Age-related alterations in immune responses to West Nile virus infection.

West Nile virus (WNV) is the most important causative agent of viral encephalitis worldwide and an important public health concern in the United States due to its high prevalence, severe disease, and the absence of effective treatments. Infection with WNV is mainly asymptomatic, but some individuals develop severe, possibly fatal, neurological disease. Individual host factors play a role in susceptibility to WNV infection, including genetic polymorphisms in key anti-viral immune genes, but age is the most well-defined risk factor for susceptibility to severe disease. Ageing is associated with distinct changes in immune cells and a decline in immune function leading to increased susceptibility to infection and reduced responses to vaccination. WNV is detected by pathogen recognition receptors including Toll-like receptors (TLRs), which show reduced expression and function in ageing. Neutrophils, monocyte/macrophages and dendritic cells, which first recognize and respond to infection, show age-related impairment of many functions relevant to anti-viral responses. Natural killer cells control many viral infections and show age-related changes in phenotype and functional responses. A role for the regulatory receptors Mertk and Axl in blood-brain barrier permeability and in facilitating viral uptake through phospholipid binding may be relevant for susceptibility to WNV, and age-related up-regulation of Axl has been noted previously in human dendritic cells. Understanding the specific immune parameters and mechanisms that influence susceptibility to symptomatic WNV may lead to a better understanding of increased susceptibility in elderly individuals and identify potential avenues for therapeutic approaches: an especially relevant goal, as the world's populating is ageing.

Normal range of bleeding scores for the ISTH-BAT: adult and pediatric data from the merging project.

Bleeding Assessment Tools (BATs) have been developed to aid in the standardized evaluation of bleeding symptoms. The Vicenza Bleeding Questionnaire (BQ), published in 2005, established a common framework and scoring key that has undergone subsequent modification over the years, culminating in the publication of the ISTH-BAT in 2010. Understanding the normal range of bleeding scores is critical when assessing the utility of a BAT. Within the context of The Merging Project, a bioinformatics system was created to facilitate the merging of legacy data derived from four different (but all Vicenza-based) BATs; the MCMDM1-VWD BQ, the Condensed MCMDM-1VWD BQ, the Pediatric Bleeding Questionnaire and the ISTH-BAT. Data from 1040 normal adults and 328 children were included in the final analysis, which showed that the normal range is 0-3 for adult males, 0-5 for adult females and 0-2 in children for both males and females. Therefore, the cut-off for a positive or abnormal BS is ≥4 in adult males, ≥6 in adult females and ≥3 in children. This information can now be used to objectively assess bleeding symptoms as normal or abnormal in future studies.

Intravascular recovery of VWF and FVIII following intraperitoneal injection and differences from intravenous and subcutaneous injection in mice.

Intravenous infusion studies in humans suggest that both von Willebrand factor (VWF) and factor VIII (FVIII) remain intravascular in contrast to other coagulation proteins. We explored whether infusion of VWF and FVIII by either intraperitoneal (i.p.) or subcutaneous (s.c.) injection would result in efficient absorption of these large proteins into the vascular circulation. FVIII(null) or VWF(null) mice were infused with plasma-derived or recombinant VWF and/or FVIII by i.p., s.c., or intravenous (i.v.) injection. Both VWF and FVIII were absorbed into the blood circulation after i.p. injection with a peak between 2 and 4 h at levels similar to those observed in mice infused intravenously. In contrast, neither VWF nor FVIII was detected in the plasma following s.c. injection. Although i.v. injection achieved peak plasma levels quickly, both human VWF and FVIII rapidly decreased during the first 2 h following i.v. injection. Following both i.v. and i.p. infusion of VWF, the multimeric structure of circulating VWF was similar to that observed in the infusate. These results demonstrate that both VWF and FVIII can be efficiently absorbed into the blood circulation following i.p., but not s.c. injection, indicating that i.p. administration could be an alternative route for VWF or FVIII infusion.

Direct demonstration of antigenic substitution of Borrelia burgdorferi ex vivo: exploration of the paradox of the early immune response to outer surface proteins A and C in Lyme disease.

The outer surface proteins (Osps) of Borrelia burgdorferi, the etiologic agent of Lyme disease, are principle targets of protective immune responses against this organism. Whereas most North American strains of B. burgdorferi in culture express an abundant amount of Osp A, antibodies to this protein are either absent or only weakly detected in the sera of naturally infected patients or experimentally infected mice. In contrast, Osp C, which has variable expression on cultured organisms; elicits an early, strong humoral response. To examine this paradox, we have studied the in vivo adaptation of a cloned population of B. burgdorferi strain N40 during the early course of experimental murine borreliosis. As in human disease, antibodies to Osp A were only weakly present in the early immune repertoire after murine inoculation with low dose (10(3)) spirochetes. In contrast, antibodies to Osp C were prominent, even though on cultured spirochetes Osp C mRNA and protein expression could not be detected by reverse transcription polymerase chain reaction (RT-PCR) or indirect immunofluorescence, respectively. These observations led us to investigate the expression of Osp A and Osp C in vivo. By direct fluorescent staining of uncultured spirochetes ex vivo and by PCR amplification of spirochetal mRNA, we show that Osp C is indeed expressed by some spirochetes after infection in the mouse. Spirochetes expressing Osp A could also be detected within the first 2 wk of infection, but not at 30 d. Osp A mRNA, although present at day 14 of infection, could not be amplified by RT-PCR at day 30, suggesting that the expression of this Osp is transient. This further implies that the late burst in Osp A antibodies in both mice and humans may be anamnestic. These results indicate that either Osp C is upregulated on spirochetes after infection, or Osp C-expressing spirochetes expand preferentially over those expressing Osp A during infection. These results have important implications for vaccine design and offer one explanation for the failure of Osp A antibodies to eradicate spirochetes from the infected host.

Unique strategies for therapeutic gene transfer in haemophilia A and haemophilia BWFH State-of-the-Art Session on Therapeutic Gene Transfer Buenos Aires, Argentina.

SUMMARY: Gene therapy of haemophilia has been initiated through a number of approaches including expression in muscle, liver and omental implanted fibroblasts, or i.v. injection of an expression construct under the control of a ubiquitous promoter. In all these approaches, the goal was to have factor VIII (FVIII) or factor IX (FIX) synthesized so that it restored the levels of the missing protein in blood. The three talks in this session are totally, or at least in part, directed at strategies that may be clinically effective even in the absence of correction of the missing plasma clotting factor, although the haematopoietic stem cell or blood outgrowth endothelial cell therapy could achieve plasma correction as well. Two of the approaches achieve localized coagulation factor expression without necessarily correcting the systemic defect--one is with synthesis of FVIII or FIX within the joint space and the other is with the local release of FVIII (or FIX) by platelets at the site of vascular injury. All of the three approaches have demonstrated efficacy in small animal models and are now the subject of larger animal studies. None has yet to progress to human trials.

von Willebrand's disease diagnosis and laboratory issues.

SUMMARY: In this paper, the recent developments in the diagnosis and laboratory issues of von Willebrand's disease (VWD) are presented. Dr. Castaman reviews the functional tests available for the diagnosis of VWD and their pathophysiological significance, focusing on which tests are best used in the diagnosis and classification of VWD. Dr Montgomery reviews an emerging issue that is accelerated clearance of von Willebrand factor (VWF) occurring in some variants of VWD. This phenotype can be suspected by the presence of an increased ratio between the VWF propeptide and the VWF antigen. These patients have typically a robust, but short-lived increase of FVIII and VWF after desmopressin. Dr Meschengieser reviews the determinants of bleeding after surgery in patients with VWD, emphasizing the role of bleeding history in predicting this risk.

On the association of glycoprotein Ib and actin-binding protein in human platelets.

Glycoprotein (GP) Ib was purified from lysates of human platelets prepared in the presence or absence of inhibitors of the endogenous calcium-activated neutral protease (CANP) by immunoaffinity chromatography, employing the GPIb-specific murine monoclonal antibody, AP1, coupled to Sepharose CL4B. When derived from lysates prepared in the presence of EDTA or leupeptin, the eluate from the AP1-affinity column contained a 240,000-260,000-mol-wt protein in addition to GPIb. In SDS PAGE, this protein was stained by Coomassie Blue R, but not by the periodic acid-Schiff reagent, and it was not labeled with 125I in intact platelets by the lactoperoxidase-catalyzed method. When derived from lysates prepared in the absence of CANP inhibitors, the eluate contained only GPIb and its proteolytic derivative, glycocalicin. A change in the electrophoretic mobility of GPIb consistent with its association with the 240,000-260,000-mol-wt protein was confirmed by crossed immunoelectrophoresis. By an immunoblot technique involving transfer of proteins eluted from the AP1-affinity column and separated by SDS PAGE onto a nitrocellulose membrane, the 240,000-260,000-mol-wt protein bound polyclonal goat antibody raised against rabbit macrophage actin-binding protein (ABP). On the basis of these results, we conclude the GPIb is tightly associated with ABP under conditions in which the endogenous CANP is inhibited, and that this apparent transmembrane complex of GPIb-ABP can be isolated in lysates of nonactivated human platelets.

Synthesis by cultured human umbilical vein endothelial cells of two proteins structurally and immunologically related to platelet membrane glycoproteins IIb and IIIa.

Human platelets participate in a number of adhesive interactions, including binding to exposed subendothelium after vascular injury, and platelet-platelet cohesion to form large aggregates. Platelet membrane glycoproteins (GP) IIb and IIIa constitute a receptor for fibrinogen that, together with fibrinogen and calcium, is largely responsible for mediating the formation of the primary hemostatic plug. Using highly specific polyclonal and monoclonal antibodies as probes, we could detect the presence of both of these glycoproteins in cultured human umbilical vein endothelial cells. Western-blot analysis showed that the endothelial cell analogues were similar in size to their platelet counterparts, and were present in cells that had been in culture for over 2 mo. Metabolic labeling of endothelium with [35S]methionine demonstrated that both GPIIb and GPIIIa were actively synthesized in culture. Using the technique of crossed immunoelectrophoresis, evidence was obtained that the endothelial cell forms of GPIIb and GPIIIa may exist complexed to one another after solubilization in Triton X-100. The presence of GPIIb-IIIa analogues in cultured endothelial cells may provide an opportunity to examine the structure, function, and synthesis of these two membrane glycoproteins, as well as provide a source of genetic material with which to begin detailed molecular genetic studies.

Toxic gases from fires.

The major lethal factors in uncontrolled fires are toxic gases, heat, and oxygen deficiency. The predominant toxic gas is carbon monoxide, which is readily generated from the combusion of wood and other cellulosic materials. Increasing use of a variety of synthetic polymers has stimulated interest in screening tests to evaluated the toxicity of polymeric materials when thermally decomposed. As yet, this country lacks a standardized fire toxicity test protocol.

von Willebrand disease (VWD): evidence-based diagnosis and management guidelines, the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel report (USA).

von Willebrand disease (VWD) is a commonly encountered inherited bleeding disorder affecting both males and females, causing mucous membrane and skin bleeding symptoms, and bleeding with surgical or other haemostatic challenges. VWD may be disproportionately symptomatic in women of child-bearing age. It may also occur less frequently as an acquired disorder (acquired von Willebrand syndrome). VWD is caused by deficiency or dysfunction of von Willebrand factor (VWF), a plasma protein that mediates platelet haemostatic function and stabilizes blood coagulation factor VIII. The pathophysiology, classification, diagnosis and management of VWD are relatively complex, but understanding them is important for proper diagnosis and management of patients with VWD. These evidence-based guidelines for diagnosis and management of VWD from the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel (USA) review relevant publications, summarize current understanding of VWD pathophysiology and classification, and present consensus diagnostic and management recommendations based on analysis of the literature and expert opinion. They also suggest an approach for clinical and laboratory evaluation of individuals with bleeding symptoms, history of bleeding or conditions associated with increased bleeding risk. This document summarizes needs for further research in VWF, VWD and bleeding disorders, including clinical research to obtain more objective information about bleeding symptoms, advancements in diagnostic and therapeutic tools, and enhancement in the education and training of clinicians and scientists in bleeding and thrombotic disorders. The NHLBI Web site (http://www.nhlbi.nih.gov/guidelines/vwd) has a more detailed document, a synopsis of these recommendations, and patient education information.

Interaction of von Willebrand factor with human platelets in the plasma milieu.

The binding of von Willebrand factor (vWf) to stimulated platelets in the plasma milieu was performed using a radiolabeled monoclonal antibody to vWf. Plasma proteins specifically inhibited the thrombin- and ADP/epinephrine-induced vWf binding to activated platelets but did not inhibit the ristocetin-induced vWf binding. When normal plasma was heat defibrinated, monoclonal-labeled vWf was bound to platelets following thrombin or ADP/epinephrine stimulation. Furthermore, monoclonal-labeled vWf from afibrinogenemic plasma bound normally to platelets. The binding of vWf to stimulated platelets in either heat-defibrinated normal plasma or afibrinogenemic plasma was specifically inhibited by the addition of normal plasma fibrinogen in a concentration-dependent manner. At levels of fibrinogen less than 1 mg/ml, however, vWf binding could be demonstrated. The inhibition by fibrinogen of vWf binding to platelets was competitive and overcome by increased concentrations of vWf. These studies show that thrombin-induced and ADP/epinephrine-induced vWf binding to platelets does not occur in the plasma milieu, although at reduced levels of fibrinogen, vWf binding to stimulated platelets can be demonstrated.

von Willebrand's disease antigen II. A new plasma and platelet antigen deficient in severe von Willebrand's disease.

Factor VIII-related antigen (VIIIag) is deficient in plasma and platelets of patients with severe von Willebrand's disease. This study reports a second von Willebrand's disease antigen (vWagII), distinct from VIIIag, that is also deficient in the platelets and plasma of patients with severe von Willebrand's disease. VIIIag and vWagII are separable by molecular exclusion chromatography, sucrose density gradient ultracentrifugation, and crossed immunoelectrophoresis. They show reactions of immunologic nonidentity with each other, and thus, do not share a precursor-product relationship. vWagII is released from normal platelets during blood clotting, accounting for a fourfold higher concentration of vWagII in serum over plasma.

Evidence for reactive nitrogen intermediates in killing of staphylococci by human neutrophil cytoplasts. A new microbicidal pathway for polymorphonuclear leukocytes.

In anucleate, granule-poor, motile fragments from human blood neutrophils (cytokineplasts; CKP), the nitric oxide synthase inhibitor N omega-monomethyl-L-arginine (NMMA) produced a modest decrease in uptake of staphylococci from supernatants (P less than 0.02, n = 7), and a marked decrease in the killing of cytoplast-associated bacteria (P less than 0.001, n = 7). After 60 min of incubation with bacteria, NMMA-treated cytoplasts had a mean of over 3.5 times as many live, CKP-associated staphylococci as did controls (51% of the inocula versus 14%), despite having taken up fewer. Effects on both uptake and killing were reversible by L-arginine but not by D-arginine. Results were the same with other granule-poor cytoplasts (U-cytoplasts, U-CYT), which, unlike CKP, retain activatable oxidase activity. Killing by intact PMN, including those from a patient with chronic granulomatous disease, was not inhibited by NMMA. Thus, the ability to discern effects of NMMA correlated with the paucity of granules, without regard to the presence or absence of activatable oxidase. We propose that the generation of reactive nitrogen intermediates serves as an additional microbial killing pathway in PMN, and that cytoplasts can be used to help delineate the spectrum of susceptible targets.

Endothelial cell synthesis of von Willebrand antigen II, von Willebrand factor, and von Willebrand factor/von Willebrand antigen II complex.

von Willebrand antigen II (vW AgII) and von Willebrand factor (vWf) are immunochemically distinct proteins that are deficient in the plasma and platelets of patients with severe von Willebrand's disease. Normal human umbilical vein endothelial cells were cultured in the presence of [35S]methionine. Crossed immunoelectrophoresis of endothelial cell supernates and detergent-solubilized endothelial cells demonstrated specific incorporation of the [35S]methionine into vW AgII. Furthermore, when endothelial cells were lysed in the presence of proteolytic inhibitors, a second, less anodal peak was identified on crossed immunoelectrophoresis. This peak represented a complex of vW AgII and vWf and demonstrated a reaction of complete identity with the vW AgII immunoprecipitate. When plasma, serum, or platelets were evaluated by crossed immunoelectrophoresis, this "complex" peak was not present. When antibodies to vWf, fibronectin, or fibrinogen were present in the first dimension of crossed immunoelectrophoresis, only the antibodies to vWf removed the complex. Radioiodinated polyclonal and monoclonal antibodies to vWf also localized vWf to this complex. Under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled immunoprecipitates indicated that the molecular weight of vW AgII is 98,000 and that vWf was present as two species of 220,000 and 260,000 mol wt, respectively. Immunofluorescent microscopy of endothelial cells demonstrated colocalization of vW AgII and vWf in endothelial cells with intense immunostaining of the same subcellular granules.

Platelets have more than one binding site for von Willebrand factor.

The binding of 125I-von Willebrand factor (125I-vWF) to platelets stimulated by thrombin, ADP, and a combination of ADP + epinephrine (EPI) is specific, saturable, and reversible. Active platelet metabolism and divalent cations are required for binding induced by these stimuli, but not by ristocetin, suggesting the existence of different mechanisms involved in the vWF-platelet interaction. A monoclonal antibody directed against an epitope of membrane glycoprotein (GP) Ib had no effect on the binding of 125I-vWF to normal platelets stimulated by thrombin or a combination of ADP + EPI, but completely blocked ristocetin-induced binding. Binding induced by thrombin to GPIb-blocked platelets was specific. Moreover, thrombin-induced binding of 125I-vWF was increased, rather than decreased, in two patients with the Bernard-Soulier syndrome whose platelets lacked GPIb. Conversely, monoclonal antibodies directed against the GPIIb/IIIa complex had no effect on ristocetin-induced binding of 125I-v-WF to normal platelets, but blocked thrombin- and ADP + EPI-induced binding. To exclude effects mediated by the platelet Fc receptor, a monoclonal IgG directed against an epitope present on human B cells and monocytes, but not expressed on resting or stimulated platelets, was used. It did not affect 125I-vWF binding induced by any of the stimuli. These studies show that platelets have more than one binding site for vWF, and that they may be exposed by different stimuli.

Diagnosis of Bernard-Soulier syndrome and Glanzmann's thrombasthenia with a monoclonal assay on whole blood.

Two hereditary platelet disorders, Bernard-Soulier syndrome and Glanzmann's thrombasthenia, are characterized by selective deficiencies of platelet membrane glycoproteins. Murine monoclonal antibodies were developed against platelet membrane glycoprotein Ib and against the glycoprotein IIb/IIIa complex. A rapid whole blood assay for the deficiency of these glycoproteins was developed and used to study whole blood samples from six patients with Glanzmann's thrombasthenia and three patients with Bernard-Soulier syndrome. Patients with type I and type II Glanzmann's thrombasthenia were easily detectable with this assay. This permits the diagnosis of these disorders on 200 microliters of whole blood within 2 h of blood sampling.

Temporal pattern of Borrelia burgdorferi p21 expression in ticks and the mammalian host.

The temporal synthesis of the P21 protein of Borrelia burgdorferi and the development of the humoral response to this antigen was assessed in infected mice. p21 is a member of the ospE-F gene family and its protein, P21, has been shown to be expressed by B. burgdorferi within infected mice but not by spirochetes cultured in vitro. P21 was not detected on B. burgdorferi in unfed or engorged Ixodes dammini (also known as I. scapularis) ticks, further supporting the postulate that P21 synthesis is specific for the mammalian host. In B. burgdorferi-infected mice, ospE mRNA and OspE antibodies were observed at 7 d, whereas p21 mRNA and P21-specific antibodies were detected at 21-28 d, suggesting that p21 is expressed later than ospE. Moreover, ospA mRNA was not discernible until day 14, indicating that ospA, like p21, is not expressed in the early stages of tick-transmitted murine Lyme borreliosis. Because p21 is expressed during infection in mice, we assessed the human humoral response to P21. 28% (34 of 122) of the patients with either early- or late-stage Lyme disease, and 33% (11 of 33) of the individuals with Lyme arthritis had P21 antibodies, suggesting that a P21 response may serve, at least partially, as a marker of infection. Active immunization with recombinant P21 did not protect C3H mice from tick-borne B. burgdorferi infection, and passive transfer of P21 antiserum to infected mice did not alter the course of disease. These data suggest that the antigenic structure of B. burgdorferi changes during the early stages of murine infection.

Effects of monoclonal antibodies against the platelet glycoprotein IIb/IIIa complex on thrombosis and hemostasis in the baboon.

To assess the hemostatic consequences and antithrombotic effectiveness of blocking the platelet glycoprotein (GP) IIb/IIIa receptor for fibrinogen and other adhesive glycoproteins in vivo, well characterized murine monoclonal antibodies against the platelet GP IIb/IIIa complex, AP-2 and LJ-CP8, were infused intravenously into baboons. Four animals each received doses of 0.2, 0.4, and 1.0 mg/kg of purified AP-2 IgG, and three animals were given 1.0 mg/kg of the F(ab)2 fragment of AP-2. Five additional animals were given 10 mg/kg LJ-CP8 IgG. At the highest dose, radiolabeled AP-2 IgG bound to an average of 33,000 sites on the circulating platelets. Serial measurements included platelet count, bleeding time, platelet aggregation (induced by ADP, collagen, and gamma-thrombin), and 111In-platelet deposition onto Dacron vascular grafts. Bleeding times were markedly prolonged after injection of 1.0 mg/kg AP-2 IgG (19.2 +/- 3.4 min), 1.0 mg/kg AP-2 F(ab)2 (16.5 +/- 1.8 min), and 10 mg/kg LJ-CP8 (greater than 30 min) vs. control studies (4.6 +/- 0.2 min), and remained prolonged for 48 h. With each antibody platelet aggregation was initially reduced or absent, with partial recovery over 48 h in a manner that was inversely related to dose. AP-2, both whole IgG and F(ab)2 fragment, but not LJ-CP8, caused a dose-dependent reduction (20-46%) in the circulating platelet count over 24 h. Neither AP-2 nor LJ-CP8 caused a reduction in intraplatelet platelet factor 4, beta-thromboglobulin, or [14C]serotonin. Graft-associated platelet thrombus formation was reduced by 73% (1.0 mg/kg AP-2 IgG and 10 mg/kg LJ-CP8) and 53% (1.0 mg/kg AP-2 F(ab)2) relative to control values. In contrast, neither heparin (100 U/kg) nor aspirin (32.5 mg/kg twice a day) showed antithrombotic efficacy in this model. Thus, antibodies that functionally alter the platelet GP IIb/IIIa complex may produce immediate, potent, and transient, antihemostatic, and antithrombotic effects.

Attachment of Borrelia burgdorferi within Ixodes scapularis mediated by outer surface protein A.

Borrelia burgdorferi outer surface protein (Osp) A has been used as a Lyme disease vaccine that blocks transmission: OspA antibodies of immune hosts enter ticks during blood feeding and destroy spirochetes before transmission to the host can occur. B. burgdorferi produce OspA in the gut of unfed Ixodes scapularis ticks, and many spirochetes repress OspA production during the feeding process. This preferential expression suggests that OspA may have an important function in the vector. Here we show that OspA mediates spirochete attachment to the tick gut by binding to an I. scapularis protein. The binding domains reside in the central region and COOH-terminus of OspA. OspA also binds to itself, suggesting that spirochete-spirochete interactions may further facilitate adherence in the gut. OspA-mediated attachment in the tick provides a possible mechanism for how stage-specific protein expression can contribute to pathogenesis during the B. burgdorferi natural cycle.

Intracellular trafficking of factor VIII to von Willebrand factor storage granules.

In plasma, von Willebrand factor (vWf) associates with Factor VIII (FVIII); however, the site at which these proteins first interact has not been defined. Administration of 1-desamino-8-D-arginine vasopressin (DDAVP) causes a rapid, concomitant elevation in plasma levels of both vWf and FVIII, suggesting the existence of a DDAVP-releasable storage pool for both proteins. To determine whether vWf and FVIII can associate intracellularly and colocalize to storage vesicles, we transfected AtT-20 cells with vWf and FVIII expression plasmids. FVIII alone was not detectable within storage granules; however, transfection of vWf cDNA into the same cell caused FVIII to alter its intracellular trafficking and to undergo granular storage, colocalizing to the vWf-containing granules. In contrast, colocalization of FVIII was not observed when these cells were transfected with plasmids encoding defective FVIII-binding vWf mutants. Transfection of bovine endothelial cells with FVIII further demonstrated vesicular storage of FVIII with vWf in Weibel-Palade bodies. Since gene therapy of hemophilia A may ultimately target endothelium or hematopoietic stem cells, the interaction between vWf and FVIII within a secretory cell is important. Thus, vWf can alter the intracellular trafficking of FVIII from a constitutive to a regulated secretory pathway, thereby producing an intracellular storage pool of both proteins.