Alisa S. Wolberg

The Effect of Temperature and pH on the Activity of Factor VIIa: Implications for the Efficacy of High-Dose Factor VIIa in Hypothermic and Acidotic Patients

BACKGROUND Recombinant coagulation factor VIIa (FVIIa) is approved for treating hemophiliacs with inhibitors. High-dose FVIIa has also been used off-label to manage hemorrhage in trauma and surgical patients, many of whom also develop hypothermia and acidosis. METHODS We examined the activity of FVIIa on phospholipid vesicles in the presence and absence of tissue factor (TF) and on platelets as a function of temperature and pH. RESULTS FVIIa activity on phospholipids and platelets was not reduced at 33 degrees C compared with 37 degrees C. The activity of FVIIa/TF was reduced by 20% at 33 degrees C compared with 37 degrees C. A pH decrease from 7.4 to 7.0 reduced the activity of FVIIa by over 90% and FVIIa/TF by over 60%. CONCLUSION FVIIa should be effective in enhancing hemostasis in hypothermic patients. However, because the activity of FVIIa is so dramatically affected by pH, its efficacy may be reduced in acidotic patients.

Thrombin generation, fibrin clot formation and hemostasis

Hemostatic clot formation entails thrombin-mediated cleavage of fibrinogen to fibrin. Previous in vitro studies have shown that the thrombin concentration present during clot formation dictates the ultimate fibrin structure. In most prior studies of fibrin structure, clotting was initiated by adding thrombin to a solution of fibrinogen; however, clot formation in vivo occurs in an environment in which the concentration of free thrombin changes over the reaction course. These changes depend on local cellular properties and available concentrations of pro- and anti-coagulants. Recent studies suggest that abnormal thrombin generation patterns produce abnormally structured clots that are associated with an increased risk of bleeding or thrombosis. Further studies of fibrin formation during in situ thrombin generation are needed to understand fibrin clot formation in vivo.

Procoagulant Activity in Hemostasis and Thrombosis: Virchow’s Triad Revisited

Virchows triad is traditionally invoked to explain pathophysiologic mechanisms leading to thrombosis, alleging concerted roles for abnormalities in blood composition, vessel wall components, and blood flow in the development of arterial and venous thrombosis. Given the tissue-specific bleeding observed in hemophilia patients, it may be instructive to consider the principles of Virchows triad when investigating mechanisms operant in hemostatic disorders as well. Blood composition (the function of circulating blood cells and plasma proteins) is the most well studied component of the triad. For example, increased levels of plasma procoagulant proteins such as prothrombin and fibrinogen are established risk factors for thrombosis, whereas deficiencies in plasma factors VIII and IX result in bleeding (hemophilia A and B, respectively). Vessel wall (cellular) components contribute adhesion molecules that recruit circulating leukocytes and platelets to sites of vascular damage, tissue factor, which provides a procoagulant signal of vascular breach, and a surface upon which coagulation complexes are assembled. Blood flow is often characterized by 2 key variables: shear rate and shear stress. Shear rate affects several aspects of coagulation, including transport rates of platelets and plasma proteins to and from the injury site, platelet activation, and the kinetics of fibrin monomer formation and polymerization. Shear stress modulates adhesion rates of platelets and expression of adhesion molecules and procoagulant activity on endothelial cells lining the blood vessels. That no one abnormality in any component of Virchows triad fully predicts coagulopathy a priori suggests coagulopathies are complex, multifactorial, and interactive. In this review, we focus on contributions of blood composition, vascular cells, and blood flow to hemostasis and thrombosis, and suggest that cross-talk among the 3 components of Virchows triad is necessary for hemostasis and determines propensity for thrombosis or bleeding. Investigative models that permit interplay among these components are necessary to understand the operant pathophysiology, and effectively treat and prevent thrombotic and bleeding disorders.

High dose factor VIIa improves clot structure and stability in a model of haemophilia B

Factor IX (FIX) deficiency results in haemophilia B and high dose recombinant activated factor VII (rFVIIa) can decrease bleeding. Previously, we showed that FIX deficiency results in a reduced rate and peak of thrombin generation. We have now used plasma and an in vitro coagulation model to examine the effect of these changes in thrombin generation on fibrin clot structure and stability. Low FIX delayed the clot formation onset and reduced the fibrin polymerisation rate. Clots formed without FIX were composed of thicker fibrin fibres than normal. rFVIIa shortened the clot formation onset time and improved the fibre structure of haemophilic clots. We also examined clot formation in the presence of a fibrinolytic challenge by including tissue plasminogen activator or plasmin in the reaction milieu. In these assays, normal FIX levels supported clot formation; however, clots did not form in the absence of FIX. rFVIIa partially restored haemophilic clot formation. These results were independent of the effects of the thrombin‐activatable fibrinolysis inhibitor. Our data suggest that rFVIIa enhances haemostasis in haemophiliacs by increasing the thrombin generation rate to both promote formation of a structurally normal clot and improve clot formation and stability at sites with high endogenous fibrinolytic activities.

Differential Contributions of Monocyte- and Platelet-derived Microparticles towards Thrombin Generation and Fibrin Formation and Stability

Summary.  Background: Microparticles (MPs) are sub‐micron vesicles shed by activated or apoptotic cells, including platelets and monocytes. Increased circulating MPs are associated with thrombosis; however, their role in thrombogenesis is poorly understood. Objective: To determine how MPs promote thrombin generation and modulate fibrin density and stability. Methods: Platelets and monocytes were isolated from healthy donors. Platelets were stimulated with calcium ionophore, thrombin receptor agonist peptide (TRAP) or TRAP/convulxin. Monocytes and human monocytic THP‐1 cells were stimulated with lipopolysaccharide (LPS). MPs were isolated, washed by high‐speed centrifugation and assessed using the following: transmission electron microscopy (TEM), Nanoparticle Tracking Analysis (NTA), flow cytometry, tissue factor (TF) activity, prothrombinase activity, thrombin generation, and clot formation, density and stability. Results: MPs from monocytes (M‐MPs) and platelets (PMPs) had similar shapes and diameters (100–300 nm). M‐MPs had TF activity (16.7 ± 2.4 pm TF per 106 MP), supported prothrombinase activity and triggered shorter thrombin generation lag times than buffer controls (5.4 ± 0.5 vs. 84.2 ± 4.8 min, respectively). Compared with controls, M‐MPs supported faster fibrin formation (0.24 ± 0.24 vs. 76.7 ± 15.1 mOD min−1, respectively), 38% higher fibrin network density and higher clot stability (3.8‐fold higher turbidity in the presence of tissue plasminogen activator). In contrast, PMPs did not have TF activity and supported 2.8‐fold lower prothrombinase activity than M‐MPs. PMPs supported contact‐dependent thrombin generation, but did not independently increase fibrin network density or stability. Interestingly, PMPs increased rates of thrombin generation and fibrin formation (1.7‐ and 1.3‐fold, respectively) when mixed with THP‐1‐derived MPs. Conclusion: MPs from platelets and monocytes differentially modulate clot formation, structure and stability, suggesting unique contributions to thrombosis.

Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability.

Fibrin is essential for hemostasis; however, abnormal fibrin formation is hypothesized to increase thrombotic risk. We previously showed that in situ thrombin generation on a cells surface modulates the 3-dimensional structure and stability of the fibrin network. Currently, we compared the abilities of extravascular and intravascular cells to support fibrin formation, structure, and stability. Extravascular cells (fibroblasts, smooth muscle) supported formation of dense fibrin networks that resisted fibrinolysis, whereas unstimulated intravascular (endothelial) cells produced coarse networks that were susceptible to fibrinolysis. All 3 cell types produced a fibrin structural gradient, with a denser network near, versus distal to, the cell surface. Although fibrin structure depended on cellular procoagulant activity, it did not reflect interactions between integrins and fibrin. These findings contrasted with those on platelets, which influenced fibrin structure via interactions between beta3 integrins and fibrin. Inflammatory cytokines that induced prothrombotic activity on endothelial cells caused the production of abnormally dense fibrin networks that resisted fibrinolysis. Blocking tissue factor activity significantly reduced the density and stability of fibrin networks produced by cytokine-stimulated endothelial cells. Together, these findings indicate fibrin structure and stability reflect the procoagulant phenotype of the endogenous cells, and suggest abnormal fibrin structure is a novel link between inflammation and thrombosis.

Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer.

Cancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In the present study, we analyzed tissue factor (TF) expression in 4 different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that 2 of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti-human TF Ab. Of the 2 TF-positive lines, only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, < 5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor-bearing and control mice in an inferior vena cava stenosis model. The results of the present study using a xenograft mouse model suggest that tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis.

Causal relationship between hyperfibrinogenemia, thrombosis, and resistance to thrombolysis in mice

Epidemiologic studies have correlated elevated plasma fibrinogen (hyperfibrinogenemia) with risk of cardiovascular disease and arterial and venous thrombosis. However, it is unknown whether hyperfibrinogenemia is merely a biomarker of the proinflammatory disease state or is a causative mechanism in the etiology. We raised plasma fibrinogen levels in mice via intravenous infusion and induced thrombosis by ferric chloride application to the carotid artery (high shear) or saphenous vein (lower shear); hyperfibrinogenemia significantly shortened the time to occlusion in both models. Using immunohistochemistry, turbidity, confocal microscopy, and elastometry of clots produced in cell and tissue factor-initiated models of thrombosis, we show that hyperfibrinogenemia increased thrombus fibrin content, promoted faster fibrin formation, and increased fibrin network density, strength, and stability. Hyperfibrinogenemia also increased thrombus resistance to tenecteplase-induced thrombolysis in vivo. These data indicate that hyperfibrinogenemia directly promotes thrombosis and thrombolysis resistance and does so via enhanced fibrin formation and stability. These findings strongly suggest a causative role for hyperfibrinogenemia in acute thrombosis and have significant implications for thrombolytic therapy. Plasma fibrinogen levels may be used to identify patients at risk for thrombosis and inform thrombolytic administration for treating acute thrombosis/thromboembolism.

A Variant of Recombinant Factor VIIa With Enhanced Procoagulant and Antifibrinolytic Activities in an In Vitro Model of Hemophilia

Objective—Recombinant factor VIIa (rFVIIa, NovoSeven) has proven efficacy in treating bleeding in hemophilia patients with inhibitors. A rFVIIa analog with mutations V158D/E296V/M298Q (NN1731) exhibits increased procoagulant activity in in vitro and in vivo models. The aim of this work was to define the effects of NN1731 toward factor X activation, platelet activation, thrombin generation, and fibrin clot formation and stability. Methods and Results—In a cell-based in vitro model of hemophilia, rFVIIa and NN1731 similarly increased factor X activation on tissue factor–bearing cells; however, NN1731 exhibited 30-fold higher factor Xa generation on platelets than similar rFVIIa concentrations. NN1731-mediated thrombin generation depended on platelet activation, but NN1731 did not directly activate platelets. NN1731 produced 4- to 10-fold higher maximal thrombin generation rates than equal rFVIIa concentrations. Both rFVIIa and NN1731 shortened clotting times in the absence of factors IX and VIII; however, NN1731 did so at 50-fold lower concentrations than were required of rFVIIa. In fibrinolytic conditions, both rFVIIa and NN1731 increased fibrin formation and stability; however, NN1731 was effective at 50-fold lower concentrations than were required of rFVIIa. Conclusions—By increasing factor Xa generation, NN1731 promotes the formation of thrombin and a stable clot to a greater degree than rFVIIa.

Factor XIII activity mediates red blood cell retention in venous thrombi

Venous thrombi, fibrin- and rbc-rich clots triggered by inflammation and blood stasis, underlie devastating, and sometimes fatal, occlusive events. During intravascular fibrin deposition, rbc are thought to become passively trapped in thrombi and therefore have not been considered a modifiable thrombus component. In the present study, we determined that activity of the transglutaminase factor XIII (FXIII) is critical for rbc retention within clots and directly affects thrombus size. Compared with WT mice, mice carrying a homozygous mutation in the fibrinogen γ chain (Fibγ390-396A) had a striking 50% reduction in thrombus weight due to reduced rbc content. Fibrinogen from mice harboring the Fibγ390-396A mutation exhibited reduced binding to FXIII, and plasma from these mice exhibited delayed FXIII activation and fibrin crosslinking, indicating these residues mediate FXIII binding and activation. FXIII-deficient mice phenocopied mice carrying Fibγ390-396A and produced smaller thrombi with fewer rbc than WT mice. Importantly, FXIII-deficient human clots also exhibited reduced rbc retention. The addition of FXIII to FXIII-deficient clots increased rbc retention, while inhibition of FXIII activity in normal blood reduced rbc retention and produced smaller clots. These findings establish the FXIII-fibrinogen axis as a central determinant in venous thrombogenesis and identify FXIII as a potential therapeutic target for limiting venous thrombosis.