Factor VIII Procoagulant Activity, Factor VIII Related Antigen and von Willebrand Factor in Newborn Cord Blood

The mean level of factor VIII procoagulant acitivity (VIII:C) and factor VIII related antigen (VIIIR:AG) was normal in 100 newborn cord plasmas, whereas that of von Willebrand factor (VIIIR:WF) activity was slightly lower than normal. On crossed immunoelectrophoresis, 20 of 50 newborn infants had an increased anodal mobility of VIIIR:AG. When the cord plasma showing an abnormal electrophoretic pattern was mixed with normal plasma, two precipitation peaks with a broad base were found. Similar mixing experiments with the abnormal cord plasma and plasma from a patient with atypical von Willebrand's disease did not normalize the electrophoretic mobility of VIIIR:AG. Gel filtration of the cord plasma with an abnormal electrophoretic pattern of VIII:AG, showed that the three activities were all detected at the position corresponding to a molecular weight of about 800 000. The results suggest the presence of qualitative abnormalities of the factor VIII molecule in half of full-term newborn cord plasma.     

Factor VIII/von Willebrand factor in subendothelium mediates platelet adhesion

The present studies were undertaken to determine whether factor VIII/von Willebrand factor (vWF) present in the vessel wall (in addition to that in plasma) may mediate the attachment of platelets to subendothelium. Subendothelium from everted rabbit aorta was exposed to human citrated blood flowing through an annular perfusion chamber at 40 mL/min (wall shear rate of 2,600 s-1 for five minutes). The vessel segments were incubated at 37 degrees C for one hour with various dilutions of either goat-anti-rabbit factor VIII/vWF serum or an IgG fraction prepared from the serum. Control segments were incubated with serum or IgG from a nonimmunized goat. Values of platelet contact (C), platelet adhesion (C + S), and thrombus formation (T) on the subendothelium were evaluated by a morphometric technique. Compared with vessels incubated with fractions prepared from a normal goat, a significant decrease in platelet adhesion (C + S), ranging from 45% to 65%, was observed on vessels incubated with various dilutions (1:5 to 1:50) of either serum or IgG fractions of goat-anti-rabbit factor VIII/vWF. A similar decrease in platelet adhesion was observed with vessels incubated with an F(ab')2 fragment against rabbit factor VIII/vWF prepared in the goat. When goat-anti-rabbit factor VIII/vWF IgG was added to rabbit blood (1:75 dilution), platelet adhesion was reduced to the same extent (65%) on normal rabbit vessels and on vessels pre-incubated with goat-anti-rabbit factor VIII/vWF. Immunofluorescence studies revealed the presence of rabbit factor VIII/vWF in the subendothelium of rabbit aorta and the continued binding of the goat-anti-factor VIII/vWF antibodies on subendothelium during the perfusion studies. No uptake of human factor VIII/vWF on the rabbit subendothelium was observed by this immunologic technique; human factor VIII/vWF was found to be entirely associated with the attached human platelets. Thus, factor VIII/vWF in the vessel wall may mediate platelet attachment to subendothelium in a manner similar to that of plasma factor VIII/vWF.     

Factor VIII/von Willebrand factor binding to von Willebrand''s disease platelets

A form of von Willebrand's disease has been described with enhanced ristocetin-induced platelet aggregation and anodal migration of the factor VIII/von Willebrand factor protein (type IIb). We studied two families with this form of von Willebrand's disease and macrothrombocytopenia. We have found that these platelets bind more of the normal and intermediate-sized multimers of the factor VIII/von Willebrand factor than normal platelets. Analysis of the binding data show an increased affinity of these vWd platelets for the factor VIII/von Willebrand factor. These findings are consistent with an increased number of platelet receptors, which, either by their native topography or migration on the platelet surface, bind factor VIII/von Willebrand factor protein with greater affinity than normal platelets, platelets of other vWd patients, and large platelets of other etiologies.     

Factor VIII and von Willebrand factor changes during normal pregnancy and puerperium.

Gestation is a challenge to haemostasis and it is associated with significant haemostatic changes. Several studies have evaluated von Willebrand factor in normal pregnancy, but none considered the personal history of bleeding. We studied a group of healthy non-bleeding women (184 pregnant, 64 puerperium, 37 non-pregnant) to evaluate normal ranges and their relationship to blood group and parity. The von Willebrand factor increased markedly from non-pregnant values up to the end of early puerperium (P < 0.0001), while factor VIII only showed a slight increase. Factor VIII and von Willebrand factor activity remained within the normal range for non-pregnant women. The return to non-pregnant factor levels occurred in late puerperium, later than previously reported. Only factor VIII was significantly lower in the O blood group (P = 0.035). As regards parity, there were no differences in factor VIII, von Willebrand factor antigen and von Willebrand factor ristocetin cofactor between primigravidae and multigravidae for any period studied (P = 0.888, 0.999, and 0.237, respectively). Our results provide reference ranges that may help to design a study in von Willebrand factor disease in pregnancy.     

Activated Factor VIII inTherapeutic Preparations:Analysis by Ultracentrifugation

Activation of factor VIII is signaled by an increase in the 1-stage factor VIII activity and release from von Willebrand factor. Ultracentrifugation in 10–40% sucrose gradients was used to identify such activation in therapeutic concentrates. Plasma-derived factor VIII lots were examined and factor VIII sedimenting independently of von Willebrand factor was identified in some of the preparations. In addition there was slower sedimentation of factor VIII by the 1-stage assay than by the chromogenic assay. These results are consistent with a factor VIII cleaved at residue 1689, a site important for von Willebrand binding. This activated form leads to some of the assay discrepancies between the 1-stage assay and the chromogenic or the 2-stage assay. There is more rapid sedimentation of the factor VIII measured by the chromogenic assay than the von Willebrand factor in some manufacturers' samples indicating that the method of fractionation may select a low molecular weight von Willebrand factor which does not bind factor VIII. Routine comparison between the 1-stage and chromogenic assays during fractionation may be able to identify such activated preparations. Other assay discrepancies may be due to structural differences between the standards and the tested product.     

Factor VIII accelerates proteolytic cleavage of von Willebrand factor by ADAMTS13

Proteolytic processing of von Willebrand factor (VWF) by ADAMTS13 metalloproteinase is crucial for normal hemostasis. In vitro, cleavage of VWF by ADAMTS13 is slow even at high shear stress and is typically studied in the presence of denaturants. We now show that, under shear stress and at physiological pH and ionic strength, coagulation factor VIII (FVIII) accelerates, by a factor of approximately 10, the rate of specific cleavage at the Tyr(1605)-Met(1606) bond in VWF. Multimer analysis reveals that FVIII preferentially accelerates the cleavage of high-molecular-weight multimers. This rate enhancement is not observed with VWF predenatured with 1.5 M guanidine. The ability of FVIII to enhance VWF cleavage by ADAMTS13 is rapidly lost after pretreatment of FVIII with thrombin. A FVIII derivative lacking most of the B domain behaves equivalently to full-length FVIII. In contrast, a derivative lacking both the B domain and the acidic region a3 that contributes to the high-affinity interaction of FVIII with VWF exhibits a greatly reduced ability to enhance VWF cleavage. Our data suggest that FVIII plays a role in regulating proteolytic processing of VWF by ADAMTS13 under shear stress, which depends on the high-affinity interaction between FVIII and its carrier protein, VWF.     

Factor VIII and von Willebrand factor interaction: biological, clinical and therapeutic importance

Summary.  The interaction of factor VIII (FVIII) with von Willebrand Factor (VWF) is of direct clinical significance in the diagnosis and treatment of patients with haemophilia A and von Willebrand disease (VWD). A normal haemostatic response to vascular injury requires both FVIII and VWF. It is well-established that in addition to its role in mediating platelet to platelet and platelet to matrix binding, VWF has a direct role in thrombin and fibrin generation by acting as a carrier molecule for the cofactor FVIII. Recent studies show that the interaction affects not only the biology of both FVIII and VWF, and the pathology of haemophilia and VWD, but also presents opportunities in the treatment of haemophilia. This review details the mechanisms and the molecular determinants of FVIII interaction with VWF, and the role of FVIII–VWF interaction in modulating FVIII interactions with other proteases, cell types and cellular receptors. The effect of defective interaction of FVIII with VWF as a result of mutations in either protein is discussed.     

Binding of Low-Molecular-Weight Canine Factor VIII Coagulant from von Willebrand Plasma to Canine Factor VIII-Related Antigen

S ummary . Plasmas having no detectable factor VIII-related antigen but moderate factor VIII coagulant were obtained from two unrelated dogs homozygous for von Willebrand's disease and with the severe clinical expression of the disease. When these plasmas were gel-filtered in a buffer at physiologic ionic strength, the factor VIII coagulant eluted in the bed volume as a single well-defined peak. Addition of protease inhibitors, including diisopropylfluorophosphate, did not change the elution pattern. Each plasma was then combined individually with plasmas from six different mutants of canine haemophilia, all of which had normal factor VIII-related antigen but no detectable factor VIII coagulant. The factor VIII coagulant elution profile of these combined plasma resembled that of normal canine plasma. Slightly over half of the recovered factor VIII coagulant coeluted in the void volume with the factor VIII-related antigen; the rest eluted as a second, distinct peak of lower molecular weight. These results demonstrated that part of the factor VIII coagulant of the von Willebrand plasmas had bound to the factor VIII-related antigen of the haemophilic plasmas. This finding supports the theory that factor VIII exists as a macromolecular complex of nonidentical components in normal citrated plasma.     

Response of Factor VIII/von Willebrand Factor to DDAVP in Healthy Subjects and Patients with Haemophilia A and von Willebrand's Disease

S ummary . These studies were designed with the purpose of providing clinico-pharmacological information relevant to the use of DDAVP in the management of mild haemophilia and von Willebrand's disease (VWD). In healthy subjects, intravenous DDAVP produced its maximal response at a dose of 0.3 μg/kg. The extent of the increase in factor VIII coagulant activity (VIII:C) and factor VIII related antigen (VIIIR:Ag) induced by this dose was not significantly different from that observed with the same dose in haemophiliacs and VWD patients. In these, the bleeding time was not shortened. DDAVP given intranasally was followed by a two-fold increase of VIII:C. This route of administration might be adopted to provide an emergency aid in bleeding patients and to yield higher VIII:C levels in blood donors. In healthy subjects, the half-disappearance time of autologous VIII:C after increase induced by i.v. DDAVP is similar to that observed in patients with VWD treated in the same conditions, whereas the response appears to be more prolonged in haemophiliacs. This study shows that the consistency of the VIII:C response tends to decrease when repeated doses are given to healthy subjects. Repeatedly-treated haemophiliacs and VWD patients showed varied patterns, ranging from no change of the response to its early abolishment.     

Alterations of Factor VIII von Willebrand Factor in Clinical Conditions Associated with an Increase in its Plasma Concentration

Factor VIII-related antigen (VIIIR:Ag) was consistently higher than factor-VIII procoagulant activity (VIII:C) in 57 patients with clinical conditions characterized by acute-phase reactions. Two different methods for measuring VIII:C (one- and two-stage assays) and VIIIR:Ag (electroimmunodiffusion and immunoradiometric assay) gave concordant results in the majority of cases. In 43% of plasma samples, crossed immunoelectrophoresis in agarose gel was characterized by the appearance of an additional, fast-moving precipitin peak which was immunologically identical with the major, slower-moving VIIIR:Ag peak. The fast-moving peak was detected in all the patients with clinical conditions typically associated with increased plasma proteolysis (DIC, acute pancreatitis, during thrombolytic therapy). It was present in a smaller proportion of cases with liver and renal failure and malignancies and in the post-operative period. The additional VIIIR:Ag peak is thought to be the result of in vivo factor VIII/von Willebrand factor fragmentation by proteolytic enzymes.     

Isolation of small molecular forms of Factor VIII/von Willebrand factor from plasma

Cryoprecipitated factor VIII/von Willebrand factor (FVIII/vWF), freed of fibrinogen by clotting with calcium and Defibrase, was chromatographed on Sepharose CL-2B. Fractions containing lower-molecular-weight forms of FVIII/vWF comprised coprecipitated plasma proteins of similar molecular weights. The major contaminants, fibronectin and IgM, were removed by affinity chromatography on gelatin- and anti-IgM-agarose, respectively. Finally, pure low-molecular-weight FVIII/vWF protein was harvested in the void volume fraction of a Sepharose CL-6B column. The smallest multimers had the size of the tetramer of the basic subunit chain of FVIII/vWF.     

Factor VIII inhibitors: role of von Willebrand factor on the uptake of factor VIII by dendritic cells

Summary  In patients with haemophilia A, factor VIII (FVIII) therapy leads to the development of anti-FVIII alloantibodies that inhibit FVIII pro-coagulant activity, in up to 25% of the cases. At a time when efficient viral screening procedures are at place, development of inhibitors poses the greatest threat to haemophilia A patients. Various risk factors, both patient and product-related, are responsible for the development of inhibitory antibodies. The role of FVIII-specific CD4+ T lymphocytes in the initiation of the humoral immune response to exogenous FVIII has been well. In view of their capacity to stimulate naïve T cells, dendritic cells (DCs) play a central role in the initiation of the primary immune response. Thus, in the context of a primary alloimmunization against FVIII, i.e. when FVIII-specific B lymphocytes are not there to take up FVIII from the circulation and to serve as antigen presenting cells (APCs), DCs are the only cell type that internalize FVIII, leading to activation of FVIII-specific CD4+ T lymphocytes. von Willebrand factor (VWF) present in plasma-derived FVIII therapeutic concentrates, is known to act as a chaperone molecule for procoagulant FVIII. In addition to its role in reducing the 'antigenicity' of FVIII, the role of VWF in the reduction of the 'immunogenicity' of therapeutic FVIII in patients with haemophilia A has also been suggested. We have recently demonstrated that VWF protects FVIII from being endocytosed by human DCs and subsequently being presented to FVIII-specific T cells. We propose that VWF may reduce the immunogenicity of FVIII by preventing, upstream from the activation of immune effectors, the entry of FVIII in professional antigen presenting cells.     

Optimizing therapy with factor VIII/von Willebrand factor concentrates in von Willebrand disease

In von Willebrand disease, the main goals of treatment are to correct the dual defect of haemostasis caused by a reduced or abnormal von Willebrand factor (vWF), i.e. the prolonged bleeding time (BT) and the deficiency of factor VIII coagulant activity (FVIII:C). The synthetic vasopressin analogue, desmopressin (DDAVP), has reduced the need for transfusions in most of the mild forms of von Willebrand disease but DDAVP is ineffective in type 3 and in other severe cases of types 1 and 2 von Willebrand disease. For many years cryoprecipitate has been the mainstay of replacement therapy but, after the introduction of virucidal methods, concentrates containing FVIII/vWF have been considered much safer than cryoprecipitate and proposed in von Willebrand disease management. FVIII/vWF concentrates have been produced and tested by many authors but there is only one report describing four virus-inactivated FVIII/vWF concentrates evaluated in a cross-over randomized trial. According to these in vitro and pharmacokinetic data, the following information can be derived: (a) no FVIII/vWF concentrate had an intact multimeric structure similar to that of normal plasma or of cryoprecipitate; (b) all FVIII/vWF concentrates were equally effective in attaining normal and sustained levels of FVIII:C postinfusion, although peak levels were more delayed in the concentrate devoid of FVIII:C; (c) no FVIII/vWF concentrate consistently normalized the BT in a sustained fashion. On the other hand, clinical haemostasis can be achieved in the management of bleeding episodes and of surgery for most of von Willebrand disease cases regardless of whether the BT is corrected; in the few rare cases with mucosal bleeding not controlled by FVIII/vWF concentrates, infusion of DDAVP or platelet concentrates can be administered in addition.     

Factor VIII alters tubular organization and functional properties of von Willebrand factor stored in Weibel-Palade bodies

In endothelial cells, von Willebrand factor (VWF) multimers are packaged into tubules that direct biogenesis of elongated Weibel-Palade bodies (WPBs). WPB release results in unfurling of VWF tubules and assembly into strings that serve to recruit platelets. By confocal microscopy, we have previously observed a rounded morphology of WPBs in blood outgrowth endothelial cells transduced to express factor VIII (FVIII). Using correlative light-electron microscopy and tomography, we now demonstrate that FVIII-containing WPBs have disorganized, short VWF tubules. Whereas normal FVIII and FVIII Y1680F interfered with formation of ultra-large VWF multimers, release of the WPBs resulted in VWF strings of equal length as those from nontransduced blood outgrowth endothelial cells. After release, both WPB-derived FVIII and FVIII Y1680F remained bound to VWF strings, which however had largely lost their ability to recruit platelets. Strings from nontransduced cells, however, were capable of simultaneously recruiting exogenous FVIII and platelets. These findings suggest that the interaction of FVIII with VWF during WPB formation is independent of Y1680, is maintained after WPB release in FVIII-covered VWF strings, and impairs recruitment of platelets. Apparently, intra-cellular and extracellular assembly of FVIII-VWF complex involves distinct mechanisms, which differ with regard to their implications for platelet binding to released VWF strings.     

In vitro and in vivo Characterization of Factor VIII Preparations

Abstract. An in vitro and in vivo comparison of nine commercial and noncommercial factor VIII preparations was made. These consisted of one lyophilized cryoprecipitate, four intermediate (IPC) and four high purity concentrates (HPC). Protein, fibrinogen, factor VIII complex, IgG, IgM and anti-A and B alloagglutinins levels were measured. These three qualities of product were defined by two ratios: units of F VIII: C per mg of protein and per mg of fibrinogen. They were, respectively, <0.5 and < l in cryoprecipitate, 0.5-1 and 1–3 in IPC, and >1 and >3 in HPC. The F VIII:C/F VIII:AG ratio ranged from 0.3 to 0.6 and the F VIII: C/F VIII: VWF ratio was always lower than 1. Varying titers of alloagglutinins were found, unrelated to either IgG or IgM levels. Seven of these preparations were injected into several classical hemophilia A patients for treatment of minor hemorrhages. The peak of F VIII: C activity was always found 1 h postinjection. The F VIII: C recovery ranged from 80 to 140% and the half-life from 8 to 15 h. No significant difference was found among these products and the clinical efficacy was similar.     

血管性血友病因子在心血管疾病中的研究进展

血管性血友病因子是一类主要由内皮细胞分泌的多聚糖蛋白,通过激活血小板,参与血栓形成。它与一系列心血管疾病如动脉粥样硬化、急性冠状动脉综合征、心房颤动等均关系密切,特异性抑制血管性血友病因子的新型抗血小板药物研发有望突破。     

Factor VIII and von Willebrand factor are ligands for the carbohydrate-receptor Siglec-5

Background

Factor VIII (FVIII) and von Willebrand factor (VWF) circulate in plasma in a tight non-covalent complex, being critical to hemostasis. Although structurally unrelated, both share the presence of sialylated glycan-structures, making them potential ligands for sialic-acid-binding-immunoglobulin-like-lectins (Siglecs).

Design and Methods

We explored the potential interaction between FVIII/VWF and Siglec-5, a receptor expressed in macrophages using various experimental approaches, including binding experiments with purified proteins and cell-binding studies with Siglec-5 expressing cells. Finally, Siglec-5 was overexpressed in mice via hydrodynamic gene transfer.

Results

In different systems using purified proteins, saturable, dose-dependent and reversible interactions between a soluble Siglec-5 fragment and both hemostatic proteins were found. Sialidase treatment of VWF resulted in a complete lack of Siglec-5 binding. In contrast, sialidase treatment left interactions between FVIII and Siglec-5 unaffected. FVIII and VWF also bound to cellsurface exposed Siglec-5, as was visualized by classical immunostaining as well as by Duolinkproximity ligation assays. Co-localization of FVIII and VWF with early endosomal markers further suggested that binding to Siglec-5 is followed by endocytosis of the proteins. Finally, overexpression of human Siglec-5 in murine hepatocytes following hydrodynamic gene transfer resulted in a significant decrease in plasma levels of FVIII and VWF in these mice.

Conclusions

Our data indicate that FVIII and VWF may act as a ligand for Siglec-5, and that Siglec-5 may contribute to the regulation of plasma levels of the FVIII/VWF complex.Key words: factor VIII, von Willebrand factor, ligand, Siglec-5, interaction     

von Willebrand factor containing factor VIII concentrates

There are several plasma derived von Wille-brand factors (vWF) containing factor (FVIII) concentrates that can be used in the treatment of von Willebrand disease (vWD). All concentrates are effective in attaining normal postinfusion levels or of FVIII:C but it is difficult to achieve normalization of the bleeding time even with concentrates containing almost all vWF multimers including those of high molecular weight. Haemate P (Centeon) may be considered as the golden standard concentrate available at present. However, the development of more purified vWF concentrates devoid of FVIII:C is the goal for future development.