Anticoagulant and Antithrombotic Properties of Three Structurally Correlated Sea Urchin Sulfated Glycans and Their Low-Molecular-Weight Derivatives.

The anticoagulant and antithrombotic properties of three structurally correlated sea urchin-derived 3-linked sulfated α-glycans and their low molecular-weight derivatives were screened comparatively through various in vitro and in vivo methods. These methods include activated partial thromboplastin time, the inhibitory activity of antithrombin over thrombin and factor Xa, venous antithrombosis, the inhibition of platelet aggregation, the activation of factor XII, and bleeding. While the 2-sulfated fucan from Strongylocentrotus franciscanus was observed to be poorly active in most assays, the 4-sulfated fucan from Lytechinus variegatus, the 2-sulfated galactan from Echinometra lucunter and their derivatives showed multiple effects. All marine compounds showed no capacity to activate factor XII and similar low bleeding tendencies regardless of the dose concentrations used to achieve the highest antithrombotic effect observed. The 2-sulfated galactan showed the best combination of results. Our work improves the background about the structure-function relationship of the marine sulfated glycans in anticoagulation and antithrombosis. Besides confirming the negative effect of the 2-sulfated fucose and the positive effect of the 2-sulfated galactose on anticoagulation in vitro, our results also demonstrate the importance of this set of structural requirements on antithrombosis in vivo, and further support the involvement of high-molecular weight and 4-sulfated fucose in both activities.

Recombinant factor VIIa addition to haemophilic blood perfused over collagen/tissue factor can sufficiently bypass the factor IXa/VIIIa defect to rescue fibrin generation.

INTRODUCTION: Factor VIII (FVIII) or factor IX (FIX)-deficient haemophilic patients display deficits in platelet and fibrin deposition under flow detectable in microfluidics. Compared to fibrin generation, decreased platelet deposition in haemophilic blood flow is more easily rescued with recombinant factor VIIa (rFVIIa), whereas rFVIIa requires FXIIa participation to generate fibrin when tissue factor (TF) is absent. AIMS: Perfusion of haemophilic whole blood (WB) over collagen/TF surfaces was used to determine whether rFVIIa/TF was sufficient to bypass poor FIXa/FVIIIa function in blood from patients with haemophilia A and B. METHODS: Whole blood treated with high-dose corn trypsin inhibitor (40 µg mL-1 ) from seven healthy donors and 10 patients was perfused over fibrillar collagen presenting low or high TF (TFlow or TFhigh ) at wall shear rate of 100 s-1 . RESULTS: With WB from healthy controls, platelet deposition and fibrin accumulation increased as TF increased. Factor-deficient WB (1-3% of normal) displayed striking deficits in platelet deposition and fibrin formation at either TFlow or TFhigh . In contrast, mildly factor-deficient WB (14-32%) supported fibrin formation under flow on TFhigh /collagen. With either TFlow or TFhigh , exogenously added rFVIIa (20 nm) increased platelet deposition and fibrin accumulation in WB from factor-deficient patients (1-3% of normal) to levels commensurate with untreated healthy WB. CONCLUSION: The absence of FIXa/FVIIIa in patients with severe haemophilia results in deficits in fibrin formation that cannot be rescued by wall-derived TF ex vivo. The effects of rFVIIa on platelet adhesion and rFVIIa/TF can act together to reinforce thrombin generation, platelet deposition and fibrin formation under flow.

A Step Toward Balance: Thrombin Generation Improvement via Procoagulant Factor and Antithrombin Supplementation.

BACKGROUND: The use of prothrombin complex concentrates in trauma- and surgery-induced coagulopathy is complicated by the possibility of thromboembolic events. To explore the effects of these agents on thrombin generation (TG), we investigated combinations of coagulation factors equivalent to 3- and 4-factor prothrombin complex concentrates with and without added antithrombin (AT), as well as recombinant factor VIIa (rFVIIa), in a dilutional model. These data were then used to develop a computational model to test whether such a model could predict the TG profiles of these agents used to treat dilutional coagulopathy. METHODS: We measured TG in plasma collected from 10 healthy volunteers using Calibrated Automated Thrombogram. TG measurements were performed in undiluted plasma, 3-fold saline-diluted plasma, and diluted plasma supplemented with the following factors: rFVIIa (group rFVIIa); factors (F)II, FIX, FX, and AT (group "combination of coagulation factors" [CCF]-AT); or FII, FVII, FIX, and FX (group CCF-FVII). We extended an existing computational model of TG to include additional reactions that impact the Calibrated Automated Thrombogram readout. We developed and applied a computational strategy to train the model using only a subset of the obtained TG data and used the remaining data for model validation. RESULTS: rFVIIa decreased lag time and the time to thrombin peak generation beyond their predilution levels (P < 0.001) but did not restore normal thrombin peak height (P < 0.001). CCF-FVII supplementation decreased lag time (P = 0.034) and thrombin peak time (P < 0.001) and increased both peak height (P < 0.001) and endogenous thrombin potential (P = 0.055) beyond their predilution levels. CCF-AT supplementation in diluted plasma resulted in an improvement in TG without causing the exaggerated effects of rFVIIa and CCF-FVII supplementation. The differences between the effects of CCF-AT and supplementation with rFVIIa and CCF-FVII were significant for lag time (P < 0.001 and P = 0.005, respectively), time to thrombin peak (P < 0.001 and P = 0.004, respectively), velocity index (P < 0.001 and P = 0.019, respectively), thrombin peak height (P < 0.001 for both comparisons), and endogenous thrombin potential (P = 0.034 and P = 0.019, respectively). The computational model generated subject-specific predictions and identified typical patterns of TG improvement. CONCLUSIONS: In this study of the effects of hemodilution, CCF-AT supplementation improved the dilution-impaired plasma TG potential in a more balanced way than either rFVIIa alone or CCF-FVII supplementation. Predictive computational modeling can guide plasma dilution/supplementation experiments.

TETIS study: evaluation of new topical hemostatic agent TT-173 in tooth extraction.

OBJECTIVES: TT-173 is a new hemostatic agent consisting of yeast-derived microvesicles containing a modified version of recombinant human tissue factor. In the present work, the procoagulant activity of TT-173 has been evaluated for the first time in humans. METHODS: This is a phase I, randomized, placebo-controlled study to evaluate the efficacy, safety, systemic absorption, and immunogenicity of TT-173 in healthy volunteers undergoing tooth extraction. Subjects received TT-173 or placebo into the alveolar cavity, just after tooth extraction. Time to clot formation, bleeding time, and adverse events were recorded. RESULTS: Treatment with TT-173 reduced the bleeding time and the time to clot formation. No adverse events related with product administration were reported. In the same way, neither systemic absorption nor immunogenic reaction against the product was detected. Our findings pave the way to evaluate the usefulness of this new topical hemostatic agent in more complex oral surgeries and in those patients affected with coagulation disorders that may compromise the realization of dental procedures. CONCLUSION: The new hemostatic agent TT-173 has proven efficacious and safe in healthy subjects undergoing tooth extraction supporting its further evaluation in more complex surgeries. CLINICAL RELEVANCE: The development of this new topical hemostatic agent could contribute to bleeding control in oral and maxillofacial surgery.

Experimental Evaluation of a New Tissue Factor-Based Topical Hemostat (TT-173) for Treatment of Hepatic Bleeding.

Background: Excessive blood loss is a relevant complication of partial liver resection. Topical hemostatic agents have proven useful to improve the control of the bleeding in this among other surgical indications. Until now all of these products have been based on the action of thrombin. In contrast TT-173 is a new topical hemostatic agent based on recombinant tissue factor naturally incorporated into membrane vesicles. This work sought to assess the efficacy and toxicity of TT-173 in an animal model of liver resection.Materials and Methods: Procoagulant activity of 0.15, 0.41, and 1 mg of TT-173 was evaluated in pigs subjected the resection of hepatic lobe margins. The most effective of these doses was also compared against thrombin. In addition, the toxicity, local tolerance, systemic absorption, and immunogenicity of the product were investigated in rats subjected to liver biopsy lesion.Results: The three doses of TT-173 evaluated significantly reduced the bleeding time in liver lesions. The highest dose of product was significantly more effective than the others and thrombin. Application of high doses of TT-173 in rats did not cause any local or systemic alterations. Absorption into blood stream was negligible and no immunogenic reaction against the product was detected.Conclusions: TT-173 shows favorable pharmacodynamic properties for improving hemostasis in partial liver resection which support further investigation of the product in this surgical indication.

Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature.

Selective occlusion of tumor vasculature was tested as a therapy for solid tumors in a mouse model. The formation of blood clots (thrombosis) within the tumor vessels was initiated by targeting the cell surface domain of human tissue factor, by means of a bispecific antibody, to an experimentally induced marker on tumor vascular endothelial cells. This truncated form of tissue factor (tTF) had limited ability to initiate thrombosis when free in the circulation, but became an effective and selective thrombogen when targeted to tumor endothelial cells. Intravenous administration of the antibody-tTF complex to mice with large neuroblastomas resulted in complete tumor regressions in 38 percent of the mice.

Construction and characterization of a truncated tissue factor­coagulation­based composite system for selective thrombosis in tumor blood vessels.

The selective induction of tumor vascular thrombosis using truncated tissue factor (tTF) delivered via a target ligand is a promising novel antitumor strategy. In the present study, an anti­neuropilin­1 (NRP­1) monoclonal antibody (mAb)­streptavidin (SA):tTF­biotin (B) composite system was established. In this system, anti­NRP­1­mAb located tTF to the tumor vascular endothelial cell surface and induced vascular embolization. Due to their high binding affinity, SA and B were used to enhance thrombogenic activity. mAb was conjugated with SA using a coupling method with water­soluble 1­ethyl­3­(3­dimethylaminopropyl) carbodiimide and N­hydroxysulfosuccinimide. Biotinylated tTF (tTF­B) was prepared using a B­labeling kit subsequent to the generation and purification of fusion protein tTF. Confocal microscopy and flow cytometry indicated that the anti­NRP­1­mAb­SA conjugate retained mAb targeting activity. The preservation of B­conjugate binding capacity was confirmed using a competitive ELISA, and factor X­activation analysis revealed that tTF­B retained the procoagulant activity exhibited by tTF. Live imaging was performed to assess mAb­SA distribution and tumor­targeting capability, and this yielded promising results. The results of in vivo studies in mice with subcutaneous xenografts demonstrated that this composite system significantly induced tumor vascular thrombosis and inhibited tumor growth, whereas these histological changes were not observed in normal organs.

Virus envelope tissue factor promotes infection in mice.

Essentials The coagulation initiator, tissue factor (TF), is on the herpes simplex virus 1 (HSV1) surface. HSV1 surface TF was examined in mice as an antiviral target since it enhances infection in vitro. HSV1 surface TF facilitated infection of all organs evaluated and anticoagulants were antiviral. Protease activated receptor 2 inhibited infection in vivo and its pre-activation was antiviral. SUMMARY: Background Tissue factor (TF) is the essential cell surface initiator of coagulation, and mediates cell signaling through protease-activated receptor (PAR) 2. Having a diverse cellular distribution, TF is involved in many biological pathways and pathologies. Our earlier work identified host cell-derived TF on the envelope covering several viruses, and showed its involvement in enhanced cell infection in vitro. Objective In the current study, we evaluated the in vivo effects of virus surface TF on infection and on the related modulator of infection PAR2. Methods With the use of herpes simplex virus type 1 (HSV1) as a model enveloped virus, purified HSV1 was generated with or without envelope TF through propagation in a TF-inducible cell line. Infection was studied after intravenous inoculation of BALB/c, C57BL/6J or C57BL/6J PAR2 knockout mice with 5 × 105 plaque-forming units of HSV1, mimicking viremia. Three days after inoculation, organs were processed, and virus was quantified with plaque-forming assays and quantitative real-time PCR. Results Infection of brain, lung, heart, spinal cord and liver by HSV1 required viral TF. Demonstrating promise as a therapeutic target, virus-specific anti-TF mAbs or small-molecule inhibitors of coagulation inhibited infection. PAR2 modulates HSV1 in vivo as demonstrated with PAR2 knockout mice and PAR2 agonist peptide. Conclusion TF is a constituent of many permissive host cell types. Therefore, the results presented here may explain why many viruses are correlated with hemostatic abnormalities, and indicate that TF is a novel pan-specific envelope antiviral target.

Specific tissue factor delivery using a tumor-homing peptide for inducing tumor infarction.

Targeting the human blood coagulation-inducing protein tissue factor (TF) to the tumor vasculature to induce infarction and disrupt the blood vessels has proven to be an effective approach for tumor therapy. In this study, we investigated the thrombogenic activity and anti-tumor potential of a novel fusion protein (tTF-CREKA) comprising the extracellular domain of human tissue factor (truncated TF, tTF) and a tumor targeting pentapeptide, Cys-Arg-Glu-Lys-Ala (CREKA). tTF is soluble and inactive in its free state, but when it is targeted to the plasma membrane of both tumor vessel endothelial cells and stromal cells by the CREKA peptide, its native coagulation-inducing activity is restored. Systemic administration of the tTF-CREKA fusion protein into tumor-bearing mice induced tumor-selective intravascular thrombosis and reduced tumor blood perfusion, consequently inhibiting tumor growth. The development of tTF-CREKA introduces a new method for treating a wide spectrum of solid tumors by selectively blocking tumor blood supply.

Factor VIII-bypassing activity of bovine tissue factor using the canine hemophilic model.

The bleeding disorder of hemophilia A currently treated by replacement therapy of the missing coagulation factor, factor VIII, is frequently complicated by the development of neutralizing antibodies. The therapeutic potential of attenuated forms of the lipid-associated glycoprotein tissue factor, a known initiator of coagulation, was investigated as a factor VIII-by-passing activity. The protein moiety of tissue factor (Apo-TF) was partially purified and exhibited minimal procoagulant activity before relipidation in vitro. In pilot studies, Apo-TF injection into rabbits previously anticoagulated with an antibody to factor VIII was found to have a procoagulant effect. The efficacy of the material was further demonstrated when injection of Apo-TF in hemophilic dogs resulted in a normalization of the cuticle bleeding time. Little or no change in the blood parameters associated with disseminated intravascular coagulation was observed at lower doses, although mild to moderate effects were seen at higher doses. These data suggest a novel role for Apo-TF preparations as a potential therapeutic agent for hemophiliacs with antibodies to factor VIII once the potential thrombogenicity of such materials is evaluated.

Infarction of solid Hodgkin's tumors in mice by antibody-directed targeting of tissue factor to tumor vasculature.

We demonstrated previously that selective thrombosis of the blood vessels of solid tumors in mice can be achieved by targeting the extracellular domain of tissue factor by means of an antibody to an experimentally induced marker on tumor vascular endothelium. In the present study, we extend this finding to a naturally occurring marker of tumor vascular endothelium, vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 is expressed by vascular endothelial cells in Hodgkin's disease and various solid tumors in mice and humans. It is absent from vascular endothelial cells in normal tissues in mice, with the exception of the heart and lungs, where it is present on venules. A monoclonal antibody to murine VCAM-1 was covalently linked to the extracellular domain of human tissue factor to create a "coaguligand." After i.v. administration to severe combined immunodeficient mice bearing human Hodgkin's tumors, the coaguligand localized selectively to VCAM-1-expressing vessels, caused thrombosis of those vessels, and retarded tumor growth. The coaguligand also localized to VCAM-1-expressing vessels in the heart and lungs of the mice but did not induce thrombosis in these sites. An immunohistochemical evaluation of the distribution of a monoclonal anti-phosphatidylserine (PS) antibody in the mice showed that the VCAM-1-expressing vessels in the tumor expressed PS, whereas the VCAM-1-expressing vessels in the heart and lungs lacked PS. The lack of thrombotic effect of the coaguligand on heart and lung vessels may be because PS is needed to provide the procoagulant surface upon which coagulation complexes can assemble. The requirement for coincident expression of the targeted marker and PS on tumor endothelium probably contributes to the selectivity of thrombotic action and the safety of coaguligands.

Is there a difference in efficacy, safety, and cost-effectiveness between 3-factor and 4-factor prothrombin complex concentrates among trauma patients on oral anticoagulants?

PURPOSE: The aim of this study was to compare the efficacy, safety, and cost-effectiveness of 3-factor prothrombin complex concentrate (3F-PCC) vs 4-factor prothrombin complex concentrate PCC (4F-PCC) in trauma patients requiring reversal of oral anticoagulants. MATERIALS AND METHODS: All consecutive trauma patients with coagulopathy (international normalized ratio [INR] ≥1.5) secondary to oral anticoagulants who received either 3F-PCC or 4F-PCC from 2010 to 2014 at 2 trauma centers were reviewed. Efficacy was determined by assessing the first INR post-PCC administration, and successful reversal was defined as INR less than 1.5. Safety was assessed by reviewing thromboembolic events, and cost-effectiveness was calculated using total treatment costs (drug acquisition plus transfusion costs) per successful reversal. RESULTS: Forty-six patients received 3F-PCC, and 18 received 4F-PCC. Baseline INR was similar for 3F-PCC and 4F-PCC patients (3.1 ± 2.3 vs 3.4 ± 3.7, P = .520). The initial PCC dose was 29 ± 9 U/kg for 3F-PCC and 26 ± 6 U/kg for 4F-PCC (P = .102). The follow-up INR was 1.6 ± 0.6 for 3F-PCC and 1.3 ± 0.2 for 4F-PCC (P = .001). Successful reversal rates in patients were 83% for 4F-PCC and 50% for 3F-PCC (P = .022). Thromboembolic events were observed in 15% of patients with 3F-PCC vs 0% with 4F-PCC (P = .177). Cost-effectiveness favored 4F-PCC ($5382 vs $3797). CONCLUSIONS: Three-factor PCC and 4F-PCC were both safe in correcting INR, but 4F-PCC was more effective, leading to better cost-effectiveness. Replacing 3F-PCC with 4F-PCC for urgent coagulopathy reversal may benefit patients and institutions.

In vivo induction of endothelial apoptosis leads to vessel thrombosis and endothelial denudation: a clue to the understanding of the mechanisms of thrombotic plaque erosion.

BACKGROUND: The mechanisms of thrombosis on plaque erosion are poorly understood. We examined the potential role of endothelial apoptosis in endothelial erosion and vessel thrombosis. METHODS AND RESULTS: Segments of New Zealand White rabbit femoral arteries were temporarily isolated in vivo. One artery was incubated with staurosporin for 30 minutes, whereas the contralateral artery was incubated with saline and served as control. Three days later, thrombosis was evaluated angiographically and histologically. TUNEL score in the endothelial layer was significantly increased in staurosporin-treated arteries compared with controls (2.43+/-0.30 versus 0.93+/-0.44, respectively; P=0.001). Large areas of endothelial denudation were detectable in staurosporin-treated vessels, whereas endothelium integrity was almost preserved in the saline group. Vessel thrombosis occurred in 58% of staurosporin-treated arteries (7 of 12) but in only 8% of saline-treated segments (P<0.01). Immunoreactivities for tissue factor, platelets, and fibrin were detectable within the thrombus. Addition of ZVAD-fmk (0.1 mmol/L) significantly reduced the occurrence of thrombosis (1 of 7 arteries or 14%, P=0.04). These results were confirmed in balloon-injured atheromatous arteries. CONCLUSIONS: In vivo induction of endothelial apoptosis leads to both vessel thrombosis and endothelial denudation. Endothelial apoptosis may be a critical step in the transition from a stable endothelialized plaque to plaque erosion and thrombosis.

Soluble tissue factor induces coagulation on tumor endothelial cells in vivo if coadministered with low-dose lipopolysaccharides.

OBJECTIVE: This study was performed to evaluate the mechanisms leading to tumor vessel occlusion by tissue factor-based drugs, which are used in vascular targeting approaches for the treatment of malignant tumors. METHODS AND RESULTS: The effects of nontargeted soluble tissue factor were evaluated in vitro and in vivo. Tumor-bearing mice were treated with (1) the extracellular portion of tissue factor (soluble tissue factor), (2) low nontoxic doses of lipopolysaccharides, or (3) a combination thereof. The combination treatment showed the best effects and resulted in selective thrombosis of tumor vessels. On the basis of our data from subsequent in vitro analyses, including surface plasmon resonance measurements and endothelial cell based coagulation assays, we propose a model on how soluble tissue factor, although lacking its membrane anchor, can promote selective tumor vessel occlusion. CONCLUSIONS: To our knowledge, this is the first report to describe the molecular mechanisms of coagulation induction by untargeted soluble tissue factor in vivo. Combination treatments including soluble tissue factor might represent an alternative vascular targeting approach for the treatment of malignant tumors.

Inhibition of localized thrombosis in P2Y1-deficient mice and rodents treated with MRS2179, a P2Y1 receptor antagonist.

Previous studies in experimental models revealed a role for the P2Y1 platelet ADP receptor in systemic vascular thromboembolism models. In the present work, we used models of localized arterial and venous thrombosis to assess the role of the P2Y1 receptor in these processes. Arterial thrombosis was induced in one mesenteric arteriole of a mouse using FeCl3, while venous thrombosis was studied in a Wessler model adapted to rats. P2Y1-deficient mice and mice treated with the P2Y1 antagonist MRS2179 displayed significantly less arterial thrombosis than their respective controls. Combination of P2Y1 deficiency with P2Y12 inhibition led to a significant additive effect. Venous thrombosis was slightly but significantly inhibited in MRS2179-treated rats. These results demonstrate a role for the P2Y1 receptor in both arterial and venous thrombosis, further establishing this receptor as a potential target for antithrombotic drugs.

Ultrastructural changes of the tissue thromboplastin after intravenous injection in rabbits.

Attempts were made to demonstrate ultrastructural changes of the tissue thromboplastin after intravenous injection, as a model experiment on the pulmonary microthrombi formation induced by the tissue thromboplastin circulating from venous return. Concentrically arranged membrane structures of the injected thromboplastin disappeared in extremely short time after the injection of the thromboplastin in rabbits. The long sheet membrane of the injected thromboplastin was frequently seen as adhered to the vascular endothelium or to the surface of blood corpuscles. Furthermore, fibrin fibres were formed in contact with the long sheet membrane of the thromboplastin. Membrane structures were not found anywhere in the control rabbits.

The turnover in normal dogs of prothrombin and its fragments; effect of induced intravascular coagulation.

When 125I-labeled canine prothrombin was given to normal adult dogs intravenously, it was calculated that 240% of the plasma prothrombin crossed the capillary barrier per day, 410% of the interstitial prothrombin returned to the blood stream daily, and 79% of the plasmatic prothrombin was catabolized per day. These data are in close agreement with those observed for bovine prothrombin in calves by Takeda (1970). When derived from normal dog prothrombin, prethrombin-1 is a mixture of 2 polypeptides, one larger than the other, and both present in about equal amounts. The longer peptide, "prethrombin-1-long," was catabolized twice as fast as prothrombin, and the shorter, "prethrombin-1-short," 4 times faster. Prothrombin fragment-1 was catabolized by the normal dog still more rapidly. The catabolism of prothrombin was not accelerated in 3 dogs receiving continuous infusions of a thromboplastic emulsion of dog brain. Nor was the level of prothrombin in their plasma remarkably altered.

Induction of vasoactive substances differs in LPS-induced and TF-induced DIC models in rats.

We have investigated the role of two vasoactive substances, nitric oxide (NO)and endothelin (ET), in the pathophysiology of disseminated intravascular coagulation (DIC), using two types of DIC models. Experimental DIC was induced by sustained infusion of 0.1, 1, 10, or 50 mg/kg lipopolysaccharide (LPS), or 3.75 U/kg thromboplastin (TF), for 4 h via the rat tail vein. Plasma levels of both NOX (metabolites of NO) and ET were significantly increased following infusion of 0.1 mg/kg or greater of LPS in the LPS-induced DIC rat model. In contrast, although a marked increase in the plasma levels of NOX was observed, only a slight increase in plasma ET levels was seen in the TF-induced DIC rat model. No significant differences in the plasma levels of platelets or thrombin-ATIII complex were observed among the TF-induced and LPS (50 mg/dl)-induced DIC models. However, plasma NOX levels rose significantly higher in the TF-induced model, relative to the LPS-induced model (p <0.01). Conversely, plasma ET levels were significantly greater after LPS-induction, compared to TF-induction, of DIC (p <0.01). Vasoconstriction, as well as depressed fibrinolytic activity, may be additional factors leading to severe organ dysfunction in the LPS-induced DIC rat model. Moreover, vasodilatation, as well as enhanced fibrinolytic activity, may help to prevent rats from severe organ dysfunction in the TF-induced DIC model. Our results suggest that modulator of vasoactive substances should be examined in the treatment of DIC.

Blockade of GPIIb/IIIa by eptifibatide and tirofiban does not alter tissue factor induced thrombin generation in human endotoxemia.

Activated platelets facilitate thrombin generation by providing a catalytic surface on which coagulation activation occurs. The glycoprotein (GP) IIb/IIIa receptor might play a major role in this process as shown by in vitro and animal experiments. However, it is controversial whether the GPIIb/IIIa receptor facilitates tissue factor-induced thrombin generation in humans as well. We therefore investigated whether two clinically used GPIIb/IIIa antagonists (tirofiban and eptifibatide) may blunt TF-induced coagulation in humans. Thirty male volunteers received 2 ng/kg endotoxin and standard doses of eptifibatide, tirofiban or placebo over 5 hours in a randomized, double-blind, placebo-controlled, double-dummy parallel-group trial. Markers of thrombin generation (prothrombin fragment 1+2, thrombin-antithrombin complexes), fibrinolysis (D-dimer, plasmin-antiplasmin complexes) as well as inflammatory markers (interleukin-6, tumor necrosis factor-alpha) were measured by enzyme linked immunoasssays, TF-mRNA expression was quantified by RT-PCR. Neither eptifibatide nor tirofiban influenced LPS-induced coagulation activation or fibrinolytic activity. Additionally, the increase of TNF-alpha and IL-6 was similar in all groups. In conclusion, GPIIb/IIIa blockade with eptifibatide or tirofiban did not influence TF-induced coagulation activation in human low grade endotoxemia.

Interpreting the International Normalized Ratio (INR) in individuals receiving argatroban and warfarin.

The effects of argatroban, a direct thrombin inhibitor, on the International Normalized Ratio (INR), activated partial thromboplastin time (aPTT) and functional factor X during warfarin co-administration were established to provide means to interpret INRs during argatroban/warfarin co-therapy. Twenty-four subjects receiving warfarin (7.5 mg, day 1; 3-6 mg/day, days 2-10) and argatroban (1-4 microg/kg/min over 5 h, days 1-11) were assessed daily for these coagulation parameters prior to argatroban infusion (warfarin "monotherapy") and at its conclusion ("co-therapy"). Argatroban increased aPTTs dose-dependently. Co-therapy INR increased linearly with monotherapy INR, with slope sensitive to argatroban dose and thromboplastin used. Prediction errors for monotherapy INRs were < or =+/- 0.4 for argatroban 1-2 microg/kg/min but > or = +/-1.0 for higher doses. Despite co-therapy INRs >7, no major bleeding occurred. Factor X remained > or =37% of normal. Therefore, the predictable effect of argatroban (< or =2 microg/kg/min only) [corrected] on INRs during warfarin co-therapy allows for reliable prediction of the level of oral anticoagulation.