Activities were compared using typical clinical coagulation assays and pharmacological assays required for potency assessment. RESULTS: The activity of semuloparin and bemiparin was comparable in FXa-based assays (anti-FXa, Heptest). However, bemiparin produced a stronger effect in the aPTT, ACT and anti-thrombin assays. Assessment of the parallelism of the concentration-response curves indicated that bemiparin and semuloparin are not equivalent in terms of anti-FIIa activity. Bemiparin had a stronger inhibitory effect on thrombin induced platelet aggregation, and a stronger interaction with HIT antibodies. CONCLUSIONS: These data demonstrate that depolymerized heparins can exhibit a range of biologic activities making them unique agents. Pharmacopoeial parameters such as anti-IIa and anti-Xa potency and molecular weight are insufficient to characterize such agents.

Upregulation of microparticles in DIC and its impact on inflammatory processes.

Disseminated intravascular coagulation (DIC) results in the catastrophic simultaneous activation of thrombotic and hemorrhagic processes. Its pathophysiology and the role of inflammation and microparticles (MPs) are not fully understood. Microparticles represent small phospholipid-expressing procoagulant vesicular fragments, released with cellular disruption and apoptosis. Functional MPs were measured in 100 random patients from a population of patients with DIC. Plasma samples from 30 normal male and female volunteers were used as control. Commercial Annexin trapping method was used to determine procoagulant activity of MPs. Mean ± SD concentration of MPs in the DIC group was 24.6 ± 14.2 nmol/L (range: 0.0-60.0 nmol/L), significantly higher than the control group: 8.5 ± 4.3 nmol/L (range: 1.3-17.4 nmol/L). Distribution curves and scattergrams showed that MPs concentration in the DIC samples was more widespread. This demonstrates that MPs are upregulated in patients with DIC and may mediate the hemostatic activation and inflammatory responses in this syndrome.

Upregulation of surrogate markers of inflammation and thrombogenesis in patients with ESRD: pathophysiologic and therapeutic implications.

Patients with end-stage renal disease (ESRD) undergoing regular hemodialysis have high annual mortality rate of around 15%. The most predominant cause of death is cardiovascular, which is not entirely explainable with conventional cardiac risk factors present in these patients. It has been postulated that ESRD is a chronic inflammatory and hypercoagulable condition with marked elevation of several markers that may explain this high mortality. In the current study, patients with ESRD on a stable regimen of hemodialysis were studied for the inflammatory and thrombogenesis markers to explain this phenomenon. The parameters studied were of thrombogenesis-thrombin-antithrombin III complex (TAT), prothrombin fragment (F1.2), D-dimer, and fibrinopeptide A (FPA) and inflammation-CD40 ligand, myeloperoxidase (MPO), tumor necrosis factor α (TNF-α), monocyte chemotactic protein-1 (MCP-1), and nitric oxide (NO), and compared to control group comprised of 100 healthy volunteers. Our study shows that ESRD patients exhibit activation of the coagulation and inflammatory processes.

Cross-reactivity of rabbit anti-bovine thrombin IgGs with human alpha-thrombin and a recombinant version of human thrombin (Recothrom).

It has been reported that patients exposed to topical bovine thrombin preparations may develop antibodies against bovine thrombin, factr V or various other proteins found in these preparations. Such antibodies can cross-react with human endogenous coagulation proteins and may lead to alterations in the coagulation laboratory parameters, hypersensitivity reactions, and severe bleeding or thrombosis. The underlying mechanisms for the coagulopathy are not fully understood yet. To better understand the cross-reactivity of anti-bovine thrombin antibodies with human corresponding coagulation proteins, bovine crude thrombin, and its purified versions, thrombin 4A (the old version of Thrombin-JMI before year 2008) and thrombin 4B (the current version of Thrombin-JMI on market), were used to generate relevant anti-bovine thrombin immunoglobulin G (IgGs) in rabbits. Using Western blotting, the cross-reactivity of each IgG with human alpha-thrombin and a recombinant version of human thrombin (Recothrom) was investigated. The results indicated that no cross-reactivity with either human alpha-thrombin or Recothrom was observed with both anti-bovine crude thrombin IgGs and thrombin 4B IgGs. However, anti-bovine thrombin 4A IgGs showed apparent cross-reactivity with human alpha-thrombin and Recothrom in a protein amount-dependent manner. Furthermore, the results revealed that the cross-reactivity of anti-bovine thrombin 4A IgGs with human alpha-thrombin and Recothrom was immunization time-dependent. The minimum concentration of 4A IgG required to exhibit cross-reactivity with human alpha-thrombin and Recothrom varied considerably among individual rabbits. These results indicate that rabbit anti-bovine thrombin IgGs can cross-react with human alpha-thrombin and Recothrom, suggesting that human antibodies against bovine thrombin may also cross-react with human recombinant thrombin. Thus, the patients who were previously exposed to bovine thrombin may also develop antibodies which can cross-react with human recombinant thrombin.

Comparative anticoagulant and platelet modulatory effects of enoxaparin and sulodexide.

Sulodexide represents a novel antithrombotic agent with multiple sites of action on blood coagulation and vascular processes. The purpose of this study was to compare sulodexide and enoxaparin on anticoagulant effects, tissue factor (TF)-induced activation of platelets, inhibition of microparticle generation and to investigate their effect on heparin-induced platelet aggregation (HIPA). Sulodexide was compared with enoxaparin at equigravimetric concentrations. When compared to enoxaparin, sulodexide produced a stronger anticoagulant effect in the prothrombin time (PT), activated partial thromboplastin time (APTT), Heptest, and thrombin time (TT) assays. In addition, sulodexide had a stronger inhibitory effect on TF-mediated microparticle generation (IC(50) = 2.8 microg/ mL), P-selectin expression (IC(50) = 4.8 microg/ml), and platelet aggregate formation (IC(50) = 8.5 microg/mL) compared to higher IC(50) values with enoxaparin. Sulodexide and enoxaparin exhibited a similar effect on heparin-induced thrombocytopenia (HIT) antibody-mediated platelet activation HIPA assays. These results suggest that sulodexide is a relatively stronger anticoagulant agent than enoxaparin. Sulodexide is subcutaneously absorbed. Its ability to inhibit TF-mediated platelet activation may contribute to the observed therapeutic effects of sulodexide in microvascular vasculopathy such as diabetic nephropathy. These results also suggest that inhibition of TF activation of platelets by sulodexide may be independent of its anticoagulant effects. These results warrant further investigation of sulodexide in additional preclinical and clinical studies.

Increased prevalence of antiheparin platelet factor 4 antibodies in patients may be due to contaminated heparin.

During the period of November 2007 to January 2008, an increased prevalence of adverse reactions to heparin was noted. These adverse events have been attributed to the presence of purposeful contaminant, oversulfated chondroitin sulfate (OSCS) from April 2007 to May 2008. An analysis of dialysis patients' plasma obtained in 2006 and 2007 consistently had a low (5%) prevalence of AHPF4 antibodies. Blood samples from 78 patients on maintenance hemodialysis, who were potentially exposed to OSCS-contaminated heparin, were analyzed for the presence of all AHPF4 antibodies using a commercially available ELISA kit from GTI. Although there was no change in the platelet count of these patients, 15 of 78 patients (19.2%) studied had an increased prevalence of AHPF4 antibodies. Subtyping of the all platelet factor 4 (PF4) antibodies documented showed a higher prevalence of immunoglobulin G antibodies as compared to their previously determined antibodies. These observations suggest that the OSCS contaminant in the recalled heparin triggers an immunogenic response not seen with OSCS-contaminated free heparin.

Reduced immunogenic potential of membrane filtered bovine thrombin preparations for hemostatic application.

There is concern that exposure to bovine thrombin can result in the development of antibodies, usually against factor V/Va, which can lead to hemostatic abnormalities. It is thought that purer preparations of bovine thrombin might be less immunogenic. Utilizing newer methods including a membrane filtration step, bovine crude thrombin is further purified into thrombin 4A and 4B preparations which exhibit a higher specific activity and are devoid of some of the protein contaminants. Bovine crude thrombin and its purified versions were administered intravenously to individual groups of rabbits using standard immunologic protocols. Antiserum was drawn from each rabbit and the pooled antisera were purified to obtain the IgGs using protein G affinity columns. The results suggest that the reported purification process, including filtration, resulted in the removal of most of the antigens found in crude thrombin, and that none of these preparations generated any detectable antibodies against bovine factor V related antigens.