Conformation of the von Willebrand factor/factor VIII complex in quasi-static flow.

Von Willebrand factor (VWF) is a plasma glycoprotein that circulates noncovalently bound to blood coagulation factor VIII (fVIII). VWF is a population of multimers composed of a variable number of ∼280 kDa monomers that is activated in shear flow to bind collagen and platelet glycoprotein Ibα. Electron microscopy, atomic force microscopy, small-angle neutron scattering, and theoretical studies have produced a model in which the conformation of VWF under static conditions is a compact, globular "ball-of-yarn," implying strong, attractive forces between monomers. We performed sedimentation velocity (SV) analytical ultracentrifugation measurements on unfractionated VWF/fVIII complexes. There was a 20% per mg/ml decrease in the weight-average sedimentation coefficient, sw, in contrast to the ∼1% per mg/ml decrease observed for compact globular proteins. SV and dynamic light scattering measurements were performed on VWF/fVIII complexes fractionated by size-exclusion chromatography to obtain sw values and z-average diffusion coefficients, Dz. Molecular weights estimated using these values in the Svedberg equation ranged from 1.7 to 4.1 MDa. Frictional ratios calculated from Dz and molecular weights ranged from 2.9 to 3.4, in contrast to values of 1.1-1.3 observed for globular proteins. The Mark-Houwink-Kuhn-Sakurada scaling relationships between sw, Dz and molecular weight, [Formula: see text] and [Formula: see text] , yielded estimates of 0.51 and -0.49 for as and aD, respectively, consistent with a random coil, in contrast to the as value of 0.65 observed for globular proteins. These results indicate that interactions between monomers are weak or nonexistent and that activation of VWF is intramonomeric.

COVID-19 Associated Hypercoagulability: Manifestations, Mechanisms, and Management.

ABSTRACT: Patients with severe coronavirus disease-2019 (COVID-19) frequently have hypercoagulability caused by the immune response to the severe acute respiratory syndrome coronavirus-2 infection. The pathophysiology of COVID-19 associated hypercoagulability is not fully understood, but characteristic changes include: increased fibrinogen concentration, increased Factor VIII activity, increased circulating von Willebrand factor, and exhausted fibrinolysis. Anticoagulant therapy improves outcomes in mechanically ventilated patients with COVID-19 and viscoelastic coagulation testing offers an opportunity to tailor anticoagulant therapy based on an individual patient's coagulation status. In this narrative review, we summarize clinical manifestations of COVID-19, mechanisms, monitoring considerations, and anticoagulant therapy. We also review unique considerations for COVID-19 patients who are on extracorporeal membrane oxygenation.

Prevention of the anti-factor VIII memory B-cell response by inhibition of Bruton tyrosine kinase in experimental hemophilia A.

Hemophilia A is a rare hemorrhagic disorder caused by the lack of functional pro-coagulant factor VIII. Factor VIII replacement therapy in patients with severe hemophilia A results in the development of inhibitory anti-factor VIII IgG in up to 30% of cases. To date, immune tolerance induction, with daily injection of large amounts of factor VIII, is the only strategy to eradicate factor VIII inhibitors. This strategy is, however, efficient in only 60-80% of patients. We investigated whether blocking B-cell receptor signaling upon inhibition of Bruton tyrosine kinase prevents anti-factor VIII immune responses in a mouse model of severe hemophilia A. Factor VIII-naïve and factor VIII-sensitized factor VIII-deficient mice were fed with the selective inhibitor of Bruton tyrosine kinase, (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxyl] phenyl)-1H pyrazole-4-carboxamide (PF-06250112), to inhibit B-cell receptor signaling prior to challenge with exogenous factor VIII. The consequences on the anti-factor VIII immune response were studied. Inhibition of Bruton tyrosine kinase during the primary anti-factor VIII immune response in factor VIII-naïve mice did not prevent the development of inhibitory anti-factor VIII IgG. In contrast, the anti-factor VIII memory B-cell response was consistently reduced upon treatment of factor VIII-sensitized mice with the Bruton tyrosine kinase inhibitor. The Bruton tyrosine kinase inhibitor reduced the differentiation of memory B cells ex vivo and in vivo following adoptive transfer to factor VIII-naïve animals. Taken together, our data identify inhibition of Bruton tyrosine kinase using PF-06250112 as a strategy to limit the reactivation of factor VIII-specific memory B cells upon re-challenge with therapeutic factor VIII.

Long-term correction of hemophilia A mice following lentiviral mediated delivery of an optimized canine factor VIII gene.

Current therapies for hemophilia A include frequent prophylactic or on-demand intravenous factor treatments which are costly, inconvenient and may lead to inhibitor formation. Viral vector delivery of factor VIII (FVIII) cDNA has the potential to alleviate the debilitating clotting defects. Lentiviral-based vectors delivered to murine models of hemophilia A mediate phenotypic correction. However, a limitation of lentiviral-mediated FVIII delivery is inefficient transduction of target cells. Here, we engineer a feline immunodeficiency virus (FIV) -based lentiviral vector pseudotyped with the baculovirus GP64 envelope glycoprotein to mediate efficient gene transfer to mouse hepatocytes. In anticipation of future studies in FVIII-deficient dogs, we investigated the efficacy of FIV-delivered canine FVIII (cFVIII). Codon-optimization of the cFVIII sequence increased activity and decreased blood loss as compared to the native sequence. Further, we compared a standard B-domain deleted FVIII cDNA to a cDNA including 256 amino acids of the B-domain with 11 potential asparagine-linked oligosaccharide linkages. Restoring a partial B-domain resulted in modest reduction of endoplasmic reticulum (ER) stress markers. Importantly, our optimized vectors achieved wild-type levels of phenotypic correction with minimal inhibitor formation. These studies provide insights into optimal design of a therapeutically relevant gene therapy vector for a devastating bleeding disorder.

The role of ultralarge multimers in recombinant human von Willebrand factor - a review of physico-and biochemical studies and findings in in vivo models and in humans with von Willebrand disease.

Ultralarge multimers (ULM) of VWF are considered to be the most active with respect to binding to platelets and to subendothelial structures and therefore are of critical importance for the function of VWF in stabilizing the primary hemostatic plug. In contrast to plasma-derived FVIII-VWF concentrates, human rVWF obtained from mammalian cell culture retains the full-spectrum of intact multimers, including ULM, as physiologically formed in the Golgi apparatus and stored in platelet α-granules and endothelial cell Weibel-Palade bodies. In the course of physico and biochemical, functional and animal studies, rVWF exhibited superiority in structure and function compared to pdVWF. These effects seemed to correlate with the multimer size and therefore might be attributed to the presence of ULM in rVWF preparations. The pharmacokinetic (PK), safety and efficacy characteristics seen in preclinical studies were further demonstrated in clinical trials.

Discoidin Domains as Emerging Therapeutic Targets.

Discoidin (DS) domains are found in eukaryotic and prokaryotic extracellular and transmembrane multidomain proteins. These small domains play different functional roles and can interact with phospholipids, glycans, and proteins, including collagens. DS domain-containing proteins are often involved in cellular adhesion, migration, proliferation, and matrix-remodeling events, while some play a major role in blood coagulation. Mutations in DS domains have been associated with various disease conditions. This review provides an update on the structure, function, and modulation of the DS domains, with a special emphasis on two circulating blood coagulation cofactors, factor V and factor VIII, and the transmembrane neuropilin receptors that have been targeted for inhibition by biologics and small chemical compounds.

The factor VIII protein and its function.

Factor VIII (FVIII), an essential blood coagulation protein, is a key component of the fluid phase blood coagulation system. Human factor VIII is a single chain of about 300 kDa consisting of domains described as A1-A2-B-A3-C1-C2. The protein undergoes processing prior to secretion into blood resulting in a heavy chain of 200 kDa (A1-A2-B) and a light chain of 80 kDa (A3-C1-C2) linked by metal ions. The role of factor VIII is to increase the catalytic efficiency of factor IXa in the activation of factor X. Variants of these factors lead frequently also to severe bleeding disorders.

[Acquired haemophilia A: two cases]. / Deux observations d'hémophilie A acquise.

BACKGROUND: Acquired haemophilia A (AH) is an uncommon bleeding disorder that presents as multiple, disseminated spontaneous subcutaneous bleeds. Diagnosis may be made on the basis of prolonged activated partial thromboplastin time (aPTT). The severity of the disease is associated with the low risk of haemoglobin levels and with potential links with other diseases. OBSERVATIONS: Two men were hospitalized for extensive and spontaneous subcutaneous hematoma. In both cases, the International Normalized Ratio (INR) was normal, but aPTT was 3 times higher than normal. Autoantibodies against coagulation factor VIII confirmed the diagnosis of AH. The patients received immunomodulatory treatment. In one patient, diffuse large B-cell lymphoma was discovered one year after successful treatment of AH. DISCUSSION: AH may be revealed by areas of bruising, subutaneous haematomas mimicking erythema nodosum, and muscle pain. APTT results alone can prompt the biologist to screen for factor VIII inhibitors. Aside from the risk of fatal bleeding, in half of all cases, the prognosis is determined by associated disorders such as blood dyscrasias, solid tumours, autoimmune diseases, use of certain medicines and pregnancy. After treatment for bleeding complications, therapy focuses on restoring the coagulation time. The aim of immunomodulatory therapy is to stem production of autoantibodies against coagulation factor VIII. CONCLUSION: AH must be considered rapidly in order to reduce the risk of bleeding emergencies and to screen for potential related diseases.

An update on the coagulopathy of trauma.

Trauma remains the leading cause of death with bleeding as the primary cause of preventable mortality during the first 24 h following trauma. When death occurs, it happens quickly, typically within the first 6 h after injury. One of four patients to arrive in the emergency department after trauma is already in the state of acute traumatic coagulopathy and shock. The principal drivers of acute traumatic coagulopathy have been characterized by tissue hypoperfusion, inflammation, and the acute activation of the neurohumoral system. Hypoperfusion leads to an activation of protein C with cleavage of activated factors V and VIII and the inhibition of plasminogen activator inhibitor 1 with subsequent hyperfibrinolysis. Endothelial damage and activation result in Weibel-Palade body degradation and glycocalyx shedding associated with autoheparinization. In contrast, there is an iatrogenic coagulopathy that occurs secondary to uncritical volume therapy leading to acidosis, hypothermia, and hemodilution. This coagulopathy then may be an integral part of the "vicious cycle" when combined with acidosis and hypothermia. The present article summarizes an update on the principal mechanisms and triggers of the coagulopathy of trauma including traumatic brain injury.

[Reference values for blood coagulation factor activity in the Mexican population].

INTRODUCTION: During the fluid phase of hemostasis, fibrinogen is converted into fibrin, but other hemostatic factors are required. Reference values of hemostatic factors are established by manufacturers producing reagents using individuals with a specific genetic background. OBJECTIVE: To establish reference values for hemostatic factors in the Mexican indigenous and Mestizo populations. MATERIAL AND METHODS: We carried out a cross-sectional, descriptive study of healthy adult Mexicans. Clotting activity was evaluated using coagulometric assays. Blood donors were informed about the nature of the study and informed consent was obtained prior to blood being drawn. The protocol was approved by the Ethics Committee of our institution. RESULTS: One hundred and twenty samples were assayed (60 females and 60 males). Fibrinogen was higher in mestizos and in females. Reference values for factor XII ranged from 40-170% in indigenous subjects and from 36-159% in mestizos. Factor VIII ranged from 57-160% in indigenous subjects and from 51-209% in mestizo subjects. Reference values for the other hemostatic factors were also clearly different from the commercial reference values. Reference values for hemostatic factors in the Mexican population are different from traditionally used commercial reference values. There were significant differences between indigenous and mestizo Mexicans in the concentration of hemostatic factors with a tendency among mestizos to have higher factor concentrations. Low levels of plasma factor XII are frequent and perhaps may represent a risk factor for thrombotic events. Using these reference values may individualize the reposition of factors in Mexican hemophiliac patients.

A novel role for factor VIII and thrombin/PAR1 in regulating hematopoiesis and its interplay with the bone structure.

Analysis of hematopoietic stem cells (HSCs) in factor VIII knockout (FVIIIKO) mice revealed a novel regulatory role for the coagulation cascade in hematopoiesis. Thus, HSCs in FVIIIKO mice had reduced proportions of CD34(low) cells within Lin(-)Sca(+)Kit(+) progenitors, and exhibited reduced long-term repopulating capacity as well as hyper granulocyte-colony-stimulating factor (G-CSF)-induced mobilization. This disregulation of HSCs is likely caused by reduced levels of thrombin, and is associated with altered protease-activated receptor 1 (PAR1) signaling, as PAR1 KO mice also exhibited enhanced G-CSF-induced mobilization. Analysis of reciprocal bone marrow (BM) chimera (FVIIIKO BM into wild-type recipients and vice versa) and the detection of PAR1 expression on stromal elements indicates that this phenotype is likely controlled by stromal elements. Micro-computed tomography analysis of distal tibia metaphyses also revealed for the first time a major impact of the FVIII/thrombin/PAR1 axis on the dynamic bone structure, showing reduced bone:tissue volume ratio and trabecular number in FVIIIKO and PAR1KO mice. Taken together, these results show a critical and novel role for the coagulation cascade, mediated in part by thrombin-PAR1 interaction, and regulates HSC maintenance and a reciprocal interplay between HSCs and the dynamic bone structure.

Coagulation potential of immobilised factor VIII in flow-dependent fibrin generation on platelet surfaces.

Coagulation factor VIII (FVIII) plays an essential role in haemostasis. To date, physiologic activity of FVIII circulating in the bloodstream (S-FVIII) is evaluated by classic coagulation assays. However, the functional relevance of FVIII (-von Willebrand factor complex) immobilised on thrombogenic surfaces (I-FVIII) remains unclear. We used an in vitro perfusion chamber system to evaluate the function of I-FVIII in the process of mural thrombus formation under whole blood flow conditions. In perfusion of either control or synthetic haemophilic blood, the intra-thrombus fibrin generation on platelet surfaces significantly increased as a function of I-FVIII, independent of S-FVIII, under high shear rate conditions. This I-FVIII effect was unvarying regardless of anti-FVIII inhibitor levels in synthetic haemophilic blood. Thus, our results illustrate coagulation potentials of immobilised clotting factors, distinct from those in the bloodstream, under physiologic flow conditions and may give a clue for novel therapeutic approaches for haemophilic patients with anti-FVIII inhibitors.

Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity.

In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients.

A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model.

Hemophilia A is a bleeding disorder resulting from coagulation factor VIII (FVIII) deficiency. Exogenously provided FVIII effectively reduces bleeding complications in patients with severe hemophilia A. In approximately 30% of such patients, however, the 'foreignness' of the FVIII molecule causes them to develop inhibitory antibodies against FVIII (inhibitors), precluding FVIII treatment in this set of patients. Moreover, the poor pharmacokinetics of FVIII, attributed to low subcutaneous bioavailability and a short half-life of 0.5 d, necessitates frequent intravenous injections. To overcome these drawbacks, we generated a humanized bispecific antibody to factor IXa (FIXa) and factor X (FX), termed hBS23, that places these two factors into spatially appropriate positions and mimics the cofactor function of FVIII. hBS23 exerted coagulation activity in FVIII-deficient plasma, even in the presence of inhibitors, and showed in vivo hemostatic activity in a nonhuman primate model of acquired hemophilia A. Notably, hBS23 had high subcutaneous bioavailability and a 2-week half-life and would not be expected to elicit the development of FVIII-specific inhibitory antibodies, as its molecular structure, and hence antigenicity, differs from that of FVIII. A long-acting, subcutaneously injectable agent that is unaffected by the presence of inhibitors could markedly reduce the burden of care for the treatment of hemophilia A.

A major determinant of the immunogenicity of factor VIII in a murine model is independent of its procoagulant function.

A main complication of treatment of patients with hemophilia A is the development of anti-factor VIII (fVIII) antibodies. The immunogenicity of fVIII potentially is a function of its procoagulant activity, which may result in danger signals that drive the immune response. Alternatively, intrinsic structural elements in fVIII may be particularly immunogenic. Finally, VWF, the carrier protein for fVIII in plasma, may play a role in immune recognition. We compared the immunogenicity of wild-type (wt) B domain-deleted fVIII and 2 inactive fVIII molecules, R372A/R1689A fVIII and V634M fVIII in fVIII(-/-) and fVIII(-/-)/VWF(-/-) mice. R372A/R1689A fVIII lacks proteolytic recognition sites and is not released from VWF. In contrast, V634M fVIII undergoes proteolytic cleavage and dissociation from VWF. No significant difference was observed in the immunogenicity of wt fVIII and V634M fVIII. R372A/R1689A fVIII was slightly less immunogenic in a subset of immunization regimens tested. High doses of wt fVIII were required to produce an immune response in fVIII(-/-)/VWF(-/-) mice. Our results indicate that a main component of the immune response to fVIII is independent of its procoagulant function, is both positively and negatively affected by its association with VWF, and may involve intrinsic elements of fVIII structure.

Engineered factor IX variants bypass FVIII and correct hemophilia A phenotype in mice.

The complex of the serine protease factor IX (FIX) and its cofactor, factor VIII (FVIII), is crucial for propagation of the intrinsic coagulation cascade. Absence of either factor leads to hemophilia, a disabling disorder marked by excessive hemorrhage after minor trauma. FVIII is the more commonly affected protein, either by X-chromosomal gene mutations or in autoimmune-mediated acquired hemophilia. Whereas substitution of FVIII is the mainstay of hemophilia A therapy, treatment of patients with inhibitory Abs remains challenging. In the present study, we report the development of FIX variants that can propagate the intrinsic coagulation cascade in the absence of FVIII. FIX variants were expressed in FVIII-knockout (FVIII-KO) mice using a nonviral gene-transfer system. Expression of the variants shortened clotting times, reduced blood loss after tail-clip assay, and reinstalled clot formation, as tested by in vivo imaging of laser-induced vessel injury. In addition, we confirmed the therapeutic efficacy of FIX variants in mice with inhibitory Abs against FVIII. Further, mice tolerant to wild-type human FIX did not develop immune responses against the protein variants. Our results therefore indicate the feasibility of using variants of FIX to bypass FVIII as a novel treatment approach in hemophilia with and without neutralizing FVIII Abs.

A comparative study of the effects of temperature, time and factor VIII assay type on factor VIII activity in cryoprecipitate in Iran.

BACKGROUND: In Iran, cryoprecipitate is an important plasma product to provide coagulation factors such as factor VIII (FVIII) in patients with factor VIII deficiency. FVIII is one of the labile coagulation factors and as such is also used as a quality marker of fresh-frozen plasma and cryoprecipitate. It is, therefore, important to optimise plasma production in order to prevent a reduction of FVIII activity. In this study we assessed the effect of temperature, time and FVIII assay type on FVIII activity in cryoprecipitate produced in Iran. METHODS: Ninety-six whole blood units were kept at two different temperatures (48 units kept at 1-6 °C and 48 kept at 20-24 °C) for periods of 4, 6, 8 or 10 hours before plasma freezing. FVIII activity was then measured by both chromogenic and one-stage clotting assays. RESULTS: At both temperatures, FVIII activity in plasma prepared after 8 and 10 hours was lower than that in plasma prepared after 4 and 6 hours. A significant decrease of FVIII activity was not seen in samples kept for 4 and 6 hours. Compared to storage between 1-6 °C, storage at 20-24 °C appears to cause a reduction in FVIII activity. There was a significant difference in apparent FVIII activity measured by the one-stage clot-based and chromogenic assays. CONCLUSION: In Iran, to improve cryoprecipitate quality, freezing should begin within 6 hours after donation and whole blood should be kept at 1-6 °C until the plasma can be frozen. In this study although a good correlation was seen between the results of the one-stage clot-based and chromogenic assays for measuring FVIII activity in cryoprecipitate, the absolute values were significantly different.

The anticoagulant effect of protamine sulfate is attenuated in the presence of platelets or elevated factor VIII concentrations.

BACKGROUND: Protamine sulfate is the antidote for heparin, but in excess it exerts weak anticoagulation. METHODS: We evaluated the effects of increasing protamine concentrations (0 to 24 microg/mL) on prothrombin time and diluted Russell's viper venom time measurements on thrombin generation in platelet-poor and platelet-rich plasma after activation by tissue factor or actin, and on thromboelastometry in platelet-poor plasma and whole blood from 6 healthy volunteers. The reversibility of excess protamine (24 microg/mL) by recombinant factor VIIa or factor VIII/von Willebrand factor concentrate was also tested. RESULTS: Protamine prolonged prothrombin time and Russell's viper venom time, concentration dependently. Protamine also increased lag time and decreased peak of thrombin generation in platelet-poor plasma after tissue factor and actin activation. In platelet-rich plasma with platelets at 50 to 200 x 10(3)/microL, protamine (24 microg/mL) prolonged the lag time, but had no effect on peak thrombin generation. The addition of factor VIII/von Willebrand factor (1.5-3.0 U/mL) to platelet-poor plasma with protamine (24 microg/mL) decreased lag time and increased peak thrombin generation with actin activation. A therapeutic concentration of recombinant factor VIIa (60 nM) only affected the lag time of thrombin generation triggered with actin. In agreement, protamine increased coagulation time evaluated by thromboelastometry significantly more in platelet-poor plasma than in whole blood. CONCLUSIONS: We demonstrated that protamine affects the propagation of thrombin generation, which is partially reversed by platelets or increased factor VIII/von Willebrand factor concentrations. The present data suggest that excess protamine might potentially increase bleeding in the case of severe thrombocytopenia or low factor VIII.