OKL-1111, A modified cyclodextrin as a potential universal reversal agent for anticoagulants.

BACKGROUND: Antithrombotic therapy is inevitably associated with a risk for bleeding and these bleeding complications can be life-threatening. Recently, specific reversal agents were developed for the direct factor Xa and thrombin inhibitors (DOACs). However, next to the fact that these agents are relatively expensive, the use of selective reversal agents complicates treatment of bleeding patients in practice. In a series of screening experiments, we discovered a class of cyclodextrins with procoagulant properties. In this study we characterize a lead compound, OKL-1111, and demonstrate its potential use as a universal reversal agent. OBJECTIVES: To assess the anticoagulant reversal properties of OKL-1111, in vitro and in vivo. METHODS: The effect of OKL-1111 on coagulation in the absence and presence of DOACs was investigated in a thrombin generation assay. Its reversal effect on a variety of anticoagulants in vivo was investigated in a rat tail cut bleeding model. A possible prothrombotic action of OKL-1111 was assessed in a Wessler model in rabbits. RESULTS: OKL-1111 concentration-dependently reversed the in vitro anticoagulant effects of dabigatran, rivaroxaban, apixaban and edoxaban in the thrombin generation assay. Also in the absence of a DOAC, OKL-1111 concentration-dependently accelerated coagulation in this assay, but did not initiate coagulation. The reversal effect was also seen for all DOACs in the rat tail cut bleeding model. In addition, when tested with other anticoagulants, OKL-1111 also reversed the anticoagulant effect of the vitamin K antagonist warfarin, the low molecular weight heparin enoxaparin, the pentasaccharide fondaparinux and the platelet inhibitor clopidogrel in vivo. OKL-1111 did not have prothrombotic effects in the Wessler model. CONCLUSION: OKL-1111 is a procoagulant cyclodextrin with a currently unknown working mechanism that has potential to become a universal reversal agent for anticoagulants and platelet inhibitors.

Nanobodies against factor XI apple 3 domain inhibit binding of factor IX and reveal a novel binding site for high molecular weight kininogen.

BACKGROUND: Factor XI (FXI) is a promising target for novel anticoagulants because it shows a strong relation to thromboembolic diseases, while fulfilling a mostly supportive role in hemostasis. Anticoagulants targeting FXI could therefore reduce the risk for thrombosis, without increasing the chance of bleeding side effects. OBJECTIVES: To generate nanobodies that can interfere with FXIa mediated activation of factor IX (FIX). METHODS: Nanobodies were selected for binding to the apple 3 domain of FXI and their effects on FXI and coagulation were measured in purified protein systems as well as in plasma-based coagulation assays. Additionally, the binding epitope of selected nanobodies was assessed by hydrogen-deuterium exchange mass spectrometry. RESULTS: We have identified five nanobodies that inhibit FIX activation by FXI by competing with the FIX binding site on FXI. Interestingly, a sixth nanobody was found to target a different binding epitope in the apple 3 domain, resulting in competition with the FXI-high molecular weight kininogen (HK) interaction. CONCLUSIONS: We have characterized a nanobody targeting the FXI apple 3 domain that elucidates the binding orientation of HK on FXI. Moreover, we have produced five nanobodies that can inhibit the FXI-FIX interaction.

Iron-Driven Alterations on Red Blood Cell-Derived Microvesicles Amplify Coagulation during Hemolysis via the Intrinsic Tenase Complex.

Hemolytic disorders characterized by complement-mediated intravascular hemolysis, such as autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived proinflammatory and oxidatively reactive mediators (e.g., extracellular hemoglobin, heme, and iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring factor Xa (FXa) and thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during hemolysis stimulate thrombin generation through a mechanism involving FVIII and FIX, the so-called intrinsic tenase complex. Iron scavenging during hemolysis using deferoxamine decreased the ability of the HMVs to enhance thrombin generation. Furthermore, the addition of ferric chloride (FeCl3) to plasma propagated thrombin generation in a FVIII- and FIX-dependent manner suggesting that iron positively affects blood coagulation. Phosphatidylserine (PS) blockade using lactadherin and iron chelation using deferoxamine reduced intrinsic tenase activity in a purified system containing HMVs as source of phospholipids confirming that both PS and iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl3 and HMVs decreased in the presence of ascorbate and glutathione indicating that oxidative stress plays a role in hypercoagulability. Overall, our results provide evidence for the contribution of iron ions derived from hemolytic RBCs to thrombin generation. These findings add to our understanding of the pathogenesis of thrombosis in hemolytic diseases.

Chemical Footprinting Reveals Conformational Changes Following Activation of Factor XI.

Coagulation factor XI is activated by thrombin or factor XIIa resulting in a conformational change that converts the catalytic domain into its active form and exposing exosites for factor IX on the apple domains. Although crystal structures of the zymogen factor XI and the catalytic domain of the protease are available, the structure of the apple domains and hence the interactions with the catalytic domain in factor XIa are unknown. We now used chemical footprinting to identify lysine residue containing regions that undergo a conformational change following activation of factor XI. To this end, we employed tandem mass tag in conjunction with mass spectrometry. Fifty-two unique peptides were identified, covering 37 of the 41 lysine residues present in factor XI. Two identified lysine residues that showed altered flexibility upon activation were mutated to study their contribution in factor XI stability or enzymatic activity. Lys357, part of the connecting loop between A4 and the catalytic domain, was more reactive in factor XIa but mutation of this lysine residue did not impact on factor XIa activity. Lys516 and its possible interactor Glu380 are located in the catalytic domain and are covered by the activation loop of factor XIa. Mutating Glu380 enhanced Arg369 cleavage and thrombin generation in plasma. In conclusion, we have identified novel regions that undergo a conformational change following activation. This information improves knowledge about factor XI and will contribute to development of novel inhibitors or activators for this coagulation protein.

Effect of Puumala hantavirus infection on human umbilical vein endothelial cell hemostatic function: platelet interactions, increased tissue factor expression and fibrinolysis regulator release.

Puumala virus (PUUV) infection causes over 5000 cases of hemorrhagic fever in Europe annually and can influence the hemostatic balance extensively. Infection might lead to hemorrhage, while a recent study showed an increased risk of myocardial infarction during or shortly after PUUV infection. The mechanism by which this hantavirus influences the coagulation system remains unknown. Therefore we aimed to elucidate mechanisms explaining alterations seen in primary and secondary hemostasis during PUUV infection. By using low passage PUUV isolates to infect primary human umbilical vein endothelial cells (HUVECs) we were able to show alterations in the regulation of primary- and secondary hemostasis and in the release of fibrinolysis regulators. Our main finding was an activation of secondary hemostasis due to increased tissue factor (TF) expression leading to increased thrombin generation in a functional assay. Furthermore, we showed that during infection platelets adhered to HUVEC and subsequently specifically to PUUV virus particles. Infection of HUVEC with PUUV did not result in increased von Willebrand factor while they produced more plasminogen activator inhibitor type-1 (PAI-1) compared to controls. The PAI-1 produced in this model formed complexes with vitronectin. This is the first report that reveals a potential mechanism behind the pro-coagulant changes in PUUV patients, which could be the result of increased thrombin generation due to an increased TF expression on endothelial cells during infection. Furthermore, we provide insight into the contribution of endothelial cell responses regarding hemostasis in PUUV pathogenesis.

A novel mutation in the F5 gene (factor V Amsterdam) associated with bleeding independent of factor V procoagulant function.

We investigated a small Dutch family with a bleeding diathesis, prolonged prothrombin, and activated partial thromboplastin times, in whom no classifying diagnosis was made. The 2 affected relatives had severely decreased in vitro thrombin generation, and levels of tissue factor pathway inhibitor (TFPI) were strongly increased. To identify the genetic cause of the bleeding diathesis, we performed whole exome sequencing analysis of all living relatives. We found a novel gain-of-function mutation in the F5 gene (c.C2588G), which leads to an aberrant splicing of F5 and ultimately to a short factor V protein (missing 623 amino acids from the B domain), which we called factor V Amsterdam. Factor V Amsterdam binds to TFPI, prolonging its half-life and concentration. This is the second report of an association between a shorter form of factor V and increased TFPI levels, resulting in severely reduced thrombin generation and a bleeding tendency.

Activation of coagulation and tissue fibrin deposition in experimental influenza in ferrets.

BACKGROUND: Epidemiological studies relate influenza infection with vascular diseases like myocardial infarction. The hypothesis that influenza infection has procoagulant effects on humans has been investigated by experimental animal models. However, these studies often made use of animal models only susceptible to adapted influenza viruses (mouse adapted influenza strains) or remained inconclusive. Therefore, we decided to study the influence of infection with human influenza virus isolates on coagulation in the well-established ferret influenza model. RESULTS: After infection with either a seasonal-, pandemic- or highly pathogenic avian influenza (HPAI-H5N1) virus strain infected animals showed alterations in hemostasis compared to the control animals. Specifically on day 4 post infection, a four second rise in both PT and aPTT was observed. D-dimer concentrations increased in all 3 influenza groups with the highest concentrations in the pandemic influenza group. Von Willebrand factor activity levels increased early in infection suggesting endothelial cell activation. Mean thrombin-antithrombin complex levels increased in both pandemic and HPAI-H5N1 virus infected ferrets. At tissue level, fibrin staining showed intracapillary fibrin deposition especially in HPAI-H5N1 virus infected ferrets. CONCLUSION: This study showed hemostatic alterations both at the circulatory and at the tissue level upon infection with different influenza viruses in an animal model closely mimicking human influenza virus infection. Alterations largely correlated with the severity of the respective influenza virus infections.

Clot lysis phenotype and response to recombinant factor VIIa in plasma of haemophilia A inhibitor patients.

Recombinant activated factor VII (rFVIIa) is a haemostatic agent that is used for the treatment of haemophilia A patients with inhibitors. However, clinical response to rFVIIa is variable and unpredictable with currently available assays. We investigated the anti-fibrinolytic effects of rFVIIa in relation to thrombin generation (TG) and other haemostatic parameters in haemophilia A patients with inhibitors. After addition of rFVIIa to plasma, the clot-lysis assay, TF-dependent TG, TF-independent TG and parameters involved in coagulation, anticoagulation and fibrinolysis were assessed. The clot-lysis test distinguished two groups of patients: a group with a normal and a group with impaired anti-fibrinolytic response to rFVIIa. Our results showed a dose-dependent increase in TF-dependent TG and TF-independent TG in all individuals. There was a significant difference in TF-independent TG parameters between the normal and impaired response groups. In addition, there was a difference between the normal and impaired response group in prothrombin time, which could be explained by significantly higher levels of coagulation factors in the normal response group, and soluble thrombomodulin. In conclusion, we observed different in vitro responses following rFVIIa addition in plasma of patients with haemophilia A and inhibitors, which could be partially attributed to levels of procoagulant proteins and soluble thrombomodulin.

Factor Xa activation of factor V is of paramount importance in initiating the coagulation system: lessons from a tick salivary protein.

BACKGROUND: Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. METHODS AND RESULTS: Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5. CONCLUSIONS: Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.

Recombinant C1-inhibitor: effects on coagulation and fibrinolysis in patients with hereditary angioedema.

BACKGROUND: Recombinant human C1-inhibitor (rhC1INH; Ruconest®) has been developed for treatment of acute angioedema attacks in patients with hereditary angioedema (HAE) due to heterozygous deficiency of C1INH. Previous reports suggest that administration of plasma-derived C1INH products may be associated with an increased risk for thromboembolic complications. OBJECTIVES: Our aim is to evaluate the effects of rhC1INH on coagulation and fibrinolysis in symptomatic HAE patients. METHODS: Levels of various coagulation and fibrinolytic parameters were determined in pre- and post-exposure plasma samples from HAE patients included in a randomized clinical trial. Patients were treated with either saline, or 50 or 100 U/kg rhC1INH for an acute angioedema attack. RESULTS: Prior to rhC1INH treatment, the majority of patients had low to normal activated partial thromboplastin times (aPTT) and increased levels of prothrombin fragment 1+2, thrombin-antithrombin complexes, D-dimers and plasmin-antiplasmin complexes, all of which indicate activation of both coagulation and fibrinolysis. Infusion of rhC1INH at doses up to 100 U/kg did not affect these parameters except for a dose-dependent prolongation of aPTT, confirming that rhC1INH is an inhibitor of the contact system, and that F1+2 levels decreased. CONCLUSION: Coagulation and fibrinolytic systems are activated in HAE patients suffering from an acute angioedema attack. Treatment with rhC1INH at 50 or 100 U/kg had no effect on parameters reflecting activation of these systems except for a significant effect on aPTT, which likely reflects a pharmacodynamic effect of rhC1INH, and a reduction on plasma levels of the prothrombin activation fragment F1+2. We conclude that these results argue against a prothrombotic effect of treatment with this rhC1INH product in HAE patients.

Identification and characterization of Ixodes scapularis antigens that elicit tick immunity using yeast surface display.

Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary antigens that react with tick-immune serum. We constructed an Ixodes scapularis nymphal salivary gland yeast surface display library and screened the library with nymph-immune rabbit sera and identified five salivary antigens. Four of these proteins, designated P8, P19, P23 and P32, had a predicted signal sequence. We generated recombinant (r) P8, P19 and P23 in a Drosophila expression system for functional and immunization studies. rP8 showed anti-complement activity and rP23 demonstrated anti-coagulant activity. Ixodes scapularis feeding was significantly impaired when nymphs were fed on rabbits immunized with a cocktail of rP8, rP19 and rP23, a hall mark of tick-immunity. These studies also suggest that these antigens may serve as potential vaccine candidates to thwart tick feeding.

Activated factor V is a cofactor for the activation of factor XI by thrombin in plasma.

The mechanism by which the intrinsic pathway of coagulation contributes to physiological hemostasis is enigmatic. Thrombin activates factor XI, a key zymogen in this pathway, which leads to increased thrombin generation. As thrombin-dependent activation of factor XI in vitro is relatively inefficient, we hypothesized that a physiological cofactor supports this reaction in a plasma environment. We therefore investigated whether the cofactors of coagulation, activated factor V, activated factor VIII, high-molecular weight kininogen, or protein S, influenced activation of factor XI by thrombin. Only activated factor V stimulated activation of factor XI by thrombin in a purified system. Binding studies demonstrated that factor XI specifically interacts with both factor V and factor Va through multiple binding sites. We further investigated this cofactor function of activated factor V in plasma. Depletion of factor V, or the addition of activated protein C, decreased the activation of the intrinsic pathway by thrombin in plasma. However, activated protein C did not exert this effect in the plasma of a homozygous carrier of the prothrombotic factor V Leiden mutation. In conclusion, we propose a role for (activated) factor V as a cofactor in the activation of factor XI by thrombin. These findings offer insights into the coagulation system in both health and disease.

Normal to increased thrombin generation in patients undergoing liver transplantation despite prolonged conventional coagulation tests.

BACKGROUND & AIMS: Patients with liver disease often show substantial changes in their hemostatic system, which may aggravate further during liver transplantation. Recently, thrombin generation in patients with stable disease was shown to be indistinguishable from controls provided thrombomodulin, the natural activator of the anticoagulant protein C system, was added to the plasma. These results indicated that the hemostatic balance is preserved in patients with liver disease, despite conventional coagulation tests suggest otherwise. METHODS: Here we examined thrombin generation profiles in serial plasma samples taken from ten consecutive patients undergoing liver transplantation. RESULTS: At all time points, the endogenous thrombin potential (ETP) was slightly lower compared to healthy volunteers, despite substantially prolonged PT and APTT values. However, when thrombin generation was tested in the presence of thrombomodulin, the ETP was equal to or even higher than that in healthy subjects. In fact, thrombin generation was hardly affected by thrombomodulin, while thrombin generation in healthy subjects decreased profoundly upon the addition of thrombomodulin. In patients undergoing liver transplantation, efficient thrombin generation in the presence of thrombomodulin may be explained by decreased levels of protein C, S, and antithrombin, and by elevated levels of FVIII. CONCLUSIONS: Thrombin generation in patients undergoing liver transplantation is equal or even superior to thrombin generation in healthy volunteers when tested in the presence of exogenous thrombomodulin. These results support the recently advocated restrictive use of plasma during liver transplantation and warrants further study of the prophylactic use of anticoagulants to reduce thromboembolic complications after transplantation.

Reduced ADAMTS13 activity in delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage.

The pathogenesis of delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (SAH) remains unknown. Besides vasospasm, microthrombosis might have an important function. As in patients with thrombotic thrombocytopenic purpura an A Disintegrin And Metalloprotease with ThromboSpondin repeats-13 (ADAMTS13) deficiency leads to higher concentrations of large von Willebrand factor (vWF) multimers resulting in microthrombosis, our purpose was to compare ADAMTS13 and vWF in patients with and without DCI after aneurysmal SAH. We measured ADAMTS13 activity, vWF antigen, vWF propeptide, and vWF ristocetin cofactor activity in plasma at standard intervals. Thirty-one patients were included. Eleven patients (35%) developed DCI. No differences were observed in baseline characteristics between patients with and without DCI. Patients with DCI had a stronger decrease in ADAMTS13 activity, and a more profound increase in vWF antigen, vWF propeptide, and vWF activity in the first few days after the hemorrhage (P-values for difference in polynomial time trend 0.0001, 0.020, 0.004, and 0.188, respectively). No indication of correlation between vWF antigen and ADAMTS13 was found (r=-0.027, P=0.736). Our results suggest that microthrombosis has a role in the pathogenesis of DCI, as a result of decreased ADAMTS13 activity and endothelium dysfunction.

Ethyl pyruvate exerts combined anti-inflammatory and anticoagulant effects on human monocytic cells.

Sepsis is characterized by a concurrent activation of inflammation and coagulation. Recently, recombinant human activated protein C was shown to decrease mortality in patients with severe sepsis presumably due to a combined anti-inflammatory and anticoagulant effect. These promising findings led to a search for other products that influence both the inflammatory and the procoagulant response to severe infection. Ethyl pyruvate (EP) was recently identified as an experimental anti-inflammatory agent during endotoxemia and sepsis. The aim of the present study was to investigate whether EP influences coagulation besides its anti-inflammatory effects. For this we investigated the effects of EP on the expression and function of tissue factor (TF), the principal initiator of coagulation activation in sepsis, in human monocytic (THP-1) cell cultures. EP dose-dependently inhibited the production of tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta by lipopolysaccharide (LPS)-stimulated THP-1 cells at mRNA and protein level, thereby confirming its anti-inflammatory properties in this in-vitro system. In addition, EP dose-dependently attenuated the increases in TF mRNA levels, TF-protein-surface expression and cell-surface-associated TF activity in LPS-stimulated THP-1 cells. These results demonstrate for the first time that EP is a compound with combined anti-inflammatory and anticoagulant effects.

The effects of continuous venovenous hemofiltration on coagulation activation.

INTRODUCTION: The mechanism of coagulation activation during continuous venovenous hemofiltration (CVVH) has not yet been elucidated. Insight into the mechanism(s) of hemostatic activation within the extracorporeal circuit could result in a more rational approach to anticoagulation. The aim of the present study was to investigate whether CVVH using cellulose triacetate filters causes activation of the contact factor pathway or of the tissue factor pathway of coagulation. In contrast to previous studies, CVVH was performed without anticoagulation. METHODS: Ten critically ill patients were studied prior to the start of CVVH and at 5, 15 and 30 minutes and 1, 2, 3 and 6 hours thereafter, for measurement of prothrombin fragment F1+2, soluble tissue factor, activated factor VII, tissue factor pathway inhibitor, kallikrein-C1-inhibitor and activated factor XII-C1-inhibitor complexes, tissue-type plasminogen activator, plasminogen activator inhibitor type I, plasmin-antiplasmin complexes, protein C and antithrombin. RESULTS: During the study period the prothrombin fragment F1+2 levels increased significantly in four patients (defined as group A) and did not change in six patients (defined as group B). Group A also showed a rapid increase in transmembrane pressure, indicating clotting within the filter. At baseline, the activated partial thromboplastin time, the prothrombin time and the kallikrein-C1-inhibitor complex and activated factor XII-C1-inhibitor complex levels were significantly higher in group B, whereas the platelet count was significantly lower in group B. For the other studied markers the differences between group A and group B at baseline were not statistically significant. During CVVH the difference in the time course between group A and group B was not statistically significant for the markers of the tissue factor system (soluble tissue factor, activated factor VII and tissue factor pathway inhibitor), for the markers of the contact system (kallikrein-C1-inhibitor and activated factor XII-C1-inhibitor complexes) and for the markers of the fibrinolytic system (plasmin-antiplasmin complexes, tissue-type plasminogen activator and plasminogen activator inhibitor type I). CONCLUSION: Early thrombin generation was detected in a minority of intensive care patients receiving CVVH without anticoagulation. Systemic concentrations of markers of the tissue factor system and of the contact system did not change during CVVH. To elucidate the mechanism of clot formation during CVVH we suggest that future studies are needed that investigate the activation of coagulation directly at the site of the filter. Early coagulation during CVVH may be related to lower baseline levels of markers of contact activation.

Predilution versus postdilution during continuous venovenous hemofiltration: a comparison of circuit thrombogenesis.

During continuous venovenous hemofiltration, predilution can prolong circuit survival time, but the underlying mechanism has not been elucidated. The aim of the present study was to compare predilution with postdilution, with respect to circuit thrombogenesis. Eight critically ill patients were treated with both predilutional and postdilutional continuous venovenous hemofiltration in a crossover fashion. A filtration flow of 60 ml/min was used in both modes. We chose blood flows of 140 and 200 ml/min during predilution and postdilution, respectively, to keep the total flow through the hemofilter constant. Extracorporeal circuit pressures were measured hourly, and samples of blood and ultrafiltrate were collected at five different time points. Thrombin-antithrombin complexes and prothrombin fragments F1 + 2 were measured by ELISA, and platelet activation was assessed by flow cytometry. No signs of thrombin generation or platelet activation were found during either mode. During postdilution, baseline platelet count and maximal prefilter pressure had a linear relation, whereas both parameters were inversely related with circuit survival time. In summary, predilution and postdilution did not differ with respect to extracorporeal circuit thrombogenesis. During postdilution, baseline platelet count and maximal prefilter pressure were inversely related with circuit survival time.

Endotoxaemia induces resistance to activated protein C in healthy humans.

Systemic inflammation activates the tissue factor/factor VIIa complex (TF/FVIIa), leading to a procoagulant state, which may be enhanced by impairment of physiological anticoagulant pathways, such as the protein C system. Besides impaired protein C activation, resistance to activated protein C (APC) may occur. We studied the effect of endotoxemia on APC resistance, analysed its determinants and evaluated the effect of TF/FVIIa inhibition on endotoxin-induced APC resistance. Sixteen healthy male volunteers participated in the study, eight receiving endotoxin alone and eight receiving the combination of endotoxin and recombinant Nematode Anticoagulant Protein c2 (rNAPc2), a potent inhibitor of TF/FVIIa. Parameters of coagulation were subsequently studied. The sensitivity to APC was determined by two tests: a test based on the endogenous thrombin potential and a test based on the activated partial thromboplastin time. In response to endotoxemia, both tests detected a transient APC resistance that was predominantly mediated by an increase in factor VIII and was not influenced by TF/FVIIa inhibition. In vitro tests confirmed that an increase in factor VIII induced APC resistance, as measured by both tests. This finding suggests that APC resistance might play a role in the procoagulant state occurring during human endotoxemia.